The Apoptosis Repressor with a CARD Domain (ARC) Gene Is a Direct Hypoxia-Inducible Factor 1 Target Gene and Promotes Survival and Proliferation of VHL-Deficient Renal Cancer Cells

Abstract

The induction of hypoxia-inducible factors (HIFs) is essential for the adaptation of tumor cells to a low-oxygen environment. We found that the expression of the apoptosis inhibitor ARC (apoptosis repressor with a CARD domain) was induced by hypoxia in a variety of cancer cell types, and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at bp −190 upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel-Lindau gene (VHL). Accordingly, our results show that ARC is not expressed in normal renal tissue but is highly expressed in 65% of RCC tumors, which also express high levels of carbonic anhydrase IX (CAIX), a HIF1-dependent protein. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.

ACKNOWLEDGMENTS

This work was supported by a gift from the Silicon Valley Community Foundation (A.J.G.) and NCI/NIH grants CA-67166 (A.J.G.), CA-088480 (A.J.G.), and T32 CA121940 (O.V.R.).

We thank Giovambattista Pani (Universitá Cattolica del Sacro Cuore, Rome, Italy) for critical reading of the manuscript, Quynh-Thu Le (Stanford) for anti-CAIX antibodies, Alejandro Sweet-Cordero (Stanford) for A549 cells, Marianne Broome-Powell (Stanford) for A375 cells, Silvestre Vicent (Stanford) for pLKO.1shGFP, Peter Chumakov (Case Western Reserve University, Cleveland, OH) for pLM-CMV-H4-puro-PL3 vector, and Aaron Puri (Stanford) for Z-VAD-FMK caspase inhibitor.