Prognosis In ER Research Articles

Sex Hormone-regulatedCMG2Is Involved in Breast and Prostate Cancer Progression

Capillary morphogenesis gene 2 (CMG2) is involved in prostate and breast cancer progression. This study aimed to investigate sex hormone receptor-mediated regulation of CMG2 in breast and prostate cancer, and its implication in disease progression. Expression of CMG2, oestrogen receptor (ER) and androgen receptor (AR) was determined in breast and prostate cancer cell lines, respectively, using real-time quantitative PCR (QPCR) and western blot. Association between CMG2 and sex hormone receptors was analysed in a number of transcriptome datasets. Immunochemical staining was performed in tissue microarrays of breast cancer (BR1505D) and prostate cancer (PR8011A). CMG2 expression was determined in 17β-oestradiol treated breast cancer cells and AR over-expressing prostate cancer cells. CMG2 was found to be inversely correlated with sex hormone receptors in breast and prostate cancer. Lower expression of CMG2 was associated with a poor prognosis in ER (+) breast cancer but not ER (-) tumours. Both ER (+) breast cancer cell lines and AR (+) prostate cancer cell lines presented lower expression of CMG2, which was increased following sex hormone deprivation. Exposure to 17-β-oestradiol and AR over-expression repressed CMG2 expression in breast cancer and prostate cancer cell lines, respectively. CMG2 is inversely correlated with ER and AR status in breast and prostate cancer, respectively. ER and AR mediate repression of CMG2 expression in corresponding cancerous cells.

Clinical characteristics and disease outcomes in ER+ breast cancer: a comparison between HER2+ patients treated with trastuzumab and HER2- patients

BackgroundTrastuzumab has changed the prognosis of HER2+ breast cancer. We aimed to investigate the prognosis of ER+/HER2+ patients treated with trastuzumab, thus to guide escalation endocrine treatment in ER+ breast cancer.MethodsER-positive early breast cancer patients operated at Ruijin Hospital between Jan. 2009 and Dec. 2017 were retrospectively included. Eligible patients were grouped as HER2-negative (HER2-neg) or HER2-positive with trastuzumab treatment (HER2-pos-T). Kaplan-Meier analysis and Cox proportional hazards model were used to compare the disease-free survival (DFS) and overall survival (OS) between these two groups.ResultsA total of 3761 patients were enrolled: 3313 in the HER2-neg group and 448 in the HER2-pos-T group. Patients in the HER2-pos-T group were associated with pre/peri-menopause, higher histological grade, LVI, higher Ki-67 level, lower ER and PR levels (all P < 0.05). At a median follow-up of 62 months, 443 DFS events and 191 deaths were observed. The estimated 5-year DFS rate was 89.7% in the HER2-neg group and 90.2% in the HER2-pos-T group (P = 0.185), respectively. Multivariable analysis demonstrated that patients in the HER2-pos-T group had a better DFS than patients in the HER2-neg group (HR 0.52, 95% CI: 0.37–0.73, P < 0.001). The estimated 5-year OS rates were 96.0% and 96.3% in the two groups, respectively (P = 0.133). Multivariate analysis found that HER2-pos-T group was still associated with significantly better OS compared with the HER2-neg group (HR 0.38, 95% CI: 0.22–0.67, P = 0.037).ConclusionER+/HER2+ breast cancer patients treated with trastuzumab were associated with superior outcome compared with ER+/HER2- patients, indicating HER2-positivity itself may not be an adverse factor for ER+ patients in the era of trastuzumab.

Reproductive Factors and Breast Cancer Prognosis: The Possible Role of Cesarean Delivery

Objective: To evaluate the possible associations between tumor pathological characteristics and reproductive factors in women with breast cancer, including method of child delivery.&#x0D; Materials and Methods: Patients diagnosed as breast cancer between January 2012 and April 2013 in Ankara University Hospitals who had at least one full term pregnancy were included. Data upon pathological prognostic features of tumors and reproductive history of patients were obtained from patient files.&#x0D; Results: 434 patients with breast cancer were included. Mean age at the time of breast cancer diagnosis was 49.2±10.7 years. History of at least one cesarean delivery was present in 17% of the patients (n=75). Axillary lymph node positivity was related to younger age at first full-term pregnancy (p=0.018), and higher number of parities (p=0.001) in estrogen receptor positive (ER+) breast cancer patients. Although extracapsular invasion was related to shorter time interval since last parity to cancer diagnosis in the whole population (p=0.029), this association disappeared when patients were categorized according to ER status. However, in ER+ patients, history of cesarean delivery was associated with extracapsular invasion (p=0.023). Also, our findings indicated that patients with history of cesarean delivery were diagnosed with breast cancer at younger ages, and after a shorter time interval since last parity (p&lt;0.001). Tumor size (p=0.001), grade (p=0.046), axillary lymph node positivity (p=0.001), lymphovascular invasion (p&lt;0.001), and shorter duration of time since last parity to breast cancer diagnosis (p=0.029) were indicators of metastatic disease.&#x0D; Conclusion: Our findings indicate that history of cesarean delivery may be associated with poor prognosis in ER+ patients with breast cancer.

Significance of Oncotype DX 21-Gene Test and Expression of Long Non-Coding RNA MALAT1 in Early and Estrogen Receptor-Positive Breast Cancer Patients.

ObjectiveTo investigate the association between the recurrence score (RS) obtained by Oncotype DX 21-gene test and long non-coding RNA (lncRNA) MALAT1 expression in early and estrogen receptor-positive (ER+) breast cancer.Materials and MethodsThe Oncotype DX 21-gene test and MALAT1 expression detection were performed in tumor samples from 76 ER+ and early breast cancer patients with the Surplex liquid chip. The RS value was calculated based on the expression of total 21 genes. The level of MALAT1 was measured in both tumor tissue and para-tumor tissue, and relatively quantified with an internal control gene. Mann–Whitney U-test or Kruskal–Wallis test were used to analyze the association between MALAT1 level and different clinical pathological characteristics, including age, tumor stage, disease grade, lymph node status, Ki-67 expression, and progesterone receptor (PR) status. The association between the RS and different characteristics was analyzed by Wilcoxon rank-sum test. Correlation between two parameters was analyzed by Spearman’s rank correlation analysis.ResultsThe expression of MALAT1 was more abundant in tumor tissue (2.992 ± 2.256) than that in adjacent normal tissue (1.641±1.438, Z=−2.594, p=0.009), and it was not correlated with any clinical pathological characteristics. According to the old criteria for RS stratification, 52.7% of patients were in low risk (RS<18), 36.8% of patients were in medium risk (18≤RS≤30), and 10.5% of patients were in high risk (RS>30). While under the new criteria, 18.4% were in low risk group (RS<11), 63.2% were in a medium risk group (11≤RS≤26), and 18.4% were in a high risk group (RS>26). The Oncotype DX 21-gene results only correlated with Ki-67 expression under both new and old criteria, and it was not related with other cancer characteristics. The expression of lncRNA MALAT1 was significantly correlated with the Oncotype DX 21-gene results under the old criteria.ConclusionMALAT1 is a novel breast cancer biomarker independent of tumor stage, disease grade and lymph node status. MALAT1 level is associated with the Oncotype DX 21-gene RS value. Therefore, combination of MALAT1 and the Oncotype DX 21-gene test may be used to predict prognosis in ER+ and early stage breast cancer.

Cross-talk between the ER pathway and the lncRNA MAFG-AS1/miR-339-5p/ CDK2 axis promotes progression of ER+ breast cancer and confers tamoxifen resistance.

Hormone receptor-positive breast cancer accounts for around 75% of breast cancers. The estrogen receptor pathway promotes tumor progression and endocrine resistance. Recently, the cross-talk between the ER signaling pathway and cell cycle regulation has been identified. It is necessary to determine the underlying molecular mechanisms involved in the ER signaling pathway and find new target genes for prognosis and drug resistance in ER+ breast cancer. In this study, lncRNA MAFG-AS1 was shown to be up-regulated and associated with poor prognosis in ER+ breast cancer. Functionally, down-regulation of MAFG-AS1 could inhibit cell proliferation and promote apoptosis. In addition, MAFG-AS1 which contained an estrogen-responsive element could promote CDK2 expression by sponging miR-339-5p. Subsequently, MAFG-AS1 and CDK2 were found to be up-regulated in tamoxifen-resistant MCF-7 cells. Cross-talk between the ER signaling pathway and cell cycle conducted by MAFG-AS1 and CDK2 could promote tamoxifen resistance. In conclusion, our study indicated that estrogen-responsive lncRNA MAFG-AS1 up-regulated CDK2 by sponging miR-339-5p, which promoted ER+ breast cancer proliferation. Cross-talk between the ER signaling pathway and cell cycle suggested that lncRNA MAFG-AS1 is a potential biomarker and therapeutic target in ER+ breast cancer. CDK2 inhibitors may be applied to endocrine resistance therapy.

Deubiquitinase USP20 promotes breast cancer metastasis by stabilizing SNAI2.

SNAI2/SLUG, a metastasis-promoting transcription factor, is a labile protein that is degraded through the ubiquitin proteasome degradation system. Here, we conducted comprehensive gain- and loss-of-function screens using a human DUB cDNA library of 65 genes and an siRNA library of 98 genes, and identified USP20 as a deubiquitinase (DUB) that regulates SNAI2 ubiquitination and stability. Further investigation of USP20 demonstrated its function in promoting migration, invasion, and metastasis of breast cancer. USP20 positively correlates with SNAI2 protein level in breast tumor samples, and higher USP20 expression is associated with poor prognosis in ER- breast cancer patients.

UBR5 over-expression contributes to poor prognosis and tamoxifen resistance of ERa+ breast cancer by stabilizing β-catenin.

Tamoxifen (TAM) resistance is a critical clinical challenge in the treatment of ERa+ breast cancer. However, the underlying mechanisms involved in TAM-resistance are not fully understood. Here we study the efficacy of UBR5 in predicting TAM-resistance in ERa+ breast cancer. Western blot RT-PCR and IHC staining were used to evaluate UBR5 protein and mRNA levels in ERa+ breast cancer cell and tissues. MTT assays and colony formation assays were used to measure cell proliferation. The xeno-graft tumor model was used for in vivo study. We performed protein stability assay and ubiquitin assay to detect β-catenin protein degradation. Immuno-precipitation assay was used to detect the interaction between UBR5 and β-catenin. The ubiquitin-based immuno-precipitation based assay was used to detect the ubiquitination of β-catenin. High UBR5 expression was correlated with poor prognosis in ER+ breast cancer. Importantly, UBR5 expression was remarkably upregulated in TAM-refractory breast cancer tissues compared with their primary paired TAM-untreated tissues. Additionally, UBR5 overexpression caused tamoxifen-resistance in vitro, whereas UBR5 knockdown increased TAM sensitivity. Mechanistic investigations revealed that UBR5 overexpression, through its ubiquitin ligase catalyzing activity, led to up-regulation of β-catenin expression and activity. Finally, our results confirmed that TAM-resistance promoting effects by UBR5 in ERa+ breast cancer cells was at least partly due to β-catenin stabilization, and inhibition of the UBR5/β-catenin signaling re-sensitizing the resistant breast cancer cells to tamoxifen in vivo. These findings suggested that UBR5/β-catenin signaling might be a potential therapeutic target for TAM-resistant ERa+ breast cancer.

Abstract 2754: Inhibition of nitric oxide synthase and cyclooxygenase potentiate immune responses that control aggressive mammary tumor in a murine model

Abstract Progression of aggressive, triple negative breast cancers (TNBC) is driven by inflammation and the presence of tumor infiltrating lymphocytes (TIL) improves survival. Recent studies show that TNBCs show high levels of TILs but these cells are exhausted as evident from decreased IFNγ, Ki67 and granzyme B. High expression of the inflammation-associated protein, inducible nitric oxide synthase (NOS2) and co-expression with high cyclooxygenase 2 (COX2) means poor prognosis in ER- breast cancer. Interestingly, NOS2 and COX2 are upregulated in different cells in the tumor microenvironment (TME). Thus, we hypothesized that the tandem use of NOS2 and COX2 inhibitors would curtail tumor progression by targeting different cell types within the TME simultaneously. As COX2 has also been reported to regulate tumor infiltration of myeloid cells, this could regulate both the lymphoid and myeloid arms of the immune system for better response. We investigated the effects of NOS2 and COX2 on tumorigenesis and pulmonary metastasis in a murine model of TNBC by injecting 4T1 cells into the fourth mammary fat pad of wild type (WT) and NOS2 knockout (NOS2-/-) BALB/c mice followed by treatment with COX inhibitor, indomethacin. Changes in infiltrating immune cells was analyzed using flow cytometry and confocal microscopy. We measured the levels of serum cytokines using bead-based, LegendPlex assay. We found that NOS2-/- mice developed fewer pulmonary metastatic lesions than WT mice while indomethacin directly reduced primary tumor growth in both WT and NOS2-/- mice. Indomethacin treated NOS2-/- mice showed better median survival (60 days compared 51 in WT mice). Two NOS2-/-, indomethacin treated mice showed complete remission and long term (5-months) tumor growth delay after 4T1 re-challenge, indicating immune activation in mice lacking NOS2 and COX2 function. Flow cytometry of primary tumors showed higher infiltration of activated CD4, CD8 cells, macrophages and reduction of Tregs in treated mice. Microscopy revealed a physical ‘walling -off' of TILs by CD11b+ cells in untreated tumors that was abrogated upon indomethacin treatment. As many TNBC patients succumb to pulmonary metastasis and because we found reduced metastasis in NOS2-/- mice, we studied the immune cells in metastatic lungs. Lung niche of NOS2-/- mice had more activated T-cells, bone marrow-derived macrophages and neutrophils that are not conducive to establishment of metastasis. Serum of NOS2-/- mice had elevated IL-6 that drove increased IL17A subsequently contributing to reduced metastasis and this effect was improved upon indomethacin treatment. IL6 is also a potential inhibitor of Tregs thus further improving immune surveillance. COX inhibition can treat aggressive TNBC directly by reducing cancer cell proliferation and indirectly by immune activation. Tandem NOS2 inhibition can render the pulmonary microenvironment unfavorable for metastatic spread and induce a favorable systemic cytokine storm that further improves outcome. Citation Format: Veena Somasundaram, Debashree Basudhar, Christopher McGinity, Robert Y. Cheng, Lisa A. Ridnour, Stephen J. Lockett, David A. Wink. Inhibition of nitric oxide synthase and cyclooxygenase potentiate immune responses that control aggressive mammary tumor in a murine model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2754.

Abstract C092: Immune characteristics of triple-negative breast cancer (TNBC) in Latin American women from Colombia

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive form of the disease with limited treatment options and poor survival. A series of retrospective studies established the presence of tumor-infiltrating lymphocytes (TILs) in most primary breast cancers, the number of which affect the response rate to chemotherapy in both neoadjuvant and adjuvant treatment contexts. A subset of TNBC is highly immunogenic, and recent studies have shown that understanding the specific immune profiles of tumors has implications both for treatment and prognosis. Most studies have been conducted in samples of women of European origin, and little is known about the immune profile of TNBCs in Hispanic/Latinas. The specific goal of our study was to test the association between immune characteristics of TNBC from Colombian patients and genetic ancestry proportions, to determine if genetic ancestry could be a relevant factor in defining the immune characteristics of TNBC. Methods: We obtained 41 TNBC tissues through collaboration with the National Cancer Institute in Colombia along with demographic and clinical information. Genetic ancestry was estimated with a panel of 106 ancestry informative markers and the program STRUCTURE. RNA was extracted from FFPE tissues, qualified using Agilent chips, and used for sequencing at the Translational Genomics Core at the Stanley S. Scott Cancer Center. Using the TNBCtype program we confirmed 33 samples as TNBC. The program xCell was used for cell-signature identification based on RNAseq data. We explored associations between genetic ancestry and scores obtained for different immune cell-types and an overall immune score using linear regression. We analyzed the association for the following specific cells: Th1 cells, Th2 cells, Treg, CD4 T cells, CD4 TEM, CD4 memory T cells, CD8 T cells, CD8 TCM, CD8 TEM, CD8 naive T cells, macrophages, macrophages M1, macrophages M2, and mast cells. Results: The overall immune score was positively associated with the proportion of African ancestry (p=0.049). Interestingly, we observed that Indigenous American ancestry was inversely associated with the Treg cell score while African ancestry was positively associated with it. Levels of these regulatory cells have been correlated with prognosis in ER- and triple-negative disease. We also found that CD4 T cell and CD4 memory T-cell scores were positively associated with African ancestry (P=0.006 and P=0.049 respectively). Conclusions: Our results suggest that genetic ancestry among Latina women from Colombia is associated with certain immune characteristics of breast cancer. We need to further test these associations in a larger sample to confirm the findings, obtain additional insights, and understand their implications for TNBC treatment and prognosis among Latin American women. Citation Format: Valentina A. Zavala, Laura C. Carrasquilla, Silvia J. Serrano-Gomez, Maria C. Sanabria-Salas, Melody C. Baddoo, Jone Garai, Jovanny Zabaleta, Laura Fejerman. Immune characteristics of triple-negative breast cancer (TNBC) in Latin American women from Colombia [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C092.

Potential roles of lymphovascular space invasion based on tumor characteristics provide important prognostic information in T1 tumors with ER and HER2 positive breast cancer

Patients presenting with lymphovascular space invasion (LVSI) had an absolute decrease in survival. In our present study, the potential roles of LVSI on tumor characteristics was explored to predict the difference in the prognosis of ER and HER2 positive T1 tumors. A total of 142 breast cancer patients diagnosed with ER+ and HER2+ tumors whose tumor size was ≤ 2cm were included in this analysis. One hundred forty-two patients were divided into four groups, group 1 (lymph nodes+ and LVSI+), group 2 (lymph nodes+ and LVSI-), group 3 (lymph nodes- and LVSI+), group 4 (lymph nodes- and LVSI-). Univariate and multivariate Cox proportional hazard models were used to identify independent prognostic factors and calculate the HR and 95% CI. Kaplan-Meier and Cox regression models were used to test the prognostic significance. LVSI positivity was significantly associated with patient age, menopausal status, tumor size, lymph node status, Ki67, PR, and tumor grade. In the univariate and multivariate model, LVSI, PR, and Ki67 were significantly associated with DFS, and LVSI, lymph node status, PR, and Ki67 were significantly associated with OS. LVSI was significantly related to increased risk of DFS and OS only in the PR-negative and low-positive subgroups. It was a prognostic factor for DFS but not for OS in women with low Ki67 and was associated with DFS and OS in high-Ki67 tumors. Furthermore, patients who presented with only LVSI had a significantly worse survival rate than those with lymph node metastasis without LVSI in small tumors. The presence of LVSI was highlighted as a variable significant to survival. In further clinical practice, patients with LVSI may need more intensive treatment in certain populations.

Abstract BL2: The Molecular Etiology of Luminal-Type Breast Cancer

Abstract Estrogen receptor positive disease is the dominant contributor to global deaths from breast cancer which now exceeds 500,000 deaths annually. Lethality is driven by endocrine therapy resistance, the development of metastasis and an inability of the immune system to mount an effective anti-tumor T cell response. While our understanding of these processes is incomplete, molecular analyses of ER+ tumor samples accrued from patients undergoing neoadjuvant endocrine therapy (NET) have generated fundamental insights into nature of the disease while providing clear benefits to patients in terms of increased use of breast conserving surgery and strategies to avoid chemotherapy1,2. From the outset, genomic analyses of ER+ breast cancer samples has demonstrated that poor outcome is associated with genomic complexity and sub-clonal diversity3,4. As endocrine therapy responsive tumors are exposed to treatment, responsive subclones regress and resistant subclones expand. Eventually clones harboring lethal mutations are selected, including ER ligand binding mutations and chromosomal translocations involving ER that generate EMT-driving chimeric transcription factors5,6. Additional genomic events include loss of tumor suppressors that deregulate ER function, such as NF1 frame-shift or nonsense mutations7, or gain of function mutations in growth factor receptors such as ErbB28. These acquired resistance mutations are druggable and targeted interventions should prove useful. But what are the DNA repair defects that underlie the lethal genomic complexity of ER+ disease? Unlike ER negative/basal like breast cancer, luminal-type breast cancer is typically not defective in homologous recombination and is TP53 wild-type. Our recent data suggest loss of components in single strand (ss) DNA repair mechanisms associate with poor prognosis in ER+ breast cancer including MLH1/PMS1/PMS2 (mismatch repair), NEIL2 (base excision repair) or ERCC1 (nucleotide excision repair)9,10. Experimental down-regulation of any of these five genes generates endocrine therapy resistance, likely by disrupting CHK2 signaling, which acts as an endogenous inhibitor of CDK4/6 function. Indeed, direct CHK2 knockdown also generates endocrine therapy resistance but CDK4/6 inhibitor sensitivity is maintained in all these experimental conditions. Essentially the CDK4/6 inhibitor therapy can be seen as replacing CHK2 for negative cell cycle regulation. Thus, we postulate that the presence of ssDNA break repair defects and/or CHK2 dysregulation could underlie the remarkably selective efficacy of CDK4/6 inhibitors in ER+ disease by targeting the intersection of ER signaling and the ssDNA repair response at the level of CHK2 activity. Furthermore, correlations between poor prognosis in ER+ disease, increased innate immunity signaling and ssDNA repair defects point towards subsets of ER+ breast cancers that might respond to immune checkpoint therapy11.

Abstract P3-08-33: Intermediate HER2 expression predicts poor prognosis in ER (+) breast cancer patients aged 55 and older

Abstract Background The antibody-drug conjugate targeting HER2, DS8201 has shown clinical activity against breast cancer with low-level HER2 expression in early clinical trial. In this study, we investigated the clinical implication and prognostic impact of intermediate HER2 expression in the prospectively collected ER (+) early breast cancer (EBC) and metastatic breast cancer (MBC) cohorts. Methods We analyzed the prospectively collected database of EBC and MBC cohort patients in Yonsei Cancer Center. We defined patients with HER2 immunohistochemistry (IHC) 0~1+ as HER2-negative group, and IHC 2+ and in situ hybridization (ISH) negative as HER2-intermediate group. Only ER (+) patients with complete HER2 IHC and ISH status were selected, excluding HER2 IHC 3+ or ISH+ patients. A total of 2,657 early breast cancer and 535 metastatic breast cancer patients were finally analyzed. The clinical and pathological characteristics and survival outcome of the patients were compared between HER2-negative and HER2-intermediate group in each cohort. Results The 654 (24.6%) EBC patients and 166 (31.0%) MBC patients were classified as HER2-intermediate group. The HER2-intermediate patients were more common in PR negative (30.4% versus 22.6%, p&amp;lt; 0.001) and higher nuclear grade (grade 2 or 3) patients (25.7% versus 17.4%, p = 0.001) in EBC cohort and in age ≥ 55 patients (36.7% versus 27.4%, p = 0.001) in MBC cohort. Other characteristics were not different between two groups in both cohorts. The HER2-intermediate patients showed significantly poorer recurrence-free survival (RFS) than HER2-negative patients in EBC patients (p = 0.044), although the prognostic impact was not significant in the multivariate analysis. Of note, intermediate HER2 expression was associated with significantly poorer RFS in EBC patients (p = 0.007) of age ≥ 55, but did not affect the RFS in patients of age &amp;lt; 55. The intermediate HER2 expression independently predicted poorer RFS of EBC patients of age ≥ 55 (Hazard ratio [HR], 1.95; 95% Confidence interval [CI], 1.03-3.73; p = 0.042) in multivariate Cox regression analysis with adjustment of other prognostic factors, T stage, N stage, PR status, and histologic grade. In line with result of EBC cohort, intermediate HER2 expression was associated with poorer overall survival (OS) in MBC patients of age ≥ 55 (p = 0.037), not affecting OS in MBC patients of age &amp;lt; 55. Intermediate HER2 expression predicted significantly poorer OS in MBC patients of age ≥ 55 (HR, 1.45; 95% CI, 1.01-2.07; p = 0.044) independent from PR status, disease status (recurrent versus de novo stage IV), and visceral metastasis. Conclusion Our analysis demonstrates intermediate HER2 expression as an independent poor prognostic factor for ER (+) breast cancer patients with age ≥ 55 in both EBC and MBC cohorts. The clinical efficacy of new HER2-targeting antibody-drug conjugates needs to be validated in this high-risk subset of ER (+) breast cancer patients. Citation Format: Min Hwan Kim, Gun Min Kim, Jee Hung Kim, Ji Ye Kim, Hyung Seok Park, Seho Park, Young Up Cho, Byeong Woo Park, Seung Il Kim, Joo-Hyuk Sohn. Intermediate HER2 expression predicts poor prognosis in ER (+) breast cancer patients aged 55 and older [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 P3-08-33.

Abstract P6-04-25: Epidermal growth factor receptor as therapeutic target in hormone receptor-positive breast cancer

Abstract Background/Aims: Despite effective therapeutic strategies for treating hormone receptor-positive (HR+) breast cancer, resistance to endocrine therapy that is either de novo or acquired still occurs. We investigated epidermal growth factor receptor (EGFR) as a therapeutic target for overcoming endocrine resistance in HR+ breast cancer models. Methods: Using clinical data from 2,166 patients who had HR+ breast tumors and received tamoxifen, we analyzed survival rates. Levels of mRNA and protein expression were analyzed by real-time PCR and western blotting, respectively. Cell viability was analyzed by MTT assays and anchorage-independent growth by soft agar colony-formation assays. Efficacy of tamoxifen and/or gefitinib was analyzed using orthotopic xenograft mouse models. Results: EGFR expression was significantly associated with more advanced stage and higher grade. EGFR expression was different in luminal A-like (Lum A, 1.3%) versus luminal B-like (Lum B, 11.4%) subtypes. On multivariate analyses for survival Lum B subtype EGFR+ tumors showed a hazard ratio (HR) of 5.22 (95% CI, 1.29-21.15, P = 0.020) for overall survival (OS) and HR of 2.91 (95% CI, 1.35-6.28, P = 0.006) for disease-free survival (DFS). Levels of EGFR inversely correlated with ER-α expression. Basal ER-α level was completely blocked by TGFA or EGF treatment. With TGFA pretreatment, ER+ breast cancer cells were resistant to 4-hydroxytamoxifen (4-OHT). Conversely, downregulation of ER-α by TGFA was reversed by gefitinib with recovered sensitivity to 4-OHT. Tumorigenicity of EGFR and ER+ breast cancer cells was significantly decreased by combined tamoxifen and gefitinib. Conclusion: Aberrant EGFR expression was associated with poor prognosis in ER+ breast cancers, especially the Lum B subtype. Loss of ER by EGFR activation induced tamoxifen resistance. Therefore, EGFR could be a therapeutic target for overcoming recurrence of ER+ breast cancer with high EGFR expression. Citation Format: Yisun Jeong, Soo Youn Bae, Daeun You, Seung Pil Jung, Hee Jun Choi, Isaac Kim, Se Kyung Lee, Jonghan Yu, Seok Won Kim, Jeong Eon Lee, Sangmin Kim, Seok Jin Nam. Epidermal growth factor receptor as therapeutic target in hormone receptor-positive 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 P6-04-25.

Abstract PD7-10: Randomized, multicenter phase II trial of weekly paclitaxel with or without the oral selective aurora kinase A (AURKA) inhibitor, Alisertib, in patients with ER+ HER2- metastatic breast cancer

Abstract Elevated expression of AURKA adversely affects prognosis in ER+ breast cancer and is associated with resistance to taxanes, endocrine therapy, and PI3K inhibitors. A recent randomized phase II trial of combined paclitaxel plus alisertib (P+A) in recurrent ovarian cancer patients (pts) showed improved progression-free survival (PFS) compared to paclitaxel (P) alone (Falchook G. JAMA Oncol 2019). We conducted a randomized phase II trial of P alone versus P+A in pts with ER+ HER2- metastatic breast cancer (MBC). Methods: The primary objective of the trial was PFS in the intent-to-treat (ITT) population with secondary endpoints of overall survival (OS), overall response rate (ORR), clinical benefit rate (CBR; CR+PR+SD&amp;gt;6 mos), safety and analysis of archival breast cancer for molecular predictors of benefit from alisertib. ER+ HER2- MBC pts who had had prior endocrine therapy, 0 or 1 prior chemotherapy regimens for MBC, &amp;gt; 12 mos treatment-free interval from neo/adjuvant taxane therapy, and who had measurable or evaluable lytic bone disease were randomized to receive P 90 mg/m2 IV D1, 8, 15 on a 28-day cycle or P 60mg/m2 D1, 8, 15 plus A 40 mg PO BID D1-3, 8-10, 15-17 on a 28-day cycle until disease progression or unacceptable toxicity. Pts were stratified by prior neo/adjuvant taxane (yes/no) and by whether they were receiving first (1L)- or second (2L)-therapy for MBC. Proportions of ORR and CBR were compared using Chi-square test between the two study arms. Kaplan-Meier estimator was applied to estimate OS and PFS and univariate Cox regression was used to assess the hazard ratio of study treatment. Results: 139 pts were randomized (69 to P+A; 70 to P) in the US Oncology Network between 2/2015 and 2/2018 and 136 pts received treatment on trial. At data cut-off, 76% of pts had had a documented PFS event and the median followup was 19.7 mos. The median age was 62 and 69% of pts received trial therapy as 1L treatment; 41% had had prior neo/adjuvant taxane therapy. Median PFS in the ITT population was 10.2 mos with P+A vs 7.1 mos with P alone, HR 0.56, 95% CI 0.37-0.84, p=0.005. Median OS was 29.8 mos with P+A vs 24.4 mos with P alone, HR 0.85, p=0.486. ORR and CBR in the response evaluable population were 31% and 67%, respectively, in the P+A arm vs 34% and 57%, respectively, in the P alone arm (p&amp;gt;0.05). Grade 3/4 adverse events (AEs) occurred in 83% of P+A pts vs 47% with P alone. The main grade 3/4 AEs with P+A vs P were neutropenia 59% vs 15%, anemia 10% vs 1%, febrile neutropenia 1.5% vs 0, diarrhea 11% vs 0, stomatitis 15% vs 0, peripheral neuropathy 1.5% vs 9%. 1 pt died of sepsis with P+A. Conclusions: Addition of oral A to weekly P significantly improved PFS, the primary endpoint, compared with P alone, and toxicity with P+A was manageable with A dose reduction. These data support further evaluation of alisertib in ER+ HER2- MBC pts. Citation Format: Joyce O'Shaughnessy, Kristi McIntyre, Sharon Wilks, Ling Ma, William Fintel, Margaret Block, David Andorsky, Michael Danso, Nicholas Koutrelakos, Tracy Locke, Amy Scales, Lauren Steckel, Yunfei Wang. Randomized, multicenter phase II trial of weekly paclitaxel with or without the oral selective aurora kinase A (AURKA) inhibitor, Alisertib, in patients with ER+ HER2- metastatic 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 PD7-10.

EGFR is a Therapeutic Target in Hormone Receptor-Positive Breast Cancer.

Despite effective therapeutic strategies for treating hormone receptor-positive (HR+) breast cancer, resistance to endocrine therapy that is either de novo or acquired still occurs. We investigated epidermal growth factor receptor (EGFR) as a therapeutic target for overcoming endocrine resistance in HR+ breast cancer models. Using clinical data from 2,166 patients who had HR+ breast tumors and received tamoxifen, we analyzed survival rates. Levels of mRNA and protein expression were analyzed by real-time PCR and western blotting, respectively. Cell viability was analyzed by MTT assays and anchorage-independent growth by soft agar colony-formation assays. Efficacy of tamoxifen and/or gefitinib was analyzed using orthotopic xenograft mouse models. EGFR expression was significantly associated with more advanced stage and higher grade. EGFR expression was different in luminal A-like (Lum A, 1.3%) versus luminal B-like (Lum B, 11.4%) subtypes. On multivariate analyses for survival Lum B subtype EGFR+ tumors showed a hazard ratio (HR) of 5.22 (95% CI, 1.29-21.15, P = 0.020) for overall survival (OS) and HR of 2.91 (95% CI, 1.35-6.28, P = 0.006) for disease-free survival (DFS). Levels of EGFR inversely correlated with ER-α expression. Basal ER-α level was completely blocked by TGFA or EGF treatment. With TGFA pretreatment, ER+ breast cancer cells were resistant to 4-hydroxytamoxifen (4-OHT). Conversely, downregulation of ER-α by TGFA was reversed by gefitinib with recovered sensitivity to 4-OHT. Tumorigenicity of EGFR and ER+ breast cancer cells were significantly decreased by combined tamoxifen and gefitinib. Aberrant EGFR expression was associated with poor prognosis in ER+ breast cancers, especially the Lum B subtype. Loss of ER by EGFR activation induced tamoxifen resistance. Therefore, EGFR could be a therapeutic target for overcoming recurrence of ER+ breast cancer with high EGFR expression.

CRABP2 regulates invasion and metastasis of breast cancer through hippo pathway dependent on ER status

BackgroundTriple Negative Breast cancer (TNBC) is incurable cancer with higher rates of relapse and shorter overall survival compared with other subtypes of breast cancer. Cellular retinoic acid binding protein 2 (CRABP2) belongs to fatty acid binding protein (FABP) family which binds with all-trans retinoic acid (RA). Previous studies from the database have reported the patients with high expression of CRABP2 showed different prognosis in ER+ and ER− breast cancer. However, its biological role and exact mechanism in breast cancer remain unknown. This aim of this study was to explore how CRABP2 regulated invasion and metastasis based on the estrogen receptor-α (herein called ER) status in breast cancer.MethodsImmunohistochemical staining method was used to analyze the expression of CRABP2 in human breast cancer tissues. Lentivirus vector-based shRNA technique was used to test the functional relevance of CRABP2 knockdown in breast tumors. Tail vein injection model was used to examine the lung metastasis. Co-immunoprecipitation, Western blotting, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were conducted to investigate the underlying mechanism that influenced the ER to the regulation of CRABP2 to Lats1.ResultsWe observed that knockdown of CRABP2 promotes EMT, invasion and metastasis of ER+ breast cancer cells in vitro and in vivo, whereas overexpression of CRABP2 yields the reverse results. In ER+ mammary cancer cells, the interaction of CRABP2 and Lats1 suppress the ubiquitination of Lats1 to activate Hippo pathway to inhibit the invasion and metastasis of ER+ mammary cancer. However, in ER− mammary cancer cells, the interaction of CRABP2 and Lats1 promote the ubiquitination of Lats1 to inactivate Hippo pathway to promote the invasion and metastasis of ER− mammary cancer.ConclusionsOur findings indicate that CRABP2 can suppress invasion and metastasis of ER+ breast cancer and promote invasion and metastasis of ER− breast cancer by regulating the stability of Lats1 in vitro and in vivo, and it provides new ideas for breast cancer therapy.

Mutual regulations and breast cancer cell control by steroidogenic enzymes: Dual sex-hormone receptor modulation upon 17β-HSD7 inhibition

Reductive 17β-hydroxysteroid dehydrogenases (17β-HSDs) and 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) play crucial roles in respectively regulating steroids and glucocorticoids for the progression of hormone-dependent breast cancer. Most studies focused on the function and individual regulation of these enzymes. However, mutual regulation of these enzymes and the induced modulation on the estrogen and androgen receptors for breast cancer promotion are not yet clear. In this study, MCF-7 and T47D cells were treated with inhibitors of 17β-HSD1, 17β-HSD7, aromatase or steroid sulfatase (STS), then mRNA levels of 17β-HSD7, STS, 11β-HSD 2, estrogen receptors α (ERα) and androgen receptor (AR) were determined by Q-PCR. ER negative cell line MDA-MB-231 was used as a negative control. Our results demonstrate that 17β-HSD7, STS and 11β-HSD2 are all regulated by the same estrogen estradiol via ERα. When the gene of ERα (ESR1) was knocked down, there was no longer significant mutual regulation of these enzymes.Our results demonstrate that important steroidogenic enzymes transcriptionally regulated by ERα, can be mutually closely correlated. Inhibition of one of them can reduce the expression of another, thereby amplifying the role of the inhibition. Furthermore, inhibition of 17β-HSD7 increases the expression of AR gene which is considered as a marker for better prognosis in ER + breast cancer, while maintaining ERα level. Thus, our mechanistic finding provides a base for further improving the endocrine therapy of ER + breast cancer, e.g., for selecting the target steroid enzymes, and for the combined targeting of human 17β-HSD7 and ERα.

Overexpression of ECT2 is a strong poor prognostic factor in ER(+) breast cancer.

Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor encoded by the ECT2 gene, which is located on the 3q26.31 chromosomal region and is directly associated with the occurrence of cancers. The aim of the present study was to examine the expression and prognostic importance of ECT2 in various breast cancer subtypes using the online tools, Gene Expression Profiling Interactive Analysis, Kaplan-Meier-plotter and bc-GenExMiner. ECT2 mRNA expression was significantly different in oestrogen receptor ER(+) breast cancer; overexpression of ECT2 was associated with poor prognosis in ER+ breast cancer. The mRNA expression levels of ECT2 were increased in basal-like breast cancer and triple negative breast cancer, but were not significant for prognostic prediction. We identified ECT2-correlated genes and their corresponding Gene Ontology (GO) enrichment terms. The results revealed that GO: 0005524 (protein binding) had the greatest number of correlated genes and also contained ECT2. This suggested that overexpression of ECT2 may be a significant prognostic factor for poor outcome in ER+ breast cancer; however, the precise role of ECT2 in breast cancer requires further investigation.

Abstract P5-04-13: Splicing factor ESRP1 controls ER-positive breast cancer progression by altering metabolic pathway genes

Abstract Background Epithelial Splicing Regulatory Protein (ESRP1)is a key splicing factor that regulates Epithelial-to-Mesenchymal Transition (EMT) splicing program. Our previous study demonstrated that high levels of ESRP1 are associated with poor prognosis in human ER-positive (ER+) breast tumors in an independent manner of EMT process. We next explored the potential mechanisms that contribute to the role ESRP1 in endocrine therapy-resistant breast cancer. Methods Probe based-Human Transcriptome Array 2.0 (HTA; Applied Biosystems/Thermo Fisher) was performed using RNAs from control and ESRP1 knockdown cells (LCC2 versus 2C3 ESRP1 and LCC9 versus 9C2 ESRP1) of endocrine resistant breast cancer. Functional enrichment analyses were performed using the DAVID functional annotation tool (http://david.abcc.ncifcrf.gov/). To confirm the functional importance of ESRP1 on regulation of cellular metabolism, we have performed experiments that analyze the metabolic substrate flux in response to ESRP1 knockdown in resistant cells (The Seahorse XFp Cell Energy Phenotype Assay). Differentially expressed genes were validated using Western blotting assay. Results Transcriptome profiling of ESRP1in 2C3 and 9C2 knockdown models revealed differentially expressed genesusing HTA 2.0 platform. In LCC2 versus 2C3 ESRP1 knockdown, expression of 1186 genes (1263 transcripts) have been altered significantly, while 413 genes (432 transcripts) have been significantly regulated in LCC9 versus 9C2 ESRP1 knockdown with FDR&amp;lt;0.1 Of these significant genes, 34 downregulated and 68 upregulated (102 genes total) were shared by both 2C3 and 9C2 ESRP1 knockdowns. Using the DAVID Functional Annotation Clustering Tool, we identified the biological processes altered significantly in response to ESRP1 knockdown. The most significant annotation clusters downregulated in ESRP1 knockdown consists of fatty acid metabolism/lipid metabolism (SCD, ACACA, FASN, ACAT2, PLCH1, and HPGD), and oxireductase processes (SCD, PHGDH, FASN, DHTKD1 and HPGD. We confirmed the altered metabolic function using the Seahorse analyzer. These analyses confirmed that ESRP1 knockdown altered the glycolysis rate (extracellular acidification rate; ECAR) in both tamoxifen-resistant and fulvestrant-resistant models. In addition, ESRP1 knockdown decreased the mitochondrial respiration in tamoxifen-resistant cells, but not fulvestrant resistant cells. Using Western blotting, we validated the altered levels of fatty acid synthase (FASN) and Stearoyl-CoA desaturase 1 (SCD1), key enzymes in fatty acid metabolism. Phosphoglycerate Dehydrogenase (PHGDH), a poor prognosis marker in cancers including breast cancer, was also downregulated in response to ESRP1 knockdown. Taken together, we have demonstrated a novel functional impact of ESRP1 on the regulation of tumor growth at the functional and molecular level independent of EMT. Conclusions For the first time, we show the role of ESRP1 in altering the cellular metabolism thereby contributing to tumor growth. The study provides a molecular evidence for the role of altered metabolism in determining adverse prognosis of ER+ breast cancer via the control of ESRP1, a splicing factor. Further studies to determine the therapeutic value are underway. Citation Format: Gokmen-Polar Y, Gu Y, Gu X, Badve SS. Splicing factor ESRP1 controls ER-positive breast cancer progression by altering metabolic pathway genes [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 P5-04-13.

Estrogen-dependent DLL1-mediated Notch signaling promotes luminal breast cancer

Aberrant Notch signaling is implicated in several cancers, including breast cancer. However, the mechanistic details of the specific receptors and function of ligand-mediated Notch signaling that promote breast cancer remains elusive. In our studies we show that DLL1, a Notch signaling ligand, is significantly overexpressed in ERα+ luminal breast cancer. Intriguingly, DLL1 overexpression correlates with poor prognosis in ERα+ luminal breast cancer, but not in other subtypes of breast cancer. In addition, this effect is specific to DLL1, as other Notch ligands (DLL3, JAGGED1, and JAGGED2) do not influence the clinical outcome of ERα+ patients. Genetic studies show that DLL1-mediated Notch signaling in breast cancer is important for tumor cell proliferation, angiogenesis, and cancer stem cell function. Consistent with prognostic clinical data, we found the tumor-promoting function of DLL1 is exclusive to ERα+ luminal breast cancer, as loss of DLL1 inhibits both tumor growth and lung metastasis of luminal breast cancer. Importantly, we find that estrogen signaling stabilizes DLL1 protein by preventing its proteasomal and lysososmal degradations. Moreover, estrogen inhibits ubiquitination of DLL1. Together, our results highlight an unexpected and novel subtype-specific function of DLL1 in promoting luminal breast cancer that is regulated by estrogen signaling. Our studies also emphasize the critical role of assessing subtype-specific mechanisms driving tumor growth and metastasis to generate effective subtype-specific therapeutics.