Targeting HER2 signaling in Invasive Lobular Cancers to improve responses to Antiestrogen Therapy

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2021-05

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The Ohio State University

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Introduction: Invasive Lobular Carcinoma (ILC) is a distinct histological and molecular subtype of breast cancer that is often estrogen receptor (ER) positive. ILC patients suffer from late recurrences and unfavorable long-term outcomes, suggesting resistance to antiestrogen therapies1. Cross talk between growth factor signaling pathways such as EGFR/HER2 and ER along with HER2 activating mutations have also been reported at the time of recurrence2. We believe that HER2 is a viable target in endocrine resistant ILC and targeting HER2 using monoclonal antibody (trastuzumab) and tyrosine kinase inhibitors (TKI) along with antiestrogen therapy (ET), would be a novel alternative treatment strategy for endocrine resistant ILC patients. Methods: We used ILC cell lines SUM44PE and MDA-MB-134-VI, along with Long Term Estrogen Deprived (LTED) and tamoxifen resistant (TamR) derivatives of each cell line in this study. We determined IC50 values of tamoxifen for each line using the MTT [[3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay. We have determined the effect of TKIs (Neratinib and Tucatinib) and Trastuzumab in combination with Tamoxifen on cell viability in in vitro cell culture system using MTT assay. We performed western blot analysis to study the effect of the drugs on downstream growth kinases such as AKT and MAPK signaling and Receptor Tyrosine Kinase (RTK) arrays to identify novel downstream signaling kinases responsive to these treatments. Results: Prolonged exposure of parental ILC cell lines to tamoxifen rendered the cells tamoxifen resistant as demonstrated by higher IC50 for tamoxifen. Specifically, IC50-TAM for MDA-MB-134-VI Parental and TamR cells are 2.27μM and 29.3μM respectively; for SUM44PE Parental and TamR cells are 7.1μM and 9.6μM respectively. Amongst the LTED cell lines, every line except for MDA-MB-134-VI LTED D has a lower IC50 value for tamoxifen. Across MDA-MB-134-VI cell lines, we observed increases in HER2 protein level in TamR100 (4.3-fold), LTED A (2.6-fold), LTED D (1.1-fold) in comparison to the parental cell line. In SUM44PE lines, we observed HER2 protein level increases in TamR100 (1.7-fold) and in LTED B (1.1-fold). ERα expression was decreased by at least 60% across all MDA-MB-134-VI LTED lines except LTED D when compared to parental cells, in addition to decreases in SUM44PE LTED A (8% decrease) and LTED B (32% decrease) compared to SUM44PE parental. The combination of tamoxifen, trastuzumab and neratinib (TTN) significantly reduced cell viability in SUM44PE TamR (p = 0.0046) and SUM44PE LTED B (p = 0.0076), but not in SUM44PE parental or LTED A when compared to tamoxifen monotherapy. Similarly, the combination of tamoxifen, trastuzumab, and tucatinib (TTT) significantly reduced cell viability in MDA-MB-134-VI-TamR cells (p = 0.047), LTED A (p = 0.026), LTED D (p = 0.011), and LTED E (p = 0.007), and TTN combination reduced cell viability in LTED A (p = 0.001) and LTED E (p = 0.035) compared to tamoxifen treatment alone. Levels of phosphoAKT and phosphoMAPK was reduced upon tamoxifen and tucatinib treatment of SUM44PE cells (82% decrease in pAKT and 41% decrease in pMAPK levels). Using RTK array we identified ACK1 as a target downstream of HER signaling in ILC cells that was inhibited upon treatment with tamoxifen or TKI. Discussion: Targeting HER2 kinase activity with small molecule inhibitor along with anti- HER2 monoclonal antibody could improve efficacy of anti-estrogen therapy in endocrine therapy resistant invasive lobular cancer. Additionally, use of broad spectrum TKI, such as neratinib is more effective than specific HER2 inhibitors such as tucatinib. ACK1 is a novel protein that could be a therapeutic target in these cancers. Further in vitro studies are underway to determine the efficacy of these drug combinations.

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