Abstract
BackgroundInvasive lobular carcinoma (ILC) of the breast typically presents with clinical biomarkers consistent with a favorable response to endocrine therapies, and over 90 % of ILC cases express the estrogen receptor (ER). However, a subset of ILC cases may be resistant to endocrine therapies, suggesting that ER biology is unique in ILC. Using ILC cell lines, we previously demonstrated that ER regulates a distinct gene expression program in ILC cells, and we hypothesized that these ER-driven pathways modulate the endocrine response in ILC. One potential novel pathway is via the Wnt ligand WNT4, a critical signaling molecule in mammary gland development regulated by the progesterone receptor.MethodsThe ILC cell lines MDA-MB-134-VI, SUM44PE, and BCK4 were used to assess WNT4 gene expression and regulation, as well as the role of WNT4 in estrogen-regulated proliferation. To assess these mechanisms in the context of endocrine resistance, we developed novel ILC endocrine-resistant long-term estrogen-deprived (ILC-LTED) models. ILC and ILC-LTED cell lines were used to identify upstream regulators and downstream signaling effectors of WNT4 signaling.ResultsILC cells co-opted WNT4 signaling by placing it under direct ER control. We observed that ER regulation of WNT4 correlated with use of an ER binding site at the WNT4 locus, specifically in ILC cells. Further, WNT4 was required for endocrine response in ILC cells, as WNT4 knockdown blocked estrogen-induced proliferation. ILC-LTED cells remained dependent on WNT4 for proliferation, by either maintaining ER function and WNT4 regulation or uncoupling WNT4 from ER and upregulating WNT4 expression. In the latter case, WNT4 expression was driven by activated nuclear factor kappa-B signaling in ILC-LTED cells. In ILC and ILC-LTED cells, WNT4 led to suppression of CDKN1A/p21, which is critical for ILC cell proliferation. CDKN1A knockdown partially reversed the effects of WNT4 knockdown.ConclusionsWNT4 drives a novel signaling pathway in ILC cells, with a critical role in estrogen-induced growth that may also mediate endocrine resistance. WNT4 signaling may represent a novel target to modulate endocrine response specifically for patients with ILC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0748-7) contains supplementary material, which is available to authorized users.
Highlights
Invasive lobular carcinoma (ILC) of the breast typically presents with clinical biomarkers consistent with a favorable response to endocrine therapies, and over 90 % of ILC cases express the estrogen receptor (ER)
WNT4 is necessary for ILC cell proliferation in culture To determine whether WNT4 is necessary for breast cancer cell proliferation, we used Small interfering RNA (siRNA) to knock down WNT4 expression in breast cancer cell lines (BCCLs)
MCF-7 cells expressed more than tenfold less WNT4 than ILC lines, while HCC1428 was the only ERpositive BCCL with higher WNT4 expression than MM134 [25, 26]; this was confirmed by quantitative PCR (qPCR) (Fig. 1a)
Summary
Invasive lobular carcinoma (ILC) of the breast typically presents with clinical biomarkers consistent with a favorable response to endocrine therapies, and over 90 % of ILC cases express the estrogen receptor (ER). ChIP-seq identified an ILC-specific estrogen receptor binding site (ERBS) at the WNT4 locus, approximately 1.5 kb downstream from the WNT4 transcription start site, an evolutionarily conserved region [9] that contains two predicted estrogen response elements (EREs) (diagrammed in Additional file 1: Figure S1). These observations suggest that direct ER binding at this site may be responsible for estrogen-induced WNT4 expression. On the basis of these observations, we hypothesized that WNT4 may play a critical role in estrogen-regulated phenotypes in ILC
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