Abstract 3565: Identification of breast cancer cells exhibiting tumor stemness phenotype
Authors: Talukdar Joyeeta , Sandhya Sorra , Pathak Lekhika , Mann Zoya , Pal Bidisha , Bhuyan Seema , Shrivastav Anurag , Mohanty Sujata , Das Bikul 
Published: September 2020

Background: Our laboratory has identified tumor stemness phenotype in several tumor types including lymphoma, oral cancer, neuroblastoma, and sarcomas. In neuroblastoma, we found that stress induction including hypoxia, chemotherapy regimens, etc., activates the tumor stemness pathways including the HIF-2alpha stemness pathway. In lymphoma, we found that MYC regulates the HIF-2alpha stemness pathway to maintain tumor stemness phenotype of lymphoma CSCs (PMID:31266772). In this study, we evaluated the tumor stemness phenotype in breast cancer.

Methods: MCF-7 breast cancer cell line was subjected to hypoxia/oxidative stress for three-days and further flow-sorted to obtain the highly tumorigenic ABCG2+ cells population. Next, these cells

were evaluated for tumor stemness phenotype (Nanog, Sox2, Oct4, p53, MYC, and HIF-2a) via quantitative PCR. Induction of EMT (epithelial-mesenchymal transition) the phenotype was evaluated via qPCR using the known markers such as Snail, Twist, and vimentin. Invitro/ invivo self-renewal capacity of ABCG2+ cells was evaluated by clonogenic assays and serial transplantation assay. siRNA treatment was performed to transiently knockdown and observe changes in phenotype.

Results: ABCG2+ cells derived from post- hypoxia treated MCF-7 indicated tumor stemness phenotype with high gene expression levels of Nanog, Sox2, Oct4, p53, MYC, and HIF-2a. Gene expression of EMT markers was markedly high in comparison to ABCG2- cell population. Hypoxia treatment amplified the self-renewal capacity in ABCG2+ cells, while the loss of HIF-2a using siRNA treatment (in-vitro) or FM19G11 treatment (potent invivo chemical HIF inhibitor) abrogated the self-renewal capacity as observed by fewer colonies and absence of tumors. Associated reduction in the stemness phenotype and EMT was observed in siRNA HIF-2a treated ABCG2+ cells. Importantly we found that in these cells, TLR-2 and Nfκβ become activated following HMGB-1 ligand binding, suggesting the pro-inflammatory phenotype of these cells. Additionally, these cells showed a high expression of the aryl hydrocarbon receptor (AhR).

Conclusion: These data suggest that MCF-7, upon exposure to hypoxia/oxidative stress undergo tumor stemness switch and activate the MYC-HIF-2alhpa stemness pathway. Our study also shows that the tumor stemness phenotype in his cell line incorporates the activation of the TLR/HGMB1 axis and also the AhR pathway. We also found that loss of HIF-2a negatively affected the tumor stemness phenotype of ABCG2+ breast cancer stem cells. Thus, targeting the MYC-HIF-2alpha stemness pathway may prove to be therapeutically beneficial.

Citation Format: Joyeeta Talukdar, Sorra Sandhya, Lekhika Pathak, Zoya Mann, Bidisha Pal, Seema Bhuyan, Anurag Shrivastav, Sujata Mohanty, Bikul Das. Identification of breast cancer cells exhibiting tumor stemness phenotype [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3565.