Abstract P4-01-24: Targeting receptor tyrosine kinases in overcoming tamoxifen resistance and dormancy in invasive lobular cancer
Authors: Ramaswamy Bhuvaneswari , Pramod Nikhil , Kulkarni Anagh , Rima Xilal Y. , Reátegui Eduardo , Shankar Eswar , Majumder Sarmila 
Published: March 2023

Abstract
Objective: The molecular hallmark of invasive lobular cancer (ILC) is the loss of E-cadherin, resulting in the unique morphology, low mitotic index and unusual metastatic spread to ovaries, gut, and peritoneum. Patients with ILC face delayed, and higher stage at diagnosis, worse disease-free and overall survival1. The most frustrating challenge is the delayed recurrence, likely due to resistance and induction of dormancy2. Despite these unique features, ILC is lumped with hormone receptor-positive invasive ductal cancers (IDC); consequently, management strategies are based on data from IDC. Hence there is an unmet need to address the unique challenges of ILC. We generated Tamoxifen-resistant (TAMR) ILC cell lines with the aim to study the distinct characteristics, dormancy, differentially activated pathways, and response to drugs targeting such pathways in the resistant cells. Methods: We used MDA-MB-134-VI and SUM44-PE cells to study the effect of tamoxifen in combination with multi-targeted receptor tyrosine kinase inhibitor, Lenvatinib. TAMR cells from both MB-134 and SUM44PE cells were generated by growing them in increasing concentrations of 4-hydroxy tamoxifen (4-HT) up to 500nM, for 6 months. Total RNA from parental and TAMR cells were subjected to RNA-seq. Whole cell extracts were analyzed for specific phospho proteins by Western Blot analysis. Growth kinetics and effect of drugs on cell viability was measured using MTT assay (Roche). Cell migration was measured using Transwell migration assay. Novel engineered 2D and 3D cultures were used to study dormancy and the effect of the drug combination. Orthotopic tumor induction in NSG mice is ongoing to determine the effect of drug combination in vivo. Results: The TAMR cell lines show distinct morphological features, small but significant increase in growth rate (p=0.05) and remarkably higher migration (MB-134TAMR:11.5-fold, p&lt; 0.005 and SUM44TAMR:&gt;100-fold, p&lt; 0.005). The IC50 for tamoxifen increased from 8.1M (MB-134) to 16.8M (MB-134TAMR) and from 11.3 M (SUM44) to 26.6 M (SUM44TAMR). RNA seq analysis revealed enrichment of PI3K-AKT, MAPK, cAMP, Rap1 signaling pathways, ECM-receptor interaction, Focal adhesion, and steroid hormone biosynthesis pathways in the TAMR cells. Upon stimulation by endothelial growth factor, the MB-134TAMR but not the parental cells showed dose dependent increase in phospho-AKT and phospho-MAPK levels. Using novel 2D/3D cultures, we show differential morphologies between IDC (MCF7) cells, MB-134TAMR, and the parental cells. MCF7 and MB-134TAMR cells are more adherent to fibronectin (p&lt; 0.0001), whereas the parental ILC cells are more adherent to COL3A1 (p&lt; 0.0001), a feature observed in dormancy3. Further, the administration of 5 M 4-HT induced a cessation of growth in the parental cell line, with a sub-population of apparent dormant cells. The combination of Lenvatinib (5 M) and 4-HT (7.5 M) synergistically inhibited both the parental cell lines by ~60%. Lenvatinib (5 M) and 4-HT (15 M) inhibited the MB-134TAMR and SUM44TAMR cell lines synergistically by 65% and 45% respectively. In vivo, the growth of MB-134 induced tumors were completely suppressed when 5mg sustained release pellet of TAM citrate was injected in the subscapular region of tumor bearing mice (tumor @100cm3). The results of the combination using in vivo models and 2D/3D cultures will be reported. Conclusion and significance: Lenvatinib with tamoxifen is active and synergistic against parental and TAMR ILC cell lines. Our novel 2D/3D cultures show distinct pattern of growth for ILC cells and a possible induction of a dormant subpopulation upon 4-HT treatment. Overcoming resistance to conventional therapies and preventing induction of dormancy will improve long-term outcomes of patients with ILC. 1. PMID:33641217 2. PMID: 34388695 3. PMID: 35121989
Citation Format: Bhuvaneswari Ramaswamy, Nikhil Pramod, Anagh Kulkarni, Xilal Y. Rima, Eduardo Reátegui, Eswar Shankar, Sarmila Majumder. Targeting receptor tyrosine kinases in overcoming tamoxifen resistance and dormancy in invasive lobular cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-24.