Regulation of PITX2 and ABCB1 in oncogenic multidrug resistance by tumorigenic HIF2 in VHL‐deficient renal cancer cells

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The tumour suppressor von Hippel‐Lindau (VHL) is involved in ubiquitination and subsequent proteasomal degradation of hypoxia‐inducible factors (HIFs). Under hypoxic conditions, HIFα‐subunits are stabilized, leading to dimerization with HIFβ and transactivation of genes for metabolism, survival and angiogenesis. Human renal cancer cells A498 are VHL‐deficient and harbor stabilized HIF‐2, whereas Caki‐1 cells are VHL‐intact and HIF‐1‐dominant. Previous studies demonstrated increased paired‐like homeodomain transcription factor 2 (PITX2) and its target gene, the ABCB1 drug transporter, in A498 cells. The relationship between VHL, HIF, PITX2 and ABCB1 remains unclear.Transient transfection of A498 cells with HIF‐1α or HIF‐2α siRNA (25 nM, 72 h, >85% protein knockdown) attenuated PITX2 protein by up to 75% by immunoblotting and PITX2 promoter activity by ~40% in a luciferase reporter gene assay. Immunoprecipitation studies did not propose direct HIF‐PITX2 protein‐protein interaction. HIF‐regulated PITX2 expression was proportional to ABCB1 expression, which was decreased at both mRNA (~20%) and protein (~70%) levels after HIF‐1α or HIF‐2α knockdown. Furthermore, HIF/PITX2 target gene CCND1 was reduced by >60%, but PITX2 targets LCN2 and WNT4 were increased >3‐fold following transient HIF downregulation. HIF‐2α siRNA was generally more effective than HIF‐1α siRNA in altering PITX2 and its target genes. Conversely, in Caki‐1 cells, HIF siRNA augmented PITX2 and ABCB1 mRNA and protein by ~1.5‐fold and ~2‐fold, respectively, with no significant changes in CCND1, LCN2 and WNT4. Polysome profiling show HIF‐2α siRNA negatively impacts active translation of PITX2 and ABCB1 mRNA. Finally, HIF2 knockdown sensitizes A498 cells to cell death by the chemotherapeutic drug and ABCB1 substrate, doxorubicin (1 µM, 24 h), as determined by trypan blue staining.In summary, these findings suggest PITX2 and ABCB1 are positively regulated by tumorigenic HIF2 in drug‐resistant A498 cells whereas they are suppressed by HIF‐1 in drug‐sensitive Caki‐1 cells. These data may provide better understanding of membrane transporters and possible upstream causes of multidrug resistance in different renal cancers.