Aromatase Inhibitor-resistant Breast Cancer Research Articles

Abstract 3863: Aminoflavone confers activity against aromatase inhibitor-resistant breast cancer cells in part via miR125b2-3p restoration

Abstract Aromatase inhibitor therapy is the most crucial adjuvant treatment option for postmenopausal women with hormone receptor-positive (HR+) non-metastatic breast cancer. However, up to 30% of these patients will experience relapse due in part to aromatase inhibitor resistance. We recently identified miR125b2-3p as a putative tumor suppressor in HR+ breast cancer. We also found that small molecule Aminoflavone (AF) induces miR125b2-3p expression in HR+ breast cancer cells. In the current study, we hypothesized that AF confers anticancer actions in breast cancer cells resistant to aromatase inhibitors via miR125b2-3p reactivation. Using the Alamar Blue and colony forming assays, we discovered that AF demonstrated anticancer activity in estrogen-deprived resistant (EDR) cells designed to mimic aromatase inhibitor resistance. AntagomiR125b2-3p enhanced, while miR125b2-3p mimics diminished colony formation of EDR cells. AF-mediated suppression of colony formation in EDR cells was attenuated during co-treatment with antagomiR125b2-3p and was enhanced during co-treatment with miR125b2-3p mimics. Both miR125b2-3p mimics and AF suppressed EDR cell migration. AntagomiR125b2-3p counteracted AF-mediated suppression of cell migration. Our in silico profiling studies identified the alpha6-integrin, a mediator of breast cancer stem cell activity and metastasis, as one miR-125b2-3p target. Immunohistochemistry of patient tumor samples revealed that alpha 6-integrin protein expression intensified following relapse on the aromatase inhibitor anastrozole. MCF-7 cells transfected to overexpress the alpha 6-integrin were less responsive to anastrozole than those transfected with empty vector. These data suggest that AF restores the tumor suppressor function of miR125b2-3p to inhibit the alpha 6-integrin and confer activity against aromatase inhibitor-resistant breast cancer. Citation Format: Eileen J. Brantley, Nicole Mavingire, Salma Khan, Gayathri Nagaraj, Shawnee Angeloni, Charles Wang, Ubaldo Soto. Aminoflavone confers activity against aromatase inhibitor-resistant breast cancer cells in part via miR125b2-3p restoration. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3863.

Abstract 5030: Treatment of aromatase inhibitor-resistant cells with polyisoprenylated cysteinyl amide inhibitors stimulates the mitogen-activated protein kinase pathway enzymes, impedes cell proliferation and causes cell death

Abstract Overexpression and hyperactivity of the estrogen receptor drives 67-80% and 90% of breast cancer in women and men, respectively. Resistance to aromatase inhibitor (AI) therapies necessitates the continuous search for novel therapies. Previous studies demonstrate AI-resistance is associated with hormone-independence, enhanced motility, and increased growth factor signaling. Here, long-term letrozole-treated (LTLT-Ca) breast cancer cells were used to evaluate polyisoprenylated cysteinyl amide inhibitors (PCAIs) as potential alternative therapies for the treatment of aromatase inhibitor-resistant breast cancer. We determined the potency of PCAIs as anticancer agents by evaluating their effects on cell viability, phosphorylation of MAPK pathway enzymes, G-proteins levels, cell migration and apoptosis. Among the PCAIs tested, NSL-YHJ-2-27 showed significant potency against cell viability with an EC50 of 3.6 µM. MEK (p-MEK1/2), ERK (p-ERK1/2), and p90RSK (p-p90RSK) phosphorylation were significantly increased by 178, 119 and 125%, respectively, over controls. In addition, of the seven G-proteins evaluated, only KRAS and NRAS showed significant increases of 49% and 97%, respectively. Cell proliferation and colony formation were impeded by 82% and 74%, respectively, after PCAIs treatment. Migration of the LTLT-Ca cells was inhibited by 80% following treatment with 5 µM of NSL-YHJ-2-27. Lastly, the PCAIs also caused the degeneration of the spheroids after 10 µM PCAIs treatment, dead cells were very apparent after AO/EB staining. Our findings suggest that the PCAIs’ suppression of cell viability and activation of the MAPK pathway causes apoptosis possibly through the activation of the proapoptotic p-p90RSK isoforms. The results reveal the ability of the PCAIs to effectively cause a negative impact on several cancer hallmarks suggesting their potency as anticancer agents. These findings also support a potential role for PCAIs’ use in treating breast cancers that have become resistant to aromatase inhibitor therapies. Citation Format: Jassy Mary S. Lazarte, Syreeta L. Tilghman, Nazarius S. Lamango. Treatment of aromatase inhibitor-resistant cells with polyisoprenylated cysteinyl amide inhibitors stimulates the mitogen-activated protein kinase pathway enzymes, impedes cell proliferation and causes cell death. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5030.

Differential Gene Expression Analysis for Aromatase Inhibitors Treatment on Breast Cancer Patients

Breast cancer is the most common global malignancy and the leading cause of cancer deaths for women. Aromatase inhibitors are clinically recommended therapy for the disorders. However, resistance to aromatase inhibitors therapies still occurs and the underlined molecular mechanisms are still poorly understood. The tumour specimens consisted of aromatase inhibitor-sensitive and aromatase inhibitor-resistant breast cancer patients, who were compared and the differentially expressed genes for aromatase inhibitor-sensitive patients were generated and related genetic network was explored. The enriched cellular process and signaling cassettes for aromatase inhibitor-sensitive patients were also in-depth investigated. Compared with the aromatase inhibitor-resistant patients, 896 genes showed a significant differential expression. 621 were significantly up-regulated and 275 were remarkably downregulated. From the interaction network, the differentially expressed genes including signal recognition particle 72, B-cell lymphoma-2 associated transcription factor 1, cathelicidin antimicrobial peptide and defensin alpha 1B could be considered as the key target genes for aromatase inhibitors treatment in breast cancer patients. These differentially expressed genes were enriched in processes involving immunoglobulin complex, cell division and others while, drug metabolism and other signalling mechanisms were enriched in aromatase inhibitors treatment of breast cancer patients. The study speculated several novel gene targets for aromatase inhibitors therapeutic treatment of breast cancer patients and initiated a beneficial direction for future aromatase inhibitors drug resistance research.

Ruxolitinib and exemestane for estrogen receptor positive, aromatase inhibitor resistant advanced breast cancer

Circulating IL-6, an activator of JAK/STAT signaling, is associated with poor prognosis and aromatase inhibitor (AI) resistance in hormone-receptor positive (HR+) breast cancer. Here we report the results of a phase 2 single-arm Simon 2-stage trial combining Ruxolitinib, an oral selective inhibitor of JAK1/2, with exemestane, a steroidal AI, in patients with HR+ metastatic breast cancer (MBC) after progression on non-steroidal AI (NSAI). Safety and efficacy were primary objectives, and analysis of inflammatory markers as predictors of response was a key secondary objective. Twenty-five subjects enrolled. The combination of ruxolitinib and exemestane was safe, though anemia requiring transfusion in 5/15 (33%) at the 25 mg dose in stage 1 led to a reduction to 15 mg twice daily in stage 2 (with no additional transfusions). Clinical benefit rate (CBR) in the overall study population was 24% (95% CI 9.4–45.1); 6/25 patients demonstrated stable disease for ≥6 months. Median progression-free survival was 2.8 months (95% CI 2.6–3.9). Exploratory biomarkers revealed high levels of systemic inflammation and 60% harbored a high-risk IL-6 genotype. Pharmacodynamics demonstrated modest on-target inhibition of phosphorylated-STAT3 by ruxolitinib at a tolerable dose. Thus, ruxolitinib combined with exemestane at a tolerable dose was safe but minimally active in AI-resistant tumors of patients with high levels of systemic inflammation. These findings highlight the need for more potent and specific therapies targeting inflammation in MBC.

Dissecting the Roles of PDCD4 in Breast Cancer.

The human programmed cell death 4 (PDCD4) gene was mapped at chromosome 10q24 and encodes the PDCD4 protein comprised of 469 amino acids. PDCD4 inhibits protein translation PDCD4 inhibits protein translation to suppress tumor progression, and its expression is frequently decreased in breast cancer. PDCD4 blocks translation initiation complex by binding eIF4A via MA-3 domains or by directly binding 5’ mRNA internal ribosome entry sites with an RNA binding domain to suppress breast cancer progression and proliferation. Numerous regulators and biological processes including non-coding RNAs, proteasomes, estrogen, natural compounds and inflammation control PDCD4 expression in breast cancer. Loss of PDCD4 expression is also responsible for drug resistance in breast cancer. HER2 activation downregulates PDCD4 expression by activating MAPK, AKT, and miR-21 in aromatase inhibitor-resistant breast cancer cells. Moreover, modulating the microRNA/PDCD4 axis maybe an effective strategy for overcoming chemoresistance in breast cancer. Down-regulation of PDCD4 is significantly associated with short overall survival of patients, which suggests that PDCD4 may be an independent prognostic marker for breast cancer.

Abstract P4-02-07: Lasofoxifene as a potential treatment for aromatase inhibitor resistant ER positive breast cancer

Abstract Postmenopausal patients with estrogen receptor positive (ERα+) primary invasive breast cancer are typically treated with aromatase inhibitors (AIs) as first line adjuvant therapy. Patients who become resistant to AIs are treated with fulvestrant and/or CDK4/6 inhibitors, such as palbociclib, as second line therapy. Lasofoxifene, a selective estrogen receptor modulator (SERM), was developed for the treatment of vaginal atrophy and osteoporosis. Our previous studies, performed in an MCF-7 xenograph metastatic breast cancer mouse model with activating ERα mutations, showed that lasofoxifene, both alone and in combination with palbociclib, was more effective than fulvestrant at inhibiting tumor growth and metastasis to the liver, lung, bone and brain. In the current study, we compared the efficacy of lasofoxifene +/- palbociclib to fulvestrant +/- palbociclib combinations in a letrozole-induced, AI-resistant breast tumor model (MCF-7 LTLT cells) that does not express ERα activating mutations. Luciferase-GFP tagged LTLT cells were injected into the mammary ducts of NSG mice (MIND model) and tumor progression was monitored by live luminescence imaging of primary tumors, as well as ex vivo imaging and histochemical analysis of metastatic sites at study endpoint. Primary tumor area was also measured at study endpoint. Lasofoxifene +/- palbociclib was significantly more effective than fulvestrant +/- palbociclib at inhibiting primary tumor growth. In addition, all treatments, except fulvestrant, inhibited bone metastasis versus vehicle. These data show that lasofoxifene is more effective than fulvestrant in this tumor model and suggest that lasofoxifene has potential as an effective therapy for AI resistant breast cancers that do not express ERα activating mutations. Citation Format: Muriel Laine, Marianne E Greene, Tiffany Leng, Justyna D Kurleto, Sophia Li, Barry Komm, Geoffrey L Greene. Lasofoxifene as a potential treatment for aromatase inhibitor resistant ER positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-07.

Development of CDK4/6 Inhibitors: A Five Years Update.

The inhibition of cyclin dependent kinases 4 and 6 plays a role in aromatase inhibitor resistant metastatic breast cancer. Three dual CDK4/6 inhibitors have been approved for the breast cancer treatment that, in combination with the endocrine therapy, dramatically improved the survival outcomes both in first and later line settings. The developments of the last five years in the search for new selective CDK4/6 inhibitors with increased selectivity, treatment efficacy, and reduced adverse effects are reviewed, considering the small-molecule inhibitors and proteolysis-targeting chimeras (PROTACs) approaches, mainly pointing at structure-activity relationships, selectivity against different kinases and antiproliferative activity.

Quantitative Proteomic Profiling Identifies a Potential Novel Chaperone Marker in Resistant Breast Cancer.

Development of aromatase inhibitor resistant breast cancer among postmenopausal women continues to be a major clinical obstacle. Previously, our group demonstrated that as breast cancer cells transition from hormone-dependent to hormone-independent, they are associated with increased growth factor signaling, enhanced cellular motility, and the epithelial to mesenchymal transition (EMT). Given the complexity of cancer stem cells (CSC) and their implications on endocrine resistance and EMT, we sought to understand their contribution towards the development of aromatase inhibitor resistant breast cancer. Cells cultured three dimensionally as mammospheres are enriched for CSCs and more accurately recapitulates tumors in vivo. Therefore, a global proteomic analysis was conducted using letrozole resistant breast cancer cells (LTLT-Ca) mammospheres and compared to their adherent counterparts. Results demonstrated over 1000 proteins with quantitative abundance ratios were identified. Among the quantified proteins, 359 were significantly altered (p < 0.05), where 173 were upregulated and 186 downregulated (p < 0.05, fold change >1.20). Notably, midasin, a chaperone protein required for maturation and nuclear export of the pre-60S ribosome was increased 35-fold. Protein expression analyses confirmed midasin is ubiquitously expressed in normal tissue but is overexpressed in lobular and ductal breast carcinoma tissue as well as ER+ and ER- breast cancer cell lines. Functional enrichment analyses indicated that 19 gene ontology terms and one KEGG pathway were over-represented by the down-regulated proteins and both were associated with protein synthesis. Increased midasin was strongly correlated with decreased relapse free survival in hormone independent breast cancer. For the first time, we characterized the global proteomic signature of CSC-enriched letrozole-resistant cells associated with protein synthesis, which may implicate a role for midasin in endocrine resistance.

Abstract OT-09-08: Solti-1502 aRIANNA: Targeting PAM50 HER2-enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative metastatic breast cancer

Abstract Background Pre-clinical evidence and retrospective studies suggest that PAM50 HER2-Enriched (HER2-E), hormone receptor-positive (HR+)/HER2-negative tumors have estrogen receptor (ER)-independency and poor prognosis, but seem to have androgen receptor (AR)-addiction (1). Enzalutamide (EZM) is a potent inhibitor of androgen receptor signaling (3). AR expression has been shown to induce resistance to both tamoxifen and aromatase inhibitors in estrogen receptor HR-expressing cell lines (4,5) The hypothesis of the ARIANNA trial is that EZM induces a significant proliferative arrest in PAM50 HER2-E, HR+/HER2-negative advanced breast cancer (BC), leading to clinical benefit in this poor prognosis population. Methods ARIANNA is an exploratory, phase II clinical trial in two independent cohorts evaluating the effect of EZM on proliferation after 2 weeks (+7 days window) on treatment in pre- or post-menopausal female or male patients with endocrine-resistant, locally advanced or metastatic HR+/HER2-negative BC. Cohort A will include 22 patients with PAM50 HER2-E HR+/HER-negative tumors and Cohort B (control group) will include 22 patients with PAM50 Luminal A/B HR+/HER2-negative tumors. Fresh tumor biopsy will be obtained at screening and sent to central laboratory for PAM50 subtyping determination to confirm the molecular subtype status prior to study treatment initiation, and to determine PAM50 11-gene proliferation-related signature. Patients with PAM50 Basal-like or Normal-like tumors will be excluded. Patients will receive EZM 160 mg once a day (QD). After 2 weeks on treatment, a tumor biopsy from the same baseline lesion (or, if not feasible, a lesion in the same organ) will be obtained for the purpose of the primary endpoint analysis. After this on-treatment biopsy, exemestane 50mg QD can be added to EZM at physician’s discretion. Tumor assessment will be performed at screening and every 8 weeks thereafter. Treatment will be continued until disease progression, unacceptable toxicity, investigator’s decision or withdrawal of consent. An optional tumor biopsy will be collected at the end of treatment. The primary objective is to evaluate the anti-proliferative effect of EZM after 2 weeks of treatment in patients with HER2-E HR+/HER2-negative tumors, measured as relative changes in the PAM50 11-gene proliferation-related signature by the PAM50 nCounter-based assay between baseline and on-treatment tumor biopsies. Secondary objectives include: anti-proliferative effect of EZM after 2 weeks of treatment in patients included in Cohort B (control group), safety, overall response rate, progression-free survival, and further correlative molecular analyses both at the tumor tissue (IHC, RNA and DNA) and ctDNA level for both cohorts. The trial will enroll patients in 8 Spanish sites and recruitment period will be 18 months. Funding was granted by Breast Cancer Research Foundation and drug was supplied by Astellas Pharma Global Development, Inc./Pfizer, Inc. Trial identification: NCT04142060 1. Cochrane DR, Bernales S, Jacobsen BM, Cittelly DM, Howe EN, D’Amato NC, et al. Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide. Breast Cancer Res. 2014;16:R7. 3. Tran C, Ouk S, Clegg NJ, Chen Y, Watson PA, Arora V, et al. Development of a Second-Generation Antiandrogen for Treatment of Advanced Prostate Cancer. Science. 2009;324:787-90. 4. Rechoum Y, Rovito D, Iacopetta D, Barone I, Andò S, Weigel NL, et al. AR collaborates with ERα in aromatase inhibitor-resistant breast cancer. Breast Cancer Res Treat. 2014;147:473-85. 5. De Amicis F, Thirugnansampanthan J, Cui Y, Selever J, Beyer A, Parra I, et al. Androgen receptor overexpression induces tamoxifen resistance in human breast cancer cells. Breast Cancer Res Treat. 2010;121:1-11 Citation Format: Mafalda Oliveira, Sonia Pernas, Mireia Margelí, Salvador Blanch, Barbara Adamo, Javier Salvador Bofill, Diana Moreno, Xavier Gonzalez-Farré, José Rios, Charles M Perou, Aleix Prat, Tomás Pascual, Juan M Ferrero-Cafiero, Patricia Villagrasa, Luis Manso. Solti-1502 aRIANNA: Targeting PAM50 HER2-enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-08.

Amphiregulin retains ERα expression in acquired aromatase inhibitor resistant breast cancer cells.

Acquired resistance to aromatase inhibitors (AIs) is a significant clinical issue in endocrine therapy for estrogen receptor (ER) positive breast cancer which accounts for the majority of breast cancer. Despite estrogen production being suppressed, ERα signaling remains active and plays a key role in most AI-resistant breast tumors. Here, we found that amphiregulin (AREG), an ERα transcriptional target and EGF receptor (EGFR) ligand, is crucial for maintaining ERα expression and signaling in acquired AI-resistant breast cancer cells. AREG was deregulated and critical for cell viability in ER+ AI-resistant breast cancer cells, and ectopic expression of AREG in hormone responsive breast cancer cells promoted endocrine resistance. RNA-sequencing and reverse phase protein array analyses revealed that AREG maintains ERα expression and signaling by activation of PI3K/Akt/mTOR signaling and upregulation of forkhead box M1 (FOXM1) and serum- and glucocorticoid-inducible kinase 3 (SGK3) expression. Our study uncovers a previously unappreciated role of AREG in maintaining ERα expression and signaling, and establishes the AREG-ERα crosstalk as a driver of acquired AI resistance in breast cancer.

Evidence for Enhanced Exosome Production in Aromatase Inhibitor-Resistant Breast Cancer Cells.

Aromatase inhibitors (AIs) represent the standard anti-hormonal therapy for post-menopausal estrogen receptor-positive breast cancer, but their efficacy is limited by the emergence of AI resistance (AIR). Exosomes act as vehicles to engender cancer progression and drug resistance. The goal of this work was to study exosome contribution in AIR mechanisms, using estrogen-dependent MCF-7 breast cancer cells as models and MCF-7 LTED (Long-Term Estrogen Deprived) subline, modeling AIR. We found that exosome secretion was significantly increased in MCF-7 LTED cells compared to MCF-7 cells. MCF-7 LTED cells also exhibited a higher amount of exosomal RNA and proteins than MCF-7 cells. Proteomic analysis revealed significant alterations in the cellular proteome. Indeed, we showed an enrichment of proteins frequently identified in exosomes in MCF-7 LTED cells. The most up-regulated proteins in MCF-7 LTED cells were represented by Rab GTPases, important vesicle transport-regulators in cancer, that are significantly mapped in “small GTPase-mediated signal transduction”, “protein transport” and “vesicle-mediated transport” Gene Ontology categories. Expression of selected Rab GTPases was validated by immunoblotting. Collectively, we evidence, for the first time, that AIR breast cancer cells display an increased capability to release exosomes, which may be associated with an enhanced Rab GTPase expression. These data provide the rationale for further studies directed at clarifying exosome’s role on endocrine therapy, with the aim to offer relevant markers and druggable therapeutic targets for the management of hormone-resistant breast cancers.

Abstract 4116: A novel phytoalexin, glyceollins, trigger anti-proliferative and apoptotic effects in aromatase inhibitor resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) are the standard endocrine treatment for postmenopausal women with estrogen-dependent metastatic breast cancer. While effective, some patients develop resistance leading to tumor relapse, metastasis, and more aggressive phenotypes. Previous research suggested that AI resistance arises as a result of increased growth factor pathways (i.e., HER2 and EGFR) and is associated with increased cellular motility. Our lab previously demonstrated that a novel phytoalexin, glyceollins, inhibits proliferation, survival, and migration of hormone independent letrozole-resistant breast cancer cells. However, many postmenopausal women with AI-resistant tumors remain hormone dependent. Therefore, there is a need to understand distinctions between estrogen receptor positive (ER+) and ER negative (ER-) AI resistant tumors and their response to therapy. We hypothesize that treating ER+ letrozole-resistant breast cancer with a combination of glyceollin and lapatinib (a dual EGFR/HER2 inhibitor) will reduce growth factor signaling, inhibit proliferation, and induce apoptosis. T47Darom cells (T47D cells which stably express the aromatase gene) and T47DaromLR cells (letrozole-resistant T47Darom cells) were characterized by measuring protein expression. As cells transition from letrozole-sensitive to letrozole-resistance ERα and aromatase expression were slightly decreased while HER2 was increased. A receptor tyrosine kinase phospho-antibody array was performed to evaluate changes in protein expression between the two cell lines. While many proteins were altered, two cell cycle regulators (p38 and p53) were significantly downregulated by in the T47DaromLR cells suggesting that T47DaromLR cells have acquired the ability to progress uncontrollably through the cell cycle, thereby causing enhanced proliferation and cell survival. To interrogate the hypothesis, both cell lines were treated with lapatinib, glyceollin, or the combination and cell viability was measured. Results demonstrated that drug treatment had no effect on the proliferation of T47Darom cells, however, glyceollin alone and glyceollin + lapatinib inhibited T47DaromLR cell viability by 46% and 59%, respectively. Glyceollin alone or the combination caused a 33% and 60% reduction in T47Darom colony formation, respectively. Interestingly, glyceollin alone or the combination of glyceollin + lapatinib significantly inhibited T47DaromLR colony formation by greater than 90%. Apoptosis studies were conducted to measure caspase 3/7 activity in both cell lines. Lapatinib alone had no effect on apoptosis, however glyceollin and the combination significantly induced apoptosis in both cell lines; the most dramatic effect was observed in T47Darom cells, suggesting that additional mechanisms may contribute to the anti-cancer effect observed in T47DaromLR cells. Taken together, these results suggest that dual inhibition using glyceollins and lapatinib may have potential as a novel combination therapy approach for postmenopausal patients with hormone-dependent, letrozole-resistant breast cancer. This work was funded by an NIH grant awarded to SL Tilghman (1SC1GM125617). Citation Format: Rashidra R. Walker, Jankiben Patel, A. Michael Davidson, Syreeta L. Tilghman. A novel phytoalexin, glyceollins, trigger anti-proliferative and apoptotic effects in aromatase inhibitor resistant breast cancer cells [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 4116.

Leptin Signaling Contributes to Aromatase Inhibitor Resistant Breast Cancer Cell Growth and Activation of Macrophages.

Obesity represents a risk factor for breast cancer development and therapy resistance, but the molecular players underling these links are unclear. Here, we identify a role for the obesity-cytokine leptin in sustaining aromatase inhibitor (AI) resistant growth and progression in breast cancer. Using as experimental models MCF-7 breast cancer cells surviving long-term treatment with the AI anastrozole (AnaR) and Ana-sensitive counterparts, we found that AnaR cells expressed higher levels of leptin and its receptors (ObR) along with a constitutive activation of downstream effectors. Accordingly, leptin signaling inhibition reduced only AnaR cell growth and motility, highlighting the existence of an autocrine loop in mechanisms governing drug-resistant phenotypes. In agreement with ObR overexpression, increasing doses of leptin were able to stimulate to a greater extent growth and migration in AnaR than sensitive cells. Moreover, leptin contributed to enhanced crosstalk between AnaR cells and macrophages within the tumor microenvironment. Indeed, AnaR, through leptin secretion, modulated macrophage profiles and increased macrophage motility through CXCR4 signaling, as evidenced by RNA-sequencing, real-time PCR, and immunoblotting. Reciprocally, activated macrophages increased AnaR cell growth and motility in coculture systems. In conclusion, acquired AI resistance is accompanied by the development of a leptin-driven phenotype, highlighting the potential clinical benefit of targeting this cytokine network in hormone-resistant breast cancers, especially in obese women.

Abstract P3-11-15: A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) are currently the standard treatment for postmenopausal women with estrogen-dependent metastatic breast cancer. While effective, patients develop resistance leading to tumor relapse, metastasis, and more aggressive phenotypes. Previous research suggested that AI resistance is due to upregulation of growth factor pathways (i.e., HER2 and EGFR) and increased cellular motility. Our lab previously demonstrated that a novel phytoalexin, glyceollins, inhibits proliferation, survival, and migration of hormone independent letrozole-resistant breast cancer cells (LTLT-Ca). However, many postmenopausal women with AI-resistance tumors remain hormone dependent. Therefore, there is a need to understand distinctions between estrogen receptor (ER+) positive and ER negative (ER-) AI resistant tumors and their response to therapy. We hypothesize that treating ER+ letrozole-resistant breast cancer using a combination approach of glyceollin and lapatinib (a dual EGFR/HER2 inhibitor) will reduce growth factor signaling, inhibit proliferation and motility. To test this hypothesis, T47Darom cells (T47D cells which stably overexpress aromatase) and T47DaromLR cells (T47Darom cells that are letrozole resistant) were treated with lapatinib, glyceollin, or the combination. To evaluate viability, resazurin assays were performed and results demonstrated that glyceollin and/or lapatinib had no effect on proliferation in the T47Darom cells. However, glyceollin alone and glyceollin + lapatinib inhibited proliferation of T47DaromLR cells by 46% and 59%, respectively. Cell survival was assessed by colony formation assays and glyceollin caused a 33% reduction in cell survival and glyceollin + lapatinib caused a 60% reduction in the colony formation of T47Darom cells. Interestingly, in T47DaromLR cells glyceollin and the combination significantly inhibited cell survival by 94% alone and 96%. To further evaluate changes that occur as AI sensitive cells transition to AI resistance, a quantitative proteomic analysis of the whole cell lysates of T47DaromLR (AI-resistant) versus T47Darom cells (AI-sensitive) was performed to identify significant protein expression changes. Results demonstrated a 3.195-fold-increase (p&amp;lt;0.01) in protein disulfide isomerase (P4HB) and a 0.484-fold decrease (p&amp;lt;0.01) in peptidyl-prolyl cis-trans isomerase (FKBP4) which was validated by immunoblotting. While the molecular mechanism by which P4HB contributes to tumorigenesis and metastasis is unknown, it has been demonstrated to be involved in the proliferation, survival, and metastasis of several types of cancer cells. FKBP4 is also of interest as it has been shown to be involved in mitotic arrest and apoptotic signaling. To interrogate the effect of combination therapy on HER effector signaling cascades, western blot analyses were performed to establish the endogenous expression of various proteins. Results demonstrated ERα expression was increased while HER2 was decreased in T47Darom cells compared to T47DaromLR cells. The protein expression findings from the T47D variant cell lines followed a similar trend as those previously reported in LTLT-Ca cells and their AI-sensitive counterparts. Taken together, these results suggest that dual inhibition using glyceollins and lapatinib may have potential as a novel combination therapy approach for postmenopausal patients with hormone-dependent, letrozole-resistant breast tumors. This work was also supported by NIH grant 1SC1GM126617 awarded to SL Tilghman. Citation Format: Rashidra R Walker, Jankiben Patel, A. Michael Davidson, Karen Gallegos, Syreeta L Tilghman. A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells [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 P3-11-15.

Abstract P1-08-01: Estrogen-induced cell cycle arrest and apoptosis in aromatase inhibitor-resistant breast cancer: Insights from single cells analysis of a patient-derived xenograft model

Abstract Background: Estradiol (E2) is known to promote breast cancer. However, several small-scale prospective clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)- resistant cases of hormone receptor-positive postmenopausal breast cancer. Considering that the practicality and safety of E2 treatments are still undefined, the objective of this study is to uncover the mechanisms of E2-induced tumor regression, which will lead to a better design of E2 therapies. Methods: While most of our ER+ breast cancer patient-derived xenograft (PDX) models are positively regulated by E2, an estrogen-suppressive model (named GS3) was established from an AI resistant ER+/PR−/HER2− brain metastatic breast cancer. E2 pellets (1mg each) or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC), RNA sequencing, and reverse phase protein array (RPPA) analyses of PDX specimens were conducted to decipher molecular changes after E2 treatments. Importantly, since the cancer tissue has a very heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist (MPP), not by ERβ antagonist (PHTPP). IHC showed that PR expression appeared, Ki-67 and CEACAM5 (CEA) expressions decreased, and the number of apoptotic cells significantly increased after E2 treatment. RNA sequencing indicated that estrogen-regulated genes, e.g., PGR, EGR3, PDZK1, and GREB1, were up-regulated, while tumor growth was repressed by E2. Single cell RNAseq analysis demonstrated that cells from E2-treated and Placebo-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes (HVGs). Importantly, after one-week, many cells of E2-treated PDXs were arrested within the G1 phase of the cell cycle (G1, 60%; S, 19%; G2M, 21%), whereas, cells of placebo-treated samples advanced to the S and G2M phases (G1, 47%; S, 27%; G2M, 26%). Gene Set Variation Analysis of cells from E2-treated PDXs revealed enrichment of genes associated with reactive oxygen species (ROS) mechanism. E2 increased cells expressing pro-apoptotic genes, such as TP53, BOK,IL24, and PGLYRP2. Gene expressions including mitogen-activated protein kinase (MAPK) signaling pathway, such as GADD45A, MAP3K5 (ASK1), MAPK9 (JNK2), and especially MAPKAPK2 were also increased in E2 treated PDXs. Conclusions: E2/ERα-mediated suppression of GS3 tumor growth was accompanying the inhibition of the cell cycle progression and increased expression of pro-apoptotic genes. It is known that classical MAPK pathway induces cell proliferation, meanwhile, JNK MAPK pathway induces p53 signaling pathway and apoptosis. We hypothesize that E2 induces ROS and promotes apoptosis of GS3 due to JNK MAPK pathway activation. This could be an unexpected outcome of AI resistance. Using this valuable estrogen-down-regulated ER+ breast cancer PDX and informative integrative omics analyses, critical molecular mechanisms on E2-mediated apoptosis are being revealed in our laboratory. The ongoing research using E2-sensitive PDXs will identify key markers to select AI resistant patients who are expected to respond effectively to estrogen-induced apoptosis treatments as well as to evaluate the efficacy of drug combination with estrogen, including CDK 4/6 inhibitors. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest and apoptosis in aromatase inhibitor-resistant breast cancer: Insights from single cells analysis of a patient-derived xenograft model [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 P1-08-01.

Suppression of Nuclear Factor-κB by Glucocorticoid Receptor Blocks Estrogen-Induced Apoptosis in Estrogen-Deprived Breast Cancer Cells.

Our clinically relevant finding is that glucocorticoids block estrogen (E2)-induced apoptosis in long-term E2-deprived (LTED) breast cancer cells. However, the mechanism remains unclear. Here, we demonstrated that E2 widely activated adipose inflammatory factors such as fatty acid desaturase 1 (FADS1), IL6, and TNFα in LTED breast cancer cells. Activation of glucocorticoid receptor (GR) by the synthetic glucocorticoid dexamethasone upregulated FADS1 and IL6, but downregulated TNFα expression. Furthermore, dexamethasone was synergistic or additive with E2 in upregulating FADS1 and IL6 expression, whereas it selectively and constantly suppressed TNFα expression induced by E2 in LTED breast cancer cells. Regarding regulation of endoplasmic reticulum stress, dexamethasone effectively blocked activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) by E2, but it had no inhibitory effects on inositol-requiring protein 1 alpha (IRE1α) expression increased by E2 Consistently, results from reverse-phase protein array (RPPA) analysis demonstrated that dexamethasone could not reverse IRE1α-mediated degradation of PI3K/Akt-associated signal pathways activated by E2 Unexpectedly, activated GR preferentially repressed nuclear factor-κB (NF-κB) DNA-binding activity and expression of NF-κB-dependent gene TNFα induced by E2, leading to the blockade of E2-induced apoptosis. Together, these data suggest that trans-suppression of NF-κB by GR in the nucleus is a fundamental mechanism thereby blocking E2-induced apoptosis in LTED breast cancer cells. This study provided an important rationale for restricting the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in patients with aromatase inhibitor-resistant breast cancer.

Interaction Between MUC1 and STAT1 Drives IFITM1 Overexpression in Aromatase Inhibitor-Resistant Breast Cancer Cells and Mediates Estrogen-Induced Apoptosis.

The human oncoprotein, mucin 1 (MUC1), drives tumorigenesis in breast carcinomas by promoting epithelial-to-mesenchymal transition (EMT), epigenetic reprogramming, and evasion of immune response. MUC1 interacts with STAT1, through JAK/STAT signaling, and stimulates transcription of IFN-stimulated genes, specifically IFN-induced transmembrane protein 1 (IFITM1). Our laboratory has previously shown that IFITM1 overexpression in aromatase inhibitor (AI)-resistant breast cancer cells promotes aggressiveness. Here, we demonstrate that differential regulation of MUC1 in AI-sensitive (MCF-7 and T-47D) compared with AI-resistant (MCF-7:5C) cells is critical in mediating IFITM1 expression. A tumor microarray of 94 estrogen receptor-positive human breast tumors correlated coexpression of MUC1 and IFITM1 with poor recurrence-free survival, poor overall survival, and AI-resistance. In this study, we investigated the effects of MUC1/IFITM1 on cell survival and proliferation. We knocked down MUC1 levels with siRNA and pharmacologic inhibitors, which abrogated IFITM1 mRNA and protein expression and induced cell death in AI-resistant cells. In vivo, estrogen and ruxolitinib significantly reduced tumor size and decreased expression of MUC1, P-STAT1, and IFITM1. IMPLICATIONS: MUC1 and IFITM1 overexpression drives AI resistance and can be targeted with currently available therapies.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/5/1180/F1.large.jpg.

Compensatory role of insulin-like growth factor 1 receptor in estrogen receptor signaling pathway and possible therapeutic target for hormone therapy-resistant breast cancer.

Hormone therapy targeting the estrogen receptor (ER) pathway is the most common treatment used for ER-positive breast cancer. However, some patients experience de novo or acquired resistance, which becomes a critical problem. Activation of the insulin-like growth factor (IGF) pathway allows breast cancer cells to proliferate and is associated with the ER pathway. Little is known about the role of the IGF pathway in hormone therapy and resistance; therefore, we investigated whether the inhibition of this pathway may represent a novel therapeutic target for overcoming hormone therapy resistance in ER-positive breast cancers. Crosstalk between the ER and IGF pathways was analyzed in breast cancer cell lines by inhibiting or stimulating either one or both pathways. We studied the effect of insulin-like growth factor one receptor (IGF1R) inhibition in aromatase inhibitor-resistant breast cancer cell lines and fulvestrant-resistant cell lines which were uniquely established in our laboratory. Under normal conditions, IGF signaling is controlled by ER signaling to promote cell growth. Temporary disruption of the estrogen supply results in attenuated ER signaling, and IGF-1 dramatically increased relative growth compared with normal conditions. In addition, IGF1R inhibitor strongly suppressd cell growth in hormone-resistant breast cancer cells where ER remains than cells where ER decreased or was almost lost. Our study suggests that inhibition of the IGF pathway may be an effective strategy for ER-positive breast cancer therapy, even in hormone therapy-resistant cases.

Abstract 4888: Disrupting MUC1/IFITM1 crosstalk reverses the aggressive phenotype of aromatase inhibitor-resistant breast cancer

Abstract Breast cancer is the most commonly diagnosed and second most lethal cancer among women, affecting over 252,000 women in 2017. The ER+/PR+ subtype of breast cancer is the most frequently diagnosed, representing approximately 70% of all cases. Estrogen depletion through aromatase inhibitors (AIs) is the standard of care to cause tumor regression in the ER+ subtype. Our research focuses on targeting the AI-resistant tumors that develop in 30% of women. The human oncoprotein, mucin 1 (MUC1), drives tumorigenesis in breast carcinomas promoting the epithelial-mesenchymal transition, epigenetic reprogramming, and evasion of the immune response. MUC1 interacts with the STAT proteins, via the JAK/STAT signaling pathway, and stimulates transcription of interferon-stimulated genes (ISGs) including IFITM1. Our laboratory has shown that IFITM1 is overexpressed in AI-resistant breast cancer cells and AI-resistant tumors and promotes an aggressive phenotype. In this study, we demonstrate that MUC1 is differentially regulated in endocrine-sensitive (MCF-7 and T-47D) compared to AI-resistant (MCF-7:5C) cells and that it plays a critical role in enhancing IFITM1 expression in the resistant cells. Analysis of a tumor microarray of 94 ER+ human breast tumors indicated that co-expression of both MUC1 and IFITM1 correlated with poor recurrence-free survival, poor overall survival, and AI resistance. To investigate how disruption of the MUC1/IFITM1 crosstalk effects cell survival and proliferation, we used an AI-resistant cell line (MCF-7:5C) that overexpresses MUC1, P-STAT1, and IFITM1. We genetically manipulated breast cancer cells by knocking down the levels of MUC1 with siRNA and observed lowered expression of IFITM1 at the mRNA and protein level and reduced IFITM1 promoter activation. Additionally, knockdown of MUC1 and IFITM1 levels with siRNA induced cell death in AI-resistant cells (MCF-7:5C). We verified this effect with the pharmacologic inhibitors GO-201 (a MUC1 inhibitor) and ruxolitinib (a JAK/STAT inhibitor) to interfere with the MUC1/IFITM1 crosstalk in vitro. Both agents significantly reduced cell proliferation in resistant cells (MCF-7:5C) but not in estrogen-sensitive cell lines (MCF-7 and T-47D). Co-IP confirmed the physical interaction between MUC1 and STAT1/2 and ruxolitinib treatment blocked this interaction by inhibiting STAT phosphorylation in both AI-resistant and -sensitive cells. In vivo studies using ruxolitinib significantly reduced tumor size in NSG mice and decreased expression of MUC1, P-STAT1, and IFITM1. The research discussed here highlights a novel mechanism by which breast cancer cells develop AI resistance and suggests that disrupting MUC1/IFITM1 crosstalk might be therapeutically beneficial to patients with AI-resistant disease. Citation Format: Taylor E. Escher, Eric Geanes, Asona Lui, Joan Lewis-Wambi. Disrupting MUC1/IFITM1 crosstalk reverses the aggressive phenotype of aromatase inhibitor-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4888.

PO-178 Androstenedione (4AD) activates an androgen receptor (AR) mediated transcriptome and AR interactions associated with cell-cell adhesion in aromatase inhibitor (AI) resistant breast cancer cells

Introduction AI’s are the front line treatment for endocrine postmenopausal breast cancer. However, response rates vary from 35%–70% and recurrence occurs in almost all advanced disease. In order to identify a target for overcoming AI treatment resistance, research has turned to the most widely expressed sex steroid receptor in breast cancer the AR. AI therapies, by their mechanism of action, create an unopposed androgenic steroid environment. 4AD is the major circulating steroid in postmenopausal women and AI therapy prevents it’s conversion to estrogens. We hypothesise that cancer cells adapt to utilise 4AD to promote resistance and survival in AI resistance. Material and methods Molecular changes in a breast cancer cell model which have transitioned from AI sensitive (MCF7 cells) to AI resistant (LetR cells) were assessed using RNA sequencing. Alterations in the steroid microenvironment occur as a result of AI therapy and responses to these steroids was confirmed using MTS, mammosphere and invasion assays. Mass spectrometry (LC–MS/MS) analysis identified androgen-mediated, AR protein interactions.The clinical relevance of AR in AI resistance was also evaluated in a cohort of primary breast cancer patients (n=375). Results and discussions LetR cell growth is AR dependent as the anti-AR drug Enzalutamide inhibits cell growth. Furthermore, Enzalutamide abrogates mammosphere formation and self-renewal of LetR cells. We have found LetR cells to be responsive to 4AD, and treatment with this steroid drives a more aggressive phenotype in vitro. RNA sequencing data highlighted a non-canonical cytoplasmic AR signalling mechanism. Our research suggests that 4AD initiates rapid second messenger signalling in the AI resistant setting. LC–MS/MS analysis identified androgen-mediated, AR protein interactors unique to our resistant model particularly associated with cell-cell adhesion and invasion. Evaluation of AR protein in a cohort of primary breast cancer patients found AR expression in 90% of patients, with 25% expressing high intensity AR staining. Analysis is ongoing into this subset and its association with clinical pathological parameters. Conclusion AR and its ligand 4AD may be a significant driving factor in AI resistance and may play a role in facilitating a more invasive phenotype. Further investigations into AR interactors identified will help elucidate mechanisms of resistance to AI therapy, and these novel AR protein partners may aid the identification of patients who would benefit from anti-AR therapy.