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A functional and prognostic study of the transcription factor SIX1 in melanoma Graziano, Laura

Abstract

This study focuses on melanoma, a cancer of pigment-producing cells within the skin (melanocytes) which insidiously spreads (metastasizes) throughout the body. This devastating disease often prevents patients from living beyond five years from diagnosis. Treatment options for melanoma are inept, which has necessitated a demand for research on the molecular mechanisms of melanoma to aid in the development of novel therapeutic targets and treatments. The gene Sineoculis homeobox homolog 1 (SIX1) encodes for a homeoprotein transcription factor which is an important developmental regulator during embryogenesis. A preliminary microarray analysis found that SIX1 was upregulated in melanoma. The purpose of this study was to pioneer the assessment of SIX1 in melanoma, including its role in the metastatic functions of melanoma in vitro as well as its clinical relevance. The specific aims included: (1) to assess SIX1 expression in melanoma cell lines and clinical samples; (2) to investigate the functional significance and pathogenic role of Six1 in vitro; and (3) to evaluate Six1’s clinical significance in relation to prognosis and clinicopathological characteristics. Cell lines and clinical samples were tested for SIX1 transcript level using qPCR and protein level using Western Blotting. Next, two malignant melanoma cell lines were transfected with shRNA plasmids to generate stable clones with low Six1 expression. These altered cell lines were used to study the functional implications of Six1 expression disparities in vitro. Furthermore, immunohistochemistry against Six1 was performed on a tissue microarray of 438 melanoma patient biopsies. We discovered that SIX1 was overexpressed in melanoma cell lines and clinical samples. Six1 knockdown cells demonstrated diminished cell growth and proliferation, increased apoptosis, and decreased migration and invasion. We also found that a profound nuclear to cytoplasmic shift of Six1 accompanied melanoma progression and correlated with poor five-year survival. Higher cytoplasmic Six1 was associated with increased tumor thickness, lower nuclear Six1 with ulceration and histological satellitosis, and both with advanced AJCC stages and nodular melanoma. To summarize, these results hint that Six1 may play a role in the execution of metastatic functions in melanoma. Furthermore, Six1 presents as a tentative candidate for a prognostic marker in patient biopsies.

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