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One arising challenge in the treatment of breast cancer is the development of therapeutic resistance to trastuzumab, an antibody targeting the human epidermal growth factor receptor-2 (HER2), which is frequently amplified in breast cancers. In this study, we provide evidence that elevated level of the receptor tyrosine kinase Eph receptor A2 (EphA2) is an important contributor to trastuzumab resistance. In a screen of a large cohort of human breast cancers, we found that EphA2 overexpression correlated with a decrease in disease-free and overall survival of HER2-overexpressing patients. Trastuzumab-resistant cell lines overexpressed EphA2, whereas inhibiting EphA2 restored sensitivity to trastuzumab treatment in vivo. Notably, trastuzumab treatment could promote EphA2 phosphorylation by activating Src kinase, leading in turn to an amplification of phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signaling in resistant cells. Our findings offer mechanistic insights into the basis for trastuzumab resistance and rationalize strategies to target EphA2 as a tactic to reverse trastuzumab resistance. Cancer Res; 70(1); 299–308.

Heregulin-mediated activation of HER4 initiates receptor cleavage (releasing an 80-kDa HER4 intracellular domain, s80HER4, containing nuclear localization sequences) and results in G2-M delay by unknown signaling mechanisms. We report herein that s80HER4 contains a functional cyclin B–like sequence known as a D-box, which targets proteins for degradation by anaphase-promoting complex (APC)/cyclosome, a multisubunit ubiquitin ligase. s80HER4 ubiquitination and proteasomal degradation occurred during mitosis but not during S phase. Inhibition of an APC subunit (APC2) using short interfering RNA knockdown impaired s80HER4 degradation. Mutation of the s80HER4 D-box sequence stabilized s80HER4 during mitosis, and s80HER4-dependent growth inhibition via G2-M delay was significantly greater with the D-box mutant. Polyomavirus middle T antigen–transformed HC11 cells expressing s80HER4 resulted in smaller, less proliferative, more differentiated tumors in vivo than those expressing kinase-dead s80HER4 or the empty vector. Cells expressing s80HER4 with a disrupted D-box did not form tumors, instead forming differentiated ductal structures. These results suggest that cell cycle–dependent degradation of s80HER4 limits its growth-inhibitory action, and stabilization of s80HER4 enhances tumor suppression, thus providing a link between HER4-mediated growth inhibition and cell cycle control. [Cancer Res 2007;67(14):6582–90]

To address the role of transforming growth factor (TGF) β in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-β on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-β1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-β1 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-β1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-β and increased metastases. Antisense-mediated inhibition of TGF-β1 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-β in hosts with established TGF-β-responsive cancers can rapidly accelerate metastatic progression.