SPARC represses E-cadherin and induces mesenchymal transition during melanoma development.

During progression of melanoma, loss of the cell-cell adhesion molecule E-cadherin contributes to uncontrolled growth and invasive behavior of transformed melanocytes. Secreted protein acidic and rich in cysteine (SPARC) is a nonstructural matricellular protein that regulates cell-matrix interactions leading to alterations in cell adhesion and proliferation. Overexpression of SPARC has been associated with progression of various cancers, including melanoma; however, its role in primary tumor development is not well defined. We show that normal human melanocytes overexpressing SPARC adopt a fibroblast-like morphology, concomitant with loss of E-cadherin and P-cadherin expression, and increased expression of mesenchymal markers. Concurrent with these changes, SPARC expression stimulates melanocyte motility and melanoma cell invasion. Expression of SPARC results in transcriptional down-regulation of E-cadherin that correlates with induction of Snail, a repressor of E-cadherin. Conversely, SPARC depletion leads to up-regulation of E-cadherin and reduces Snail levels, and SPARC-null cells exhibit a marked change in their mesenchymal phenotype. Finally, analysis of SPARC, Snail, and E-cadherin levels in melanocytes and malignant melanoma cell lines further supports the functional relationship among these proteins during melanoma progression. Our findings provide evidence for the role of SPARC in early transformation of melanocytes and identify a novel mechanism, whereby tumor-derived SPARC promotes tumorigenesis by mediating Snail induction and E-cadherin suppression.

Spleen tyrosine kinase functions as a tumor suppressor in melanoma cells by inducing senescence-like growth arrest.

Loss of tumor-suppressive pathways that control cellular senescence is a crucial step in malignant transformation. Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase that has been recently implicated in tumor suppression of melanoma, a deadly skin cancer derived from pigment-producing melanocytes. However, the mechanism by which Syk suppresses melanoma growth remains unclear. Here, we report that reexpression of Syk in melanoma cells induces a p53-dependent expression of the cyclin-dependent kinase (cdk) inhibitor p21 and a senescence program. We first observed that Syk expression is lost in a subset of melanoma cell lines, primarily by DNA methylation-mediated gene silencing and restored after treatment with the demethylating agent 5-aza-2-deoxycytidine. We analyzed the significance of epigenetic inactivation of Syk and found that reintroduction of Syk in melanoma cells dramatically reduces clonogenic survival and three-dimensional tumor spheroid growth and invasion. Remarkably, melanoma cells reexpressing Syk display hallmarks of senescent cells, including reduction of proliferative activity and DNA synthesis, large and flattened morphology, senescence-associated beta-galactosidase activity, and heterochromatic foci. This phenotype is accompanied by hypophosphorylated retinoblastoma protein (Rb) and accumulation of p21, which depends on functional p53. Our results highlight a new role for Syk tyrosine kinase in regulating cellular senescence and identify Syk-mediated senescence as a novel tumor suppressor pathway the inactivation of which may contribute to melanoma tumorigenicity.

Tumor-derived fibronectin is involved in melanoma cell invasion and regulated by V600E B-Raf signaling pathway.

Melanomas are malignant tumors of melanocytes that, if not detected early, are highly aggressive and poorly treatable. Activation of extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase signaling is commonly found in melanomas mainly through oncogenic mutations of B-Raf. We previously reported that activation of ERK/MAP kinase stimulates synthesis of fibronectin by upregulating the transcription factor early growth response-1 (Egr-1). To further analyze the link between ERK/MAP kinase pathway and fibronectin in melanoma, we have studied the regulation and role of fibronectin produced by melanoma cells bearing oncogenic B-Raf mutation. We show that fibronectin is expressed in situ during tumor progression and that high fibronectin and Egr-1 levels are found in cells expressing this mutation. Expression of active mutants of B-Raf induces fibronectin, whereas endogenous fibronectin is inhibited by small interfering RNA (siRNA)-mediated depletion of B-Raf or Egr-1. In contrast, stimulation of ERK pathway is insufficient to promote fibronectin upregulation in normal melanocytes. Finally, we show that suppression of fibronectin by siRNA leads to decreased melanoma cell invasiveness in vitro. These results reveal a tumor-specific regulation of fibronectin by constitutive ERK/MAP kinase signaling and indicate that self-production of fibronectin may play a role in melanoma tumorigenesis, by promoting tumor cell invasion.