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Preclinical model systems are essential research tools that help us understand the biology of invasive lobular carcinoma of the breast (ILC). The number of well-established ILC models is increasing but remain limited. Lower incidence of ILC, underrepresentation of patients with ILC in clinical trials, and intrinsic ILC tumor characteristics all contribute to this challenge. Hence, there is significant need to continually develop better model systems to recapitulate the essential characteristics of ILC biology, genetics, and histology, and empower preclinical therapeutic studies to be translated back into the clinic. In this Perspective, we highlight recent advances in in vivo experimental models, which recapitulate key features of ILC biology and disease progression and potentially reshape the future of ILC translational research. We assert that all existing in vitro and in vivo ILC preclinical models have their strengths and weaknesses, and that it is necessary to bridge key deficiencies in each model context as we move forward with ILC research. Thus, unlocking the mysteries of ILC will be best achieved by choosing the right combination of preclinical model systems.

Invasive lobular carcinoma (ILC) is the second most common histologic subtype of breast cancer. Though >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.

Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer, affecting ~30,000 U.S. women annually. Over 90% of ILC are estrogen receptor (ER)-positive, however, endocrine therapy may have poorer efficacy in a subset of ILC patients compared to invasive ductal carcinoma (IDC) patients. Based on these observations, we assessed genome-wide ER-mediated gene expression and ER genomic binding in ILC cell lines MDA MB 134VI (MM134) and SUM44PE (44PE), to identify novel mediators of ER signaling and putative therapeutic targets specifically in ILC. Among ILC-specific estrogen-regulated genes, the most strongly induced was the Wnt ligand WNT4. In parallel, we identified an ILC-specific ER binding site (ERBS) at WNT4, suggesting that WNT4 is directly ER-controlled in ILC cells. We hypothesized that this would be an analog to progesterone-controlled WNT4 in mammary gland expansion, and assessed whether WNT4 is necessary for estrogen-induced growth in ILC cells. Using siRNAs, knockdown of WNT4 completely blocked estrogen-induced growth in ILC cells, but not IDC cells. Consistent with this, we found that the WNT4 ERBS is only occupied in ILC cells that strongly upregulate WNT4 in response to estrogen, whereas progesterone-regulated WNT4 expression in T47D cells was not associated with ER binding at the WNT4 ERBS. These data suggest that, via an ILC-specific ERBS at WNT4, ILC cells can drive estrogen-regulated proliferation by hijacking a developmental Wnt pathway. Canonical Wnt pathways activate β-catenin; however, we observed β-catenin dysfunction in ILC cells, and that WNT4 cannot activate β-catenin in cell lines. Thus, WNT4 regulates estrogen-induced growth in ILC cells via a novel non-canonical pathway. Using long-term estrogen-deprived (LTED) variants of MM134 and 44PE (4 and 2 lines, respectively), we assessed WNT4 in ILC endocrine resistance. WNT4 is over-expressed but uncoupled from ER in all MM134-LTED; expression is greatly reduced in 44PE-LTED, but weakly estrogen-regulated. Consistent with regulation, ER occupies the WNT4 ERBS only in 44PE-LTED cells. However, 44PE-LTED (low WNT4) are resistant to growth inhibition by WNT4 siRNA, while MM134-LTED (high WNT4) are growth-inhibited. Taken together, uncoupling and upregulating WNT4 may be necessary for LTED growth in MM134. Clinical observations suggest that ER regulates unique downstream pathways in ILC. We identified WNT4 as a putative downstream effector of endocrine signaling in ILC, having critical roles in both estrogen-induced growth and endocrine resistance. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Citation Format: Matthew J. Sikora, Amir Bahreini, Caroline M. Alexander, Steffi Oesterreich. WNT4 signaling mediates endocrine response and resistance in invasive lobular carcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A35.