Abstract 2765: Cytoplasmic expression of the aryl hydrocarbon receptor elicits sensitivity and nuclear expression resistance to aminoflavone prodrug
Authors: Stark Karri , Nguyen Binh , Shelton Phillip , Polin Lisa , LoRusso Patricia M. , Burger Angelika M. 
Published: February 2011

Aminoflavone Prodrug (AFP464) is a novel anticancer agent currently in Phase I clinical trials in the US and Europe. Its target is the aryl hydrocarbon receptor (AhR). Upon binding to AhR in the cytoplasm, the receptor/AFP464 complex dimerizes with the AhR nuclear translocator and is shuttled into the nucleus where it induces xenobiotic response via the cytochrome P (CYP) system. CYP1A1 is critical to the activity of AFP464 in cells as it converts the drug into metabolites that cause DNA double strand breaks and cell death. Previous screening of AFP464 in tumor cell lines in vitro has revealed differential cytotoxicity with some lines being sensitive at nanomolar concentrations, while others were resistant even at &gt;100μM. Here, we have investigated the cause of intrinsic resistance of tumor cells to AFP464. We used a panel of 15 breast, ovarian and pancreas cancer cell lines and compared their sensitivity to AFP464 with the cellular localization of the AhR. Five day MTT assays were used to determine in vitro antiproliferative activity and nude mouse xenografts to assess in vivo efficacy. Immunofluorescence and immunoperoxidase staining of fixed tumor cells and archival tissue were employed to analyze AhR expression. Five breast cancer cell lines (MCF-7, T47D, MDA-MB-468, MCF-7TAM1, MCF-7Her2-18), 2 ovarian (OVCAR-3, IGROV-1), and 2 pancreatic (ASPC-1 and Hs766T) lines exhibited cytoplasmic AhR; their inhibitory concentration 50% (IC50) ranged from 20nM (MDA-MB-468) to 10μM (Hs766T). The IC50s for breast (MDA-MB-231, Hs578T), ovarian (OVCAR-8) and pancreatic (PANC-1, MIAPaCa2, Capan-1) cell lines with nuclear AhR were between 13μM (OVCAR-8) and &gt;100μM (pancreatic lines). These in vitro results translated into in vivo efficacy: MDA-MB-468 xenografts responded to single agent AFP464 at a dose of 35 mg/kg (given i.v. 4 cycles x [q2dx3]) with 62% growth inhibition compared to vehicle control, while MDA-MB-231 xenografts were resistant to AFP464, even at the maximal tolerated dose of 70mg/kg. To test whether primary human tumors would also show nuclear or cytoplasmic AhR and to assess the extent to which AhR was expressed, we analyzed 165 archival human tissues comprising breast, pancreas, ovarian and renal cell cancers. We found the 59% of all cases had detectable AhR, amongst those 78% exhibited cytoplasmic AhR and 22% nuclear AhR. Pancreatic (70%) and breast cancers (46%) showed the highest percentage of cytoplasmic AhR.

Together our data indicate that AhR has a distinct distribution pattern in tumor cells. Cytoplasmic AhR expression elicits sensitivity to the AhR ligand AFP464. If AhR is located in the nucleus, xenobiotic response is impaired and AFP464 cannot be activated. Thus, immunohistological analysis of AhR should be considered as a tool in the upcoming Phase II trials in breast and pancreatic cancer to select patients that are most likely to benefit from AFP464 treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2765.