Introduction. Tumor-initiating (TIC) phenotype is potent inducer of dissemination of cancer cells from early lesions showing self-renewal, multi-lineage differentiation, chemo-resistance, and are defined as CD44+/CD24-/ALDHA+. Here, we demonstrate that BRCA1-IRIS overexpression (IRISOE) promotes TNBC formation by enhancing basal, EMT and TIC phenotype. In regards to TIC phenotype, we show that IRISOE elevates CD44 expression and ALDHA activity while prevents CD24 surface presentation. We present a novel pathway for CD24 internalization and confirm these data in animal model and tumor samples. Finally, we evaluated in pre-clinical models the ability of IRISOE to promote dissemination from early lesion and potential utility of IRIS inhibition to deplete TICs in TNBCs.

Methods. We used western blot, qRT/PCR, immunofluorescence (IF) and IHC to establish association of IRISOE with basal, EMT and TIC phenotype. FACS analysis to determine IRISOE effect on the TIC phenotype. 2D and 3D culture to evaluate the utility of IRIS inhibition + a ligating anti-CD24 antibody on TICs. In vivo mice models to study early dissemination, metastasis formation and the effect of IRIS peptide on TIC tumor growth. Finally, patient cohort to correlate IRISOE to BRCA1 expression, stem and dissemination biomarkers in breast cancer patients.

Results. We demonstrate an association between IRISOE and high basal and EMT phenotype. IRISOE leads to significant increase in basal biomarkers (EGFR, CK5, CK17, CDH3) and EMT markers (N-cadherin, vimentin, fibronectin, FOXC2, twist and snailI). Within a cohort of breast cancer patients (n=326), including subcohort of TNBC patients (n=72), IRISOE was associated with loss of BRCA1. Association between IRISOE and increase in TIC phenotype, in vitro and in vivo was documented. We propose EGFR-c-Src-cortactin signaling pathway induced by IRISOE to be involved in CD24 internalization and thus TIC phenotype. In support of that both western blot and IF analysis showed that while IRIS silencing in TNBC cells led to complete re-direction of cytoplasmic CD24 to the membrane, silencing of members of this pathway led to partial effect. Conversely, their overexpression also partially reversed the effect of IRIS silencing on the TIC phenotype. IRISOE could also induce tumorigenesis and metastasis in non-tumorigenic luminal A, MCF7 cells by enhancing TIC phenotype in them. Not only that, mice injected with low numbers of MCF7/IRISOE cells generate larger tumors, most likely, due to enhanced interactions with the stroma, an effect most likely not possible when large number of cells is injected instead. These tumors also showed high dissemination in the form of increased CTCs and DTCs in the blood and bone marrow, respectively. Inhibiting IRIS with specific peptide led to 80% regression of tumors developed using TICs in NSG mice and the effect was even more dramatic on the Sox2+-population remained within these tumors.

Conclusion. This study reveals that IRISOE cells acquire TIC, basal and EMT phenotypes along with lack of BRCA1 expression and possess high dissemination and metastasis ability. Our results support inhibiting IRIS in TNBC patients to prevent early dissemination and metastasis.

Citation Format: Sinha A, Paul BT, ElShamy WM. BRCA1-IRIS overexpression promotes and maintains the tumor initiating phenotype in TNBC cells: Implications for breast cancer early lesions [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-05.