Cancer Therapy: Preclinical Inhibition of Mammalian Target of Rapamycin Is Required for Optimal Antitumor Effect of HER2 Inhibitors against HER2-Overexpressing Cancer Cells

Purpose: A significant fraction of HER2-overexpressing breast cancers exhibit resistance to the HER2 antibody trastuzumab. Hyperactivity of the phosphatidylinositol 3kinase (PI3K)/AKT pathway confers trastuzumab resistance, and mammalian target of rapamycin (mTOR) is a major downstream effector of PI3K/AKT. Therefore, we examined whether mTOR inhibitors synergize with trastuzumab. Experimental Design: Immunocompetent mice bearing HER2 mammary tumors were treated with trastuzumab, the mTOR inhibitor rapamycin, or the combination. Mice were imaged for tumor cell death using an optical Annexin-V probe and with [F] FDG positron emission tomography. The signaling and growth effects of the mTOR inhibitor RAD001 on HER2 cells treated with trastuzumab or lapatinib were evaluated. Results: Treatment of mice with trastuzumab plus rapamycin was more effective than single-agent treatments, inducing complete regression of 26 of 26 tumors. The combination induced tumor cell death (Annexin-V binding) and inhibited FDG uptake. Rapamycin inhibited mTOR and tumor cell proliferation as determined by phosphorylated S6 and Ki-67 immunohistochemistry, respectively. In culture, the combination of RAD001 plus trastuzumab inhibited cell growth more effectively than either drug alone. Trastuzumab partially decreased PI3K but not mTOR activity. Knockdown of TSC2 resulted in HER2-independent activation of mTOR and dampened the response to trastuzumab and lapatinib. Treatment with the HER2 inhibitor lapatinib decreased phosphorylated S6 and growth in TSC2-expressing cells but not in TSC2-knockdown cells. Conclusions: Inhibition of PI3K and mTOR are required for the growth-inhibitory effect of HER2 antagonists. These findings collectively support the combined use of trastuzumab and mTOR inhibitors for the treatment of HER2 breast cancer. (Clin Cancer Res 2009;15(23):7266–76) Amplification and/or overexpression of the HER2/erbB2 oncogene occurs in 20% to 30% of human breast cancers and is associated with poor prognosis (1, 2). The humanized monoclonal antibody trastuzumab (Herceptin), which binds the extracellular region of human HER2 (3), is approved for the treatment of HER2 breast cancer. Adjuvant trastuzumab therapy significantly improves patient outcome overall. However, in the metastatic setting, only a fraction of patients with HER2 disease responded to trastuzumab therapy (4–8), implying that many advanced cancers are trastuzumab-resistant. HER2 breast tumors showed increased caspase-3 activation following 1 week of neoadjuvant trastuzumab treatment (9), indicative of Authors' Affiliations: Departments of Medicine, Radiology and Radiological Sciences, Biomedical Engineering, Neurosurgery, Pathology, Cancer Biology, and Chemistry, Program in Chemical and Physical Biology, Vanderbilt University Institute of Imaging Science, and Breast Cancer Research Program, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University, Nashville, Tennessee Received 6/28/09; revised 9/14/09; accepted 9/27/09; published OnlineFirst