EXPLORING NEW THERAPEUTIC AVENUES AGAINST HER2-POSITIVE BREAST CANCER

Abstract

HER2-positive breast cancer (BC) is a challenging disease characterized by poor prognosis, relapses, and aggressive behavior in the tumor. HER2 oncoprotein dimerization with other growth factor receptor family members (EGFR, HER2, HER3, and IGF-1R) is the crucial driver of cancer cell growth. Thus, targeting HER2 and its family members is an effective strategy to tackle this disease. However, currently available treatments face several limitations, mainly acquired resistance and severe side effects. In this study, we explored a novel treatment strategy to target HER2-positive BC using a combination of neratinib (NER) and metformin (MET). We used a panel of HER2-positive BC cell lines in addition to a non-cancerous control. We investigated the effect of NER and MET combination on cell morphology, cell viability, colony formation, cell migration, cell invasion, and cell cycle. Western blotting was used to investigate changes in the protein levels upon treatment. Further, a tissue microarray followed by immunohistochemistry (IHC) was used to identify the presence of drug targets in HER2-positive BC tissue blocks. Our data revealed a significant dose-dependent decrease in HER2-positive BC cell viability after monotreatment with NER and MET (P<0.05) (down to 13.7%±0.43%, 25.49%±2.53%, 26.92%±0.15%, and 29.8%±1.82% in SKBR3, HCC1954, ZR75, and MDA-MB-453, respectively). The combination treatment demonstrated a further reduction in cell viability compared to individual treatments at the same doses, showing mostly a synergistic effect (combination index ≤1). NER and MET combination treatment inhibited colony formation (by ~ 60%), cell migration, and invasion (by ~ 80%) significantly compared to controls and individual treatment (P<0.01). Cell cycle deregulation was also observed upon treatment with NER and MET combination. The underlying mechanisms of action were found to be downregulating the expression patterns of EGFR, HER2, HER3, and IGF-1R proteins, in addition to ERK1/2, p38 MAPK, mTOR, AKT, and c-Jun and other EMT and apoptotic markers. Using a cohort of 55 HER2-positive BC samples, we revealed that HER2 and IGF-1R are co-expressed in most cases. We also established NER-resistant cell lines from parental SKBR3 and HCC1954; these two cell lines were named SNR and HNR, respectively. The resistant cell lines exhibited significantly elevated levels of EGFR, HER2, HER3, and IGF-1R. Interestingly, treatment with NER combined with MET re-sensitized the resistant cell lines to NER treatment. Finally, the effect of NER and MET on angiogenesis was investigated using the chorioallantoic membrane (CAM) of the chicken embryo as a model. Our findings revealed that NER and MET combination inhibit the angiogenesis of the CAM compared to controls by significantly reducing vessel percentage area and average vessel length (P<0.05). Our findings introduce a promising new combination treatment strategy against HER2-positive BC using NER and MET. This approach offers a fresh perspective by targeting multiple key proteins, including IGF-1R, and reducing tumor angiogenesis. Future directions include exploring the therapeutic potential of combining MET with other HER2-targeted agents and validating predictive biomarkers of response in in-vivo and patient-derived models.