Porcupine O-acyltransferase (PORCN) is considered essential for Wnt secretion and signaling. However, we observed that PORCN inhibition does not phenocopy the effects of WNT4 knockdown in WNT4-dependent breast cancer cells. This suggests a unique relationship between PORCN and WNT4 signaling. To examine the role of PORCN in WNT4 signaling, here we overexpressed WNT4 or WNT3A in breast cancer, ovarian cancer, and fibrosarcoma cell lines. Conditioned medium from these lines and co-culture systems were used to assess the dependence of Wnt secretion and activity on the critical Wnt secretion proteins PORCN and Wnt ligand secretion mediator (WLS). We observed that WLS is universally required for Wnt secretion and paracrine signaling. In contrast, the dependence of WNT3A secretion and activity on PORCN varied across the cell lines, and WNT4 secretion was PORCN-independent in all models. Surprisingly, WNT4 did not exhibit paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell-autonomous Wnt signaling activation by WNT4 and WNT3A, independent of PORCN or Wnt secretion. The PORCN-independent, cell-autonomous Wnt signaling demonstrated here may be critical in WNT4-driven cellular contexts or in those that are considered to have dysfunctional Wnt signaling.

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.

Wnt ligand WNT4 is critical in female reproductive tissue development, with WNT4 dysregulation linked to related pathologies including breast cancer (invasive lobular carcinoma, ILC) and gynecologic cancers. WNT4 signaling in these contexts is distinct from canonical Wnt signaling yet inadequately understood. We previously identified atypical intracellular activity of WNT4 (independent of Wnt secretion) regulating mitochondrial function, and herein examine intracellular functions of WNT4. We further examine how convergent mechanisms of WNT4 dysregulation impact cancer metabolism. In ILC, WNT4 is co-opted by estrogen receptor α (ER) via genomic binding in WNT4 intron 1, while in gynecologic cancers, a common genetic polymorphism (rs3820282) at this ER binding site alters WNT4 regulation. Using proximity biotinylation (BioID), we show canonical Wnt ligand WNT3A is trafficked for secretion, but WNT4 is localized to the cytosol and mitochondria. We identified DHRS2, mTOR, and STAT1 as putative WNT4 cytosolic/mitochondrial signaling partners. Whole metabolite profiling, and integrated transcriptomic data, support that WNT4 mediates metabolic reprogramming via fatty acid and amino acid metabolism. Furthermore, ovarian cancer cell lines with rs3820282 variant genotype are WNT4 dependent and have active WNT4 metabolic signaling. In protein array analyses of a cohort of 103 human gynecologic tumors enriched for patient diversity, germline rs3820282 genotype is associated with metabolic remodeling. Variant genotype tumors show increased AMPK activation and downstream signaling, with the highest AMPK signaling activity in variant genotype tumors from non-White patients. Taken together, atypical intracellular WNT4 signaling, in part via genetic dysregulation, regulates the distinct metabolic phenotypes of ILC and gynecologic cancers. Significance: WNT4 regulates breast and gynecologic cancer metabolism via a previously unappreciated intracellular signaling mechanism at the mitochondria, with WNT4 mediating metabolic remodeling. Understanding WNT4 dysregulation by estrogen and genetic polymorphism offers new opportunities for defining tumor biology, precision therapeutics, and personalized cancer risk assessment.

Invasive lobular carcinoma (ILC) is a common subtype of breast cancer, molecularly defined by genetic loss of CDH1, and subsequent loss of cell adhesion protein E-cadherin, in ∼95% of ILC. Though CDH1 loss occurs early in ILC oncogenesis, it is unclear how this facilitates transformation. We modeled early CDH1 loss using “normal” human mammary epithelial cells (HMEC), i.e. finite lifespan cells reflecting early hyperplasia, and targeted E-cadherin signaling using antibodies versus causing genetic CDH1 loss using siRNA or CRISPR/Cas9-knockout. Transcriptome analysis across four HMEC models showed that the mode of E-cadherin targeting is critical for the subsequent phenotype. Antibody-mediated inhibition of cell-cell contacts induced gene signatures of epithelial-mesenchymal transition (EMT), consistent with the role of E-cadherin suppression during the EMT process. Conversely, genetic CDH1 loss – as in ILC oncogenesis – repressed EMT signatures, and instead remodeled gene expression toward a luminal epithelial phenotype. RNA-seq, single cell transcriptomics, flow cytometry, microscopy, and ATACseq analyses support that CDH1 loss induces lineage remodeling to a luminal state, which is mirrored in transcriptomic analysis of clinical ILC precursor lesions. By isolating luminal versus basal cells prior to CDH1 knockout, we found that CDH1 loss led to remodeling of lineage identity in both populations, converging on a new lineage homeostasis with a luminal progenitor-like phenotype. Consistent with the shift to a luminal progenitor phenotype, CDH1 loss enhanced proliferative capacity over the finite lifespan of the HMECs, highlighting a feature of early CDH1 loss that may contribute to clonal advantage during tumor initiation. Moreover, CDH1 loss enhanced anoikis resistance, a defining feature of ILC cells. Our findings support that genetic loss of CDH1 in mammary epithelial cells induces transcriptional and phenotypic changes consistent with lineage identity remodeling toward a luminal progenitor-like state, which may underpin the mechanism by which early CDH1 loss mediates ILC oncogenesis.

Invasive lobular carcinoma of the breast (ILC) is typically estrogen receptor α (ER)-positive and presents with biomarkers of anti-estrogen sensitive disease, yet patients with ILC face particularly poor long-term outcomes with increased recurrence risk, suggesting endocrine response and ER function are unique in ILC. ER is co- regulated by the DNA repair protein Mediator of DNA Damage Checkpoint 1 (MDC1) specifically in ILC cells, driving distinct ER activity. However, this novel MDC1 activity is associated with dysfunctional canonical DNA repair activity by MDC1, but without typical features of DNA repair deficiency. To understand reciprocal activities of MDC1, we profiled the MDC1 interactome and found MDC1-associated proteins in ILC cells mirror a “BRCA-like” state lacking key homologous recombination (HR) proteins, consistent with HR dysfunction but distinct from classic “BRCAness”. HR dysfunction in ILC cells is supported by single-cell transcriptome and DNA repair activity analyses, with DNA repair signaling and functional data, showing dysfunctional induction and resolution of HR. In parallel, ILC tumor data are consistent with a distinct form of HR dysfunction via impaired HR resolution, lacking BRCA-like genomic scarring but showing elevated signatures of PARP inhibitor sensitivity. We demonstrate this HR dysfunction can be exploited using PARP inhibition, and found that talazoparib treatment produced a durable growth suppression both in vitro and in multiple ILC xenografts in vivo . ILC-specific ER:MDC1 activity creates a new context for ER and MDC1 function in ILC, at the cost of a DNA repair dysfunction, which may be therapeutically targetable. Significance: ILC is rarely associated with biomarkers of HR deficiency, and as such patients are rarely eligible for treatment with PARP inhibitors. Our work suggests ILC presents with a previously unappreciated form of HR dysfunction, linked to ILC-specific genomic activity of ER and MDC1, which imparts sensitivity to PARP inhibition.

Invasive lobular carcinoma of the breast (ILC) is typically estrogen receptor α (ER)-positive and presents with biomarkers of antiestrogen sensitive disease. Unfortunately, patients with ILC face particularly poor long-term outcomes with high recurrence risk, suggesting a divergent endocrine response and ER function in ILC compared with other breast cancers. ER is coregulated by the DNA repair protein mediator of DNA damage checkpoint 1 (MDC1), specifically in ILC cells, driving distinct ER activity. In this study, we profiled the MDC1 interactome to examine how MDC1 regulates ER activity and DNA repair function in ILC. MDC1-associated proteins in ILC cells mirrored a “BRCA-like” state lacking key homologous recombination (HR) proteins, consistent with HR dysfunction but distinct from classic “BRCAness.” Single-cell transcriptome and DNA repair activity analyses, along with DNA repair signaling and functional data, substantiated dysfunctional induction and execution of HR in ILC cells. In parallel, ILC tumor data were consistent with a form of HR dysfunction distinct from overt HR deficiency, lacking BRCA-like genomic scarring but showing elevated signatures of PARP inhibitor sensitivity. Treatment with the PARP inhibitor talazoparib produced a durable growth suppression both in vitro and in multiple ILC xenografts in vivo. Together, these findings reveal that ILC-specific ER:MDC1 activity comes at the cost of DNA repair dysfunction, which may be therapeutically targetable. Significance: Association between ER and MDC1 in lobular breast cancer cells induces a form of dysfunctional homologous recombination, distinct from “BRCAness,” that creates the mechanistic context for synthetic lethal interaction with PARP inhibition.