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 3-kinase (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 [(18)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.

Conditional overexpression of active transforming growth factor beta1 in vivo accelerates metastases of transgenic mammary tumors.

To address the role of transforming growth factor (TGF) beta in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-beta on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-beta1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-beta1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-beta1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-beta and increased metastases. Antisense-mediated inhibition of TGF-beta1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-beta in hosts with established TGF-beta-responsive cancers can rapidly accelerate metastatic progression.

Early changes in protein expression detected by mass spectrometry predict tumor response to molecular therapeutics.

Biomarkers that predict therapeutic response are essential for the development of anticancer therapies. We have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to directly analyze protein profiles in mouse mammary tumor virus/HER2 transgenic mouse frozen tumor sections after treatment with the erbB receptor inhibitors OSI-774 and Herceptin. Inhibition of tumor cell proliferation and induction of apoptosis and tumor reduction were predicted by a >80% reduction in thymosin beta4 and ubiquitin levels that were detectable after 16 hours of a single drug dose before any evidence of in situ cellular activity. These effects were time- and dose-dependent, and their spatial distribution in the tumor correlated with that of the small-molecule inhibitor OSI-774. In addition, they predicted for therapeutic synergy of OSI-774 and Herceptin as well as for drug resistance. These results suggest that drug-induced early proteomic changes as measured by MALDI-MS can be used to predict the therapeutic response to established and novel therapies.