Abstract A39: Estrogen-driven noncanonical WNT4 signaling is essential for proliferation and survival in lobular carcinoma cells
Authors: Rao Deviyani M. , Ferguson Rebecca , Sikora Matthew J. 
Published: August 2018

Abstract
Invasive lobular carcinoma (ILC) is the second most common histologic subtype of breast cancer. Though &gt;90% of ILC tumors are estrogen receptor (ER)-positive, ILC patients have poorer outcomes than others with ER-positive tumors, suggesting ER biology is unique in ILC cells. We previously identified the Wnt ligand WNT4 as an ILC-specific ER transcriptional target that is critical for both estrogen-induced growth and antiestrogen resistance in ILC cells. We hypothesize that characterizing the WNT4 signaling network in ILC cells will identify novel biomarkers and therapeutic targets for ILC patients.
WNT4 signals in a paracrine manner in normal mammary gland and in breast cancer tissues, via secretion from luminal cells to nearby basal or basal-like cells. Inhibiting Wnt secretion with Porcupine-inhibitors (PORCNi) in these contexts blocks WNT4-driven phenotypes. However, we have observed that PORCNi have no effect on ILC cell proliferation. Further, PORCNi combined with siRNA knockdown of PORCN completely suppress secretion of overexpressed WNT3A but not of WNT4. These observations suggest that WNT4 engages its signaling cascade via an atypical mechanism in ILC cells, where WNT4 signals in an autocrine or intracellular manner or is secreted via PORCN-independent mechanisms. Understanding how WNT4 activates signaling is vital to developing appropriate therapeutic strategies, especially as PORCNi have been tested clinically for ILC patients.
In both the normal mammary gland and in breast cancer cells, WNT4 engages the canonical Wnt pathway and activates β-catenin-mediated gene regulation. However, ILC cells and tumors lack β-catenin protein (related to loss of E-cadherin); WNT4 must be activating an alternative signaling cascade in ILC cells. To define downstream WNT4 signaling in ILC cells, we utilized reverse phase protein arrays (RPPA) and identified ER-mediated protein changes blocked by concurrent WNT4 knockdown. These experiments identified putative targets that link WNT4 signaling to control of proliferation and cell cycle as well as apoptosis and cell survival. In the former, coordinate suppression of p21 and induction of FOXM1 control G1/S and G2/M transitions, respectively, and WNT4 knockdown engages both checkpoints to halt cellular proliferation. In the latter, WNT4 activates distinct components of Akt/mTOR signaling in ILC versus IDC cells, as E2 treatment increases phosphorylation of mTOR/Rictor but not of Akt in ILC cells. Activation of mTOR correlates with post-transcriptional regulation of levels of the anti-apoptotic BCL2-family protein MCL-1; E2-mediated suppression of MCL-1 protein levels via mTOR may regulate cell survival and sensitivity to apoptotic stimuli.
WNT4 mediates a critical ER-driven pathway in ILC cells, but the WNT4 signaling network has not been characterized to identify putative therapeutic approaches for ILC patients. Our data suggest that targeting FOXM1, mTOR, or BCL2-family proteins may modulate ER/WNT4 signaling and that ILC cells are unlikely to respond to PORCNi. Characterization of this unique signaling network will drive new predictive models and therapeutic approaches for ILC patients.
Citation Format: Deviyani M. Rao, Rebecca Ferguson, Matthew J. Sikora. Estrogen-driven noncanonical WNT4 signaling is essential for proliferation and survival in lobular carcinoma cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A39.