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Invasive lobular carcinoma of the breast (ILC) is strongly estrogen-driven and represents a unique context for estrogen receptor (ER) signaling. In ILC, ER controls the expression of the Wnt ligand WNT4, which is critical for endocrine response and anti-estrogen resistance. However, signaling mediated by WNT4 is cell type- and tissue-specific, and has not been explored in ILC. We utilized reverse phase protein array (RPPA) to characterize ER and WNT4-driven signaling in ILC cells and identified that WNT4 mediates downstream mTOR signaling via phosphorylation of S6 Kinase. Additionally, ER and WNT4 control levels of MCL-1, which is associated with regulation of mitochondrial function. In this context, WNT4 knockdown led to decreased ATP production and increased mitochondrial fragmentation. WNT4 regulation of both mTOR signaling and MCL-1 were also observed in anti-estrogen resistant models of ILC. We identified that high WNT4 expression is associated with similar mTOR pathway activation in ILC and serous ovarian cancer tumors, suggesting that WNT4 signaling is active in multiple tumor types. The identified downstream pathways offer insight into WNT4 signaling and represent potential targets to overcome anti-estrogen resistance for patients with ILC.

Twitter: @mjsikoraAuthors' contributions MJS, DMR, and SO conceived of the project and experiments. MJS, DMR, EKB, and HMH designed and performed experiments. MJS and DMR developed models for the project. All authors contributed to data analysis and interpretation. MJS wrote the draft manuscript; all authors read and revised the manuscript and have read and approved of this version of the manuscript. AbstractInvasive lobular carcinoma of the breast (ILC) is strongly estrogen-driven, and represents a unique context for estrogen receptor (ER) signaling. In ILC, ER controls the expression of the Wnt ligand WNT4, which is critical for endocrine response and anti-estrogen resistance, yet signaling mediated by WNT4 is poorly understood. We utilized reverse phase protein array (RPPA) to characterize ER and WNT4-driven signaling in ILC cells, and identified WNT4 as a mediator of downstream mTOR signaling via p70-S6K. Independent of mTOR/p70-S6K, ER and WNT4 control levels of MCL-1, which is associated with mitochondrial function. In this context, knockdown of WNT4 caused accumulation of reactive oxygen species and decreased ATP production that precede cell death. WNT4 regulation of both mTOR signaling and MCL-1 levels was also observed in anti-estrogen resistant models of ILC.Further, we identified that high WNT4 expression is associated with similar mTOR pathway activation in serous ovarian cancer tumors, suggesting that this WNT4 pathway is important in multiple tumor types.The identified downstream pathways represent potential targets to inhibit WNT4 signaling in ovarian cancer and overcome anti-estrogen resistance for patients with ILC.

Invasive lobular carcinoma of the breast (ILC) has a distinct metabolic phenotype among breast cancer, characterized by relative metabolic quiescence and limited glucose uptake. However, recent work suggests ILC preferentially use fuels such as lipids and amino acids, and that ILC metabolism is linked to estrogen receptor α (ER) signaling. We previously reported that in ILC, estrogen-induced expression of Wnt ligand WNT4 activates an atypical intracellular WNT4 pathway that regulates cellular metabolism and mitochondrial dynamics. However, mechanisms by which intracellular WNT4 regulates mitochondria are unknown. To address this, we performed proximity-dependent biotinylation with mass spectrometry (BioID) to profile WNT4 trafficking, localization, and intracellular functions. BioID showed that whereas canonical Wnt ligand WNT3A trafficked through the endoplasmic reticulum for secretion, WNT4 is predominantly in the cytosol and at the mitochondria. We also identified DHRS2, mTOR, and STAT1 as putative WNT4 cytosolic/mitochondrial signaling partners. These findings support a role for intracellular WNT4 regulating mitochondrial function and metabolism. We further investigated regulation of metabolism by ER-WNT4 using global metabolomics, following WNT4 knockdown versus over-expression compared to siRNA or small molecule inhibition of ER-WNT4 signaling. We found WNT4 signaling regulates oxidative phosphorylation (OXPHOS), with WNT4 knockdown suppressing OXPHOS as well as fatty acid and glutamate metabolism pathways, but not glycolytic activity. Taken together, WNT4 has atypical intracellular localization and signaling activity directly at the mitochondria that mediates ER regulation of cellular metabolism. ER-WNT4 signaling may play a key role in regulating the distinct metabolic phenotype of ILC by regulating mitochondrial activity and fuel usage.