Date of Award
MD (Medical Doctor)
Biological Markers, Tumor Markers Biological, S100 Proteins
Cutaneous melanoma is an aggressive disease, which is recognised as the most common fatal skin cancer worldwide. Approximately 450 cases are diagnosed in Ireland per year. Between 1994 and 2004, melanoma demonstrated a higher rate of increase in mortality than any other cancer in this country. Although the prognosis for early melanoma is favorable, less than 20 percent of patients with metastatic melanoma survive for five years.
S100B is a calcium sensor protein that modulates biological activity via calcium binding, which is routinely used in histological diagnosis of malignant melanoma and which is also a well-recognized serum marker of the disease. HOX proteins are members of the homeodomain family of transcription factors, which are involved in a host of cellular functions including organogenesis, cellular differentiation, cell cycle and apoptosis. As transcription factors, HOX proteins require co-activator proteins to achieve their full function. SRC-1 is one such coactivator and our group has extensively explored its function. In particular, functional interactions between SRC-1 and HOXCI 1 in breast cancer cell lines and tissue have been described. HOXCI 1 is known to enhance expression of the secreted serum marker S100B. Given the strong association between S100B and malignant melanoma, we believe that this pathway may also have a role in melanoma tumour genesis.
S100B is a well-described biomarker in melanoma and serum levels have been shown to correlate with disease stage and response to treatment. In spite of this and the availability other prognostic indicators, many patients go on to develop an unpredictable disease course. For this reason, identification of novel biomarkers is an active area of melanoma research. In the second part of this work, an autoantibody microarray screen was undertaken to identify differentially expressed biomarkers in sera from patients with melanoma.
Production of S100B in malignant melanoma is regulated by the transcription factor HOXCI 1, in cooperation with coactivator SRC-1. Protein microarray technology may provide a useful means of identification of autoantibody biomarkers in serum from patients with melanoma.
To define the molecular role of HOXC11 and SRC-1 in the transcriptional control of S100B in malignant melanoma.
To characterise the effect of manipulation of HOXC11 and SRC-1 on S100B expression.
To identify new biomarkers in sera from patients with malignant melanoma.
Expression of SIOOB, HOXC11 and SRC-1 protein in primary (SKMe128) and metastatic (MeWo) melanoma cell lines was confirmed by Western blotting and quantitative Real Time PCR (qRT-PCR) analysis. Colocalisation of HOXCI 1 and SRC-1 in melanoma cells was confirmed by immunofluorescence. Co-immunoprecipitation was carried out and demonstrated interaction of HOXC11 and SRC-1 in cell lysates. Paraffinembedded melanoma and nevi samples were examined by immunofluorescence and a significantly higher nuclear expression of HOXCI 1 and SRC-1 was observed in the melanoma cohort. Colocalisation of the two proteins was also demonstrated in a series of melanoma primary culture specimens.
Chromatin-immunoprecipitation was employed to confirm recruitment of HOXCI 1 to the promoter region of the S100B gene. To determine the ability of HOXCI 1 to regulate expression of S1 OOB, HOXCI 1 was transfected into the SKMe128 cell line and it was found to significantly increase the expression of the target gene S100B. When concomitant HOXCI 1 and SRC- 1 knockdowns were performed, a significant reduction in the presence of S100B was noted. Treatment of cell lines with the phospho-Src inhibitor, dasatinib, resulted in decreased coassociation between HOXCI 1 and SRC-1 in both primary and metastatic cell lines as well as decreased expression of S100B in SKMe128 cells.
Protein microarray analysis of sera from patients with melanoma and control patients was carried out. A series of differentially expressed autoantibodies was identified and the non-receptor tyrosine kinase, BMX was chosen for further study. Elevated expression of anti-BMX autoantibody in sera from a larger cohort of melanoma patients was confirmed. Furthermore, expression of BMX protein in melanoma cell lines and frozen tissue samples was confirmed by Western blotting.
In the absence of effective treatment for advanced melanoma, elucidation of novel signalling pathways and therapeutic targets remains at the forefront of molecular research. In this work, translational techniques have provided an insight in to the transcriptional regulation of S100B in melanoma. Furthermore, protein microarray analysis has been utilised to identify potentially useful autoantibody biomarkers. These findings constitute a small fragment of all the potential genetic aberrations that may be implicated in melanoma turnourgenesis. Advanced melanoma is likely to present a significant therapeutic challenge to clinicians and academics for many years to come. What is certain is that translational research methods, as have been employed here, are essential in pushing forward the boundaries of our molecular understanding of this fascinating disease.
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de Blacam C. Transcriptional regulation of S100B and identification of novel melanoma biomarkers [MD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2010.