Finally, we have generated gastric cells resistant to a MET specific inhibitor Lapatinib IC50 and, upon ruling out the presence of MET gene amplification or mutations in either MET itself or other downstream signalling molecules such as RAS, Raf or PI3K, we found that the levels of HER2 and HER3 were significantly increased in these resistant cells. Moreover, HER3 silencing led to reversion of the resis tance to MET inhibitors and to decreased cell viability. These data suggest that a molecular mechanism exploited by addicted cells to overcome the pro apoptotic effect of MET inhibition may be the increased expression of HER family members, enhancing the sensibility to their cog nate growth factors, which Inhibitors,Modulators,Libraries are usually available in the tumour microenvironment.

Conclusions In our work we studied the molecular mechanisms that could Inhibitors,Modulators,Libraries cause resistance to therapies targeting MET in gas tric cancer. Altogether our data suggest that even in the cellular contexts that are more likely to respond Inhibitors,Modulators,Libraries to treat ment with MET inhibitors, activation of HER family receptors which is rather frequent in gastric tumors can impair the biological response to treatment and can con cur to the appearance of resistance. This should be taken in consideration in light of using new drugs or new asso ciation schemes that could concomitantly inhibit both these receptors and act synergistically. Methods Cell culture and compounds SNU 5, NCI H1993 cell lines were from ATCC, EBC 1 from JCRB. GTL16 cells were described in. EGF was from Sigma, HRG1 B1 and IGF 1 from R D. MSP was produced as in.

LY294002 was from Calbiochem, U0126 from Promega, PHA 665752 from Tocris Bioscience, Gefitinib from Sequoia Research Prod uct. The EGFR L858R vector was kindly provided by Dr. Yarden. TGF was cloned in p156RRLsin. PPTh CMV. MCS. pre. The MET shRNA was described in, the HER2 shRNA in . the HER3 siRNA Inhibitors,Modulators,Libraries was from Sigma. Virus preparation, cell transduction and electoporation Lentiviruses were produced as in. Cells were trans duced using 40 ng/ml of p24. Electroporation was per formed with siRNAs 2 nM using the Cell line Nucleofector Inhibitors,Modulators,Libraries Kit V and Nucleofector II machine. Western blot analysis Cells were starved in serum free medium for 24 hours and then treated with EGF or HRG1 B1 for 10 minutes. Cells were lysed in LB buffer. Primary antibodies anti phospho Tyrosine . anti Actin, anti HER3 and anti HER2 . anti vinculin .

anti MET DL21 . anti phospho MET Tyr1234/ 1235 . anti AKT, anti phos pho AKT, anti p42/44 MAPK and anti phos pho p42/44 MAPK Thr202/Thr204. Secondary antibodies were from Amer sham. Detection was performed with ECL system. Biological assays Growth curves experiments were performed as in . viability was evaluated on day. 4, as in. Growth in soft agar was GSI-IX performed as in, and was quantified with the Alamar Blue indicator dye. Measurements were recorded using a DTX 880 Multi mode plate reader. Real Time PCR analysis Total RNAs were extracted using TriReagent lysis buffer.