Deficiency Overcoming Trastuzumab Resistance Caused by PTEN Preclinical Testing of Clinically Applicable Strategies for Updated

Purpose:We have previously shown that PTEN loss confers trastuzumab resistance in ErbB2overexpressing breast cancer using cell culture, xenograft models, and patient samples. This is a critical clinical problem because trastuzumab is used in a variety of therapeutic regimens, and at the current time, there are no established clinical strategies to overcome trastuzumab resistance. Here, we did preclinical studies on the efficacy of clinically applicable inhibitors of the Akt/ mammalian target of rapamycin (mTOR) pathway to restore trastuzumab sensitivity to PTENdeficient cells. Experimental Design: Cell culture and xenograft models were used to test a panel of clinically applicable, small-molecule inhibitors of the Akt/mTOR signal transduction pathway, a critical pathway downstream of ErbB2, and identify compounds with the ability to restore trastuzumab sensitivity to PTEN-deficient cells. Results:When trastuzumab was combined with the Akt inhibitor triciribine, breast cancer cell growthwas inhibited andapoptosis was induced. In a xenograftmodel, combination therapywith trastuzumab and triciribine dramatically inhibited tumor growth.The combination of trastuzumab and the mTOR inhibitor RAD001also slowed breast cancer cell growth in vitro and in vivo. Conclusions: Combining trastuzumab with inhibitors of the Akt/mTOR pathway is a clinically applicable strategy and combinations of trastuzumab with triciribine or RAD001are promising regimens for rescue of trastuzumab resistance caused by PTEN loss. Breast cancer patients whose tumors overexpress the ErbB2 receptor tyrosine kinase have poor overall survival and shorter time to relapse (1). ErbB2 (HER2, Neu) activates the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, which promotes proliferation and invasion and inhibits apoptosis. Trastuzumab (Herceptin; Genentech) is a humanized monoclonal antibody that targets ErbB2. When given with adjuvant chemotherapy, trastuzumab significantly improves disease-free survival following surgical removal of ErbB2-positive breast tumors (2, 3). However, the overall response to trastuzumab is limited. Less than 30% of patients with ErbB2-positive tumors have a durable response to trastuzumab as a single agent and 2% to 5% will suffer from clinical cardiac dysfunction as a side effect (4, 5). We previously investigated the mechanisms of trastuzumab resistance and discovered an important novel mechanism. Trastuzumab treatment increased PTEN phosphatase activity and PTEN was required for the antitumor activity of trastuzumab (6). Moreover, PTEN loss predicted poor clinical response to trastuzumab-based chemotherapy in patients (6). Because PTEN opposes the actions of PI3K, we proposed a strategy to overcome trastuzumab resistance using PI3K inhibitors. We showed a synergistic inhibition of tumor growth following combination treatment of trastuzumab and wortmannin or LY294002 in vitro and in vivo (6). However, these drugs have poor clinical potential and are unlikely to become available to clinical oncologists. Both act nonspecifically on other PI3K family members (7). Wortmannin has a short half-life (8) and LY294002 causes an untoward patchy dermatitis in animal models (9). Thus, they have not been used in clinical trials despite antitumor activities in vitro and in vivo (10). In the current study, we focused on compounds with clinical potential, limiting our study to inhibitors that were either in clinical trials, under development for clinical trials, or previously tested in patients. We obtained six small-molecule inhibitors of the PI3K pathway and examined their preclinical Cancer Therapy: Preclinical Authors’ Affiliations: Departments of Molecular and Cellular Oncology, MolecularTherapeutics, and Breast Medical Oncology,The University of Texas M. D. Anderson Cancer Center, Houston,Texas Received11/29/06; revised1/12/07; accepted 2/15/07. Grant support:NIHgrants P30-CA16672 (M.D. AndersonCancer Center),1RO1CA109570, 1RO1-CA119127, PO1-CA099031project 4, and P50 CA116199 01 project 4 (D.Yu) and Department of Defense Center of Excellence grant DAMD1702-1-0694 (G.B. Mills and G.N. Hortobagyi). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). C-H. Lu and S.L. Wyszomierski contributed equally to this work. Current address for C-H. Lu: Department of Obstetrics and Gynecology,Taichung Veterans General Hospital, Taichung, Taiwan. Current address for L-M. Tseng: Department of Surgery, Taipei Veterans General Hospital and National Yang-Min University,Taipei,Taiwan. Current address for K.H. Lan: Department of Oncology, NationalTaiwan University Hospital,Taipei,Taiwan. Requests for reprints:DihuaYu, Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Unit 108, 1515 Holcombe Boulevard,Houston,TX77030. Phone: 713-792-3636; E-mail: dyu@mdanderson.org. F2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-2837 www.aacrjournals. org Clin Cancer Res 2007;13(19) October1, 2007 5883 Research. on July 3, 2013. © 2007 American Association for Cancer clincancerres.aacrjournals. org Downloaded from efficacy in combination with trastuzumab for treating PTENdeficient breast tumors. We identified two compounds with the ability to rescue trastuzumab sensitivity in PTEN-deficient cells. Materials andMethods Cell lines and cell cultures. The tumorigenic BT474.m1 subline was a gift from Dr. Dajun Yang (Georgetown University, Washington, DC). It was maintained in DMEM/Ham’s F-12 (1:1) with 8% to 10% fetal