Wnt family member 4 (WNT4) and WNT3A activate cell-autonomous Wnt signaling independent of porcupine O-acyltransferase or Wnt secretion.

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 media 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 (WLS) mediator. 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.

SNAIL is induced by tamoxifen and leads to growth inhibition in invasive lobular breast carcinoma.

PURPOSE: Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer that is predominantly estrogen receptor alpha (ER)-positive (+) and is thus treated with endocrine therapies. Herein, we sought to understand the molecular underpinnings of the 4-hydroxytamoxifen (4OHT) resistance in ILC by assessing the potential role of the epithelial-to-mesenchymal transition transcription factor (EMT-TF) SNAIL (SNAI1). METHODS: Using a series of breast cancer cell lines, we measured the basal, estrogen and 4OHT-induced expression of SNAIL and other EMT-TF family members by quantitative reverse transcription-polymerase chain reaction and immunoblotting. Chromatin immunoprecipitation experiments were performed to assess ER binding to the SNAIL promoter. Cell proliferation, cell cycle and apoptosis were assessed in 2D cultures. 3D growth was assessed in Matrigel and Collagen I cultures. RESULTS: Estrogen and 4OHT induced SNAIL expression, but not that of the other EMT-TF family members SLUG (SNAI2) and SMUC (SNAI3), with the 4OHT effect being specific to the lobular but not the ductal subtype. We observed estrogen and 4OHT-induced ER recruitment to the SNAI1 promoter and high endogenous basal levels of SNAIL and several EMT-TFs in ILC cell lines. While SNAIL knockdown had a minor impact on the 4OHT partial agonism in estrogen-depleted conditions, it led to a surprising increase in cell proliferation in full serum. In complementary experiments, inducible SNAI1 overexpression caused decreased proliferation, associated with a cell cycle arrest in G0/G1. Additionally, apoptosis was observed in BCK4 cells. CONCLUSION: These data suggest a previously unrecognized role for SNAIL in ILC, substantiating a context-dependent behavior for this EMT-TF.

Activation of Wnt signaling promotes olaparib resistant ovarian cancer.

Epithelial ovarian cancer (EOC) has one of the highest death to incidence ratios among all cancers. High grade serous ovarian carcinoma (HGSOC) is the most common and deadliest EOC histotype due to the lack of therapeutic options following debulking surgery and platinum/taxane-based chemotherapies. For recurrent chemosensitive HGSOC, poly(ADP)-ribose polymerase inhibitors (PARPi; olaparib, rucaparib, or niraparib) represent an emerging treatment strategy. While PARPi are most effective in homologous recombination DNA repair-deficient (HRD) HGSOCs, recent studies have observed a significant benefit in non-HRD HGSOCs. However, all HGSOC patients are likely to acquire resistance. Therefore, there is an urgent clinical need to understand PARPi resistance and to introduce novel combinatorial therapies to manage PARPi resistance and extend HGSOC disease-free intervals. In a panel of HGSOC cell lines, we established matched olaparib sensitive and resistant cells. Transcriptome analysis of the matched olaparib-sensitive vs -resistant cells revealed activation of the Wnt signaling pathway and consequently increased TCF transcriptional activity in PARPi-resistant cells. Forced activation of canonical Wnt signaling in several PARPi-sensitive cells via WNT3A reduced olaparib and rucaparib sensitivity. PARPi resistant cells were sensitive to inhibition of Wnt signaling using the FDA-approved compound, pyrvinium pamoate, which has been shown to promote downregulation of ß-catenin. In both an HGSOC cell line and a patient-derived xenograft model, we observed that combining pyrvinium pamoate with olaparib resulted in a significant decrease in tumor burden. This study demonstrates that Wnt signaling can mediate PARPi resistance in HGSOC and provides a clinical rationale for combining PARP and Wnt inhibitors.

Key regulators of lipid metabolism drive endocrine resistance in invasive lobular breast cancer.

BACKGROUND: Invasive lobular breast carcinoma (ILC) is a histological subtype of breast cancer that is characterized by loss of E-cadherin and high expression of estrogen receptor alpha (ERα). In many cases, ILC is effectively treated with adjuvant aromatase inhibitors (AIs); however, acquired AI resistance remains a significant problem. METHODS: To identify underlying mechanisms of acquired anti-estrogen resistance in ILC, we recently developed six long-term estrogen-deprived (LTED) variant cell lines from the human ILC cell lines SUM44PE (SUM44; two lines) and MDA-MB-134VI (MM134; four lines). To better understand mechanisms of AI resistance in these models, we performed transcriptional profiling analysis by RNA-sequencing followed by candidate gene expression and functional studies. RESULTS: MM134 LTED cells expressed ER at a decreased level and lost growth response to estradiol, while SUM44 LTED cells retained partial ER activity. Our transcriptional profiling analysis identified shared activation of lipid metabolism across all six independent models. However, the underlying basis of this signature was distinct between models. Oxysterols were able to promote the proliferation of SUM44 LTED cells but not MM134 LTED cells. In contrast, MM134 LTED cells displayed a high expression of the sterol regulatory element-binding protein 1 (SREBP1), a regulator of fatty acid and cholesterol synthesis, and were hypersensitive to genetic or pharmacological inhibition of SREBPs. Several SREBP1 downstream targets involved in fatty acid synthesis, including FASN, were induced, and MM134 LTED cells were more sensitive to etomoxir, an inhibitor of the rate-limiting enzyme in beta-oxidation, than their respective parental control cells. Finally, in silico expression analysis in clinical specimens from a neo-adjuvant endocrine trial showed a significant association between the increase of SREBP1 expression and lack of clinical response, providing further support for a role of SREBP1 in the acquisition of endocrine resistance in breast cancer. CONCLUSIONS: Our characterization of a unique series of AI-resistant ILC models identifies the activation of key regulators of fatty acid and cholesterol metabolism, implicating lipid-metabolic processes driving estrogen-independent growth of ILC cells. Targeting these changes may prove a strategy for prevention and treatment of endocrine resistance for patients with ILC.

WNT4 mediates estrogen receptor signaling and endocrine resistance in invasive lobular carcinoma cell lines.

BACKGROUND: 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). 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. METHODS: The 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. RESULTS: ILC 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. CONCLUSIONS: WNT4 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.

The CYP17A1 inhibitor abiraterone exhibits estrogen receptor agonist activity in breast cancer.

Cytochrome P450 17A1 (CYP17A1) is the requisite enzyme for synthesis of sex steroids, including estrogens and androgens. As such, inhibition of CYP17A1 is a target for inhibiting the growth of hormone-dependent cancers including prostate and breast cancer. Abiraterone, is a first in class potent and selective CYP17A1 inhibitor that has been approved for the treatment of castration-resistant prostate cancer. Given that, androgens are the precursors for estrogen production, it has been proposed that abiraterone could be an effective form of treatment for estrogen receptor (ER)-positive breast cancer, though its utility in this context has yet to be established. Abiraterone has a core steroid-like chemical structure, and so we hypothesized that it may bind to nuclear steroid receptors including ER and have estrogenic activity. We tested this hypothesis by investigating abiraterone's ability to directly modulate ER signaling in breast cancer cell line models. We show that abiraterone directly activates ER, induces ER-target gene expression, and elicits estrogen-response-element reporter activity in the ER-positive cell lines MCF-7 and T47D. Abiraterone also induced cell proliferation by ~2.5-fold over vehicle in both MCF-7 and T47D cells. Importantly, abiraterone-induced cell proliferation and ER-activity was blocked by the selective estrogen receptor downregulator (SERD) fulvestrant, confirming that abiraterone directly acts at the ER. These data suggest that abiraterone should be combined with other ER antagonists when used for the clinical management of ER-positive breast cancer.

High expression of orphan nuclear receptor NR4A1 in a subset of ovarian tumors with worse outcome.

OBJECTIVE: Nuclear receptors (NRs) play a vital role in the development and progression of several cancers including breast and prostate. Using TCGA data, we sought to identify critical nuclear receptors in high grade serous ovarian cancers (HGSOC) and to confirm these findings using in vitro approaches. METHODS: In silico analysis of TCGA data was performed to identify relevant NRs in HGSOC. Ovarian cancer cell lines were screened for NR expression and functional studies were performed to determine the significance of these NRs in ovarian cancers. NR expression was analyzed in ovarian cancer tissue samples using immunohistochemistry to identify correlations with histology and stage of disease. RESULTS: The NR4A family of NRs was identified as a potential driver of ovarian cancer pathogenesis. Overexpression of NR4A1 in particular correlated with worse progression free survival. Endogenous expression of NR4A1 in normal ovarian samples was relatively high compared to that of other tissue types, suggesting a unique role for this orphan receptor in the ovary. Expression of NR4A1 in HGSOC cell lines as well as in patient samples was variable. NR4A1 primarily localized to the nucleus in normal ovarian tissue while co-localization within the cytoplasm and nucleus was noted in ovarian cancer cell lines and patient tissues. CONCLUSIONS: NR4A1 is highly expressed in a subset of HGSOC samples from patients that have a worse progression free survival. Studies to target NR4A1 for therapeutic intervention should include HGSOC.

The spatial structure of the tumor immune microenvironment can explain and predict patient response in high-grade serous carcinoma.

Despite ovarian cancer being the deadliest gynecological malignancy, there has been little change to therapeutic options and mortality rates over the last three decades. Recent studies indicate that the composition of the tumor immune microenvironment (TIME) influences patient outcomes but are limited by a lack of spatial understanding. We performed multiplexed ion beam imaging (MIBI) on 83 human high-grade serous carcinoma tumors - one of the largest protein-based, spatially-intact, single-cell resolution tumor datasets assembled - and used statistical and machine learning approaches to connect features of the TIME spatial organization to patient outcomes. Along with traditional clinical/immunohistochemical attributes and indicators of TIME composition, we found that several features of TIME spatial organization had significant univariate correlations and/or high relative importance in high-dimensional predictive models. The top performing predictive model for patient progression-free survival (PFS) used a combination of TIME composition and spatial features. Results demonstrate the importance of spatial structure in understanding how the TIME contributes to treatment outcomes. Furthermore, the present study provides a generalizable roadmap for spatial analyses of the TIME in ovarian cancer research.

WNT4 Regulates Cellular Metabolism via Intracellular Activity at the Mitochondria in Breast and Gynecologic Cancers.

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.

EstroGene database reveals diverse temporal, context-dependent and directional estrogen receptor regulomes in breast cancer.

As one of the most successful cancer therapeutic targets, estrogen receptor-α (ER/ESR1) has been extensively studied in decade-long. Sequencing technological advances have enabled genome-wide analysis of ER action. However, reproducibility is limited by different experimental design. Here, we established the EstroGene database through centralizing 246 experiments from 136 transcriptomic, cistromic and epigenetic datasets focusing on estradiol-treated ER activation across 19 breast cancer cell lines. We generated a user-friendly browser ( https://estrogene.org/ ) for data visualization and gene inquiry under user-defined experimental conditions and statistical thresholds. Notably, documentation-based meta-analysis revealed a considerable lack of experimental details. Comparison of independent RNA-seq or ER ChIP-seq data with the same design showed large variability and only strong effects could be consistently detected. We defined temporal estrogen response metasignatures and showed the association with specific transcriptional factors, chromatin accessibility and ER heterogeneity. Unexpectedly, harmonizing 146 transcriptomic analyses uncovered a subset of E2-bidirectionally regulated genes, which linked to immune surveillance in the clinical setting. Furthermore, we defined context dependent E2 response programs in MCF7 and T47D cell lines, the two most frequently used models in the field. Collectively, the EstroGene database provides an informative resource to the cancer research community and reveals a diverse mode of ER signaling.

The EstroGene Database Reveals Diverse Temporal, Context-Dependent, and Bidirectional Estrogen Receptor Regulomes in Breast Cancer.

As one of the most successful cancer therapeutic targets, estrogen receptor-α (ER/ESR1) has been extensively studied over the past few decades. Sequencing technological advances have enabled genome-wide analysis of ER action. However, comparison of individual studies is limited by different experimental designs, and few meta-analyses are available. Here, we established the EstroGene database through unified processing of data from 246 experiments including 136 transcriptomic, cistromic, and epigenetic datasets focusing on estradiol (E2)-triggered ER activation across 19 breast cancer cell lines. A user-friendly browser (https://estrogene.org/) was generated for multiomic data visualization involving gene inquiry under user-defined experimental conditions and statistical thresholds. Notably, annotation of metadata associated with public datasets revealed a considerable lack of experimental details. Comparison of independent RNA-seq or ER ChIP-seq data with the same design showed large variability and only strong effects could be consistently detected. Temporal estrogen response metasignatures were defined, and the association of E2 response rate with temporal transcriptional factors, chromatin accessibility, and heterogeneity of ER expression was evaluated. Unexpectedly, harmonizing 146 E2-induced transcriptomic datasets uncovered a subset of genes harboring bidirectional E2 regulation, which was linked to unique transcriptional factors and highly associated with immune surveillance in the clinical setting. Furthermore, the context dependent E2 response programs were characterized in MCF7 and T47D cell lines, the two most frequently used models in the EstroGene database. Collectively, the EstroGene database provides an informative and practical resource to the cancer research community to uniformly evaluate key reproducible features of ER regulomes and unravels modes of ER signaling. SIGNIFICANCE: A resource database integrating 246 publicly available ER profiling datasets facilitates meta-analyses and identifies estrogen response temporal signatures, a bidirectional program, and model-specific biases.

Mechanisms of estrogen-independent breast cancer growth driven by low estrogen concentrations are unique versus complete estrogen deprivation.

Despite the success of the aromatase inhibitors (AIs) in treating estrogen receptor positive breast cancer, 15-20 % of patients receiving adjuvant AIs will relapse within 5-10 years of treatment initiation. Long-term estrogen deprivation (LTED) of breast cancer cells in culture mimics AI-induced estrogen depletion to dissect mechanisms of AI resistance. However, we hypothesized that a subset of patients receiving AI therapy may maintain low circulating concentrations of estrogens that influence the development of endocrine resistance. We expanded established LTED models to account for incomplete suppression of estrogen synthesis during AI therapy. MCF-7 cells were grown in medium with charcoal-stripped serum supplemented with defined concentrations of 17ß-estradiol (E2) or the estrogenic androgen metabolite 5α-androstane-3ß,17ß-diol (3ßAdiol), an endogenous selective estrogen receptor modulator. Cells were selected in concentrations of E2 or 3ßAdiol that induce 10 or 90 percent of maximal proliferation (EC(10) and EC(90), respectively), or estrogen deprived. Estrogen independence was evaluated during selection by assessing cell growth in the absence or presence of E2 or 3ßAdiol. Following >7 months of selection, estrogen independence developed in estrogen-deprived cells and EC(10)-selected cells. Functional analyses demonstrated that estrogen-deprived and EC(10)-selected cells developed estrogen independence via unique mechanisms, ERα-independent and dependent, respectively. Estrogen-independent proliferation in EC(10)-selected cells could be blocked by kinase inhibitors. However, these cells were resistant to kinase inhibition in the presence of low steroid concentrations. These data demonstrate that further understanding of the total estrogen environment in patients on AI therapy who experience recurrence is necessary to effectively treat endocrine-resistant disease.

Unlocking the Mysteries of Lobular Breast Cancer Biology Needs the Right Combination of Preclinical Models.

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.

Mutual exclusivity of ESR1 and TP53 mutations in endocrine resistant metastatic breast cancer.

Both TP53 and ESR1 mutations occur frequently in estrogen receptor positive (ER+) metastatic breast cancers (MBC) and their distinct roles in breast cancer tumorigenesis and progression are well appreciated. Recent clinical studies discovered mutual exclusivity between TP53 and ESR1 mutations in metastatic breast cancers; however, mechanisms underlying this intriguing clinical observation remain largely understudied and unknown. Here, we explored the interplay between TP53 and ESR1 mutations using publicly available clinical and experimental data sets. We first confirmed the robust mutational exclusivity using six independent cohorts with 1,056 ER+ MBC samples and found that the exclusivity broadly applies to all ER+ breast tumors regardless of their clinical and distinct mutational features. ESR1 mutant tumors do not exhibit differential p53 pathway activity, whereas we identified attenuated ER activity and expression in TP53 mutant tumors, driven by a p53-associated E2 response gene signature. Further, 81% of these p53-associated E2 response genes are either direct targets of wild-type (WT) p53-regulated transactivation or are mutant p53-associated microRNAs, representing bimodal mechanisms of ER suppression. Lastly, we analyzed the very rare cases with co-occurrences of TP53 and ESR1 mutations and found that their simultaneous presence was also associated with reduced ER activity. In addition, tumors with dual mutations showed higher levels of total and PD-L1 positive macrophages. In summary, our study utilized multiple publicly available sources to explore the mechanism underlying the mutual exclusivity between ESR1 and TP53 mutations, providing further insights and testable hypotheses of the molecular interplay between these two pivotal genes in ER+ MBC.

Making an IMPACT on career development for early- and mid-career faculty.

Nuclear receptors are critically important in normal and disease physiology. Recent advances have created opportunities to expand our success in nuclear receptor (NR) basic and translational research, but this field lacks a platform to lay the collaborative groundwork for aspiring and upcoming leaders in the field. Nuclear Receptor IMPACT (Interdisciplinary Meeting for Progress And Collaboration Together) is a new collaborative group designed specifically for early- and mid-career faculty who study nuclear receptors in their many forms. A unique goal of NR IMPACT is to also directly address career challenges for early- and mid-career faculty. NR IMPACT held an inaugural conference in September 2020 and developed a roadmap identifying five major structural and science policy challenges facing early- and mid-career faculty. NR IMPACT identified potential best practices, resources needed, and key action items to address these issues. NR IMPACT is a first-of-its-kind cohort dedicated to building a foundation for the scientific and professional growth of investigators studying nuclear receptors, and supporting new collaborations that will advance new paradigms in NR biology. Our unique focus on career development will enhance the success of current faculty and remove hurdles for new faculty, creating a robust pipeline of investigators with exciting new ideas to advance NR biology. The growth of NR IMPACT will build a strong peer-mentoring cohort that can be a unique resource for researchers and a prototype peer group for other disciplines.

WNT4 Balances Development vs Disease in Gynecologic Tissues and Women's Health.

The WNT family of proteins is crucial in numerous developmental pathways and tissue homeostasis. WNT4, in particular, is uniquely implicated in the development of the female phenotype in the fetus, and in the maintenance of müllerian and reproductive tissues. WNT4 dysfunction or dysregulation can drive sex-reversal syndromes, highlighting the key role of WNT4 in sex determination. WNT4 is also critical in gynecologic pathologies later in life, including several cancers, uterine fibroids, endometriosis, and infertility. The role of WNT4 in normal decidualization, implantation, and gestation is being increasingly appreciated, while aberrant activation of WNT4 signaling is being linked both to gynecologic and breast cancers. Notably, single-nucleotide polymorphisms (SNPs) at the WNT4 gene locus are strongly associated with these pathologies and may functionally link estrogen and estrogen receptor signaling to upregulation and activation of WNT4 signaling. Importantly, in each of these developmental and disease states, WNT4 gene expression and downstream WNT4 signaling are regulated and executed by myriad tissue-specific pathways. Here, we review the roles of WNT4 in women's health with a focus on sex development, and gynecologic and breast pathologies, and our understanding of how WNT4 signaling is controlled in these contexts. Defining WNT4 functions provides a unique opportunity to link sex-specific signaling pathways to women's health and disease.

Mediator of DNA Damage Checkpoint 1 (MDC1) Is a Novel Estrogen Receptor Coregulator in Invasive Lobular Carcinoma of the Breast.

Invasive lobular carcinoma (ILC) is the most common special histologic subtype of breast cancer, and nearly all ILC tumors express estrogen receptor alpha (ER). However, clinical and laboratory data suggest ILC are strongly estrogen-driven but not equally antiestrogen-sensitive. We hypothesized ILC-specific ER coregulators mediate ER functions and antiestrogen resistance in ILC, and profiled ER-associated proteins by mass spectrometry. Three ER+ ILC cell lines (MDA MB 134VI, SUM44PE, and BCK4) were compared with ER+ invasive ductal carcinoma (IDC) line data, and we examined whether siRNA of identified proteins suppressed ER-driven proliferation in ILC cells. This identified mediator of DNA damage checkpoint 1 (MDC1), a tumor suppressor in DNA damage response (DDR), as a novel ER coregulator in ILC. We confirmed ER:MDC1 interaction was specific to ILC versus IDC cells, and found MDC1 knockdown suppressed ILC cell proliferation and tamoxifen resistance. Using RNA-sequencing, we found in ILC cells MDC1 knockdown broadly dysregulates the ER transcriptome, with ER:MDC1 target genes enriched for promoter hormone response elements. Importantly, our data are inconsistent with MDC1 tumor suppressor functions in DDR, but suggest a novel oncogenic role for MDC1 as an ER coregulator. Supporting this, in breast tumor tissue microarrays, MDC1 protein was frequently low or absent in IDC, but MDC1 loss was rare in ER+ ILC. ER:MDC1 interaction and MDC1 coregulator functions may underlie ER function in ILC and serve as targets to overcome antiestrogen resistance in ILC. IMPLICATIONS: MDC1 has novel ER coregulator activity in ILC, which may underlie ILC-specific ER functions, estrogen response, and antiestrogen resistance.

Estrogen Regulation of mTOR Signaling and Mitochondrial Function in Invasive Lobular Carcinoma Cell Lines Requires WNT4.

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.

The Capacity of the Ovarian Cancer Tumor Microenvironment to Integrate Inflammation Signaling Conveys a Shorter Disease-free Interval.

PURPOSE: Ovarian cancer has one of the highest deaths to incidence ratios across all cancers. Initial chemotherapy is effective, but most patients develop chemoresistant disease. Mechanisms driving clinical chemo-response or -resistance are not well-understood. However, achieving optimal surgical cytoreduction improves survival, and cytoreduction is improved by neoadjuvant chemotherapy (NACT). NACT offers a window to profile pre- versus post-NACT tumors, which we used to identify chemotherapy-induced changes to the tumor microenvironment. EXPERIMENTAL DESIGN: We obtained matched pre- and post-NACT archival tumor tissues from patients with high-grade serous ovarian cancer (patient, n = 6). We measured mRNA levels of 770 genes (756 genes/14 housekeeping genes, NanoString Technologies), and performed reverse phase protein array (RPPA) on a subset of matched tumors. We examined cytokine levels in pre-NACT ascites samples (n = 39) by ELISAs. A tissue microarray with 128 annotated ovarian tumors expanded the transcriptional, RPPA, and cytokine data by multispectral IHC. RESULTS: The most upregulated gene post-NACT was IL6 (16.79-fold). RPPA data were concordant with mRNA, consistent with elevated immune infiltration. Elevated IL6 in pre-NACT ascites specimens correlated with a shorter time to recurrence. Integrating NanoString (n = 12), RPPA (n = 4), and cytokine (n = 39) studies identified an activated inflammatory signaling network and induced IL6 and IER3 (immediate early response 3) post-NACT, associated with poor chemo-response and time to recurrence. CONCLUSIONS: Multiomics profiling of ovarian tumor samples pre- and post-NACT provides unique insight into chemo-induced changes to the tumor microenvironment. We identified a novel IL6/IER3 signaling axis that may drive chemoresistance and disease recurrence.

Differential Regulation and Targeting of Estrogen Receptor α Turnover in Invasive Lobular Breast Carcinoma.

Invasive lobular breast carcinoma (ILC) accounts for 10% to 15% of breast cancers diagnosed annually. Evidence suggests that some aspects of endocrine treatment response might differ between invasive ductal carcinoma (IDC) and ILC, and that patients with ILC have worse long-term survival. We analyzed The Cancer Genome Atlas dataset and observed lower levels of ESR1 mRNA (P = 0.002) and ERα protein (P = 0.038) in ER+ ILC (n = 137) compared to IDC (n = 554), and further confirmed the mRNA difference in a local UPMC cohort (ILC, n = 143; IDC, n = 877; P < 0.005). In both datasets, the correlation between ESR1 mRNA and ERα protein was weaker in ILC, suggesting differential post-transcriptional regulation of ERα. In vitro, 17ß-estradiol (E2) decreased the rate of degradation and increased the half-life of ERα in ILC cell lines, whereas the opposite was observed in IDC cell lines. Further, E2 failed to induce robust ubiquitination of ERα in ILC cells. To determine the potential clinical relevance of these findings, we evaluated the effect of 2 selective estrogen receptor downregulators (SERDs), ICI 182,780 and AZD9496, on ERα turnover and cell growth. While ICI 182,780 and AZD9496 showed similar effects in IDC cells, in ILC cell lines, AZD9496 was not as effective as ICI 182,780 in decreasing ERα stability and E2-induced proliferation. Furthermore, AZD9496 exhibited partial agonist activity in growth assays in ILC cell lines. Our study provides evidence for a distinct ERα regulation by SERDs in ILC cell lines, and therefore it is important to include ILC models into preclinical and clinical testing of novel SERDs.