Using Proteins to Inhibit Tumor Growth Research Paper

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…