Osteopontin is a downstream effector of the PI3-kinase pathway in melanomas that is inversely correlated with functional PTEN

The tumor suppressor PTEN antagonizes phosphatidylinositol 3-kinase (PI3K), which contributes to tumorigenesis in many cancer types. While PTEN mutations occur in some melanomas, their precise mechanistic consequences have yet to be elucidated. We sought to identify novel downstream effectors of PI3K using a combination of genomic and functional tests. Microarray analysis of 53 melanoma cell lines identified 610 genes differentially expressed (P<0.05) between wild-type lines and those with PTEN aberrations. Many of these genes are known to be involved in the PI3K pathway and other signaling pathways influenced by PTEN. Validation of differential gene expression by qRT-PCR was performed in the original 53 cell lines and an independent set of 18 melanoma lines with known PTEN status. Osteopontin (OPN), a secreted glycophosphoprotein that contributes to tumor progression, was more abundant at both the mRNA and protein level in PTEN mutants. The inverse correlation between OPN and PTEN expression was validated (P<0.02) by immunohistochemistry using melanoma tissue microarrays. Finally, treatment of cell lines with the PI3K inhibitor LY294002 caused a reduction in expression of OPN. These data indicate that OPN acts downstream of PI3K in melanoma and provides insight into how PTEN loss contributes to melanoma development.

Loss-of-function fibroblast growth factor receptor-2 mutations in melanoma

We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer.

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.