Rapamycin and lifespan extension

Will a pill containing the immunosuppressant drug rapamycin someday extend human lifespan a few years? In spite of the hopeful research announcements that appeared a few days ago, I wouldn't recommend getting one's hopes up just yet.

This is a topic I've discussed before: Calorie restriction, TOR signaling, and aging. And for related stuff on mTOR: here.

The executive summary is that inhibition of mTOR signaling has been shown to extend lifespan in yeast, roundworms, and fruit flies. Mice can now be added to this list, in experiments that included rapamycin in their diet.

Here's the press release:

Easter Island Compound Extends Lifespan Of Old Mice: 28 To 38 Percent Longer Life (7/8/09)

On July 8, in the journal Nature, The University of Texas Health Science Center at San Antonio and two collaborating centers reported that the Easter Island compound – called "rapamycin" after the island's Polynesian name, Rapa Nui – extended the expected lifespan of middle-aged mice by 28 percent to 38 percent. In human terms, this would be greater than the predicted increase in extra years of life if cancer and heart disease were both cured and prevented.

Although rapamycin and some related compounds have been investigated as anti-cancer therapies, the hypothesized lifespan-extending benefits are thought to be related to the by now well-documented benefits of calorie restricted diets. (For very recent news on that front, see here, for example.)

Aging researchers currently acknowledge only two life-extending interventions in mammals: calorie restriction and genetic manipulation. Rapamycin appears to partially shut down the same molecular pathway as restricting food intake or reducing growth factors.

It does so through a cellular protein called mTOR (mammalian target of rapamycin), which controls many processes in cell metabolism and responses to stress.

A decade ago, Dr. [Dave] Sharp proposed to his colleagues that mTOR might be involved in calorie restriction. "It seemed like an off-the-wall idea at that time," Dr. Richardson said.

Experiments were performed in parallel at three separate research centers and consisted of feeding hundreds of mice, starting at an age of 20 months, a diet containing a special formulation of rapamycin designed to evade breakdown in the digestive system. It was found that the age at which 90% of mice had died rose from 1,078 days to 1,179 days in male mice, compared to controls, and from 1,094 days to 1,245 days in females. The total lifespan extension, on average, was therefore 9.4% in males and 13.8% in females.

Note that some accounts of the research claim lifespan extensions of 28% to 38%, but this is misleading, since those figures represent the extension of the "old age" period of mouse life beginning at 20 months. They do not mean that the mice lived up to almost 40% longer in total. (Some pretty shoddy reporting going on here....) And there was no particular evidence to indicate that extensions of such size would have occurred if the special diet began at an earlier age. However, in experiments still going on, there is evidence for some extension when addition of rapamycin to the diet begins for mice 270 days old.

Of course, even an extension of human lifespan in the 10% range – 7 or 8 years – would be quite an accomplishment, provided quality of life in the final years remained about where it is today. (Which is a big if.)

But there are various reasons to suspect that even a 10% extension in humans is rather optimistic. Some reasons:

1. Rapamycin is an immunosuppressant, currently used therapeutically to prevent organ transplant rejection. The experimental mice were maintained under conditions that carefully protected them from infection – conditions that would not be realistic for humans.
   
2. Although mice and humans are both mammals, their genetics are not all that similar. The complete sequence of the mouse genome was recently announced (see here), and it turns out that about 20% of mouse genes are different from human analogs, or not found in humans at all. (It's been 90 million years since the last common ancestor of mice and humans.)
   
3. Rapamycin is known to inhibit an important protein kinase called mTOR (mammalian target of rapamycin). mTOR plays a key role in regulating cell growth, proliferation, and survival, so it's not all that surprising that rapamycin might affect cell biology relevant to aging and longevity. This same property of rapamycin makes it interesting as an anti-cancer agent. Rapamycin and similar compounds that inhibit mTOR have in fact been found to have anti-cancer properties in animal models. Several analogs of rapamycin have been investigated as anti-cancer therapies, and one has even been approved for human use (Torisel). But even in the anti-cancer setting, mTOR inhibitors haven't yet been slam-dunk successes.
   
4. It is not clear that rapamycin in these experiments was working the same way as calorie restriction. None of the rapamycin-fed mice lost body weight, and calorie restriction usually works best when started relatively early in life.
   
5. Experimental mice that received rapamycin got a dose of 2.24 mg per kg of body weight. That's quite a lot – about 30 to 60 times (per kg) what would be given to a 60 kg human for immunosuppression.
   

The unfortunate truth is that cell signaling pathways that affect cell growth, proliferation, and survival are rather complicated, and any interventions in such pathways are very likely to not have the expected effects and/or to have various unexpected side-effects. Here's a diagram of just some of the important pathways mTOR is involved in. Imagine that were an electrical circuit and you made ad hoc changes to important components of the circuit.... Perhaps you can see how trying to affect mTOR in order either to control cancer or enhance longevity might be a dicey proposition.

In spite of all the reservations, there are still promising signs for the role of mTOR inhibition in lifespan extension. The mechanism of action need not be the same as calorie restriction, even though that hasn't been ruled out either. For example, TOR is known from yeast and nematode studies to promote protein production in ribosomes and to inhibit protein degradation via autophagy. Invertebrate studies have shown that reversal of these TOR effects can increase lifespan. And TOR signaling is also known to influence cell growth, cell-cycle progression, mitochondrial metabolism, and insulin-analog signaling.

Remember what we said about the diversity of effects of mTOR signaling? That's definitely a sword that can cut both ways – it's powerful, but hard to predict and control. We need to understand a lot more of the biological details – otherwise we're just swinging the sword in the dark.

Harrison, D., Strong, R., Sharp, Z., Nelson, J., Astle, C., Flurkey, K., Nadon, N., Wilkinson, J., Frenkel, K., Carter, C., Pahor, M., Javors, M., Fernandez, E., & Miller, R. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice Nature DOI: 10.1038/nature08221

Further reading:

Tests raise life extension hopes (7/8/09) – BBC news story

Immune drug boosts lifespan (7/8/09) – TheScientist.com

Fountain of Youth on Easter Island? (7/8/09) – ScienceNOW

Cancer Drug Delays Aging in Mice (7/8/09) – Wired.com

A pill for longer life? (7/8/09) – Nature.com

Ageing: A midlife longevity drug? (7/8/09) – Nature.com PDF

Rapamycin extends life in mice, raising hopes of life-prolonging drug for humans (7/9/09) – The Times (UK)

What Does Life-Extending Drug Mean for Humans? (7/9/09) – Time

New clues in search for elixir of youth (7/9/09) – New Scientist

Antibiotic Delayed Aging in Experiments With Mice (7/8/09) – New York Times

First Drug Shown to Extend Life Span in Mammals (7/8/09) – Technology Review

Longevity pill on the horizon? (7/10/09) – press release

Rapamycin: “An anti-aging drug today”? (3/6/07) – Ouroboros blog post

Tags: rapamycin, biogerontology, aging, longevity

mTOR, MAPK, and cancer

Recent studies are making it increasingly apparent that cancer is really many different diseases – hundreds actually – in the sense that there are hundreds of distinct problems at a molecular level that can result in the symptoms of cancer in a large variety of tissue types. It is necessary to regard all these problems as distinct diseases, because different techniques will be necessary to deal effectively with each one.

One of the things we are now understanding is that it is not simply mutations in a few specific genes that account for most different cancers. Instead, each different type of cancer can be attributed to mutations in one or several genes randomly chosen from a larger set that collectively defines some specific signaling "pathway" in a cell. Or perhaps even several interrelated pathways.

See here for one account of some of the latest research on this. I'd like to discuss the papers that cover this research, but first I'd like to discuss some slightly earlier research that provides a simpler look at the issue.

I've already written about one particular pathway of special importance, the one associated with mTOR. That discussion, from last April, is here.

As you recall, mTOR is a serine/threonine kinase. The pathway in which it plays a prominent part regulates the growth, proliferation, motility, and survival of cells. And also angiogenesis. From that list it should be obvious why malfunctions in the pathway can give rise to cancer. The pathway, in turn, integrates input from a number of upstream pathways, such as those involving intercellular signaling molecules like insulin, IGF-1, and mitogens.

The name mTOR is short for mammalian target of rapamycin. Rapamycin, also known as sirolimus, is a bacterial product that was originally of interest for its antifungal properties. It was subsequently found to have immunosuppressive and antiproliferative properties. These properties, in turn, are a consequence of the fact that rapamycin binds to a protein complex called mTOR complex 1 (mTORC1). The antiproliferative properties, of course, are due to the importance of mTOR in regulating cell proliferation and motility.

All this stuff is well known to cancer biologists and not new. In particular, much research has been devoted to finding useful inhibitors of mTOR. Unfortunately, however, the research hasn't been as successful at actually treating cancer as might have been hoped:

A Role For MAPK Inhibitors Combined With MTORC1 Inhibitors (8/21/08)

Nearly a decade ago, while it was being tested as an immunosuppressive agent to prevent organ rejection in transplant patients, the drug rapamycin was also discovered to have anti-tumor properties. Since then, several rapamycin analogs known as mTOR (mammalian target of rapamycin) inhibitors have been tested in clinical trials for the treatment of various types of cancer.

But despite promising early results, mTOR inhibitors have proven less successful than originally expected.

The problem is that the mTOR inhibitors that have been tried as anti-cancer drugs also seem to stimulate another pathway that promotes cell growth and proliferation:

Now research led by scientists at Beth Israel Deaconess Medical Center (BIDMC) identifies a previously unrecognized problem faced by these agents when it comes to attacking cancers. ... [T]he new findings show that at the same time that rapamycin analogs are halting tumor growth by inhibiting the mTOR protein complex 1 (mTORC1), they are activating the MAPK (mitogen-activated protein kinase) pathway -- thereby encouraging cancer cell survival.

The MAPK pathway has also been under intensive investigation in connection with cancer. As the name implies, kinases in this pathway are activated by mitogens – external signals that promote mitosis. These kinases also affect cell survival and apoptosis. So it's reasonable to guess that adding a MAPK inhibitor to an mTOR inihibitor might counteract the MAPK-stimulating effect of the mTOR inhibitors.

Are you with me? Anyhow, what the new research does is show how there's a feedback loop that connects mTOR inhibition with MAPK activation.

Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer

Numerous studies have established a causal link between aberrant mammalian target of rapamycin (mTOR) activation and tumorigenesis, indicating that mTOR inhibition may have therapeutic potential. In this study, we show that rapamycin and its analogs activate the MAPK pathway in human cancer, in what represents a novel mTORC1-MAPK feedback loop. ... We further show that rapamycin-induced MAPK activation occurs in both normal cells and cancer cells lines and that this feedback loop depends on an S6K-PI3K-Ras pathway.

PI3K is another important signaling kinase about which there is a lot of other interesting current research – which we'll get around to discussing at some point. Ras is a G protein known to be very important in cancer because it activates MAPK pathways.

Are you beginning to get the picture of how complicated cancer can be, due to the interaction of pathways?

Fortunately, the research also shows that MAPK inhibition can offset problems due to mTOR inhibition:

[P]harmacological inhibition of the MAPK pathway enhanced the antitumoral effect of mTORC1 inhibition by rapamycin in cancer cells in vitro and in a xenograft mouse model. Taken together, our findings identify MAPK activation as a consequence of mTORC1 inhibition and underscore the potential of a combined therapeutic approach with mTORC1 and MAPK inhibitors.

Another research group has already confirmed the same thing, using the same MAPK inhibitor (PD0325901):

Anti-tumor Effects Are Enhanced By Inhibiting Two Pathways Rather Than One (8/21/08)

In the second study, Cory Abate-Shen and colleagues, at Columbia University College of Physicians and Surgeons, New York, and the University of Medicine & Dentistry of New Jersey, Piscataway, show that simultaneous inhibition of the mTOR and MAPK signaling pathways inhibited the in vitro growth of prostate cancer cell lines and the in vivo growth of prostate tumors in a mouse model of prostate cancer.

Here's their research paper:

Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model

The AKT/mammalian target of rapamycin (AKT/mTOR) and ERK MAPK signaling pathways have been shown to cooperate in prostate cancer progression and the transition to androgen-independent disease. We have now tested the effects of combinatorial inhibition of these pathways on prostate tumorigenicity by performing preclinical studies using a genetically engineered mouse model of prostate cancer. We report here that combination therapy using rapamycin, an inhibitor of mTOR, and PD0325901, an inhibitor of MAPK kinase 1 (MEK; the kinase directly upstream of ERK), inhibited cell growth in cultured prostate cancer cell lines and tumor growth particularly for androgen-independent prostate tumors in the mouse model.

AKT is yet another family of serine/threonine kinases, often associated with mTOR, that is deeply involved in tumorigenicity. There's a lot of recent research on it that should also be discussed... some other time.

Further reading:

From Metabolism to Oncogenes and Back - Part II – 3/21/08 blog post that discusses many cancer-related signaling pathways, including mTOR, AKT, PI3K, Ras, and their connection with metabolism

Arkaitz Carracedo, Li Ma, Julie Teruya-Feldstein, Federico Rojo, Leonardo Salmena, Andrea Alimonti, Ainara Egia, Atsuo T. Sasaki, George Thomas, Sara C. Kozma, Antonella Papa, Caterina Nardella, Lewis C. Cantley, Jose Baselga, Pier Paolo Pandolfi (2008). Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer Journal of Clinical Investigation DOI: 10.1172/JCI34739

Tags: cancer, mTOR

Cancer stem cells II

Since we've just had a discussion of generalities about cancer stem cells (here), it seems like it would be fun to have a summary of research on CSCs that was presented at the recent meeting of the American Association for Cancer Research in San Diego, or reported since then. So here it is.

As general background, keep in mind that cell surface proteins CD44 and CD24 are considered to be markers of cancer stem cells in various (but not necessarily all) types of cancer. Also, certain cell signaling pathways are thought to be especially important in the activity of cancer stem cells. The list includes Wnt, Sonic hedgehog, Notch, and Bmi1.

Stem Cell-Like Cancer Cells Resistant To Standard Therapy, Responsive To Targeted Therapy (4/29/08)

Previous research had identified a subset of cells in breast tumors that have the ability to form colonies in culture and give rise to tumors in mouse models. Such cells are thought to be cancer stem cells. They express the cell surface glycoprotein CD44, but not CD24, and they appear to be resistant to standard chemotherapeutic agents. However, the drug lapatinib, which inhibits the HER2 pathway, seems to selectively kill these cells.

Getting To The Roots Of Breast Cancer (4/29/08)

This is another report on the research described in the previous item. It provides additional details on the research protocol.

Stem Cell Type Supposed To Be Crucial For Angiogenesis And Cancer Growth Does Not Exist? (4/22/08)

This study casts doubt on the existence of a certain type of bone-marrow derived stem cell that has been suspected of circulating in the blood and acting as a precursor to endothelial cells that make up blood vessel walls. Such cells, if they existed, would be an important target for inhibiting angiogenesis in tumors. The researchers showed, using advanced techniques with mouse models, that endothelial differentiation is not a typical function of bone-marrow derived stem cells.

Ovarian Cancer Stem Cells Identified, Characterized (4/17/08)

Researchers have identified, characterized and cloned ovarian cancer stem cells and have shown that these stem cells may be the source of ovarian cancer's recurrence and its resistance to chemotherapy. They isolated cells from samples of either peritoneal fluid or solid tumors. The cancer stem cells that were identified had traditional cancer stem cell markers including CD44 and MyD88 (which interacts with toll-like receptors to activate NF-κB).
The cells also showed a high capacity for repair and self-renewal. Such cells, when isolated, were capable of forming tumors 100 percent of the time. Within those tumors, 10 percent of the cells were CD44 positive, while 90 percent were CD44 negative, indicating that some cancer stem cells had undergone differentiation.

Stem Cells: The Role Of Cancer-initiating Cells In Diagnosis And Treatment (4/15/08)

This press release describes research presented at the AACR meeting related to stem cells and pancreatic, bladder, ovarian, and breast cancer, and glioma.

Research in pancreatic cancer found that in addition to CD44 and CD24, the enzyme aldehyde dehydrogenase was expressed in a small population of tumor cells. Cells expressing aldehyde dehydrogenase had greater growth capacity than those that didn't, and they were also associated with poorer overall survival.

In a study of breast cancer and glioma, surface markers were not found be sufficient as markers of stem cell activity. However, cells with low proteasome activity did have notably greater capacity for self-renewal and tumor production capacity. (Proteasomes are large protein complexes that degrade unneeded or damaged proteins.)

Researchers studying bladder transitional cell carcinomas found, in 40% of cases, CD44+ cells with other stem cell self-renewal patterns. In these cells, 85% had active Gli1, a part of the Hedgehog pathway, originally discovered in human glioblastoma. A relatively small percentage had active Bmi1, Stat3, or β-catenin (part of the Wnt pathway). None had active Oct4 or Nanog (pluripotent stem cells markers).

The research on ovarian cancer (noted above) involving CD44 and MyD88 markers is referenced again here.

Stem Cell Marker Controls Two Key Cancer Pathways (4/14/08)

Research into breast cancer stem cells has identified, for the first time, another gene that may be involved, Msi1. The investigators showed that Msi1 activated Wnt and Notch signaling. Other studies have shown that Msi1 is a marker of human adult stem cells in general because it has been found in human breast, colon, brain, skin, and other cells. Msi1 was found to affects mammary cells to influence whether they develop into muscle, milk duct linings, etc. Further, Msi1 was found to be expressed in particularly aggressive tumors.

Stem Cells And Cancer: Scientists Investigate A Fine Balancing Act (4/11/08)

This is a report of a general talk about how the mechanisms normally involved in balancing different functions of stem cells may also contribute to cancer. For example, research shows that Bmi1 is important for maintaining stocks of stem cells, and without it stocks of stem cells are depleted. But also Bmi1 is overactive in various cancers including brain tumors.

Secrets of cellular signaling shed light on new cancer stem cell therapies (4/10/08)

Researchers are beginning to study inhibition of signaling pathways that seem to be active in tumors fed by cancer stem cells. In this case, inhibition of the Notch pathway is being investigated as part of a treatment, together with chemotherapy, for metastatic breast cancer. An important question is whether cancer stem cells are sufficiently different from normal adult stem cells so that inhibition of Notch signaling is not harmful. Results of testing in mice indicate that Notch signals are not required for the maintenance of blood-forming stem cells in adult mice.

Stem cells and cancer: cancer pathways that also control the adult stem cell population (4/10/08)

Apc (adenomatosis polyposis coli) is a tumor-suppressing protein that controls β-catenin and hence affects Wnt signaling. When intestinal crypts are damaged and need to be regenerated, Wnt signaling directs stem cells to generate replacement cells. Apc normally turns off Wnt signaling of stem cells when it is no longer needed. The research here showed that if Apc is lost or damaged, Wnt signaling may continue and result in tumor formation

Cancer Stem Cells Created With New Technique (4/9/08)

One of the most important unresolved questions about cancer stem cells is how they originate to begin with. For instance, are they mutations of existing stem cells, or instead precancerous cells that have acquired stem-cell-like capabilities? The research here supports the latter scenario. Starting with normal skin cells, the researchers activated three genes associated with embryonic stem cells. The result closely resembled known cancer stem cells. And they also had more resemblance to normal embryonic stem cells than to normal adult stem cells. One of the genes was Myc, which has also been used to create pluripotent stem cells from skin cells. In addition to the scientific significance of this work, it should also facilitate study of cancer stem cells, which are otherwise hard to locate.

Module Map Links Embryonic Stem Cells And Cancer Stem Cells (4/9/08)

The researchers involved in the work described in the preceding report have additional related findings. They systematically compared gene expression patterns between embryonic stem cells and multiple types of human cancer cells. Gene expression patterns in diverse human epithelial cancers were much like patterns in embronic stem cells. Further, presence of these patterns in cancer cells strongly predicted metastasis and death. On the other hand, normal adult tissue stem cells had an opposite pattern, which was repressed in various human cancers compared to normal tissues. The researchers additionally demonstrated that c-Myc, but not other oncogenes, was sufficient to reactivate the ESC-like program in normal and cancer cells.

And here's some earlier research that features the Nanog and Bmi1 proteins:

To Evade Chemotherapy, Some Cancer Cells Mimic Stem Cells (9/19/07)

Anti-cancer treatments often effectively shrink the size of tumors, but some might have an opposite effect, actually expanding the small population of cancer stem cells believed to drive the disease, according to new findings.

"Our experiments suggest that some treatments could be producing more cancer stem cells that then are capable of metastasizing, because these cells are trying to find a way to survive the therapy," said one of the study's investigators, Vasyl Vasko.

When the researchers applied various anti-cancer drugs to experimental cancer cells, they found that surviving cells expressed more Nanog and Bmi1:

They selected a rare form of cancer, mesenchymal chondrosarcoma (MCS), which has not been well described and for which there is no effective treatment. The researchers first determined that Nanog and BMI1 stem cell markers were more highly expressed in metastatic tumors compared to primary tumors. ...

They then applied various therapies - from VEGF inhibitors such as Avastin to the proteasome inhibitor Velcade - in mice implanted with human MSC, and analyzed the effects on tumors. Some of the treatments seemed to work, because they led to a dramatic decrease in the size of the tumors, Dr. Vasko said. But analysis of stem cell expression before and after treatment revealed that even as some anti-cancer treatments shrank tumors, they increased expression of Nanog and BMI1. "These treatments were not enough to completely inhibit tumor growth, and the cancer stem cell markers were still present," Dr. Vasko said.

Tags: cancer, stem cells, cancer stem cells

TOR signaling and cancer

Another recent development pertinent to the discussion of TOR signaling and cancer (see here), is the announcement of preclinical findings about a potential anti-cancer drug that may act against ovarian cancer. The drug works by inhibiting the mTOR signaling pathway. (mTOR is the mammalian form of TOR.)

This is not at all the first anti-cancer drug that's come along with a similar mechanism of action. But it's still interesting, because any drug that affects TOR signaling has the potential of also causing unwanted side effects, since TOR signaling is involved in so many cell processes. Presumably some effort has been made to find reasons why the effect of the drug should be limited to cancer cells.

The drug is called NV-128, and has been developed by an Australian biotech company called Novogen. Since the drug hasn't yet entered clinical trials in humans, it could take a decade or so (as usual) to perform enough testing to determine that NV-128 is actually effective, and relatively safe.

Anyhow, here's the news release:

Drug Compound Leads To Death Of Ovarian Cancer Cells Resistant To Chemotherapy (4/17/08)

In a discovery that may be useful for maintaining remission in chemo-resistant ovarian cancer, Yale scientists report that pre-clinical studies have shown the drug compound NV-128 can induce the death of ovarian cancer cells by halting the activation of a protein pathway called mTOR.

Many traditional cancer drugs work by triggering cell death via apoptosis. Unfortunately, apoptosis needs enzymes called caspases to work, as explained here. And cancer cells may develop a circumvention of this mechanism by turning down the production of caspases, which are needed to allow mitochondria to respond to apoptosis signals. NV-128, however, is able to overcome this problem by triggering caspase-independent cell death.

In cancer cells, mTOR signals enhance tumor growth and may be associated with resistance to conventional therapies. Inhibition of mTOR could shut down many of these survival pathways, including proteins that protect the mitochondria of cancer cells.

Here's the Novogen press release:

Novogen’s NV-128 shown to target the akt-mTOR receptor in chemoresistant cancer cells (4/15/08)

NV-128 is unique in that it does not induce caspase-mediated apoptosis which can be non-functional in chemoresistant cancer cells due to accumulated mutations in tumour suppressor/promoter genes and over-expression of anti-apoptotic proteins. Rather, NV-128 uncouples the akt-mTOR­P70S6K signal transduction cascade which has a key role in driving protein translation and uncontrolled cancer cell proliferation. Further, NV-128 induces mitochondrial depolarization via a novel pathway involving the autophagy protein Beclin-1 and Bcl-2, thereby resulting in endonuclease G translocation to the nucleus and cell death.

The same research group that presented the findings just mentioned has also done work on ovarian cancer itself, and been able to locate cancer stem cells for this type of cancer:

Ovarian Cancer Stem Cells Identified, Characterized (4/17/08)

Researchers at Yale School of Medicine have identified, characterized and cloned ovarian cancer stem cells and have shown that these stem cells may be the source of ovarian cancer's recurrence and its resistance to chemotherapy.

As already mentioned, NV-128 is not the only drug under investigation for attacking cancer by targeting the TOR pathway. In fact, almost a year ago, the first anti-cancer mTOR-inhibitor received FDA approval. It's Toricel (temsirolimus), an intravenous drug from Wyeth Pharmaceuticals, for kidney cancer. Novartis has an oral drug (everolimus) for kidney cancer in Phase III trials. (It's already been approved by the FDA as an immunosuppressant to prevent rejection of organ transplants.) Interestingly, and unsurprisingly, everolimus is a derivative of Rapamycin (sirolimus) – an anti-fungal and immunosuppressive compound – which led to the original discovery of mTOR. Everolimus works similarly to Rapamycin as an mTOR inhibitor.

The American biotech company Ariad Pharmaceuticals has a small molecule anti-cancer mTOR inhibitor called deforolimus in intermediate clinical trials for a variety of solid cancers, such as sarcomas, endometrial, prostate, breast and non-small cell lung cancers. The company describes the drug as "a novel small-molecule inhibitor of the protein mTOR, a “master switch” in cancer cells. Blocking mTOR creates a starvation-like effect in cancer cells by interfering with cell growth, division, metabolism, and angiogenesis." Last summer Ariad entered into a major partnership with Merck to develop and test the drug, so this is an indication that the drug has definite promise.

Ariad has a nice video you can download, which explains a bit about how their drug works, and about TOR signaling in general. I highly recommend having a look at it, since it covers upstream signals that activate mTOR (growth factors, amino acids, oxygen, energy), downstream effects (synthesis of proteins for cell growth, cell division, metabolism, and angiogenesis). It notes that certain other signaling proteins (PTEN, Akt, PI3K) cause overactivation of mTOR, and it points out that mTOR stimulates the production of the cyclin D cell division protein.

Tags: cancer, TOR signaling

Calorie restriction, TOR signaling, and aging

Now that I've given some pointers to information about how TOR signaling is involved with metabolism (see here), it seems like an opportune time to mention a recent research announcement in this general area.

How Dietary Restriction Slows Down Aging (4/17/08)

University of Washington scientists have uncovered details about the mechanisms through which dietary restriction slows the aging process. Working in yeast cells, the researchers have linked ribosomes, the protein-making factories in living cells, and Gcn4, a specialized protein that aids in the expression of genetic information, to the pathways related to dietary response and aging.

Here's the key background:

Previous research has shown that the lifespan-extending properties of dietary restriction are mediated in part by reduced signaling through TOR, an enzyme involved in many vital operations in a cell. When an organism has less TOR signaling in response to dietary restriction, one side effect is that the organism also decreases the rate at which it makes new proteins, a process called translation.

The researchers investigated various strains of yeast cells that had low rates of protein production, but increased lifespan. They found that a common characteristic of such cells was mutations to one part of the cell's ribosomes, the complex of RNA and certain proteins which manufactures all new proteins in the cell. The result of these ribosome changes was a decrease in the production of most proteins, except for one, called Gcn4, a transcription factor, whose production increased. The effect seems to depend on the same pathway affected by reduced TOR signaling. Gcn4 is associated with control of amino acid synthesis, and is activated when a cell is starved for amino acids.

To make the link between Gcn4 and longevity, the scientists then asked whether preventing the increase of Gcn4 would block life span extension. In every case, cells lacking Gcn4 did not respond as strongly as Gcn4-positive cells.

"The increased production of Gcn4 in long-lived yeast strains, combined with the requirement of Gcn4 for full life-span extension, makes a compelling case for Gcn4 as an important downstream factor in this longevity pathway," Kaeberlein said.

One might speculate that increased Gcn4 production somehow helps the cell cope with lack of nutrients, and one effect is that the cell takes steps to conserve its resources and slow the rate of aging.

Since reduction of TOR signaling is one way to bring about this effect, TOR inhibitors might help slow aging and increase lifespan, at least in yeast. However, since TOR affects so many other cell functions, the chance for harmful side effects of reduced TOR signaling is high.

"The role of TOR and translation in aging is known to be conserved across many different species, so it's plausible that this function of Gcn4 is conserved as well," Kennedy said. Future research will be aimed at testing this hypothesis.

"Clearly TOR signaling is one component, and perhaps the major component, of the beneficial health effects associated with dietary restriction," said Kaeberlein. "The difficulty with TOR as a therapeutic target, however, is the potential for negative side effects. As we learn more of the mechanistic details behind how TOR regulates aging, we will hopefully be able to identify even better targets for treating age-associated diseases in people."

Tags: calorie restriction, TOR signaling

Cancer, metabolism, and oncogenes

I want to call attention (somewhat belatedly) to a series of three very good tutorial blog posts at The Daily Transcript. Although they are nominally about changing views regarding cancer and its causes, they actually provide a nice overview of a number of important topics in molecular biology. Reading these posts will be a big help in understanding a lot of things written about here, in particular topics such as:

• cancer, and how it is "caused" by various factors like metabolism and genetic mutations, and indirectly affected by other biological systems like the immune system
  
• metabolism in general, and how problems with metabolism lead to disease conditions like diabetes and metabolic syndrome, perhaps even Alzheimer's disease
  
• calorie restriction, and how it seems to play a role in longevity
  
• stem cells – what makes them special, how they function biologically and may play a role in the process of cancer
  
• important processes in cell biology, such as apoptosis, autophagy, and (of course) the cell cycle itself
  
• general topics in molecular biology, such as growth factors, transcription factors, signaling cascades, and cell surface receptors
  

So here are the links, with a brief summary of each:

From Metabolism to Oncogenes and Back - Part I (3/17/08)

Historical introduction to the subject. Explains how Otto Warbug had the idea, 100 years ago, that the way to understand cancer was through metabolism. Somewhat later, the discovery of the Rous Sarcoma Virus (1916), and much later, after the revolutionary understanding of DNA and modern molecular biology came about, the focus shifted to the role of oncogenes, tumor suppressors, and genetic mutations in cancer.

From Metabolism to Oncogenes and Back - Part II (3/21/08)

More detailed look at the molecular biology of cancer, protein signaling pathways in general, and TOR signaling in particular. This part includes a great diagram of some of the more important signaling pathways as far as metabolism and cancer are concerned. Besides TOR, it clearly emphasizes the importance of the MAP kinase Ras, and the phosphoinositide signaling proteins PI3K, PTEN, and AKT.

From Metabolism to Oncogenes and Back - Part III (4/2/08)

An even more technical summary of recent discoveries about metabolism, and the peculiar kind of metabolic activity found in cancer cells. It appears that a type of enzyme called pyruvate kinase, which occurs in various forms, plays a big role in cell metabolism and whether a cell uses available energy for making sugars, fats, or DNA.

Tags: cancer, TOR signaling

FoxO transcription factors

Transcription factors are proteins that help regulate genes. This regulation may involve either enabling the expression of a gene or preventing expression. In the first case, the transcription factor is an "activator", and in the second case a "repressor".

Transcription factors perform their function by binding to a particular portion of DNA that is specific to a given gene. When bound to the appropriate DNA segment, a transcription factor affects gene expression by either facilitating (activator) or inhibiting (repressor) the operation of RNA polymerase in transcribing the affected gene into messenger RNA. Usually more than one transcription factor must be present to affect gene transcription, and additional proteins (called "cofactors") may also be required.

To make things even more interesting, transcription factors usually affect multiple genes, which may be otherwise unrelated to each other.

A particularly important family of related transcriptions factors comprises what are called "forkhead box" proteins, or Fox proteins, for short. (The name refers to a sequence of 80 to 100 amino acids that are part of the protein and bind to DNA, and which was originally discovered in fruit flies (Drosophila).)

Among the genes that Fox proteins are involved with are genes related to cell growth, proliferation, differentiation, longevity, and embryonic development. So there are Fox proteins that are important for things like cancer and stem cells – and thus it's quite useful to know about them.

An important subfamily of Fox proteins are the FoxO proteins, and we'll discuss some recent examples in this note.

To begin with, perhaps the most recent example is this:

Molecular Signal That Helps Muscle Regenerate Discovered (12/19/07)

Muscle regeneration after injury is complex and requires a coordinated interplay between many different processes. Key players in regeneration are muscle stem cells, so-called satellite cells. They divide and produce many new muscle cells to fix the damage incurred by injury. A crucial regulator of muscle function and repair is a signalling molecule called calcineurin. It is activated by injury and controls the activity of other key proteins involved in differentiation and the response to damage.

It turns out that calcineurin works by inhibiting FoxO.

Using sophisticated molecular techniques, the scientists revealed that calcineurin accomplishes its effect on muscle by inhibiting another protein called FoxO. FoxO is a transcription factor, a protein that plays a crucial role in skeletal muscle atrophy through the induction of genes involved in cell cycle repression and protein degradation. Suppressing the effects of FoxO, calcineurin ensures that proliferating cells stay alive and keep dividing to produce enough cells to repair muscle damage.

In this case, the normal function of FoxO is to inhibit cell proliferation (as a check on cancer), but this needs to be bypassed (temporarily) to enable muscle regeneration.

This result follows the discovery a few months earlier of the way a specific FoxO protein (FoxO1) cooperates with another important developmental protein (Notch) to control muscle cell differentiation:

Building Muscle Requires Foxo1 (8/25/07)

The mechanisms by which Foxo proteins regulate metabolism are relatively well characterized. However, little was known about the mechanisms by which these same proteins regulate cellular differentiation.

New data generated by Domenico Accili and colleagues at Columbia University, New York, now indicates that Foxo1 cooperates with Notch to control muscle cell differentiation in vitro.

Overexpression of either a constitutively active form of Foxo1 or a constitutively active form of Notch was found to inhibit the in vitro differentiation of a mouse myoblast cell line.

Note that the preceding alludes to the involvement of FoxO proteins in regulation of metabolism. This comes about because they affect the insulin signaling pathway, and hence also glucose and lipid metabolism.

This function is what allows yet another well-known protein, mTOR, to play a role in "metabolic syndrome" – a group of disorders that includes insulin resistance, heart disease and high lipid levels. (mTOR is short for "mammalian target of rapamycin". It's a protein kinase that modifies other proteins by phosphorylation.) The same mechanism appears relevant also to the "Atkins diet" and the effects of calorie restriction.

Fly Genetics Reveal Key Workings Of Atkins Diet (8/8/06)

Using fruit flies bred with a newly created mutant form of the gene TOR (short for target of rapamycin), Oldham and his colleagues were able to determine how the TOR pathway interacted with other important regulators of insulin, glucose and lipid metabolism.

TOR is an ancient gene, found in nearly all animal and plant cells. The researchers discovered that their new mutant fly reduced TOR function, allowing them to observe what happens when TOR's influence is removed.

Reductions in TOR function lowered glucose and lipid levels in the body. They also blocked the function of another important insulin regulator, a factor called FOXO, which is known to be a critical mediator of insulin signals and therefore glucose and lipid metabolism.

As if all that weren't enough, FoxO proteins are also involved with cancer and stem cells:

Gene Knockouts Reveal FoxOs' Vital Functions In Cancer Defense, Health Of Stem Cells (1/25/07)

In an elegant, multiple-gene knockout experiment, a team of Boston scientists has discovered that a trio of molecules, called FoxOs, are fundamentally critical in preventing some cancers, maintaining blood vessel stability, and in keeping blood-forming stem cells healthy. ...

The researchers at Brigham and Women's found that mice engineered to lack genes for the FoxO1, FoxO3, and FoxO4 molecules had serious blood abnormalities. Without the FoxO gene-regulating molecules, the rodents' blood stem cells -- master cells that give birth to working blood cells while also renewing themselves -- divided too fast and "burned out." ...

In the companion paper, lead author Ji-Hye Paik, PhD, of Dana-Farber and colleagues from the DePinho lab report that the three FoxO molecules, known as transcription factors, normally function as tumor suppressors that override maverick cells threatening to grow too fast and form tumors. When FoxOs are eliminated, it may allow cancer to develop.

And even that's not the end of it. FoxO proteins are also involved in the increased levels of inflammation often associated with the aging process. (This phenomenon has been tagged with the neologism "inflammaging".) It has been hypothesized that inflammaging results from the effect of phosphorylated FoxO on another notorious transcription factor, NF-κB (which is heavily involved in inflammation). Some of the effects of calorie restriction may also be due to FOXO phosphorylation. Reference: Restricting inflammaging (11/12/07)

FoxO is also regulated (as is P53) by SIRT1 – so this is yet another relationship to calorie restriction. Reference: Unlocking the Secrets of Longevity Genes

Additional references (for the seriously interested):

An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans.

Ageing: When Less Is More

Tags: Fox proteins, transcription factor, longevity, aging, calorie restriction