Inhibition of EGFR-AKT Axis Results in the Suppression of Ovarian Tumors In Vitro and in Preclinical Mouse Model Sivakumar Loganathan1., Prabodh K. Kandala2., Parul Gupta2, Sanjay K. Srivastava1,2* 1 Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh, Pennsylvania, United States of America, 2 Department of Biomedical Sciences and Cancer Biology Center, Texas Tech University Health Sciences Center, Amarillo, Texas, United States of America Abstract Ovarian cancer is the leading cause of cancer related deaths in women. Genetic alterations including overexpression of EGFR play a crucial role in ovarian carcinogenesis. Here we evaluated the effect of phenethyl isothiocyanate (PEITC) in ovarian tumor cells in vitro and in vivo. Oral administration of 12 mmol PEITC resulted in drastically suppressing ovarian tumor growth in a preclinical mouse model. Our in vitro studies demonstrated that PEITC suppress the growth of SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells by inducing apoptosis in a concentration-dependent manner. Growth inhibitory effects of PEITC were mediated by inhibition of EGFR and AKT, which are known to be overexpressed in ovarian tumors. PEITC treatment caused significant down regulation of constitutive protein levels as well as phosphorylation of EGFR at Tyr1068 in various ovarian cancer cells. In addition, PEITC treatment drastically reduced the phosphorylation of AKT which is downstream to EGFR and disrupted mTOR signaling. PEITC treatment also inhibited the kinase activity of AKT as observed by the down regulation of p-GSK in OVCAR-3 and TOV-21G cells. AKT overexpression or TGF treatment blocked PEITC induced apoptosis in ovarian cancer cells. These results suggest that PEITC targets EGFR/AKT pathway in our model. In conclusion, our study suggests that PEITC could be used alone or in combination with other therapeutic agents to treat ovarian cancer. Citation: Loganathan S, Kandala PK, Gupta P, Srivastava SK (2012) Inhibition of EGFR-AKT Axis Results in the Suppression of Ovarian Tumors In Vitro and in Preclinical Mouse Model. PLoS ONE 7(8): e43577. doi:10.1371/journal.pone.0043577 Editor: Joseph Alan Bauer, Bauer Research Foundation, United States of America Received January 13, 2012; Accepted July 26, 2012; Published August 27, 2012 Copyright: 2012 Loganathan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported in part by R01 grants CA106953 and CA129038 (to S.K.S.) awarded by the National Cancer Institute. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sanjay.srivastava@ttuhsc.edu . These authors contributed equally to this work. Introduction Ovarian cancer is one of the leading causes of gynecologic cancer-related deaths among women in western countries [1]. The cause of ovarian cancer is not clear and it is often detected at an advanced stage. The overall prognosis of ovarian cancer is very poor despite significant advances in surgical and therapeutic management [2]. The current standard of care includes cytoreduction followed by cytotoxic chemotherapy. However, recurrence remains a significant problem [3]. The most common form of ovarian cancer arises from ovarian surface epithelium. Epidermal growth factor receptor (EGFR) is commonly expressed in ovarian surface epithelium [4]. Activation of various tyrosine kinases including EGFR is important in ovarian cancer pathogenesis. Approximately 70% of ovarian tumors express activated EGFR [5]. EGFR is a trans- membrane receptor whose activation is a highly conserved process. Various ligands such as EGF and TGF activate EGFR. EGFR plays a significant role in neural development and formation of skin. However, in cancer cells, EGFR is involved in various pro-survival and anti-apoptotic pathways [6–8]. Further- more, EGFR is also involved in cell migration, metastasis, angiogenesis and EMT [9–11]. One of the major downstream pathways that are regulated by EGFR is AKT. Activation of EGFR leads to the activation of AKT by its phosphorylation at Ser-473 [12,13]. AKT is frequently activated or overexpressed in ovarian tumors [14,15] and plays a major role in ovarian carcinogenesis. The overexpression of AKT is frequently associated with poor prognosis and more aggressive phenotype. Like EGFR, AKT also plays a major role in angiogenesis, metastasis and anti-apoptosis. Since EGFR and AKT are involved in various aspects of cancer growth ranging from tumor initiation, angiogenesis, and metastasis, EGFR-AKT axis represents an attractive target for therapeutic intervention. PEITC is a major isothiocyanate present in cruciferous vegetables [16]. Accumulating epidemiological evidence indicates an inverse relationship between intake of cruciferous vegetables and the risk of ovarian cancer [17,18]. Several studies, including those from our laboratory suggested that various isothiocyanates possess chemo- preventive and therapeutic properties [16,19,20]. PEITC in particular was shown to be effective against prostate, cervical and lung cancers [21–23]. Interestingly, PEITC is in clinical trials for lung cancer. In the present study, we investigated the mechanism by which PEITC inhibits the proliferation of ovarian cancer cells and evaluated its efficacy in vivo in a tumor xenograft model. Materials and Methods Chemicals Antibodies against Cl-caspase3, Cl-PARP, p-EGFR (Tyr-1068), EGFR, p-AKT (Ser-473), p- mTOR (Ser 2481), Raptor and AKT PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43577