Abstract 3480: CD44 V6 and V9 isoforms act in resistance to Fas-mediated apoptosis in breast cancer initiating cells

Abstract

Breast cancer initiating cells (BCIC) are CD44+/CD24 low/ESA+ cells derived from breast tumors that highly tumorigenic and resistant to apoptosis and to chemotherapy. To determine anti-apoptotic mediator (s) to both Fas death receptor signaling and chemotherapeutic agents induced apoptosis in BCIC, we detest the expression of V5, V6 and V9 of CD44 isoforms in BCIC derived from breast cancer cell lines MCF-7, MDA-MB-231, SUM159 and SUM1315, and assess their effects on both Fas death receptor signaling and chemotherapeutic agents induced apoptosis. Methods: 1). We measured relative expression of the various CD44 isoforms in parental and BCIC populations using quantitative real time-PCR. 2).We then determined which CD44 isoforms play a role in BCIC's resistance to apoptosis. SUM159 was chosen to use due to the overexpression of all CD44 isoforms. 3). After isolation of cells expressing the BCIC markers from SUM159 cells (CD44+/CD24 low/ESA+), we knocked down CD44 V5, V6 and V9 by siRNA in SUM159 BCIC cells and challenged them with increasing doses of the chemotherapeutic agent doxorubicin and FasL. 4).Tissue microarrays of human breast cancer (www.tissue-array.com) were also employed to assess association between the CD44 V9 expression and clinical parameters such as nodal metastasis, histological status, ER status, PgR status, HER2 status and survival in 40 cases of primary breast cancers. Results: 1).CD44 isoforms V5, V6 and V9 are differentially expressed in BCIC compared to their parental cell lines. V9 is markedly overexpressed in all 4 BCIC tested. 2). BCIC cells treated with siRNA targeting isoforms V5, V6 and V9 demonstrated no significant effect on doxorubicin IC50 when compared to controls. The existence of CD44 V9 and CD44 V6 in breast cancer cells confer resistance to apoptosis induced by FasL, knockdown of CD44 V6 resulted in 39% increase in caspase 3/7 activity when compared to controls while knockdown of CD44V9 caused a 49% increase. Further study showed that this is through the inhibition of the extrinsic apoptotic pathway by suppressing caspases-8 activation, thereby blocking downstream apoptotic events. 3). 40 cases of primary breast cancers provided assessable cores paired with corresponding uninvolved tissue from the same patient. The analysis showed a tendency between CD44V6 expression and HER2 status. We found that approximately 72.8% of HER2-positive patients express CD44V9 compared to 48.3% of HER2-negative patients expressing CD44V9. It did not reach statistically significance probably because of small size of samples. Conclusions: Loss of expression of the V6 and V9 but not V5 isoforms in SUM159 BCIC resulted in an increased sensitivity to apoptosis in response to the Fas L but not to the chemotherapeutic agent doxorubicin. These findings establish a role for CD44 V6 and V9 isoforms in aptoptotic resistance of BCIC and suggest that they may serve as targets for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3480. doi:1538-7445.AM2012-3480