HIF1α and HIF2α independently activate SRC to promote melanoma metastases

Malignant melanoma is characterized by a propensity for early lymphatic and hematogenous spread. The hypoxia-inducible factor (HIF) family of transcription factors is upregulated in melanoma by key oncogenic drivers. HIFs promote the activation of genes involved in cancer initiation, progression, and metastases. Hypoxia has been shown to enhance the invasiveness and metastatic potential of tumor cells by regulating the genes involved in the breakdown of the ECM as well as genes that control motility and adhesion of tumor cells. Using a Pten -deficient, Braf -mutant genetically engineered mouse model of melanoma, we demonstrated that inactivation of HIF1α or HIF2α abrogates metastasis without affecting primary tumor formation. HIF1α and HIF2α drive melanoma invasion and invadopodia formation through PDGFRα and focal adhesion kinase–mediated (FAK-mediated) activation of SRC and by coordinating ECM degradation via MT1-MMP and MMP2 expression. These results establish the importance of HIFs in melanoma progression and demonstrate that HIF1α and HIF2α activate independent transcriptional programs that promote metastasis by coordinately regulating cell invasion and ECM remodeling

Intrinsic Genomic Differences Between African American and White Patients With Clear Cell Renal Cell Carcinoma

IMPORTANCE: There are well-documented racial disparities in outcomes for African American patients with clear cell renal cell carcinoma (ccRCC). Despite a dramatic improvement in overall survival in white patients since the advent of targeted therapy, survival for African Americans with advanced ccRCC has not changed. There is little known about potential racial differences in tumor biology of ccRCC. OBJECTIVE: To determine if there are racial differences in the somatic mutation rate and gene expression of ccRCC tumors from white and African American patients. DESIGN, SETTING, AND PARTICIPANTS: Overall, 438 patients with ccRCC were identified through The Cancer Genome Atlas (TCGA) clear cell kidney (KIRC) dataset (419 white and 19 African American patients). The GSE25540 dataset containing 135 patients (125 white and 10 African American patients) was used for validation. Tumor samples were collected from numerous cancer centers and were examined for racial differences in somatic mutation rates and RNA expression. Racial differences in somatic mutation rates and RNA expression were examined. MAIN OUTCOMES AND MEASURES: The comparison of somatic mutation rates and differences in RNA expression in white and African American patients with ccRCC. RESULTS: Overall, 419 ccRCC tumor data sets from non-Hispanic white patients and 19 from non-Hispanic African American patients were identified through the publically available TCGA KIRC data set, and a validation set of 125 white and 10 African American ccRCC patient tumors was identified from the publicly available GSE25540 data set. African American patients were significantly less likely than white patients to have VHL mutations (2 of 12 [17%] vs 175 of 351 [50%], respectively; P = 04) and were enriched in the ccB molecular subtype (79% in African American vs 45% in white patients; P = 005), a molecular subtype that carries a worse prognosis. It was found that RNA expression analysis revealed relative down-regulation of hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF)-associated pathways in African American patients compared with white patients. CONCLUSIONS AND RELEVANCE: African American patients have less frequent VHL inactivation, are enriched in the ccB molecular subtype, and have decreased up-regulation of HIF-associated gene signatures than white patients. These genomic differences would predict decreased responsiveness to VEGF-targeted therapy and are a biologically plausible contributing factor to the worse survival of African American patients with ccRCC, even in the targeted therapy era

Novel renal cell carcinoma cell lines lack VHL and overexpress HIF.

<p>(<b>A</b>) Photomicrographs of H&E stains (left panels) and bright field images (right panels) of UNC-R1 and UNC-R2 PDX derived cell lines. (<b>B</b>) Whole cell extracts from UNC-R1 and UNC-R2s were immunoblotted with the indicated antibodies. RCC4 2-1 (VHL null) and RCC4 3–14 (VHL positive) were included as controls.</p

Combined mTOR and MEK inhibition attenuates cellular proliferation and increases the apoptotic response.

<p>(<b>A</b>) The indicated cells were treated for 24 hrs. with rapamycin or BEZ235 and immunoblotted with the indicated antibodies. (<b>B</b>) 786-0 and RCC4 cells were treated increasing doses of GSK212 for 24 hrs. and immunoblotted with the indicated antibodies. (<b>C</b>) 786-0 and RCC4 cells were treated for 24 hrs with rapamycin and BEZ235 in the presence of Edu. Edu incorporation was assessed by flow cytometry. (<b>D</b>) 786-0 and RCC4 cells were treated with the indicated compounds for 24 hrs. Whole cell extracts were immunoblotted for the cell cycle related proteins indicated. (<b>E</b>) 786-0 and RCC4 cells were treated with indicated drugs and assessed for viability on day 4 using CellTiter-Glo 4. (<b>F</b>) 786-0 and RCC4 cells were plated, allowed to attach, and treated with the indicated drug(s). Photographs of wells containing 786-0 (day 11) and RCC4 (day 17) cells fixed with 4% PFA and stained with 0.1% crystal violet. (<b>G</b>) 786-0 and RCC4 cells were treated with the indicated compounds for 24 hrs. Whole cell extracts were immunoblotted with the indicated antibodies.</p

Catalytic mTOR inhibition attenuates proliferation and induces apoptosis better than allosteric mTOR inhibition.

<p>(<b>A</b>) The indicated cell lines were assessed for viability on the indicated days using CellTiter-Glo. Statistical significance was determined by comparing rapamycin and BEZ235 treated groups. (<b>B</b>) The indicated cell lines were treated with rapamycin and BEZ235 for 48 hours and immunoblotted with the indicated antibodies. (<b>C</b>) The indicated cell lines were treated with rapamycin and BEZ235 for 48 hours and assessed for apoptosis by flow cytometry analysis of the Annexin V+/PI – fraction. (<b>D</b>) 786-0 cells were stably infected with shRNAs targeting Raptor (mTORC1) or Rictor (mTORC2) and confirmed for knock-down by western blot. (<b>E</b>) Whole cell extracts from 786-0 shNS, shRaptor, and shRictor cells were immunoblotted with the indicated antibodies.</p