Neurocan as a prognostic marker in Merkel Cell Carcinoma

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Title: Neurocan as a prognostic marker in Merkel Cell Carcinoma
Author: Salmikangas, Marko Erkki Kristian
Contributor: University of Helsinki, Faculty of Medicine
Publisher: Helsingin yliopisto
Date: 2020
Thesis level: master's thesis
Abstract: Background Merkel Cell Carcinoma (MCC) is a rare neuroendocrine tumor that is associat-ed with old age and immunosuppressive condition. It has two distinct sub-groups differentiated with their Merkel Cell Polyomavirus (MCPyV) positivi-ty/negativity. While both groups are considered aggressive, the Merkel cell Pol-yomavirus negative group has significantly worse prognosis. Traditionally MCC cases have been diagnosed based on their physiological appearance and im-munohistochemical markers such as cytokeratin 20 and tumor transcription factor-1, which differentiate MCCs from small cell carcinomas. It still requires skilled personnel such as dermatologists and pathologists to identify MCCs. More precise and effective biomarkers are required to improve MCC diagnos-tics, to enhance patient survival and in the development of personalized medi-cine for MCC. Neurocan (NCAN) is a chondroitin sulphate proteoglycan that is found mainly in central nervous tissue in adults. The core protein of Neurocan is formed of 3 domains, G1 containing a single immunoglobulin domain, the glycosamino-glycan binding backbone and G3 domain containing regulatory protein-like sequences and epidermal growth factor/lectin-like domains. It is produced mainly by reactive astrocytes and its main function is to guide the growth of ax-ons and to participate in the formation of neural extracellular matrix. Neurocan is linked to inhibition of axonal regeneration and glial scarring in case of neu-ral injury. There are few mentions of Neurocan changes related to cancer out-side of the central nervous system, however, there is clear evidence of chon-droitin sulphate proteoglycan involvement in tumor invasiveness and potential-ly promotion of malignant tumor phenotype. Aim of the study and experimental design This thesis is a part of a project studying novel biomarkers and therapeutic tar-gets for MCC. The aim of the study was to identify a novel cancer specific gene (Neurocan) that would be either a potential biomarker or therapeutic target, and to set up the pipeline for further expanding the parent project. Neurocan was first identified from outlier gene detection methods applied to MCC sample series containing samples of 141 MCC patients. After this Neu-rocan expression levels were studied at protein level using immunohistochem-istry for MCC sample series. NCAN expression levels in MCC cell lines were investigated at mRNA and protein level with qPCR and Western blotting re-spectively. Functional studies of Neurocan such as an effect on cell prolifera-tion were performed with siRNA knockdown assays, and analyzed with West-ern blotting and qPCR. Results 144 FFPE samples in TMA (tissue microarray) format were stained for Neu-rocan protein expression; 31 samples expressed NCAN at low level, 60 at in-termediate level and 53 cases had high NCAN expression. The low NCAN ex-pression correlated with poor MCC specific survival (5-year survival 44%) when compared to intermediate and high expression groups (5-year survival 73% and 65% respectively). Kaplan-Meier survival analysis also implicated a signifi-cant difference in survival between the groups, p-value 0.044. NCAN expres-sion levels had a strong association with Merkel Cell Polyomavirus (MCPyV) status (Pearson Chi-square, p-value = 0.006) with 83% of high NCAN expres-sion cases being MCPyV positive, where as 55% of low NCAN expression cases were MCPyV negative. Cox proportional hazards model revealed that NCAN is unlikely to be an independent variable in patient survival. NCAN expression correlated with the MCPyV status of 9 tested MCC cell lines (Student’s t-test, p-value = 0.041). Protein level studies were inconclusive due to lack of specific antibodies and testing methods. 4 cell lines were tested for NCAN functionality in cell cultures. siRNA knock-down of NCAN did not affect the survival of MCC cell lines, however, it had a reducing effect on Large T-antigen expression of the MCPyV positive cell lines. Likewise, siRNA knockdown of Large T-antigen reduced the expression of NCAN mRNA in MCPyV positive cell lines. No such interactions were found in the MCPyV negative cell lines. siRNA knockdown of sT-antigen significantly reduced the growth of MCPyV positive WaGa cell line (Student’s t-test, p-value = 0.01). NCAN and large T-antigen targeting siRNAs had only a minor growth reducing effect on WaGa cell lines, and MKL1 cell line saw only minor growth reduction with all of the different siRNA treatments. These were not statistically significant findings. Whether Neurocan expression is directly controlled by MCPyV T-antigens, or whether the regulation is due to a signaling cascade of sorts, is still unknown.
Subject: biomarker
Merkel cell carcinoma
Merkel cell polyomavirus

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