Bhinge, Kaustubh N.

A combinatorial cell microarray platform to map the impact of both extracellular matrix proteins and genotype on the drug responses of tumor cells.

Abstract Lung cancer accounts for about 27% of the cancer related deaths in the USA annually. Pathologically, it is a very complex disease broadly classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLCs are further classified into squamous cell carcinoma, large cell carcinoma and adenocarcinoma. Achaete-scute homolog 1 (ASCL1), an important transcription factor essential in the development of neuroendocrine cells (NE) in lungs is shown to be specifically expressed in lung NE cancers and 10-20% of adenocarcinomas (AD) with NE differentiation (NED), thus suggesting a role in the pathogenesis of these tumors. Our previous study showed that ASCL1 is a regulator of the RET oncogene in AD with NED. RET is a receptor tyrosine kinase with two isoforms in humans: RET9 (short) and RET51 (long). We performed survival analysis to study implications of RET isoforms in ASCL1+ tumors and found that elevated expression of the long RET mRNA was associated with poor survival. Subsequent in vitro experiments demonstrated that treatment with EGF robustly induced phosphorylation of RET in HCC1833 and H1755 cell lines which have high endogenous levels of ASCL1 and RET but not in VMRC-LCD cell line which has high level of ASCL1 but low level of RET. EGF induced phosphorylation of RET was diminished by gefitinib and by EGFR siRNA. Immunoprecipitation results indicated direct binding between EGFR and RET in presence of EGF. Furthermore, a high throughput drug screening found 8 EGFR inhibitors that were 10 - 250 fold more cytotoxic in ASCL1+ compared with ASCL1- AD cells. These results implicate EGFR as a key regulator of RET activation in ASCL1+ AD and suggest that EGFR inhibitors may be therapeutic for this population of patients. Citation Format: Kaustubh N. Bhinge, Yang Lin, Hamed Rahi, Marie Christine Aubry, Aaron Mansfield, Irina Kovtun, Stephen Murphy, Ping Yang, Dennis Wigle, Joanne (Eunhee) Yi, Aqsa Nasir, Simone Terra, Julian Molina, George Vasmatzis, Farhad Kosari. EGFR-mediated activation of RET in ASCL1+ lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1129.

Abstract ERRFI1, a negative regulator of the EGFR family, is known to inhibit EGFR activation along with downstream of EGFR signaling. It is recognized as a tumor suppressor. However, ERRFI1 has multiple functions, and here, we present a novel function of ERRFI1. We found that it can directly interact with AKT and positively regulate AKT activity through regulation of AKT473 site by inhibiting PHLPP, the phosphatase access to AKT. The effect of this regulation on cell proliferation and on response to chemotherapy is cell context dependent, e.g. EGFR levels. We hypothesize that in cancer cells with high EGFR expression, down regulation of ERRFI1 enhances EGFR signaling, while on the other hand, increases AKT dephosphorylation. The strength of these two opposing signals will determine the eventual cellular phenotypes. In cancer cells with low EGFR expression, the direct effect of ERRFI1 on AKT is dominant, resulting in activation of AKT. To test these hypotheses, we knockdown ERRFI1 in multiple human cancer cell lines with different EGFR levels. Knockdown of ERRFI1 in high EGFR expressing cells resulted in an increase of cell proliferation and resistance to gemcitabine treatment, and this effect was due to the dominant negative effect of ERRFI1on EGFR, which was confirmed by increased EGFR signaling. Knockdown of ERRFI1 in cell lines with moderate level of EGFR also increased cell proliferation and resistance to gemcitabine, although biochemically, down regulation of ERRFI1 reduced AKT pS473 levels, but increased ERK/MAPK signaling. The addition of EGFR inhibitor, gefitinib, overcame gemcitabine resistance due to downregualtion of ERRFI1. In contrary, in cell lines with low level of EGFR, ERRFI1 positively regulated AKT signaling via directly binding to AKT and blocking PHLPP access to AKT. Therefore, down-regulation of ERRFI1 inhibited cell proliferation and sensitized cells to gemcitabine. In summary, we identified a novel function of ERRFI1 as a positive AKT regulator and its effect on cell proliferation and response to therapy is cell context dependent, e.g. EGFR levels. These results provide us insights into the dual roles of ERRFI1 in regulation of EGFR and AKT pathway and would help us to better individualize cancer therapy. Citation Format: Junmei Hou, Kaustubh N. Bhinge, Liewei Wang. The effect of ERRFI1 as a novel AKT regulator on cell proliferation and response to therapy is cell context dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2782. doi:10.1158/1538-7445.AM2014-2782