Search for novel STAT3-dependent genes reveals SERPINA3 as a new STAT3 target that regulates invasion of human melanoma cells.

Transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many cancers and promotes uncontrolled tumor growth and progression through multiple mechanisms. Compelling evidence shows tissue and cell-specific sets of STAT3 targets. Transcriptional targets of STAT3 in melanoma cells are largely unknown. Malignant melanoma is a deadly disease with highly aggressive and drug-resistant behavior. Less than 10% of patients with advanced melanomas reach the 5-year survival, partly due to the aggressive character of the tumor and ineffectiveness of current therapeutics for treating metastatic melanoma. STAT3 is constitutively activated in melanoma cells and plays important roles in its growth and angiogenesis in tumor xenograft studies. Moreover, highly metastatic melanoma cells have higher levels of active STAT3 than poorly metastatic ones. To identify genes that are driven by STAT3 in human melanoma cells, we performed JAK/STAT signaling specific and global gene expression profiling of human melanoma cells with silenced STAT3 expression. For selected genes, we performed computational identification of putative STAT3-binding sites and validated direct interactions STAT3 with defined promoters by using chromatin immunoprecipitation followed by qPCR. We found that STAT3 knockdown does not affect human melanoma cell viability, proliferation, or response to chemotherapeutics. We show that STAT3 regulates a discrete set of genes in melanoma cells, including SERPINA3, a novel STAT3 target gene, which is functionally involved in regulation of melanoma migration and invasion. Knockdown of STAT3 impaired cell migration and invasion, in part via regulation of its transcriptional target SERPINA3. Our results present novel targets and functions of STAT3 in melanoma cells.

Knockdown of STAT3 targets a subpopulation of invasive melanoma stem-like cells.

Transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many cancers, including melanomas. Active, phosphorylated STAT3 contributes to tumor growth and formation of the immunosuppressive tumor microenvironment. Recent evidence suggests an important role of STAT3 in self-renewal of cancer stem-like cells (CSCs). In the present study, we aimed to determine the expression and role of active STAT3 in melanoma CSCs. We found the increased levels of phosphorylated (Y705) STAT3 in CSC sphere cultures derived from three human and murine melanoma cells. Knockdown of STAT3 did not affect basal proliferation, but reduced sphere forming capacity of two human melanoma cell lines. Moreover, the level of active STAT3 was elevated in rhodamine 123 negative subpopulations of CSCs sorted from three melanoma cell lines. We found that focal adhesion kinase (FAK) and AKT signaling pathways, implicated in the regulation of cell migration and invasion, were up-regulated in melanoma CSCs. Moreover, expression of SERPINA3, which regulates melanoma invasion, was increased in melanoma CSCs sphere cultures, which correlated with augmented cell invasion in Matrigel. Our findings show that STAT3 is activated and supports maintenance of melanoma CSCs. It suggests that STAT3 could serve as a potential target to impair tumor progression or recurrence.

Silencing of the transcription factor STAT3 sensitizes lung cancer cells to DNA damaging drugs, but not to TNFα- and NK cytotoxicity.

Transcription factor STAT3 (Signal Transducers and Activators of Transcription 3) is persistently active in human tumors and may contribute to tumor progression. Inhibition of STAT3 expression/activity could be a good strategy to modulate tumor cell survival and responses to cancer chemotherapeutics or immune cytotoxicity. We silenced STAT3 expression in human A549 lung cancer cells to elucidate its role in cell survival and resistance to chemotherapeutics, TNFα and natural killer (NK)-mediated cytotoxicity. We demonstrate that STAT3 is not essential for basal survival and proliferation of A549 cancer cells. Stable silencing of STAT3 expression sensitized A549 cells to DNA damaging chemotherapeutics doxorubicin and cisplatin in a p53-independent manner. Sensitization to DNA damage-inducing chemotherapeutics could be due to down-regulation of the Bcl-xL expression in STAT3 depleted cells. In contrast, knockdown of STAT3 in cancer cells did not modulate responses to TNFα and NK-mediated cytotoxicity. We found that STAT3 depletion increased the NFκB activity likely providing the compensatory, pro-survival signal. The treatment with TNFα, but not doxorubicin, enhanced this effect. We conclude that STAT3 is not crucial for the control of basal cell proliferation and survival of lung carcinoma cells but modulates susceptibility to DNA damaging chemotherapeutics by regulation of intrinsic pro-survival pathways.

A sensitized RNA interference screen identifies a novel role for the PI3K p110γ isoform in medulloblastoma cell proliferation and chemoresistance.

Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor samples and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110γ (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110γ phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma.