Elucidation of a SOX10-dependent pathway in melanoma development

Abstract

Melanoma is the most lethal skin cancer in the world, which is malignantly transformed from neural crest derived melanocyte under the combined effects of genetic mutations and extracellular stimulation. Although therapies targeting oncogenetic mutations and immune deficiency have been developed rapidly, the 5-year survival rate of patients suffering from metastatic melanoma is as low as 15 % to 20 %, and they develop drug resistance and other side effects. Previous studies have demonstrated that Sry-related HMG box containing family subgroup E (SOXE) proteins are crucial for melanoma development. SOX10 plays vital roles in contributing to various aspects of melanoma progression including tumor initiation, cell proliferation, invasion as well as drug resistance; whereas SOX9 appears to be an anti-tumorigenic and antagonizes SOX10 expression. Therefore, understanding the molecular pathways of melanoma development mediated by SOX10 would pave the way for novel therapeutic strategies. To further investigate this issue, the SOX10 and SOX9 expression levels were first examined in the human melanoma microarray and two human melanoma cell lines, A375 and SK-ME-28, with aberrant SOXE genes expression were employed in this study. Our data showed that SOX10 expression was elevated in the metastatic melanoma patient samples and the two cell lines, but the SOX9 levels were relatively low. By using shRNA mediated knockdown (KD) of SOX10, we showed that reduced level of SOX10 expression resulted in functional defects in melanoma survival, proliferation and in vivo metastatic ability, coupling with significant upregulation of SOX9 expression, which was consistent with previous studies. By contrast, SOX9 overexpression only reduced cell migration capacity, but not proliferation which was contradictory with previous studies. Among several candidate genes, Neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9), a melanoma metastatic gene, was identified as a novel target regulated by SOX10 and SOX9. The luciferase reporter assay results further revealed that NEDD9 was directly regulated by SOX10 through direct binding to the NEDD9 promoter region. Additional expression NEDD9 was able to rescue the functional defects of melanoma survival, proliferation and migratory capacity in SOX10 KD A375 cells. Consistently, co-expression of SOX10 and NEDD9 was detected in the metastatic melanoma patient samples, indicating their regulatory relationship in contributing to melanoma progression In addition, upregulation of SOX9 expression in SOX10 KD was not sufficient to restore NEDD9 expression unless exogenous expression of SOX9 was introduced into SOX10 KD, suggesting that SOX9 functions in a dosage-dependent manner in regulating NEDD9 expression. Consistently, ectopic SOX9 expression in the SOX10-KD cells also rescued the functional defects in melanoma survival, proliferation and migratory capacity. NEDD9 has been reported as a positive regulator of small GTPase RAC1, its function is important for cell motility and tumor cell plasticity. We found that SOX10 and SOX9 could positively regulate the RAC1 activation, probably through the modulation of NEDD9. The discovery of the novel SOX10/SOX9-NEDD9-RAC1 regulatory axis advances the understanding of molecular events in melanoma pathology and may provide comprehensive insights for novel drug development.