Purpose: We have investigated mechanisms of acquired resistance to the HER2 antibody trastuzumab in BT-474 human breast cancer cells. Experimental Design: BT-474 xenografts established in athymic nude mice were eliminated by trastuzumab. Continuous cell lines (HR for Herceptin resistant) were generated from tumors that recurred in the presence of continuous antibody therapy. Results:The isolated cells behaved resistant to trastuzumab in culture as well as when reinjected into nude mice. They retained HER2 gene amplification and trastuzumab binding and were exquisitely sensitive to peripheral blood mononuclear cells ex vivo in the presence of the antibody. The HR cells exhibited higher levels of phosphorylated epidermal growth factor receptor (EGFR) and EGFR/HER2 heterodimers. Phosphorylation of HER2 in HR cells was inhibited by the EGFR tyrosine kinase inhibitors erlotinib and gefitinib. Gefitinib also inhibited the basal association of p85 with phosphorylated HER3 in HR cells. Both inhibitors as well as the dual EGFR/HER2 inhibitor, lapatinib, induced apoptosis of the HR cells in culture. Growth of established HR5 xenografts was inhibited by erlotinib in vivo. In addition, the HR cells overexpressed EGFR, transforming growth factor a, heparin-binding EGF, and heregulin RNAs compared with the parental trastuzumabsensitive cells. Conclusions: These results are consistent with the inability of trastuzumab to block the heterodimerization of HER2 and suggest that amplification of ligand-induced activation of ErbB receptors is a plausible mechanism of acquired resistance to trastuzumab that should be investigated in primary mammary cancers.HER2/neu (ErbB2) is a member of the ErbB family of transmembrane receptor tyrosine kinases, which also includes the epidermal growth factor (EGF) receptor (EGFR; ErbB1), HER3 (ErbB3), and HER4 (ErbB4). Binding of ligands to the extracellular domain of EGFR, HER3, and HER4 induces the formation of kinase-active homodimers and heterodimers to which activated HER2 is recruited as a preferred partner (1). Although HER2 does not bind any of the ErbB ligands directly, its catalytic activity can potently amplify signaling by ErbBcontaining heterodimers via increasing ligand binding affinity and/or receptor recycling and stability (2 -5).Amplification of the HER2/neu (ERBB2) gene occurs in f25% of invasive breast cancers and is associated with poor patient outcome (6). Trastuzumab (Herceptin), a humanized monoclonal IgG1 that binds to the ectodomain of HER2, induces clinical responses in HER2-overexpressing breast cancers and prolongs patient survival when combined with chemotherapy (7 -13). The clinical efficacy of trastuzumab seems limited to breast cancers that overexpress HER2 as measured by intense membrane staining in the majority of tumor cells with HER2 antibodies (3+ by immunohistochemistry) or excess copies of the HER2 gene determined by fluorescence in situ hybridization. Therefore, HER2 overexpression by immunohistochemistry and/or fluorescence in situ hybridization is...

Sustained and complete inhibition of HER3 and its output to PI3K/Akt are required for the optimal antitumor effect of therapeutic inhibitors of the HER2 oncogene. Here, we show that, after inhibition of the HER2 tyrosine kinase with lapatinib, there is PI3K/ Akt and FoxO3a-dependent up-regulation of HER3 mRNA and protein. Up-regulated HER3 was then phosphorylated by residual HER2 activity, thus partially maintaining P-Akt and limiting the antitumor action of lapatinib. Inhibition of HER3 with siRNA or a neutralizing HER3 antibody sensitized HER2+ breast cancer cells and xenografts to lapatinib both in vitro and in vivo. Combined blockade of HER2 and HER3 inhibited pharmacodynamic biomarkers of PI3K/Akt activity more effectively than each inhibitor alone. These results suggest that because of HER3-mediated compensation, current clinical inhibitors of HER2 and PI3K/Akt will not block the PI3K pathway completely. They also suggest that therapeutic inhibitors of HER3 should be used in combination with HER2 inhibitors and PI3K pathway inhibitors in patients with HER2-and PI3K-dependent cancers.T he HER (ErbB) transmembrane receptor tyrosine kinase family is comprised of four members: EGF receptor (ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). HER2 is amplified in approximately 25% of human breast cancers (1) and is associated with poor prognosis (2). HER2/HER3 heterodimers are the most transforming of this receptor network (3). HER3, which lacks intrinsic kinase activity (4), is able to potently activate the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway (5) via its six docking sites for the p85 adaptor subunit of PI3K (6). HER2-mediated transformation of mammary epithelial cells has been attributed to a large degree to activation of the PI3K-Akt survival pathway. Trastuzumab, a monoclonal antibody directed against the ectodomain of HER2, and the EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib are approved for the treatment of HER2-overexpressing breast cancer. Although these therapies work by different mechanisms, it has been proposed that, to exert an antitumor effect, they should inhibit phosphorylation of HER3 and disable the PI3K/ Akt pathway (7,8).The HER3 coreceptor plays an essential role in HER2-mediated transformation, tumor progression, and drug resistance. In HER2-dependent cells, loss of HER3 results in reduced signaling through PI3K and cell proliferation (9, 10), suggesting that HER2 may be dependent on HER3 to drive growth and survival of breast cancer cells. As it applies to drug resistance, inhibition of HER2 phosphorylation by TKIs targeting EGFR and HER2 in HER2+ breast cancer cells is followed by feedback upregulation of activated HER3, thus limiting the inhibitory effect of HER TKIs (11,12). These studies point to a central role for HER3 in the survival of HER2+ cells that potentially limit the full action of HER2 antagonists. ResultsInhibition of the HER2 Tyrosine Kinase Is Followed by Up-Regulation of HER3 and P-HER3. We hypothesized that sustained and complete inhibiti...

Although some cancers are initially sensitive to EGFR tyrosine kinase inhibitors (TKIs), resistance invariably develops. We investigated mechanisms of acquired resistance to the EGFR TKI gefitinib by generating gefitinibresistant (GR) A431 squamous cancer cells. In GR cells, gefitinib reduced phosphorylation of EGFR, ErbB-3, and Erk but not Akt. These cells also showed hyperphosphorylation of the IGFI receptor (IGFIR) and constitutive association of IRS-1 with PI3K. Inhibition of IGFIR signaling disrupted the association of IRS-1 with PI3K and restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit GR cell growth. Gene expression analyses revealed that GR cells exhibited markedly reduced IGF-binding protein 3 (IGFBP-3) and IGFBP-4 RNA. Addition of recombinant IGFBP-3 restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit cell growth. Finally, gefitinib treatment of mice with A431 xenografts in combination with an IGFIR-specific monoclonal antibody prevented tumor recurrence, whereas each drug given alone was unable to do so. These data suggest that loss of expression of IGFBPs in tumor cells treated with EGFR TKIs derepresses IGFIR signaling, which in turn mediates resistance to EGFR antagonists. Moreover, combined therapeutic inhibition of EGFR and IGFIR may abrogate this acquired mechanism of drug resistance and is thus worthy of prospective clinical investigation.

We examined the effects of an inhibitor of PI3K, XL147, against human breast cancer cell lines with constitutive PI3K activation. Treatment with XL147 resulted in dose-dependent inhibition of cell growth and levels of pAKT and pS6, signal transducers in the PI3K/AKT/TOR pathway. In HER2-overexpressing cells, inhibition of PI3K was followed by up-regulation of expression and phosphorylation of multiple receptor tyrosine kinases, including HER3. Knockdown of FoxO1 and FoxO3a transcription factors suppressed the induction of HER3, InsR, IGF1R, and FGFR2 mRNAs upon inhibition of PI3K. In HER2 + cells, knockdown of HER3 with siRNA or cotreatment with the HER2 inhibitors trastuzumab or lapatinib enhanced XL147-induced cell death and inhibition of pAKT and pS6. Trastuzumab and lapatinib each synergized with XL147 for inhibition of pAKT and growth of established BT474 xenografts. These data suggest that PI3K antagonists will inhibit AKT and relieve suppression of receptor tyrosine kinase expression and their activity. Relief of this feedback limits the sustained inhibition of the PI3K/AKT pathway and attenuates the response to these agents. As a result, PI3K pathway inhibitors may have limited clinical activity overall if used as single agents. In patients with HER2-overexpressing breast cancer, PI3K inhibitors should be used in combination with HER2/HER3 antagonists.signaling | targeted therapy P I3K transmits signals from ligand-activated receptor tyrosine kinases (RTKs) to intracellular molecules that regulate growth, metabolism, cell size, motility, and survival. In turn, PI3K catalyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate to produce the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3) (1, 2). Several pleckstrin homology domaincontaining proteins, including AKT and PDK1, bind to PIP3 at the plasma membrane. Phosphorylation of AKT at T308 by PDK1 and at S473 by a complex involving mTOR/Rictor (i.e., TORC2) results in the full activation of this enzyme. AKT facilitates survival and cell cycle entry by phosphorylation of proteins including GSK3α/β, FoxO transcription factors, MDM2, BAD, and p27 KIP1 (3). In addition, AKT regulates protein synthesis and cell growth via activation of the mTOR/Raptor (i.e., TORC1) complex (4, 5).PI3K/AKT is arguably the most commonly altered pathway in human cancers (6, 7). Gain-of-function mutations in PIK3CA, the gene encoding the class I A PI3K catalytic subunit p110α, are frequently present in multiple human tumors (8). Second, the PIP3 phosphatase PTEN is a tumor suppressor frequently inactivated by mutation, gene deletion, and promoter methylation (9). Further, PI3K is potently activated by oncogenes like mutant Ras and tyrosine kinases such as Bcr-Abl, HER2 (ErbB2), MET, and KIT, among others (1). Therefore, a large group of tumors with molecular alterations in the PI3K/AKT pathway is therapeutically targetable with PI3K inhibitors.Several PI3K pathway antagonists have been developed. These include ATP mimetics that bind reversibly in the ATP...

We have investigated transforming growth factor beta (TGF-␤)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-␤ via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, ␣-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-␤ induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-␤ induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-␤ induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-␤ induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-␤ control of cell motility, and the loss of this TGF-␤ response is a critical step in the acquisition of metastatic phenotype by tumor cells. INTRODUCTIONThere is solid evidence that the transforming growth factor beta (TGF-␤) signaling pathway is a major cellular growth inhibitory and proapoptotic pathway in epithelial, endothelial, hematopoeitic, and other cell types (Roberts and Wakefield, 2003). However, clinical and experimental studies indicate that metastatic cancers of the breast and other tissues express elevated levels of TGF-␤ that appears to support the metastatic behavior of the tumor cells (Saito et al., 2000;Derynck et al., 2001). This apparent paradox has been associated with a progressive decline in the antitumorigenic function and a gain of protumorigenic activities of TGF-␤, including induction of epithelial to mesenchymal transition (EMT) and tumor cell migration and invasion (Derynck et al., 2001;Wakefield and Roberts, 2002). Oncogenic Ras, Src, and ErbB2 as well as alterations in TGF-␤ signaling mediated by Smads, mitogen-activated protein kinases (Mapks), Rho kinases, and Akt/PKB are thought to contribute to the metastatic phenotype (Derynck and Zhang, 2003;Roberts and Wakefield, 2003).The actin cytoskeleton plays a central role in the regulation of cellular processes linked to metastasis including cell proliferation, apoptosis, anchorage-independent cell growth, and cell migration and invasion (Pawlak and Helfman, 2001;Jaffe and Hall, 2002). TGF-␤ induces a rapid reorganization of the actin cytoskeleton, leading to membrane ruffling at the cell edges in both nontumorigenic and tumorigenic epithelial cells, whereas a prolonged incubation with TGF-␤ results in the formation of stress fibers (Bakin et al., 2002;Edlund et a...