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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

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.

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.

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, 17β-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.

Retrospective analysis. Analysis of a standardized, pre-surgical psychological evaluation program for complex spine surgery. Adult spinal deformity (ASD) patients have a high rate of comorbid mental health conditions. Although there is a body of literature demonstrating the impact of psychological factors, including anxiety and depression, on spine surgery outcome, it is estimated that spine surgeons utilize a psychological assessment only about one third of the time prior to a patient’s spine surgery. At this time, there is not a widely reported pre-surgical psychological evaluation program for ASD patients. 129 consecutive complex spine surgery candidates receiving a pre-surgical psychological evaluation were analyzed between January 1st 2014 and December 31st 2018. Based on the available literature and professional experience in our facility, a color code for patients was developed from Green (low psychological or psychosocial co-morbidity) to Red (high psychological or psychosocial co-morbidity). Univariate analysis was used to evaluate between color grades and demographics, mental health disorders and outcomes. 83% of complex spine patients had at least one psychological disorder or psychosocial barrier. Only 17% had a combination of realistic expectations for surgery, a good support plan, and were without a history of mental illness. The pre-surgical psychological color criteria were validated in showing higher rates of major depression, anxiety disorder, and bipolar disorder in moderate to severe color grades (p

STUDY DESIGN Single-institution retrospective cohort study. OBJECTIVE To determine whether prescribing practices at discharge are associated with opioid dependence (OD) in patients undergoing discectomy or laminectomy procedures for degenerative indications. SUMMARY OF BACKGROUND DATA Long-term opioid use in spine surgery is associated with higher healthcare utilization and worse postoperative outcomes. The impact of prescribing practices at discharge within this surgical population is poorly understood. METHODS A query of an administrative database was conducted to identify all patients undergoing discectomy or laminectomy procedures at our high-volume tertiary referral center between 2007 and 2016. For patients included in the analysis, opioid prescription data on admission and discharge were manually abstracted from the electronic health record, including opioid type, frequency, route, and dose, and then converted to daily morphine equivalent dose (MED) values. We defined OD as a consecutive narcotic prescription lasting for at least 90 days within the first 12 months after the index surgical procedure. RESULTS Of the 819 total patients, 499 (60.9%) patients had an active opioid prescription before surgery. Postoperatively, 813 (99.3%) received at least one narcotic prescription within 30 days of index surgery, and 162 (19.8%) continued with sustained opioid use in the 12 months after surgery. In adjusted analysis, patients with OD had a higher incidence of preoperative depression (P = 0.012) and preoperative opioid use (P

Invasive lobular carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. To study acquired resistance to endocrine therapy in ILC, we have recently performed RNA-Sequencing on long-term estrogen deprived cell lines and identified FGFR4 overexpression as a top druggable target. Here, we show that FGFR4 expression also increases dramatically in endocrine-treated distant metastases, with an average fold change of 4.8 relative to the paired primary breast tumor for ILC, and 2.4-fold for invasive ductal carcinoma (IDC). In addition, we now report that FGFR4 hotspot mutations are enriched in metastatic breast cancer, with an additional enrichment for ILC, suggesting a multimodal selection of FGFR4 activation. These data collectively support the notion that FGFR4 is an important mediator of endocrine resistance in ILC, warranting future mechanistic studies on downstream signaling of overexpressed wild-type and mutant FGFR4.

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 poorly understood. 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 S6K. Additionally, independent of mTOR/S6K, ER and WNT4 control levels of MCL-1, which alters mitochondrial function. In this context, WNT4 knockdown caused decreased ATP production and increased mitochondrial fragmentation. WNT4 regulation of both mTOR signaling and MCL-1 were also observed in antiestrogen resistant models of ILC. Further, we identified that highWNT4expression is associated with similar mTOR pathway activation in serous ovarian cancer tumors, suggesting that WNT4 signaling is important in multiple tumor types. The identified downstream pathways offer insight in to WNT4 signaling and represent potential targets to overcome anti-estrogen resistance for patients with ILC.

Although general risk of deep brain stimulation (DBS) therapy has been previously described, application of risk prediction at the individual patient level is still largely at the discretion of a treating physician or a multidisciplinary team. To explore associations between potentially modifiable patient characteristics and common adverse events following DBS surgery, we retrospectively reviewed consecutive adult patients who had undergone new DBS electrode placement surgeries at two high-volume tertiary referral centers between October 1997 and May 2018. Among 501 patients included in the analysis (mean age (SD), 64.6 (10.4) years), 165 (32.9%) were female, 67 (13.4%) had diabetes, 231 (46.1%) had hypertension, 25 (5.0%) were smokers, 27 (5.4%) developed an infection, 15 (3.0%) had intracranial or intraventricular hemorrhage, and 53 (10.6%) had an unplanned return to the operating room. Patients who developed a surgical site infection were more likely to report history of smoking before DBS surgery (16% vs 5%, p = 0.04). There was a trend for patients with hypertension to be at risk for intracranial hemorrhage (p = 0.11). In conclusion, this multicenter study demonstrated an association between preoperative smoking and increased risk of infection following new DBS implantation surgery. Counseling about this risk should be considered in preoperative evaluation of patients who are considering undergoing a DBS procedure.

Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer following invasive ductal carcinoma (IDC). To identify potential genetic drivers of ILC progression, we used NanoString nCounter technology to investigate the DNA copy number (CN) in 70 well-curated primary ILC samples. We confirmed prior observations of frequent amplification of CCND1 (33%), and MYC (17%) in ILC, but additionally identified a substantial subset of ILCs with ESR1 and ERBB2 (19%) amplifications. Of interest, tumors with ESR1 CN gains (14%) and amplification (10%) were more likely to recur compared to those with normal CN. Finally, we observed that MDM4 (MDMX) was amplified in 17% of ILC samples. MDM4 knockdown in TP53 wild-type ILC cell lines caused increased apoptosis, decreased proliferation associated with cell cycle arrest, and concomitant activation of TP53 target genes. Similar effects were seen in TP53 mutant cells, indicting a TP53-independent role for MDM4 in ILC. To conclude, amplification of ESR1 and MDM4 are potential genetic drivers of ILC. These amplifications may represent actionable, targetable tumor dependencies, and thus have potential clinical implications and warrant further study.

Invasive lobular carcinoma (ILC) is an understudied breast cancer subtype. Clinical features of ILC suggest exquisite dependence on estrogen and the estrogen receptor (ER), and that ILC patients should have good outcomes with anti-estrogen treatment. However, relative to other breast cancers, patients with ILC have poorer long-term outcomes and poorer anti-estrogen response. Advances in ILC treatment are hindered by our poor understanding of the distinct biology of ILC, especially regarding ER signaling. We previously identified the Wnt ligand WNT4 as an ILC-specific ER transcriptional target critical for estrogen-induced growth and anti-estrogen resistance in ILC cells. We hypothesize that characterizing the WNT4 signaling network in ILC will identify novel biomarkers and therapeutic targets for ILC patients. Wnt signaling typically requires the enzyme PORCN, leading us to test whether PORCN inhibitors blocked growth of WNT4-dependent ILC cells. However, PORCN inhibition did not phenocopy WNT4 knockdown, suggesting an atypical role for PORCN in WNT4 signaling. WNT4 was over-expressed in a diverse cell line panel including ILC. Conditioned medium was assessed for Wnt secretion and activity, and dependence on Wnt secretion proteins PORCN and WLS. WLS was universally required for Wnt secretion and paracrine signaling. In contrast, WNT4 secretion was PORCN-independent in all models. Surprisingly, secreted WNT4 did not present paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell autonomous non-canonical Wnt signaling activation, independent of secretion, by WNT4. To define downstream cell-autonomous WNT4 signaling in ILC cells, we utilized reverse phase protein arrays (RPPA) and identified ER-mediated protein changes blocked by concurrent WNT4 knockdown. These experiments identified putative WNT4 signaling targets including Akt/mTOR signaling; estrogen treatment activated mTOR signaling, but not Akt, in ILC cells in a WNT4-dependent manner. Consistent with this, ILC cell lines were sensitive to mTOR-targeting inhibitors (eg everolimus) but not PI3K- or Akt-targeting inhibitors. Activation of mTOR by WNT4 was associated with atypical intracellular activation of DVL proteins (Wnt second messengers), as we identified that WNT4 can directly activate DVL independent of membrane receptors. Estrogen regulates ILC cell proliferation and survival by upregulating WNT4, subsequently initiating a novel cell-autonomous WNT4 signaling pathway that leads to mTOR activation. Further understanding of this pathway will develop strategies to target WNT4 signaling therapeutically, as currently available drugs (eg targeting PORCN) are ineffective. PORCN-independent cell-autonomous Wnt signaling may be critical in contexts otherwise considered to have dysfunctional Wnt signaling.

Epithelial ovarian cancer (EOC) has one of the highest deaths 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 versus -resistant cells revealed activation of 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 β-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.

Charcoal-stripped bovine serum (CSS) is a critical reagent in the study of steroid hormones. However, CSS has high lot-to-lot variability, including residual growth factor and steroid hormone content. Assessing and reporting this variability is challenging but may affect experimental outcomes and data reproducibility. We hypothesized that CSS lot variability would affect endocrine response phenotypes in breast cancer cells, and we tested the effects of five individual CSS lots on endocrine response in MCF-7 and MDA MB 134VI (MM134) cells. Based on the effects of antiestrogens on MCF-7 cell proliferation, we defined CSS lots as having complete vs partial hormone deprivation. In partial deprivation CSS, the absolute effects of residual estrogens on cell proliferation were modest, but these effects masked the partial agonist activity of 4-hydroxytamoxifen in MM134 cells. Importantly, this effectively reversed the interpretation of tamoxifen-resistance in MM134 cells. Variable effects of CSS lots on endocrine resistance phenotypes were also observed in MCF-7 cells. In this context, we observed that partial vs complete deprivation CSS allowed for the development of unique early endocrine resistance phenotypes that correlated with the presence or absence of residual estrogenic hormones. We evaluated the methods of CSS preparation and identified factors contributing to the extent of hormone deprivation. Our observations suggest that CSS lot-to-lot variability has substantial effects on endocrine response phenotypes and that this ubiquitous factor in study methodology may confound reproducibility. Renewed vigilance in testing and reporting CSS phenotypes will greatly aid in interpreting and reproducing endocrine response and resistance data by the community.

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.

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 G(0)/G(1). 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.

Epithelial ovarian cancer (EOC) has one of the highest deaths to incidence ratios. High grade serous ovarian carcinoma (HGSOC) is the most common and deadliest EOC histotype because of the lack of secondary 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, more recent studies have observed a significant clinical benefit in non-HRD HGSOCs. However, all HGSOC patients are likely to acquire resistance to PARPi. Therefore, there is an urgent clinical need to better understand PARPi resistance, and to introduce novel combinatorial therapies to overcome PARPi resistance and extend HGSOC disease-free intervals. Utilizing a two BRCA2-mutated and one BRCA-wildtype HGSOC cell lines that are olaparib sensitive, we established resistant cells. Transcriptome analysis of the matched olaparib-sensitive versus resistant cells did not detect BRCA2 reversion mutations, but revealed activation of Wnt/TCF signaling pathway, as TCF transcriptional activity was significantly increased in PARPi-resistant cells. In parallel, forced activation of Wnt signaling in PARPi-sensitive cells via WNT3A stimulation reduced response to PARPi. In a recurrent-HGSOC PARPi insensitive patient-derived xenograft model there was an increase in a Wnt/TCF transcriptional target. PARPi resistant cells were sensitive to inhibition of Wnt signaling using the FDA-approved compound, pyrvinium pamoate, which has been shown to inhibit Wnt signaling. We observed that combining pyrvinium pamoate with olaparib resulted in a significant decrease in tumor burden and number of tumor nodules. This study demonstrates that Wnt signaling can mediate PARPi resistance in HGSOC and provides a clinical rationale for combining PARPi and Wnt inhibitors.

Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, accounting for 10-15% of cases. Though ILC represents a minority of breast cancer patients, ILC affects over 30,000 women annually in the US, making ILC the 6th most common women’s cancer. Despite this incidence, no ILC-specific therapeutic options exist. The majority of ILC are estrogen receptor (ER)-positive (>90%); this and other biomarkers suggest that ILC patients are ideal candidates for endocrine therapy. However, the efficacy of endocrine therapy in ILC is poorly understood since no ILC-specific prospective clinical trial data exist. Recent retrospective analyses suggest that a subset of ILC patients may not benefit from endocrine therapy. Understanding the unique mechanisms of endocrine response and resistance in ILC is critical to improving ILC patient outcomes. We recently identified estrogen-mediated gene expression specific to the ILC cell lines MDA MB 134VI and SUM44PE. ILC-specific ER target genes were enriched for repression by estrogen. Further, tamoxifen regulated estrogen-repressed genes as an agonist in MDA MB 134VI cells, in parallel with tamoxifen-induced growth. Thus, ER-mediated repression of target genes may be critical in maintaining cell growth and survival. Our laboratory previously identified that ER-mediated gene repression in breast cancer cells is mediated by histone deacetylase 7 (HDAC7). E2-induced repression requires specifically HDAC7, not HDAC1-6/8-10. In a panel of breast cancer cell lines, HDAC7 was most strongly expressed in MDA-MB-134VI cells, the only cell line among the panel derived from an ILC. Increased HDAC7 expression in ILC versus invasive ductal carcinoma (IDC) can also be observed in the Cancer Genome Atlas; mean HDAC7 expression in ER-positive tumors is 2-fold higher in ILC (n=148) versus IDC (n=508) (p To model HDAC7 inhibition, we generated stable cell lines carrying inducible HDAC7 shRNA constructs from MCF-7 (IDC) and MDA MB 134VI (ILC). These lines are being used to examine the effects of HDAC7 depletion on estrogen- and tamoxifen-mediated gene expression, cell proliferation, anoikis resistance, and endocrine resistance. Parallel studies using the class IIa HDAC inhibitor MC1568 are being used as proof-of-principle for using HDAC7-specific inhibitors. Additionally, we identified novel splice variants of HDAC7 in breast cancer cells. Based on the mouse homolog Hdac7, these splice variants may alter the ability of HDAC7 to interact with transcription factors, or change its activity as a transcriptional repressor. Our observations suggest that HDAC7 may mediate ER-mediate gene repression in ILC cells, which may be necessary for cell proliferation and endocrine resistance in this breast cancer subtype. Preclinical studies using ILC model systems will identify the role of HDAC7 and specific splice variants in controlling ER-driven gene expression. Further studies using HDAC7-specific inhibitors will determine whether targeting HDAC7 in ILC patients can improve endocrine response and inhibit endocrine resistance. Citation Format: Matthew J Sikora, Steffi Oesterreich. Histone deacetylase HDAC7 is a putative therapeutic target in invasive lobular carcinoma [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-06-01.

Background: Patients with invasive lobular carcinoma (ILC) would be expected to have favorable outcomes compared to patients with invasive ductal carcinoma (IDC) given that ILC is more often hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative, of lower grade, and displays decreased proliferation markers. Based on our preclinical studies showing differential hormone response in HR+ ILC vs. IDC and on recent studies suggesting differences in endocrine treatment response between patients with ILC vs. IDC, we designed a biomarker-driven, neoadjuvant window trial for newly diagnosed women with HR+, HER2-negative ILC. We hypothesize that Ki67 will be reduced by 85% in the fulvestrant arm compared with 60% and 75% reduction in the tamoxifen and anastrozole arms, respectively, and that Ki67 reduction will correlate with alterations in expression of ER and ER-regulated genes. Differential Ki67 effect will serve as a surrogate for outcome of patients with ILC on endocrine therapy. Trial Design: This multicenter study (NCT02206984) will enroll 150 women with HR+ and HER2-negative ILC. A mandatory research breast tumor biopsy will be performed at baseline. Fifty patients will be randomized to each of three open-label treatment arms for 21 days: fulvestrant (two 250 mg IM injections on both day 1 and day 14), anastrozole (1mg orally daily), or tamoxifen (20 mg orally daily). Biomarkers of response will be assessed on baseline and post-treatment tumor tissue. Patients will proceed to definitive surgery on day 21 after study drug exposure, or they will undergo a second research breast core biopsy if further neoadjuvant treatment is planned. Eligibility Criteria: Eligible patients include postmenopausal women with newly diagnosed, HR+, HER2-negative ILC (excluding pleomorphic subtype) measuring ≥ 1cm, with adequate organ function, ECOG PS ≥ 2, and agreeable to baseline research breast tumor biopsy. Specific Aims: The primary endpoint is percent change from baseline to post-treatment Ki67 values in ILC tissue after 21 days of endocrine treatment. Comparisons across study arms will be made using a general linear model adjusting for institutional effect, with 80% power estimated for pairwise comparisons of log2(% staining) between treatment arms, allowing for 10% attrition. Secondary endpoints include post-therapy Ki67, and change in ER and PR protein expression by IHC. Finally, planned correlative studies include evaluation of gene expression, epigenetic markers, and DNA sequence variants in ILC tissues in an effort to identify biomarkers of endocrine response and putative drivers of endocrine resistance in ILC. Target Accrual: This study will be open to enrollment by August 2015 at the University of Pittsburgh. Additional sites will be opened through the Translational Breast Cancer Research Consortium (TBCRC). We anticipate an accrual rate of 8 patients per month. (Funding from Susan G. Komen® and AstraZeneca). Citation Format: Jankowitz RC, McAuliffe PF, Sikora MJ, Butler L, Ahrendt G, Johnson R, Diego E, Bonaventura M, Puhalla S, Lembersky B, Clark B, Brufsky A, Kurland BF, Davidson NE, Dabbs DJ, Oesterreich S. A neoadjuvant window trial of endocrine response in women with invasive lobular carcinoma. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-14.

Background: Invasive lobular breast carcinoma (ILC) accounts for 10-15% of breast cancers diagnosed annually. ILCs are more likely to be positive (90-95%) for ER compared to IDC (60-70%), and there is some evidence that endocrine treatment response might be different in patients with IDC vs ILC. We asked the question whether there were differences in ER protein steady state levels, and/or turn-over rates. Methods: We utilized TCGA dataset to compare ESR1 mRNA and ER protein levels between ER+ ILC (n=137) and IDC (n=554). ER H-scores and ESR1 mRNA levels were analyzed from patients with ER+ ILC (n=143) and IDC (n=877) seen at UPMC Magee Women's Hospital. Correlation analysis with Pearson's (r) and Spearman's rank order coefficient (ρ) was used to study the relationship between mRNA and protein levels. Basal and ligand induced ESR1 mRNA and ER protein expression and turn-over were determined in a panel of estrogen responsive ER+ IDC (MCF-7, T47D and ZR75-1) and ILC (BCK-4, MDA-MB-134 VI (MM134), SUM44PE) cell lines to identify potential mechanisms that can contribute to differential expression of ERα protein. Results: TCGA database analysis revealed significantly lower ESR1 mRNA and ER protein levels in ER+ ILC compared to IDC tumors. Analysis of data from our Magee hospital showed similar ER IHC H-scores for ER+ ILCs and IDCs despite having significantly lower ESR1 mRNA in ILC. In both the study sets, the correlation between ER mRNA and protein levels were found to be significantly weaker in ER+ ILC than IDC suggesting subtype specific increased synthesis and/or stability of the receptor protein. ILC cell lines MM134 and SUM44 have increased levels of ER protein compared to IDC cell lines. Estradiol decreased the levels and half-life of ER protein in all IDC cell lines tested. In MM134 and SUM44PE ILC cell lines, estradiol decreased the rate of degradation of ER and increased its half-life. In MM134 cells, treatment with estradiol induced a dose- and time-dependent increase in ER, which is associated with a sustained level of elevated phosphorylation at Ser 118. Conclusions and ongoing/future studies: The estradiol-induced ER protein stability in a subset of ILC cell lines suggest a possible mechanism leading to weaker ER mRNA-protein correlation in ILC tumors. We currently do not know if and how this observation might be linked to antiestrogen response in ILC. We have recently initiated a TBCRC-supported trial in which we will compare the effect of different endocrine therapies in patients with ILC. Access to pre and post therapy samples will provide the opportunity to study ER levels and its downstream signaling as a function of antiestrogen treatment. Citation Format: Jankowitz RC, Sreekumar S, Levine KM, Meier C, Sikora MJ, Basudan A, Boone D, Dabbs DJ, Jacobsen B, Lee AV, Oesterreich S. Differential regulation of ER protein-turnover in invasive lobular carcinoma cells [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 P4-05-02.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer following invasive ductal carcinoma (IDC) and accounts for 10-15% of all cases. Unlike the masses or lumps formed by IDCs, ILCs grow as small, dyscohesive cells in a single-file pattern within dense layers of extracellular matrix. Paradoxically, while patients with ILC display favorable prognostic and predictive factors (Estrogen Receptor [ER]+, Progesterone Receptor+, HER2-, low Ki67), they present with more frequent long-term recurrences compared to those with IDC, indicative of endocrine resistance. Thus, there is urgent need to investigate genetic drivers of ILC disease progression in order to develop more effective therapeutic strategies and improve patient outcome. To this end, we used the Nanostring platform to measure the expression of 577 copy number variation-associated genes in 131 primary ILC tumors with long-term clinical data. This analysis identified CTTN (Cortactin; cortical actin binding protein) as a candidate ILC driver that exhibited higher expression in tumors from patients with subsequent recurrent (n=33) versus non-current disease (n=98). We further validated high CTTN mRNA/protein expression in human ILC cell lines, tumors and patient-derived xenografts, and CTTN locus (11q13.3) amplification in clinical ILC metastases to the brain, bone and ovaries. In follow-upin vitro studies, RNAi-mediated inhibition of CTTN diminished the adhesion and haptotaxis of human ILC cell lines to Collagen I, as well as impairing their growth in 3D Collagen I culture. To assess the functional role of CTTN in ILC tumor growth and metastasis, we transplanted control and CTTN knockdown human ILC cell lines into the mammary fat pads and ducts of immuno-compromised mice and monitored disease progression via bioluminescence imaging. While CTTN inhibition did not lead to a substantial impact on measurable tumor burden, both CTTN shRNAs resulted in a significant survival benefit in the fat pad model. Ongoing work is aimed at pinpointing the underlying mechanisms of CTTN's role in mediating growth in 3D Collagen I culture in vitro and disease progression in vivo. Collectively, the results of these studies will advance our understanding of ILC disease mechanisms and serve as a pre-clinical basis for improving the clinical outcome of patients with this understudied subtype of breast cancer. Citation Format: Tasdemir N, Sikora MJ, Zhu L, Levine KM, Scott J, Basudan A, Sflomos G, Sreekumar S, Bossart EA, Elishaev E, Chandran UR, Tseng GC, Jankowitz RC, Dabbs DJ, McAuliffe PF, Brisken C, Davidson NE, Oesterreich S. Investigating cortactin as a genetic driver of disease progression in invasive lobular carcinoma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-03.

Invasive lobular carcinoma (ILC) represents ∼10% of newly diagnosed breast tumors, or ∼30,000 cases annually in the US. However, ILC-specific signaling and endocrine responsiveness are not well characterized. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients with similar biomarkers, and that ILC patients may not benefit from adjuvant tamoxifen. We hypothesize that estrogen receptor-alpha (ER) regulated gene expression is unique in ILC cells and drives endocrine resistance. The ER-positive ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of cell growth and ER-regulated gene expression in response to estradiol (E2). To examine the ER-regulated transcriptome, we performed gene expression microarray analyses and ER ChIP-Seq following E2 treatment. In parallel, E2 response was assessed in vivo in the primary ILC xenograft HCI-013. Response to endocrine therapies, tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI), were also examined in ILC cell lines. We observed that E2 induced growth and ER target gene expression in MDA MB 134VI and SUM44PE. We compared our ILC microarray data to published data from ER-positive IDC cell lines (MCF-7, T47D, BT474), and identified 254 genes that were E2-regulated in all 5 cell lines (e.g. GREB1, MYC). 915 genes were E2-regulated only in both ILC cell lines. Consistent with this, roughly half of ER binding sites identified in MDA MB 134VI ChIP-Seq were unique versus published MCF-7 data. We chose a subset of ILC-specific and common E2-regulated genes (n = 107) to assess in vivo using Nanostring gene expression analyses of HCI-013. E2-regulation was observed for 32/107 target genes (30%), suggesting that these genes may be E2-regulated in vivo in ILC patient tumors. Consistent with clinical data, both ILC cell lines presented de novo tamoxifen resistance. SUM44PE were growth-inhibited by ICI, but unaffected by Tam, 4OHT, or Bx. Similarly, ICI blocked E2-induced growth in MDA MB 134VI, but Tam, 4OHT, and Bx acted as partial agonists, inducing ∼25% growth. Partial agonism was not limited to tamoxifen, as other SERMs (e.g. raloxifene) also induced growth. We then measured ER-regulated gene expression in MDA MB 134VI following tamoxifen treatment. Tam, 4OHT, and Bx acted as agonists for 38/107 genes, whereas ICI acted as an antagonist. All 38 genes were E2-repressed targets (e.g. CCNG2), suggesting that ER-mediated gene repression may be critical to tamoxifen-resistance in ILC. Finally, we observed that FGFR1, frequently amplified in ILC, may be critical for ILC cell survival in the presence of tamoxifen. These data support the hypothesis that unique ER-mediated gene expression in ILC cells drives endocrine resistance. The de novo tamoxifen resistance observed in ILC cells may correlate with the worse outcomes in ILC patients recently reported. We hypothesize that genes regulated by tamoxifen as an agonist may play a role in tamoxifen-induced growth or serve as biomarkers of resistance. Targeting growth factor signaling using FGFR1 inhibitors may block survival pathways required by ILC cells and reverse tamoxifen resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-03.

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.

Antiestrogen therapies targeting the function of estrogen receptor (ER) have been the cornerstone of therapy for ER+ breast cancer for decades. However, as long as these therapies have been in use, it has also been evident that response to antiestrogen therapy is not based solely on ER expression but that other factors modify breast cancer antiestrogen response. Such factors may include ER's relatives in the steroid hormone receptor (HR) family, androgen receptor (AR), progesterone receptor (PR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR). A series of recent studies has demonstrated that these HRs are not bystanders in ER signaling but rather can alter ER genomic binding and subsequent control of target gene expression. For example, PR and GR may "reprogram" ER binding to DNA toward PR/GR sites; androgen receptor may reverse ER gene regulation functions or regulate ER DNA binding. Accordingly, modulation of HR function concurrently with antiestrogen therapy can either improve antiestrogen response or mediate antiestrogen resistance. This highlights the critical need to better understand how other HRs influence ER function, in particular in the context of antiestrogen therapy. This review discusses recent insights into the mechanisms by which HRs can modify ER function and antiestrogen response, as well as pharmacological implications for antiestrogen therapies and potential combined endocrine therapies.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0748-7) contains supplementary material, which is available to authorized users.

Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII), and esr2b (ERβI), by the genome-editing technology clustered regularly interspaced short palindromic repeats and its associated nuclease (CRISPR/Cas9). Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double, and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male. In addition, the current study also revealed a unique role for Esr2a in follicle cell proliferation and transdifferentiation, follicle growth, and chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a, and esr2b in fish reproduction.

Background: Invasive lobular carcinoma (ILC) accounts for 10-15% of invasive breast cancers diagnosed annually. There is increasing evidence that endocrine treatment response might differ between Invasive Ductal Carcinoma (IDC) and ILC, and that patients with ILC have worse long-term survival when other prognostic factors are taken into account. One such factor is ER status, which is more likely to be positive in ILC (90-95%) compared to IDC (60-70%). There are few studies that have directly compared mRNA and protein levels between ER+ ILC and ER+ IDC. Hypothesis: Differences in ER protein steady state levels, and/or turn-over rates contribute to differences in endocrine treatment response between patients with ILC vs IDC. Methods: We utilized publicly available TCGA data to compare ER mRNA and protein levels between ER+ ILC (n=184) and IDC (n=534). Correlation analysis with Spearman's rank order coefficient (ρ) was used to study the relationship between mRNA and protein levels. METABRIC data were analyzed to compare ER mRNA levels between ER+ ILC (n=130) and IDC (n=1152). ER H-scores and mRNA levels were also analyzed from patients with ER+ ILC (n=180) and IDC (n=1183) seen at our local UPMC Magee Womens Hospital. Finally, ER mRNA and total protein levels, and RNA and protein turn-over rates were determined in 2 IDC and 2 ILC breast cancer cell lines, using qRT-PCR and immunoblots analysis. Results: Analysis of ESR1 gene expression in the TCGA database revealed significantly lower levels of ER mRNA (Mann-Whitney, p Conclusion: We have provided functional and in silico data that collectively suggest altered ER protein turn-over in ILC compared to IDC. We are currently testing if and how this affects sensitivity of ILC cells to SERDs, and underlying mechanisms. Citation Format: Sreekumar S, Levine K, Sikora MJ, Boone D, Dabbs DJ, Lee AV, Jankowitz RC, Oesterreich S. Differential turnover of estrogen receptor alpha in invasive lobular carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-02.

Objectives Efavirenz-based HIV therapy is associated with breast hypertrophy and gynaecomastia. Here, we tested the hypothesis that efavirenz induces gynaecomastia through direct binding and modulation of the oestrogen receptor (ER). Methods To determine the effect of efavirenz on growth, the oestrogen-dependent, ER-positive breast cancer cell lines MCF-7, T47D and ZR-75-1 were treated with efavirenz under oestrogen-free conditions in the presence or absence of the anti-oestrogen ICI 182,780. Cells treated with 17β-oestradiol in the absence or presence of ICI 182,780 served as positive and negative controls, respectively. Cellular growth was assayed using the crystal violet staining method and an in vitro receptor binding assay was used to measure the ER binding affinity of efavirenz. Results Efavirenz induced growth in MCF-7 cells with an estimated effective concentration for half-maximal growth (EC50) of 15.7 μM. This growth was reversed by ICI 182,780. Further, efavirenz binds directly to the ER [inhibitory concentration for half maximal binding (IC50) of ∼52 μM] at a roughly 1000-fold higher concentration than observed with 17β-oestradiol. Conclusions Our data suggest that efavirenz-induced gynaecomastia may be caused, at least in part, by drug-induced ER activation in breast tissues.

Background and aims: Biomarker use for pancreatic cancer diagnosis has been impaired by a lack of samples suitable for reliable quantitative RT-PCR (qRT-PCR). Fine needle aspirates (FNAs) from pancreatic masses were studied to define potential causes of RNA degradation and develop methods for accurately measuring gene expression. Methods: Samples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phosphate dehydrogenase, and effects on qRT-PCR were determined by using a 150-bp and a 80-bp amplicon for RPS6. Potential causes of and methods to circumvent RNA degradation were studied by using FNAs from a pancreatic cancer xenograft. Results: RNA extracted from pancreatic mass FNAs was extensively degraded. Fragmentation was related to needle bore diameter and could not be overcome by alterations in aspiration technique. Multiplex PCR for glyceraldehyde-3-phosphate dehydrogenase could distinguish samples that were suitable for qRT-PCR. The use of short PCR amplicons (

Objectives: To investigate changes in estrogen receptor–alpha (ERα) signaling during the progression of endometriosis to endometriosis-associated ovarian cancer (EAOC) as a putative driver of malignant transformation. Methods: We procured tissue samples of normal endometrium (n=23), endometriosis (benign, n=19; atypical, n=11; concurrent with EAOC, n=9), and EAOC (n=21). In this cohort, we evaluated expression of a 236-gene signature of estrogen signaling. ANOVA and unsupervised clustering were used to identify distinct gene expression profiles across disease states. These profiles were compared to public profiles of estrogen regulation in preclinical 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. Results: ANOVA revealed 158 differentially expressed genes (q Conclusions: Gene expression data suggest that classical ERα signaling becomes inactivated throughout the progression of endometriosis to EAOC. Rather, the gene expression pattern in EAOC is more consistent with profiles of endocrine resistance. De-repression of ERα target genes such as FGF18 may contribute to evolution of endometriosis into EAOC. Citation Format: Michelle Boisen, Courtney Andersen, Matt Sikora, Tianzhou Ma, George Tseng, Anda Vlad, Esther Elishaev, Uma Chandran, Robert Edwards, Steffi Oesterreich. The evolution of estrogen receptor signaling in the progression of endometriosis to endometriosis-associated ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B23.

Members of the cytochrome P450 (P450) family of drug-metabolizing enzymes are present in the human brain, and they may have important roles in the oxidation of endogenous substrates. The polymorphic CYP2D6 is one of the major brain P450 isoforms and has been implicated in neurodegeneration, psychosis, schizophrenia, and personality traits. The objective of this study was to determine whether the endocannabinoid arachidonoylethanolamide (anandamide) is a substrate for CYP2D6. Anandamide is the endogenous ligand to the cannabinoid receptor CB1, which is also activated by the main psychoactive component in marijuana. Signaling via the CB1 receptor alters sensory and motor function, cognition, and emotion. Recombinant CYP2D6 converted anandamide to 20-hydroxyeicosatetraenoic acid ethanolamide and 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid ethanolamides (EET-EAs) with low micromolar K(m) values. CYP2D6 further metabolized the epoxides of anandamide to form novel dioxygenated derivatives. Human brain microsomal and mitochondrial preparations metabolized anandamide to form hydroxylated and epoxygenated products, respectively. An inhibitory antibody against CYP2D6 significantly decreased the mitochondrial formation of the EET-EAs. To our knowledge, anandamide and its epoxides are the first eicosanoid-like molecules to be identified as CYP2D6 substrates. Our study suggests that anandamide may be a physiological substrate for brain mitochondrial CYP2D6, implicating this polymorphic enzyme as a potential component of the endocannabinoid system in the brain. This study also offers support to the hypothesis that neuropsychiatric phenotype differences among individuals with genetic variations in CYP2D6 could be ascribable to interactions of this enzyme with endogenous substrates.

We showed that tumor cells with wild-type p53 and high levels of Bcl-x(L) are cisplatin resistant but are induced to undergo apoptosis by (-)-gossypol, making this a promising agent for overcoming cisplatin resistance. However, some cells in a population with this phenotype are not killed and continue to survive. Conversely, tumor cells with low Bcl-x(L) expression and either wild type or mutant p53 are relatively cisplatin sensitive and do not exhibit such high levels of apoptosis. However, these do undergo progressive loss of viability after (-)-gossypol that may not be tumor specific. We sought to elucidate the basis for these observations using cDNA microarray analysis of (-)-gossypol treated cisplatin sensitive and resistant cells. Genes in the reactive oxygen species (ROS) pathway were highly upregulated in response to (-)-gossypol. The upregulation was of much greater magnitude in cisplatin sensitive than resistant cells. Staining with an oxidation reporter dye confirmed differential induction of ROS in tumor cells with low Bcl-x(L). As (-)-gossypol is known to undergo oxidative metabolism in vivo, ROS generation may be responsible for both off-target cytotoxicity and inactivation of the drug. In agreement with this hypothesis, oxidation of (-)-gossypol by pre-treatment with hydrogen peroxide eliminated its activity. Combined treatment with the antioxidant N-acetyl-cysteine (NAC) to block ROS increased (-)-gossypol-induced cytotoxicity to tumor but not normal cells. Furthermore, NAC increased the induction of apoptosis as measured by the sub-G(1) population, in both cisplatin sensitive and resistant cells. We postulate that concurrent treatment with antioxidant to block ROS prevents oxidative inactivation of (-)-gossypol and limits off-target toxicity allowing more potent (-)-gossypol-induced anti-tumor activity.

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.

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.

Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer representing 10-15% of newly diagnosed breast tumors. Over 90% of ILC are estrogen receptor (ER)-positive, however, endocrine response and estrogen signaling are not well understood in ILC. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients, and that ILC patients may not benefit from adjuvant tamoxifen. Based on these observations, we hypothesize that ER regulates unique signaling pathways in ILC cells that control growth and endocrine response. To identify putative targets that regulate endocrine response in ILC, we assessed genome-wide ER-mediated gene expression and ER genomic binding in the ILC cell lines MDA MB 134VI (MM134) and SUM44PE (SUM44). Among ILC-specific estrogen-regulated genes, the most strongly induced was the secreted ligand WNT4. Additionally, we identified an ILC-specific ER binding site (ERBS) at WNT4, suggesting that WNT4 is directly controlled by ER in ILC cells. Direct ER-regulation of WNT4 is in contrast to control of WNT4 by progesterone receptor (PR) during mammary gland development; however, further ER ChIP experiments suggest that ILC cells place WNT4 under ER control via unique use of the WNT4 ERBS. Thus, ILC cells may hijack a tightly regulated developmental program to drive estrogen-regulated cell phenotypes. Based on the role of WNT4 in mammary gland growth and expansion, we hypothesized that WNT4 is required for estrogen-induced growth in ILC cells. To test this, we used siRNA to knock down WNT4 in ILC and IDC cell lines. Using either of two siRNAs, WNT4 knockdown completely blocked estrogen-induced growth in ILC cells, but not IDC cells. Consistent with this, WNT4 knockdown abrogated estrogen-regulation of a subset of ER-target genes in MM134 cells, suggesting that these genes are downstream of WNT4 signaling. Wnt signaling typically acts via the canonical, β-catenin-dependent pathway; however, we observed that β-catenin is dysfunctional in ILC cells, and WNT4 cannot activate β-catenin signaling in cancer cell lines. This suggests that WNT4 regulates estrogen-induced growth in ILC cells via a novel non-canonical signaling pathway. Clinical observations suggest that ER regulates unique downstream pathways in ILC. We identified WNT4 as a putative downstream effector of ER signaling in ILC, as WNT4 is strongly-induced and directly regulated by ER specifically in ILC cells. Further, WNT4 is necessary for estrogen-induced growth in ILC cells, and likely signals via a non-canonical signaling pathway. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Future studies will focus on characterizing the signaling pathway controlled by WNT4 in order to identify putative therapeutic targets. Citation Format: Sikora MJ, Oesterreich S. Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-04-02.

Invasive lobular carcinoma (ILC) represents only 5-15% of all breast cancer. However, if it were an independent cancer type, it would rank as the sixth most common cancer in women, as common as non-Hodgkin lymphoma or melanoma. ILC is chronically understudied as a breast cancer subtype, but compared to invasive ductal carcinoma (IDC) ILC is more often HR-positive, HER2-negative, and lower histologic grade. Despite these favorable tumor characteristics, recent studies suggest outcome in ILC is not consistent with these favorable characteristics. We herein describe our single institution experience with ILC. Using cancer registry data, we identified 709 women over the age of 18 (including 85 with recurrences) diagnosed at our institution between 1990 and 2011 with HR-positive, early-stage (I-III), primary ILC and potentially available banked tissue samples. We implemented a case-cohort design to investigate molecular tumor characteristics associated with ILC prognosis. Patients were excluded for a history of synchronous bilateral breast cancer at the time of diagnosis, a prior history of invasive breast cancer, or a diagnosis of non-breast malignancy within 5 years prior to their diagnosis of ILC. Originally, a sample of 200 subjects was selected for further study and molecular characterization. However, tissue acquisition, subject eligibility and verification of recurrences yielded a sample of in total 131 subjects (including 33 with recurrences), and the case-cohort design was recreated for this final sample. All available information regarding patient and tumor characteristics, treatment history, and outcome was collected from medical records. ER, PR, and Ki67 protein (IHC) and RNA (Nanostring analysis) levels were analyzed with respect to outcome of patients. PR H-score, but not ER H-score, was significantly associated with survival in our overall ILC cohort. In the case-cohort analysis there was a weak correlation between ER, PR and Ki67 RNA levels (measured by nanostring) and ER, PR and Ki67 protein levels (measured by IHC) in the ILC tumor samples. After the Nanostring data was normalized, box plots were constructed for ESR1, PR and MKI67 so that a simple t-test could look at gene expression in recurrences and non-recurrences. There was no significant difference in ER expression between non-recurrences and recurrences. The recurrence cases had significantly lower PR expression levels. We will present quantitative ER and PR data, as well as detailed clinical characteristics for this cohort in relation to outcome. Citation Format: Rachel C. Jankowitz, Kevin Levine, Maryam Zamanian, Matthew Sikora, Nilgun Tasdemir, Priscilla McAuliffe, David Dabbs, Nancy Davidson, Brenda Diergaarde, Steffi Oesterreich. Associations between biomarkers and outcome in a patient cohort with invasive lobular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5645. doi:10.1158/1538-7445.AM2017-5645