Human epidermal growth factor receptor-2 (HER2)-targeted therapies prolong survival in HER2-positive breast cancer patients. Benefit stems primarily from improved control of systemic disease, but up to 50% of patients progress to incurable brain metastases due to acquired resistance and/or limited permeability of inhibitors across the blood-brain barrier. Neratinib, a potent irreversible pan-tyrosine kinase inhibitor, prolongs disease-free survival in the extended adjuvant setting, and several trials evaluating its efficacy alone or combination with other inhibitors in early and advanced HER2-positive breast cancer patients are ongoing. However, its efficacy as a first-line therapy against HER2-positive breast cancer brain metastasis has not been fully explored, in part due to the lack of relevant pre-clinical models that faithfully recapitulate this disease. Here, we describe the development and characterisation of a novel syngeneic model of spontaneous HER2-positive breast cancer brain metastasis (TBCP-1) and its use to evaluate the efficacy and mechanism of action of neratinib. TBCP-1 cells were derived from a spontaneous BALB/C mouse mammary tumour and characterised for hormone receptors and HER2 expression by flow cytometry, immunoblotting and immunohistochemistry. Neratinib was evaluated in vitro and in vivo in the metastatic and neoadjuvant setting. Its mechanism of action was examined by transcriptomic profiling, function inhibition assays and immunoblotting. TBCP-1 cells naturally express high levels of HER2 but lack expression of hormone receptors. TBCP-1 tumours maintain a HER2-positive phenotype in vivo and give rise to a high incidence of spontaneous and experimental metastases in the brain and other organs. Cell proliferation/viability in vitro is inhibited by neratinib and by other HER2 inhibitors, but not by anti-oestrogens, indicating phenotypic and functional similarities to human HER2-positive breast cancer. Mechanistically, neratinib promotes a non-apoptotic form of cell death termed ferroptosis. Importantly, metastasis assays demonstrate that neratinib potently inhibits tumour growth and metastasis, including to the brain, and prolongs survival, particularly when used as a neoadjuvant therapy. The TBCP-1 model recapitulates the spontaneous spread of HER2-positive breast cancer to the brain seen in patients and provides a unique tool to identify novel therapeutics and biomarkers. Neratinib-induced ferroptosis provides new opportunities for therapeutic intervention. Further evaluation of neratinib neoadjuvant therapy is warranted.

[10]-gingerol induces apoptosis and inhibits metastatic dissemination of triple negative breast cancer in vivo [10]-gingerol induces apoptosis and inhibits metastatic dissemination of triple negative breast cancer in vivo

Human epidermal growth factor receptor-2 (HER2)-targeting therapies provide clinical benefits for patients with HER2-positive breast cancer. However, the resistance to monotherapies invariably develops and leads to disease relapse and treatment failure. Previous studies have demonstrated a link between the potency of HER2-targeting tyrosine kinase inhibitors (TKIs) and their ability to induce an iron-dependent form of cell death called ferroptosis. The aim of this study was to understand the mechanisms of resistance to TKI-induced ferroptosis and identify novel approaches to overcome treatment resistance. We used mouse and human HER2-positive models of acquired TKI resistance to demonstrate an intimate link between the resistance to TKIs and to ferroptosis and present the first evidence that the cell adhesion receptor αvβ3 integrin is a critical mediator of resistance to TKI-induced ferroptosis. Our findings indicate that αvβ3 integrin-mediated resistance is associated with the re-wiring of the iron/antioxidant metabolism and persistent activation of AKT signalling. Moreover, using gene manipulation approaches and pharmacological inhibitors, we show that this “αvβ3 integrin addiction” can be targeted to reverse TKI resistance. Collectively, these findings provide critical insights into new therapeutic strategies to improve the treatment of advanced HER2-positive breast cancer patients.

The human epidermal growth factor receptor-2 (HER2) subtype of breast cancer is aggressive and associated with a high propensity to metastasize to the brain. Whilst the introduction of HER2-targeted therapies has significantly improved survival outcomes in affected patients, effective treatment of brain metastasis remains a challenge due to acquired resistance and limited drug permeability across the blood brain barrier. Moreover, progress in identifying more effective treatments, particularly against therapy-resistant brain-metastatic HER2 breast cancer, has been hampered by the lack of robust and clinically relevant preclinical models. Here, we introduce a new Balb/c syngeneic model of spontaneous breast cancer brain metastasis that naturally expresses HER2 (RCSI-Her2). In vivo metastasis assays confirmed that this model gives rise to high incidence of spontaneous brain metastases from mammary tumors or experimental brain metastases following intra-cardiac inoculation. RT-PCR, western blotting and immunohistochemical analyses of RCSI-Her2 cells, tumors or brain metastases confirmed the lack of hormone receptor expression but high levels of HER2. Accordingly, RCSI-Her2 cell proliferation is inhibited by HER2 inhibitors, lapatinib or neratinib, but not by anti-estrogens, indicating phenotypic and functional similarities to human HER2 breast cancer. Importantly, short term in vitro proliferation assays show that the combination of neratinib with αvβ3 integrin inhibitors (Cilengitide or DisBa-01) potently and synergistically inhibits the proliferation of RCSI-HER2 or brain-metastatic triple negative breast cancer cells (4T1Br4). Moreover, treatment of neratinib-resistant RCSI-Her2 variants with Cilengitide or DisBa-01 restores sensitivity to neratinib. Preliminary in vivo experiments indicate that neratinib is well-tolerated in immune-competent mice, inhibits RCSI-HER2 primary tumour growth and extends survival. Transcriptomic analysis (RNAseq) of neratinib-sensitive and resistant variants treated or not with neratinib to identify signaling pathways implicated in resistance is underway. The results from this analysis will be presented. To our knowledge, RCSI-Her2 is the only model that fully recapitulates the spontaneous spread of HER2 breast cancer to the brain in immune-competent hosts. This model provides a unique tool to validate the efficacy of neratinib/αvβ3 integrin inhibitors in vivo against advanced brain-metastatic and therapy-resistant HER2 breast cancer. Citation Format: Normand Pouliot, Aadya Nagpal, Miriam Fuentes, Effie Mouhtouris, Francesca Avogadri-Connors, Alshad S. Lalani, Heloisa Sobreiro Selistre de Araujo. A novel approach to overcome resistance to HER2 targeted therapy in a new syngeneic model of breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1971.