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Preclinical model systems are essential research tools that help us understand the biology of invasive lobular carcinoma of the breast (ILC). The number of well-established ILC models is increasing but remain limited. Lower incidence of ILC, underrepresentation of patients with ILC in clinical trials, and intrinsic ILC tumor characteristics all contribute to this challenge. Hence, there is significant need to continually develop better model systems to recapitulate the essential characteristics of ILC biology, genetics, and histology, and empower preclinical therapeutic studies to be translated back into the clinic. In this Perspective, we highlight recent advances in in vivo experimental models, which recapitulate key features of ILC biology and disease progression and potentially reshape the future of ILC translational research. We assert that all existing in vitro and in vivo ILC preclinical models have their strengths and weaknesses, and that it is necessary to bridge key deficiencies in each model context as we move forward with ILC research. Thus, unlocking the mysteries of ILC will be best achieved by choosing the right combination of preclinical model systems.

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

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.

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

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.

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.

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.

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.

Background Invasive lobular breast carcinoma (ILC) is a histological subtype of breast cancer that is characterized by loss of E-cadherin and high expression of estrogen receptor alpha (ERα). In many cases, ILC is effectively treated with adjuvant aromatase inhibitors (AIs); however, acquired AI resistance remains a significant problem. Methods To identify underlying mechanisms of acquired anti-estrogen resistance in ILC, we recently developed six long-term estrogen-deprived (LTED) variant cell lines from the human ILC cell lines SUM44PE (SUM44; two lines) and MDA-MB-134VI (MM134; four lines). To better understand mechanisms of AI resistance in these models, we performed transcriptional profiling analysis by RNA-sequencing followed by candidate gene expression and functional studies. Results MM134 LTED cells expressed ER at a decreased level and lost growth response to estradiol, while SUM44 LTED cells retained partial ER activity. Our transcriptional profiling analysis identified shared activation of lipid metabolism across all six independent models. However, the underlying basis of this signature was distinct between models. Oxysterols were able to promote the proliferation of SUM44 LTED cells but not MM134 LTED cells. In contrast, MM134 LTED cells displayed a high expression of the sterol regulatory element-binding protein 1 (SREBP1), a regulator of fatty acid and cholesterol synthesis, and were hypersensitive to genetic or pharmacological inhibition of SREBPs. Several SREBP1 downstream targets involved in fatty acid synthesis, including FASN, were induced, and MM134 LTED cells were more sensitive to etomoxir, an inhibitor of the rate-limiting enzyme in beta-oxidation, than their respective parental control cells. Finally, in silico expression analysis in clinical specimens from a neo-adjuvant endocrine trial showed a significant association between the increase of SREBP1 expression and lack of clinical response, providing further support for a role of SREBP1 in the acquisition of endocrine resistance in breast cancer. Conclusions Our characterization of a unique series of AI-resistant ILC models identifies the activation of key regulators of fatty acid and cholesterol metabolism, implicating lipid-metabolic processes driving estrogen-independent growth of ILC cells. Targeting these changes may prove a strategy for prevention and treatment of endocrine resistance for patients with ILC. Electronic supplementary material The online version of this article (10.1186/s13058-018-1041-8) contains supplementary material, which is available to authorized users.

The enzyme PORCN is considered essential for Wnt secretion and signaling, however, we observed PORCN inhibition did not phenocopy the effects of WNT4 knockdown in WNT4-dependent breast cancer cells. This suggests a unique relationship between PORCN and WNT4 signaling. To examine the role of PORCN in WNT4 signaling, WNT4 or WNT3A were over-expressed in breast and ovarian cancer, and fibrosarcoma cell lines. Conditioned medium from these lines, and co-culture systems, were used to assess the dependence of Wnt secretion and activity on critical Wnt secretion proteins PORCN and WLS. We observed that WLS was universally required for Wnt secretion and paracrine signaling. In contrast, the dependence of WNT3A secretion and activity on PORCN varied across cell lines, and WNT4 secretion was PORCN-independent in all models. Surprisingly, WNT4 did not present paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell autonomous Wnt signaling activation by WNT4 and WNT3A, independent of PORCN or secretion. The PORCN-independent, cell-autonomous Wnt signaling demonstrated herein may be critical in WNT4-driven cellular contexts, or those that are otherwise considered to have dysfunctional Wnt signaling.Summary StatementWnt proteins can mediate an atypical mode of cell-autonomous signaling, distinct from paracrine signaling, that is independent of both palmitoylation by PORCN and Wnt secretion.

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: 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 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 (

Invasive lobular carcinoma (ILC) is the second most common histologic subtype of breast cancer. Though >90% of ILC tumors are estrogen receptor (ER)-positive, ILC patients have poorer outcomes than others with ER-positive tumors, suggesting ER biology is unique in ILC cells. We previously identified the Wnt ligand WNT4 as an ILC-specific ER transcriptional target that is critical for both estrogen-induced growth and antiestrogen resistance in ILC cells. We hypothesize that characterizing the WNT4 signaling network in ILC cells will identify novel biomarkers and therapeutic targets for ILC patients. WNT4 signals in a paracrine manner in normal mammary gland and in breast cancer tissues, via secretion from luminal cells to nearby basal or basal-like cells. Inhibiting Wnt secretion with Porcupine-inhibitors (PORCNi) in these contexts blocks WNT4-driven phenotypes. However, we have observed that PORCNi have no effect on ILC cell proliferation. Further, PORCNi combined with siRNA knockdown of PORCN completely suppress secretion of overexpressed WNT3A but not of WNT4. These observations suggest that WNT4 engages its signaling cascade via an atypical mechanism in ILC cells, where WNT4 signals in an autocrine or intracellular manner or is secreted via PORCN-independent mechanisms. Understanding how WNT4 activates signaling is vital to developing appropriate therapeutic strategies, especially as PORCNi have been tested clinically for ILC patients. In both the normal mammary gland and in breast cancer cells, WNT4 engages the canonical Wnt pathway and activates β-catenin-mediated gene regulation. However, ILC cells and tumors lack β-catenin protein (related to loss of E-cadherin); WNT4 must be activating an alternative signaling cascade in ILC cells. To define downstream 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 targets that link WNT4 signaling to control of proliferation and cell cycle as well as apoptosis and cell survival. In the former, coordinate suppression of p21 and induction of FOXM1 control G1/S and G2/M transitions, respectively, and WNT4 knockdown engages both checkpoints to halt cellular proliferation. In the latter, WNT4 activates distinct components of Akt/mTOR signaling in ILC versus IDC cells, as E2 treatment increases phosphorylation of mTOR/Rictor but not of Akt in ILC cells. Activation of mTOR correlates with post-transcriptional regulation of levels of the anti-apoptotic BCL2-family protein MCL-1; E2-mediated suppression of MCL-1 protein levels via mTOR may regulate cell survival and sensitivity to apoptotic stimuli. WNT4 mediates a critical ER-driven pathway in ILC cells, but the WNT4 signaling network has not been characterized to identify putative therapeutic approaches for ILC patients. Our data suggest that targeting FOXM1, mTOR, or BCL2-family proteins may modulate ER/WNT4 signaling and that ILC cells are unlikely to respond to PORCNi. Characterization of this unique signaling network will drive new predictive models and therapeutic approaches for ILC patients. Citation Format: Deviyani M. Rao, Rebecca Ferguson, Matthew J. Sikora. Estrogen-driven noncanonical WNT4 signaling is essential for proliferation and survival in lobular carcinoma cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A39.

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.

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) is a histological subtype of breast cancer representing 10-15% of newly diagnosed breast tumors. Over 90% of ILC are ER-positive, however, endocrine response and estrogen signaling are not well described in ILC. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than similar invasive ductal carcinoma (IDC) patients, and that ILC patients may not benefit from adjuvant tamoxifen. Additionally, we recently identified ILC-specific ER-target genes and de novo tamoxifen resistance driven by ER in ILC model systems. Based on these observations, we hypothesize that ILC-specific signaling pathways driven by ER mediate growth and endocrine resistance in ILC cells. Among ILC-specific estrogen-regulated genes in the ILC cell lines MDA MB 134VI (MM134) and SUM44PE (SUM44), Wnt signaling genes were highly differentially expressed. The secreted ligand WNT4 was the most strongly estrogen-induced gene in ILC cells. The frizzled receptor FZD7 is also strongly induced in ILC cells, but only transiently induced in the ER-positive IDC cell line MCF-7. Among IDC cell lines, either WNT4 or FZD7 is over-expressed in ER-positive or ER-negative cells, respectively. Conversely, MM134 and SUM44 over-express both WNT4 and FZD7. Also, we identified an ILC-specific ER binding site at WNT4; located in intron 1, this site contains a predicted estrogen response element. Direct WNT4 regulation and parallel regulation of pathway genes suggests that ER controls a WNT4 signaling pathway in ILC cells. In samples from the Cancer Genome Atlas, WNT4 and FZD7 are each over-expressed in ER-positive ILC versus IDC; co-expression is also enriched only in ILC. These observations suggest that a WNT4 signaling pathway may be specifically active in ILC tumors. To assess whether WNT4 is necessary for estrogen-induced growth, we used siRNA to knock down WNT4. Using either of two siRNAs, WNT4 knockdown completely blocks 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; induction or repression was inhibited by WNT4 knockdown prior to estrogen treatment. Thus, a subset of estrogen-induced gene expression changes is mediated by WNT4 signaling. Though Wnt signaling typically acts via the canonical, β-catenin-dependent pathway, we observed that β-catenin signaling is dysfunctional in ILC cells. Additionally, WNT4 over-expression or recombinant protein cannot activate canonical Wnt signaling in breast cancer cell lines. This suggests that WNT4 signaling mediates estrogen-induced growth in ILC cells via a novel non-canonical signaling pathway. Wnt signaling pathway genes including WNT4 are uniquely regulated in ILC cell lines, and are over-expressed in ILC tumors, suggesting that a WNT4-driven pathway may be active specifically in ILC. WNT4 is necessary for estrogen-mediated growth in ILC cells, and likely signaling via a novel non-canonical signaling pathway. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Future studies will focus on identifying the signaling pathway controlled by WNT4 in order to identify novel therapeutic targets. Citation Format: Matthew J Sikora, Amir Bahreini, Caroline M Alexander, Steffi Oesterreich. Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth [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 P3-04-05.

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

Invasive lobular carcinoma (ILC) is a breast cancer subtype affecting ∼30,000 U.S. women annually. Over 90% of ILC are estrogen receptor (ER)-positive; however, endocrine therapy may have poorer efficacy in a subset of ILC patients versus invasive ductal carcinoma (IDC) patients. This prompted us to assess global ER activity in ILC cell lines MDA MB 134VI (MM134) and SUM44PE (44PE) to identify novel mediators of ER signaling. These analyses identified the Wnt ligand WNT4 as an ILC-specific ER target gene, with an ILC-specific ER binding site (ERBS) at the WNT4 locus. Considering the critical role of WNT4 in normal mammary gland expansion, we hypothesize that ILC cells utilize WNT4 signaling to drive endocrine response and resistance. We assessed whether WNT4 is necessary for ILC cell growth using siRNA. WNT4 knockdown completely blocked estrogen-induced growth in ILC cells but not IDC cells. In parallel, the WNT4 ERBS was only occupied in ILC cells in response to estrogen, but progesterone-induced WNT4 in IDC was not associated with this ERBS. This suggests that, via the ILC-specific WNT4 ERBS, ILC cells drive estrogen-regulated proliferation by hijacking a developmental Wnt pathway. Wnt pathways typically activate â-catenin; however, we observed â-catenin dysfunction in ILC cells and that WNT4 cannot activate â-catenin. Thus, WNT4 signals in ILC cells via a novel non-canonical pathway. Using long-term estrogen-deprived (LTED) variants of MM134 and 44PE (4 and 2 lines, respectively), we assessed WNT4 in ILC endocrine resistance. WNT4 is over-expressed, but uncoupled from ER, in all MM134-LTED. Conversely, WNT4 is reduced in 44PE-LTED but remains ER-regulated; ER occupies the WNT4 ERBS only in 44PE-LTED cells and not MM134-LTED. Using siRNA, MM134-LTED (high WNT4) are growth-inhibited by WNT4 knockdown, while 44PE-LTED (low WNT4) are insensitive. However, WNT4 knockdown sensitizes 44PE-LTED to endocrine therapy. Taken together, uncoupling and upregulating WNT4 or WNT4/ER cross-talk may represent convergent endocrine resistance mechanisms in ILC. To assess the role of WNT4 in patient ILC, we examined WNT4 protein in archival breast tumors and observed that WNT4 is frequently expressed in ILC and IDC tumors. We also examined WNT4 regulation and endocrine response in patient tumor explants. We observed ER regulation of WNT4 directly in ILC tissues that correlated with sensitivity to fulvestrant but resistance to tamoxifen; this may mimic clinical endocrine resistance. Ongoing studies are assessing WNT4 as a biomarker and mediator of endocrine resistance in ILC. Clinical observations suggest that ER regulates unique pathways in ILC. We identified WNT4 as a downstream effector of endocrine signaling in ILC, with critical roles in both estrogen-induced growth and endocrine resistance. WNT4 signaling may represent a novel target to modulate endocrine response specifically for ILC patients. Citation Format: Matthew J. Sikora, Courtney L. Andersen, Caroline M. Alexander, Priscilla M. McAuliffe, Steffi Oesterreich. WNT4 mediates endocrine response and resistance in invasive lobular carcinoma cell lines and patient tumor explants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 862.

Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer, affecting ~30,000 U.S. women annually. Over 90% of ILC are estrogen receptor (ER)-positive, however, endocrine therapy may have poorer efficacy in a subset of ILC patients compared to invasive ductal carcinoma (IDC) patients. Based on these observations, we assessed genome-wide ER-mediated gene expression and ER genomic binding in ILC cell lines MDA MB 134VI (MM134) and SUM44PE (44PE), to identify novel mediators of ER signaling and putative therapeutic targets specifically in ILC. Among ILC-specific estrogen-regulated genes, the most strongly induced was the Wnt ligand WNT4. In parallel, we identified an ILC-specific ER binding site (ERBS) at WNT4, suggesting that WNT4 is directly ER-controlled in ILC cells. We hypothesized that this would be an analog to progesterone-controlled WNT4 in mammary gland expansion, and assessed whether WNT4 is necessary for estrogen-induced growth in ILC cells. Using siRNAs, knockdown of WNT4 completely blocked estrogen-induced growth in ILC cells, but not IDC cells. Consistent with this, we found that the WNT4 ERBS is only occupied in ILC cells that strongly upregulate WNT4 in response to estrogen, whereas progesterone-regulated WNT4 expression in T47D cells was not associated with ER binding at the WNT4 ERBS. These data suggest that, via an ILC-specific ERBS at WNT4, ILC cells can drive estrogen-regulated proliferation by hijacking a developmental Wnt pathway. Canonical Wnt pathways activate β-catenin; however, we observed β-catenin dysfunction in ILC cells, and that WNT4 cannot activate β-catenin in cell lines. Thus, WNT4 regulates estrogen-induced growth in ILC cells via a novel non-canonical pathway. Using long-term estrogen-deprived (LTED) variants of MM134 and 44PE (4 and 2 lines, respectively), we assessed WNT4 in ILC endocrine resistance. WNT4 is over-expressed but uncoupled from ER in all MM134-LTED; expression is greatly reduced in 44PE-LTED, but weakly estrogen-regulated. Consistent with regulation, ER occupies the WNT4 ERBS only in 44PE-LTED cells. However, 44PE-LTED (low WNT4) are resistant to growth inhibition by WNT4 siRNA, while MM134-LTED (high WNT4) are growth-inhibited. Taken together, uncoupling and upregulating WNT4 may be necessary for LTED growth in MM134. Clinical observations suggest that ER regulates unique downstream pathways in ILC. We identified WNT4 as a putative downstream effector of endocrine signaling in ILC, having critical roles in both estrogen-induced growth and endocrine resistance. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Citation Format: Matthew J. Sikora, Amir Bahreini, Caroline M. Alexander, Steffi Oesterreich. WNT4 signaling mediates endocrine response and resistance in invasive lobular carcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A35.

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

The aromatase inhibitors (AIs) are used to treat estrogen receptor-positive (ER+) breast tumors in post-menopausal women, and function by blocking the conversion of adrenal androgens to estrogens by the enzyme CYP19 aromatase. Breast cancer patients receiving AI therapy have circulating estrogen levels below the level of detection; however, androgen concentrations remain unchanged. We were interested in studying the effects of androgens on breast cancer cell proliferation under profound estrogen-deprived conditions. Using in vitro models of estrogen-dependent breast cancer cell growth we show that the androgens testosterone and 5alpha-dihydrotestosterone induce the growth of MCF-7, T47D and BT-474 cells in the absence of estrogen. Furthermore, we demonstrate that under profound estrogen-deprived conditions these breast cancer cells up-regulate steroidogenic enzymes that can metabolize androgens to estrogens. Lastly, we found that the downstream metabolite of 5alpha-dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol (3betaAdiol), is estrogenic in breast cancer cells, and induces growth and ER-signaling via activation of ERalpha. In conclusion, our results show that breast cancer cells deprived of estrogen up-regulate steroidogenic enzymes and metabolize androgens to estrogen-like steroids. The generation of estrogen-like steroids represents a potential mechanism of resistance to aromatase inhibitors.

Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer representing ∼10% of newly diagnosed breast tumors. Over 90% of ILC cases are ER-positive, however, endocrine response and estrogen signaling are not well described in ILC. 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. Additionally, we have recently identified ILC-specific ER-target genes and de novo tamoxifen resistance driven by ER in ILC model systems. Based on these observations, we hypothesize that ILC-specific signaling pathways driven by ER mediate growth and endocrine resistance in ILC cells. We focused on three putative mechanisms of endocrine response and resistance in ILC cells. First, we investigated the role of the Wnt signaling protein WNT4 in driving E2-induced growth. E2 induced ER binding at the WNT4 promoter and up-regulated expression specifically in ILC cells (versus IDC cells). As WNT4 has also been previously implicated in normal mammary gland growth and development, we hypothesize that ER-driven WNT4 expression of mediates E2-induced growth of ILC cells. Second, we investigated the role of the transcriptional repressor SNAI1 in ER-mediated gene expression and tamoxifen-resistance. SNAI1 gene expression was induced by both E2 and tamoxifen specifically in ILC cells, which paralleled unique patterns of E2-mediated gene repression in ILC cells. Thus, SNAI1 may mediate E2-driven gene repression and tamoxifen resistance. Third, our prior identification of ILC-specific ER-target genes and ER-mediated tamoxifen resistance suggested that novel co-factors may associate with ER in ILC. We hypothesize that identification of these co-factors would provide novel targets for therapy in ILC. Cell lines MDA MB 134VI and SUM44PE are used as in vitro models of ILC versus MCF-7 and T47D as models of IDC. siRNA-mediated knockdown of gene expression is utilized to assess the function of WNT4 and SNAI1. Cell proliferation and ER-target gene expression are assessed in both ILC and IDC models following knockdown. To identify novel ER co-factors in ILC, we co-immunoprecipitate ER with associated factors, and identify bound proteins by mass spectrometry. Preliminary experiments have demonstrated that knockdown of either WNT4 or SNAI1 can suppress the growth of ILC cells, and that this effect is specific to ILC cells. Additionally, Co-IP/mass spec identifies co-factors that are specifically associated with ER in ILC cells. These observations suggest that unique ER-mediated signaling pathways drive endocrine response and resistance in ILC cells, and that further understanding of these pathways may present novel therapeutic targets in ILC. Citation Format: Matthew J. Sikora, Amir Bahreini, Steffi Oesterreich. Estrogen receptor mediates novel mechanisms of estrogen-induced growth and tamoxifen resistance in invasive lobular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4755. doi:10.1158/1538-7445.AM2014-4755

Invasive lobular carcinoma (ILC) represents ∼10% of newly diagnosed breast tumors, accounting for ∼30,000 cases annually in the US. However, ILC has been understudied as a breast cancer subtype. ILC-specific signaling pathways and responses to endocrine therapies are not well characterized. Recent retrospective analyses of luminal-type breast cancers suggest that ILC patients treated with endocrine therapy have poorer disease-free survival and overall survival than invasive ductal carcinoma (IDC) patients with similar biomarkers. We hypothesize that unique transcriptional control of estrogen receptor-alpha (ERα) by estrogens and anti-estrogens in ILC cells drive a differential response of ILC tumors to endocrine therapies. The ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of ILC tumors to examine responses to estradiol (E2) and the anti-estrogens tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI). We examined cell growth and expression of canonical ERα-regulated genes in response to E2. Cells were also treated with anti-estrogens +/− E2 to examine their ability to inhibit E2-induced growth. To establish a genome-wide profile of ERα-mediated gene regulation in ILC cells, ILC cells were subjected to gene expression microarray analysis following 0–24hrs treatment with 1nM E2. In parallel to these in vitro studies, in vivo models of ILC are being assessed for endocrine responsiveness, including MDA MB 134VI xenografts and the primary human tumor xenograft HCI-013. We observed that both MDA MB 134VI and SUM44PE are growth-induced by E2 treatment. However, both cell lines also present de novo resistance to Tam, 4OHT, and Bx. While ICI treatment completely blocked E2-induced growth in MDA MB 134VI, Tam, 4OHT, and Bx acted as partial agonists. Treatment with these compounds alone induced ∼25% growth relative to E2, and E2-induced growth could only be inhibited ∼75%. Similarly, SUM44PE cells are strongly growth-inhibited by ICI treatment, whereas growth is not reduced by Tam, 4OHT, or Bx treatment. Consistent with these observations, novel patterns of gene expression are induced by E2 treatment in ILC cells. E2-treatment induced canonical targets (e.g. GREB1, IGFBP4) in both ILC cell lines. However, in MDA MB 134VI cells only ∼35% of genes regulated by E2 at 24hrs overlap with those regulated in the IDC cell line MCF-7. Further, a subset of the overlapping genes is differentially regulated between cell types, including ESR1 (1.25-fold and 0.67-fold versus control in MDA MB 134VI and MCF-7, respectively), HOXC6, PMP22, and L1CAM. Examination of these observations in in vivo models is currently ongoing. These data are consistent with the hypothesis that E2 and anti-estrogens differentially regulate ERα-mediated gene expression in ILC cells versus IDC cells. The de novo resistance to tamoxifen observed in ILC cells may correlate with the worse outcomes in ILC patients observed in retrospective analyses. We hypothesize that elucidation of the mechanisms responsible for differential ERα regulation may reveal novel therapeutic targets for treating ILC patients and overcoming endocrine resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-20.