Abstract P2-06-04: Proteosome inhibitor bortezomib inhibits NFκB and effectively overcomes cancer stem cell escape triggered by Wnt inhibitor therapy in FOXC1+ basal-Like/claudin-low breast cancer
Authors: Ray Partha S , Tsai Connie Y , Ray Tania , Kim Bomy , Jensen Tor W 
Published: May 2015

Abstract
Cancer stem cells (CSCs) are considered to be an important contributing factor towards treatment failure, cancer recurrence, and mortality. CSCs are known to be more enriched in the basal-like and claudin-low subtypes of breast cancer. The Wnt/β catenin signaling pathway is well known as a regulator of embryonic development and stem cell biology, and is prominently active in basal-like and claudin-low breast cancers. We have previously reported that transcription factor FOXC1 plays a critical role in mediating aggressive cell traits in basal-like/claudin-low breast cancer. In this study, we sought to investigate the link between Wnt signaling and FOXC1 and its potential in regulating CSC biology in basal-Like/claudin-low breast cancer. We observed that exposure of the MDA-MB-231 basal-like/claudin-low cell line (low constitutive FOXC1 expressor) to Wnt3a (a canonical Wnt signaling ligand), resulted in increased expression of FOXC1. Reciprocally, overexpression of FOXC1 in MCF10A human mammary epithelial cells led to a pronounced increase in Wnt signaling activity, strongly suggestive of a direct or indirect positive feedback loop between Wnt signaling and FOXC1. More importantly, BT549 and HS578t basal-like/claudin-low cells (high constitutive FOXC1 expressors) proved to be more sensitive to treatment with the Wnt inhibitor ICRT3 as evidenced by decreased cell viability when compared to MCF7 (luminal) or SKBR3 (HER2) breast cancer cell lines. Furthermore the decrease in cell viability appeared to be proportionate to the level of FOXC1 expression. Upon pharmacological inhibition with ICRT3 and biological inhibition with siRNA knockdown of LRP6, (a canonical Wnt signaling cell surface receptor) a decrease in FOXC1 expression level was observed in a dose and time dependent manner. This effect was particularly pronounced in mammosphere cultures enriched for BT549 cancer stem-like cells. Inhibition of Wnt signaling reduced mammosphere formation efficiency of BT549 cells, suggesting that Wnt inhibition targets cancer stem cells (CSCs) in the basal-like/claudin-low breast cancer subtype. More importantly, however, after an initial 4 day incubation period, some cells are observed to persist and later display renewed enhancement of mammosphere formation ability. Profiling of such cells interestingly revealed depletion of FOXC1+ve cells but persistence of cells displaying pronounced up regulation of stereotypical embryonic stem cell Transcription Factors OCT4, SOX2 and NANOG, strongly suggestive of a potential primitive stem cell/quiescent cell state escape mechanism. qRT-PCR based pathway activation analysis revealed marked activation of NFκB signaling in the residual cells that withstood Wnt inhibition. Simultaneous pharmacologic inhibition with Wnt inhibitor ICRT3 and the proteasome inhibitor Bortezomib (known to inhibit NFκB signaling) effectively targeted BT549 cancer stem cells in mammosphere culture and prevented the persistence/emergence of any residual cells. Taken together, our findings suggest that combination therapy approaches are likely required to effectively target breast cancer stem cells.
Citation Format: Partha S Ray, Connie Y Tsai, Tania Ray, Bomy Kim, Tor W Jensen. Proteosome inhibitor bortezomib inhibits NFκB and effectively overcomes cancer stem cell escape triggered by Wnt inhibitor therapy in FOXC1+ basal-Like/claudin-low breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-04.