Merkel Cell Carcinoma

Griffiths, D., et al. (2013). Merkel Cell Polyomavirus Small T. Antigen Targets

the EMO Adaptor Protein To Disrupt Inflammatory Signaling. Journal of Virology. 87 (24), 13853-67.

Merkel cell carcinoma is a relatively rare disease in which malignant cells form in the skin, usually in individuals who have a weak immune system or extensive exposure to the sun. Merkel cells are found in the top (epidermis) layer of the skin, close to the nerve endings that house responders to touch. Merkel cell carcinoma is also known as neuroendocrine carcinoma of the skin, sometimes as trabecular cancer. It forms when the Merkel cells grow rapidly and out of control, usually starting in areas of the skin that have more exposure to the sun (neck, arms, legs, etc.). The cancer tends to grow robustly and metastasize at a relatively early stage. Typically, it spreads to nearby lymph nodes and then the lymphatic system, which then may affect the lungs, brain, bone or other organs.

In 2008, researchers identified the Merkel cell polyomavirus in 80% of Merkel cell carcinomas (MCC). This polyomavirus is one of the most recently discovered viruses contributing to cancer, and shows a strong linkage to tumor production and virulence. Research has shown that a monoclonal viral integration pattern is found in most MCC, which then indicates that the virus uses RNA and other cellular mechanisms to expand into other, non-infected, cell structure (13853). Recent studies have identified a new function to inhibit production of the virus by using small T. antigen (ST) as a way to limit transcription. In effect, this limiting interacts with the virus and the Nf-kB essential modulator protein (NEMO) and reduces the viruses’ ability to replicate, eventually subverting the virus completely.

Experiments Performed

Essentially, 293 cells were used as a base for experimentation. The McPyV ST was taken from MCC tumor DNA using a specific methodology from base pair genetics and then cloned to generate additional cells. The cells were maintained in Dulbecco’s Eagle’s medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were then broken down in an RIPA buffer and supplemented with a protease inhibitor cocktail, then separated out. Total RNA was then harvested and cellular structures were analyzed. Assays were performed, as well as immunofluorescence. In order to delineate the actions of the protein and its ability to inhibit cellular growth, SILAC-based immunopreciptations and NF-XB pathway actions were induced, with the results then calculated (13854-5).

Hypothesis

Recent evidence from this study, and others in the field, suggests that MCPyV ST is oncogenic. Yet ST alone seems sufficient to reduce viral growth in certain cancerous rodent cells, which suggests the same effect may be true in human cells. The authors believe that “specific depletion of MCPyV ST is sufficient to inhibit MCPyV-positive MCC cell growth” (13853). In addition, the authors have identified an essential role for NF-kB activation in both the antiviral and the inflammatory response, which then makes it a perfect target for viral subversion. Since virus proteins antagonize all stages of the NF-kB signaling pathway, the authors posit that an interaction between MCPyV ST and the NF-kB protein will be a robust new way to more effectively treat MCC by targeting specific cellular mechanisms. This may have more positive patient-outcomes with fewer side effects than traditional chemotherapy.

Results

The study confirmed the important function of MCPyV ST in viral replication and transformation. In order to define the actual functions of the ST, it was necessary to use expression-profiling analysis. Through the arrays performed using cDNA and RNA there was a clear reduction of genes associated with the immune response and a clear tend to activating other protein pathways within the cellular structure.

The results, in fact, indicate that “MCPyV ST prevents NF-kB activation by a mechanism independent of activated receptor complexes” (13856). Additional interactions indicate that the MCPyV ST interacts with another target protein, which was identified and tagged and became known as an additional way that NEMO interferes with viral growth and reproduction.

Additional data shows that MCPyV ST does not promote any degradation of NEMO, which then allows an even greater and more robust response to viral cells. Further, “nuclear levels of p50 and RelA were also markedly lower, approximately 50% in TNF-activated cells expressing MCPyV ST, compared with uninduced cells” (13859). This also confirmed that MCPyV ST inhibits the IKKa/IKK? phosphorylation. This is crucial because IKK plays an essential role in cellular signaling, and the prevention of the IKKa/IKK? also works to inhibit viral reproduction. In addition, it appears that the MCPyV ST inhibits a number of chemical reactions within the viral cell that increase the virility of the MCC cell, particularly the ability for it to grow so rapidly and spread so quickly. Overall, the data suggest that the MCPyV ST and NF-kB transcriptional repression is done through specifically identified cellular phosphates and that dephosphorylating of IKK is an important target for this inhibition.

Mechanism

The chemical mechanism that seems to be working within this study is an NF-kB pathway inhibition that targets certain protein structures within the virus itself, rendering it unable to build cytokines, chemokine’s and other proteins involved in viral growth. By doing this, the virus is limited in its ability to replicate, and thus reduces its virulence within the skin cells. Typically, viruses gain advantage by stimulating or inhibiting the NF-kB pathway, increasing cell proliferation, and then provides additional molecular components that aid the virus in reproducing.

The findings of this study, however, suggest that MCPyV ST binds NEMO and negatively regulates NF-kB, deregulating the NF-kB’s ability to allow the MCPyV to evade the body’s antiviral response. Thus, MCPyV ST prevents phosphorylation of IkBa, which then means the NF-kB becomes inactive within the cellular cytoplasm and is thus unable to move into the nucleus and activate any transcription targets. In essence, the McPyV ST interrupts the cellular process by interfering with protein communication that would allow the virus to move into the nucleus and trick the cell into becoming another virus cell.

In addition to preventing initial cellular growth of the virus, the MCPyV ST also appears to work on tumors and regions in which the MCC has advanced. It does this by working to interrupt additional cellular functions so that, in essence, the tumor cells starve because they are unable to complete their necessary metabolic processes in order to grow. Thus, from an immunological standpoint, the MCPyV ST increases cellular resistance to the virus, prevents it from reproducing in new nucleus portions of the cell, and acts to limit the spread of already expressed tumors.

Significance of the Paper

This is the first immunomodulatory function ascribed to a polyomavirus ST that may allow for greater protection against the MCPyV life cycle and concurrent tumor development. The techniques used within the paper to both identify and analyze (tabulate) specific sets of protein interactions were novel and important to future immunological research. The findings identify a new and important way to subvert viral replication as well as the innate immune response that allows persistent infection within the cell. Instead, by blocking protein expression, MCC cells can be limited in growth.

Conclusions

In the United States alone, there are just under 2,000 new cases of MCC diagnosed. Further, MCC occurs at an alarmingly high rate among people with chronic lymphocytic leukemia. Metastatic MCC may respond slightly to chemotherapy, but is usually not curative or completely effective in more than shrinking the tumor and improving the options for surgery. In addition, MCC tumors are often located in difficult or dangerous places near the spinal cord or brain, making surgery risky or even prohibitive. Instead, by using a therapy that uses an antigen to target certain cellular proteins, there is a greater chance for a positive patient outcome with fewer side effects, since the increase in T-Antigens are not caustic or poisonous to the cellular structure, and act in a manner that is relatively specific to viral cell reproduction. In fact, using this type of therapy has broad applications not only to MCC, but also to any cancer that is virally related. Additionally, this type of technique may be expanded to provide better options for the treatment of other cancers, by helping researchers understand the complex interrelationships that exist within the cell and are expressed through protein regulation.