What does marathon running do to an athlete's cells?

If you've ever taken up running as a form of exercise, or even thought about it, there's a certain paradox that may have occurred to you. The health benefits of aerobic exercise are well-documented. (See here, for example.) In particular such exercise has been shown to reduce risks of cardiovascular disease, diabetes, and some forms of cancer. Beneficial physiological effects include reduction of high blood pressure, better control of blood sugar, and reducing blood levels of low-density lipoprotein while raising levels of high-density lipoprotein.

On the other hand, exercise necessarily increases a person's rate of metabolism, as food is processed to provide energy expended through exercise. An inevitable side-effect of metabolism is the production of reactive oxygen species (ROS) and "free radicals" that can damage DNA and other cellular constituents. This cellular damage can lead to either cancer or accelerated aging due to cell senescence and cell death.

The paradox, then, is that the health benefits of exercise do not seem to be canceled out by the side-effects of higher rates of metabolism. It's an important issue not just for humans who are trying to stay healthy, but even more important in animals like birds that may need to expend energy continuously over significant periods of time.

So what's going on here? Perhaps this research has some of the answer:

The effect of marathon on mRNA expression of anti-apoptotic and pro-apoptotic proteins and sirtuins family in male recreational long-distance runners

Background

A large body of evidence shows that a single bout of strenuous exercise induces oxidative stress in circulating human lymphocytes leading to lipid peroxidation, DNA damage, mitochondrial perturbations, and protein oxidation.

In our research, we investigated the effect of physical load on the extent of apoptosis in primary cells derived from blood samples of sixteen healthy amateur runners after marathon (a.m.).

Results

Blood samples were collected from ten healthy amateur runners peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and bcl-2, bax, heat shock protein (HSP)70, Cu-Zn superoxide dismutase (SOD), Mn-SOD, inducible nitric oxide synthase (i-NOS), SIRT1, SIRT3 and SIRT4 (Sirtuins) RNA levels were determined by Northern Blot analysis. Strenuous physical load significantly increased HSP70, HSP32, Mn-SOD, Cu-Zn SOD, iNOS, GADD45, bcl-2, forkhead box O (FOXO3A) and SIRT1 expression after the marathon, while decreasing bax, SIRT3 and SIRT4 expression (P < 0.0001).

Conclusion

These data suggest that the physiological load imposed in amateur runners during marathon attenuates the extent of apoptosis and may interfere with sirtuin expression.

There are two main findings here, related to apoptosis and sirtuin expression. Let's take them in order.

Apoptosis is a form of programmed cell death that has several purposes. The invocation of a cell's apopotosis program isn't necessarily an indication that something is wrong. For example, it occurs normally during embryonic development. Early in the development process embryos of all tetrapods have tissues between what will become the fingers and toes of their hands and feet. But since animals that have left an aquatic environment are usually better off without this extra tissue, evolution has led to signals at a certain stage of embryonic development that cause apoptosis in the cells of the relevant tissue. This is an example of what's known as the "extrinsic" apoptotic pathway.

But for our present purposes there's a second pathway – the "intrinsic" pathway – which is used whenever a cell either detects internal damage (usually to its DNA) or some stressful condition, such as an excessive level of reactive oxygen species. A ROS is a chemically-reactive molecule containing oxygen, including what are sometimes called "free radicals".

This condition of excess ROS is called oxidative stress. It can occur for various reasons, including exposure to high levels of heat or ultraviolet radiation – or abnormally rapid cell metabolism due to vigorous exercise. Cells recognize the condition of oxidative stress indirectly though signaling involving various other molecules that are produced in response to particular ROS molecules. Among such indicators are proteins called heat shock proteins. Two members of this family that were measured in the research under discussion were HSP70 and HSP32.

Signals of oxidative stress trigger the second, "intrinsic" apoptotic pathway, which involves a cell's energy-producing organelles, the mitochondria. The main players in the intrinsic pathway are proteins called, generically, "caspases" – short for "cysteine-rich aspartate proteases". Caspases are enzymes that cleave proteins at aspartate units. (Cysteine and aspartate are two of the 21 amino acids that normally make up proteins.)

Caspases are fairly active enzymes, so they don't ordinarily occur at significant concentrations within cells. Instead, they are produced when needed from other protein enzymes called procaspases. One of these, procaspase-9 is found normally within mitochondria, along with another protein, cytochrome c. Most of the time these proteins are confined within the mitochondria. However, under certain conditions some channels in a mitochondrion's membrane can open and allow the release of procaspase-9 and cytochrome c. Once these proteins enter the cytosol (cell fluid) outside a mitochondrion, they can team up with another protein (Apaf-1: "apoptotic protease activating factor 1") to convert the procaspase-9 into the caspase known as caspase-9. The latter is an active enzyme that leads to the production of other caspases, with cell apoptosis as the eventual result.

Since a cell does not want to have apoptosis going on normally, the process must be tightly regulated. This is done (partly) by another pair of proteins, Bcl-2 and Bax. These two proteins have structural similarities and are considered to be in the same family, the Bcl-2 family. They are always present in the cytosol, and the relative concentration between Bcl-2 and Bax is what controls whether mitochondrial membrane channels will allow release of procaspase-9 and cytochrome c. If the ratio favors Bcl-2, the channels are essentially closed – the normal case – but if the ratio favors Bax, the channels open... and apoptosis may follow.

The present research measured the levels of certain proteins in 10 individuals before and after a marathon run. (The measurement was done indirectly by measuring levels of mRNA transcripts of the associated genes.) A key finding was that the ratio of Bcl-2 to Bax shifted in favor of Bcl-2 from the before to the after measurement. In other words, there was an anti-apoptotic effect, which countered the pro-apoptotic effects of ROS molecules produced by vigorous exercise. Although ROS levels were not measured (since there was no corresponding mRNA), levels of superoxide dismutase (SOD) antioxidants (Mn-SOD and Cu-Zn-SOD) increased after the marathons, reflecting ROS production.

Analysis of the results indicates that apoptosis actually was inhibited, though less in some experimental subjects than others. An increase in levels of procaspase-9 was not observed. Further, in 7 of the 10 experimental subjects, there was little evidence of DNA fragmentation (a consequence of apoptosis). In the other 3 subjects, there was some evidence of DNA fragmentation, but also smaller changes in the Bcl-2 to Bax ratios.

Most interestingly, there was a significant positive correlation in after marathon measurements between levels of Bcl-2 and both HSP70 and HSP32. This suggests that the expected increases of HSP70 and HSP32 may play some part in increased Bcl-2 levels. There was also a positive correlation post-marathon between HSP70 and Mn-SOD levels.

These findings, especially given the small sample size, certainly aren't conclusive. But, as the paper says, "Here, we have found a significant relationship between HSP70 and bcl-2 RNA ... following marathon, but the underlying cellular and molecular mechanisms involved in this [sic] exercise induced adaptations in apoptosis and HSP70 are unknown and require further investigation."

Expression of the sirtuins SIRT1, SIRT3, and SIRT4 pre- and post-marathon were also measured. (We've discussed the sirtuins on a number of occasions.) There's an extensive history of research on SIRT1, concerning its connections with such things as cellular metabolism, cell survival under stress, and antioxidant activity. Research on other sirtuins like SIRT3 and SIRT4 is less extensive. However, members of this family have various things in common. All are enzymes. SIRT1 and SIRT3 are histone deacetylases (HDACs), so have epigenetic roles in affecting gene expression. SIRT3 and SIRT4 occur in mitochondria.

Although it's possible to make various speculations about how sirtuins could be involved with apoptosis and metabolic consequences of exercise, not all that much is known about specific molecular mechanisms. Nevertheless, it's interesting that the present research does show an effect of strenuous exercise on SIRT1, SIRT3, and SIRT4 expression. The paper notes that "the RNA contents of SIRT1 increased substantially in the group after marathon.... On the other hand, the RNA contents of SIRT3 and SIRT4 decreased in the group after marathon."

Further research into these connections could be very interesting.

Marfe, G., Tafani, M., Pucci, B., Di Stefano, C., Indelicato, M., Andreoli, A., Russo, M., Sinibaldi-Salimei, P., & Manzi, V. (2010). The effect of marathon on mRNA expression of anti-apoptotic and pro-apoptotic proteins and sirtuins family in male recreational long-distance runners BMC Physiology, 10 (1) DOI: 10.1186/1472-6793-10-7

Further reading:

Running a marathon halts cellular suicide (5/11/10)


Articles related to sirtuins:

Sirtuin proteins (11/16/07)

The discovery of sirtuins, part 1 (11/17/07)

The discovery of sirtuins, part 2 (11/20/07)

Sirtuin news (1/21/08)

SIRT1 and cancer (10/26/08)

New anti-cancer role for p53

I suppose that just about everyone knows of the important role the p53 protein plays in protecting cells from becoming cancerous. The protein was identified 30 years ago and its gene (TP53) cloned soon thereafter. What's not so widely known is just how complex the operation of p53 in protecting against cancer really is. And very recent research shows the complexity is even more than previously thought.

However, the complexity is to be expected, because evolution doesn't "design" cellular mechanisms to work in a straightforward way. The mechanisms are simply the result of about a billion years of trial and error. Being pretty and elegant was not a criterion for success.

Nature is "hairy", knowing nothing of Occam's Razor, and caring even less. Simplicity is for wimps.

But one thing is clear: p53 plays a large role in preventing, or at least suppressing, the development of cancer. In many types of cancer, p53 is found to have mutations more than 50% of the time. Even if p53 isn't mutated, cancer cells generally have other p53 abnormalities, such as low levels of the protein or the presence of various factors that interfere with its activity.

Until the latest research, there have been two principal ways known in which p53 works against cancer, and several additional minor ways. The two main ways p53 has been known to act are binding to DNA as a transcription factor, and binding directly to certain proteins. And each of these mechanisms can lead to either of two main types of tumor suppression: apoptosis (cell death) and temporary or permanent suspension of the cell cycle, which is the process a cell goes through in order to divide and proliferate.

P53 is primarily a transcription factor. In this role it is found in a cell nucleus and binds to various specific DNA gene promoter regions, in order to direct transcription of the associated gene – the first step in production of proteins from a gene.

The proteins that are expressed as a result of this p53 activity can play a part in either apopotosis or cell cycle control (as well as other functions not directly related to cancer – see here, here, here). Which function is invoked depends on the type of signal that activates the p53. Among the possible conditions that may be signaled are detection of correctable or uncorrectable damage to DNA and detection of chromosome telomeres that are too short.

In addition to binding to DNA as a transcription factor, p53 is also capable of binding directly to other proteins in order to control their behavior. Mainly these proteins are involved with apoptosis, such as members of the Bcl2 family.

P53 itself is actually a family of proteins – there are at least 9 different RNA transcripts that can be derived from the TP53 gene. But one thing that each of these family members have in common is a segment, called the DNA binding domain. It is this part of the p53 that is capable of binding to either DNA or other proteins. (In general, a protein domain is a more-or-less self-sufficient component of a protein. Often the same domain appears in different members of a family of proteins.)

One indication of the importance of this p53 domain is the fact that point mutations (errors involving only a single nucleotide pair) in the part of TP53 that code for the binding domain are the only type of point mutations of p53 that are commonly found in tumors. Errors that affect portions of p53 outside of the binding domain are not associated with cancer.

There's one more thing to note about p53's role as a transcription factor. Namely, the RNA that is transcribed under the direction of p53 is not always messenger RNA (mRNA) that will eventually code for the production of a protein. P53 can also initiate the transcription of genes that code for microRNA (miRNA), which is a single-stranded RNA molecule that's normally only 21 to 23 nucleotides in length. Over 500 different types of miRNA have been found in human cells.

MicroRNA is never translated into a protein. Instead, miRNA molecules regulate the translation of messenger RNA for many different proteins (by binding with the mRNA to prevent translation). It has been known for some time that p53 acts as a transcription factor for the miRNA family known as miR-34. It has also been learned that among the proteins regulated by miR-34 are some found in pathways that lead to apoptosis or cell cycle arrest. The net effect is that miR-34 has tumor-suppressing properties, so this is another way that p53, as a transcription factor, helps suppress tumors.

Many other miRNA molecules, on the other hand, are found at high levels in cancer cells. Such miRNAs most likely inhibit expression of tumor suppressing genes, whose proteins might otherwise control cell proliferation or migration. We've discussed a number of miRNAs associated with cancer, mostly of the sort that promote cancer, here and here.

Nevertheless, there are miRNAs besides miR-34 that have anti-cancer effects. Three in particular are miR-16-1, miR-143, and miR-145. It has been observed that these miRNAs, and several others, are found at higher levels in cells where p53 has been activated as a result of DNA damage. (Normally, p53 formed in non-cancer cells is either quickly degraded or else inhibited by certain proteins, especially MDM2, so as not to unnecessarily promote apoptosis or cell cycle arrest. The presence of DNA damage results in the removal of these inhibitions on p53.)

It therefore appears that p53 is doing something to help produce a number of miRNAs, some of which are tumor suppressors. The curious thing, though, is that it can be shown that p53 is not a transcription factor for the genes that encode these miRNAs.

So what is it that p53 is doing instead to help produce these miRNAs? New research published in the July 23, 2009 issue of Nature answers this question – and it uncovers an entirely new mechanism through which p53 (and its binding domain, in particular) acts as a tumor suppressor. Here's the research abstract:

Modulation of microRNA processing by p53

MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression, involved in diverse physiological and pathological processes. Although miRNAs can function as both tumour suppressors and oncogenes in tumour development, a widespread downregulation of miRNAs is commonly observed in human cancers and promotes cellular transformation and tumorigenesis. This indicates an inherent significance of small RNAs in tumour suppression. However, the connection between tumour suppressor networks and miRNA biogenesis machineries has not been investigated in depth. Here we show that a central tumour suppressor, p53, enhances the post-transcriptional maturation of several miRNAs with growth-suppressive function, including miR-16-1, miR-143 and miR-145, in response to DNA damage. ... These findings suggest that transcription-independent modulation of miRNA biogenesis is intrinsically embedded in a tumour suppressive program governed by p53. Our study reveals a previously unrecognized function of p53 in miRNA processing, which may underlie key aspects of cancer biology.

To understand what's going on, it's necessary to explain a few things about how miRNAs are produced. It's not a simple 1-step process of transcribing an miRNA gene into the final short piece of RNA.

There are, instead, three steps. The first step is transcription, done just as is done for any other gene. The RNA produced in this step is many nucleotides long, and is called the "primary transcript" or pri-miRNA. This pri-miRNA is then cut into smaller pieces having a hairpin shape, called pre-miRNA. The pre-miRNA, in turn, is further processed to produce the final "mature" miRNA.

The intermediate step that converts pri-miRNA to pre-miRNA is performed by a protein complex known as the "microprocessor complex" (having nothing to do with computers, of course). One of the key proteins in this complex is an enzyme called Drosha. The final step, which is performed by another enzyme called Dicer, splits the pre-miRNA apart to yield the mature miRNA.

The main contribution of p53 in this process is to facilitate the action of Drosha. It seems that, although Drosha can do the job by itself (since miRNAs are needed even if p53 isn't active), p53 helps by binding (via its binding domain) with parts of the microprocessor complex. This is indicated by the observation that mutations in the binding domain disable p53 binding to the complex, resulting in lower levels of miRNA production.

So there you have it: an essentially novel way that p53 acts as a tumor suppressor, by facilitating production, non-transcriptionally, of tumor-suppressing miRNAs.

Suzuki, H., Yamagata, K., Sugimoto, K., Iwamoto, T., Kato, S., & Miyazono, K. (2009). Modulation of microRNA processing by p53 Nature, 460 (7254), 529-533 DOI: 10.1038/nature08199

Further reading:

Protein plays three cancer-fighting roles (7/22/09) – Science News article on the research

Link between p53 and miRNA – editor's summary in Nature of the research

Cancer: Three birds with one stone (7/23/09) – Nature news article on the research

Tags: p53, microRNA, cancer

Inflammation, microRNA, and cancer

If there's just one single point worth making about the biology of cancer, it would have to be "it's complicated".

Cells in general, and animal cells in particular, are extremely intricate Rube-Goldberg-like mechanisms. Their correct functioning depends on the integrity of 20,000 or so genes (in the case of humans), and at least 5 times as many proteins whose form is specified by the genes. Damage to even one of a few thousand important genes can put a cell on the road to becoming cancerous. So the first fact about cancer isn't really all that hard to understand: cancer (in all of its many forms) is a disease that begins with damage to the DNA of one or more genes.

This damage, which is necessary but not sufficient, can occur in many ways. Sometimes it happens because of the action of external agents, like carcinogenic chemicals or high-energy radiation (including ultraviolet light). Other times it happens simply because of occasional errors made in copying DNA during the process of cell division. These are just a few of many ways in which DNA can suffer damage. It's estimated that from 10,000 to a million DNA mutations can occur in a single human cell per day.

Fortunately, only a few percent of the 3 billion fundamental units (base pairs) of DNA actually occur within genes – everything else is "noncoding DNA". Although much of this noncoding DNA serves some useful purpose, we have little idea at present what that might be. However, it's certainly less critical to cell function than the DNA of actual genes. Even so, 10,000 or so genes in every cell could suffer mutations every day.

Of course, complex multicellular life couldn't exist unless nature had evolved some means for coping with all this random genetic damage. And so, there are a large number of ways that cells have of detecting and repairing the damage that does occur. Then in the relatively small number of cases where damage cannot be repaired, cells have additional fail-safe mechanisms to avoid malfunctions which lead to unlimited proliferation – i. e. cancer. One such mechanism is for a cell to enter a state of "senescence", where it ceases to be able to divide at all. A more drastic, but common, mechanism is for the cell to undergo "apoptosis" – orderly cell death.

A necessary condition, therefore, for a cell to become cancerous, even after DNA damage remains unrepaired (perhaps because of damage to part of the repair mechanism), is that the damage occurs in a gene that codes for proteins needed for one of the various fail-safe mechanisms. Consequently, in almost every case of cancer where a tumor has begun to form, one finds problems in some part of the cell's anti-proliferation machinery.

We'll look at a recent piece of research that identifies one particular way this can happen, and it's interesting for the variety of different cell processes that become involved.

Many of the known "causes" of cancer are fairly easy to understand. Certainly, the cancer risk from DNA-damaging carcinogenic chemicals is obvious enough. And once one understands how important a key protein known as p53 is in crucial cellular processes such as detection of unrepaired DNA damage and invocation of apoptosis if necessary, it's not hard to understand why more than 50% of human tumors have mutated genes for p53.

But there are other factors which have been found, in epidemiological studies, to be statistically associated with cancer development. One of these is inflammation, which is a very normal part of the body's immunological defenses against infection. Inflammation itself is a highly complex process – too complex to outline here. Chronic infections by various agents can cause a state of persistent inflammation. An example is the result of H. pylori bacterial infections. In addition to being responsible for stomach ulcers, such infections are also found in cases of stomach cancer. Obesity is also known as an epidemiological factor in various cancers, and the reason is now thought to be the state of chronic inflammation that obesity often causes.

What is not clear is exactly what mechanism connects inflammation with cancer. There's undoubtedly a variety of mechanisms, given how complicated cellular processes turn out to be when you get down to the finer details. The recent research mentioned above illustrated one such mechanism, in one single type of cancer.

Anti-inflammatory drugs may defeat a treatment-resistant type of cancer (6/24/09)

The research focused on a type of non-Hodgkin lymphoma called diffuse large B-cell lymphoma. In some patients with the disease, chemotherapy works well. In a recent study of 40 patients more than 75 percent of patients with one form of this type of lymphoma survived five years or longer.

But that study also identified a group of patients whose cancer proved difficult to treat. Their tumors failed to respond to chemotherapy, and only 16 percent of patients with this form of lymphoma survived more than five years after they were diagnosed.

Several molecular flags mark this treatment-resistant lymphoma, but the links between them were unknown until now. The new paper reports that tumor cells isolated from these patients have depressed levels of a protein called SHIP1, which was known to suppress tumors. In fact, patients with the lowest levels of SHIP1 are the least likely to survive.

SHIP1 is a phosphatase enzyme. That means it removes phosphate groups from proteins. So a phosphatase has the opposite effect of enzymes known as kinases, which attach phosphate groups to proteins. Having a phosphate group attached at the right place on a protein is what enables the protein to take part in a signaling pathway, which is the basic communication mechanism in a cell responsible for making things happen. Therefore, phosphatases disrupt pathways, and stop things from happening. This can be beneficial, for example, if what's happening is the excessive cell division that occurs in cancer. Accordingly, SHIP1 has been found to be a tumor suppressing protein.

In the case of diffuse large B-cell lymphoma (DLBCL), it is found that SHIP1 levels are abnormally low. It's not that the SHIP1 is defective; there's just not enough of it. So the question is why. Is there some other defective gene that's responsible?

Apparently, there is not. Instead, it's the presence of inflammation that's responsible, and in an interesting way. Inflammation is a perfectly normal product of the body's immune system, and it exists to counteract harmful agents such as bacteria. The immune system initiates and regulates the process of inflammation by means of signaling molecules called cytokines. One of the more common and important of these cytokines is TNFα.

Now, TNFα normally goes about its business without causing cancer or other lasting ill effects. In fact, under the right conditions it can induce apoptosis or inhibit tumor formation in other ways. But for some reason, in DLBCL, TNFα suppresses SHIP1, and thus promotes cancer. The research in question also discovered the mechanism of SHIP1 suppression. It turns out that the real culprit here is a small piece of microRNA called miR-155. This little bugger was already known to be involved with leukemia in mice, and with other cancers. (See references in here.)

The resistant type of lymphoma cells also have elevated levels of miR-155, a specific example of a type of genetic material called microRNA, the team found. They demonstrated that miR-155 suppresses SHIP1 by sticking to the template for the protein, preventing its manufacture. ...

The final clue came from earlier reports that an inflammatory molecule called TNFα could boost levels of miR-155. Additional laboratory work confirmed the observation for this type of lymphoma cell.

Some anti-inflammatory drugs, used for diseases such as arthritis and inflammatory bowel disease, where inflammation gets out of hand, work by suppressing TNFα. So it was hypothesized that such a drug might be beneficial in treating DLBCL. And voilà:

The anti-inflammatory drugs etanercept and infliximab, which are currently used to treat arthritis and inflammatory bowel disease, work by suppressing TNFα, suggesting a new way to curb the malignancy of this type of lymphoma.

The team tested the idea in mice that had been injected with aggressive lymphoma cells and found that nascent tumors shrank in six days.

However, mice are not humans, so the drugs need to be tested in human DLBCL patients. Patients are already being recruited for clinical studies.

Now, there are plenty of questions remaining. More needs to be understood about just what pathways SHIP1 disrupts in order to suppress tumors. This should also help in understanding why inflammation and the resulting TNFα do not, fortunately, cause cancer more often. Baby steps. But perhaps significant ones.

Here's the research abstract:

Onco-miR-155 targets SHIP1 to promote TNFα-dependent growth of B cell lymphomas

Non-coding microRNAs (miRs) are a vital component of post-transcriptional modulation of protein expression and, like coding mRNAs harbour oncogenic properties. However, the mechanisms governing miR expression and the identity of the affected transcripts remain poorly understood. Here we identify the inositol phosphatase SHIP1 as a bonafide target of the oncogenic miR-155. We demonstrate that in diffuse large B cell lymphoma (DLBCL) elevated levels of miR-155, and consequent diminished SHIP1 expression are the result of autocrine stimulation by the pro-inflammatory cytokine tumour necrosis factor alpha (TNFα). Anti-TNFα regimen such as eternacept or infliximab were sufficient to reduce miR-155 levels and restored SHIP1 expression in DLBCL cells with an accompanying reduction in cell proliferation. Furthermore, we observed a substantial decrease in tumour burden in DLBCL xenografts in response to eternacept. These findings strongly support the concept that cytokine-regulated miRs can function as a crucial link between inflammation and cancer, and illustrate the feasibility of anti-TNFα therapy as a novel and immediately accessible (co)treatment for DLBCL.

Pedersen, I., Otero, D., Kao, E., Miletic, A., Hother, C., Ralfkiaer, E., Rickert, R., Gronbaek, K., & David, M. (2009). Onco-miR-155 targets SHIP1 to promote TNFα-dependent growth of B cell lymphomas EMBO Molecular Medicine, 1 (5), 288-295 DOI: 10.1002/emmm.200900028

Tags: cancer, inflammation

MicroRNA and cancer II

We haven't recently discussed the role of microRNA in cancer. Last time (February 2008) is here. There have been some relatively recent research announcements, so let's have a look.

If you want a refresher on the subject, here's a good introductory overview from Cancer Research UK: Micro RNAs and cancer. Although this piece is fairly elementary, it does have many good links to actual research papers.

Now let's jump into a few summaries of recent research.

What's Feeding Cancer Cells? (2/17/09)

Cancer cells grow and multiply rapidly, so they need lots of nutrients. Much is already known about how cancer cells use blood sugar, but other nutrients are also needed. One of these is the amino acid glutamine. This research found that the transcription factor Myc is able to enhance the expression of the enzyme glutaminase (GLS) in cellular mitochondria. GLS is the first enzyme that processes glutamine to produce energy in mitochondria. (Overexpression of Myc is frequently found in cancer – see here.)

The research found that depriving cancer cells of GLS slowed their growth significantly. It was suspected that Myc could directly up-regulate the GLS gene, but it was not that simple. Instead, it appears that Myc down-regulates genes for two types of microRNA: mi-R23a and mi-R23b. Since these mircoRNAs interfere with the GLS messenger RNA, the net effect of Myc is to enhance GLS production.

Research abstract: c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism

A new discovered mutation can hold the key to treat a large number of different cancers (2/17/09)

Since microRNA normally inhibits production of certain proteins, if the proteins affected promote cancer, the inhibitory miRNA will counteract this. This research examined cells of twelve different cancer types.

The basic finding was that mutations of the gene TARBP2 disrupts a pathway that produces anti-oncogenic microRNAs. Mutated TARBP2 diminishes TRBP protein expression, resulting in a defect in the processing of miRNAs. Specifically, the DICER1 protein, which is necessary for miRNA production, is adversely affected.

Research abstract: A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function

Micro RNA Plays A Key Role In Melanoma Metastasis (2/15/09)

Metastasis is the main process by which cancer becomes deadly, and it is especially problematic in melanoma. In order for cancer cells to metastasize (spread to another body location) they must become able to migrate and establish themselves in the new location. This research finds that the microRNA miR-182 assists in this process.

MiR-182 is frequently up-regulated in human melanoma, usually because melanoma cellular DNA contains extra copies of the miR-182 gene. This up-regulation was shown to assist metastasis. Conversely, down-regulation impedes invasion and triggers apoptosis. Over-expressed miR-182 is shown to repress the expression of two tumor suppressors, FOXO3 and MITF, which are both transcription factors. (For more on FOXO3, see here.)

Research abstract: Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor

New Genes Involved In Acute Lymphoblastic Leukemia Play Fundamental Role In Prognosis Of The Disease (2/6/09)

This investigation found that 13 microRNAs were epigenetically regulated in an abnormal way in many patients with acute lymphoblastic leukaemia (ALL). This means that instead of having actual gene mutations, certain parts of the DNA were methylated in an unusual way, so that the underlying genes, which coded for microRNAs, were down-regulated. More precisely, certain histones of the cell's chromatin were methylated, so that genes located on the DNA wrapped around those histones would not be expressed. The genes involved coded for microRNAs that, evidently, are important for suppressing cancer. When approriate steps were taken to reverse abnormal epigenetic regulation of the affected genes, expression levels rose, confirming that the abnormal methylation patterns were responsible for down-regulation.

65% of 352 ALL patients had one or more methylation abnormalities affecting microRNA under investigation. There was a highly significant positive correlation between patient survival at 14 years after diagnosis and absence of such abnormalities. Consequently, tests for methylation problems with the appropriate microRNA genes should be good predictors of survival prospects.

Research abstract: Epigenetic regulation of microRNAs in acute lymphoblastic leukemia

Researchers Identify Another Potential Biomarker For Lung Cancer (1/13/09)

The research showed that smoking impacts bronchial airway gene expression. Various miRNAs were found that were differently expressed in bronchial airway epithelial cells, mostly down-regulated. Messenger RNAs were also identified, whose expression was inversely correlated to the miRNA expression (so that the corresponding genes appear to be down-regulated by the miRNA.)

MiR-218 was especially noteworthy. It is known to be strongly affected by smoking. The conclusion is that miR-218 levels modulate airway epithelial gene expression response to cigarette smoke, suggesting a role for miRNAs in regulating response to environmental toxins.

Research abstract: MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium

Molecule Linked To Muscle Maturation, Muscle Cancer (12/31/08)

The study clarified the role of MiR-29 in myogenesis (muscle cell formation) and found that its down-regulation is associated with rhabdomyosarcoma (RMS), a cancer caused by the proliferation of immature muscle cells. While miR-29 is required for maturation of myoblasts (immature muscle cells), it is also found to be mostly absent from RMS cells.

The study found, further, that the transcription factor NF-κB is responsible for down-regulating miR-29. (NF-κB is an old friend of ours. See here for a small part of the story about its role in inflammation. There's also much more to be said about the role of NF-κB in cancer, where it provides an important connection between inflammation and cancer.)

NF-κB acts to repress miR-29 through another transcription factor, YY1, and Polycomb-group proteins (which remodel chromatin to block transcription factors from DNA promoter sequences).

During myogenesis, NK-κB and YY1 are down-regulated, permitting expression of miR-29, which then further down-regulates YY1 and accelerates cell differentiation. However, in RMS the NF-κB–YY1 pathway remains active, silencing miR-29 and inhibiting differentiation. But reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation,

Research abstract: NF-κB–YY1–miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

Harnessing MiRNA Natural Gene Repressors For Anticancer Therapy (12/1/08)

This research investigates the potential therapeutic use of miR-181a through its ability to repress expression of selected genes. If successful, this would provide a very clever kind of immunotherapy for cancer and possibly other diseases.

In immune system T cells miR-181a is highly expressed in developing T cells, but is markedly down-regulated in mature T cells. Mouse bone marrow cells were engineered to express desired therapeutic genes only when miR-181a is down-regulated. These cells were transplanted into mice and allowed to develop into mature T cells. The proteins repressed by miR-181a would therefore not be found in the immature cells, but would show up in the mature T cells. And so when the genes repressed by miR-181a corresponded to proteins that direct T cells to attack tumor cells expressing the protein hCD19, mice with the engineered bone marrow cells were able to reject tumors expressing hCD19.

Research article (open access): Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras

Molecule Linked To Aggressive Cancer Growth And Spread Identified (11/13/08)

EZH2 is a polycomb group protein, which helps maintain transcriptional repression of genes over successive cell generations. It contributes to the epigenetic silencing of target genes and enables the survival and metastasis of cancer. The research indicates that miR-101 inhibits the expression and function of EZH2 in cancer cells.

The researchers found that miR-101 is significantly underexpressed in a variety of cancers, including prostate and breast cancer. In human prostate tumors miR-101 expression decreases as cancer progresses and expression of EZH2 increases. MiR-101 is coded for at two locations in cell DNA. One or both of those locations is found to be defective in 37.5% of localized prostate cancer cells and in 66.7% of metastatic cells. This suggests that that underexpression of miR-101 is responsible for overexpression of EZH2 and consequent cancer progression.

More: here (11/13/08)

Research abstract: Genomic Loss of microRNA-101 Leads to Overexpression of Histone Methyltransferase EZH2 in Cancer

Further reading:

MicroRNA—implications for cancer – excellent open access review article

Tags: cancer, microRNA

MicroRNA and stem cells II

MicroRNA and stem cells are both pretty hot topics these days. But curiously, there haven't been a whole lot of reports that involve the two together. My last discussion of both, back in March, is here.

However, the combination of microRNA and stem cells just was back in the news, as briefly noted here.

As you recall, microRNA refers to small single-stranded RNA molecules, generally about 21 to 23 nucleotides in length. Each different microRNA (miRNA for short) is transcribed from a DNA gene like any other gene, but the resulting RNA isn't translated into a protein. Instead, the typical miRNA functions by downregulating the expression of another gene that codes for a protein.

An embryonic stem cell (ESC) has the property of pluripotency, which means that it is capable of giving rise to essentially any type of cell in the body of a multicellular organism. Whenever an ESC divides, the resulting daughter cells may also by ESCs (hence pluripotent) or they may be more specialized cells that will eventually give rise to some type of adult body cell.

In any particular ESC, the determination of remaining pluripotent or instead heading down the path to a more specialized cell type depends on what set of genes are expressed. Since miRNAs downregulate gene expression, they can keep an ESC in its pluripotent state, if they are active and suppress a gene that would make an ESC more specialized. But then if such a miRNA is blocked from being expressed, the ESC can start to become more specialized.

On the other hand, as we shall see, some miRNAs may block transcription factors that are needed to maintain a pluripotent state. Such an miRNA needs to be silent in an ESC, so some other protein needs to suppress its expression. (The miRNA called miR-21, discussed later, is an example.)

So the name of the game in studying ESCs, as far as miRNA is concerned, is to figure out what causes a miRNA gene to be expressed or not. Like other genes in an ESC, which genes are expressed is strongly controlled by a few master transcription factors.

There are four such transcription factors which seem to be especially important in ESCs: Oct4, Sox2, Nanog, and Tcf3. As discussed here, the first three of these factors have been found capable of playing a role in turning an ordinary adult cell into a pluripotent stem cell (called an "induced pluripotent stem cell").

Currently there are 336 mature mouse miRNAs known, and 441 mature human miRNAs. It is simple (given the known, complete sequences of mouse and human genomes) to locate the genes for each miRNA. However, in order to determine when a transcription factor regulates the miRNA gene, the promoter for the gene (a separate portion of DNA) must also be located. In order for a gene to be expressed, the right transcription factors have to bind to the gene's promoter.

Finding promoters is a lot harder, but there are techniques that involve searching for methylation of histone proteins that make up the nucleosomes around which cellular DNA is wrapped.

It was known, before the recent research we're discussing, that there were 14,230 sites in the genome where all four of the named master transcription factors could bind simultaneously. Most of those sites were not promoters of some miRNA, but it was straightforward to identify those that were. Of those miRNAs that appeared to be regulated by Oct4, Sox2, Nanog, and Tcf3, it was found that most are in fact preferentially expressed in ESCs. This set of miRNAs would seem to be good candidates for maintaining ESC pluripotency by downregulating other genes.

On the other hand, some of the miRNAs mediated by the transcription factors are silent in ESCs. Subsequent research found that another type of proteins (polycomb proteins) also bind to the miRNA promoters. These proteins were already known to block transcription by binding to gene promoters. But it turns out that some of these silenced miRNAs become active once the ESC loses its pluripotency and begins to differentiate.

The next step will be to figure out what each of these miRNAs regulated by Oct4, Sox2, Nanog, and Tcf3 actually does – either in the ESC or a differentiated cell. That should be very interesting, as the press release suggests:

Putting microRNAs on the stem cell map (8/7/08)

“We now have a list of what microRNAs are important in embryonic stem cells,” says Alex Marson, co-lead author on the paper and an MD/PhD student in the Young lab. “This gives us clues of which microRNAs you might want to target to direct an embryonic stem cell into another type of cell. For example, you might be able to harness a microRNA to help drive an embryonic stem cell to become a neuron, aiding with neurodegenerative disease or spinal cord injury.”

Moreover, the results give scientists a better platform for analyzing microRNA gene expression in cancer and other diseases. “We and others are finding that the overall gene circuitry for embryonic stem cells and cancer cells is very similar,” notes Marson. “Now that we have connected the circuitry to microRNAs, we can begin to compare microRNAs that are regulated in embryonic stem cells to those in cancer cells.”

Here's a somewhat more detailed description of the research: Stem Cell microRNA, Transcription Factor Interplay Uncovered (8/8/08)

Other research on miRNA and ESCs that has appeared since the previous discussion (here) gives a small taste of what may be learned about the miRNAs silenced in ESCs:

Protein Protects Embryonic Stem Cells' Versatility And Self-renewal (3/23/08)

A protein known as REST blocks the expression of a microRNA that prevents embryonic stem cells from reproducing themselves and causes them to differentiate into specific cell types, scientists at The University of Texas M. D. Anderson Cancer Center report in the journal Nature.

Researchers show RE1-silencing transcription factor (REST) plays a dual role in embryonic stem cells, said senior author Sadhan Majumder, Ph.D., professor in M. D. Anderson's Department of Cancer Genetics. "It maintains self-renewal, or the cell's ability to make more and more cells of its own type, and it maintains pluripotency, meaning that the cells have the potential to become any type of cell in the body."

The details are particularly interesting:

In studies using mouse embryonic stem cells, the researchers found that REST disarms a specific microRNA called microRNA-21 or miR-21. MicroRNAs are tiny pieces of RNA that control gene expression by binding to the gene's messenger RNA.

The team found that MiR-21 suppresses embryonic stem cell self-renewal and is associated with a corresponding loss of expression of critical self-renewal regulators, such as Oct4, Nanog, Sox2 and c-Myc. REST counters this by suppressing miR-21 to preserve the cells' self-renewal and pluripotency.

The researchers discovered the roles of REST and miR-21 in a series of experiments using cultured mouse embryonic stem cells in either a self-renewal state or a differentiating state. They found that REST expression was significantly higher in the self-renewal state. Withdrawing REST reduced the stem cells' ability to reproduce themselves and started differentiation -- even when the cells were grown under conditions conducive to self-renewal. Adding REST to differentiating cells maintained their self-renewal.

These experiments also revealed that REST is bound to the gene chromatin of a set of microRNAs with the potential to target self-renewal genes. REST controls transcription of 11 microRNAs.

Is anything special known about miR-21? Yes, in fact – it is known to play a role in cancers of the colon, liver, and thyroid. (See here.)

Tags: microRNA, stem cells, embryonic stem cells

Pluripotency and Lin28

As we discussed here, pluripotent stem cells have been obtained by "reporogramming" various kinds of adult cells. In one case, a set of 4 transcription factors – Oct3/4, Sox2, c-Myc, and Klf4 – were used for the reprogramming. Another research team used a slightly different set – Oct3/4, Sox2, Nanog, and Lin28.

Two of the transcription factors are the same in these two sets. Of those that are different, c-Myc and Nanog are very familiar to molecular biologists for a variety of reasons. (We discussed some of what's known about c-Myc here, and Klf4 is discussed here.)

So what, if anything, is special about Lin28? Quite a lot, it turns out. Apparently Lin28 not only promotes pluripotency, but it also interacts with a very well-known type of microRNA called let-7. As we saw here, let-7 does several things that help suppress cancer. For one thing, let-7 regulates the oncogene Ras, apparently by binding to the mRNA encoding Ras, thereby inhibiting protein expression. (See here.) For another thing, and more to the point, let-7 tends to negate some of the "stemness" of stem cells, and pushes them onto a path for differentiation into more specialized cell types. (See here.) This helps inhibit cancer by reducing the ability of suspected cancer stem cells to proliferate. Let-7 has also been mentioned as an inhibitor of oncogenicity of c-Myc.

Lin28, on the other hand, seems to regulate let-7, and therefore it helps preserve "stemness", but at the same time it may raise the risk for development of cancer. A paper in the April 4 issue of Science describes the research that indicates such activity:

Selective Blockade of MicroRNA Processing by Lin28

Here we show that Lin28, a developmentally regulated RNA binding protein, selectively blocks the processing of pri-let-7 miRNAs in embryonic cells. Using in vitro and in vivo studies, we found that Lin28 is necessary and sufficient for blocking Microprocessor-mediated cleavage of pri-let-7 miRNAs. Our results identify Lin28 as a negative regulator of miRNA biogenesis and suggest that Lin28 may play a central role in blocking miRNA-mediated differentiation in stem cells and in certain cancers.

Some people are suggesting that perhaps at least some "cancer stem cells" are actually more ordinary cancer cells that have been reprogrammed (in part by Lin28) to be capable of more stemcell-like behavior. What's really going on here still seems a bit speculative at this point.

This blog post of 3/25/08 goes into a lot of detail on mircoRNA, let-7, Lin28, and the whole ball of wax: bring 'em all together: cancer, stem cells, miRNAs.

Further reading:

Deconstructing Pluripotency – overview article in April 4, 2008 Science that discusses two stem cell papers, including the one cited above

Let7 miRNAs, Lin-28, Cancer and Stem Cells – 3/24/08 blog post that discusses this research

Lin-28 is Master of Let-7 miRNA Processing – 3/25/08 tongue-in-cheek blog post that discusses the previous blog post and the subject more generally

Tid Bits – 3/28/08 blog post on related topics

Tags: stem cells, induced pluripotent stem cells, microRNA, cancer

Memory and BDNF

Following up on part of this note, where I discussed relationships between memory and stress, it turns out that there are some interesting things known, related to this, which involve a "neurotrophic factor" called BDNF.

In fact, there's quite a lot to say. Let's begin with an explanation of some terms, a little about BDNF, and a look at some research from the past several years on the relationship between BDNF and memory. Later we'll take up more on how stress and depression enter the picture.

A neurotrophin is a type of protein that promotes the survival of neurons – which is in general a pretty good thing. (We'll get to examples in a moment.) One type of neurotrophin, known as a "neutotropic factor", is a growth factor that affects neurons in particular.

More generally, a growth factor is a proteins that signals certain types of cells to survive, differentiate, or grow. A growth factor that helps a cell survive does so by inhibiting programmed cell death. Other growth factors promote cell division, which results in growth of the tissue that contains the affected cells. Yet other growth factors may induce cells to differentiate into cells of a more specialized type.

An important example of a general growth factor is IGF-1, also known as "insulin-like growth factor 1", which we'll be looking at more extensively in upcoming posts.

In this post we're going to consider the specific neurotrophic factor known as BDNF, the brain-derived neurotrophic factor.

Research has shown that BDNF plays a role in memory formation and in the connection between stress and depression. For example, in rats the stress hormone corticosterone seems to decrease the expression of BDNF, and if stress is persistent, this eventually leads to the atrophy of the hippocampus. Since the hippocampus plays an important role in long term memory, this is one way in which stress can negatively impact memory.

Atrophy of the hippocampus has also been found in humans suffering from chronic depression. There is evidence that suggests a deficiency of BDNF may be at least in part implicated in such depression. For example, various factors (such as the neurotransmitter glutamate, exercise, calorie restriction, and antidepressant drugs) are known to stimulate expression of BDNF – and often ameliorate depression as well.

There's a lot of science behind all this. Let's just look at a few research announcements from the past several years to get a feel for the interactions of BDNF and memory.

Key Pathway In Synaptic Plasticity Discovered (5/23/07)

The researchers studied a major developmental event in newborn rodents. A rapid increases in synapse strength and visual circuit refinement occurs quickly after the animal's eyes first open. It was already known that the PSD-95 protein rushes to visual system synapses soon after eye opening. PSD-95 is a scaffold protein that anchors several types of receptors. Some of these receptors are for the neurotransmitter glutamate, and there is also the TrkB receptor for BDNF (and other neurotrophins).

A positive feedback loop is initiated, in which the NMDA glutamate receptor activates BDNF. BDNF then triggers a signaling pathway involving the kinases PI3 and Akt. This pathway leads to more PSD-95 production, completing the loop. The net result is to make synapses more responsive to BDNF, followed by production of additional PSD-95. Once this loop is started at just a few of a neuron's synapses, the rush of PSD-95 to other excitory synapses of the neuron is on. In this way a few very active synapses can prime larger regions of a neuron for long-term synaptic strengthening in response to subsequent stimulation in the newborn animal.

Proteins Necessary For Brain Development Found To Be Critical For Long-term Memory (9/5/06)

This research indicates that BDNF, which is crucial for the growth of brain cells during development, is also equally important for the formation of long-term memories. The study was performed on the common marine snail Aplysia. When the snails are electrically shocked, the neurotransmitter serotonin is released and promotes the formation of long-term memories associated with the shocks. But when the researchers blocked interaction between BDNF and its TrkB receptor, long-term memories did not form, even though serotonin was still released at synapses. This indicates that serotonin alone was not sufficient for long-term memory formation. Short-term memory formation was not affected. Further investigation showed that interfering with the BDNF receptors blocked long-term enhancement of the connections between the brain cells in the reflex circuit normally induced by the shock treatment.

Drug Triggers Body's Mechanism To Reverse Aging Effect On Memory Process (7/27/06)

A class of drugs known as "ampakines" (so-called because they target AMPA receptors) has been under study and development since the early 1990s to deal with neurological conditions, such as schizophrenia, problems of attention span and alertness, and memory impairment associated with dementia and Alzheimer's disease. The research reported here was conducted by a team that included Gary Lynch, who has long been associated with investigation of the biological bases of learning and memory. (See here for more about Lynch and long-term memory.)

In this study, rats were treated for four days with an ampakine drug. Of particular interest was the effect of the drug on the hippocampus of the brain, because of its known importance in the formation of long-term memories. In the hippocampus areas of rats treated with the drug, it was found that (compared to controls) there was a significant increase both of levels of BDNF and of long-term potentiation (LTP) of synapses (an indicator of memory formation). Further, even though the drug had a known half-life of only 15 minutes, elevated levels of BDNF and LTP were observed as long as 18 hours after drug administration was stopped.

Tiny RNA Molecules Fine-tune The Brain's Synapses (1/24/06)

Synapses between two neurons are formed between locations at the tip of an axon of one neuron (the "presynaptic" neuron) and a dendrite on the body of another neuron (the "postsynaptic" neuron). In order to form a complete synapse, it is necessary for there to be protrusions called "dendritic spines" on dendrites of the postsynaptic neuron. In the process of synaptic signaling, it is these spines that absorb neurotransmitter molecules released by the axon of the presynaptic neuron. Consequently, any mechanism that affects the density of spines on dendrites will affect the total number of synapses that can form between neurons.

It had previously been established that BDNF activates a protein kinase called Limk1, which in turn promotes the growth of dendritic spines and hence the ability of synapses to form. This research on rats studied the effect of the microRNA miR-134 on growth of dendritic spines of hippocampal neurons. It was found that when neurons were exposed to miR-134, spine volume significantly decreased, and synapses weakened. Conversely, when miR-134 was inhibited, spines increased in size, strengthening synapses. However, increased levels of BDNF negated the effects of miR-134, indicating that miR-134 achieved its effect by suppressing Limk1.

More: Spine control

Tags: BDNF, memory

MicroRNA and stem cells

I've written about both microRNA and stem cells recently. But there is more news that reports on connections between the two.

Following are summaries of the announcements.

Role Of Tiny RNAs In Controlling Stem Cell Fate Identified (3/6/08)

The microRNAs miR-1 and miR-133 have been known to be associated with muscle development. The new research shows that they actively encourage heart muscle development and suppress genes that could cause pluripotent embryonic stem cells to turn into undesired cells like neurons or bone. The two miRNAs turn on genes that encourage mesoderm formation. They also turn off genes that cause stem cells to become ectodermal or endodermal cells.

Research abstract: MicroRNA Regulation of Cell Lineages in Mouse and Human Embryonic Stem Cells.
Blog post: here.

Short RNA Strand Helps Exposed Skin Cells Protect Body From Bacteria, Dehydration And Even Cancer (3/2/08)

In a wide range of vertebrates, from zebrafish to chickens and humans, miR-203 is found only in very specific types of skin – the outer layers of stratified epithelial tissues. In the 13th day of mouse embryo development, the embryo's skin is primarily composed of undifferentiated stem cells, and there is very little miR-203 in the cells. During the next two days expression of miR-203 rises rapidly, and the cells begin to differentiate into cells that form the outermost, protective layer of skin.

When miR-203 was artificially caused to be expressed too early, the normal rapid proliferation of stem cells was significantly slowed. The effect was attributed to inhibition of the p63 gene, which normally encourages stem cell proliferation, by miR-203. On the other hand, when miR-203 was suppressed, cells in the outer layer proliferated significantly more than normally, because p63 was not being inhibited.

p63 is a master regulatory gene, which maintains pluripotency in skin stem cells. It is often found to be overexpressed in cancerous cells. Future research will explore whether low expression of miR-203 is associated with cancer, and if so, whether increasing miR-203 expression is helpful.

More: here.

MicroRNA Pathway Essential For Controlling Self-renewal Of Stem Cells (2/15/07)

The gene Dicer-1 (Dcr-1) has been known to affect expression of specific miRNAs in fruit flies (Drosophila). It is essential for generating mature miRNAs from their corresponding precursors. This research shows that unmutated Dcr-1 is necessary for controlling self-renewal or maintenance of germline stem cells and somatic stem cells in Drosophila ovaries. The researchers infer that lack of miRNAs, due to mutant Dcr-1, is responsible for failure of self-renewal of stem cells, but specific miRNAs that are affected are yet to be determined.

Research abstract: Dcr-1 Maintains Drosophila Ovarian Stem Cells.
Blog post: here.

Additional reading:

MicroRNA-134 Modulates the Differentiation of Mouse Embryonic Stem Cells, Where It Causes Post-Transcriptional Attenuation of Nanog and LRH1 – abstract of research paper published online 10/4/07

Tags: microRNA, stem cells, cancer

MicroRNA and cancer

MicroRNAs are 18 to 25 nucleotide, noncoding RNA molecules that have been found to regulate a wide variety of cellular processes. Unusual levels of varions miRNAs have been shown to be diagnostic of pathology in a number of different cancers, such as chronic lymphocytic leukemia, lung cancer and pancreatic cancer.

The first microRNA was discovered in 1993, in C. elegans, but the term microRNA was not introduced until 2001. It took seven years for the second miRNA to be found, also in C. elegans, but many others then followed. Over 500 microRNAs have been identified in the human genome. Over a third of the human genome appears to be regulated by miRNAs orginally found is some mammal or another.

In some cases, changes to gene expression caused by miRNA seem to promote cancer. But in other cases, a miRNA may repress genes that promote cancer or its metastasis.

The p53 anti-cancer gene is affected by some miRNAs, but we will cover that in a separate note. Some miRNAs also seem to be related to cancer stem cells, which is a large topic that merits broader coverage. (See here, here.)

We've already looked at one example (here, and see below) where a gene associated with cancer can affect levels of some miRNAs. Unlike most other findings, this is a case where cancer-related pathology seems to affect miRNA expression levels, rather than the reverse.

One miRNA, in particular, stands out for the variety of genes it may affect. This miRNA is let-7, and it is known to be expressed in the later stages of animal development. Some estimates put the number of human genes affected by let-7 in the hundreds, though most of those may not be related to cancers. Ras is an important cancer-related protein that is suppressed by let-7 (See here, here.) (This report has more on let-7. See also here, here, here, here.)

The miRNA miR-21 has been associated with cancers of the colon, liver, and thyroid.

It's difficult to summarize the following research findings – they involve a variety of cancer types and many different miRNAs. Unusually high or low levels of some miRNAs seem to promote cancer in some cases, but suppress cancer in others. MiRNAs also work in a variety of different ways to affect gene expression and protein activity.

This diversity of effects due to miRNAs may well be the most interesting current finding to come out of research in this area.

Molecules may help predict survival in liver cancer (1/30/08)

In a long-term study of patients with liver cancer, it was found that those with the poorest survival history also had lower levels of 19 specific microRNAs in cancer cells compared to nearby noncancer cells than did patients with significantly better survival. This result is out of a total of 196 different microRNAs whose levels were measured.

Expression Patterns Of MicroRNAs Appear Altered In Colon Cancer, And Associated With Poor Outcomes (1/29/08)

In one cohort of 84 patients with colon cancer 37 different microRNAs were differently expressed in cancer cells compared with noncancer cells. 5 of these miRNAs could reliably discriminate between tumorous and nontumor tissue. The same 5 miRNAs had similar discriminatory powers in a different cohort of 113 patients. For the specific miRNA known as miR-21, high expression levels were associated with poor survival outcomes in both patient cohorts. (High levels of miR-21 are also associated with thyroid cancer, see here, and with liver cancer, see here.)

Two MicroRNAs Promote Spread Of Tumor Cells (1/28/08)

MicroRNAs have been shown in many studies to block translation of tumor suppressor genes. In this study, two specific miRNAs (out of 450 tested) have been shown to transform non-invasive human breast cancer cells into cells that rapidly metastasized in cell cultures and laboratory mice. One of the miRNAs, miR-373, was previously identified as a possible oncogene in testicular cancer. The other miRNA, miR-520c, hasn't previously been associated with cancer, but is similar to miR-373. Both miRNAs are found only in cancer cells. There is evidence that they downregulate the CD44 gene, and that in turn leads to metastasis of non-metastatic tumor cells. In human patients, metastatic cells are found to have higher levels of miR-373 and lower expression of CD44 than non-metastatic tumor cells.

Molecules Might Identify High-risk Acute-leukemia Patients (1/15/08)

In a study of leukemia cells from 122 patients with high- and intermediate-risk acute myeloid leukemia (AML) the same miRNAs could be found in both normal and leukemic cells, but there were differences in levels of various miRNAs present. Two specific miRNAs (miR-191 and miR-199a) were present at abnormally high levels that were clearly associated with patient survival. The same two miRNAs have been previously found to be associated with cancers of the lung, prostate, colon, stomach and breast. Another miRNA (miR-155) was associated with a gene mutation, and high levels of it have been reported in other cancers (see here, here) and to cause leukemia in mice.

Small Molecule Can Prevent Spread Of Breast Cancer, Study Suggests (1/9/08)

Three miRNAs have been found that prevent breast cancer metastasis by interfering with the expression of genes that give cancer cells the ability to proliferate and migrate. Researchers found lower levels of a few miRNAs (miR-335, miR-126 and miR-206) in metastatic cells compared to non-metastatic tumor cells. Testing in mice showed that raising levels of these miRNAs inhibited metastasis. Further analysis showed that miR-126 influences the proliferation rate of metastatic cells, while miR-335 and miR-206 influence the cancer cells' ability to migrate into lungs or bone. miR-335 was found to inhibit expression of SOX4 and TNC genes, which affect cell migration.

More: here, here

Virus Discovered Using Same Tools As Host Cell (12/17/07)

A microRNA expressed in the genome of the Kaposi's Sarcoma Associated Herpesvirus (KSHV) appears to be very similar in stucture and function to a miRNA associated with lymphoma – miR-155, mentioned above. KSHV itself causes a rare skin cancer that disproportionately affects HIV-infected individuals. Both miR-155 and the KSHV miRNA regulate the same genes, including several associated with B cell function and cell cycle regulation. Hence KSHV may promote B cell tumors by repressing one or more of these genes, as miR-155 seems to do.

Scientists Identify And Repress Breast Cancer Stem Cells In Mouse Tissue (12/17/07)

Since 2001 stem-like cells that appear to initiate cancer development have been discovered in breast, lung, brain and colon tissues, as well as in the blood. A microRNA (let-7) has now been found that can help to identify such cancer stem cells in breast cancer tissue of mice. Further tests indicate that let-7 can attack and eliminate these cancer progenitors.

MicroRNA Regulates Cancer Stem Cells: Could Lead To Treating Cancer As A Whole (12/13/07)

Another study involving the interaction of miRNA let-7 and cancer stem cells was conducted independently and published just before the one described above. In this study, researchers found a way to grow large quantities of tumor stem cells by growing human breast cancer cells in immunosuppressed mice. They found that these stem cells contained low amounts of several miRNAs, compared to more mature tumor cells or stem cells that had differentiated in culture. When let-7 was activated in these cells, they lost their ability to self-renew and began to differentiate. They also became less able to form tumors in mice or to metastasize. It appears that let-7 did this by switching off two cancer-related genes: the oncogene Ras, and HMG2A (see here, here).

Silencing Small But Mighty Cancer Inhibitors (12/10/07)

As discussed here, the important transcription factor Myc, which is overexpressed in many cancers, can also stop the production of at least 13 microRNAs. Some of these miRNAs have an inhibiting effect on cancer. In some cases re-introducing repressed miRNAs into Myc-containing cancer cells suppressed tumor growth in mice. So repression of miRNAs may be another pathway through which overexpression of Myc promotes cancer. The research involved lymphoma cells in mice, and showed that Myc repressed the miRNAs by directly attaching to the DNA at the miRNA genes.

Scientists discover new role for miRNA in leukemia (12/10/07)

A microRNA has been found to play a new role in the development of cancer, in this case chronic myeloid leukemia (CML). The miRNA is miR-328, and normally it is able to directly bind to a certain protein, inhibiting the activity of that protein. But if levels of miR-328 become abnormally low, the protein prevents white blood cells maturing as they should. The result is the build-up of immature white blood cells and entrance to what is called the "blast-crisis" phase of the disease.

Cellular Pathway Identified That Makes Prostate Cancer Fatal (11/27/07)

Expression levels of microRNAs were measured in samples of prostate cancer cells. Five different miRNAs were found to have unusual expression levels. One of these, miR-125b, was found at high levels in both androgen-dependent and the more dangerous androgen-independent prostate cancer cells.

Earlier research reports:

• MicroRNA Convicted Of Triggering Metastasis (9/26/07) – More: here – breast cancer, miR-10b
  
  
• Molecule Blocks Gene, Sheds Light On Liver Cancer (8/1/07) – liver cancer, miR-21
  
  
• New Genetic Marker Characterizes Aggressiveness Of Cancer Cells (6/25/07) – various cancers, let-7
  
  
• Tiny Genes May Increase Cancer Susceptibility, Scientists Find (5/23/07)
  
  
• Newly Identified Mechanism For Silencing Genes Points To Possible Anti-cancer Strategies (5/16/07)
  
  
• Pancreatic Cancer Markers Identified, May Predict Survival (5/1/07) – pancreatic cancer
  
  
• Biomarker Discovery Bodes Well For Better Cancer Diagnostics, Pharmacologist Says (5/1/07) – pancreatic cancer
  
  
• MicroRNAs Can Be Tumor Suppressors (4/25/07) – various cancers, let-7
  
  
• Leukemia Drug Turns Mini-molecules Up, Cancer Genes Down (3/2/07) – leukemia, miR-15b, miR-16-1, let-7
  
  
• MicroRNA Helps Prevent Tumors (2/22/07) – various cancers, let-7
  
  
• Micro Molecules Can Identify Pancreatic Cancer (1/10/07) – pancreatic cancer
  
  
• Pair Of MicroRNA Molecules Controls Major Oncogene In Most Common Leukemia (12/15/06) – leukemia, miR-29, miR-181 – more on miR-29 here
  
  
• MiRNA Fingerprint Identified In Platelet Formation (3/16/06) – leukemia
  
  
• Role Of MicroRNA Identified In Thyroid Cancer (12/23/05) – thyroid cancer, miR-21, miR-146, miR-181a, miR-221, miR-222
  
  
• Unusual RNAs Linked To Chronic Leukemia, May Help Treat It (9/18/05) – leukemia, miR-15, miR-16
  
  
• Yale Scientists Find MicroRNA Regulates Ras Cancer Gene (3/25/05) – lung cancer and others, let-7
  
  
• Small Molecule May Help Pinpoint Some Cancers (3/9/05) – lymphoma, miR-155
  

Tags: cancer, microRNA, cancer stem cell

Cancer and Myc

Myc is a gene that has been studied for over 20 years and is found to be overexpressed in many human cancers. The protein it codes for (which for simplicity we'll also refer to as Myc) is a transcription factor, like members of the FoxO family, which means it can enable or disable the expression of many other genes. How many? The estimate is about 15% of all human genes. Since there are at least 20,000 genes, the number regulated by Myc is 3000 or more. That's a lot of leverage.

Obviously, Myc is essential for cell function or it wouldn't have so much influence. The biological functions Myc affects include cell proliferation, cell growth, apopotosis, cell differentiation, and stem-cell self-renewal. It is possible for a single transcription factor to have such varied effects because other transcription factors (coactivators or corepressors) must also be present in order to activate or repress the expression of some specific other gene. That is, the expression of any particular gene depends on the presence (or absence) of a particular set of transcription factors.

Since Myc affects the functions mentioned above, it's hardly surprising to find that Myc is overexpressed in many cancers. In such cases, the difference between a normal cell and a cancer cell is the overexpression of Myc in the latter and possibly the abnormal presence of other coactivators or corepressors. The result in a cancer cell is excessive proliferation and/or avoidance of cell death by apoptosis.

Surely you've wondered why development of cancer therapies has taken so long. A large part of the reason is that so many genes involved in cancer interact with so many other genes that have essential functions. So it's not possible to interfere with the cancer-related genes without disrupting vital biological functions elsewhere in the body.

The expression of Myc itself is triggered by signals that are usually external to the cell and normally serve to stimulate cell growth and proliferation when needed, as in lymphocyte production, normal growth, or wound healing. Such signals are called mitogens, because they can initiate mitosis. Wnt, Shh, and EGF are mitogens that can lead to Myc expression.

It is probably almost impossible to hope to combat cancer by directly affecting Myc or its related mitogens, because all of these have essential normal functions themselves. Instead, anti-cancer research needs to examine how Myc may be overexpressed or complemented in harmful ways by other transcription factors.

One promising line of research involves cancer stem cells – cells that, like normal stem cells, can proliferate and differentiate into other cell types, and that also carry cancer-causing mutations. There is now thought to be a "signature" consisting of 11 genes that may be characteristic of cancer stem cells. One of these genes has an effect on Myc:

Scientists Uncover Role Of Cancer Stem Cell Marker: Controlling Gene Expression (1/18/08)

Scientists at Jefferson's Kimmel Cancer Center in Philadelphia have made an extraordinary advance in the understanding of the function of a gene previously shown to be part of an 11-gene "signature" that can predict which tumors will be aggressive and likely to spread. The gene, USP22, encodes an enzyme that appears to be crucial for controlling large scale changes in gene expression, one of the hallmarks of cancer cells. ...

In one example, they looked at the relationship between MYC and USP22. MYC, which is among the most commonly overexpressed genes in cancer, encodes a protein that controls the expression of thousands of other genes. The scientists showed that USP22 is a critical partner of MYC and that by depleting cells of USP22, they could prevent MYC from working properly, stopping it from inducing the invasive growth of cancer cells.

It turns out that Myc affects not only 3000 or so ordinary genes, but also a number of DNA sequences that code for microRNA – and some of these microRNAs play an important role in suppressing cancer. In fact, Myc can stop the production of at least 13 microRNAs:

Silencing Small But Mighty Cancer Inhibitors (12/12/07)

Researchers from Johns Hopkins and the University of Pennsylvania have uncovered another reason why one of the most commonly activated proteins in cancer is in fact so dangerous. As reported in Nature Genetics recently, the Myc protein can stop the production of at least 13 microRNAs, small pieces of nucleic acid that help control which genes are turned on and off.

Furthermore, additional observations showed that some of these microRNAs have an inhibiting effect on cancer, a striking result in itself:

[I]n several instances, re-introducing repressed miRNAs into Myc-containing cancer cells suppressed tumor growth in mice, raising the possibility that a sort-of gene therapy approach could be effective therapy for treating certain cancers.

Since the microRNAs repressed by Myc affect many other genes, there could be thousands of other genes indirectly affected by Myc:

"This study expands our understanding of how Myc acts as such a potent cancer-promoting protein," says Mendell. "We already knew that it can directly regulate thousands of genes. Through its repertoire of miRNAs, Myc likely influences the expression of thousands of additional genes. Activation of Myc therefore profoundly changes the program of genes that are expressed in cancer cells."

More: Researchers zero in on the tiniest members in the war on cancer (12/13/07)

Myc is actually a member of a family of genes, the Myc family. Most members of the family have similar functions. Separate members undoubtedly appeared in the course of evolution from the duplication of earlier members and later mutation. One member, called N-myc, plays a role in both normal development of the retina, as well as cancers of the retina (retinoblastoma).

Recent research has shown that N-myc seems to be responsible for the surprising fact that the retinas in all vertebrates have about the same thickness, regardless of the size of the entire animal or its eyes:

Eye Cancer Gene's Role In Retinal Development Defined (1/18/08)

"A series of complex developmental processes must be carefully orchestrated for the eye to form correctly," said Michael Dyer, Ph.D., associate member in the St. Jude Department of Developmental Neurobiology. "One important aspect of this coordination is that retinal thickness be the same, irrespective of eye size. For example, the mouse eye is about 5,000 times smaller than that of the elephant eye, but the retinal thickness in these two species is comparable."

Working with mice, the researchers found that a gene called N-myc coordinates the growth of the retina and other eye structures to ensure the retina has the proper thickness necessary to convert light from the lens into nerve impulses that the brain transforms into images.

Tags: cancer, Myc, microRNA