The identification of molecular pathways in cancer cells is important for understanding the cells' underlying biology and for designing effective cancer therapies. We demonstrate that the expression of aryl hydrocarbon receptor nuclear translocator (ARNT) is critical during the development of cisplatin resistance. The reduced expression of ARNT was correlated with cisplatininduced cell death in drug-sensitive cells. In addition, suppression of ARNT reversed the characteristics of cisplatin-resistant cells, making these cells cisplatin-sensitive, and significantly enhanced caspase-3 activation, DNA fragmentation, and apoptosis. The inhibition of colony formation, regulated by cisplatin, was more significant in ARNT-knockdown cells than in parental cells. In a xenograft analysis of severe combined immunodeficiency mice, cisplatin also efficiently inhibited ARNT-deficient c4 tumors but not ARNT-containing vT2 tumor formation. Furthermore, the downregulation of multidrug resistance 1 (MDR1) expression and retention of drugs in cells caused by suppression of ARNT, resulting in the resensitization of drug-resistant cells to cisplatin, was observed. When overexpressed, ARNT interacted with Sp1 to enhance the expression of MDR1 through Sp1-binding sites on the MDR1 promoter, resulting in a reversal of the effect of cisplatin on cell death. In addition, ARNT-induced MDR1 expression was inhibited in Sp1-knockdown cells. These results reveal previously unrecognized, multifaceted functions of ARNT in establishing the drug-resistant properties of cancer cells by the upregulation of MDR1, highlighting ARNT's potential as a therapeutic target in an important subset of cancers.

The (ARNT) belongs to the basic-helix-loop-helix (bHLH) transcription factors containing Per-Arnt-Sim (PAS) domain. In addition to forming heterodimers with many other bHLH-PAS proteins, including the aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α, ARNT can also form homodimers. Our previous study had shown that ARNT is a new factor involved in EGF regulated COX-2 gene expression under normoxia involving tumorigenesis in cervical cancer tissues. Resistence to cisplatin is often observed in cervical cancer therapy. Here we showed that ARNT expression is critical for cisplatin resistance. Specifically, we showed that in HeLa cervical cancer lines, cisplatin decreased ARNT expression level. Over-expression of ARNT rendered HeLa cells resistant to cisplatin. In addition, inhibition of ARNT by small interfering RNA silencing increased cisplatin induced cell death by activation of caspase 3 and sensitized drug resistant cells to cisplatin. Using ARNT deficient and ARNT fully expressed cell lines showed that induction of apoptosis by cisplatin correlates with ARNT. Importantly down regulation of ARNT decreased drug efflux pump protein MDR1 expression, suggesting that MDR1 gene expression depends on ARNT activity. In conclusion, ARNT mediates the efflux of cisplatin and has a functional role in cervical cancer cell sensitivity to cisplatin. In a xenograft analysis of SCID mice, cisplatin also efficiently inhibited ARNT-deficient tumor formation. These results suggest that targeting ARNT could overcome cisplatin resistance in human cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4228. doi:1538-7445.AM2012-4228