PLX4032 is a promising experimental new cancer drug in phase III clinical trials for treatment of melanoma.  PLX4032 selectively targets tumor cells with activating mutations in the protein, B-RAF (V600E). The drug has shown an exceptional anti-tumor response rate of 80% in patients positive for the mutated B-RAF, however resistance often develops after 7 months of treatment, even for patients whose tumors seem to have disappeared. A recent news article discussed the findings of two separate groups of scientists who have independently discovered mechanisms behind the resistance to PLX4032. These mechanisms include the activation of other kinases to bypass the inhibited B-RAF, thus reactivating the downstream pathway.  Also, cells resistant to the B-RAF inhibitor compensate by up-regulation of B-RAF independent growth factor receptor pathways. These findings provide starting points for development of combined therapies to overcome the resistance to PLX4032.

B-RAF mutations are found in about 60% of malignant melanomas, in high frequencies in thyroid and colon cancers, and in approximately 8% of all cancers. B-RAF, a serine-threonine protein kinase, normally functions in B-RAF/B-RAF homodimers or in heterodimers with a related protein, RAF1 kinase, generally in response to upstream signaling molecules, including the activation of the protein RAS. B-RAF signals through the MAPK/ERK signaling pathway, and when mutated, functions as a monomer rather then a dimer, thus activating downstream signals in the absence of upstream input.  This signaling drives tumor growth through the activation of the MAPK pathway.  PLX4032 inhibits the mutated B-RAF by occupying the pocket containing the mutation.  This alters the mutated B-RAF’s 3-dimensional structure and thereby disrupts the monomer activity. The drug is exquisitely specific for B-RAF mutant tumors, which is important because it is effective only in high doses and such specificity should mean fewer undesirable side-effects.

The mechanisms of resistance to PLX4032 follow the common cancer drug resistance theme of bypassing the oncogenic pathway that has been shutdown.  However, the expectation was to see compensating mutations in B-RAF leading to resistance, rather than activation of alternative signaling pathways.

One group of scientists sequenced the entire genome of 16 tumor samples from 12 patients who acquired resistance to PLX4032 during their treatment in phase I or II clinical trials. They found one patient acquired resistance via an additional mutation in N-RAS which reactivated the MAPK pathway. In 5 other patients samples, the scientists found that a receptor-tyrosine kinase (RTK)- survival pathway was activated as a result of over-expression of a platelet-derived growth factor receptor (PDGFRβ). They could not find mechanisms of resistance in the remaining patients’ samples.

The other group of researchers expressed roughly 600 different kinase protein open reading frames (ORFs) to identify which ones allowed the cells to grow in the presence of PLX4032.  They found that the protein C-RAF, known to form dimers with normal B-RAF, and the protein COT, encoded by the gene MAP3K8, conferred resistance to the drug.  Both kinases do this by independently activating the MAPK/ERK cell-growth pathway, similar to B-RAF and to N-RAS.

The findings are promising in that recognition of mechanisms of resistance suggests additional drug targets and could lead to therapies with drug combinations such as PLX4032 paired with another drug that inhibits a protein further downstream in the cell growth signaling pathway, such as ERK or MEK. In experimental models the RAF and MEK inhibitor combination was able to thwart resistance to a single RAF inhibitor.  Some patients whose cancer relapsed after treatment with PLX4032 are enrolled in a phase II clinical trial of an MEK inhibitor. Additionally, PLX4032 will likely be tested in combination with this MEK inhibitor in clinical trials in the near future.

The finding that PDGF receptor overexpression can lead to resistance is analogous to the finding that insulin-like growth factor receptor (IGF-1R) overexpression results in resistance to a different B-RAF(V600E) inhibitor.  Both IGF-1R and the PDGF receptor activate the same pathway, involving PI3 kinase and AKT. Apparently, this pathway could be another drug target.

The above, recent two studies included only a total of 17 patient samples. With the heterogeneity exhibited in the resistance mechanisms in such a small number of patients, larger studies will likely be needed to determine all possible resistance mechanisms.

In summary, these studies support the idea that combinatorial drug treatments, such as those that have revolutionized HIV treatment, will be important in cancer therapies. Further, screening techniques used by the researchers could show ways to stratify patients into distinct treatment groups, as is currently done for breast cancer.  However, several questions will have to be answered in the process of developing drug combinations.   For example, how many drugs are needed simultaneously and which can be tolerated in combination?

Abstract of article describing resistance mechanisms to PLX4032

Abstract from another article describing other resistance mechanisms to PLX4032

An alternative approach is targeting conserved viral antigens or the generation of so-called “universal” influenza vaccines that would provide immunity in a strain and subtype independent manner. Conserved antigen vaccines  do not provide the sterilizing immunity (which implies complete prevention of infection)  afforded by the HA-specific, neutralizing antibody vaccines. However, universal vaccines would lead to a more mild, shorter infection and reduce morbidity and mortality by speeding up viral clearance and  reducing transmission of the virus. Additionally, the mild, transient infection would boost immunity and  lead to the production of neutralizing antibodies against the specific strain in circulation, thereby preventing re-infection. Ideally, these heterosubtypic, “universal” vaccines would, in the case of an unexpected pandemic, provide a stop gap measure that could limit the disease and reduce the impact of newly emergent Influenza strains until sufficient supplies of a specific vaccine has been produced.

The M2 channel, or matrix 2 protein, of influenza A is the target of the amantane-derived antiviral drugs (e.g. amantadine and rimantadine). These drugs are now ineffective because virtually all circulating strains have acquired mutations in the channel that confer drug resistance. A recent article discussed new developments made studying the structure of the M2 channel by solid-state nuclear magnetic resonance produced images. Clarification of functional and structural mechanisms underlying proton transport in the M2 channel is critical in development of new antiflu drugs.

The M2 channel is a single-pass integral membrane protein that associates into tetrameric, proton-selective ion pores on the cell membranes of infected cells and in lower levels on virion membranes.  Acting as an ion channel that modulates the pH of intraviral compartments with exceptional proton-selectivity—essential for viral uncoating—the M2 protein is essential to the life cycle of the Influenza A virus and is therefore highly conserved. Additionally, it is small (97 amino acids), and the N-terminal ectodomain, M2e, is especially conserved (23 amino acids).  Thus, immune-escape mutations are not as likely. The article reporting the new images of the M2 virus stated that the scientists behind the study knew all of the “handful” of possible changes to the channel that could occur and still allow it to function. All of these factors give the matrix 2 protein channel great potential as a “universal” vaccine target.  How much this potential comes to fruition is yet to be determined.

There are many aspects in vaccine development: types of vaccines (e.g recombinant vector vaccines, adjuvant killed vaccines, prime-boost regimens, live-attenuated viruses, etc): methods of application (intramuscular vaccination or intranasal vaccination); and additional variables (e.g. adjuvants used, hapten-carrier conjugates, targeting multiple antigens, etc.) These issues complicate new vaccine development.   And, there are  other factors that will have to be assessed, such as the stability of the vaccine , vaccine doses, and the effects of different genetic backgrounds on effectiveness of immunization.

The M2 targeting vaccines have shown some significant progress and promising results. Preliminary results in phase I clinical trials with a M2e -HBc fusion particle vaccine showed 90% seroconversion rates after two doses. While these results are indicative of the efficacy of M2 vaccines in humans, it has been suggested that the number of individuals in the study is not sufficient to mitigate concerns that M2 vaccines alone may not be effective in all of the population, because of different genetic backgrounds and major histocompatibility (MHC) types. The concern regarding different genetic backgrounds is based on the differences in immune responses (including differences in antibody production, T cell responses, and protection against challenge) observed when an M2 vaccine was given to different inbred mouse strains representing different MHC types. 

The small size of M2 and the limited number of epitopes  it represents makes it likely that M2 would not have a productive epitope for every human MHC type. In addition to use of hapten-carrier conjugates and adjuvants, one very promising idea is to increase the proportion of the population that the vaccine protects by the addition of another influenza epitope.  Additional antigens could include nucleoprotein and matrix 1 protein. Clinical trials of such combinations are underway.

Influenza A M2

Universal Influenza Vaccine

Immune response to M2

Clinical trials:

Intramuscular/subcutaneous universal Influenza vaccine comparison

Recombinant M2e Influenza A vaccine

Cellular/Humoral immune response to Influenza vaccine

Phase I Flagellin-HuM2 Influenza vaccine

Bisphosphonates have a core molecular structure similar to a naturally occurring compound, pyrophosphate, essentially a diphosphate moiety.  However, in a bisphosphonate, a carbon atom replaces an oxygen atom linking the two phosphates groups in pyrophosphate.  The carbon atom stabilizes this “diphosphate”, which would otherwise be rapidly broken down by cellular enzymes.  The carbon atom also provides two other bonds to which many different chemical groups can be linked.  Numerous chemical groups have been linked to the carbon atom, particularly chemical groups with a nitrogen atom, in an attempt to generate bisphosphonates with greater and greater efficacy.

Bisphosphonates have been used primarily to minimize bone destruction in many diseases, especially osteoporosis, but also including cancer metastases to the bone, very common in prostate cancer.  The two phosphate groups of the bisphosphonates are highly negatively charged and therefore bisphosphonates migrate to and accumulate at the bone mineral, where there are many positive charges, particularly from the calcium ions.  The bisphosphonates are then taken up by osteoclasts, cells that absorb bone.  Inside the osteoclasts, the bisphophonates inhibit an enzyme termed, farnesyl pyrophosphate synthase.  This enzyme is important for generating a small lipid molecule that is covalently attached to proteins, called Ras proteins, that are required for cell division.  The lipid molecule facilitates the positioning of the Ras proteins to the inside of the cell membrane, which is mostly constituted with lipids itself.  Thus, the lipid molecule attached to the Ras protein essentially dissolves into the cell membrane, bringing the Ras protein in very close proximity to other proteins that are also located just inside the cell membrane and that are required for normal Ras function.  Without the lipid molecule, termed a prenyl group, the Ras protein is not positioned properly for interactions with its partner proteins and cell division does not occur properly.  The osteoclasts thus undergo programmed cell death, termed apoptosis.  The fewer number of osteoclasts means less bone resorption.

As might be obvious from the above, interfering with Ras prenylation could reduce cell proliferation in other cases, such as cancer.  In fact, there is very active field of cancer biology related to the development of Ras prenylation inhibitors and these drugs are entering clinical trials now or very soon.  However, the bisphosphonates have been used extensively and much is known about the related safety issues.  Thus, the authors of the British Journal of Cancer article, among others, have considered the possible usefulness of the bisphosphonates preventing cancer.  The answer to this question is not obvious, in particular because the bisphosphonates accumulate at the bone mineral and may not have a big impact on distant tissues.

The authors of the British Journal of Cancer article reviewed the bisphosphonate use among women who were diagnosed with breast cancer during 2003-2006.  Their results indicated that the risk of being diagnosed with breast cancer, during the use of bisphosphonates, over 3 months to several years, was reduced by a maximum of 30%.  Furthermore, the greater the period of bisphosphonate usage, the greater the reduction in breast cancer diagnosis risk.

While the results are encouraging, particularly given the understanding that bisphosphonate can reduce Ras stimulation of cell proliferation, the study has many limitations that need to be researched before the true usefulness of bisphosphonates in reduced breast cancer occurrence can be understood.  In fact, there are so many limitations to a report of this kind that, no matter how encouraging from a basic research point of view,  they cannot all be listed here.  However, here are a few.  First, the risk reduction was seen among current bisphosphonate users; the sample size of previous users was too small to allow conclusions.  A larger study with patients who are no longer taking bisphosphonates can now be justified.  Second, there is no information about overall mortality, either due to breast cancer or due to unintended consequences of bisphosphonate usage, during such a short time period.  Finally, many mechanistic issues remain unresolved.  For example, T-cell proliferation could be inhibited by bisphosphonates, and in several human conditions where T-cell proliferation is impaired, cancers develop, for example Kaposi’s sarcoma in AIDS.

Recent British Journal of Cancer article on bisphosphonates and breast cancer

Excellent background material on bisphosphonates

In Pompe disease, the GAA defficiency leads to a build up and enlargement of the intracellular storage vesicles for glycogen, and it is the abnormal enlargement of these vesicles that interferes with the normal muscle fiber structure, leading to muscle ineffectiveness, particularly ineffectiveness of the diaphragm muscle.

The replacement of a defective gene with a wild-type gene is referred to as gene therapy, and it has been heralded for two decades as an approach with many applications in medicine.  Unfortunately, gene therapy approaches to cures to the disease have been few and far between, with a few notable exceptions.

There are many technical problems limiting gene therapy success.  For example, in the disease osteogenesis imperfecta (OI), a collagen protein is not made properly.  Scientists have known for years which gene is defective, but replacing the gene at just the right time in development and in the proper tissues, has been impossible. 

One of the opportunities with a gene therapy approach to Pompe disease is the fact that restoration of the diaphragm function could restore normal lung function.  However, this still leaves the difficulty of delivering the normal gene for GAA into enough diaphragm cells to have an effect on the diaphragm function.  In general delivery modes for normal genes have been highly inefficient.

Nevertheless, there are some conditions where an inefficient delivery system is not an impediment, or at least not an impediment that cannot be readily overcome.  For example, in some cases, it is desirable to deliver a gene for a viral protein, to establish immunity.  In this case, very few cells need to express that gene and thereby generate the viral protein.  This type of gene therapy can be a desirable approach to vaccination for several reasons.  For example, gene vaccines would be extremely inexpensive to produce compared to a conventional vaccine.  Once a gene vaccine, or what is more commonly referred to as a DNA vaccine, is established and tested, enough gene vaccine could be produced to vaccinate everyone on the planet for a few thousand dollars.  Also, a DNA vaccine could be transported easily and safely, and stored conveniently, forever, anywhere on the globe.

However, the general inefficiency of gene therapy means that diseases can persist due to an insufficient number of cells being able to receive and properly express the normal gene.  This is particularly difficult with solid tissues in the body, as opposed to blood cells.  Solid tissues have layers and layers of cells with all sides of the cells in contact with other cells, making each individual cell difficult to access. 

The authors of the Molecular Therapy report took advantage of mice that have the gene for GAA artificially destroyed, or “knocked out”.  While often mice, with gene defects that cause diseases in humans, have different or no health problems, in this case the GAA knockout mice have an incapacitated diaphragm and decreased lung function, making it reasonable to study the therapeutic replacement of the GAA in these mice to improve lung function.

Thus, the authors from the University of Florida reported the success of modifying a gene therapy approach that represented the highly efficient transfer of the normal gene for GAA into diaphragm muscle cells in mice.  Their approach is a modification of the use of what are termed, viral vectors.  Viral vectors are virus capsids containing a normal, therapeutic gene in place of the normal viral proteins.  These capsids can be produced in a laboratory in specialized cells engineered to supply all the proteins necessary to generate the viral capsid in a way that the viral capsid encapsulates the normal GAA gene rather, than the virus genetic material.  Because the pathological components of the virus genetic material are not present, a viral vector does not cause a viral infection or the spread of virus internally.  The viral vector used in the University of Florida study is termed, AAV for adeno-associated virus, derived from a harmless virus.  This viral vector is highly efficient at entering cells.

Nevertheless, the AAV viral vector is not able to deliver the normal GAA gene to enough of the diaphragm cells to improve the function of the diaphragm without a gel first described by the University of Florida team in a 2004 article, also in Molecular Therapy.  The gel is derived from a relatively ordinary chemical called glycerol, and the original motivation for testing the gel was the tiny size of the mouse diaphragm, making it less amenable to other modes of applying the virus.  By suspending the virus in the glycerol gel and spreading the gel over the diaphragm muscle, in mice that have been anesthetized and where the muscle has been surgically exposed, the authors demonstrated a highly efficient transfer of the normal GAA gene throughout the diaphragm.  In the more recent article, the authors demonstrate that this extensive transfer of the gene leads to increased diaphragm muscle contractile strength and increased lung function.  However, the use of the glycerol gel does not lead to muscle and lung function maintained over certain, relatively long periods.

What are the benefits of this research?  The primary benefit is that the authors have shown that improving the efficiency of transferring the normal gene into muscle cells has a positive effect, even if the disease has progressed prior to treatment.  The disease progression factor is important, because in the human cases, there will be disease progression prior to an opportunities for treatment, particularly in cases of experimental treatments.  Whether the gel used by the authors will be necessary or useful in humans remains to be seen.  It is possible that more conventional approaches will be sufficient in humans.  In fact, the same University of Florida group is in the process of starting a human clinical trial with the GAA gene in an AAV viral vector, but in this trial, the AAV viral vector will be used without the gel.

2010 article describing the GAA gene transfer into mice

2004 article describing the use of the glycerol based gel to improve the efficiency of AAV-based gene therapy

GAA clinical trial information

General gene therapy info

It has been known for many years that tumor cells have mutations, when compared to the DNA of a patient’s normal cells.  However, by determining the sequence of the entirety of the DNA, referred to as the genome, researchers are able to identify mutations without bias involved in the previous approaches to identifying cancer mutations.  For example, cancer is a disease of abnormal cell proliferation, leading scientists to focus on genes that encode proteins that regulate cell proliferation.  And indeed, many of these types of genes are mutated in cancer, thus explaining the abnormal cell proliferation, at least in part.  However, cancer has other characteristics besides abnormal cell proliferation, for example, metastasis and immune system evasion.  Thus, by sequencing the entire genome of a cancer cell, still other characteristics of cancer may be discovered where previous approaches failed.  This type of approached is referred to as “discovery-based”, as opposed to a hypothesis based approach where a specific idea, hopefully well-grounded in science, guides the collection of data.

The technology for obtaining the entire sequence of the A, T, G, and C bases that make up the DNA sequence has evolved over the last decade, in particular, allowing for the sequences of the bases to be obtained more rapidly with advancing technology.  The authors of the above report used a technology marketed by Illumina. 

In addition to the improved technology, the knowledge of a basic human genome sequence also provides efficiency in assembling a new version, via statistical algorithms that have been developed to match a new sequence with its likely position in the previously obtained, reference sequence. 

In determining the sequences of two different types of cancer cells in the two different reports, the researchers learned several things.  First, the number of mutations in the cancer genome, compared to the normal genome, was far higher than expected.  The melanoma had over 32,000 mutations and the lung cancer cell had over 22,000 mutations.  In both cases, about 99% of these mutations were in sections of the genome that are considered to be inert and presumably none, or at best only a few of these mutations could conceivably play a role in tumor development.  The presumed-inert mutations are in intergenic regions or in spaces within genes that are not used for coding for a protein.  However, the large number of mutations that have occurred in these cells indicates that over the course of a lifetime, the cells that make up the body undergo an unexpectedly large number of DNA base changes. 

Only about 1% of these mutations occurred in regions of the DNA that code for protein.  While it is conceivable that mutations in regions of the DNA that do not code for protein could stimulate cancer, almost all known mutations that are involved in cancer development occur in protein coding regions.  However, data presented in these reports, although preliminary, indicate that even most of the new mutations in the protein coding regions are irrelevant.  Although many more complete cancer genomes will be needed to verify this result, the implication of the result that most of the mutations of the protein coding DNA are not likely to be involved in tumor development is that mutations that actually drive cancer development are very rare.  This could be hopeful for the treatment of cancer, because it would suggests that most of the targets for treatment have been identified.

A somewhat more esoteric value to the work is its verification of the way in which mutations accumulate in the lung cancer and in melanoma.  In the lung cancer cells, the type of mutations are consistent with smoking; in the melanoma cells, the types of mutation are consistent with ultraviolet radiation, i.e., sun exposure.  How can the researchers make these types of determinations?  Here is an example: The DNA base, C, or cytosine, occurs in the genome in two chemical forms, methylated and unmethylated.  That is, cytosine can have an additional methyl group bonded to its ring structure.  The methylated cytosine facilitates the shut down of gene expression; gene expression generally requires that the cytosines in the region of DNA that is being transcribed into RNA be unmethylated.  The cellular regulation of the cytosine methylation involves methylating, or not methyling the cytosines that occur next to G’s, or guanines.  Thus, the symbol CpG is used to refer to C’s that are candidates for methylation, with the “p” indicating the phosphate linkage between the DNA bases.  Furthermore, the frequency of C methylation is much higher for CpG’s that are isolated in the genome than for CpG’s that occur in clusters.  Tobacco carcinogens have been shown in laboratory experiments to very efficiently mutate methylated C’s to the base, T, or thymine.  The results of the DNA sequence determination for the lung cancer cell are that most of the C’s that have been mutated to T’s have occurred in isolated CpG’s, consistent with the idea that methylated C’s, in the body, are in fact highly vulnerable to tobacco carcinogens.

What was not learned and what are possible next steps?  First, as indicated above, there was relatively little new discovered regarding gene mutations that are relevant to cancer development.  Second, it is apparent that other DNA alterations that do play a role in cancer development, besides single base mutations, are only inefficiently identified by the technology employed.  For example, cancer development often involves chromosomal breaks that abnormally juxtapose pieces of genes, referred to as cancer fusion genes, which were not identified as efficiently as the DNA mutations.  Furthermore, the cancer cells used in the analyses were immortalized cell lines, meaning that these cells have been propagated outside the body in artificial culture processes.  While it seems unlikely that there will be significant differences between what could be discovered using an actual cancer cell versus cells cultured in the laboratory, based on previous information, this possibility cannot be ruled out and will be a concern for many scientists until it is addressed.

As with all important basic research reports, the most important next step is repeating the work.  For example, a determination of the entire DNA sequence of another 20 or so cancer types could confirm the preliminary indications that very few mutations are relevant to cancer development.  This confirmation in turn could have a big impact on the greater medical community’s expectation for personalized treatment of patient tumors.  For example, if every patient had to have the entire sequence of his tumor determined, this could be more complicated than if the tumors of almost all patients could be completely characterized, with regard to genome mutations involvement in cancer development, by the very rapid determination of only a subset of the genome sequence.

Melanoma genome

Lung cancer genome

Cytosine methylation

Illumina Corporation

Technical info regarding Illumina sequencing technology

Tamoxifin is what is referred to as an anti-estrogen and thereby blocks estrogen binding to the estrogen receptor. This prevents the estrogen receptor from signaling to the cell to proliferate and leads eventually to tumor size reductions.

The protein E-cadherin is a membrane protein present on the surface of breast cancer cells, other types of cancer cells, and normal cells. E-cadherin links cells together, and thus it is often absent from metastatic tumor cells, presumably because the loss of E-cadherin allows the cancer cells to separate from one another and disseminate throughout the body.

In the report in the journal Breast Cancer Research (linked below) scientists studied a single breast cancer cell line, termed MCF-7. MCF-7 is referred to as a cell line because it is propagated in the laboratory, presumably with an unlimited capacity to be propagated outside the body, using laboratory techniques. Other sources of cells in laboratory cultures may have known, limited life spans, in which case the term “line” would not be used. The MCF-7 cell line was derived from patient breast cancer cells decades ago. It is considered one of many experimental models for breast cancer because it has many characteristics of breast cancer cells found in the body of patients.

The authors of the report first determined whether tamoxifen affected the level of E-caherin, because one possible explanation for tamoxifen’s role in stimulating metastasis would be reducing E-cadherin dependent cell-cell adhesiveness. The authors concluded that tamoxifin had no effect on E-cadherin levels in the MCF-7 cells. However, the authors considered the possibility that tamoxifen had a way of decreasing the cell-cell adhesion in the absence of E-cadherin. In other words, losing E-cadherin would reduce cell-cell adhesiveness, but there is the presumption that multiple factors play a role in lack of cell-cell adhesiveness. Thus, loss of E-cadherin in addition to another, unknown tamoxifen effect could lead to even more efficient cell dissemination throughout the body than the loss of E-cadherin alone. So, the authors artificially eliminated the E-cadherin from the MCF-7 cells and then performed a series of laboratory tests to determine if tamoxifen made the cells even more invasive and motile than loss of E-cadherin alone, which was indeed the case.

So what is the significance of this report to patients? At this point, the report has virtually no significance for patients, although as a basic research advance, it could be highly significant.

Why is the information in the report not currently useful? First, the authors did their study with a single, immortalized cell line. Many studies leading to highly useful basic research are done this way, because of the efficiency of working with a cell line. But cell lines can change over time in culture, and lose important properties that are relevant to cancer development in the body. Also, at best a cell line can represent only one patient’s cancer. It is clear that even cancers representing a single tissue type can have many molecular variations. Second, the assays used in the report to discover the increase in MCF-7 motility, with tamoxifen treatment, were highly artificial. And third, the mechanism of the effect was not studied to any great degree. For example, the role of E-cadherin in cell-cell cohesiveness is highly understood, and thus it is no surprise that cells that metastasize lack E-cadherin. But for now, there is no “connect all the dots” type of explanation for why tamoxifen would make E-cadherin-negative cells even more motile.

Why is this latter concern regarding mechanism important? Why not just conclude, it works and move on to patients, regardless of how it works? Mechanism is important because the increase in motility may turn out to have nothing to do with the development of the tumor in the body. For example, the tamoxifen mediated increase in motility could conceivably occur only when using the artificial assay in the lab.

However, from a basic research point of view, the study is highly significant because it will lead to the following questions: (i) Can the tamoxifen mediated increase in cell motility, in the absence of E-cadherin, be reproduced in more relevant settings, such as in animal models of tumor development with many different sources of breast cancer cells? Many different sources of cells and highly sophisticated animal models are readily available, and so given the relatively modest financial resources required, this question can be answered very quickly. (ii) Can the molecular mechanism of the tamoxifen effect be determined? Answering this question will provide the credibility needed to ultimately apply the ideas of the basic report for the highly expensive and long-term testing in patients. However, the authors have provided a somewhat inspiring albeit highly incomplete view of mechanism by reporting that at least one protein, termed src, for which there are known inhibitors, is involved in the tamoxifen mediated increase in cell motility. Thus, there is the hope that one day, once some of the other, above concerns have been addressed, that these src-inhibitors can be used in combination with tamoxifen to reduce the metastasis.

Report indicating tamoxifen mediated, increased MCF-7 cell motility in the absence of E-cadherin

E-cadherin and cancer

Article with some background information on Tamoxifen

Another article on Tamoxifen

C-reactive protein (CRP) was discovered many years ago as one of the first proteins that rises to a high level in the blood of persons infected with bacteria. Work over the decades has shown that this protein binds to a chemical termed phosphocholine that is present on the bacterial cell wall. Once CRP is bound to this chemical, i.e., to the bacterial cell wall, it provides a binding site for yet another protein that in turn recruits a series of proteins to the bacteria, referred to as the complement proteins. The complement proteins have several functions. These proteins can punch holes in the bacterium, essentially killing it. The complement proteins also recruit macrophage which in turn can ingest and digest the dead bacteria. In other ways, this ingestion process contributes to a further immune response against the bacteria, finally ridding the body of the infection, if all goes right.

Phosphocholine is also found on lipids that are present in atherosclerotic plaques, and thus CRP may be responsible for the presence of macrophage in the plaques. More experimentation is needed to confirm this. However, it is known that reduction in the level of CRP, caused by taking the statin drugs, is associated with a reduction in atherosclerosis related deaths independently of the reduction in the level of cholesterol that is a constituent of these plaques.

Why does reducing CRP reduce the apparent danger of atherosclerotic plaques? There is no conclusive answer to this, however there are several possibilities. First, CRP may bind to the phosphocholine parts of lipids that are present in the plaques, thereby attracting macrophage and ultimately leading to series of events where a plaque bursts and leads to a blocked artery. Second, phosphocholine is also present on the surface of dying immune function cells, and CRP can set off the same series of events by binding to these dying immune system cells as it does for bacteria. Presumably, this is a natural process, in that this process facilitates the normal recycling of the immune system. However, high CRP levels may indicate an immune imbalance that for unknown reasons is more likely to lead to a heart attack or stroke.

And why do the statins lower CRP? Here again, there is next to nothing known, though there are several tantalizing possibilities that will be investigated. CRP expression is increased by an immune function hormone termed, interleukin-6 (IL-6), and IL-6 is decreased in patients receiving statins. And, IL-6 is increased by a protein termed AP-1, which activates the transcription of the IL-6 gene. And finally, AP-1 is activated, indirectly, by a protein called Ras, which in turn is chemically modified by one of the cholesterol biosynthesis pathway products for proper function. Thus, the statins, which inhibit cholesterol biosynthesis are also able to inhibit Ras function.

Protective effect for Crestor even in the absence of high cholesterol

Heart attacks related to CRP but independent of LDL levels

C-reactive protein basics

Low density lipoproteins and atherosclerotic plaques

Example of IL-6 and AP-1 relationship

Ras and statins

However, like skin color, SNP’s can be associated with diseases. For example, fair skinned individuals are more susceptible to certain skin cancers. Thus, it is possible to collect the DNA from hundreds or thousands of individuals with a disease, such as lung cancer, and from controls, who do not have the disease, and determine whether any of the SNP’s in the human genome are associated with lung cancer. The technology for making this determination is subject to a certain failure rate, and the increased association of any given SNP with lung cancer may be low. In this case, one of the SNP’s occurred 60% more often in the lung cancer cases than in the controls. Because of the failure rate of the approach and the relatively modest increases in association of a SNP with lung cancer patients, and because of the million or so SNP’s in the human genome, a very large number of patient and control samples must be used and analyzed for statistical significance. In this case, about 5900 patient samples and 9300 control samples were analyzed leading to a very high degree of statistical significance for the results.

The SNPs in the human genome are defined by formal identifiers that have no biological reference. They are essentially identified by their position in the genome. However, SNPs can have an association with a gene. For example, some SNPs can be located inside a gene or next to a gene. If inside a gene, a SNP may affect the amino acid sequence of the protein that is encoded by the gene and thereby affect the function of that protein. If located in a “noncoding” region within or next to a gene, the SNP may affect the regulation of the expression of the gene. In other words, the SNP could cause the gene to be expressed at a higher level, compared with a different nucleotide at that position in another person.

In this case, the authors of the report noted that two genes, previously associated with tumor development, were located very close to SNPs associated with lung cancer. One of these genes encodes the TERT protein (telomerase reverse transcriptase). This protein is required for re-synthesizing the ends of chromosomes, termed telomeres, following every round of cell division. TERT uses an RNA template to synthesize the telomeric DNA, hence the phrase “reverse transcriptase”. In human cells, ordinarily, DNA is used as a template for RNA.

As cells age and die, TERT expression is reduced or ended altogether. This is a normal process, because a dying cell has no need for DNA replication. However, tumor cells cannot survive without continued TERT expression. Thus, the discovery of a SNP in the noncoding part of the TERT gene raises two issues. First, the SNP associated with TERT and with lung cancer may indicate that the nucleotide at that point in the DNA affects the regulation of the TERT, and in particular, that some individuals have a higher risk of an anomalous regulation of TERT, thereby facilitating lung cancer. Although much experimental work would be needed to prove this, high levels of TERT expression have been strongly implicated in tumorigenesis in many experimental settings.

A second question becomes, can the TERT SNP be used to identify persons at greater or lesser risk of lung cancer? It would indeed be a very simple matter, now that the SNP has been classified, to detect the SNP in any individual. However, the increased risk of having a certain nucleotide at the position of this SNP is only 60%, and it applies only to Northern Europeans. By comparison, cigarette smoking increases the risk of lung cancer by 10-20 times and it applies to everyone. Thus, the greater bang for the public health buck at this point remains the anti-smoking campaign.

What is the greatest usefulness of this work? Most likely, the greatest value is in the discovery that a SNP associated with lung cancer is part of the TERT gene. The authors used a completely unbiased approach that could have identified a SNP in any region of the genome, which has more than 25,000 genes. As it turns out, the SNP is associated with the TERT gene, a gene already related to tumorigenesis because of completely independent scientific approaches. Thus, this provides very strong evidence that expression of this gene is an important part of lung cancer in general. There are already numerous clinical trials related to TERT, and this recent work will further support that effort.

Abstract of article regarding new lung cancer genes

Review article about telomerase and cancer

Acyclovir is related to the guanine nucleotide that is a subunit of DNA. The basic theory behind its mode of inhibition of Herpes virus infections is that this altered DNA subunit is incorporated into DNA as the DNA replicates. However, since the acyclovir subunit is chemically different from the guanine nucleotide, continuation of DNA replication is impossible. Because of its chemical differences, the acyclovir will not permit the addition of the next DNA nucleotide to be incorporated into the growing DNA molecule.

However, for acyclovir to be incorporated into DNA, and block further DNA synthesis, it has to be phosphorylated, i.e., have phosphate groups added to its structure. This is because DNA polymerase, an enzyme that replicates DNA, does not add unphosphorylated subunits to a growing DNA chain. DNA polymerase can only make use of a triphosphate nucleotide. In the case of acyclovir, one phosphate group is added by an enzyme encoded by a gene of the Herpes virus termed, Herpes thymidine kinase. Thymidine is another DNA subunit and “kinase” is a general term for enzymes that add phosphate groups to biochemicals or other proteins. Once the first phosphate group is added by the Herpes enzyme, two other phosphate groups are added to acyclovir by cellular enzymes. This process makes the acyclovir specific for inhibiting DNA replication in Herpes infected cells because only these cells will have the Herpes thymidine kinase protein.

In a certain sense, it is not a big surprise that the triphospate form of acyclovir would inhibit reverse transcriptase, as reverse transcriptase is a type of DNA polymerase that use RNA as a template for making DNA. And, the authors of the recent report demonstrate quite elegantly that the phosphorylated form of acyclovir can be incorporated into the growing DNA chain that is being synthesized by reverse transcriptase.

The bigger issue for this type of discovery is, how does the phosphorylated form of acyclovir move from the Herpes infected cells to the HIV infected cells, keeping in mind that these viruses infect two different types of cells. In fact, the authors of the recent study demonstrate clearly that this type of transfer must be occurring at a certain level, or there must some other unappreciated mechanism of producing the phosphorylated acyclovir in the T-cells where HIV replicates, because these authors conducted their experiments with tissues that contained multiple cell types allowing replication of both viruses, including tonsil tissue.

The transfer of the phosphorylated version of acyclovir, while not well understood, is not without precedent. Others have observed this effect. However, the movement of a phosphorylated compound through cell membranes is not automatic, because cell membranes are hydrophobic and movement of charged molecules through hydrophobic membranes is as inefficient as moving water through oil. In fact, a great deal of drug development involves figuring out how to make drugs hydrophobic, so the drugs can enter cells, while not reducing the effectiveness or increasing the toxicity of the drugs. In the case of acyclovir that has been phosphorylated by Herpes thymidine kinase, transfer may occur by unknown membrane proteins that facilitate the transfer or by cell-fusions that occur at a sufficient frequency to have an effect on HIV replication.

More importantly, the knowledge that HIV infection can be inhibited by phosphorylated acyclovir will inspire new attempts to make this compound particularly available to HIV infected T-cells. For example, in the laboratory, it would be a very simple matter to provide HIV infected T-cells with the Herpes thymidine kinase gene, which can be prepared and inserted into T-cells artificially, without any other components of the Herpes virus. If this approach can be developed in a way that is suitable for patients, it would likely lead to a more efficient clinical effect for acyclovir than has been seen so far.

Abstract of report indicating that HIV reverse transcriptase is inhibited by acyclovir

Abstract and link to full-length article describing acyclovir and related drugs

Example abstract indicating an effect of acyclovir treatment of Herpes and HIV infected patients on HIV load

Attempting to answer this question has a high probability of success due to the complete de-coding of the human genome. Almost a decade ago, the human genome project led to a complete, ordered sequence of the nucleotide bases that comprise the three billion base pair, DNA-set in humans. The bases are often abbreviated as A, T, G, and C, and thus we know the order, from one end to the other, of the A’s, T’s, G’s and C’s for the length of three billion base pairs. Anyone can access this sequence on the web, along with a plethora of related information.

The base sequence dictates the codes for proteins and also the regulation of when these proteins are expressed. With a complete knowledge of the entire code, it is possible to create laboratory tools that detect the “expression”, or lack of expression, for every single part of the genome, essentially every single gene in the genome. This is possible because a string of bases will form bonds with what is termed a “complementary” string of bases, as dictated by the simple principle of Watson-Crick base pairing every middle school student now learns: G’s bond with C’s; A’s with T’s. And, the first level of expression is to produce messenger RNA (mRNA), which, like DNA, is made from nucleotide bases. Thus, a string of mRNA bases will form a double stranded molecule with a string of DNA bases, if the order of the bases is the same, for at least 20-25 bases. That number of complementary bases, all in a row, is stable enough for DNA-RNA binding to occur.

Thus, there exist small platforms, termed DNA microarrays, with each section human genome represented by a string of DNA nucleotides in a different part of the platform. The microarray can be divided up into hundreds of thousands of sections, with each section containing a short string of DNA nucleotides and identifiable by scanning technology and a computer. Each short string of DNA nucleotides can represent a gene or a part of a gene that would in turn correspond to an mRNA.

For the melanoma study, mRNA was taken from the melanoma cells of patients who had a poor survival rate following detection of the melanoma in the lymph nodes and from patients who had lived longer. The mRNA was then placed on the DNA micro-array to determine which mRNAs were present in the different melanoma samples, i.e., which parts of the microarray could detect mRNA through Watson-Crick complementarity. The scanner then scans the microarray and the computer notes the results for the hundreds of thousands of DNA molecules in the different positions in the microarray.

The amount of mRNA for the vast majority of the 25,000 or so genes is the same in both sets of samples. This not a big surprise, because mRNA was taken from melanoma cells that share many of the same functions, regardless of the patients’ time of survival. In fact, some scientists, without knowing the results, might even argue that there would be no significant differences, because it is possible that patient survival differences have nothing to do with the melanoma cells themselves. Instead, the survival differences could be related to patient immune systems or to environmental factors.

As it turns out, there was more mRNA for 14 human genes in the good prognosis set compared to the poor prognosis set; and there was less mRNA for another 7 genes in the good prognosis set compared to the poor prognosis set. A note of caution: The mRNA differences most likely indicate differences in the level of protein, which is generated from the mRNA and which actually carries out the cellular functions. But, mRNA level differences do not always represent differences in protein levels.

Most of the differences did not indicate any obvious reason why good prognosis cells would be less aggressive or less deadly. A couple of the differences were related to the immune response against the tumor, which could conceivably explain why good prognosis cells did not grow as fast. But to be sure, a great deal of experimental tests, using animal models, would be necessary to verify this idea.

So what good is this kind of information about melanoma if it does not readily explain the differences between poor prognosis and good prognosis patients? There are two answers to this question. First, the genes that were identified as being expressed differently in one type of melanoma or another can now be further researched, as with the genes mentioned above that are related to the immune response against melanoma. It is possible that a higher level of mRNA expression for one or more of these genes will quickly be shown to be directly responsible for a good prognosis. If so, it may be possible to augment the expression of these genes in patients with a poor prognosis.

But there is a second value to this knowledge that is far more immediate. The differences in expression levels can be used as a marker, or fingerprint of one type of melanoma versus another. While this may be of little comfort to those with poor prognosis, it will provide both those with good and bad prognoses more accurate information about their health.

Genes that distinguish good and bad prognosis for melanoma

A related study regarding differences in gene expression in different forms of melanoma