Sun, Zhifu

Abstract Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. We have shown that estrogen receptor beta (ERβ) is expressed in approximately 20% of TN breast tumors and that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion and migration in vitro and in vivo. Therefore, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. RNAseq analysis of ERβ-expressing MDA-MB-231 cells demonstrated that ERβ significantly downregulates NFκB signaling in the presence of E2. ChIPseq for ERβ in these cells revealed that ERβ primarily associated with estrogen response elements, but 12% of all ERβ binding sites were located at NFκB consensus motifs. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. RNAseq analysis of MDA-MB-231-ERβ cells treated with TNFα or E2+TNFα revealed substantial global inhibition of TNFα regulated genes in the presence of E2. ChIPseq for NFκB demonstrated that ERβ significantly alters NFκB’s cistrome whereby it can both diminish NFκB binding and redistribute NFκB throughout the genome. ChIPseq also demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). The addition of H3K27Me3 at these loci was shown to occur through the recruitment of the histone methyltransferase, EZH2. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Knockdown of NFκB or Mutation of ERβ’s DNA binding domain rendered ERβ incapable of associating with DNA, recruiting EZH2, methylating NFκB target gene loci, repressing NFκB target gene expression and inhibiting proliferation. Interestingly, ERβ was shown to elicit more potent anti-cancer effects in TNBC cells expression a constitutively active form of NFκB. These finding suggest that a primary mechanism by which ERβ functions as a tumor suppressor is through inhibition of NFκB pathway activity. Our studies have also revealed that ERβ functions as a molecular switch for EZH2 and repurposes it for tumor suppressive activities, as EZH2 has previously been reported to enrich NFκB signaling in TNBC. These findings could address the paradox that high EZH2 expression is associated with worse TNBC patient outcomes, while high H3K27Me3 expression is associated with improved patient outcomes. Currently, a Mayo Clinic Breast Cancer SPORE prospective phase II clinical trial is underway to investigate the efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ, EZH2 and NFκB signaling.

Abstract Background: Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. Using well-validated antibodies, we have found that estrogen receptor beta (ERβ) protein is expressed in approximately 20% of TN breast tumors. We have demonstrated that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. As such, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. Methods: RNAseq and ChIPseq were used to identify ERβ and NFκB regulomes and cistromes in TNBC cells. The global effects of ERβ on H3K27Ac and H3K27Me3 were also assessed via ChIPseq. The influence of ERβ on NFκB signaling was determined using co-immunoprecipitation, luciferase assays transcriptional readouts and ChIPseq. The involvement of EZH2 in mediating ERβ’s suppression of NFκB signaling and cancer cell phenotypes was determined using pharmacological approaches. Results: Pathway analysis of ERβ-regulated genes identified NFκB and inflammatory signaling as being highly suppressed in response to E2 treatment. Numerous NFκB target genes were among the most down-regulated genes by ERβ. ChIPseq revealed that ERβ primarily associated with estrogen response elements (EREs), but was also highly enriched at NFκB binding sites following E2 treatment. However, ERβ had no impact on TNFα-mediated NFκB phosphorylation or nuclear localization, but was shown to physically associate with NFκB protein. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. Globally, RNAseq identified 373 genes to be significantly regulated by TNFα in TNBC cells, of which 107 were blocked in the presence of E2+TNFα. Interestingly, E2+TNFα treatment induced novel NFκB binding sites compared to TNFα alone as identified by ChIPseq for NFκB. ChIPseq also demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). These modifications were shown to occur through the recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Finally, knockdown of NFκB expression inhibited the anti-cancer effects of ERβ while expression of a constitutively active form of NFκB enhanced ERβ’s inhibitory effects. Conclusions: Our data suggest that ERβ elicits its anti-cancer effects, in part, via formation of a novel co-repressor complex consisting of ERβ, NFκB, and EZH2. These data are in keeping with prior observations of the importance of NFκB signaling as it relates to TNBC cell proliferation and invasion, and that decreased expression of NFκB target genes is associated with improved outcomes in TNBC patients. Currently, a Mayo Breast SPORE prospective study is underway to investigate the efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling. Citation Format: Kirsten GM Aspros, Adam W Nelson, Zhenqing Ye, Zhifu Sun, Igor Chernukhin, Jason S Carroll, James N Ingle, Matthew P Goetz, John R Hawse. Estrogen receptor beta repurposes EZH2 to inhibit oncogenic NFκB signaling in triple negative breast cancer [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 P6-05-02.

1037 Background: Folate receptor alpha (FRA the product of the FOLR1 gene) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense expression of FRA in breast tumors and poor prognosis, yet little is known about FOLR1/FRA expression across clinically relevant breast cancer subtypes. Methods: 131 breast cancer tumors including 4 benign, 33 ER+, 26 HER2+, and 68 triple negative (TN) were constructed into tissue microarrays (TMAs). FRA expression was analyzed by immunohistochemistry (IHC) using a high affinity FRA antibody. Tumor membrane staining intensity was scored by a pathologist as negative (0), weak (1+), moderate (2+) and strong (3+). The percent of cells within each tissue core stained at each intensity was recorded to calculate an H-score. The H-score is a weighted score that captured both the proportion of positive staining and intensity for each tumor. H-score values can range from zero (no membrane staining) to a maximum of 300 (100% membrane staining at 3+). H-scores for each patient sample were averaged over 3 TMA cores. The mean H-scores for each tumor subtype and the percentage of 3+ staining in >30% of tumor cells were compared by a Mann-Whitney test. The distribution of FOLR1 mRNA was completed using a TCGA RNA-seq dataset from 691 breast tumors classified as ER+, HER2+ and TN. FOLR1 levels of TN versus ER+ and HER2+ were compared by a Mann-Whitney test. Results: The mean H-score for the benign tumors was 0, ER+ (13.31), HER2+ (39.36), TN (119.02). The median H-score for the benign tumors was 0, ER+ (0), HER2+ (7.5), TN (127.5). The TN tumors mean and median H scores were significantly higher than benign, ER+ or HER2+ (p<0.001). The largest percentage of 3+ staining in >30% of tumor cells was observed in TN tumors (36.7%) and lowest in ER+ tumors (0%) (p<0.001). TN tumors had significantly higher levels of FOLR1 mRNA compared to ER+ and HER2+ subtypes (p<0.0001). Conclusions: Our data indicate that expression of FRA is highly prevalent in TN tumors and is supported by FOLR1 mRNA levels. Anti-FRA therapy may represent an important therapeutic intervention in TNBC who to this point have no active targeted treatment options.

13013 Background: The glutathione metabolic pathway is directly involved in the inactivation of platinum compounds. Our earlier work indicated that genotypes corresponding to varied glutathione-related enzyme activities may predict survival in patients with advanced lung cancer. Herein, we evaluated the role of glutathione-related genotypes on clinical outcomes in stage IIIB/IV non-small cell lung cancer patients who were stable or responding from one prior platinum-based chemotherapy. Methods: DNA samples from patients who had tumor regression or stable disease after treatment with one platinum based chemotherapy regimen were analyzed using 6 polymorphic DNA markers that encode 5 important enzymes in the glutathione metabolic pathway. The contrasting genotypes used in the analysis were GCLC (homozygous repeat 77 vs. heterozygous 7*), GPX1 (CC vs. other), GSTP1-I105V (AA vs. other), GSTP1-A114V (CC vs. other), GSTM1 (null vs. present), and GSTT1 (null vs. present). Multivariable Cox proportional hazards models adjusted for age, gender, treatment arm, ECOG performance status, stage, and prior tumor response, were used to evaluate the prognostic significance of the genotypes at each locus. Results: Data from 112 patients with a median follow-up of 10.6 (range: 0.4–56.3) months was used. No significant difference in time to disease progression was observed for any of the genotypes. Among the 6 genomic DNA markers, the GCLC genotype was an important prognostic factor for overall survival after adjusting for other factors. In particular, patients carrying a 77 repeat genotype (homozygous) had a significantly worse survival (Hazard ratio = 1.67, 95% CI: 1.05–2.63). Conclusions: Genotypes of glutathione-related enzymes, especially GCLC, may be used as host factors in predicting patients’ prognosis after platinum-based chemotherapy. Further investigation to define and measure the effects of these genes in chemotherapeutic regimens is needed. (This work was partly supported by NIH research grants CA77118, CA80127, and CA84354.) No significant financial relationships to disclose.

Abstract Background Human epidermal growth factor receptor ( EGFR ) is an oncogenic gene and one of top targets of precision therapy in lung cancer with EGFR mutations. Although there are many reports for some individual cancers, comprehensive profiling of EGFR mutations, overexpression, amplification, DNA methylation, and their clinical associations across many different cancers simultaneously was not available. This study aimed to fill the gap and provide insights to the alteration spectrum of EGFR and its therapeutic and prognostic implications. Methods The Cancer Genome Atlas (TCGA) datasets for 32 cancer types involving 11,314 patients were analyzed for alterations (mutations and amplification/deletion), abnormal expression and DNA methylation in EGFR gene. Mutation frequency, genomic location distribution, functional impact, and clinical targeted therapy implication were compared among different cancer types, and their associations with patient survival were analyzed. Results EGFR alteration frequency, mutation sites across functional domains, amplification, overexpression, and DNA methylation patterns differed greatly among different cancer types. The overall mutation frequency in all cancers combined was relatively low. Targetable mutations, mainly in lung cancer, were primarily found in the Pkinase_Tyr domain. Glioblastoma multiforme had the highest rate of alterations, but it was dominated by gene amplification and most mutations were in the Furin‐like domain where targeted therapy was less effective. Low‐grade glioma often had gene amplification and increased EGFR expression which was associated with poor outcome. Colon and pancreatic adenocarcinoma had very few EGFR mutations; however, high EGFR expression was significantly associated with short patient survival. Squamous cell carcinoma regardless of their sites (the head and neck, lung, or esophagus) exhibited similar characteristics with an alteration frequency of about 5.0%, was dominated by gene amplification, and had increased EGFR expression generally associated with short patient survival. DNA methylation was highly associated with EGFR expression and patient outcomes in some cancers. Conclusions EGFR aberration type, frequency, distribution in functional domains, and expression vary from cancer to cancer. While mutations in the Pkinase_Tyr domain are more important for treatment selection, increased expression from amplification or deregulation affects more tumor types and leads to worse outcome, which calls for new treatment strategies for these EGFR‐ driven tumors.

Abstract Purpose: The burden of esophageal cancer continues to rise, and noninvasive screening tools are needed. Methylated DNA markers (MDM) assayed from plasma show promise in detection of other cancers. For esophageal cancer detection, we aimed to discover and validate MDMs in tissue, and determine their feasibility when assayed from plasma. Experimental Design: Whole-methylome sequencing was performed on DNA extracted from 37 tissues (28 EC; 9 normal esophagus) and 8 buffy coat samples. Top MDMs were validated by methylation specific PCR on tissue from 76 EC (41 adeno, 35 squamous cell) and 17 normal esophagus. Quantitative allele-specific real-time target and signal amplification was used to assay MDMs in plasma from 183 patients (85 EC, 98 controls). Recursive partitioning (rPART) identified MDM combinations predictive of esophageal cancer. Validation was performed in silico by bootstrapping. Results: From discovery, 23 candidate MDMs were selected for independent tissue validation; median area under the receiver operating curve (AUC) for individual MDMs was 0.93. Among 12 MDMs advanced to plasma testing, rPART modeling selected a 5 MDM panel (FER1L4, ZNF671, ST8SIA1, TBX15, ARHGEF4) which achieved an AUC of 0.93 (95% CI, 0.89–0.96) on best-fit and 0.81 (95% CI, 0.75–0.88) on cross-validation. At 91% specificity, the panel detected 74% of esophageal cancer overall, and 43%, 64%, 77%, and 92% of stages I, II, III, and IV, respectively. Discrimination was not affected by age, sex, smoking, or body mass index. Conclusions: Novel MDMs assayed from plasma detect esophageal cancer with moderate accuracy. Further optimization and clinical testing are warranted.

Abstract Background The aetiology of Crohn’s disease [CD] involves immune dysregulation in a genetically susceptible individual. Genome-wide association studies [GWAS] have identified 200 loci associated with CD, ulcerative colitis, or both, most of which fall within non-coding DNA regions. Long non-coding RNAs [lncRNAs] regulate gene expression by diverse mechanisms and have been associated with disease activity in inflammatory bowel disease. However, disease-associated lncRNAs have not been characterised in pathogenic immune cell populations. Methods Terminal ileal samples were obtained from 22 CD patients and 13 controls. RNA from lamina propria CD4+ T cells was sequenced and long intergenic non-coding RNAs [lincRNAs] were detected. Overall expression patterns, differential expression [DE], and pathway and gene enrichment analyses were performed. Knockdown of novel lincRNAs XLOC_000261 and XLOC_000014 was performed. Expression of Th1 or Th17-associated transcription factors, T-bet and RORγt, respectively, was assessed by flow cytometry. Results A total of 6402 lincRNAs were expressed, 960 of which were novel. Unsupervised clustering and principal component analysis showed that the lincRNA expression discriminated patients from controls. A total of 1792 lincRNAs were DE, and 295 [79 novel; 216 known] mapped to 267 of 5727 DE protein-coding genes. The novel lincRNAs were enriched in inflammatory and Notch signalling pathways [p &lt;0.05]. Furthermore, DE lincRNAs in CD patients were more frequently found in DNA regions with known inflammatory bowel disease [IBD]-associated loci. The novel lincRNA XLOC_000261 negatively regulated RORγt expression in Th17 cells. Conclusions We describe a novel set of DE lincRNAs in CD-associated CD4+ cells and demonstrate that novel lincRNA XLOC_000261 appears to negatively regulate RORγt protein expression in Th17 cells.

Abstract Background: Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. Using well-validated antibodies, Estrogen Receptor Beta (ERβ) protein has been shown to be expressed in approximately 25% of TNBCs and is associated with improved patient outcomes. Using multiple ERβ +/- TNBC cell lines and PDX models, we have demonstrated that ligand-mediated activation of ERβ by estradiol (E2) decreases cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. Methods: To determine the mechanisms by which ERβ elicits these anti-cancer effects, we elucidated the ERβ transcriptome and cistrome via Microarray and ChIPseq, respectively, in TNBC cells stably expressing ERβ in a doxycycline-inducible manner. We also performed gene expression and luciferase assays to assess the impact of ERβ on NFκB signaling, followed by ChIP-PCR and ChIPseq to assess how ERβ modifies chromatin architecture near NFκB target genes. Results: Pathway analysis of ERβ-regulated genes identified NFκB signaling as one of the most suppressed pathways in response to E2 treatment. Indeed, numerous NFκB target genes were among the most down-regulated genes following E2 treatment but only in the presence of ERβ expression. Chromatin Immunoprecipitation followed by sequencing (ChIPseq) revealed that ERβ primarily associated with estrogen response elements (EREs), but was also enriched around NFκB binding sites following E2 treatment. In fact, 12% of all ERβ binding sites were enriched for NFκB response elements and ERβ was shown to physically associate with NFκB protein. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. Globally, RNAseq identified 200 genes to be significantly regulated by TNFα in TNBC cells, of which 81 were significantly altered in the presence of E2+TNFα. ChIPseq demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). These modifications are also associated with recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of HDAC and EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Conclusions: Our data suggest that ERβ may elicit its anti-cancer effects in part via formation of a novel co-repressor complex consisting of ERβ, NFκB, and EZH2. These data are in keeping with prior observations of the importance of NFκB signaling as it relates to TNBC cell proliferation and invasion, and that decreased expression of NFκB target genes is associated with improved outcomes in TNBC patients. Currently, a Mayo Breast SPORE prospective study is underway to investigate the role of estradiol in ERβ expressing TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling in TNBC. Citation Format: Aspros K, Nelson A, Ye Z, Sun Z, Chernukhin I, Carroll J, Ingle J, Goetz M, Hawse J. Estrogen receptor beta elicits anti-cancer effects in triple negative breast cancer through suppression of NFκB signaling [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 P5-05-03.