One theory of aging is that the genome and other DNA of an organism accumulate increasing numbers of errors with age and that these errors are responsible for the macroscopic phenomenon we call “aging.” See the second theory of aging covered in my treatise, Cell DNA Damage. After a discussion of background I focus in this blog entry on a special topic related to very-recent research news, the role of the sirtuin SIRT6 in assuring genomic stability. I also touch on other possible roles of SIRT6 in assuring longevity.

Background on genomic stability, aging and DNA repair

The DNA in a healthy organism is hardly static. “In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day.^[1] (ref )” If there are on the order of 30 trillion cells in the body, it takes a calculator with a lot more decimal places than mine has to show the number of such daily molecular DNA damage events. “Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell’s ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell’s genome, which affect the survival of its daughter cells after it undergoes mitosis(ref ).” So, the health of the genome is driven by the dynamic interaction between an ongoing onslaught of DNA damage on the one hand and the body’s DNA repair and cell-policing machinery on the other hand. Damage can include:

*  Double strand DNA breaks discussed in the blog entry DNA repair cleanup failure – a root cause for cancers. These are “breaks that can occur naturally in cell differentiation or that are created by radiation and certain chemicals.”  The current view is that most spontaneous chromosomal rearrangements result from DSBs created mainly during DNA replication as a result of broken, stalled or collapsed replication forks(ref).”

*  Single strand DNA breaks.  These breaks constitute the most commonly-occurring form of DNA damage and ares discussed in the April 2010 blog entry More on DNA repair strategies.

*  Aneuploidy– extra or missing chromosomes. Aneuploidy can occur during cell divisions

*  Errors introduced inthe DNA repair process itself or in the process of repair cleanup. Some of these are discussed in the two aforementioned blog entries.

*  Other types of DNA errors such as those listed here.

DNA damage can impede normal cell differentiation and division, can lead to cancers, Alzheimer’s Disease and numerous other diseases, and produce the phenotype of aging. Somatic mutation rate in Drosophila (fruit flies) correlates with aging(ref). Further, chromosomal and DNA damage is known in mice as well as Drosophila to increase with aging and vary by organ. For example, the amount of aneuploidy gain in chromosome 18 in mouse brains increases with age. (F. Faggioli, unpublished data). And, spontaneous mutation frequency in mouse intestines increases drastically with age but remains essentially flat with age in mouse spleen, testis and brains(ref).

The body has developed sophisticated mechanisms for detecting DNA damage and DNA repair. Discussion of some of the central DNA repair mechanisms are provided in the two aforementioned blog entries (ref)(ref). If the topic fascinates you, you could also look at these representative articles and their “related citations” lists: Dancing on damaged chromatin: functions of ATM and the RAD50/MRE11/NBS1 complex in cellular responses to DNA damage, Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template, DNA damage and repair in Alzheimer’s disease, and DNA repair, mitochondria, and neurodegeneration. 

And of course, if a cell decides that DNA damage has occurred beyond that which can be repaired, if it has not already turned cancerous it brings its apoptotic mechanisms into play which is to say, commits suicide. See this blog entry on the P53 “guardian of the genome.”

In previous blog entries I have discussed how Sirtuins, SIRT1 in particular, might affect aging through their impact on the insulin/Igf1-like signaling pathway(ref)(ref)(ref)(ref), the pathway involved in calorie restriction life extension(ref). “Limited overexpression of the Sir2 gene (in humans known as SIRT1) results in a lifespan extension of about 30%^[10], if the lifespan is measured as the number of cell divisions the mother cell can undergo before cell death(ref).” Other Sirtuins also affect aging through different pathways. Here, I will be concerned with the role of SIRT6 in a DNA repair pathway known as homologous recombination.

SIRT6 and DNA repair

As far back as 2006 it was recognized that SIRT6 plays a critical role in DNA repair. The 2006 publication Certainly can’t live without this: SIRT6 summarized the situation: “Cellular metabolic rates might regulate aging by impinging on genomic stability through the DNA repair pathways. A new study published in Cell (Mostoslavsky et al., 2006) reports that deficiency in one of the mammalian Sir2 homologs, SIRT6, results in genome instability through the DNA base excision repair pathway and leads to aging-associated degenerative phenotypes.” The Mostoslavsky paper Genomic instability and aging-like phenotype in the absence of mammalian SIRT6 reported “Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.” Exactly how SIRT6 worked to support DNA damage repair was not known at that time.

A 2008 publication SIRT6 in DNA repair, metabolism and ageing looked more carefully at the role of SIRT6 as well as the other sirtuins in DNA repair and promoting longevity. “Overexpression or hyperactivity of sirtuins in many organisms – including yeast, worms, flies, and potentially fish and mammals – promotes longevity [2]. Mammals possess at least seven sirtuins, termed SIRT1–SIRT7 [3, 4]. Sirtuins exert their effects via NAD⁺-dependent enzymatic modification of other proteins: — SIRT6 deficiency causes a degenerative syndrome with progeroid features — From the standpoint of ageing research, SIRT6 deficiency causes the most striking phenotype among all the sirtuin knockouts. At the cellular level, SIRT6 deficiency leads to slow growth and increased sensitivity to certain forms of genotoxic damage. In addition, SIRT6-deficient cells show increased spontaneous genomic instability, characterized by numerous non-clonal chromosomal aberrations [1]. These findings suggest a defect in the ability of SIRT6-deficient cells to cope with DNA damage.” This paper goes on to speculate how SIRT6 may affect DNA damage repair, but more clarity on this subject is provided by very-recent publications cited here directly below.

Exactly how SIRT6 impacts on DNA repair is characterised in a September 2010 publication Human SIRT6 Promotes DNA End Resection Through CtIP Deacetylation. “We found that human SIRT6 has a role in promoting DNA end resection, a crucial step in DNA double-strand break (DSB) repair by homologous recombination. SIRT6 depletion impaired the accumulation of replication protein A and single-stranded DNA at DNA damage sites, reduced rates of homologous recombination, and sensitized cells to DSB-inducing agents. We identified the DSB resection protein CtIP [C-terminal binding protein (CtBP) interacting protein] as a SIRT6 interaction partner and showed that SIRT6-dependent CtIP deacetylation promotes resection. A nonacetylatable CtIP mutant alleviated the effect of SIRT6 depletion on resection, thus identifying CtIP as a key substrate by which SIRT6 facilitates DSB processing and homologous recombination. These findings further clarify how SIRT6 promotes genome stability.” Double-strand breaks (DSBs)and DNA repair via homologous recombination are simply described in the blog entry DNA repair cleanup failure – a root cause for cancers. DNA resection is a critical step in the repair process for DSBs . “DNA-end resection, the first step in recombination, is a key step that contributes to the choice of DSB repair. Resection, an evolutionarily conserved process that generates single-stranded DNA, is linked to checkpoint activation and is critical for survival. Failure to regulate and execute this process results in defective recombination and can contribute to human disease(ref).”

An editor of Science summarized the important finding in the September 2010 Editor’s Choice article under the caption UnSIRT6ain Repair. He wrote “Efficient and accurate repair of double-strand DNA breaks is critical for genome stability and involves a process known as homologous recombination. During repair of the sheared ends, the DNA must be resected by trimming one of the two strands on either side of the break. For the repair to be accurate, the remaining single-stranded DNA (ssDNA) has to be bound by the ssDNA-binding protein, RPA, after which the ssDNA can then bind homologous sequences. Kaidi et al. found that the mammalian deacetylase, SIRT6 (which has been implicated in maintaining genome stability), was critical for resection. At sites of DNA damage, SIRT6 deacetylated and activated CtIP (a protein important for resection), ensuring that resection occurred at the appropriate place and time.”

Other longevity-related roles of SIRT6

I have previously pointed out that SIRT6 appears to also have other important health and possibly longevity-producing effects, particularly the inhibition of NF-kappaB signaling. See the 2009 article SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span.

Also, SIRT6 appears to exercise control over critical glucose-metabolic pathways which could affect lifespan regulation. SIRT6 may also play an important role in repressing cancers. The Massachusetts General Hospital 2010 news release Lack of cellular enzyme triggers switch in glucose processing discusses SIRT6. “In a series of experiments in mouse cells, the researchers showed that SIRT6-deficiency hypoglycemia is caused by increased cellular uptake of glucose and not by elevated insulin levels or defects in the absorption of glucose from food. They then found increased levels of glycolysis and reduced mitochondrial respiration in SIRT6-knockout cells, something usually seen when cells are starved for oxygen or glucose, and showed that activation of the switch from cellular respiration to glycolysis is controlled through SIRT6’s regulation of a protein called HIF1alpha. Normally, SIRT6 represses glycolytic genes through its role as a compactor of chromatin – the tightly wound combination of DNA and a protein backbone that makes up chromosomes. In the absence of SIRT6, this structure is opened, causing activation of these glycolytic genes. — Elevated glycolysis also is commonly found in tumor cells, suggesting that a lack of SIRT6 could contribute to tumor growth.” The same Raul Mostoslavsky is still studying sirtuins and DNA repair. “The Mostoslavsky Laboratory at Massachusetts General Hospital is interested in understanding the influence of chromatin on DNA repair, and the relationship between the DNA damage response and the metabolic adaptation of cells. We focus on the study of a group of proteins called SIRTs, the mammalian homologues of the yeast Sir2. Sir2 is a chromatin silencer that functions as an NAD-dependent histone deacetylase to inhibit DNA transcription and recombination(ref).”

There is still more to be said about SIRT6 and the other sirtuins. And there is more that can be said about DNA repair. Also, I have not touched here on another important topic related to genomic stability: changes in the epigenome that occur in aging. There appears to be no end to possible topics for future blog entries.

A small biotech company, ImmunoGen, has been developing targeted therapies for cancers, therapies based on attaching anticancer drug payloads to antibodies that home in on cancer cells.  This is another “guided missile” strategy that promises to increase the efficacy and reduce the toxicity of treatments for several cancers.  The impact on the “war on cancer” is likely to be important.

Guided-missile cancer therapies

These are therapies that are designed to home in on cancer cells and tumors, in contrast to the usual chemotherapy and radiation therapies that affect multiple healthy body tissues as well as cancerous ones.  One example was given in the blog entry Trojan-horse stem cells might offer an important new cancer therapy.  “The therapeutic concept is simple and based on two observations.  The first observation is that for some reason mesenchymal stem cells (MSCs which are normally found in bone marrow) circulating in the body seek out cancer cells.  I conjecture that this is because cancers excrete signaling molecules that cause the circulating MSCs to home in on them, a strategy cancers use to achieve rapid growth(ref). The second observation is that it is possible to attach a payload molecule to mesenchymal stem cells which cause them to kill cancer cells but not normal cells, a molecule called TRAIL (TNF-related apoptosis-inducing ligand in case you wanted to know).” 

Another example of a guided missile anti-cancer therapy is discussed in the blog entry Terminator stem cells in the early pipeline.  “The concept here is engineering stem cells so they differentiate into body cells that target, go after and kill “bad” cells, such as cells infected with HIV or cancer cells.“

A third example is given in the blog entry Progress in stem cell oncolytic virotherapy.   In this case “The basic idea is to go after cancer cells with viruses that kill them.  To help the viruses escape the immune system, they are packaged in stem cells that are expected to snuggle up to the cancer cells.  It is hoped that the approach will go after cancer stem cells as well as mature cancer cells and therefore possibly provide a basic cure for the cancer concerned.  The broader area, oncolytic virotherapy is an approach to curing cancers that has been intensely researched for a number of years.  What is new is using stem cells or other human cells for safely getting the viruses to and into the target cancer cells.”

Targeted Antibody Payload technology

ImmunoGen is a 200-person biotech company located in Waltham Massachusetts near where I live.  ImmunoGen has been developing yet-another guided-missile anti-cancer approach, Targeted Antibody Payload (TAP) technology.  This morning there was news that ImmunoGen is cutting a deal with drug giant Novartis.    ImmunoGen is riding very high.  “Under a collaboration agreement to discover and develop antibody drug conjugates (ADCs) for cancer, Novartis will pay ImmunoGen a US$45 million fee upfront for exclusive rights to combine the TAP technology with antibodies to several as yet unnamed antigen targets. —  For each of these targets that results in an anticancer therapeutic, ImmunoGen will be entitled to receive milestone payments potentially totalling US $200.5 million, as well as royalties on any ensuing product sales.  — ImmunoGen also stands to get financial compensation for research and any manufacturing it does on behalf of Novartis. The Swiss company is responsible for the development, manufacturing and marketing of any products that emerge from the collaboration. — As ImmunoGen pointed out, the new agreement means it now has partnerships with the pharmaceutical industry’s top three oncology antibody-based therapeutic companies, Roche/Genentech, Sanofi-Aventis and Novartis(ref).”

TAP technology involves attaching cancer-killing substances to antibodies that home in on cancer cells.  TAP technology consists of three components, the cancer cell killing agent, the antibodies and what are called linkers that link the two together.

Killing agents

Because the cancer-killing drugs will only affect cancer cells, they can be vastly more powerful than the usual chemotherapy agents.  From the Immunogen website “Our cancer-cell killing agents (CKAs) are 1,000- to 10,000-fold more potent than traditional chemotherapy drugs. We developed them specifically for attachment to antibodies for targeted delivery to cancer cells. — The CKAs used in the TAP compounds in clinical testing act by interfering with tubulin and kill cancer cells when they attempt to undergo cell division. We continue to expand our portfolio of CKAs to further extend the utility of our technology, and unveiled our IGN family of DNA-acting agents at a scientific conference last year.”

Antibodies

“Each TAP compound contains an antibody that binds specifically to an antigen found on cancer cells. Each different TAP compound contains a different antibody, enabling different cancers to be targeted. For example, T-DM1, IMGN901, and SAR3419 are in development for HER2+ cancers, CD56+ cancers, and CD19+ cancers, respectively, as their antibodies target these different types of cancers(ref).”

Linkers

Immunogen has developed what it calls “linkers” to make sure the CKAs do what they are supposed to do and only what they are supposed to do.  “Our linkers serve to keep our CKAs attached to the antibody until the TAP compound has entered a cancer cell. They then control the release of the CKA to kill the cancer cell. — Just as different cancers respond better to some drugs than others, we have found that different linkers work better for some cancers than others. Therefore, we have developed a portfolio of linkers to enable us, and our partners, to achieve the best product design for the cancer target. Our modular approach – separate linkers and CKAs – enables rapidly evaluation of different product designs(ref).”

Preliminary clinical results

A press release from the European Society for Medical Oncology describes initial results of a clinical trial using the TAP technology to treat HER2-positive metastatic breast cancer. “Principal investigator Edith Perez, MD, Mayo Clinic in Florida, presented the results of the first ever randomized trial of trastuzumab-DM1 (T-DM1) as a first-line treatment for metastatic breast cancer.  — T-DM1 is the first of a new type of cancer medicine known as an antibody-drug conjugate. It binds together two existing cancer drugs with the aim of delivering both drugs specifically to cancer cells: trastuzumab, a monoclonal antibody that targets cells that overproduce the protein HER2; and DM1, a chemotherapy agent that targets microtubules.   — “This is the first ever presentation of an anti-HER2 antibody-drug conjugate used as first-line therapy for patients with advanced breast cancer,” said Professor Perez. “We are encouraged by the results. The study demonstrated that T-DM1 has very good anti-tumor activity as well as much lower toxicity when evaluated side by side to the older ‘standard’.” –  T-DM1 has shown promising activity in preclinical studies. Other clinical trials have also shown it to be effective in patients whose advanced cancer has not responded to other treatments. “This trial represents the logical step –moving the drug up to patients with newly diagnosed HER2-positive metastases,” Prof Perez said. — In the trial, researchers randomly assigned 137 women to treatment with either trastuzumab plus the chemotherapy drug docetaxel, or T-DM1. All participants had HER2-positive metastatic cancer, with no prior chemotherapy for their metastatic disease.  — After a median of approximately 6 months of follow-up, the researchers found an overall response rate of 48% in patients administered T-DM1, compared to 41% in the trastuzumab .  Perhaps the most significant finding was a drastic decrease in adverse effects due to the therapy.  “Importantly, the rates of clinically relevant adverse events were significantly lower in the T-DM1 arm (37%) compared to the rate in women given traztuzumab plus docetaxel (75%).”

The press release goes on: “This trial is ongoing and the positive outcomes are generating enthusiasm for a larger Phase-III trial which is now underway — The results are important for two reasons, commented Dr Fabrice André from Institut Gustave Roussy in Villejuif, France.  “Firstly, they confirm that in coming years chemotherapy could be replaced by a less toxic compound. Indeed, in the present study, the rates of serious adverse events were much lower in patients given T-DM1 compared to the chemotherapy arm. These results suggest that, with the same efficacy, T-DM1 could dramatically reduce the toxicities related to chemotherapy.” — The second important implication of this study is that it proves the concept that linking a monoclonal antibody to a cytotoxic drug leads to an anticancer effect. “This could have several implications beyond drugs that target HER2,” Dr André said.”

The future of TAP

Going back to today’s press release, “Seven such compounds are now in clinical trials through ImmunoGen’s own product programmes and those of its partners, which also include Amgen, Bayer Schering Pharma, Biogen and Biotest. ImmunoGen sees these partnerships as a vital revenue source for its own efforts. — The most advanced TAP compound is T-DM1, currently in Phase III clinical trials under ImmunoGen’s collaboration with Genentech/Roche. — In July, Roche filed a Biologics Licence Application (BLA) with the US Food and Drug Administration for T-DM1, an ADC that combines Genentech’s HER2-targeting antibody, trastuzumab (Herceptin), with ImmunoGen’s DM1 cancer cell-killing agent to treat patients with advanced HER2-positive breast cancer who have previously received multiple HER2-targeted medicines and chemotherapies. Unusually, the submission was based on the results of a 110-women Phase II clinical trial, which showed that T-DM1 shrank tumours in 33% of women who had already received seven drugs on average for advanced HER2-positive breast cancer. In August, however, Roche announced that the FDA had issued a Refuse to File letter denying the trastuzumab-DM1 combination accelerated approval. The company now expects to file T-DM1 worldwide in mid-2012. — In the meantime, last Friday ImmunoGen reported positive interim clinical data with T-DM1 for the first-line treatment of HER2-positive metastatic breast cancer at the 35th European Society for Medical Oncology (ESMO) meeting in Milan, Italy.”

The TAP story will no-doubt have many future chapters.  It is a good example of how a relatively tiny but highly innovative biotech company has been able to create a potentially important new technology for combating aggressive cancers, something the drug-company giants have rarely been able to achieve despite spending tens of billions of dollars on conventional approaches to drug discovery.

Incidentally, I still stand by my position that the best way to deal with cancers is not to have them happen in the first place.  Further, the best overall way to prevent or combat many cancers is to discover how to delay aging significantly. 

A newly-reported breakthrough in technology for generating high-fidelity induced pluripotent stem cells (iPSCs) suggests that these cells will soon be available and safe for use for in people.  The implications for regenerative medicine and extending human longevity may be profound.   

Background on iPSCs

If you are already familiar with iPSCs and their potentials you may skip this section.

An iPSC is a stem cell created from a normal adult body cell like a skin or blood cell through introduction of transcription factors that hopefully reverts the cell to the epigenomic state of an embryonic stem cell.  That epigenomic state involves pluripotency, a condition where that cell can differentiate into any of the hundreds of different body’s cell types.  See the March 2009 blog entry Rebooting cells and longevity for my first post on the initial discovery of how to make iPSCs.

If reliable and safe iPSCs that are fully pluripotent could be generated in adequate quantities, the potential for their use in regenerative medicine and for creating significant human longevity could be incredible:

–         They might be used to cure genetic diseases.  See the blog entries A simple treatment for human genetic diseases and Treating genetic diseases with corrected induced pluripotent stem cells.

–         They could be used for all the therapies human embryonic stem cells (hESCs) are being considered for.  iPSCs and would be superior to hESCs because they are made from the patient’s own cells and immunologically identical with them, obviating all the possible complications of graft-vs-host-disease which occurs in medical procedures where other people’s cells are used for therapeutic purposes.

–         iPSCs might even be used to create extremely longevity through closing the loop in the stem cell supply chain.  I have often referred to the blog entries The stem cell supply chain – closing the loop for very long lives, and the follow-up entry Progress in closing the stem cell supply chain loop.

–         Finally, iPSCs are free from the religious, moral and political uproars associated with applications using hESCs.  The right-to-life people say “Why not use iPSCs instead?”  And, speaking as a scientist who believes hESC research should continue, I have to say that they are probably right about this.

The original approach to creating iPSCs, introduction of four cell transcription factors, Oct4, Sox2, Klf4, and c-Myc, had a number of serious problems associated with it including:

–         The viral vectors originally used to introduce the transcription factors left traces of their DNA in the resulting cells.

–         Other random genetic damage to the cell could be created in the process of cell reversion; there was risk of genomic recombination or insertional mutagenesis.

–         The processes of cell reversion were slow and extremely inefficient, converting only a tiny fraction of the cells treated to iPSC status.

–         Careful examination of the iPSCs indicated that they were not epigenetically the same as embryonic stem cells and therefore possibly not as pluripotent. 

–         The problem remained of how to introduce iPSCs into the body so that they differentiate into cell types associated with a particular objective, e.g. to make neural cells to help a Parkinson’s Disease patient, to make heart cells to repair a heart muscle defect, etc.   This problem had been identified much earlier with hESCs.  If pluripotent cells are simply injected into a body tissue, a teratoma could result which is a hodgepodge tumor of varied tissue types including hair, teeth and bone.

There has been much reported subsequent progress at addressing these issues by new and improved techniques for cell reversion, however none of the approaches overcame all the issues and produced cells sufficiently safe and reliable to be used in humans.  Some commercially-available iPSCs, for example, were reported to have short telomeres. See my April 2010 blog entry Induced pluripotent stem cells – second-rate stem cells so far.  It looked like iPSCs were good enough for testing drugs but not safe for use in humans.  In June 2010 I wrote the blog entry A near-term application for iPSCs – making cell lines for drug testing and in that entry I said “a number of technical challenges must be overcome including: a) obtaining iPSCs that are free of DNA contamination, and that have long telomeres and full hESC pluripotency, b) developing reliable means for assuring differentiation into adult stem cells of various types, and c) developing reliable and safe means for introducing  those cells into their respective body niches. 

The July 2010 blog entry Induced pluripotent stem cells – developments on the road to big-time utilization reported significant progress in the technology for generating iPSCs and by that time several alternative approaches were known.  None, however overcame all of the problems identified above with sufficient reliability to yield iPSCs that could be used in humans, even for experimental purposes. “The search for ways to induce pluripotency without incurring genetic change has thus become the focus of intense research effort. Toward this end, iPSCs have been derived via excisable lentiviral and transposon vectors or through repeated application of transient plasmid, episomal, and adenovirus vectors (Chang et al., 2009,Kaji et al., 2009,Okita et al., 2008,Stadtfeld et al., 2008,Woltjen et al., 2009,Yu et al., 2009). iPSCs have also been derived with two DNA-free methods: serial protein transduction with recombinant proteins incorporating cell-penetrating peptide moieties (Kim et al., 2009,Zhou et al., 2009) and transgene delivery using the Sendai virus, which has a completely RNA-based reproductive cycle (Fusaki et al., 2009)(ref).”  None of these approaches completely abrogated the problems identified above, particularly  the problem  of potential genetic damage or contamination in the resulting iPSC cells. It “become increasingly apparent that all iPSCs are not created equal with respect to epigenetic landscape and developmental plasticity)(ref).” 

The new breakthrough development

The required breakthrough is reported in the September 30, 2010 publication Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA.  “Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine.  — Here we demonstrate that repeated administration of synthetic messenger RNAs incorporating modifications designed to bypass innate antiviral responses can reprogram differentiated human cells to pluripotency with conversion efficiencies and kinetics substantially superior to established viral protocols. Furthermore, this simple, nonmutagenic, and highly controllable technology is applicable to a range of tissue-engineering tasks, exemplified here by RNA-mediated directed differentiation of RNA-iPSCs (RiPSCs) to terminally differentiated myogenic cells.”

Going on, “By using a combination of RNA modifications and a soluble interferon inhibitor to overcome innate antiviral responses, we have developed a technology that enables highly efficient reprogramming of somatic cells to pluripotency and can also be harnessed to direct the differentiation of pluripotent cells toward a desired lineage. Although it is relatively technically complex, the methodology described here offers several key advantages over established reprogramming techniques. By obviating the need to perform experiments under the stringent biological containment required for virus-based approaches, modified RNA technology should make reprogramming accessible to a wider community of researchers. More fundamentally, because our technology is RNA based, it completely eliminates the risk of genomic integration and insertional mutagenesis inherent to all DNA-based methodologies, including those that are ostensibly nonintegrating. Moreover, our approach allows protein stoichiometry to be exquisitely regulated within cultures while avoiding the stochastic variation of expression typical of integrating vectors, as well as the uncontrollable effects of viral silencing(ref).”

In other words, it appears that this new approach that uses modified mRNA to reset cells instead of directly applying transcription factors addresses most of the main issues that have bedeviled human use of iPSCs up to this point.   As reported in the popular press “After tinkering with the mRNA molecules in the laboratory to make signals that the cells would not destroy as dangerous invaders, the researchers found that a daily cocktail of their creations were surprisingly fast and efficient at reprogramming the cells. The approach converted the cells in about half the time of previous methods – only about 17 days – with surprising economy – up to 100 times more efficient than the standard approach.  — Moreover, detailed tests indicated the cells had not experienced any disturbing changes in their DNA caused by previous methods and were virtually identical to embryonic stem cells. In addition, the researchers went one step further and showed that they could use the approach to then coax the iPS cells they created into a specific type of cell, in this case muscle cells.”

We have to wait for confirming research to be sure there are not other limitations or nasty surprises associated with iPSCs created this new way.  And faithful directed differentiation of the iPSCs was actually demonstrated for only one type of muscle cell.  But for the moment it looks like there is a real breakthrough.  In the June 2010 blog entry I stated that although much research is being devoted to iPSCs, 10-20 years are likely to be required before the stem cell supply chain can truly be closed in humans, the problems being mainly bioengineering in nature. 

I still believe the challenges are of a bioengineering nature but there has been so much progress reported since June that I now want to cut my 10-20 year estimate in half.  I expect that within 3-5 years we will see experiments with mammals, no-doubt mice to start with, involving use of iPSCs directed to differentiate so as to renew adult stem cells in their niches, the first experiments at closing the loop in the stem cell supply chain.   We will also see the first regenerative animal experiments using iPSCs before then, for example the use of iPSCs to regenerate spinal cord tissues and damaged heart valves.  And so, in 5-8 years we could see approved human regenerative iPSC therapies.  And just possibly, in less than 10 years we will see the first therapies where iPSCs are used to renew adult stem cells in their niches, the initial implementations of the longevity intervention: closing the loop in the stem cell supply chain.

Marios Kyriazis has invited me to engage with him in a dialog about the possibility of indefinite life extension and our first e-mail exchange is included here.  Dr. Kyriazis is a well-known physician and researcher in the field of anti-aging medicine with a long history of research, scientific and popular publications in this field.   You can find the Wikipedia entry on him here and get a sense of some of his accomplishments from this google search.  This dialog will appear here and on Dr. Kyriazis’ web site www.elpistheory.info 

My comments, starting with a response to Dr. Kyriazis’ original e-mail appear in this blue font preceded by VG and his original e-mail comments are in this black font preceded by MK. 

VG: Thank you for entering this dialog.  There is much food for thought in your comments and it seems clear to me that we are very much in alignment both on a deep level of ontology and in intention.  I comment on your points as they are listed.

MK: Yes, I am based in London and so it will be necessary to communicate by email. I read your treatise On Being and Creation, but I need some time to digest its contents, particularly as some parts are directly relevant to my interests.

VG: Yes, I think aspects of that treatise are likely to be quite relevant to what you want to see accomplished.  I would very much appreciate any further comments.  The framework in that treatise about being able to create my own reality is what empowered me to get into my present career of longevity science.  And faith in that framework is why I am willing to enter with enthusiasm into a dialog with you designed to lead to a historically “impossible” objective, very significantly increasing human life spans, perhaps making them indefinite. 

MK: Basically, I am exploring ways to achieve human biological immortality. My current line of thought is as follows.

MK: As you rightly say,” Creations are the result of Source and more directly the operation of the normal laws of reality”.  These laws operate from a simple level to a more complex one. Biologically, first there was the formation of organic matter, then more complex matter, then primitive cells, then fully formed bacteria etc etc until we see the creation of complex animals and finally, humans. We contain much more complexity in out biological and other systems than say, a primitive cell. This emergence of higher levels of complexity is seen through the universe.

VG: I absolutely agree. 

MK: It is difficult to avoid the conclusion that these normal laws of reality, operate in a way that constantly and progressively creates higher complexity and sophistication, particularly neural tissue sophistication that eventually results in intelligence, consciousness and wisdom.

VG: Yes, I believe this is so.  As I see it though, biological evolution has been supported strongly by human social evolution, without which the potentials of our brains could never be realized.  And the social evolution is now bringing us a new distributed form of memory and kind of intelligence particularly via the Internet (which, by the way, I played a role in forming) and its distributed computers and devices, vast memory and instant communications. 

MK: So, I say: what is the purpose of aging and death within this scenario?  I can accept the view that the ‘purpose’ of nature is to evolve the complexity of the DNA and this can only be achieved through Darwinian evolution.

VG: Yes, that was and probably still is the objective of biological evolution.  With our intelligence we have evolved the complexity of silicon chips and distributed electronic networks and now are moving on to quantum computing, theories-of-everything and if we can get our way, life extension.

MK: This in turn, must operate within the cycle of death-birth that we experience at present.

VG: Yes.  Of course the accumulated knowledge and society survives the life of any individual.

MK: The DNA must evolve (as everything obeys the universal laws of evolution towards higher complexity). In order to evolve, it must be mixed with other DNA and hope that something more complex will result. In the process, due to limited energy resources, the currying body must die and a new one created. This is the basis of aging and death by aging.

VG: Yes. As Darwin put it, nature favors the species, not the individual.   The issue at hand is whether:

1.     VG: Additional complexity will primarily be achieved in the social/distributed intelligence  sphere depending on accumulated networks, instant communication, brains connecting easily with other brains as we are now doing, and brains connecting with computers with little biological DNA evolution,

2.     VG: Evolution in DNA will accompany 1., leading to ever-longer life spans to keep up with the social evolution.

3.     VG: We can radically speed up biological evolution by significantly extending life spans.

VG: I think 2. Is already happening and that is why, as a colleague recently put it, “Every day, in advanced Western countries average lifespan increases 4 hours.”  You and I want to do 3.  I want to do it because I think effective social evolution will require great wisdom, and we are not going to get that easily from young people or from computers for a good while. 

VG: I comment that since our genomes are fairly stable and change very slowly, the evolution must be epigenomic, not in the genes but in the DNA that determines gene activation patterns. 

VG: I don’t think our bodies die because of limited energy resources any more.  Rather, I would phrase it that evolution created programs so older members of a species assuredly die off in well-defined time frames to enable younger members of the species to have access to resources like food. Perhaps this is what you are getting at.

MK: BUT. I am proposing that we have now reached such an advanced level of neural sophistication (we are homo sapiens sapiens) that it may be possible to avoid the above scenario. It may be possible for our brain to continue evolving without the need for DNA to continue its evolution. In effect it is the brain that matters, and not the DNA.  It is now more energy-efficient for our brains to evolve via increased input of information.  (http://www.ncbi.nlm.nih.gov/pubmed/15929717)  PLUS increased use of technology (such as the internet, nanotechnology and AI).

VG: I am convinced that overall evolution of intelligence via electronic augmentation of our brains is already happening and has been the basis for much of our progress over the last several years.  As to hormesis as mentioned in the citation, have you seen my blog entry Hormesis and age retardation?  While hormesis can extend lifespans, however, and while hard thinking can augment intelligence, I think we will need additional interventions to get to the really extraordinary longevity we want.

MK: I claim that this is a more efficient way for achieving higher intellectual sophistication, rather than wait for Darwinian evolution to create more sophisticated brains through trial and error. If this is correct then, it follows that humans must remain alive for an indefinite number of years, so that their brain can evolve (through self- input).

VG: Yes, yes and yes.  However, if we are truly gaining 4 hours of average longevity per day right now in Western countries, there has to be something else already at work that is more profound than Darwinian evolution.  I think it is a rapid capability of the epigenome to respond to social and environmental stimuli. 

MK: Thus aging will become redundant and immortality will ensue. (Immortality=indefinite lifespans, not indestructability)

VG: The process of life extension is very slow now.  I think if we want to see the possibility of immortality while we are still alive, we must conceive or foster the creation of interventions to make that possible.  I do agree with your definition of immortality.

MK: Then, if this is the case, I ask: can we do something now to see if we could bring this process forward?   I have started studying possible interventions, for example with transposons, but I am quite willing to accept other less tangible suggestions.

VG: I see two possible kinds of interventions that could radically expand life spans, perhaps indefinitely, because they assure constant renewal of the soma.  One is what I call closing the loop in the stem cell supply chain, and the other is discovering epigenetic means for resetting cells to earlier states – and both approaches are actually equivalent.  To start, you can view my presentation at the 2010 American Aging Society meeting Towards a Systems Theory of Aging.   This presentation describes what I think  are the deepest mechanisms of aging that are susceptible to interventions – exhaustion of the pools of adult stem cells that replenish practically every cell type, and age-related epigenomic silencing or activation of longevity-related genes.

VG: The stem cell supply chain theory of aging is my own creation, covered in my treatise of aging here.

VG: I have written several essays on the topic in my blog.  You could start with The stem cell supply chain – closing the loop for very long lives, and then go on to Progress in closing the stem cell supply chain loop .  Three days ago, breakthrough results were published which brings the possibility of closing the stem cell supply chain a step closer.  I plan to start generating a blog entry on this topic later today.

MK: In summary:

1.      MK:  Everything must become more complex. 

VG:  YES

2.     MK:   Until now, Darwinian evolution was the way to go. 

 VG:  YES. IN CONJUNCTION WITH MUCH SOCIETAL EVOLUTION INCLUDING THE INTERNET

3.       We have now achieved high neural sophistication AND high technology.

VG: YES, YES

4.   MK:     The best way to evolve from now on is via long-term input of information into our brain.  

VG: I WOULD SAY WE NEED TO AUGMENT OUR ALREADY  RAPIDLY- EVOLVING EXTERNAL KNOWLEDGE NETWORKS OF COMPUTERS, INTERNET ETC. WITH SIGNIFICANTLY LONGER LIFE SPANS. 

5.    MK:    Our brain (and us) must now stay alive for an undetermined amount of time for point 4 to succeed. 

VG: YES.  WE NEED TO CONSERVE OUR HUMAN CAPITAL.  OUR SOCIETIES CAN ONLY SURVIVE THROUGH ACCUMULATED WISDOM AND WE CAN’T AFFORD TO HAVE IT DIEING OFF

6.   MK:     This is going to happen anyway, but can we make it happen soon?

VG: YES, EXACTLY 

MK:   So, these are some of my initial thoughts. Please feel free to comment or criticise. If you agree, I can put this dialogue on my website www.elpistheory.info) and you on yours.  Best wishes 
Marios 

VG: I agree about putting the dialog on our websites.  The site has up to 1,800 serious daily visitors and expect we will get a lot of comments.  The dialog will go up today, and I hope we can continue it.

Vince

Readers, please feel free to wade in with comments.

The stage for this blog entry was set by the recent one If we can multiply lifespans of nematodes by seven, why have we not been able to get anywhere with significant human lifespan extension?  The way things are going it is highly unlikely we are going to see a radical upward increase in human lifespan for many years.  I asserted that perhaps, if we are very lucky, we will see a 15% increase in expected human lifespan for people who follow certain interventions within ten years.  But there will be nothing approaching the lifespan increase of 60% or more that we have seen to be possible in mice.   I have laid out the reasons progress in anti-aging science and practice is likely to be slow and incremental for the next 15 years or so.  Unless, that is, we can manage to launch a War On Aging.  Such a war is what this blog entry is about.  I deal first with how to justify such a war, second with what such a war could look like, and third, steps to getting the war started.

Justifying the War On Aging

There will be no War On Aging (WOA) unless there is a massive transformation in the perceptions of the public about aging, the great benefits of waging a War on Aging, and the costs of not waging such a war.  A massive educational campaign is needed to get the following points across and embedded in everyone’s conscience:

1.      A war on aging is winnable. Significant extension of human lives is possible.  We have multiplied the lifespans of lower animals and we can do it for us humans if we put our minds to it and our resources behind it.

2.     The result will increase health and decrease health care costs.  Lifespan and healthspan go hand-in-hand, so we are talking about people having lots more healthy productive years, not about expensive interventions to keep very-sick people from dying.  The result of victory in a WOA will be an increase in the ratio of healthy productive years to infirm unproductive years.

3.     The war would produce an immense increase in national wealth.  In a highly complex post-industrial world, human capital is the most important resource representing trillions of dollars spent on education and work experience.  Keeping that educated and experiences human capital around and working 10, 20 or more years represents trillions of dollars in conserved national wealth.

4.     Enormous productivity and economic benefits could be realized.  The result would be an immense increase of productivity and economic benefits due to healthy experienced older people working longer.  An increase of healthspan of only 10 years would not only cut healthcare costs immensely, but also produce more than enough trillions of dollars of productivity benefit to wipe out our national debt and put our economy into the black.

5.     It is the right war to fight.  We have been spending hundreds of billions of dollars a year on wars on diseases of aging like Alzheimer’s disease, cancers of aging and Parkinson’s disease.  And we have mostly not been winning those wars because we have not been attacking the root cause which is the aging process itself.  If the war on aging increases healthspan by 15 years, the average time of onset of all diseases of aging will be postponed 15 years.  For a massive increase in public health, the WOA is the right one to fight.  

6.     It is a war that can impact directly on you and your family.  Victories in this war can keep you and your loved ones around and healthy for many more long years.

7.     The war does not have to be that expensive.  I venture to guess that a properly-organized NIH budget for the two WOA missions I described below that builds up to around the $600 million level in 3-4 years and stays at that level would probably produce significant results within 7-15 years.  This figure is under 2% of the NIH total budget.  “The NIH invests over $31.2* billion annually in medical research for the American people(ref).”  

What the WOA would look like

The War on Aging (WOA) will have to be a partnership of the media, academia, government, the health care industry and biotech/pharmaceutical businesses.  Each of these would play important roles, not-for-profits and government in the earlier stages, businesses in the later stages.

The aging-research programs funded by the National Institute for Aging (NIA) and organizations like the Ellison Medical Foundation have produced valuable results and mostly merit continuation.  However, these programs are primarily basic-research oriented and do not have human longevity as a goal.  Human life and healthspan extension is not part of the mission on NIA and must be the  first and foremost goal in whatever government organization takes the lead initiative in the WOA.  Mission-oriented programs with time-specified objectives are required such as those at NASA.  For example, I suggest two such mission-oriented programs here.

The 20% life extension mission – 8 years to full public availability

–       The target objective of this mission is to establish reliable and safe interventions to increase human lifespan by 20% – so that instead of expected lifespan being around 80 as it is now in the US, it can be expected to go up to about 96 as the fruits of the program are realized.  There should be a targeted average increase of at least 15 productive years for members of developed societies who benefit themselves from the interventions of this program.

–         The focus of this mission is not so much on new basic science breakthroughs as it is on providing safe “engineering solutions” for limited lifespan extension.

–         This program would focus on interventions affecting known longevity pathways and genes where there is already a significant base of science and animal experimentation: mTOR, IGF-1, SIRT-1, human counterparts of INDY in fruit flies and DAF-2 in nematodes, etc.

–         Within 5 years, starting with mouse and working up to simian models, establish the science and probable feasibility of 20% lifespan extension in humans.   Determine the best combination of interventions to achieve this objective.  Favor non-invasive lifestyle, nutritional, and natural-supplement interventions to the maximum extent possible.    Conduct programs to also establish the safety of the interventions in simians.

–         It is too soon to say whether stem cell or epigenomic interventions will play a role in this mission.

–         Dietary supplements like curcumin and resveratrol may play a role in this mission as well as drugs, but drugs are likely to be ones already in existence or under development, e.g. SIRT1 activators, rapamycin analogs or metformin.

–         In the third year of the program adjust the lifespan target for the Mission upwards (to 25%) or downward (to 15%) depending on research progress.

–         By year 7 of the mission, have established the safety of the anti-aging interventions in humans and have developed biomarkers of efficacy.  If regulatory hurdles are in the way, ways around them must be discovered.

–         Within the next year – 8 years total –products  and lifestyle regimens for humans  will be on the market with probable capability for extending lives by an average of 20%.  The 20% figure is a rough aggregate.  Probable life extension would not be the same for everyone and would vary depending on health and genetic makeup of the individual, age when the interventions are initiated, and lifestyle factors.

I have no way to prove it, but I strongly suspect that the current anti-aging lifestyle regimen and supplement regimen together may already have the capacity to extend average human lifespan an average of 10% to 15%.  So I see a 20% average extension within 8 years as an objective that is quite possibly within reach given all that is already known about aging pathways and already-available interventions.

The 60% life extension mission – 15 years to availability of longevity products

–         The aim of this mission is to establish strong feasibility for extending human lifespans by 60%, and to do this so that interventions aimed at this objective are available to the public within 15 years.  If the program is successful, the average human lifespan maximum for those benefiting from the intervention would go up to about 128 years, with good healthspan of over 115 years, maximum human lifespan of about 190 years.

–         This 60% lifespan increase program can be run in parallel with the program designed to produce 20% lifespan increase and both programs can benefit from what is learned in the other program. 

–         The program for this mission will require a hefty research component as well as a daunting subsequent engineering component.  It will require new kinds of interventions beyond those in the 20% lifespan extension program.

 –         Stem cell science is likely to play a major role in this mission, probably based on perfection of technologies for creating reliable high-fidelity autologous induced pluripotent stem cells (iPSCs), learning how to get them to differentiate into any tissue desired, and developing a host of therapeutic approaches for using them.  A news item appeared yesterday signaling an important new breakthrough in iPSCs and I will explore the ramifications of this development soon in another post.  I have a lot of faith in the longevity potential of closing the loop in the stem cell supply chain.  See my blog posts IPSCs, telomerase, and closing the loop in the stem cell supply chain,  and The stem cell supply chain – closing the loop for very long lives. Also the discussion in my treatise of the Stem Cell Supply Chain Breakdown theory of aging is applicable. 

–         Another stream of technology that could figure heavily in this mission is epigenetic regulation of longevity genes.  See the discussion in my treatise for the Programmed Epigenomic Changes theory of aging.  I expect to produce a new blog entry soon with up-to-date news on research on epigenetic regulation of aging.  Also you could review my 2010 AAAS presentation Towards a Systems Theory of Aging.

–         In the earlier stages, all research will be on animals to establish feasibility, starting with mice and working up to pigs and simians to establish safety and efficacy.  There can and should be human trials for safety of interventions but it obviously will take many decades to establish the degree of efficacy in ensuring such long lives. 

–         Like the 20% mission, this mission would probably not countenance use of genetic interventions such as knockout of genes or insertion of multiple gene copies – the kinds of interventions that have led to significant life extension in lower species.   The social environment would have to be prepared to allow even experimental genetic modifications of humans, and I don’t think that is possible in the near future.  Ethical, moral and religious scruples would have to be dealt with, and an enormous controversy could ensue making the present battle over using embryonic stem cells seem like an outpost skirmish.  That controversy could kill or hobble the War On Aging before it even gets started. 

–         I think it both highly desirable and possible to sidestep such a controversy and achieve the goals of this mission without altering human genes.  We probably don’t need new human genes or to get rid of existing ones to extend lives by 60%.  We just have 1.  To develop safe and reliable means for turning certain of our existing longevity-related genes off and on and/or 2. Develop a means for continuing renewal of human cells with aging.  Again, the preferable approaches would involve use of induced pluripotent stem cells and epigenomic manipulation of activation and silencing of selected longevity-related genes.

–         We will need different ground rules for human experiments than clinical trials to move along with this research.  The time frames and costs of clinical trials and the need to cloak them as trials of medical interventions would slow research so much as to make a 15-year goal for this mission impossible. 

–         If there is to be a third future mission in the WOA with objective to double human lifespans, however, at that point the issue of human genetic alterations will have to be squarely faced.

Social aspects of the war on aging

Significant social changes will be required if the WOA is to be successful making the social engineering of life extension as important as the biological engineering.

–         If people are to live, say, 16 years longer and are expected to work that much longer, there needs for increasing emphasis on lifetime learning, on work as opportunity for self-fulfillment and play, on the contributions older people are uniquely qualified to make, and on the multiple adventures life can offer.  There will be no productivity benefits to longevity if people at 55 or 65 continue to move to Florida where they will live in retirement communities, play bridge, golf and bingo and live their extra years in general boredom until they finally die.

–         As longevity increases, so will the general business retirement age and the social security retirement age have to be raised.  The message is not just “you have to work a lot longer.”  Simple economics says that if people work longer, they should be able to retire with more money.   

Getting the War On Aging started

Like all wars, the war on aging will have to be concertedly and skillfully sold – sold to policymakers and to the public  – and this is a matter of communications, media and social organization not a matter of scientists talking to other scientists.  The two usual major selling points for wars have to emphasized:

–         Absolutely terrible things will happen if the war is not fought.  This one is easy.  A hundred million or more US citizens will die prematurely if there is no War on Aging.

–         There is a large payoff to the war, and the war is the right thing to do.  This one is also easy, just looking at the economic benefits involved.  And think of how wonderful it will be to keep your parents and grandparents around and healthy so much longer.

In other words, the moral high ground goes with fighting the war.

So the messages required for starting a WOA are fairly clear.  The key questions is “Who will deliver these messages to whom in a way that gets things going?” and here the situation is very murky.  For now, I will share a few general ideas:

–         The arena for selling the War on Aging is the public media, not the scientific literature. 

–         Before the WOA can be started as mission-oriented programs, the possibility of WOA must be started as a broad dialog among leaders from every sphere including economists, social scientists, politicians, community leaders and religious leaders.

–         Getting major TV exposure for the concept of WOA will be very helpful for getting the dialog going.

–         It would be also very helpful if prestigious life-scientists came out of the closet and supported the idea of a WOA.  We need TV showcasing of Methuselah mice.

–         It would be good for a high-profile science series like Nova to do a series on life extension.

–         Emphasis needs to be put on the points listed above: the economic benefits of fighting the WOA; the fact the war can be won; how this war gets to the root causes of a lot of other expensive wars we are fighting  against the diseases of old age, and the personal benefits of longer lives.

–         It would be helpful; if there were more high-profile studies by economists that quantify the economic value of life extension by 10, 20 or 30 years

–         It would be very helpful if some visionary foundation put its resources behind starting a War on Aging.

–         A good first step would be a high-level conference of policymakers, economists, scientists and political leaders examining the prospects for a War on Aging.

I plan to return to this topic.

Back in a July 2009 blog post Life extension by a factor of 10, I described how radical life extension has been achieved in baker’s yeast.  The most-recent blog entry posted two days ago New extraordinary longevity lessons from the nematode chronicles how researchers over 20 years have managed to discover interventions that multiply the lifespans of nematode worms (C-elegans) by a factor of seven.  This raises the question explored in the present blog entry: Why have researchers over the same 20 years essentially gotten nowhere in significantly extending human lifespan?  This blog post examines what has held us back and also sounds a note of optimism based on what is happening.  My opinions about what we would have to do to move forward more concertedly will be treated in yet-another blog entry.  I start out on a positive note. 

Why nematodes are a wonderful model organism for study of aging 

Virtually everything related to genetic pathways affecting longevity in mammals and humans was discovered first in primitive organisms like baker’s yeast and the nematode.   I am talking about the major pathways known to have a potential for human life extension including mTOR, SIRT1 and FOXO/DAF16.  Nematodes are ideal organism for first-pass studies of longevity because, put simply, you can freely mess with them.  In more detail: 

·        As I pointed out in the blog post MicroRNAs in cancers and aging, and back-to-the-nematode “Nematodes are “–  simple enough to be studied in great detail. Strains are cheap to breed and can be frozen. When subsequently thawed they remain viable, allowing long-term storage.”

·        A whole supplier industry has grown up for C-elegans.  You can buy C-elegans DNA here, and if you want the worms themselves you can buy them from the C-elegans Genetics Center for $7 per strain if you are in a university or not-for profit lab; otherwise cost is $100.

·         “C. elegans is transparent, facilitating the study of cellular differentiation and other developmental processes in the intact organism.”

·        Nematodes are cheap to maintain, feed and breed.  Unlike people, they don’t need fancy housing, education equal opportunity or health care and they don’t vote.  And they don’t vocalize religious or ethical scruples about what is done to them.  They don’t yell or cry.

·        Nematodes don’t hire lawyers so you can do things to them you can’t do to humans, like modify them genetically.  If you are a nematode researcher, you don’t need to worry about consent forms, the FDA or the Animal Rescue League.

·        Nematode longevity research is roaring ahead in a worldwide community of thousands of nematode researchers.  See the Caenorhabditis elegans WWW Server for WormBase, C-elegans meetings, jobs, software, recent papers, worm genomes, Wormatlas, Wormbook, C elegans movies and much more. Life-extension progress reported in the previous blog entries is just a start.  “There’s careers, money and fame in them there worms.”

·        The developmental fate of every single somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped out(ref).^[8][9]“ 

·        The little critters have very short normal lifespans (around 20 days), so in a couple of weeks interventions affecting lifespans can be studied.  In a couple of months it is possible to perform longevity and generational experiments that would take centuries in humans.

·        The critters have evolved very effective stress-management strategies to get them through their inactive dauer stage, and most of those strategies are conserved right up through the evolutionary hierarchy and work, albeit somewhat differently, in us.  As stated in the 2010 publication An overview of stress response and hypometabolic strategies in Caenorhabditis elegans: conserved and contrasting signals with the mammalian system, “Caenorhabditis elegans, undergoes a state of hypometabolism called the ‘dauer’ stage. This period of developmental arrest is characterized by a significant reduction in metabolic rate, triggered by ambient temperature increase and restricted oxygen/ nutrients. C. elegans employs a number of signal transduction cascades in order to adapt to these unfavourable conditions and survive for long times with severely reduced energy production. The suppression of cellular metabolism, providing energetic homeostasis, is critical to the survival of nematodes through the dauer period. This transition displays molecular mechanisms that are fundamental to control of hypometabolism across the animal kingdom. In general, mammalian systems are highly inelastic to environmental stresses (such as extreme temperatures and low oxygen), –“

·        C-elegans provides an excellent working model for examining how a limited number of transcriptional regulators can mediate to influence downstream aging lifespan effector genes.

·        “However, there is a great deal of conservation between the signal transduction pathways of nematodes and mammals. Along with conserving many of the protein targets in the stress response, many of the critical regulatory mechanisms are maintained, and often differ only in their level of expression. Hence, the C. elegans model outlines a framework of critical molecular mechanisms that may be employed in the future as therapeutic targets for addressing disease states(ref).” 

The 2006 mini-review Recent aging research in Caenorhabditis elegans summarizes some of the conserved pathways studied in C-elegans. “Evidence gathered over the past 15 years shows that the nematode Caenorhabditis elegans is excellently suited as a model to study aging processes in the entire organism. Genetic approaches have been used to identify and elucidate multiple mechanisms and their corresponding genes that limit the life span of C. elegans. These highly conserved pathways include the well-studied insulin/IGF-1 receptor-like signaling pathway, which is thought to be a central determinant of life span, since several other mechanisms depend or converge on the insulin/IGF-1 pathway transcription factor DAF-16/FoxO. In this review we focus on new insights into the molecular mechanisms of aging in C. elegans, including new genes acting in the insulin/IGF-1 pathway and germline signaling. In addition, stress response pathways and mitochondrial mechanisms, dietary restriction, SIR2 deacetylase activity, TOR and TUBBY signaling, as well as telomere length contribution are discussed in relation to recent developments in C. elegans aging research.”

Why human beings are a terribly difficult model for the study of aging.

Put simply, you can’t mess with people except extremely carefully.  In detail:

–         Obviously, we are a lot more complicated than nematodes and what works to keep them young might not work for us and, in fact, could harm us.

–         Since human life is regarded to be sacred, you can’t ethically, morally or legally do things to people like knock out or add to their genes.  The consequences for a researcher doing such things could be being thrown out of his university, driven out of his profession, ending up in jail, or facing billion- dollar lawsuits.

–         People yell, cry, complain, see lawyers or can get guns and shoot you.

–         It is extremely expensive to research anything involving large numbers of humans.  Big pharma companies spend hundreds of millions of dollars on very specific clinical trials.  They won’t spend that kind of money on longevity treatments unless they can see multi-billion dollars payoffs in a few years.

–         Carefully-designed longevity clinical trials, if there were such things, would take centuries.  And it makes no sense to conduct such a clinical trial because in 5-10 years research progress will make the original interventions being examined in such a trial obsolete.

–         Besides, you can’t conduct a government-recognized clinical trial on a “cure” for aging in people because aging is not regarded by the government to be a disease and is not recognized as an indication for clinical trials.

–         A small university laboratory with a modest budget and hard-working research associates can study and produce significant research results about nematodes; it takes a big pharma company spending hundreds of million dollars to conduct a single clinical trial with very limited objectives on people.

–         There is no big money in human longevity research.  The NIH spends a pittance on aging research compared to what it spends on cancer or HIV research, and pharma companies generally won’t touch it.

–         The social context in the US at least is strongly against human genetic modification.  And, I opine that vociferous fundamentalist religious groups will probably be against radical life extension by any means.  The people who are pro-life for fetuses may well turn out to be anti-life when it comes to older people and take the viewpoint “We should not mess with God’s plan for people growing older and passing away when their time comes.”  And as a consequence “We should not spend a single penny of government money on life extension.”  They forget that God’s original plan for humans was to have most of us pass away by age 25.  The blog entry Getting the world ready for radical life extension examines what would probably go sour if someone went public with an effective “cure” for aging tomorrow morning.

–         Above all, what is missing is a general framework of thought and shared values that says radical human life extension would be a very good thing and something that should be pursued systematically with significant resources.  Such a perception may exist in the minds of a few visionaries, selected researchers and readers of this blog.  But it does not exist out there in the general public or even in the programs of the agencies that fund aging research.  Instead, the general image evoked by extending life span is more doddering unproductive retirees kept alive by expensive drugs fed through tubes and filling up nursing homes and hospitals, creating terrible auto accidents, driving social security broke ever-more quickly, and running health care costs even more over the top.  Most people don’t get the essential point that increasing lifespan and increasing healthspan are the same thing and that the biggest risk factor for the costly diseases of aging like Alzheimer’s, Parkinson’s and most cancer is aging itself.  The point is obvious when you think about it, but most people don’t think about it.

I will say more about these last points in a next blog entry after this one Preparing for the war on aging.  But first I want to review what might happen with life extension if we keep going as we have been going.

Is there hope for radical life extension in humans?

The bottom line is that if radical life extension in human’s is to take place in humans the way things are going, it will be something that we blunder into rather than be the result of a specific R&D program.  There are reasons, however, for hope that we may well blunder at least part of the way:

–         Nematode research provides a good start for going on to higher animals   The research on nematodes has told us a lot about the operation of genes and pathways that are largely conserved in mammals and humans, and provides well-defined guidelines for research that leads to radical extension of lifespans in mice as the next step.  We know there is remarkable similarity among “longevity” genes and related pathways across a wide spectrum of species ranging from yeast to worms to flies to humans.  See the blog entry Longevity Genes and two Fantasies.  As far as mice goes there appears to be several interventions that increase their lifespans by around 30% and “A few transgenic species of mice have been created that have maximum life spans greater than that of wild-type or laboratory mice. The Ames and Snell mice, which have mutations in pituitary transcription factors and hence are deficient in Gh, LH, TSH, and secondarily IGF1, have extensions in maximal lifespan of up to 65%(ref)” The next step is to double up like what was done with nematodes and then find out how to quadruple mouse lifespans.  We are much more like mice than like nematodes.  But mice don’t complain or sue much more than nematodes do.

–         Medical and drug research for diseases of old age will probably yield anti-aging therapies.  A lot of money is being spent on cancer research, Alzheimer’s Disease research, diabetes research,  and research on other diseases of old age.  Not curiously, potential therapies that can slow down or stop such diseases are also potential anti-aging interventions.  For example, inhibition of the mTOR pathway via rapamycin both wards off cancers and Alzheimer’s disease in mice and extends their lives.  See the 2010 publications Rapamycin extends maximal lifespan in cancer-prone mice and Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-beta levels in a mouse model of Alzheimer’s disease.  According to an April 2010  article in Gen, “This marks the second report linking rapamycin to AD treatment within the last month or so. The previous study, published February 23 in The Journal of Biological Chemistry (JCB), highlighted an interrelation between mammalian target of rapamycin (mTOR) signaling and A-beta.  Using a different animal model of AD, the group found that pharmacologically restoring mTOR signaling with rapamycin rescues cognitive deficits and ameliorates A-beta and tau pathology by increasing autophagy. — Additionally, in July 2009, a different group of Barshop Institute researchers and colleagues at two other institutions reported that microencapsulated rapamycin extended the life span of mice, possibly by delaying aging. — “The fact that we are seeing identical results in two vastly different mouse models of Alzheimer disease,” Dr. Galvan added, in reference to the February JCB paper, “provides robust evidence that rapamycin treatment is effective and is acting by changing a basic pathogenic process of Alzheimer that is common to both mouse models. This suggests that it may be an effective treatment for Alzheimer in humans, who also have very diverse genetic makeup and life histories.”

This last description makes me smile because the “basic pathogenic process of Alzheimer that is common to both mouse models” is probably just the complex process of aging itself.  The same basic pathogenic process of aging invites age-related cancers, Parkinson’s disease, advanced Type 2 diabetes, coronary heart disease, macular degeneration, etc. – all those things that cripple and eventually kill us when we grow old.

–         Certain dietary supplements may be life-extending.  Despite lack of hard evidence such as provided by clinical trials, certain dietary supplements like several listed in my combined anti-aging firewalls regimen may be life extending.  Regarding resveratrol, for example, see the blog entries SIRT1, mTOR, NF-kappaB and resveratrol, Visit with Leonard Guarante, and What does resveratrol do?  Regarding curcumin, you could review Curcumin, cancer and longevity.  And I have discussed many other dietary substances and supplements in this blog, ones which convey important health benefits and with potential probably-mild life-extending properties: (ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref).

–         Social, economic, technical, business, infrastructure and knowledge factors are operating together to ever accelerate the discovery of anti-aging interventions, albeit from a very low current base.  See the blog entry Factors that drive Giuliano’s Law.

–         A whole DNA supplier industry has developed to facilitate longevity as well as medical research.  You can find links to buy DNA for mice, guinea pigs, rats, chickens, fish, cats, rabbits, zebrafish, dogs, cows and simians here.

–         The databases of basic knowledge related to genomics, epigenomics and related ‘omics” are rapidly increasing in size and sophistication as the cost of whole-genome sequencing plummets(ref). 

–         Personalized Predictive Preventative Participatory Medicine (PPPPM) is slowly being adopted and will in time transform the practice of medicine to where it is much more science-based and likely to open the door to more and more interventions that are designed to prevent diseases and, in the process, extend lifespans.  See the blog entries Harnessing the engines of finance and commerce for life-extension, Personalized medicine – reducing the cost and improving the effectiveness of health care, and Transformed State of Medicine – 2025.

–         Genome-wide association studies are telling us more and more about what creates the diseases of old age, even, cancers, and how they can be averted.  See the blog entry Genome-wide association studies.

–         Social evolution and epigenomic changes are driving longer lifespans.  See the blog entries Average US life expectancy up 73 days in one year, Antagonistic pleiotropy revisited – for the last time, and Ever-increasing longevity– is epigenomics involved?

In summary, multiple social, economic, demographic, scientific, and technical factor are interacting to create an exponential growth in knowledge related to longevity, and, eventually, longevity treatments.  The problem is that we are starting from a low base.  In 40-50 years we will be improving practical longevity prospects at a furious rate.  We are improving them now, but progress is relatively snail-paced.

One Scenario for the emergence of longevity drugs

One possible scenario for the development of anti-aging therapies could conceivably go like this:  a drug is developed for Alzheimer’s disease –  say rapamycin itself or a rapamycin analog – that also delays aging.  As the word gets around that the drug delays aging and slows down the onset of other diseases like cancers, people who do not have AD or cancer will also start to demand that drug. 

And there are a number of other drug candidates that could also get the longevity ball rolling.  In the previous blog entry where I discussed nematode aging pathways, I discussed how PDEF is being investigated as a negative regulator of certain human cancers while its nematode counterpart ETS-4 is a known lifespan regulator.  An anti-cancer treatment that is life-extending might be based on sestrin proteins.  See the blog entry Sestrins, longevity and cancers. On another front yet, Sirtris pharmaceuticals is “creating revolutionary medicines for the diseases of aging.” “Our research focus is on modulating the sirtuins, a recently discovered class of enzymes involved in the aging process.”  To the extent that Sirtris’ products will work, since they are based on activating the SIRT1 gene they will almost certainly be life-extending.   Yet-another possibility are drugs in Phase III clinical trials that mimic the effects of variants of the CEPT gene, variants that are protective against cardiovascular diseases, memory decline and dementia and that are found in centenarians and thought to confer longevity.  See the blog entry CETP gene longevity variants. And you can bet that once one longevity drug starts to reach blockbuster status, other big-pharma companies will start pouring billions into longevity drug R&D.

In short, there are a number of drugs being researched or developed for diseases of old age that could also confer important longevity benefits.  And that is how the first longevity-enhancing drug could get on the marketplace 

The prospect for breakthrough human longevity

We are likely to see only slow incremental shifts in our longevity, even though the pace of research and knowledge is picking up.  I do not expect anything like the factor of 7 increase in lifespan over 20 years as was the result of research on nematodes.  Perhaps, if we are very lucky, we will see a 15% increase in expected human lifespan for people who follow certain interventions within ten years.  This is because our society is simply not prepared now to invest significant resources in really prolonging life – say doubling lifespans.  And, if researchers seriously tried to do that they would probably be fiercely resisted and burned at the academic, moral and legal stake.  See the blog entry Getting the world ready for radical life extension. “The idea of people living hundreds of years has about as much credibility today as the idea of the world not being at the center of the universe had in 1540.  Intellectually and in terms of our laws, institutions and actions, we are just not ready for radical life extension.”  I illustrate this point with a story of what could well happen if a good life-extension drug were developed right now. And I concluded “Above all, there is a need for a major shift in general perspective regarding life extension FROM more and more doddering, sick, non-functional, non-contributing individuals drawing social security, filling nursing homes, driving their grown children crazy, having automobile accidents and driving health care costs ever-higher, TO more and more healthy, creative, fully-functional working individuals in their 70s, 80s, 90s and beyond who are not getting the diseases of old age, and who are more than doing their part to contribute to our society in every way.”

Until that shift in perception changes, we may continue to get nowhere towards the goal of real breakthrough longevity. Being a visionary though, in a subsequent blog entry I will outline how I think a big breakthrough possibly could be achieved within the next five years.  Stay tuned!

Genetic lifespan regulation has been studied extensively in the nematode roundworm Caenorhabditis elegans (C-elegans) since the 1980s.  Hundreds of research papers have been written on this topic.  And I have generated a number of blog entries on longevity pathways known through nematode-based research, including calorie restriction, FOXO/DAF-16, IGF-1, SIRT1, and mTOR.  And important new studies adding to the knowledge in this area are continuing to appear in the literature.  One showed up only two days ago.  As a result of this collective research, genetic interventions are now known that can extend the lifespans of nematodes by a factor of about seven.  If this were true for people, we could live to the age of 570. 

This blog entry reports on recent findings not discussed in this blog  before related to the ETS and PDEF transcription factors, the AGE protein, TUBBY, WWP-1, and GATA activation factors.  A follow-up blog entry will deal with why nematode life extension has gotten so far during the last 20 years while, during the same period, there has been virtually no progress in creating significant life extension in humans. 

Before I get into the newer findings, I need to review what has been discovered over the years about nematode longevity as related to well-studied pathways.  In the process I will note a few benchmark points relating to life extension in C-elegans.   

AGE-1 

Back in 1988 it was noticed that there is a gene called AGE-1 that, when mutated, extends the life of C-elegans by around 50% and longer in hermaphrodite worms.   As described in the publication A Mutation in the age-1 Gene in Caenorhabditis elegans Lengthens Life and Reduces Hermaphrodite Fertility,  “age-1(hx546) is a recessive mutant allele in Caenorhabditis elegans that results in an increase in mean life span averaging 40% and in maximal life span averaging 60% at 20{deg}; at 25{deg} age-1(hx546) averages a 65% increase in mean life span (25.3 days vs. 15.0 days) and a 110% increase in maximum life span (46.2 days vs. 22.0 days for wild-type hermaphrodites).” 

FOXO/DAF-16/IGF-1 pathway, insulin-like signaling  

A subsequent benchmark for radical life extension in C-elegans was telegraphed in a 1993 publication A C. elegans mutant that lives twice as long as wild type.  This time the FOXO/DAF-16/IGF-1 pathway was involved.  The FOXO transcription-factor protein DAF-16 is an important regulator of longevity that I have discussed in a number of previous blog postings and specifically in the April 2010 post Another piece of DAF-16 research.  According to the 1993 publication, “We have found that mutations in the gene daf-2 can cause fertile, active, adult Caenorhabditis elegans hermaphrodites to live more than twice as long as wild type. This lifespan extension, the largest yet reported in any organism, requires the activity of a second gene, daf-16. Both genes also regulate formation of the dauer larva, a developmentally arrested larval form that is induced by crowding and starvation and is very long-lived. Our findings raise the possibility that the longevity of the dauer is not simply a consequence of its arrested growth, but instead results from a regulated lifespan extension mechanism that can be uncoupled from other aspects of dauer formation.  Daf-2 and daf-16 provide entry points into understanding how lifespan can be extended.” 

It did not take researchers very long to figure out how to go from doubling nematode lifespans to nearly quadrupling it based on Daf mutations and associated modifications in insulin-like signaling.  The 1995 publication Genes that regulate both development and longevity in Caenorhabditis elegans reported: “The increased life spans are suppressed completely by a daf-16 mutation and partially in a daf-2; daf-18 double mutant. A genetic pathway for determination of adult life span is presented based on the same strains and growth conditions used to characterize Daf phenotypes. Both dauer larva formation and adult life span are affected in daf-2; daf-12 double mutants in an allele-specific manner. Mutations in daf-12 do not extend adult life span, but certain combinations of daf-2 and daf-12 mutant alleles nearly quadruple it. This synergistic effect, which does not equivalently extend the fertile period, is the largest genetic extension of life span yet observed in a metazoan.” 

As further outlined in the 2006 publication Worming pathways to and from DAF-16/FOXO “In Caenorhabditis elegans, the insulin/IGF-1 signaling pathway controls many biological processes such as life span, fat storage, dauer diapause, reproduction and stress response.  This pathway is comprised of many genes including the insulin/IGF-1 receptor (DAF-2) that signals through a conserved PI 3-kinase/AKT pathway and ultimately down-regulates DAF-16, a forkhead transcription factor (FOXO).”   See the publications An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans (1998),  Regulation of C. elegans life-span by insulinlike signaling in the nervous system(2000), and Insulin-like signaling, metabolism, stress resistance and aging in Caenorhabditis elegans(2001).   

MicroRNAs and EGF signaling 

The list of microRNAs and pathways implicated in C-elegans lifespans appears to be ever increasing(ref)(ref).  The August 2010 paper Novel EGF pathway regulators modulate C. elegans healthspan and lifespan via EGF receptor, PLC-gamma, and IP3R activation  reports “Further analysis revealed a potent role of EGF signaling, acting via downstream phospholipase C-gammaplc-3 and inositol-3-phosphate receptor itr-1, to promote healthy aging associated with low lipofuscin levels, enhanced physical performance, and extended lifespan. This study identifies HPA-1 and HPA-2 as novel negative regulators of EGF signaling and constitutes the first report of EGF signaling as a major pathway for healthy aging. Our data raise the possibility that EGF family members should be investigated for similar activities in higher organisms.” 

TUBBY 

The TUBBY gene tub-1 is another that seems to play a critical role in nematode aging as described in the 2005 paper C. elegans tubby regulates life span and fat storage by two independent mechanisms.  “Here, we show that mutation in tub-1 also leads to life span extension dependent on daf-16/FOXO. Interestingly, function of tub-1 in fat storage is independent of daf-16. –. Taken together, we define a role for tub-1 in regulation of life span and show that tub-1 regulates life span and fat storage by two independent mechanisms.”  

GATA 

The 2008 publication An elt-3/elt-5/elt-6 GATA Transcription Circuit Guides Aging in C. elegans identifies another piece of transcriptional circuitry and begins to clarify some of the major issues involved in nematode aging. “To define the C. elegans aging process at the molecular level, we used DNA microarray experiments to identify a set of 1294 age-regulated genes and found that the GATA transcription factors ELT-3, ELT-5, and ELT-6 are responsible for age regulation of a large fraction of these genes. Expression of elt-5 and elt-6 increases during normal aging, and both of these GATA factors repress expression of elt-3, which shows a corresponding decrease in expression in old worms. elt-3 regulates a large number of downstream genes that change expression in old age, including ugt-9, col-144, and sod-3. elt-5(RNAi) and elt-6(RNAi) worms have extended longevity, indicating that elt-3, elt-5, and elt-6 play an important functional role in the aging process. These results identify a transcriptional circuit that guides the rapid aging process in C. elegans and indicate that this circuit is driven by drift of developmental pathways rather than accumulation of damage.”  

WWP-1 

There appears to be no end to discovery of critical new genes in nematodes that play a role in aging.  The 2010 publication WWP-1 is a novel modulator of the DAF-2 insulin-like signaling network involved in pore-forming toxin cellular defenses in Caenorhabditis elegans relates “Here we reveal that reduction of the DAF-2 insulin-like pathway confers the resistance of Caenorhabditis elegans to cytolitic crystal (Cry) PFTs produced by Bacillus thuringiensis. In contrast to the canonical DAF-2 insulin-like signaling pathway previously defined for aging and pathogenesis, the PFT response pathway diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and appears to feed into a novel insulin-like pathway signal arm defined by the WW domain Protein 1 (WWP-1). In addition, we also find that WWP-1 not only plays an important role in the intrinsic cellular defense (INCED) against PFTs but also is involved in innate immunity against pathogenic bacteria Pseudomonas aeruginosa and in lifespan regulation. Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 insulin/IGF-1 signaling network to regulate fundamental cellular responses in C. elegans.”  Here we see an example of an important point:  the same pathway that confers longevity confers bacterial resistance and health. 

ETS/PDEF 

A very new publication, Sept 23 2010, ETS-4 is a Transcriptional Regulator of Life Span in Caenorhabditis elegans, points to yet another important set of transcription factors affecting nematode longevity. “Animal life span is regulated in response to developmental and environmental inputs through coordinate changes in gene expression. Thus, longevity determinants include DNA-binding proteins that regulate gene expression by controlling transcription. Here, we explored the physiological role of the transcriptional regulator, ETS-4, in the roundworm Caenorhabditis elegans. Our data showed that worms that lack ETS-4 lived significantly longer, revealing ETS-4s role in the transcription network that regulates life span. We identified 70 genes whose expression was modulated by ETS-4 that function in lipid transport, lipid metabolism and innate immunity. Some of the ETS-4-regulated genes were also controlled by two other regulators of aging, the FOXO and GATA factors. We concluded that a common set of transcriptional targets orchestrate the network of physiological factors that affect aging. ETS-4 is closely related to the human ETS protein SPDEF that exhibits aberrant expression in breast and prostate tumors. Because the genetic pathways that regulate aging are well conserved in other organisms, including humans, our findings could lead to a better understanding of SPDEF function and longevity regulation in mammals.”

The human ortholog of ETS-4 is SPDEF, quite possibly also a longevity determinant in us humans.  Interestingly, SPDEF may play an important role in preventing/treating cancers.  See PDEF is a negative regulator of colon cancer cell growth and migration, Prostate-derived Ets transcription factor (PDEF) downregulates survivin expression and inhibits breast cancer cell growth in vitro and xenograft tumor formation in vivo, Prostate-derived Ets transcription factor as a favorable prognostic marker in ovarian cancer patients and the 2010 paper PDEF and PDEF-induced proteins as candidate tumor antigens for T cell and antibody-mediated immunotherapy of breast cancer.   

I cannot begin here to cover the hundreds of additional papers on factors affecting C-elegans longevity.  Some researchers have been responsible for multiple discoveries related to nematode longevity over the years.  In particular, Cynthia Kenyon of the University of California, a lead author of the 1993 paper describing a doubling of nematode lifespans, has authored or co-authored some 80 relevant papers including these most-recent ones:

·         The somatic reproductive tissues of c. elegans promote longevity through steroid hormone signaling.

·         A pathway that links reproductive status to lifespan in Caenorhabditis elegans.

·         Widespread protein aggregation as an inherent part of aging in C. elegans. 

·         Insulin/IGF-1 signaling mutants reprogram ER stress response regulators to promote longevity.

·         Glucose shortens the life span of C. elegans by downregulating DAF-16/FOXO activity and aquaporin gene expression.

·         A transcription elongation factor that links signals from the reproductive system to lifespan extension in Caenorhabditis elegans.

·         Stimulation of movement in a quiescent, hibernation-like form of Caenorhabditis elegans by dopamine signaling

·         Regulation of the longevity response to temperature by thermosensory neurons in Caenorhabditis elegans.

·         A regulated response to impaired respiration slows behavioral rates and increases lifespan in Caenorhabditis elegans.

·         A role for autophagy in the extension of lifespan by dietary restriction in C. elegans.

·         Distinct activities of the germline and somatic reproductive tissues in the regulation of Caenorhabditis elegans’ longevity.

·         Tissue entrainment by feedback regulation of insulin gene expression in the endoderm of Caenorhabditis elegans.

·         DAF-16/FOXO targets genes that regulate tumor growth in Caenorhabditis elegans.

·         On why decreasing protein synthesis can increase lifespan.

·         Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans.

·         A steroid hormone that extends the lifespan of Caenorhabditis elegans.

·         My adventures with genes from the fountain of youth.

·         Mutations that increase the life span of C. elegans inhibit tumor growth.

·         Worming pathways to and from DAF-16/FOXO.

·         Enrichment of regulatory motifs upstream of predicted DAF-16 targets. 

A list of many additional nematode-related publications coming from the Kenyon lab can be found here.  And oh yes, some of the nematodes living in Dr. Kenyon’s lab are now capable of living seven times as long as their normal wild-type counterparts.   From the Kenyon lab website  – Quicktime movie of two active 144 day old worms. [normal life span 21 days]. 

Some lessons from the nematode

·        Very significant lifespan extension is possible in genetically complex organisms that share many common gene activation pathways with humans.

·        Well over a thousand genes affect aging or are age-regulated in nematodes and probably a larger number in humans.

·        Nematode studies strongly supports the hypothesis that aging is an extremely complex process involving the interactions of multiple pathways  and an immense number of genes and is not the result of accumulated damage, oxidative damage or other damage.  [My own theory is that the two smoking guns implicated in aging are accumulated epigenetic changes (e.g. in DNA methylation and histone acetylation) and exhaustion of the stem cell supply chain.  See my presentation Towards a Systems Theory of Aging offered at the 2009 American Aging Society meeting.]

·        C-elegans provides an excellent working model for examining how a limited number of transcriptional regulators can mediate to influence downstream aging  lifespan effector genes.

·        After twenty years of research, the job of studying longevity in nematodes is hardly complete.  Important new transcription factors, pathways and genes are still being identified.  In this blog entry I have been able to cover only highly-selected topics in the nematode research domain.

·        Metabolic regulation is essential for life extension.  The 2010 publication Regulation of metabolism in Caenorhabditis elegans longevity, reports “The nematode Caenorhabditis elegans is a favorite model for the study of aging. A wealth of genetic and genomic studies show that metabolic regulation is a hallmark of life-span modulation. A recent study in BMC Biology identifying metabolic signatures for longevity suggests that amino-acid pools may be important in longevity.”

·        There appear to be certain proteins that both extend longevity in nematodes and combat cancers in humans, ETS-3 and its human homolog SPDEF being a good example.  In general, factors that increase longevity do so by increasing healthspan.

·        In terms of regulation of aging-related genes, some genes act upstream of others, and downstream genes can be regulated by several different upstream pathways.  For example, the FOXO, GATA and Ets-4 transcription factors all regulate common genes.  In fact, it appears that Ets-4 functions in parallel to the insulin/IGF-1 receptor, daf-2 and akt-1/2 kinases. Many pathways impact on many genes to slow down/accelerate aging.  This explains why a similar pattern of age postponement (or acceleration) can be generated by quite different upstream interventions such as calorie restriction, feeding rapamycin, and knocking out of various gene combinations.

There is incredibly good life extension news, if you are happen to be a nematode. The following blog entry will examine why we have not been able to make similar progress with human life extension.

The October 2009 blog entry Klotho anti-aging gene in the news describes how the Klotho protein exhibits anti-aging effects in mice when over-expressed and accelerates aging when under-expressed, Klotho’s role with respect to the vitamin D receptor (VDR),  and how defects in Klotho expression is correlated with a number of disease processes.  I said “As to how Klotho may impact on longevity: a) I have already mentioned its actions in averting tissue glycation, b) the IGF-1 pathway (affected by Klotho) has long been known to be associated with longevity and is that affected by calorie restriction, and c) Klotho promotes the body’s antioxidant defenses.”  Research published in the last year reveals an important new link of Klotho to longevity involving phosphate clearance.  I discuss that link and possible implications for consummate drinkers of cola drinks in this blog entry.  Also, increasing evidence exists that Klotho functions as tumor suppressor, and I also discuss that topic. 

Defects in Klotho expression can lead to underexpression of FGF23 and accumulation of phosphates 

The 2010 paper Klotho introduces the main topic of this blog entry: “The klotho gene was identified as an “aging-suppressor” gene in mice that accelerates aging when disrupted and extends life span when overexpressed. It encodes a single-pass transmembrane protein and is expressed primarily in renal tubules. The extracellular domain of Klotho protein is secreted into blood and urine by ectodomain shedding. The two forms of Klotho protein, membrane Klotho and secreted Klotho, exert distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors and functions as an obligate co-receptor for FGF23, a bone-derived hormone that induces phosphate excretion into urine. Mice lacking Klotho or FGF23 not only exhibit phosphate retention but also display a premature-aging syndrome, revealing an unexpected link between phosphate metabolism and aging.” 

The link between Klotho, phosphate retention and aging was telegraphed in the October 2009 paper Klotho and aging.  “The klotho gene encodes a single-pass transmembrane protein that forms a complex with multiple fibroblast growth factor (FGF) receptors and functions as an obligatory co-receptor for FGF23, a bone-derived hormone that induces negative phosphate balance. Defects in either Klotho or Fgf23 gene expression cause not only phosphate retention but also a premature-aging syndrome in mice, unveiling a potential link between phosphate metabolism and aging.” 

The 2010 review Klotho as a regulator of fibroblast growth factor signaling and phosphate/calcium metabolism also describes the situation.  “PURPOSE OF REVIEW: This review summarizes the most recent findings on Klotho in the regulation of fibroblast growth factor-23 (FGF23) signaling and phosphate/calcium homeostasis.  — RECENT FINDINGS: The klotho gene encodes a single-pass transmembrane protein and functions as an aging-suppressor gene, which extends life span when overexpressed and accelerates the development of aging-like phenotypes when disrupted in mice. FGF23 is a hormone that suppresses phosphate reabsorption in renal proximal tubules. Recent studies have shown that Klotho mice and Fgf23 mice exhibit identical phenotypes including hyperphosphatemia and hypercalcemia in addition to the aging-like syndrome. This may be explained by the fact that Klotho binds to multiple FGF receptors and increases their affinity to FGF23.”

The mechanism of operation of Klotho with respect to FGF23 is further detailed in the 2010 publication Regulation of ion channels by secreted Klotho: mechanisms and implications.  “Klotho is an anti-aging protein predominantly expressed in the kidney, parathyroid glands, and choroid plexus of the brain. It is a single-pass transmembrane protein with a large extracellular domain. The extracellular domain of Klotho is cleaved and released into extracellular fluid, including blood, urine, and cerebrospinal fluid. The membrane-bound full-length Klotho and secreted extracellular domain of Klotho have distinct functions. Membrane Klotho interacts with fibroblast growth factor (FGF) receptors to form high-affinity receptors for FGF23. Secreted Klotho functions as a humoral factor that regulates several ion channels and transporters, and other processes, including insulin and insulin-like growth factor signaling.”

FGF23 and its relationship to Klotho are linked to a number of bone and joint diseases, such as described in the 2010 publication [Bone and joint diseases in children. Phosphaturic hormone, FGF23, and bone metabolism].   “Fibroblast growth factor 23 (FGF23) belongs to FGF19 subfamily, whose members function like endocrine factors, and has a phosphaturic effect, leading to hypophosphatemia associated with rickets or osteomalacia when its concentration in blood is elevated. FGF23 is involved in the pathogenesis in many forms of hypophosphatemia including the autosomal dominant and recessive types, the X-linked type and the tumor-induced type. Alpha klotho, originally discovered as an anti-aging factor, along with the FGF receptor type 1 makes a specific receptor for FGF23.”

 Accumulated phosphates can accelerate aging

The link between accumulated phosphates and aging is detailed in the 2010 publication Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging. “Identifying factors that accelerate the aging process can provide important therapeutic targets for slowing down this process. Misregulation of phosphate homeostasis has been noted in various skeletal, cardiac, and renal diseases, but the exact role of phosphate toxicity in mammalian aging is not clearly defined. Phosphate is widely distributed in the body and is involved in cell signaling, energy metabolism, nucleic acid synthesis, and the maintenance of acid-base balance by urinary buffering. In this study, we used an in vivo genetic approach to determine the role of phosphate toxicity in mammalian aging. Klotho-knockout mice (klotho(-/-)) have a short life span and show numerous physical, biochemical, and morphological features consistent with premature aging, including kyphosis, uncoordinated movement, hypogonadism, infertility, severe skeletal muscle wasting, emphysema, and osteopenia, as well as generalized atrophy of the skin, intestine, thymus, and spleen. Molecular and biochemical analyses suggest that increased renal activity of sodium-phosphate cotransporters (NaPi2a) leads to severe hyperphosphatemia in klotho(-/-) mice. Genetically reducing serum phosphate levels in klotho(-/-) mice by generating a NaPi2a and klotho double-knockout (NaPi2a(-/-)/klotho(-/-)) strain resulted in amelioration of premature aging-like features. The NaPi2a(-/-)/klotho(-/-) double-knockout mice regained reproductive ability, recovered their body weight, reduced their organ atrophy, and suppressed ectopic calcifications, with the resulting effect being prolonged survival. More important, when hyperphosphatemia was induced in NaPi2a(-/-)/klotho(-/-) mice by feeding with a high-phosphate diet, premature aging-like features reappeared, clearly suggesting that phosphate toxicity is the main cause of premature aging in klotho(-/-) mice. The results of our dietary and genetic manipulation studies provide in vivo evidence for phosphate toxicity accelerating the aging process and suggest a novel role for phosphate in mammalian aging.”

Other effects of Klotho 

Of course Klotho does other things beyond binding to FGF23 as outlined in my earlier blog entry and in the 2008 paper Klotho as a regulator of oxidative stress and senescence.  “The klotho gene encodes a single-pass transmembrane protein that binds to multiple fibroblast growth factor (FGF) receptors and functions as a co-receptor for FGF23, a bone-derived hormone that suppresses phosphate reabsorption and vitamin D biosynthesis in the kidney. In addition, the extracellular domain of Klotho protein is shed and secreted, potentially functioning as a humoral factor. The secreted Klotho protein can regulate multiple growth factor signaling pathways, including insulin/IGF-1 and Wnt, and the activity of multiple ion channels. Klotho protein also protects cells and tissues from oxidative stress, yet the precise mechanism underlying these activities remains to be determined. Thus, understanding of Klotho protein function is expected to provide new insights into the molecular basis for aging, phosphate/vitamin D metabolism, cancer and stem cell biology.” 

Klotho and cancer processes

The 2010 publication Klotho inhibits growth and promotes apoptosis in human lung cancer cell line is one of several dealing with the anti-cancer properties of Klotho.  “Recently, published studies suggest that klotho can also serve as a potential tumor suppressor. The aim of this study is to investigate the effects and possible mechanisms of action of klotho in human lung cancer cell line A549. — CONCLUSIONS: Klotho can inhibit proliferation and increase apoptosis of A549 cells, this may be partly due to the inhibition of IGF-1/insulin pathways and involving regulating the expression of the apoptosis-related g nes bax/bcl-2. Thus, klotho can serve as a potential tumor suppressor in A549 cells.” 

The 2010 publication The anti-aging gene KLOTHO is a novel target for epigenetic silencing in human cervical carcinoma suggests that turning the Klotho gene off by means of epigenetic silencing may be an important survival strategy used by certain cancers.  “Recently, KLOTHO was reported to function as a secreted Wnt antagonist and as a tumor suppressor. Epigenetic gene silencing of secreted Wnt antagonists is considered a common event in a wide range of human malignancies. Abnormal activation of the canonical Wnt pathway due to epigenetic deregulation of Wnt antagonists is thought to play a crucial role in cervical tumorigenesis. In this study, we examined epigenetic silencing of KLOTHO in human cervical carcinoma. — Methylation-specific PCR and bisulfite genomic sequencing analysis of the promoter region of KLOTHO revealed CpG hypermethylation in non-KLOTHO-expressing cervical cancer cell lines and in 41% (9/22) of invasive carcinoma cases. Histone deacetylation was also found to be the major epigenetic silencing mechanism for KLOTHO in the SiHa cell line. — CONCLUSIONS: Epigenetic silencing of KLOTHO may occur during the late phase of cervical tumorigenesis, and consequent functional loss of KLOTHO as the secreted Wnt antagonist may contribute to aberrant activation of the canonical Wnt pathway in cervical carcinoma.”

Statins promote expression of Klotho

Finally, I hearken back to a 2004 publication which suggests that use of statins promotes Klotho expression, HMG-CoA reductase inhibitors up-regulate anti-aging klotho mRNA via RhoA inactivation in IMCD3 cells.  The conclusion is “Statins inactivate the RhoA pathway, resulting in overexpression of klotho mRNA, which may contribute to the novel pleiotropic effects of statins towards vascular protection.” Other substances may also activate the expression of Klotho, but discovering them will take more research on my part.

Soft drinks, phosphoric acid and aging

Sixteen years ago, I would head down the corridor in my software company twice or three times a day to the vending machine for a diet coke.  I loved the stuff and I still like its taste and short-term impact on me.  And I love diet Dr. Pepper for the same reason too.  Back then, little did I think I might be pursuing a pro-aging strategy.  These and some other soft drinks are strong sources of phosphoric acid, a species of phosphates.  If the above-described research is correct, and if I did not have strong Klotho expression to activate FGF23 to clear the phosphates out, accelerated aging could well have been a consequence of my soft drink addiction. 

There has been serious questions posed as to whether consistent consumption of diet colas leads to osteoporosis(ref). “New research indicates that there may be more to the soda and osteoporosis connection than simply replacing the good stuff with the useless stuff. — Researchers at Tufts University, studying several thousand men and women, found that women who regularly drank cola-based sodas — three or more a day — had almost 4% lower bone mineral density in the hip, even though researchers controlled for calcium and vitamin D intake. But women who drank non-cola soft drinks, like Sprite or Mountain Dew, didn’t appear to have lower bone density. Soda and Osteoporosis: Possible Culprits. — Phosphoric acid, a major component in most sodas, may be to blame, according to lead study author Katherine Tucker, PhD. — Phosphorus itself is an important bone mineral. But if you’re getting a disproportionate amount of phosphorus compared to the amount of calcium you’re getting, that could lead to bone loss.” 

Now to that concern I add another based on the research cited here.  Is loading up on phosphoric acid due to frequent drinking of cola sodas robbing of longevity?  And If there is a life-shortening effect due to chronic ingestion of drinks containing phosphoric acid, does it apply to everybody or only to people with defective Klotho or FGF23 expression?

Smurf2 is a fascinating gene and enzyme that plays a number of key roles throughout life in people, ranging from roles in embryonic development and stem cell differentiation to ones relating to cell senescence and accelerated (or delayed) aging.  It is also implicated in cancers and osteoarthritis.   I strive here to summarize some of the key properties of this substance and point out why it is particularly interesting from the viewpoint of aging. I was made aware of the importance of this substances by a presentation by Hong Zhang at the recent Ellison Medical Foundation’s Colloquium on the Biology of Aging.  Zhang is a researcher at the University of Massachusetts Medical School

What is Smurf2?

The biochemical activities and genetic activation pathway related to Smurf2 are very complex.  In super-simplified terms, Smurf2 has a lot to do with key processes that go in cells including cell reproduction, apoptosis and differentiation.    Smurf2 stands for SMAD specific E3 ubiquitin protein ligase 2, an enzyme that in humans is encoded by the SMURF2 gene.  One of the first documents discussing Smurf2, published in 2000, was Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-beta signaling. Decoded, this means that Smurf2 links up with ubiquitin (a small regulatory protein found in almost all cells with nuclei that directs proteins for breakdown and recycling) for breakdown of Smad2  in the proteasomes (large protein complexes in cells that break down and recycle unwanted proteins) as part of transforming growth factor-beta  (a protein that controls proliferation, cellular differentiation, and other functions in most cells) signaling.  “SMAD2 mediates the signal of the transforming growth factor (TGF)-beta, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation(ref)” Whew! 

The 2001 document Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase expands further on the early understanding of the actions of Smurf2.  “Smad proteins are key intracellular signaling effectors for the transforming growth factor-beta superfamily of peptide growth factors. Following receptor-induced activation, Smads move into the nucleus to activate transcription of a select set of target genes. The activity of Smad proteins must be tightly regulated to exert the biological effects of different ligands in a timely manner. Here, we report the identification of Smurf2, a new member of the Hect family of E3 ubiquitin ligases.  Smurf2 selectively interacts with receptor-regulated Smads and preferentially targets Smad1 for ubiquitination and proteasome-mediated degradation.”       

Telomere length attrition, Smurf2 and cell senescence

There are a number of publications further detailing actions of Smurf2, but I am focusing this discussion on longevity-related issues so I move now to a December 2004 publication co-authored by Zhang Smurf2 up-regulation activates telomere-dependent senescence.  “Progressive telomere shortening activates replicative senescence, which prevents somatic cells from being propagated indefinitely in culture. The limitation of proliferative capacity imposed by replicative senescence is thought to contribute to both organismal aging and the prevention of tumor development. Here we report that up-regulation of Smurf2, an E3 ubiquitin ligase previously implicated in TGF-β signaling, is a specific consequence of telomere attrition in human fibroblasts and that such up-regulation is sufficient to produce the senescence phenotype.”  In other words, telomere attrition leads to upregulation of Smurf2 which in turn drives the cell into senescence.  Smurf2 upregulation is an intermediary between telomere attrition and cell senescence.

Telomere attrition can be due to DNA damage, oxidative stress, oncogenic activation or aging.  Continuing to quote from the same 2004 article, “We show that the senescence-inducing actions of Smurf2 occur in the absence of detectable DNA damage or stress response, that Smurf2’s effects require a novel function distinct from its E3 activity, that Smurf2 recruits the Rb and p53 pathways for senescence induction, and that while p21 is elevated by Smurf2, Smurf2-mediated senescence is independent of p21. Smurf2 is the first gene found to be both up-regulated by telomere attrition and sufficient to induce senescence.”   

Expression of Smurf2 in fibroblasts appears to depend on telomere attrition, not on how many times a cell reproduces.  “Importantly, fibroblasts immortalized by adventitious expression of hTERT and analyzed after multiple passages in culture showed no increase in Smurf2 expression (Fig. 1C), indicating that up-regulation of Smurf2 is not the result of extended cell passage per se but, rather, is a consequence of telomere shortening.”  And exposing immortalized fibroblasts with long and stable telomeres to Smurf2 drives them directly into a senescent state.  “Smurf2 expression produces senescence in hTERT-immortalized cells —  Expression of hTERT in primary cultures of human fibroblasts precludes the progressive shortening of telomeres that activates events leading to senescence, resulting in the immortalization of cell populations (Bodnar et al. 1998; Vaziri and Benchimol 1998; Dickson et al. 2000). In strong support of the conclusion that Smurf2 expression is sufficient to produce the senescence phenotype, we found that adventitious expression of Smurf2 to the level normally observed during replicative senescence induced by telomere attrition (cf. Figs. ​Figs.5A5A and ​and1B) reversed hTERT-mediated immortalization of human fibroblasts. — Collectively, our findings support the argument that Smurf2 up-regulation mediates one of the multiple cellular pathways that have been proposed to lead to senescence (Pereira-Smith and Smith 1988).” 

These findings are clearly relevant to the 12^th theory of aging laid out in my treatise Telomere Shortening and Damage.   Long telomeres and even extraordinary expression of telomerase cannot protect a cell from senescence if Smurf2 is also strongly present in that cell. 

Zhang has been concerned with the overall process of cell senescence as well as with the specific role of Smurf2 and has generated a number of publications  on the topic including a comprehensive 2007review paper Molecular signaling and genetic pathways of senescence: Its role in tumorigenesis and aging.  His discussions in that paper of senescence and aging, senescence as a tumor suppression mechanism, and senescence and tissue microenvironment are worth reading though there has been much relevant subsequent research.   

The 2008 paper Suppression of human tumor cell proliferation by Smurf2-induced senescence, again co-authored by Zhang, continues to tell the story, this time extending the research from fibroblasts cells to a wide variety of cell types including cancer cells.    “Here we report that Smurf2 up-regulation induced senescence in a wide variety of human cell types, including highly neoplastic cell lines. Consistent with our previous findings, the ability of Smurf2 to arrest cell proliferation did not require its ubiquitin ligase activity. Furthermore, expression of the cyclin-dependent kinase inhibitor p21 was increased in tumor cells undergoing Smurf2-induced senescence, and such increase occurred independently of the transactivation function of p53. Our results, which reveal a previously unsuspected tumor suppression function for Smurf2-induced senescence, suggest that modulation of Smurf2 action may be a useful strategy for inhibition of cancer cell growth.”   

From an evolutionary viewpoint, it appears that one role of Smurf2 is protection against cancers.  Cells experiencing telomere attrition are driven into senescence by Smurf2 rather than being exposed to the possibility of oncogenesis due to DNA damage that could be incurred in further cycles of replication.  The upside of cell senescence is limiting the accumulation of additional DNA mutations and limiting the population of cells at risk for neoplastic transformation.  The downside is limiting the renewal capacity of stem and progenitor cells and negative changes in gene expression and cell secretions.  Senescent cells make bad neighbors and contribute to organismal aging.  Quoting from my treatise  “It appears that cellular senescence initiates a self-amplifying cycle between mitochondrial and telomeric DNA damage.  The telomere shortening theory of aging suggests that when a substantial number of cells in an organ approach the Hayflick limit and cell senescence, integrity of that organ can no longer be assured and that virtually all of the conditions and diseases of old age are thus traceable to cell senescence.” 

At his presentation at the Ellison colloquium, Zhang described another round of Smurf2 research, this time based on breeding a new strain of Smurf2-knockout mice.  In culture, the cells in these mice generally survived longer with more population doublings,  and were more prone to turning cancerous when compared with similar cells from  normal; “wild type” mice. The Smurf2 knockout mice showed a significant increase in B cell proliferation.  Conclusions of the slide presentation were “1.  Smurf2-deficient MEFs exhibit delayed senescence and enhanced potential to immortalize in culture, 2.  Smurf2 deficient mice develop tumors including lymphomas, soft tissue sarcomas, small intestine andocarcinomas and hepatocellular carcinomas, suggesting that Smurf2 is a tumor suppressor, and 3. Smurf2 deficient mice have increased bone marrow and LT-HSC populations, suggesting a beneficial effect of Smurf2 deficiency during aging.”   

So, we have another example of a familiar story, in aging Smurf2 is strongly protective against cancers but at a cost of limiting stem and progenitor cell differentiation and the increased life span that would result from this.  Smurf2 is similar to P16/Ink4a in this regard.  As I said in my treatise, “Unfortunately there is a paradox in that the same mechanisms that promote neurogenesis, like expression of Bcl-2 and NF-kappaB, can also promote carcinogenesis(ref)., The Ink4a proteins which are increasingly active with age suppress those mechanisms leading to increased protection against cancers with age, but at the cost of decreased neurogenesis and decreased proliferation of other somatic stem cell types. Sorting out the differences between the biomolecular programs that promote stem cell expression and the programs that promote cancers, assuming there are some differences, is a major challenge that must be overcome if substantial life extension is to be made possible.”  Smurf2 is another Dr Jekyll and Mr. Hyde protein. 

Smurf2 and cancers 

A number of past and relatively recent papers point out ugly things Smurf2 does in cancers, like the 2002 publication High-level expression of the Smad ubiquitin ligase Smurf2 correlates with poor prognosis in patients with esophageal squamous cell carcinoma.  The 2009 publication Smad ubiquitination regulatory factor 2 promotes metastasis of breast cancer cells by enhancing migration and invasiveness states “Overexpression of Smurf2 promotes metastasis in a nude mouse model and increases migration and invasion of breast cancer cells. Moreover, expression of Smurf2CG, an E3 ligase-defective mutant of Smurf2, suppresses the above metastatic behaviors. These results establish an important role for Smurf2 in breast cancer progression and indicate that Smurf2 is a novel regulator of breast cancer cell migration and invasion.” 

Not all actions of Smurf2 are negative with respect to cancers, however.  The 2008 publication Smurf2 induces ubiquitin-dependent degradation of Smurf1 to prevent migration of breast cancer cells states “In the present study, we show the post-translational regulation of Smurf1 by Smurf2 and the functional differences between Smurf1 and Smurf2 in the progression of breast cancer cells. Smurf2 interacted with Smurf1 and induced its ubiquitination and degradation, whereas Smurf1 failed to induce degradation of Smurf2. Knockdown of Smurf2 in human breast cancer MDA-MB-231 cells resulted in increases in the levels of Smurf1 protein, and enhancement of cell migration in vitro and bone metastasis in vivo. — These results indicate that two related E3 ubiquitin ligases, Smurf1 and Smurf2, act in the same direction in TGF-beta family signaling but play opposite roles in cell migration.” 

Smurf2 and Osteoarthritis 

Smurf2 also plays a negative role in certain other age-related disease processes, specifically, osteoarthritis.  The 2009 publication Smurf2 induces degradation of GSK-3beta and upregulates beta-catenin in chondrocytes: a potential mechanism for Smurf2-induced degeneration of articular cartilage has to say: “We have previously demonstrated that Smurf2 is highly expressed in human osteoarthritis (OA) tissue, and overexpression of Smurf2 under the control of the type II collagen promoter (Col2a1) induces an OA-like phenotype in aged Col2a1-Smurf2 transgenic mice, suggesting that Smurf2 is located upstream of a signal cascade which initiates OA development. — Furthermore, we discovered that ectopically expressed Smurf2 interacted with GSK-3beta and induced its ubiquitination and subsequent proteasomal degradation, and hence upregulated beta-catenin in Col2a1-Smurf2 transgenic chondrocytes ex vivo. It is therefore likely that Smurf2-mediated upregulation of beta-catenin through induction of proteasomal degradation of GSK-beta in chondrocytes may activate articular chondrocyte maturation and associated alteration of gene expression, the early events of OA.”  The 2010 publication β-catenin, cartilage, and osteoarthritis states flatly “Overexpression of Smurf2, an E3 ubiquitin ligase, also induces an OA-like phenotype through upregulation of β-catenin signaling.” 

From the Arthritis Foundation’s website:  “A new clinical trial seeks to predict who is most likely to experience osteoarthritis, and to test whether an experimental treatment can prevent it altogether. Physicians are setting their sights on people who sustain a knee injury, seeking to understand why nearly half of them will later go on to develop osteoarthritis. — Initial research has shown an enzyme that controls the response of cells to growth factors may in fact be a major cause of osteoarthritis. The enzymes are called “Smad Ubiquitination Regulatory Factors,” or smurfs; but unlike the small, loveable blue cartoon characters, researchers believe that a particular form of these regulatory enzymes, smurf2, might be responsible for America’s leading cause of disability. — We believe that smurf2 controls whether or not a cartilage cell matures and calcifies into hard bone, which is a very good thing when ‘turned on’ in those areas of the body where we are supposed to have hard bone,” said Randy Rosier, MD, PhD, professor of Orthopaedics and director of Research Translation in Orthopaedics at the University of Rochester Medical Center in New York. “But when smurf2 is active in joint cartilage, it may set off a chain reaction that leads to the steady deterioration of the smooth gliding surface tissue, or cartilage, which comprises the joint surface. When this occurs, the cartilage breaks down and severely damages the weight-bearing surface of a joint. Or, put another way, activation of smurf2 in the joint cartilage appears to significantly contribute to the onset of osteoarthritis.”  The clinical trial referenced is entitled Chondrocyte Maturation and Cartilage Loss Following Meniscal Injury and is currently recruiting participants. 

Smurf2 is another odd-shaped piece of the longevity and health jigsaw puzzle with possible future implications both for treatment of diseases of the aged and pro-longevity interventions. 

The July 2010 blog entry HSP70 to the rescue describes how heat shock protein 70 (HSP70) works to promote survival of cells under stress and provides examples of the positive hormetic effects of this chaperone protein.  For example HSP70 is neuroprotection in case of cerebral ischemia.  HSP-70 is also protective of cancer cells which gives it its good-guy – bad-guy characteristics.   This blog entry briefly reviews the actions of HSP70 again, its role in cancer cells, and research efforts aimed at turning it off in cancer cells.

Roles of HSP70 in cells

One of HSP70’s key job is to act as a cell’s protein-folding officer, detecting unfolded or improperly folded proteins, refolding them properly if possible and, if a protein can’t be folded properly, signaling the cell to commit apoptosis.  Unfolded or improperly folded proteins can lead to dysfunctional cells and a number of disorders.  In my treatise I have discussed incorrect protein folding as a theory of aging. To protect themselves against folded proteins, cells have evolved what is known as the unfolded protein response (UPR).  The UPR process is described in the review paper Signal integration in the endoplasmic reticulum unfolded protein response. “The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways — cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.” 

“Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. — Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100(ref).” 

So, HSP70 keep cells alive by refolding badly-folded proteins and preventing excess aggregation of proteins in cells.  And it helps transportation of proteins to their proper intracellular locations.    HSP70 also keeps cells alive by other means.  “In addition to the well-studied role of Hsp70 as a molecular chaperone assisting in correct protein folding, several new mechanisms by which Hsp70 can prevent cell death have been described. Hsp70 is now known to regulate apoptotic cell death both directly by interfering with the function of several proteins that induce apoptotic cell death as well as indirectly by increasing levels of the anti-death protein bcl-2(ref).”

HSP70 family members and other heat shock proteins are thought to play a role in delaying aging.  Widespread protein aggregation in cells is thought to play a prominent role in aging, at least in lower species and quite possibly in humans(ref).  The 2009 publication The shock of aging: molecular chaperones and the heat shock response in longevity and aging–a mini-review concludes “Molecular chaperones play an important role in the deterrence of protein damage during aging and their expression is required for longevity. Chemical stimulation of HSP synthesis might therefore be a significant strategy in future design of antiaging pharmaceuticals.”  The previous blog post Valproic acid – The phoenix drug arises again pointed out that valproic acid appears to induce HSP70 in cortical neurons and this may be responsible for some of the neuroprotective and therapeutic effects of valproic acid.

A more complete discussion of HSP70’s protective roles can be found in the blog entry HSP70 to the rescue. 

HSP70 in cancer cells

Recently, awareness has been developing that the same mechanisms that make HSP70 protective of normal stressed cells makes it protective of cancer cells. 

For some time it has been known that HSP70 is highly expressed in certain cancer cells, although its exact role was unknown.  The 2000 paper Hsp70 plasma membrane expression on primary tumor biopsy material and bone marrow of leukemic patients stated “With the exception of mammary carcinomas, an Hsp70 plasma membrane expression was found on freshly isolated human biopsy material of colorectal, lung, neuronal, and pancreas carcinomas, liver metastases, and leukemic blasts of patients with acute myelogenous leukemia.  Since normal tissues and bone marrow of healthy human individuals do not express Hsp70 on the cell surface, Hsp70 can be considered as a tumor-selective structure in vivo.”

The 2004 article Heat shock protein 70 promotes cancer cell viability by safeguarding lysosomal integrity started to zoom in on the cancer-specific cell-protective effects of HSP70.  “The major heat-inducible Hsp70 is a potent survival protein that confers cytoprotection against numerous death-inducing stimuli and increases the tumorigenicity of rodent cells. The depletion of Hsp70 by adenovirus-mediated transfer of antisense cDNA induces caspase-independent death of tumorigenic cells while non-tumorigenic cells are unaffected, suggesting that Hsp70 has cancer-specific function(s). We have recently demonstrated that the depletion of Hsp70 in cancer cells results in a cysteine cathepsin-dependent death, which is preceded by lysosomal destabilization and release of lysosomal constituents to the cytosol. In line with this, Hsp70 localizes to the membranes of lysosomes in human colon carcinoma cells and immortalized murine embryonic fibroblasts (MEFs) and prevents lysosomal membrane permeabilization and cell death induced by tumor necrosis factor (TNF), etoposide and H2O2. These findings identify Hsp70 as the first survival protein that functions by stabilizing the lysosomal membrane.”

It is increasingly clear that mobilization of HSP70 as well as other heat shock proteins is a survival strategy employed in several cancer lines.  The 2010 publication Heat shock proteins in breast cancer progression – A suitable case for treatment?  reports “Heat shock proteins (HSP) and heat shock factor 1 (HSF1), key factors in the heat shock response (HSR) have been implicated in the etiology of breast cancer. At least two members of the HSP family, Hsp27 and Hsp70 undergo significant increases in cellular concentration during the transformation of mammary cells. These changes result in HSP-mediated inhibition of tumour cell inactivation through blockade of the apoptosis and replicative senescence pathways. The increases in HSP thus mediate two of the common hallmarks of cancer and favour cell birth over cell death.”

“The cancer microenvironment exposes malignant cells to a variety of stressful conditions that promote protein misfolding. HSP70 helps cancer cells deal with this stress. Unlike normal cells, which typically express little, if any, of HSP70, cancer cells contain high levels of this protein all of the time. Indeed, HSP70 has been termed a cancer-critical survival factor, since cancer cells probably require the actions of this protein to survive the protein-altering adverse conditions(ref).”

A July 2010 report in Science Daily states “Professor Afshin Samali, lead author of the study and head of the Department of Biochemistry at NUI Galway, and his team have discovered that under cell stress conditions Hsp70 interacts with a receptor in the cell, activating survival mechanisms and preventing apoptosis, the normal cell death mechanism. — Professor Samali states: “Our results have identified a novel protein-protein interaction that helps cancer cells to survive stressful growth conditions. By interfering with this interaction we hope to develop a new class of anticancer drugs.”  The source publication for the study mentioned in this quote deals with the mechanism of action of HSP72: “Here, we report that Hsp72, a stress-inducible cytosolic molecular chaperone, can bind to and enhance the RNase activity of IRE1α, providing an important molecular link between the heat shock response and the ER stress response. Importantly, increased production of active XBP-1 was necessary for Hsp72 to exert its prosurvival effect under conditions of ER stress. Our results suggest a mechanism whereby Hsp72 overexpression helps cells adapt to long-term ER stress in vivo by enhancing the pro-survival effects of the IRE1α/XBP1 branch of the UPR.”  HSP72 is the same as HSP70 as related to the HSPA1A gene.

Inhibiting HSP70 in cancer cells

The growing awareness of the importance of HSP70 as a survival factor in cancers has motivated a search for new molecules that can inhibit the expression of HSP70.

HSP70 inhibition via anti-sense Hsp70 cDNA

A page from the Sloan-Kettering Institute web site discusses inhibitors of HSP70 expression.  One of those is anti-sense Hsp70 cDNA.  “It is documented that inhibition of Hsp70 expression by anti-sense Hsp70 cDNA resulted in inhibition of tumor cell proliferation and induction of apoptosis. Depletion of Hsp70 by Ad.asHsp70 led to massive cell death of all tumorigenic cell lines tested (carcinomas of breast, colon, prostate and liver as well as glioblastoma). In spite of an effective depletion of Hsp70, Ad.asHsp70 had no effect on the survival or growth of fetal fibroblasts or non-tumorigenic epithelial cells of breast or prostate.”

HSP70 inhibition via quercetin

The same Sloan-Kettering web page mentions use of quercetin as a HSP70 inhibitor.  “Inhibition of hsp70 gene expression has been documented after pharmacological intervention with the flavanoid quercetin. The agent induced apoptosis in several tumor cell lines. In addition, inhibition of hsp70 accumulation by quercetin made cells more susceptible to apoptotic inducers. Quercetin also sensitized cells to hyperthermia, chemotherapy and radiation. Inhibition of hsp70 synthesis as well as induction of apoptosis by treatment with quercetin combined with hyperthermia was reported to be confined to leukemic cells, and not to normal hematopoietic progenitor cells.”  The ability of quercetin to kill tumor cells via a mechanism involving HSP70 has been known for some time.  The 1994 paper Induction of Apoptosis by Quercetin: Involvement of Heat Shock Protein stated “Quercetin, a widely distributed bioflavonoid, inhibits the growth of tumor cells. The present study was designed to investigate the possible involvement of apoptosis and heat shock protein in the antitumor activity of quercetin. — These results suggest that quercetin displays antitumor activity by triggering apoptosis and that HSP70 may affect quercetin-induced apoptosis.”

The same Sloan-Kettering web page goes on to say “In spite of its evident utility in cancer treatment, quercetin is not potent enough to grant its clinical use. Since the introduction of anti-sense mRNA or siRNAs into humans will be problematic because the extent of inhibition cannot be modulated, and the effects of quercetin are likely pleiotropic, small molecules that directly compromise but not completely inhibit the activities of Hsp70 chaperones will prove clinically valuable to combat cancer. In addition, the above data suggest that an Hsp70 inhibitor concentration can be identified that will not be toxic to healthy cells. To date, however, Hsp70 inhibitors have not been tested in cell or animal cancer models, and very few Hsp70 inhibitors have been identified. — We are interested in identifying both inhibitors of Hsp70 activity and expression and efforts in this regard are currently underway.”

HSP70 inhibition via PES

Late last year there was a report of a possibly promising substance, PES.  The October 2009 publication A Small Molecule Inhibitor of Inducible Heat Shock Protein 70 reports “The multifunctional, stress-inducible molecular chaperone HSP70 has important roles in aiding protein folding and maintaining protein homeostasis. HSP70 expression is elevated in many cancers, contributing to tumor cell survival and resistance to therapy. We have determined that a small molecule called 2-phenylethynesulfonamide (PES) interacts selectively with HSP70 and leads to a disruption of the association between HSP70 and several of its cochaperones and substrate proteins. Treatment of cultured tumor cells with PES promotes cell death that is associated with protein aggregation, impaired autophagy, and inhibition of lysosomal function. Moreover, this small molecule is able to suppress tumor development and enhance survival in a mouse model of Myc-induced lymphomagenesis. The data demonstrate that PES disrupts actions of HSP70 in multiple cell signaling pathways, offering an opportunity to better understand the diverse functions of this molecular chaperone and also to aid in the development of new cancer therapies.”

According to a November 2009 Science Daily article on this research Inhibitor Of Heat Shock Protein Is A Potential Anticancer Drug, Study Finds,  “The inhibitor, called PES, interferes with the HSP70 activities that the cancer cell needs to survive, so by targeting HSP70, one can target the cancer cell. — The investigators showed that PES interacts with HSP70 by blocking its stress-relieving functions. It also induces HSP70-dependent cell death by disrupting the cell’s ability to remove damaged components. Paradoxically for a compound first identified for blocking the cell-death pathway of apoptosis, PES does kill cells, but by a different mechanism. — PES seems to be specifically targeting HSP70, a protein that is differentially expressed in normal versus cancerous cells, and “one that the cancer cell seems to require to survive” says George. “It’s still early days — we don’t know what it will do in a human. But, the exciting part is that this is a pathway and a protein target that clearly is important for cancer cells. — Given the extreme heterogeneity of cancer cells, simultaneously disabling networks of signaling pathways may be important. Indeed, PES was more or less equally effective in every type of cancer cell tested, she says, “which is unusual and supports the idea that it is targeting a protein that is required for the functioning of multiple pathways.”

The article goes on “”We found several known HSP70-interacting proteins that were no longer interacting properly when the cells were exposed to the small molecule,” Leu notes. — Among those were proteins that help HSP70 refold misfolded proteins and proteins that abet its protein trafficking functions. — When they then studied the effect that loss of those functions had on the cell, the team discovered that PES blocks the cell’s ability to get rid of the proteins damaged by cellular stress in a process called autophagy, a process in which cells were basically eating themselves to death. In mice, Murphy and her students Julia Pimkina and Amanda Frank found that PES could inhibit tumor formation and significantly extend survival. — “That was one of the highlights from our perspective, because PES has potential to be developed as a therapeutic,” says Murphy.’

Wrapping it up:

·        HSP70 assures proper folding of proteins in cells and, via this and other mechanisms, is strongly protective of cells under stress and possibly plays a positive role with respect to longevity.

·        HSP70 also plays a major role in protecting cancer cells, setting off a search for substances that can inhibit HSP70 expression in cancer cells.

·        Quercetin, a substance in the anti-aging firewalls regimen, inhibits the expression of HSP70 in cancer cells, but its effects may be too weak for its use as a clinical therapy.

·        A substance called PES shows promise as a possible drug candidate for inhibiting HSP70 expression in cancer cells.

·        The research in this area still involves experimentation with laboratory animals and may or may not lead to clinical trials in humans.