BACKGROUNDSpecific tyrosine kinase receptors such as c‐MET mediate epithelial‐mesenchymal (EMT) transition, leading to phenotypic alterations associated with increased cell motility. It was hypothesized that RON, a tyrosine kinase receptor related to c‐MET, would be expressed in human pancreatic cancer cells, induce EMT, and would thus serve as a target for therapy in a preclinical model.METHODSRON expression in human pancreatic cancer specimens was assessed by immunohistochemistry. In pancreatic cancer cell lines, RON expression was assessed by reverse‐transcriptase polymerase chain reaction (PCR) and Western blot analysis. The human pancreatic cancer cell line L3.6pl, with high RON expression, was exposed to macrophage stimulating protein (MSP), the RON ligand, and assessed for cell migration, invasion, and changes associated with EMT. Western blot analysis and immunofluorescent staining were used to assess alterations in protein expression and cellular location, respectively. A RON monoclonal antibody (MoAb) was used to block ligand‐induced activation of RON.RESULTSImmunohistochemical staining revealed RON overexpression in 93% of human pancreatic cancer specimens relative to nonmalignant ductal tissue. RON mRNA and protein was expressed in 9 of 9 human pancreatic cancer cell lines. Treatment of L3.6pl cells with MSP increased Erk phosphorylation, cell migration, and invasion (P < .001). RON activation led to a decrease in membrane‐bound E‐cadherin in association with nuclear translocation of β‐catenin. RON MoAb inhibited downstream signaling as well as cell migration and invasion. In nude mice, RON MoAb inhibited subcutaneous and orthotopic tumor growth by about 60%.CONCLUSIONSRON activation induced molecular and cellular alterations consistent with EMT. Inhibition of RON activation inhibited tumor growth in vivo. Novel antineoplastic therapies designed to inhibit RON activity may hinder mechanisms critical for pancreatic tumor progression. Cancer 2007 © 2007 American Cancer Society.

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e13515 Background: HER2+ breast cancers (BC) account for 20–25% of invasive BC and are associated with an aggressive phenotype and poor patient outcome. The development of trastuzumab and other HER2-targeted therapy dramatically improved outcomes for HER2-positive BC, but most patients with advanced HER2+ BC will eventually become resistant to treatment, underlying the importance of developing alternative or combination treatments. Alterations in the PI3K/mTOR/Akt pathways are cited as contributors to the development of trastuzumab resistance, however targeting these kinases as single agents has yielded less than expected clinical results. This suggests that combinational treatment with other kinase pathway inhibitors may be required, including those targeting the Ras/MAPK pathway, which is typically not mutationally activated in breast cancer. Methods: Trastuzumab resistant HER2+ BC cell lines were subjected to dose responses with the mTOR inhibitor everolimus or the AKT inhibitor MK-2206 alone or in combination with the MEK 1/2 inhibitor GSK212 and changes in EC50s determined by MTT assay. Western blot analysis was performed to assess changes in the mTOR/AKT/ MAPK pathways or apoptotic regulators accompanying single or combination treatments. Results: In 4 of 5 trastuzumab-resistant HER2+ BC cell lines, each lacking activating mutations in the Ras/Raf pathway, combination treatment with everolimus or MK-2206 with GSK212 had significantly greater efficacy than by either inhibitor alone. Furthermore, combinational treatment targeting the mTOR and AKT pathways showed only an additive effect, and was much less effective than targeting both the MEK and PI3K pathways. Western analysis showed that AKT and mTOR inhibition caused a transient increase in ERK 1/2 activity in sensitive cell lines, suggesting that treatment by mTOR or AKT inhibitors activated critical survival pathways in the MAPK signaling that were blocked by GSK212. Conclusions: Treatment with mTOR or AKT inhibitors in combination with MEK inhibitors can act in a synergistic manner with greater efficacy than each inhibitor alone. Moreover, ERK 1/2 activity may serve as a predictive biomarker in trastuzumab-refractory patients treated with mTOR/MEK or AKT/MEK doublet therapy.

609 Background: Thetargeting of HER2/neu oncoprotein with trastuzumab (Herceptin) has altered the natural history of HER2+ breast cancer, however, its clinical benefit is limited by de novo and/or acquired resistance which almost always develops in the advanced setting. While several mechanisms of resistance have been postulated, none are validated for clinical use to best select patients for treatment. Our study aims were to identify predictive biomarkers based on reproducible differences in HER2 initiated signaling pathway observed in trastuzumab-resistant, HER2-overexpressing human breast cell line models. Methods: Exposing HER2+ breast cancer cells BT474 and SKBR3 to 200 µg/ml of trastuzumab for 12 months, we established two trastuzumab-resistant, HER2 overexpressing breast cancer models BtRT and SkRT. MTT assay was used to characterize drug sensitivities of the cells. Immunocytochemistry and immunoblotting were used to assess the expression levels of HER2 signaling pathway proteins. The impact of trastuzumab on cell cycle process was analyzed by FACS. EdU incorporation was adapted to measure cell proliferation. Results: Trastuzumab-resistant cells had a higher proliferation rate and altered expression levels of HER2 signaling pathway proteins such as p-mTOR, p-S6k1, p-Akt, p-S6, and p-4EBP1, in comparison with parental cell lines. A downstream effector of HER2/Akt/mTOR pathway, phosphorylated ribosomal protein S6 (p-S6), was highly expressed and could not be suppressed by trastuzumab in the resistant cells. When the resistant cells were treated with selected HER2/Akt/mTOR pathway targeted drugs including lapatinib, erlotinib, linsitinib, AZD2014, MK-2206, everolimus and BEZ235, the level of p-S6 in these cells was found to be inversely correlated to the drug induced growth inhibition of these cells. Conclusions: p-S6 is a feasible and easy to measure molecular readout of HER2-targeted therapy resistance. This marker is a good candidate for further clinical validation to predict or efficiently measure a patient’s response to HER2/Akt/mTOR targeted drugs and to test novel agents that would reverse resistance and improve the outcome of trastuzumab refractory, HER2 overexpressing breast cancer.