13PAXL as a potential primary and secondary trastuzumab resistance mechanism in breast cancer cells with HER2 overexpression

Background: Breast cancer (BC) is a heterogeneous disease, HER2+ represents between 15-30% of all subtypes. Trastuzumab (T), a monoclonal antibody able to inhibit HER2 activation, has been successfully employed in HER2 amplified tumors both in adjuvant and in metastatic settings, conferring an improvement in DFS, PFS and OS. Despite these results, many patients experience primary or secondary resistance to therapy. The mechanism of resistance is unclear, our aim is to assess AXL, a receptor tyrosine kinases (RTK) implicated in epithelial-to-mesenchymal transition, as potential mechanisms of resistance. Methods: We used two cell lines to investigate possible mechanisms of primary and secondary resistance to T in HER2+ and hormone receptor negative BC. AU565 sensitive to T (AU565-S), and HCC1954 a primary T-resistant cell line. A third cell line with acquired resistance to T (AU565-R) was generated by treating AU565-S cells with constant dose of T (15mg/mL) for 4 months. Cell viability was estimated by MTT assay. We explored the expression of AXL by Western blot (WB) and quantitative reverse transcriptase PCR (qRT-PCR). Results: The cell viability analysis at 7 days assay confirmed AU565-S as sensible to T, HCC1954 as primary resistant and the development of a secondary resistance to T (AU565-R) (50% of increased viability from AU565-S). HER2 overexpression in all three cells lines were confirmed by WB and FISH. qRT-PCR indicated an important up-regulation of AXL at mRNA levels in AU565-R and HCC1954 compared to AU565-S (p < 0.05). In the same line, WB analyses showed a significantly increase in AXL protein expression in AU565-R and HCC1954 (2.03 and 7.37 fold, respectively). Finally, a selective AXL inhibitor (TP-0903) has demonstrated reduction of viability in all cell lines and significant restoration of sensitivity to T in AU565-R (p < 0.01). Conclusions: Our results suggest: 1) AXL could be a potential mechanism of both primary and secondary resistance to T; 2) combination therapy with AXL inhibitor plus T restored T sensitivity in in vitro model with AXL overexpressed. These results merit further study and to explore this RTK as possible therapeutic targets in case of anti-HER2 treatment failure. Legal entity responsible for the study: Hospital Clinico Universitario of Valencia. Biomedical Research Institute INCLIVA Funding: None Disclosure: J.A. Perez Fidalgo: Received fees from AstraZeneca, Ipsen, Novartis, Pfizer and Roche for participation in speaker bureaus. Had travel/accomodation expenses paid/reimbursed by AstraZeneca, Roche and Sandoz. All other authors have declared no conflicts of interest.