Hypoxia-Responsive Elements

Abstract: The hypoxia inducible factor (HIF) signaling pathway is known as the main renal carcinogenetic pathway. MUC1, an O-glycoprotein membrane-bound mucin, is overexpressed in clear renal cell carcinomas (cRCC) with correlation to two major prognostic factors: tumor-node-metastasis stage and nuclear Furhman grade. We questioned whether there is a direct link between the HIF pathway and MUC1 overexpression in renal tumors. Interestingly, we observed concomitant increase of HIF-1alpha and MUC1 in metastatic cRCC group versus nonmetastatic cRCC group. Using different renal cell models and small interfering RNA assays targeting either HIF-1alpha or YC-1, a HIF-1 pharmacologic inhibitor, we showed induction of MUC1 expression under hypoxia by a HIF-dependent mechanism. Chromatin immunoprecipitation assay showed a direct binding of HIF-1alpha at the MUC1 promoter. In addition, combined site-directed mutagenesis and gel shift assay allowed the identification of two functional putative hypoxia responsive elements at -1488/-1485 and at -1510/-1507 in the promoter. Using a rat kidney model of ischemia/reperfusion, we confirmed in vivo that clamping renal pedicle for 1 hour followed by 2 hours of reperfusion induced increased MUC1 expression. Furthermore, MUC1 knockdown induced significant reduction of invasive and migration properties of renal cancer cells under hypoxia. Altogether, these results show that MUC1 is directly regulated by HIF-1alpha and affects the invasive and migration properties of renal cancer cells. Thus, MUC1 could serve as a potential therapeutic target in cRCC.

Abstract: Hypoxia is a common feature of solid tumours that promotes invasion and metastatic dissemination. Invadopodia are actin-rich membrane protrusions that direct extracellular matrix proteolysis and facilitate tumour cell invasion. Here, we show that CSRP2, an invadopodial actin bundling protein, is upregulated by hypoxia in various breast cancer cell lines, as well as in pre-clinical and clinical breast tumour specimens. We functionally characterized two hypoxia responsive elements within the proximal promoter of CSRP2 gene which are targeted by hypoxia-inducible factor-1 (HIF-1) and required for promoter transactivation in response to hypoxia. Remarkably, CSRP2 knockdown significantly inhibits hypoxia-stimulated invadopodium formation, ECM degradation and invasion in MDA-MB-231 cells, while CSRP2 forced expression was sufficient to enhance the invasive capacity of HIF-1α-depleted cells under hypoxia. In MCF-7 cells, CSRP2 upregulation was required for hypoxia-induced formation of invadopodium precursors that were unable to promote ECM degradation. Collectively, our data support that CSRP2 is a novel and direct cytoskeletal target of HIF-1 which facilitates hypoxia-induced breast cancer cell invasion by promoting invadopodia formation.

Abstract: Despite being an adverse prognostic factor in radiotherapy, hypoxia represents a physiological difference that can be exploited for selective cancer gene therapy. In this study gene therapy vectors responsive to both hypoxia and ionizing radiation (IR) were developed. Gene expression was regulated by novel, synthetic promoters containing hypoxia responsive elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase 1 (PGK1) and the vascular endothelial growth factor (VEGF) genes, and IR-responsive CArG elements from the early growth response (Egr) 1 gene. All chimeric promoters could be activated by hypoxia and/or IR-treatment, and selectively control marker gene expression in human T24 bladder carcinoma and MCF-7 mammary carcinoma cells. Importantly, enhancers containing combinations of HREs and CArG elements were able to respond to both triggering treatments, with the Epo HRE/CArG combination proving to be the most responsive and robust. The Epo HRE/CArG enhancer could effectively control a suicide gene therapy strategy by selectively sensitizing hypoxic and/or irradiated cells expressing the enzyme horseradish peroxidase (HRP) to the prodrug indole-3-acetic acid (IAA). These data indicate that the use of such chimeric promoters may effectively regulate therapeutic gene expression within the tumor microenvironment in gene therapy strategies aimed at addressing the problem of hypoxia in radiotherapy.