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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.

Histological description of tumors and Cancer antigen 125 (CA125) levels for each patient over time. Units = U/mL

Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin–mediated adherens junctions. Despite displaying unique histologic and clinical features, ILC still remains a chronically understudied disease, with limited knowledge gleaned from available laboratory research models. Here we report a comprehensive 2D and 3D phenotypic characterization of four estrogen receptor–positive human ILC cell lines: MDA-MB-134, SUM44, MDA-MB-330, and BCK4. Compared with the IDC cell lines MCF7, T47D, and MDA-MB-231, ultra-low attachment culture conditions revealed remarkable anchorage independence unique to ILC cells, a feature not evident in soft-agar gels. Three-dimensional Collagen I and Matrigel culture indicated a generally loose morphology for ILC cell lines, which exhibited differing preferences for adhesion to extracellular matrix proteins in 2D. Furthermore, ILC cells were limited in their ability to migrate and invade in wound-scratch and transwell assays, with the exception of haptotaxis to Collagen I. Transcriptional comparison of these cell lines confirmed the decreased cell proliferation and E-cadherin–mediated intercellular junctions in ILC while uncovering the induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion channels, drug metabolism, and alternative cell adhesion molecules such as N-cadherin, some of which were differentially regulated in ILC versus IDC tumors. Altogether, these studies provide an invaluable resource for the breast cancer research community and facilitate further functional discoveries toward understanding ILC, identifying novel drug targets, and ultimately improving the outcome of patients with ILC.Significance: These findings provide the breast cancer research community with a comprehensive assessment of human invasive lobular carcinoma (ILC) cell line signaling and behavior in various culture conditions, aiding future endeavors to develop therapies and to ultimately improve survival in patients with ILC. Cancer Res; 78(21); 6209–22. ©2018 AACR.

Lists of upregulated and downregulated pathways in ILC versus IDC cell lines.

Purpose: High-grade serous ovarian cancer (HGSOC) is an aggressive disease with few available targeted therapies. Despite high expression of estrogen receptor-alpha (ERα) in approximately 80% of HGSOC and some small but promising clinical trials of endocrine therapy, ERα has been understudied as a target in this disease. We sought to identify hormone-responsive, ERα-dependent HGSOC.Experimental Design: We characterized endocrine response in HGSOC cells across culture conditions [ two-dimensional (2D), three-dimensional (3D), forced suspension] and in patient-derived xenograft (PDX) explants, assessing proliferation and gene expression. Estrogen-regulated transcriptome data were overlapped with public datasets to develop a comprehensive panel of ERα target genes. Expression of this panel and ERα H-score were assessed in HGSOC samples from patients who received endocrine therapy. Time on endocrine therapy was used as a surrogate for clinical response.Results: Proliferation is ERα-regulated in HGSOC cells in vitro and in vivo, and is partly dependent on 3D context. Transcriptomic studies identified genes shared by cell lines and PDX explants as ERα targets. The selective ERα downregulator (SERD) fulvestrant is more effective than tamoxifen in blocking ERα action. ERα H-score is predictive of efficacy of endocrine therapy, and this prediction is further improved by inclusion of target gene expression, particularly IGFBP3.Conclusions: Laboratory models corroborate intertumor heterogeneity of endocrine response in HGSOC but identify features associated with functional ERα and endocrine responsiveness. Assessing ERα function (e.g., IGFBP3 expression) in conjunction with H-score may help select patients who would benefit from endocrine therapy. Preclinical data suggest that SERDs might be more effective than tamoxifen. Clin Cancer Res; 23(14); 3802–12. ©2017 AACR.

Detailed Clinical History. Supplemental figure legend Supplemental table legends.

Supplementary Text

Purpose: The current study evaluated associative effects of breast cancer cells with the tumor microenvironment and its influence on tumor behavior.Experimental Design: Formalin-fixed, paraffin-embedded tissue and matched protein lysates were evaluated from two independent breast cancer patient datasets (TCGA and MD Anderson). Reverse-phase protein arrays (RPPA) were utilized to create a proteomics signature to define breast tumor subtypes. Expression patterns of cell lines and normal breast tissues were utilized to determine markers that were differentially expressed in stroma and cancer cells. Protein localization and stromal contents were evaluated for matched cases by imaging.Results: A subtype of breast cancers designated “Reactive,” previously identified by RPPA that was not predicted by mRNA profiling, was extensively characterized. These tumors were primarily estrogen receptor (ER)-positive/human EGF receptor (HER)2-negative, low-risk cancers as determined by enrichment of low-grade nuclei, lobular or tubular histopathology, and the luminal A subtype by PAM50. Reactive breast cancers contained high numbers of stromal cells and the highest extracellular matrix content typically without infiltration of immune cells. For ER-positive/HER2-negative cancers, the Reactive classification predicted favorable clinical outcomes in the TCGA cohort (HR, 0.36; P < 0.05).Conclusions: A protein stromal signature in breast cancers is associated with a highly differentiated phenotype. The stromal compartment content and proteins are an extended phenotype not predicted by mRNA expression that could be utilized to subclassify ER-positive/HER2-negative breast cancers. Clin Cancer Res; 22(20); 5068–78. ©2016 AACR.

Classification of RPPA platform antibodies as "stromal" or ER-pos "cancer" enriched (Excel sheet)

Supplementary File 3 contains the available pre- and post-treatment CA-125 data for our patient cohort.

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.

Supplementary File 1 contains the genes included in the "EndoRx" NanoString code set.

This document contains the supplementary figures for the manuscript.

Proteins differentially expressed in Reactive subtype (Excel sheet).

XLSX file - 20K, Description and specifics regarding the ILC E2 geneset.

PDF file - 89K, Expression of genes in the ILC E2 geneset in MCF-7 and MM134 cells following treatment with anti-estrogens.

PDF file - 53K, E2 regulation of ER target genes in ILC cells.

PDF file - 137K

PDF file - 165K, Cell viability assays following kinase inhibition.

Invasive lobular carcinoma (ILC) is a histologic subtype of breast cancer that is frequently associated with favorable outcomes, as approximately 90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that patients with ILC receiving adjuvant endocrine therapy may not benefit as much as patients with invasive ductal carcinoma. On the basis of these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome via gene expression microarray analyses and ER ChIP-Seq, and to examine response to endocrine therapy. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. We identified 915 genes that were uniquely E2 regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also E2 regulated in vivo in HCI-013. MM134 cells were de novo tamoxifen resistant and were induced to grow by 4-hydroxytamoxifen, as well as other antiestrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required fibroblast growth factor receptor (FGFR)-1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance. Cancer Res; 74(5); 1463–74. ©2014 AACR.

XLSX file - 2042K, Lists for genes regulated by E2 in MM134 and SUM44 cells, and for categories of genes based on cell type specificity.

PDF file - 52K, Motif analysis of ER binding sites identified in MM134 by ChIP-seq.

XLSX file - 17K, Expression data from estrogen deprivation studies using PDX HCI-013.

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.

Objective: Review a single institution’s vestibular schwannoma (VS) microsurgery experience to determine (1) correlations between demographics, comorbidities, and/or surgical approach on hospital length of stay (LOS) and discharge disposition and (2) trends in surgical approach over time. Methods: Retrospective case series from a multidisciplinary skull base program at a tertiary care, academic hospital. All adult (>18 years) patients undergoing primary microsurgery for VS between 2008 and 2018 were included. Results: A total of 147 subjects were identified. Surgical approach was split between middle fossa (MF) (16%), retrosigmoid (RS) (35%), and translabyrinthine (TL) (49%) craniotomies. For the 8% of patients had other than routine (OTR) discharge. Mean LOS was significantly longer for patients undergoing RS than either MF or TL. Brainstem compression by the tumor was associated with longer LOS as were diagnoses of chronic obstructive pulmonary disease (COPD) and peripheral vascular disease (PVD). For all discharges, the 40 to 50- and 50 to 60-year-old subgroups had significantly shorter LOS than the 70-years-and-older patients. For the 92% of patients routinely discharged, there was a significantly shorter LOS in the 40 to 50-year-olds compared to the 70-years-and-older patients. There was a significant shift in surgical approach from RS to TL over the study period. Conclusion: Over 90% of VS microsurgery patients were routinely discharged with a median hospital LOS of 3.2 days, both of which are consistent with published data. There is an inverse relationship between age and LOS with patients older than 70 years having significantly longer LOS. Brainstem compression, COPD, PVD, and the RS approach negatively affect LOS. Level of Evidence 4

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.

Quality improvement with before and after evaluation of the intervention.To evaluate postoperative opioid utilization at a high-volume tertiary referral center following implementation of an opioid reduction protocol for simple outpatient neurosurgical procedures.The opioid epidemic has been well-publicized both in the scientific and lay press over the last few years. As a response to this crisis many state-wide and national medical groups have sought to develop opioid prescribing guidelines for both acute and chronic pain states. Some guidelines have studied opioid prescribing in orthopedic procedures but have primarily limited their recommendations to simple outpatient orthopedic joint procedures. Although, it is not clear that these opioid prescribing reductions are directly translatable to neurosurgical procedures.We implemented an opioid reduction protocol geared towards the postoperative management for simple outpatient neurosurgical procedures and measured the effect on number of pills and total morphine equivalent dose (MED) prescribed, postoperative readmissions, refill requests, and conversion to long-term opiate use.Our study population was 246 patients, with 109 patients in the pre-intervention (PRE) group and 137 patients in the post-intervention (POST) group. The vast majority of patients in both groups were discharged with an opioid prescription (93% PRE, 91% POST, P = 0.87). The POST group had significantly lower total discharge opioid medication quantity (52 tabs PRE, 27 tabs POST, P 0.001), discharge day MED (51.3 PRE, 45.3 POST, P = 0.01), and total discharge MED (287 PRE, 149 POST, P 0.001).A standardized discharge protocol for postoperative neurosurgery can lead to significant reductions in opioid discharge quantity without compromising patient safety or increasing the utilization of hospital resources through readmissions, refill requests, or clinic phone calls. This study provides an example of a feasible and effective discharge prescription regimen that may be generalizable to some of the most common outpatient neurosurgical procedures.3.

OBJECTIVE: To report the 1-year pre and postoperative analgesic use in patients undergoing primary surgery for adult spinal deformity (ASD) and assess risk factors for chronic postoperative opioid use.METHODS: Patients > 18 years undergoing primary instrumented surgery for ASD in Denmark between 2006 and 2016 were identified in the Danish National Patient Registry. Information on analgesic use were obtained from the Danish National Health Service Prescription Database. Use of analgesics was calculated one year before and after surgery for each patient, per quarter (-Q4 to -Q1 before and Q1 to Q4 after). Users were defined as patient with one or more prescriptions in the given quarter.RESULTS: We identified 892 patients. Preoperatively, 28% (n = 246) of patients were opioid users in -Q4 and 33% (n = 295) in -Q1. Postoperatively, 85% (n = 756) of patients were opioid users in Q1 and 31% (n = 280) in Q4. Proportions of users of other analgesics (paracetamol, antidepressants, and anticonvulsants) were stable before and after surgery. Use of nonsteroidal anti-inflammatory drug decreased postoperatively by 40% (-Q1 vs. Q4). 26% of patients had chronic preoperative opioid use (one or more prescriptions in each -Q2 and -Q1) and 24% had chronic postoperative use (prescription each of Q1-Q4). Multivariate logistic regression analysis showed age increment per 10 years and preoperative chronic opioid use as risk factors for chronic postoperative opioid use.CONCLUSION: One year after ASD surgery, opioid use was not reduced compared to preoperative usage.

Invasive lobular breast carcinoma (ILC) accounts for 10-15% of breast cancers diagnosed annually. Evidence suggests that endocrine treatment response might differ between invasive ductal carcinoma (IDC) and ILC, and that patients with ILC have worse long-term survival. ILCs are more likely to be estrogen receptor alpha (ER/ERα) positive (90-95%) compared to IDCs (60-70%). We investigated whether there are differences in ER protein steady state levels and/or turn-over rates in ILC that might explain differences in response to endocrine therapy. Analysis of the TCGA dataset revealed lower levels of ESR1 mRNA (P = 0.002) and ERα protein (P = 0.038) in ER+ ILC (n = 137) compared to ER+ IDC (n = 554). Supporting this observation, analysis of tumors from patients treated at UPMC Magee-Women’s Hospital (ILC, n = 143; IDC, n = 877), showed lower ESR1 mRNA (P < 0.005) in ILC and similar ERα protein levels in both subtypes. In both datasets, the correlation between ESR1 mRNA and ERα protein was significantly weaker in ILC suggesting differential post-transcriptional regulation of ER. This was confirmed in in vitro studies showing 17β-estradiol (E2) decreased the rate of degradation and increased half-life of ERα in MDA-MB-134-VI and SUM44PE ILC cell lines, whereas the opposite was observed in IDC cell lines. Further analysis of MDA-MB-134-VI cells revealed that E2 downregulated ubiquitination pathway genes and failed to induce ubiquitination of ERα. To determine potential clinical relevance of our findings, we evaluated the effect of two selective estrogen receptor down-regulators (SERDs) on ERα turnover and E2-induced proliferation of IDC and ILC cell lines. While fulvestrant and AZD9496, a novel orally bioavailable SERD, showed similar effects in IDC cell lines, AZD9496 was less effective than fulvestrant in decreasing ERα protein stability and E2-induced proliferation in ILC cells. Furthermore, AZD9496 exhibited partial agonist activity in growth and gene expression assays in the absence of ligand. Collectively, our data provide evidence for distinct ligand-induced ERα turnover in ILC cell lines. In addition, the novel SERD AZD9496 displays partial agonist activity in ILC cells, and further studies should address whether these observations are causally linked. These results provide a strong rationale for inclusion of ILC subgroups into preclinical and clinical testing of SERDs. Citation Format: Jennifer M Atkinson, Sreeja Sreekumar, Kevin M Levine, Matthew J Sikora, Jian Chen, David J Dabbs, Carolin Meier, Ahmed Basudan, Nilgun Tasdemir, David Boone, Priscilla F McAuliffe, Rachel C Jankowitz, Adrian V Lee, Steffi Oesterreich. Unique estrogen receptor alpha turnover, regulation and targeting in invasive lobular breast carcinoma [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-21-03.

Background: There is growing awareness of the unique etiology, biology, clinical presentation and progression of Invasive lobular breast cancer (ILC), but additional research is needed to assure translation of findings into management and treatment guidelines. We performed a survey to: 1) analyze the landscape of the current understanding of ILC, and 2) identify consensus research questions on ILC. Methods: The IRB-approved survey was developed with input from representatives of three major stakeholder groups - breast cancer clinicians/researchers, laboratory-based researchers, and advocates/patients. We fielded the survey from March to May 2022 using targeted email and via social media. Results: 1,774 participants answered at least one question and 1,310 finished the survey. Participants are from 66 countries from all continents (except Antarctica). Respondents self-identified as clinicians (mostly medical oncologists and surgeons) (N=413), researchers (N=376), and breast cancer patients (1,121), with some belonging to more than one category. 26% of the patients who participated in the survey belong to advocate groups. Only 46% of clinicians reported being confident in describing the differences between ILC and no special type (NST) (invasive ductal) breast cancer. Knowledge of histology was seen as important (73%), affecting their treatment decisions (51%), and refined treatment guidelines would be valuable for patients with ILC in the future (76%). 85% of clinicians have never powered a clinical trial to allow subset analysis for histological subtypes, but the majority would consider it. 88% would participate in a consortium to conduct clinical trials on ILC. The top two most important research questions were: 1) determining mechanisms of endocrine resistance, and, 2) identifying novel therapeutic targets, repurposing existing drugs and progressing them to clinical trials. Of the researchers, 48% reported being confident in describing differences between ILC and NST. They reported that ILCs are inadequately presented in large genomic data sets (52%), and that ILC models are insufficient (42%). Only 13% of respondents have inadequate access to tissue or blood from patients with ILC. The top two most important research questions identified by the laboratory researchers overlapped with those identified by the clinicians, i.e. understanding of endocrine resistance and identifying novel drugs that can be tested in clinical trials. The majority of patients (52%) thought that their health care providers did not explain unique features of ILC, and that in general communication was limited. When asked about top research question, they chose: 1) Improvement of ILC screening/early detection, and, 2) Identifying new and specific imaging tools for ILC. When comparing top priority topics across six research domains, there was a high degree of consistency, especially among clinicians and researcher, but less so when compared with the breast cancer patients (Table 1). Conclusion: In summary, we have gathered timely and representative information from an international community of clinicians, researchers, and patients/advocates that we expect will lay the foundation for a community-informed collaborative research agenda, with the goal of improving the management and personalizing treatment for patients with ILC. Table 1. Ratings by all three stakeholder groups of the most critical and impactful ILC research topics. Top box scores between stakeholder groups were compared using chi-square analysis. Citation Format: Steffi Oesterreich, Leigh Pate, ADRIAN V. LEE, Rachel C. Jankowitz, Patrick Derksen, Rita Mukhtar, Otto Metzger, Matthew J. Sikora, Christopher Li, Christos Sotiriou, Gary Ulaner, Jorge Reis-Filho, Nancy E Davidson, Karen Van Baelen, Laurie Hutcheson, Siobhan Freeney, Flora Migyanka, Claire Turner, Todd Bear, Christine Desmedt. An international survey on invasive lobular breast cancer (ILC) reveals gaps in knowledge and top priority research areas [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-05-10.

BothTP53andESR1mutations 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 betweenTP53andESR1mutations in metastatic breast cancers; however, mechanisms underlying this intriguing clinical observation remain largely understudied and unknown. Here, we explored the interplay betweenTP53andESR1mutations 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.ESR1mutant tumors do not exhibit differential p53 pathway activity, whereas we identified attenuated ER activity and expression inTP53mutant 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 ofTP53andESR1mutations 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 betweenESR1andTP53mutations, providing further insights and testable hypotheses of the molecular interplay between these two pivotal genes in ER+ MBC.

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.

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.

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.

Invasive lobular carcinoma (ILC) is the most common histological 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 sensitive to anti-estrogen therapies. We hypothesized that ILC-specific ER transcriptional co-regulators mediate ER functions in ILC and anti-estrogen resistance, and profiled ER-associated proteins by mass spectrometry. Three ER+ ILC cell lines, MDA MB 134VI, SUM44PE, and BCK4, were compared to published data from ER+ invasive ductal carcinoma (IDC) cell lines, and we examined whether siRNA knockdown of identified proteins suppressed ER-driven proliferation in ILC cells. This approach found mediator of DNA damage checkpoint 1 (MDC1), a key tumor suppressor in DNA damage response (DDR), as a putative novel ER co-regulator in ILC. We confirmed ER:MDC1 interaction was specific to ILC cell lines versus IDC cells, and found MDC1 knockdown suppressed ILC cell proliferation and suppressed tamoxifen resistance in MDA MB 134VI. Using RNA-sequencing, we found that in ILC cells, MDC1 knockdown broadly dysregulates the estrogen-driven ER transcriptome, with ER:MDC1 target genes enriched for hormone-response-elements in their promoter regions. Importantly, our data are inconsistent with MDC1 regulating ER via MDC1 DDR and tumor suppressor functions, but instead suggest a novel oncogenic role for MDC1 in mediating ER transcriptional activity as a co-regulator. Supporting this, in breast tumor tissue microarrays MDC1 protein was frequently low or absent in IDC or ER-ILC, but MDC1 loss is rare in ER+ ILC. ER:MDC1 interaction and MDC1 co-regulator functions may underlie cell type-specific ER functions in ILC, and serve as important biomarkers and therapeutic targets to overcome anti-estrogen resistance in ILC.

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.

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.

Invasive Lobular Carcinoma (ILC) is the 2nd most common histotype of breast cancer, but is critically understudied. ~95% of ILC are estrogen receptor (ER) positive, and previous studies demonstrate the importance of estrogen in ILC etiology. However, retrospective studies show that anti-estrogens are substantially less effective in ILC than in ER+ Invasive Ductal Carcinoma (IDC). This strongly suggests that regulation of ER function is unique in ILC, and we hypothesize that this is due to an ILC-specific cohort of ER co-regulators. We performed Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) to determine ILC-specific ER-interacting proteins, and identified Mediator of DNA Damage Checkpoint 1 (MDC1) as a novel ER co-regulator in ILC cells. We confirmed ER:MDC1 interaction by co-immunoprecipitation and proximity ligation assays (PLA); interaction was specifically observed in ILC cell lines but not IDC cell lines. Consistent with co-regulator function, we found MDC1 is essential for ER-driven proliferation of ILC cells. MDC1 knockdown dysregulates transcription of ER target genes in ILC cells (e.g. IGFBP4, WNT4). Moreover, RNA-seq analysis showed that in ILC cell line MDA MB 134VI, >50% of ER target genes require MDC1 for their regulation. To understand how MDC1 controls ER transcriptional activity, we performed ChIP-qPCR and found that MDC1 controls ER binding to DNA in ILC cells. Further, MDC1 controls binding of the pioneer factor FOXA1 to DNA, and Dual PLA studies of ER:MDC1 and ER:FOXA1 interaction revealed that MDC1 knockdown decreased ER:FOXA1 interaction. MDC1 canonically functions in DNA damage response, but our preliminary data suggest MDC1 is decoupled from its canonical role in DDR in the context of ER co-regulator activity in ILC cells. Together, these data suggest MDC1, independent of its role in DDR, acts as a novel ER co-regulator in ILC and regulates ER:DNA binding and ER transcriptional function to drive ILC cell proliferation and survival.

Ovarian cancer has one of the highest deaths to incidence ratios across all cancers. Initial chemotherapy is typically effective, but most patients will develop chemo-resistant disease. Mechanisms driving clinical chemo-response and -resistance in ovarian cancer are not well understood. However, achieving optimal surgical cytoreduction improves survival, and cytoreduction is improved by neoadjuvant platinum/taxane-based chemotherapy (NACT). NACT offers a window to profile pre-versus post-therapy tumor specimens, which we used to identify chemotherapy-induced changes to the tumor microenvironment. We hypothesized changes in the immune microenvironment correlate with tumor chemo-response and disease progression. 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 (NanoString), and performed reverse phase protein array (RPPA) on a subset of matched tumors. We examined cytokine levels in additional pre-NACT ascites samples (n=39) by multiplex ELISA. A tissue microarray with 128 annotated ovarian tumors expanded the transcriptional, RPPA, and cytokine data by multi-spectral immunohistochemistry. In NanoString analyses, transcriptional profiles segregated based on pre- and post-NACT status. The most upregulated gene post-NACT wasIL6(17.1-fold, adjusted p = 0.045). RPPA data were highly concordant with mRNA, consistent with elevated immune infiltration. Elevated IL-6 in pre-NACT ascites specimens correlated with a shorter time to recurrence. Integrating NanoString, RPPA, and cytokine studies identified an activated inflammatory signaling network and inducedIL6andIER3(Immediate Early Response 3) post-NACT, associated with poor chemo-response and decreased time to recurrence. Taken together, multi-omic profiling of ovarian tumor samples before and after NACT provides unique insight into chemo-induced changes to the tumor and microenvironment. We integrated transcriptional, proteomic, and cytokine data and identified a novel IL-6/IER3 signaling axis through increased inflammatory signaling which may drive ovarian cancer chemo-resistance.

© 2019 Elsevier Inc. Background: Deep brain stimulation (DBS) surgery is an option for patients experiencing medically resistant neurologic symptoms. DBS complications are rare; finding significant predictors requires a large number of surgeries. Machine learning algorithms may be used to effectively predict these outcomes. The aims of this study were to 1) investigate preoperative clinical risk factors and 2) build machine learning models to predict adverse outcomes. Methods: This multicenter registry collected clinical and demographic characteristics of patients undergoing DBS surgery (n = 501) and tabulated occurrence of complications. Logistic regression was used to evaluate risk factors. Supervised learning algorithms were trained and validated on 70% and 30%, respectively, of both oversampled and original registry data. Performance was evaluated using area under the receiver operating characteristics curve (AUC), sensitivity, specificity, and accuracy. Results: Logistic regression showed that the risk of complication was related to the operating institution in which the surgery was performed (odds ratio [OR] = 0.44, confidence interval [CI] = 0.25–0.78), body mass index (OR = 0.94, CI = 0.89–0.99), and diabetes (OR = 2.33, CI = 1.18–4.60). Patients with diabetes were almost 3× more likely to return to the operating room (OR = 2.78, CI = 1.31–5.88). Patients with a history of smoking were 4× more likely to experience postoperative infection (OR = 4.20, CI = 1.21–14.61). Supervised learning algorithms demonstrated high discrimination performance when predicting any complication (AUC = 0.86), a complication within 12 months (AUC = 0.91), return to the operating room (AUC = 0.88), and infection (AUC = 0.97). Age, body mass index, procedure side, gender, and a diagnosis of Parkinson disease were influential features. Conclusions: Multiple significant complication risk factors were identified, and supervised learning algorithms effectively predicted adverse outcomes in DBS surgery.

Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin–mediated adherens junctions. Despite displaying unique histologic and clinical features, ILC still remains a chronically understudied disease, with limited knowledge gleaned from available laboratory research models. Here we report a comprehensive 2D and 3D phenotypic characterization of four estrogen receptor–positive human ILC cell lines: MDA-MB-134, SUM44, MDA-MB-330, and BCK4. Compared with the IDC cell lines MCF7, T47D, and MDA-MB-231, ultra-low attachment culture conditions revealed remarkable anchorage independence unique to ILC cells, a feature not evident in soft-agar gels. Three-dimensional Collagen I and Matrigel culture indicated a generally loose morphology for ILC cell lines, which exhibited differing preferences for adhesion to extracellular matrix proteins in 2D. Furthermore, ILC cells were limited in their ability to migrate and invade in wound-scratch and transwell assays, with the exception of haptotaxis to Collagen I. Transcriptional comparison of these cell lines confirmed the decreased cell proliferation and E-cadherin–mediated intercellular junctions in ILC while uncovering the induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion channels, drug metabolism, and alternative cell adhesion molecules such as N-cadherin, some of which were differentially regulated in ILC versus IDC tumors. Altogether, these studies provide an invaluable resource for the breast cancer research community and facilitate further functional discoveries toward understanding ILC, identifying novel drug targets, and ultimately improving the outcome of patients with ILC. Significance: These findings provide the breast cancer research community with a comprehensive assessment of human invasive lobular carcinoma (ILC) cell line signaling and behavior in various culture conditions, aiding future endeavors to develop therapies and to ultimately improve survival in patients with ILC. Cancer Res; 78(21); 6209–22. ©2018 AACR.

To investigate changes in estrogen receptor alpha (ERα) signaling during progression of endometriosis to endometriosis-associated ovarian cancer (EAOC) as a driver of malignant transformation. We procured tissue samples of normal endometrium, endometriosis (benign, atypical, concurrent with EAOC), and EAOC. We evaluated expression of a 236-gene signature of estrogen signaling. ANOVA and unsupervised clustering were used to identify gene expression profiles across disease states. These profiles were compared to profiles of estrogen regulation in cancer models from the Gene Expression Omnibus (GEO). Gene Set Enrichment Analysis (GSEA) was performed to determine whether gene expression in EAOC was consistent with ERα activity. ANOVA revealed 158 differentially expressed genes (q

Background Invasive Lobular Carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. Distinctive properties of ILC include growth patterns, metastatic behavior, and receptor status (almost universally estrogen receptor (ER) positive). Our lab recently generated six long-term estrogen deprivation (LTED) models of ILC cells and performed RNA-Sequencing to identify differentially expressed genes compared to their parental cells cultured with estrogen. We overlapped these results with a previously published microarray dataset and found that FGFR4 is the most consistently overexpressed gene in the setting of acquired resistance to endocrine therapy in ILC cells. Hypothesis FGFR4 is an important mediator of resistance to endocrine therapy in ILC. Methods To study the role of FGFR4 in vitro, we used multiple shRNAs and specific small molecule inhibition for growth assays of ILC cells. To study the role of FGFR4 in de novo resistance to endocrine therapy, we collected 129 well curated ER+ ILC tumor specimens and performed gene expression analysis on the pre-treatment samples using a custom NanoString panel. To study the role of FGFR4 in acquired resistance, we collected over 50 pairs of primary-metastatic ER+ tumors and performed exon capture based RNA-Sequencing. Results FGFR4 inhibition decreases parental and LTED ILC cell growth in classic 2D conditions, in the setting of ultra-low attachment, and in colony formation assays. The LTED cells, with higher FGFR4 expression, are more sensitive to its inhibition. For the parental cells, combination FGFR4 and ER-targeting drugs results in synergistic decreases in growth. In our database of primary ILC clinical samples, increased expression of FGFR4 is predictive of shorter time to distant recurrence. For our collection of 50 paired, primary-metastatic ER+ tissues, FGFR4 expression increases on average >2.5 fold in the metastatic setting, with large gains even in ductal carcinoma cases. Finally, in analyzing recently published cohorts of metastatic tumors, there is a significant enrichment of hotspot FGFR4 mutations in tumors originating in the breast, with >2% of metastatic ILC tumors containing such a mutation. Conclusion FGFR4 may play an important role in both acquired and de novo resistance to endocrine therapy in ILC. Citation Format: Levine KM, Chen J, Sikora MJ, Tasdemir N, Priedigkeit N, Tseng GC, Puhalla SL, Jankowitz RC, Dabbs DJ, McAuliffe PF, Lee AV, Oesterreich S. Combination FGFR4 and ER-targeted therapy for invasive lobular carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-09.

A number of studies have tested the hypothesis that breast cancer patients with low-activity CYP2D6 genotypes achieve inferior benefit from tamoxifen treatment, putatively due to lack of metabolic activation to endoxifen. Studies have provided conflicting data, and meta-analyses suggest a small but significant increase in cancer recurrence, necessitating additional studies to allow for accurate effect assessment. We conducted a retrospective pharmacogenomic analysis of a prospectively collected community-based cohort of patients with estrogen receptor-positive breast cancer to test for associations between low-activity CYP2D6 genotype and disease outcome in 500 patients treated with adjuvant tamoxifen monotherapy and 500 who did not receive any systemic adjuvant therapy. Tumor-derived DNA was genotyped for common, functionally consequential CYP2D6 polymorphisms (*2, *3, *4, *6, *10, *41, and copy number variants) and assigned a CYP2D6 activity score (AS) ranging from none (0) to full (2). Patients with poor metabolizer (AS = 0) phenotype were compared to patients with AS > 0 and in secondary analyses AS was analyzed quantitatively. Clinical outcome of interest was recurrence free survival (RFS) and analyses using long-rank test were adjusted for relevant clinical covariates (nodal status, tumor size, etc.). CYP2D6 AS was not associated with RFS in tamoxifen treated patients in univariate analyses (p > 0.2). In adjusted analyses, increasing AS was associated with inferior RFS (Hazard ratio 1.43, 95% confidence interval 1.00–2.04, p = 0.05). In patients that did not receive tamoxifen treatment, increasing CYP2D6 AS, and AS > 0, were associated with superior RFS (each p = 0.0015). This population-based study does not support the hypothesis that patients with diminished CYP2D6 activity achieve inferior tamoxifen benefit. These contradictory findings suggest that the association between CYP2D6 genotype and tamoxifen treatment efficacy is null or near null, and unlikely to be useful in clinical practice.