Abstract P3-04-05: Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth
Authors: Sikora Matthew J , Bahreini Amir , Alexander Caroline M , Oesterreich Steffi 
Published: May 2015

Abstract
Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer representing 10-15% of newly diagnosed breast tumors. Over 90% of ILC are ER-positive, however, endocrine response and estrogen signaling are not well described in ILC. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than similar invasive ductal carcinoma (IDC) patients, and that ILC patients may not benefit from adjuvant tamoxifen. Additionally, we recently identified ILC-specific ER-target genes and de novo tamoxifen resistance driven by ER in ILC model systems. Based on these observations, we hypothesize that ILC-specific signaling pathways driven by ER mediate growth and endocrine resistance in ILC cells.
Among ILC-specific estrogen-regulated genes in the ILC cell lines MDA MB 134VI (MM134) and SUM44PE (SUM44), Wnt signaling genes were highly differentially expressed. The secreted ligand WNT4 was the most strongly estrogen-induced gene in ILC cells. The frizzled receptor FZD7 is also strongly induced in ILC cells, but only transiently induced in the ER-positive IDC cell line MCF-7. Among IDC cell lines, either WNT4 or FZD7 is over-expressed in ER-positive or ER-negative cells, respectively. Conversely, MM134 and SUM44 over-express both WNT4 and FZD7. Also, we identified an ILC-specific ER binding site at WNT4; located in intron 1, this site contains a predicted estrogen response element. Direct WNT4 regulation and parallel regulation of pathway genes suggests that ER controls a WNT4 signaling pathway in ILC cells. In samples from the Cancer Genome Atlas, WNT4 and FZD7 are each over-expressed in ER-positive ILC versus IDC; co-expression is also enriched only in ILC. These observations suggest that a WNT4 signaling pathway may be specifically active in ILC tumors.
To assess whether WNT4 is necessary for estrogen-induced growth, we used siRNA to knock down WNT4. Using either of two siRNAs, WNT4 knockdown completely blocks estrogen-induced growth in ILC cells, but not IDC cells. Consistent with this, WNT4 knockdown abrogated estrogen-regulation of a subset of ER-target genes in MM134 cells; induction or repression was inhibited by WNT4 knockdown prior to estrogen treatment. Thus, a subset of estrogen-induced gene expression changes is mediated by WNT4 signaling. Though Wnt signaling typically acts via the canonical, β-catenin-dependent pathway, we observed that β-catenin signaling is dysfunctional in ILC cells. Additionally, WNT4 over-expression or recombinant protein cannot activate canonical Wnt signaling in breast cancer cell lines. This suggests that WNT4 signaling mediates estrogen-induced growth in ILC cells via a novel non-canonical signaling pathway.
Wnt signaling pathway genes including WNT4 are uniquely regulated in ILC cell lines, and are over-expressed in ILC tumors, suggesting that a WNT4-driven pathway may be active specifically in ILC. WNT4 is necessary for estrogen-mediated growth in ILC cells, and likely signaling via a novel non-canonical signaling pathway. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Future studies will focus on identifying the signaling pathway controlled by WNT4 in order to identify novel therapeutic targets.
Citation Format: Matthew J Sikora, Amir Bahreini, Caroline M Alexander, Steffi Oesterreich. Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth [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 P3-04-05.