Development Of Endocrine Therapy Resistance Research Articles

Analysis of TLR2 in Primary Endocrine Resistant of Breast Cancer.

Previous clinical studies have suggested that Toll-like receptor (TLR)2 had predictive function for endocrine resistance in HER2-positive breast cancer (BCa). Nevertheless, it remains unclear whether TLR2 would relate to development of endocrine therapy resistance in triple-positive breast cancer (TPBC). Bioinformatic analysis of TLR2 was carried out through a database. Ten tumor tissues were obtained from TPBC patients who underwent surgery, with five patients displaying primary resistance to tamoxifen (TAM) with the remaining 5 being sensitive. Different levels of proteins were identified through mass spectrometry analysis and confirmed through reverse transcription polymerase chain reaction (RT-PCR) and western blot. TAM-resistant cell lines (BT474-TAM) were established by continuous exposure to TAM, and TAM resistance was assessed via IC50. Additionally, TLR2 mRNA was analyzed through western blot and RT-PCR in BT474, BT474-TAM, MCF-7, and MCF10A cells. Furthermore, TLR2-specific interference sequences were utilized to downregulate TLR2 expression in BT474-TAM cells to elucidate its role in TAM resistance. TLR2 had a correlation with decreased relapse-free survival in BCa patients from the GSE1456-GPL96 cohort, and it was involved in cancer development predominantly mediated by MAPK and PI3K pathways. TLR2 protein expression ranked in the top 5 proteins within the TAM-resistant group, and was 1.9 times greater than that in the sensitive group. Additionally, TLR2 mRNA and protein expression increased significantly in the established TAM-resistant BT474/TAM cell lines. The sensitivity of TAM was restored upon TLR2 downregulation in BT474/TAM cells. TLR2 might have a therapeutic value as it was involved in the TAM resistance in TPBC, with potential to be a marker for primary endocrine resistance.

A functional genetic screen for metabolic proteins unveils GART and the de novo purine biosynthetic pathway as novel targets for the treatment of luminal A ERα expressing primary and metastatic invasive ductal carcinoma.

Targeting tumor cell metabolism is a new frontier in cancer management. Thus, metabolic pathway inhibitors could be used as anti-estrogen receptor α (ERα) breast cancer (BC) drugs. Here, the interplay among metabolic enzyme(s), the ERα levels and cell proliferation was studied. siRNA-based screen directed against different metabolic proteins in MCF10a, MCF-7 and MCF-7 cells genetically resistant to endocrine therapy (ET) drugs and metabolomic analyses in numerous BC cell lines unveil that the inhibition of GART, a key enzyme in the purine de novo biosynthetic pathway, induces ERα degradation and prevent BC cell proliferation. We report here that a reduced GART expression correlates with a longer relapse-free-survival (RFS) in women with ERα-positive BCs. ERα-expressing luminal A invasive ductal carcinomas (IDCs) are sensitive to GART inhibition and GART expression is increased in receptor-positive IDCs of high grade and stage and plays a role in the development of ET resistance. Accordingly, GART inhibition reduces ERα stability and cell proliferation in IDC luminal A cells where it deregulates 17β-estradiol (E2):ERα signaling to cell proliferation. Moreover, the GART inhibitor lometrexol (LMX) and drugs approved for clinical treatment of primary and metastatic BC (4OH-tamoxifen and the CDK4/CDK6 inhibitors) exert synergic antiproliferative effects in BC cells. In conclusion, GART inhibition by LMX or other inhibitors of the de novo purine biosynthetic pathway could be a novel effective strategy for the treatment of primary and metastatic BCs.

437 Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ Breast Cancer to Mitigate Therapy Resistance

OBJECTIVES/GOALS: Estrogen receptor-positive (ER+) breast cancers (BC) comprise >70% of all BC and cause the most BC-related deaths in women worldwide. Despite available therapies against ER+ BC, recurrence often arises due to development of endocrine therapy resistance. Thus, our goal is to identify novel biomarkers that could serve as therapeutic targets for ER+ BC. METHODS/STUDY POPULATION: We have identified a neuroimmune molecule, Semaphorin 7a (SEMA7A), as a potential biomarker for endocrine therapy resistance, relapse, and poor survival in ER+ BC patients. SEMA7A promotes tumor growth, angiogenesis, epithelial-to-mesenchymal transition, metastasis, and endocrine therapy resistance in our pre-clinical models. Specifically, using in vivo models we have shown that SEMA7A+ MCF7 tumors result in lung metastases that do not respond to fulvestrant, in part via downregulation of ER, posing the need to identify novel, druggable targets for SEMA7A+ ER+ BC patients. SEMA7A is a membrane-bound protein that can inhibit tumor cell death via integrin-mediated PI3K/Akt pro-survival signaling. Thus, we hypothesized that SEMA7A+ ER+ BC may be sensitive to PI3K inhibition in combination with fulvestrant. RESULTS/ANTICIPATED RESULTS: Our preliminary studies confirmed that high SEMA7A expression associates with increased phospho-Akt levels and decreased apoptosis of tumor cells in forced suspension conditions. We also observed that human MCF7 ER+ SEMA7A overexpressing (OE) cells are sensitive to the PI3K (P110ï ¡ï€©ï€ inhibitor, Alpelisib. Also, the combination of Alpelisib with fulvestrant inhibited tumor cell viability in MCF7 cells, which was further enhanced in the SEMA7A OE counterparts. The combination also decreased proliferation and tumor sphere formation. There are currently no therapies that directly target SEMA7A, and here I propose an innovative hypothesis that PI3K inhibition will block SEMA7A signaling. DISCUSSION/SIGNIFICANCE: The role of SEMA7A has been studied in several cancer types, but its function in ER+ BC remains less well understood. In future studies, we will explore the mechanisms by which SEMA7A signals in the cell and to promote tumor cell survival. Delineating these mechanisms will help optimize treatment combinations to improve BC patient survival.

Abstract GS3-04: GS3-04 Capivasertib and fulvestrant for patients with aromatase inhibitor-resistant hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer: results from the Phase III CAPItello-291 trial

Abstract Background: AKT pathway activation has been implicated in the development of endocrine therapy resistance in hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2–) advanced breast cancer (ABC). In the Phase II, placebo (PBO)-controlled FAKTION trial, the addition of the pan-AKT inhibitor capivasertib to fulvestrant significantly improved progression-free survival (PFS) and overall survival in postmenopausal women with aromatase inhibitor (AI)-resistant HR+/HER2– ABC. The Phase III, randomized, double-blind, PBO-controlled CAPItello-291 trial (NCT04305496) investigated the efficacy and safety of capivasertib + fulvestrant in patients with AI-resistant HR+/HER2– ABC. Methods: Eligible pre/peri or postmenopausal women or men with HR+/HER2– ABC that had recurred or progressed on or after AI therapy with or without a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor were randomized 1:1 to receive fulvestrant (per standard dosing schedule) with either PBO or capivasertib (400 mg twice daily; 4 days on, 3 days off). Randomization was stratified by the presence of liver metastases, prior use of CDK4/6 inhibitors, and geographic location. AKT pathway alteration status (at least one qualifying PIK3CA, AKT1, or PTEN alteration) was determined using next-generation sequencing in tumor tissue. The dual primary endpoint was investigator-assessed PFS in the overall population and in patients with AKT pathway-altered tumors. Results: A total of 708 patients were randomized: 355 to capivasertib + fulvestrant and 353 to PBO + fulvestrant. Overall, 41% of patients had AKT pathway-altered tumors (48% [n=289/602] of patients with tumor sequencing results), 22% were pre/perimenopausal and 77% postmenopausal, with 1% male. Prior therapy for advanced disease included: 87% of patients with ≥1 line of prior treatment, 69% with a prior CDK4/6 inhibitor, and 18% with prior chemotherapy. Demographic and baseline characteristics were broadly balanced between the overall and altered populations and by treatment groups. At primary analysis (data cut-off Aug 15, 2022), 551 and 236 PFS events had occurred in the overall and pathway-altered populations, respectively. Overall, the median PFS was 7.2 months with capivasertib + fulvestrant and 3.6 months with PBO + fulvestrant (hazard ratio [HR] 0.60; 95% confidence interval [CI] 0.51–0.71; p<0.001). In patients with AKT pathway-altered tumors, median PFS was 7.3 months with capivasertib + fulvestrant and 3.1 months with PBO + fulvestrant (HR 0.50; 95% CI 0.38–0.65; p<0.001). The objective response rate in patients with measurable disease was 22.9% for capivasertib + fulvestrant vs 12.2% for PBO + fulvestrant overall and 28.8% vs 9.7% in the AKT pathway-altered population. The most frequent all-grade adverse events (AEs) with capivasertib + fulvestrant were diarrhea (72.4% vs 20.0% PBO + fulvestrant arm), rash (group term of rash, rash macular, rash maculo-papular, rash papular, rash pruritic; 38.0% vs 7.1%) and nausea (34.6% vs 15.4%). The most frequently reported grade ≥3 AEs were rash (group term; 12.1% vs 0.3%), diarrhea (9.3% vs 0.3%), and hyperglycemia (2.3% vs 0.3%); grade ≥3 stomatitis was 2.0% vs 0%. AEs leading to discontinuation of capivasertib/placebo were reported in 13.0% and 2.3% of patients, respectively. Conclusions: Capivasertib + fulvestrant significantly improved PFS compared to fulvestrant alone in the overall population, and in patients with AKT pathway-altered tumors, and may become a future treatment option in this setting. The safety profile of capivasertib + fulvestrant was generally manageable and consistent with prior data. Funding: CAPItello-291 is sponsored by AstraZeneca. Editorial acknowledgment: AstraZeneca-funded medical writing support was provided by Suzanne Patel, Ph.D., from BOLDSCIENCE Inc. Capivasertib was discovered by AstraZeneca subsequent to a collaboration with Astex Therapeutics (and its collaboration with the Institute of Cancer Research and Cancer Research Technology Limited). Citation Format: Nicholas Turner, Mafalda Oliveira, Sacha J. Howell, Florence Dalenc, Javier Cortés, Henry Gomez, Xichun Hu, Komal Jhaveri, Sibylle Loibl, Serafin Morales Murillo, Zbigniew Nowecki, Meena Okera, Yeon H. Park, Masakazu Toi, Lyudmila Zhukova, Chris Yan, Gaia Schiavon, Andrew Foxley, Hope Rugo. GS3-04 Capivasertib and fulvestrant for patients with aromatase inhibitor-resistant hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer: results from the Phase III CAPItello-291 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS3-04.

Abstract P3-11-03: Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ Breast Cancer

Abstract Endocrine receptor-positive (ER+) breast cancers (BC) comprise over 70% of all breast cancers and are the leading cause of BC-related deaths in women. Despite available targeted therapies against ER+ BC, recurrence arises primarily due to the development of endocrine therapy resistance and metastases. Thus, there is an unmet need to identify novel biomarkers for treating ER+ BC patients with metastases. We have identified a neuroimmune molecule, Semaphorin 7a (SEMA7A), as a potential biomarker for endocrine therapy resistance, increased relapse and decreased overall survival in ER+ BC patients. We have also found that SEMA7A promotes tumor growth, angio- and lymphangiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis and endocrine therapy resistance in pre-clinical models. Specifically, we have shown that SEMA7A+ ER+ MCF7 tumors result in lung metastases that are resistant to the endocrine therapy, fulvestrant, in part, via downregulation of ER in vivo – posing the need to identify novel, druggable targets for SEMA7A+ ER+ BC. SEMA7A is a membrane-bound protein that we have shown can inhibit tumor cell death via integrin-mediated activation of PI3K/Akt pro-survival signaling. Thus, we hypothesized that that SEMA7A+ ER+ BC cells may be sensitive to PI3K inhibition in combination with fulvestrant. Our results demonstrate that SEMA7A+ MCF7 cells are sensitive to the combination of fulvestrant and PI3K inhibition by alpelisib, which is FDA approved for ER+ BC patients with activating mutations in PI3K; this combination also inhibited tumor cell growth and decreased tumor sphere formation. In complementary studies, we identified that Singleminded 2s (SIM2s), which is expressed in breast epithelial cells and inhibits EMT, is downregulated SEMA7A+ MCF7 tumors. Additionally, our studies show that while ER-mediated signaling turns on SEMA7A expression prolonged expression of SEMA7A results in downregulation of ER, and the loss of SIM2s causes downregulation of ER expression and upregulation of SEMA7A in MCF7 cells. Further, while SEMA7A-overexpressing (OE) tumor cells exhibit increased tumor-promoting PI3K/Akt survival signaling, we found that SIM2s plays a role in suppression of PI3K/Akt survival signaling. Therefore, we are investigating the interactions between SIM2s, SEMA7A and the survival signaling pathways involved in their crosstalk. Our recent results show that MCF7 SIM2-knockout (KO) cells exhibit increased SEMA7A promoter activity, SEMA7A expression, activation of Akt signaling, EMT phenotypes and decreased ER expression. Also, addition of exogenous SEMA7A protein reduces SIM2s expression and promoter activity. Since SIM2s correlates with inhibition of PI3K/Akt signaling and decreased SEMA7A, and SEMA7A promotes survival signaling, we are investigating additional therapeutic strategies to prevent endocrine therapy resistance via direct inhibition of SEMA7A and/or restoration of SIM2s, including inhibition of COX-2 and Akt signaling. Citation Format: Rachel Steinmetz, Garhett Wyatt, Traci Lyons, Weston Porter, Lyndsey Crump. Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-03.

18β-glycyrrhetinic Acid Modulated Autophagy is Cytotoxic to Breast Cancer Cells.

The development of endocrine therapy resistance in the luminal A subtype of breast cancer is related to the appearance of protective autophagy. The bioactive component from the root of licorice, 18β-glycyrrhetinic acid (18β-GA), has many antitumor properties. Whether 18β-GA can modulate autophagy to inhibit proliferation of the luminal A subtype is still unclear. The proportion of apoptosis caused by 18β-GA in MCF-7 and T-47D cells was determined using flow cytometry. The autophagy marker, LC3-II conversion, was investigated using Western blotting, and a PremoTM Tandem Autophagy Sensor Kit. We found that the concentration (150-μM) of 18β-GA caused caspase-dependent apoptosis and LC3-II accumulation or blocked autophagic flux. Moreover, 18β-GA-mediated apoptosis was improved using rapamycin but reversed by 3-methyladenine (3-MA) addition. The phosphorylation level of Jun-amino-terminal kinase (JNK) was increased significantly in the 18β-GA treatment and combined incubation using rapamycin. A JNK inhibitor (SP600125) significantly inhibited 18β-GA-mediated apoptosis, LC3-II accumulation and rescued the numbers of MCF-7 and T-47D colony formation. Especially, 18β-GA can inhibit xenograft tumor growth in BALB/c nude mice. These data indicate the combination of 18β-GA with rapamycin or 3-MA can sensitize or decrease MCF-7 and T-47D cells to 18β-GA-induced apoptosis, respectively. 18β-GA modulated autophagy is cytotoxic to luminal A subtype breast cancer cells through apoptosis promotion and JNK activation.

Exploring new pathways in endocrine-resistant breast cancer.

The most common breast cancer (BC) subtypes are hormone-dependent, being either estrogen receptor-positive (ER+), progesterone receptor-positive (PR+), or both, and altogether comprise the luminal subtype. The mainstay of treatment for luminal BC is endocrine therapy (ET), which includes several agents that act either directly targeting ER action or suppressing estrogen production. Over the years, ET has proven efficacy in reducing mortality and improving clinical outcomes in metastatic and nonmetastatic BC. However, the development of ET resistance promotes cancer survival and progression and hinders the use of endocrine agents. Several mechanisms implicated in endocrine resistance have now been extensively studied. Based on the current clinical and pre-clinical data, the present article briefly reviews the well-established pathways of ET resistance and continues by focusing on the three most recently uncovered pathways, which may mediate resistance to ET, namely receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK), nuclear factor kappa B (NFκB), and Notch. It additionally overviews the evidence underlying the approval of combined therapies to overcome ET resistance in BC, while highlighting the relevance of future studies focusing on putative mediators of ET resistance to uncover new therapeutic options for the disease.

Abstract P1-21-05: A novel long-acting insulin for cancer therapy reduces xenograft tumor growth

Abstract A link between obesity, metabolic dysfunction, and an increased risk in cancer mortality has been well established in several cancer types including, breast, colon, pancreatic, and prostate cancers. A likely mechanism driving this elevated risk is the associated increase in circulating insulin and insulin-like growth factor (IGF), which are known promoters of cancer growth and have been linked to shorter progression-free survival, and increased risk of metastatic disease and death in breast cancer patients. IGF receptor (IGF1R) targeting has been unsuccessful as insulin receptor (IR) also is functional in breast cancers. To address IR targeting, Akston Biosciences developed a novel insulin-Fc fusion protein (AKS-130) with the goal of lowering patient blood glucose levels without causing clinical hypoglycemia. AKS-130 was also found to downregulate IR expression in models of colon cancer (HCT-116) and malignant melanoma (WM266.4). Further, AKS-130 suppressed xenograft growth in fed and fasted states. We examined if AKS-130 had similar effects on estrogen receptor (ER) positive breast cancer cells as ER+ breast cancers may have elevated expression or activation of IGF1R and IR. Data from our lab show that development of endocrine therapy resistance in ER+ breast cancer cell lines is associated with loss of IGF1R expression and subsequently increased sensitivity to insulin signaling, which supports overlapping pro-tumorigenic signaling pathways to IGF1R. To test the hypothesis that resistance to endocrine therapy increases the reliance of ER+ breast cancer cells on IR signaling, we used the ER+ breast cancer cell line, MCF-7L, and a Tam resistant derivative, MCF-7L TamR, to assess IR/IGF1R activation and growth in response to AKS-130. Similar to HCT-116 and WM266.4 cells, MCF-7L parental and TamR cells showed that AKS-130 was an agonist of IR. But after 72 hours of exposure, a marked reduction in IR was seen and associated with partially suppressed downstream signaling to Akt. Interestingly, AKS-130 induced proliferation at similar levels to insulin treatment in MCF-7L parental cells at three and five days post treatment. However, pre-treatment of cells with AKS-130 suppressed further insulin-stimulated growth in MCF-7L TamR cells. Ongoing studies are evaluating the effects of AKS-130 on ER+ breast cancer xenografts in vivo to determine if the ability of the drug to downregulate IR expression results in decreased tumor growth. Taken together these data show that novel agents, such as AKS-130, can target IR function in cancer cells. AKS-130 is a drug which may serve as way to disrupt IR in cancer cells without affecting host glucose metabolism. Targeting of IR signaling may represent a new strategy to overcome endocrine therapy resistance in breast cancer. Citation Format: Lynsey M Fettig, Xihong Zhang, Kelly LaPara, Sylaja Murikipudi, Andrea R Delpero, Thomas M Lancaster, Todd C Zion, Douglas Yee. A novel long-acting insulin for cancer therapy reduces xenograft tumor growth [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-21-05.

Randomized phase-II evaluation of letrozole plus dasatinib in hormone receptor positive metastatic breast cancer patients

The non-receptor tyrosine kinase Src activation plays a role in the malignant progression of breast cancer, including development of endocrine therapy resistance and survival of bone metastases. This study investigated whether adding Src kinase inhibitor dasatinib to aromatase inhibitor (AI) therapy improved outcomes in estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer (MBC). Postmenopausal patients with ER-positive, HER2-negative MBC (0–1 prior chemotherapies and no prior AI for MBC) were eligible for this non-comparative, parallel group, phase-II study. Patients were randomized to letrozole (2.5 mg/day PO) alone or with dasatinib (100 mg/day PO). Patients with disease progression on letrozole alone could crossover to dasatinib plus continued letrozole. The primary endpoint was clinical-benefit-rate (CBR; complete response + partial response + stable disease ≥6 months). A total of 120 patients were randomized. The CBR of 71% (95% CI 58–83%) was observed with letrozole + dasatinib versus the projected CBR of the combination of 56%. The CBR of 66% (95% CI 52–77%) with letrozole alone also exceeded the projected CBR of 39% with letrozole alone. The CBR was 23% in the crossover arm of letrozole plus dasatinib in patients progressing on letrozole alone. Median progression-free survival with the combination was 20.1 months and 9.9 months with letrozole alone. Letrozole plus dasatinib was well tolerated, although 26% of patients required dasatinib dose reductions. In this non-comparative phase-II trial, the CBR of 71% and the median PFS of 20.1 months with letrozole + dasatinib are encouraging and suggest that dasatinib may inhibit the emergence of acquired resistance to AI therapy.

The Central Contributions of Breast Cancer Stem Cells in Developing Resistance to Endocrine Therapy in Estrogen Receptor (ER)-Positive Breast Cancer.

Breast cancer stem cells (BCSC) play critical roles in the acquisition of resistance to endocrine therapy in estrogen receptor (ER)-positive (ER + ve) breast cancer (BC). The resistance results from complex alterations involving ER, growth factor receptors, NOTCH, Wnt/β-catenin, hedgehog, YAP/TAZ, and the tumor microenvironment. These mechanisms are likely converged on regulating BCSCs, which then drive the development of endocrine therapy resistance. In this regard, hormone therapies enrich BCSCs in ER + ve BCs under both pre-clinical and clinical settings along with upregulation of the core components of “stemness” transcriptional factors including SOX2, NANOG, and OCT4. SOX2 initiates a set of reactions involving SOX9, Wnt, FXY3D, and Src tyrosine kinase; these reactions stimulate BCSCs and contribute to endocrine resistance. The central contributions of BCSCs to endocrine resistance regulated by complex mechanisms offer a unified strategy to counter the resistance. ER + ve BCs constitute approximately 75% of BCs to which hormone therapy is the major therapeutic approach. Likewise, resistance to endocrine therapy remains the major challenge in the management of patients with ER + ve BC. In this review we will discuss evidence supporting a central role of BCSCs in developing endocrine resistance and outline the strategy of targeting BCSCs to reduce hormone therapy resistance.

Long Non-Coding RNA H19 Acts as an Estrogen Receptor Modulator that is Required for Endocrine Therapy Resistance in ER+ Breast Cancer Cells

Background/Aims: Blocking estrogen signaling with endocrine therapies (Tamoxifen or Fulverstrant) is an effective treatment for Estrogen Receptor-α positive (ER⁺) breast cancer tumours. Unfortunately, development of endocrine therapy resistance (ETR) is a frequent event resulting in disease relapse and decreased overall patient survival. The long noncoding RNA, H19, was previously shown to play a significant role in estrogen-induced proliferation of both normal and malignant ER⁺ breast epithelial cells. We hypothesized that H19 expression is also important for the proliferation and survival of ETR cells. Methods: Here we utilized established ETR cell models; the Tamoxifen (Tam)-resistant LCC2 and the Fulvestrant and Tam cross-resistant LCC9 cells. Gain and loss of H19 function were achieved through lentiviral transduction as well as pharmacological inhibitors of the Notch and c-Met receptor signaling pathways. The effects of altered H19 expression on cell viability and ETR were assessed using three-dimensional (3D) organoid cultures and 2D co-cultures with low passage tumour-associated fbroblasts (TAFs). Results: Here we report that treating ETR cells with Tam or Fulvestrant increases H19 expression and that it’s decreased expression overcomes resistance to Tam and Fulvestrant in these cells. Interestingly, H19 expression is regulated by Notch and HGF signaling in the ETR cells and pharmacological inhibitors of Notch and c-MET signaling together significantly reverse resistance to Tam and Fulvestrant in an H19-dependent manner in these cells. Lastly, we demonstrate that H19 regulates ERα expression at the transcript and protein levels in the ETR cells and that H19 protects ERα against Fulvestrant-mediated downregulation of ERα protein. We also observed that blocking Notch and the c-MET receptor signaling also overcomes Fulvestrant and Tam resistance in 3D organoid cultures by decreasing ERα and H19 expression in the ETR cells. Conclusion: In endocrine therapy resistant breast cancer cells Fulvestrant is ineffective in decreasing ERα levels. Our data suggest that in the ETR cells, H19 expression acts as an ER modulator and that its levels and subsequently ERα levels can be substantially decreased by blocking Notch and c-MET receptor signaling. Consequently, treating ETR cells with these pharmacological inhibitors helps overcome resistance to Fulvestrant and Tamoxifen.

Clinical relevance of Cyr61 expression in patients with hormone-dependent breast cancer.

Tumor resistance to endocrine therapy triggers estrogen-independent cancer progression, which is a major obstacle to the successful treatment of hormone receptor positive breast cancer (BC). The underlying molecular mechanisms of endocrine resistance are not fully understood yet. The matricellular protein cysteine-rich angiogenic inducer 61 (Cyr61) is associated with tumor invasiveness and the induction of tumorigenesis in various malignancies in vivo and the induction of estrogen-independence and endocrine therapy resistance in BC. The present study evaluated the potential effects and clinical relevance of Cyr61 expression levels in 67 patients with primary non-metastatic BC. Immunohistochemical analysis of formalin-fixed paraffin-embedded tissue sections was performed, and the association between Cyr61 protein expression and clinicopathological factors and survival was analyzed. Cyr61 overexpression was revealed to be significantly associated with a positive estrogen receptor (ER)/progesterone receptor (PR) status (P=0.016) and to the molecular subtype of BC (P=0.039). Compared with patients without Cyr61 overexpression, patients with Cyr61 overexpression exhibited an increased recurrence rate (30.6 vs. 22.6%) and decreased long-term survival (10-year overall survival, 62.9 vs. 69.7%); however, these associations did not reach statistically significant levels in Cox regression model analysis. Similar results were identified in the subgroup analysis of patients with ER/PR positive BC. These results indicate that Cyr61 serves a role in the development of endocrine therapy resistance in BC and is thus a potential therapeutic target to overcome endocrine therapy resistance. However, additional long-term survival analyses with large patient populations are required.

Abstract P3-04-25: Role of H19, a long non-coding RNA, in development of resistance to endocrine therapy in breast cancer cells

Abstract Introduction: Majority of breast cancer tumors are Estrogen receptor positive (ER+) where antiestrogen therapies (endocrine therapies) are the best therapeutic strategy to treat this type of tumors. However, eventually over 30% of patients will develop resistance to endocrine therapies resulting in disease relapse. We recently showed that the long noncoding RNA, H19, is an estrogen target gene that plays a significant role in estrogen-induced proliferation of the normal and malignant ER+ cells. We therefore hypothesize that H19 expression is also important to the proliferation of endocrine therapy resistant cells. In this study, we examined if estrogen-independent H19 expression is important to the development of endocrine therapy resistance. Objective: The overall objective of this project is to use therapy sensitive (MCF-7) and therapy-resistant (LCC9) breast cancer cells as model systems to examine the role of long non-coding RNA H19 in development and maintenance of resistance to endocrine therapy. Methodology and Results: We examined the expression of H19 in ER+ breast cancer cells (MCF7) that under the selective pressure of fulvesterant (ICI, ER down regulator) acquire resistance to ICI. We observed that while H19 expression was initially decreased as expected, its expression subsequently increased in the ICI-resistant MCF7 cells. Interestingly, H19 knockdown in MCF7 cells significantly decrease their proliferation as determined by Flowcytometry and made them more sensitive to ICI. We also examined H19 expression in the ICI-resistant LCC9 cells and found that ICI treatment increased H19 expression. Interestingly, H19 knockdown in the LCC9 cells decreased their proliferation and surprisingly made them sensitive to ICI treatment. Previous observations indicate that NOTCH4 receptor (NR4) may be involved in endocrine therapy resistance. Interestingly we found that in presence of ICI, NR4 expression is increased and that forced activation of NR4 markedly increases H19 expression in LCC9 cells. Conclusion: Altogether these observations suggest that H19 plays an important role in the development of endocrine therapy resistance and further our understanding of the cellular and molecular mechanisms involved in endocrine therapy resistance. These and similar studies could potentially lead to the development of new therapies to treat therapy resistant tumor cells. Further experiments would reveal if signalling pathways that regulate H19 expression independent of estrogen are useful therapies against endocrine therapy resistant tumors. Citation Format: Basak P, Chatterjee S, Bhat V, Jin H, Su A, Murphy LC, Raouf A. Role of H19, a long non-coding RNA, in development of resistance to endocrine therapy in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-25.

Abstract A08: A patient-derived xenograft model to study the effects of postmenopausal obesity on endocrine therapy resistance

Abstract Introduction: Obesity affects more than one-third of adults in the U.S. and increases the risk for and worsens outcomes of individuals with cancer. Obese women have a greater risk of postmenopausal hormone-receptor positive breast cancer and are more likely to relapse after endocrine therapy. The prevailing dogma is that elevated circulating estrogen, produced by aromatase in abundant adipose tissue, drives breast cancer formation and progression. While estrogen is clearly an important factor in the obesity-breast cancer relationship, increasing the dose of aromatase inhibitors provides no added benefit to obese women with breast cancer. A switch from hormone- to growth factor-dependence has been implicated in the development of endocrine therapy resistance. Hypothesis and Study Objective: We hypothesized that obese, postmenopausal women have elevated growth factor production in adipose tissue, which supports the progression of breast tumors that were previously dependent on estrogen. The purpose of our study was to develop a transplant-competent mouse model of obesity and postmenopausal breast cancer using estrogen receptor (ER) positive breast cancer patient-derived xenografts (PDX) from peri- and postmenopausal women (ages 45-71). Methods: Rag1-null mice on the C57Bl/6 background were fed either a low fat (LFD) or high fat (HFD) diet for 10 weeks. Thermoneutral housing was used to facilitate development of the obese phenotype. Animal weights and percent body fat were monitored. After weight separation (approximately 10 weeks), mice were ovariectomized and implanted with an ER+ estrogen-dependent PDX derived from a 54 year old woman bilaterally into the orthotopic site (inguinal mammary fat pad). Mice were given slow release estrogen pellets and tumor growth was monitored. After 8 weeks of tumor growth, a subset of animals received estrogen withdrawal (EWD) to mimic the menopausal transition. Results: HFD-fed mice weighed more, and had significantly increased body fat percentages, as well as subcutaneous, and gonadal fat pad weights, compared to LFD-fed mice. After EWD, tumors regressed in LFD-fed mice; however, the HFD prevented tumor regression in the absence of estrogen. Circulating estrogen levels in both LFD and HFD EWD groups were not significantly different. Tumor cellularity and Ki67 were greater in HFD- compared to LFD-fed mice in the absence of estrogen. Phospho-protein analysis of tumors suggested increased human epidermal growth factor receptor 2 (HER2) and fibroblast growth factor receptor (FGFR) signaling in HFD-EWD versus LFD-EWD mice. Conclusions: We have developed a novel, transplant-competent mouse model of obesity and menopause using a specialized HFD and thermoneutral housing. Using this model, we determined that HFD supports the growth of a previously estrogen-dependent breast cancer PDX after EWD, potentially through increased signaling through HER2 and FGFR pathways. Our mouse model, in combination with PDX breast tumors, will offer novel insights into the relationships between obesity, menopause, and ER+ breast cancer progression. Citation Format: Elizabeth Wellberg, Britta Jacobsen, Peter Kabos, Paul MacLean, Carol Sartorius. A patient-derived xenograft model to study the effects of postmenopausal obesity on endocrine therapy resistance. [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 A08.

Molecular and clinical implications of the crosstalk between growth factor receptors and estrogen receptor in the development of endocrine therapy resistance

Molecular and clinical implications of the crosstalk between growth factor receptors and estrogen receptor in the development of endocrine therapy resistance

Abstract 1733: Therapeutic significance of ERα—PELP1 axis in blocking endocrine therapy resistance

Abstract Breast cancer therapies target the estrogen receptor-alpha (ERα) with letrozol an aromatase inhibitor or anti-estrogens such tamoxifen to limit circulating estrogen levels and alter ERα functions. Many women suffering from recurrent or advanced disease develop resistance to current targeted endocrine treatments. Emerging data suggests therapeutic resistance is precipitated through dysfunction of ERα coregulatory proteins and crosstalk between ERα and growth factor signaling. Deregulated expression of proline-, glutamic acid- and leucine-rich protein 1 (PELP1), an ERα coregulator, was observed in a subset of human breast tumors. Recently, PELP1 was identified as an independent prognostic biomarker of shorter survival in ERα positive/luminal-like breast cancer patients. Epigenetic changes of target gene promoters are modulated by PELP1 via an interaction with lysine specific demethylase 1 (KDM1). Additionally, PELP1 promotes crosstalk between ERα and Src kinase leading to enhanced activation of extranuclear signaling pathways. Therapeutic potential of targeting the ERα-PELP1 axis in vivo was examined using a pharmacological approach in well-established preclinical models of endocrine therapy resistant breast cancer. We utilized (1) siRNA liposomes to down regulate PELP1 expression; (2) pargyline and NCL-1, KDM1 inhibitors, to block PELP1-mediated epigenetic modifications and (3) dasatinib, a Src kinase inhibitor, to prevent PELP1-mediated extranuclear actions in both pre- and post-menopausal conditions. In xenograft-based assays, treatment of ERα positive breast cancer cells with liposomes containing PELP1-siRNA, dasatinib or KDM1 inhibitors significantly reduced proliferation and tumor growth. Combinatorial therapies of letrozol or tamoxifen with PELP1 axis blockers substantially impeded xenograft tumor growth by blocking both ERα nuclear and extranuclear signaling and promoting apoptosis in therapy resistant cells. Taken together, our results suggest targeting the ERα-PELP1 axis in combination with current endocrine therapies will enhance therapeutic efficacy and may inhibit or delay development of endocrine therapy resistance. This study is funded by Komen grant KG090447 and NIH grant CA095681. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2011-1733

Importance of PI3-kinase pathway in response/resistance to aromatase inhibitors

Endocrine therapy has been the most effective treatment modality for hormone receptor positive breast cancer. However, its efficacy has been limited by either de novo or acquired resistance. Recent data indicates that activation of the phosphatidylinositol 3-kinase (PI3K) signaling is associated with the poor outcome luminal B subtype of breast cancer and accompanied by the development of endocrine therapy resistance. Importantly, inhibition of PI3K pathway signaling in endocrine resistant breast cancer cell lines reduces cell survival and improves treatment response to endocrine agents. Interestingly, mutations in PIK3CA, the alpha catalytic subunit of the class IA PI3K, which renders cells dependent on PI3K pathway signaling, is the most common genetic abnormality identified in hormone receptor positive breast cancer. The synthetic lethality observed between estrogen deprivation and PI3K pathway inhibition in estrogen receptor positive (ER+) breast cancer cell lines provides further scientific rational to target both estrogen receptor and the PI3K pathway in order to improve the outcome of ER+ breast cancer.

Feasibility trial of letrozole in combination with bevacizumab in patients with metastatic breast cancer.

Preclinical models suggest that the use of anti-vascular endothelial growth factor (anti-VEGF) therapy with antiestrogens may prevent or delay the development of endocrine therapy resistance. We therefore performed a feasibility study to evaluate the safety of letrozole plus bevacizumab in patients with hormone receptor-positive metastatic breast cancer (MBC). Patients with locally advanced breast cancer or MBC were treated with the aromatase inhibitor (AI) letrozole (2.5 mg orally daily) and the anti-VEGF antibody bevacizumab (15 mg/kg intravenously every 3 weeks). The primary end point was safety, defined by grade 4 toxicity using the National Cancer Institute Common Toxicity Criteria, version 3.0. Secondary end points included response rate, clinical benefit rate, and progression-free survival (PFS). Prior nonsteroidal AIs (NSAIs) were permitted in the absence of progressive disease. Forty-three patients were treated. After a median of 13 cycles (range, 1 to 71 cycles), select treatment-related toxicities included hypertension (58%; grades 2 and 3 in 19% and 26%), proteinuria (67%; grades 2 and 3 in 14% and 19%), headache (51%; grades 2 and 3 in 16% and 7%), fatigue (74%; grades 2 and 3 in 19% and 2%), and joint pain (63%; grades 2 and 3 in 19% and 0%). Eighty-four percent of patients had at least stable disease on an NSAI, confounding efficacy results. Partial responses were seen in 9% of patients and stable disease >or= 24 weeks was noted in 67%. Median PFS was 17.1 months. Combination letrozole and bevacizumab was feasible with expected bevacizumab-related events of hypertension, headache, and proteinuria. Phase III proof-of-efficacy trials of endocrine therapy plus bevacizumab are in progress (Cancer and Leukemia Group B 40503).

Reactive oxygen species induce phosphorylation of serine 118 and 167 on estrogen receptor alpha

Estrogen receptor alpha (ERalpha) is a well-known target for signaling pathways originating from growth factor receptors. Reactive oxygen species (ROS) can induce activation of extracellular response kinase 1/2 (Erk1/2) and protein kinase B (Akt). Both kinases have been implicated in the phosphorylation of serine 118 and serine 167 on ERalpha, respectively. This activity may lead either to ligand-independent activation of ERalpha or down-regulation of ERalpha and may contribute to development of the resistance to endocrine therapy. Treatment of MCF-7 human breast cancer cells with glucose oxidase (GO, 0.1 un/ml) induced transient phosphorylation of serine 118 and serine 167. The increase in expression of p-ser118-ERalpha was 355 +/- 98% (mean +/- SD) and of p-ser167-ERalpha was 632 +/- 355%. These effects were enhanced in Her2 over-expressing MCF7 cells. ERalpha expression declined to 63 +/- 20% within the first 90 min of treatment and was below 10% 24 h later. ROS induced phosphorylation of ERalpha resulted in decreased expression of pS2 and progesterone receptor. Activation of Erk1/2 and Akt was transient with highest levels of Erk1/2 being 595 +/- 143% and p-Akt 311 +/- 125%. Inhibition of Erk1/2 by U0126 (10 microM) decreased p-ser118-ERalpha by 51.7 +/- 8.5% and decreased p-ser167-ERalpha by 41.9 +/- 16.9% whereas inhibition of Akt by LY294002 (20 microM) and wortmannin (500 nM) or by siRNA knock-down, had no effect on p-ser167-ERalpha expression. Our data show for the first time that ROS can induce post-translational modifications of ERalpha at serine 118 and serine 167, and may lead to ERalpha down-regulation in human breast cancer cells. Both the phosphorylation and consequent down-regulation of ERalpha may be a mechanism associated with development of endocrine therapy resistance.

NF-κB inhibition markedly enhances sensitivity of resistant breast cancer tumor cells to tamoxifen

Studies show that high Akt activity in breast carcinoma is associated with endocrine therapy resistance. Breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions, and are resistant to the growth inhibitory effects of tamoxifen. Understanding the targets of Akt signaling mediating tamoxifen resistance is of clinical significance. One possible target is nuclear factor kappa B(NF-κB), a transcription factor that plays a critical role in resistance to apoptosis and the induction of angiogenesis and invasion. In the present study, we found that Akt activity correlated with phosphorylation of IκB (the negative regulator of NF-κB), NF-κB DNA binding and tamoxifen resistance in vivo. Importantly, we found that co-treatment with the NF-κB inhibitor, parthenolide, or overexpression of IκB superrepressor restored tamoxifen sensitivity to our refractory Akt MCF-7 cells. These data suggest that activation of NF-κB via the PI3K/Akt signaling pathway may be a significant mechanism for development of endocrine therapy resistance in breast cancer, and that inhibition of NF-κB may be an effective treatment strategy to limit the progression of this disease.