Merkel cell carcinoma (MCC) is a very aggressive neuroendocrine cancer. The overall survival (OS) in MCC patients who have localized disease is about 50%. But those with distant metastasis have an abysmal OS of only about 10-14%. A neuroendocrine carcinoma, MCC presents as firm reddish nodules on the skin of elderly patients. In a small proportion of patients, the skin lesions are absent but the tumor is localized in the lymph node. The risk of MCC is higher in immunosuppressed individuals. Significantly, Most MCCs are infected with recently identified virus, Merkel cell Polyoma virus (MCPyV). It is not clear whether the MCPyV infection which underlies the MCC development is the cause or consequence of immunosuppression. Previously, we have shown that viruses can alter cellular miRNAs to alter immune checkpoints like PD-L1 in lymphoma. In the present proposal, we would like to examine an Italian cohort of MCC for the presence of MCPyV, expression of immune checkpoints (IC) and IC targeting miRNAs. Using both MCPyV positive and negative MCC cell lines, first we will establish miRnome of these cell lines to understand how the presence of MCPyV affects cellular miRNAs. Subsequently, we will analyze expression of PD-L1, PD-L2 and other IC proteins in MCC which are infected in vitro. We will identify virally encoded proteins which influence IC expression. To better understand, molecular mechanisms behind IC alteration, we will investigate IC targeting miRNAs, like miR-34a, miR-24, miR-200c. Towards identification of novel biomarkers for MCC, we propose to investigate deregulated cellular miRNAs in sera from MCC patients and healthy controls.
MCC is a rare but deadly cancer. Immunosuppression underlies its development (1). As mentioned earlier, MCPyV is frequently present in most MCCs (2). Very little, if anything at all, is known how the presence of this polyoma virus in MCC influences immune checkpoints (IC) like PD-L1, PD-L2 and ICOSL (3). It is also not known how cellular miRNAs targeting ICs are affected by the presence of the virus. We want to tackle these issues in two different ways. First, we will establish in vitro model system, where we will compare MCPyV positive and negative cell lines for immune checkpoints expression. Once we have established the role of the virus in deregulating PD-L1, PD-L2 and ICOSL, we will transfect MCPyV negative MCC cell lines with virally encoded small and large T proteins. MCC cell lines infected with MCPyV and transfected with single virally encoded proteins will be analyzed for miRNA expression by microarrays. Such analysis will give us a broader picture of how cellular miRNAs are altered in the presence of the entire virus or transfected viral genes. We will then proceed to identify miRNAs which target IC proteins. Specifically, we will analyze expression of miR-34a, miR-200c, miR-24 by real time qPCR. We will also construct luciferase reporter constructs of PD-L1, PD-L2 and ICOSL 3' UTR. These constructs will be transfected into MCPyV infected cells to establish specificity of miRNAs and their IC targets. The 3' UTR of IC genes will be mutated in the seed sequences where the miRNAs bind to demonstrate unequivocally that MCPyV indeed affects IC Proteins through alteration of cellular miRNAs. The in vitro studies above will set the stage for the second major question that we will address is in clinical samples of MCC derived from patients. Through our collaborators at Verona University hospital (Prof. Michele Millela), we have access to a rather large cohort of MCC samples. The MCC tumor samples from patients will be analyzed for PD-L1, PD-L2 and ICOSL expression. The clinical material will also be tested for single miRNAs mentioned above. We will also test expression of IC targeting miRNAs in circulation. To this end, we have access to sera samples of all MCC patients in the Italian cohort from the Verona University Hospital collaborators. Over the last three years or so, we have established a novel 3D microfluidic chip based system to test and reconstitute tumor immunogenicity. We will employ this method to evaluate tumor immunogenicity of MCPyV infected MCCs and test if combination of miRNAs and IC blocking antibodies could render the MCC cells more immunogenic. Overall, the present proposal will provide answers to some lingering questions as to how MCPyV affect cellular miRNAs to influence IC proteins and make tumors invisible to the immune system. I strongly feel that tumors like MCC need urgent attention and since cancer immunotherapy with IC blocking antibodies alone has proven successful only in a fraction of cases, it is time that we investigate novel miRNA based approaches to further strengthen the immune system. The data obtained from this proposal will open new avenues of diagnosis, in terms of miRNA based biomarkers and improved prognosis for MCC patients.
References:
1: Becker, J.C et al., Nat. Rev. Dis. Primers 2017, 3, 17077
2: Liu, W et al. Curr. Opin. Virol. 2016, 20, 20-27
3: Nagase et al, Curr. treat. opt. Oncol. 2018, 19: 57.