PO-500 NRF2 represents a convergent point of acquired resistance in HER2 positive gastric cancer models

Introduction Prognosis of patients diagnosed with advanced gastric (GC) cancer is still poor. Personalised medicine for gastric cancer patients still represents a big challenge. HER2 is the only actionable target and only trastuzumab (T) is able to improve median overall survival in selected patients. Lapatinib (L), when used in I or in II line, was not able to achieve a clinical benefit. Nevertheless, acquired resistance is an inevitable event. Material and methods OE 19 and NCI N87, HER2 positive GC cell lines, were continuously treated with increasing doses of L and T to obtain resistant clones. Resistance was confirmed by MTT viability assays. Genome-wide expression profiles of OE 19 and derived clones was evaluated by using Clariom S microarray from Affymetrix. Data were analysed using Partek Genomics Suite v6.6 software and Pathway Studio v10 (Elsevier). Protein expression was analysed by immunofluorescence (IF) and, after subcellular fractionation, by Western Blot. siRNA of NRF2 was performed. mRNA expression was performed by qRT PCR Results and discussions L-resistant clones were obtained Resistance was confirmed by MTT analysis. Data obtained by microarray were analysed by principal component analysis to determine the significant sources of variability in the data sets. Gene expression changes are clearly observed between two resistant clones versus parental line. Impressively, 132 genes were significant differentially expressed among resistant clones versus parental line. Unsupervised hierarchical clustering of 132 genes revealed a robust classification between three different groups. When analysed in details, it was possible to identify a large number of genes regulated by NFR2, a master transcriptional regulator that activates genes involved in oxidative stress response, detoxification, and drug resistance. NRF2 expression was evaluated by Western Blot analysis among both L and T resistant cells. After subcellular fractionation, it was possible to observe a nuclear overexpression among resistant cell lines, this data was confirmed by IF. When siRNA of NRF2 was performed, a decrease of cell growth among resistant cell lines was observed. When treatment with L was administered in knockdown cells, it was possible to restore sensitivity. Conclusion NRF2 is identified as a new mechanism of resistance to antiHER2 inhibition in GC. The evaluation of its expression among xenograft models and patients, who experienced a disease progression, are ongoing.