Letrozole-resistant Cells Research Articles

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.

Acquisition of Letrozole Resistance Through Activation of the p38/MAPK Signaling Cascade.

Previous reports identified a global proteomic signature of estrogen-independent letrozole resistant breast cancer cells, however, it remains unclear how letrozole-resistance is impacted when cells remain estrogen receptor positive (ER+). To capture the protein expression profile associated with ER+ Aromatase inhibitor (AI) resistance, a global proteomic analysis was conducted using the letrozole-sensitive (T47Darom cells) and letrozole-resistant cells (T47DaromLR cells). To examine the molecular features associated with this phenotype Kaplan- Meier analysis, phospho-antibody arrays, proliferation and apoptosis assays were conducted. MAP3K6 was up-regulated in the T47DaromLR cells by 3.2-fold (p<0.01) which was associated with a decrease in relapse-free survival among breast cancer patients (p=0.0019). Members of the MAPK/p38 pathway (i.e., phospho-MKK6, phospho-p38, phospho-RSK1, phospho-RSK2, and p70S6K MAPK) were also increased in the T47DaromLR cells, while inhibiting p38 led to decreased proliferation and induction of apoptosis. Activation of the p38/MAPK pathway leads to ER+ AI-resistance.

Abstract 4365: Estrogen receptor β agonists reverses letrozole therapy resistance of breast cancer through FOXO3 and FOXM1 mediated actions

Abstract Introduction: Breast cancer (BC) is the most common cancer in women worldwide and leading cause of cancer death. More than 70% of cancers are estrogen receptor alpha (ERα) positive. Despite effective therapeutic strategies for treating hormone receptor-positive (HR+) breast cancer, resistance to endocrine therapy that is either de novo or acquired still occurs. Understanding the molecular pathways that contribute to BC resistance will enable to develop new treatments for improving the efficacy of endocrine therapy. Studies have shown that estrogen receptor beta (ERβ) and its agonists inhibits the different types of cancers including BC. Herein, we investigated mechanism(s) for therapeutic efficacies of ERβ agonists in breast cancer. Methods: To investigate the mechanisms of ERβ agonists (S-Equol and LY500307), in the treatment of breast cancer, we used therapy sensitive (MCF7-Aro) and letrozole resistant (MCF7-Aro-LTLT) cells and ERβ knock down cell lines, generated by CRISPR-Cas9 technology. The wild type and ERβ knock down cells with or without treatment of ERβ agonists were analyzed, for cell proliferation, protein (Western), Gene expression array analysis, RNA-seq analysis, data-independent acquisition (DIA) mass spectrometry analyses and RNA (RT-qPCR) analysis. Xenograft and patient derived BC explants (PDEX) were used to test the antiproliferative activity of ERβ agonists. Results: Our results demonstrated that treatment with ERβ agonists, S-Equol and LY500307 inhibited the growth of both endocrine therapy sensitive and resistant breast cancer cells. Gene expression array analysis of LY500307 treated breast cancer cells showed the modulation of signaling molecules involved in cell death and cell cycle pathways. RNA-seq analysis showed that treatment with S-equol modulated the ERβ target genes involved in tumor progression and resistance to hormone therapies. Mass spectrometry based DIA analysis and Reactome pathway analysis revealed that S-equol treatment modulated the key proteins involved in DNA replication and cell cycle. Both ERβ agonists S-Equol and LY500307 inhibited the cell proliferation of parental cells whereas they could not elicits the inhibitory effect on the knock out cells. By using ERα positive PDX tumors, we have demonstrated that both the ERβ agonists inhibited the proliferation of human BC tumors. Our mechanistic studies confirmed that increased FOXO3 activation by S-equol in LTLT cells can reverse letrozole resistance. Our results showed that inhibiting FOXM1 by ERβ agonists significantly reduce the proliferation of letrozole-resistant cells. These changes were not observed in ERβ knockdown cells confirming the anti-proliferative role of ERβ in breast cancer progression. Conclusions: Collectively, our results suggest that ERβ agonists may offer a new targeted therapy for endocrine therapy resistant breast tumors. (Supported by BCRP 151884/DOD grant W81XWH-16-1-0294 and P30 CA-54174 NCI-Mays cancer center grant). Citation Format: Kumaraguruparan Ramasamy, Suryavathi Viswanadhapalli, M.A. Sureshkumar, Susan Weintraub, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. Estrogen receptor β agonists reverses letrozole therapy resistance of breast cancer through FOXO3 and FOXM1 mediated actions [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 4365.

Abstract P6-04-19: Estrogen receptor β agonist S-Equol promotes letrozole sensitivity of endocrine therapy resistant breast cancer cells by upregulating FOXO3 expression

Abstract Background: Breast cancer (BC) is the most frequently diagnosed cancer and the primary leading cause of cancer deaths in women worldwide. Although the clinical outcome of BC patients has been considerably improved, resistance to endocrine and chemotherapy treatments contributes to BC relapse and mortality. Understanding the molecular pathways that contribute to BC resistance will enable to develop new treatments for improving the efficacy of endocrine therapy. Recent studies suggested that estrogen receptor beta (ERβ) and its ligand S-Equol promote suppression of BC progression. However, the mechanisms by which S-equol inhibits growth of BC cells and enhance therapy response remains unknown. Here, we examined the mechanisms by which S-equol contribute to BC growth inhibition in hormone therapy sensitive and resistant BC cells. Methods: We evaluated the therapeutic efficacy of S-equol in therapy sensitive (MCF7-Aro) and letrozole resistant (MCF7-Aro-LTLT) cells. Expression levels of target genes were evaluated in both cell lines by global RNA-seq analysis, data-independent acquisition (DIA) mass spectrometry analyses, qRT-PCR and Western blot. ERβ knock down cell lines were generated by CRISPR-Cas9 technology. Xenograft and patient derived BC explants (PDEX) were used for preclinical evaluation of the antiproliferative activity of S-equol. Results: S-equol reduced the growth of endocrine sensitive and resistant BC models in vitro and in vivo. Treatment of ER-positive PDEX with S-equol decreased the proliferation compared to untreated tumors. RNA-seq analysis of S-equol treated BC cells showed modulation of ERβ target genes involved in tumor progression and resistance to hormone therapies. Mass spectrometry based DIA analysis showed that S-equol treatment upregulated 438 proteins and downregulated 429 proteins in MCF7-Aro cells compared to controls. Whereas, DIA analysis in Letrozole-resistant cells revealed that S-equol upregulated 572 proteins and downregulated 481 proteins compared to controls. Reactome pathway analysis revealed that S-equol treatment modulated the proteins involved in DNA replication, cell cycle, proteins regulating p53 activity and VEGF-VEGFR2 pathway. Our mechanistic studies confirmed that treatment with S-equol upregulated FOXO3 and downregulated FOXM1. Using CRISPR-Cas9 mediated ERβ knockout cells, we confirmed that S-equol could not modulate forkhead box transcription factors in BC cells lacking ERβ compared to parental cells. Conclusions: Collectively, our results suggest that S-equol exhibits its antitumor activity by targeting forkhead box transcription factors and that S-equol might be an effective treatment strategy in treating / preventing breast cancer recurrence. (Supported by BCRP 151884/DOD grant W81XWH-16-1-0294). Citation Format: Kumaraguruparan Ramasamy, Shaorong Chen, Suryavathi Viswanadhapalli, Rong Li, Ratna K Vadlamudi, Rajeshwar R Tekmal. Estrogen receptor β agonist S-Equol promotes letrozole sensitivity of endocrine therapy resistant breast cancer cells by upregulating FOXO3 expression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-19.

Use of dual mTOR inhibitor MLN0128 against everolimus-resistant breast cancer.

HR+/HER2- aromatase inhibitor-resistant metastatic breast cancer can be treated with everolimus and a second AI until the cancer recurs. Targeting these everolimus-resistant patients with the latest standard of care, CDK4/6 inhibitors, has not been clearly addressed. Understanding the signaling transduction pathways, which everolimus resistance activates, will elucidate the mechanisms and offer treatment strategies of everolimus resistance. To mimic the clinical setting, letrozole-resistant cells were used to generate an everolimus-resistant model (RAD-R). Reverse phase protein array (RPPA) was performed to reveal changes in the signaling transduction pathways, and expression levels of key proteins were analyzed. Inhibitors targeting the major signaling pathways, a CDK4/6 inhibitor palbociclib and a mTORC1/2 inhibitor (MLN0128), were evaluated to establish resistance mechanisms of RAD-R. RPPA results from RAD-R indicated changes to significant regulatory pathways and upregulation of p-AKT expression level associating with everolimus resistance. MLN0128, that inhibits the AKT phosphorylation, effectively suppressed the proliferation of RAD-R cells while treatment with palbociclib had no effect. Among the many signaling transduction pathways, which are altered post everolimus resistance, targeting dual mTORC1/2 is a possible option for patients who have recurrent disease from previous everolimus treatment.

Abstract 2053: Mechanisms of resistance to palbociclib and aromatase inhibitors in hormone receptor positive breast cancer

Abstract Estrogen receptor-positive (HR+) breast cancer is the most common form of breast cancer that is accountable for the majority of breast cancer mortality. Currently, the cyclin dependent kinase inhibitor palbociclib in combination with endocrine therapy represents the new standard first and second line therapy for patients with metastatic HR+ breast cancer. While palbociclib has been shown to significantly improved progression free survival in combination with aromatase inhibitor (AI) and fulvestrant, resistance will inevitably occur in patients with metastatic breast cancer. However, the mechanisms of resistance to the combination of palbociclib and AI remain largely unknown. It is also unclear whether resistance mechanisms would be the same if palbociclib was given as first line treatment in combination with letrozole vs. if palbociclib was given as second line treatment after letrozole resistance had already occurred. To address this, previously well established and well characterizied letrozole-sensitive MCF7Ca cells and letrozole-resistant LTLTCa cells were subjected to continuous, long term treatment with increasing doses of letrozole and palbociclib until resistance to both drugs was achieved (MCF7Calet+palb and LTLTCapalb, respectively). Preliminary MTT cell viability assays indicate palbociclib IC50s of 750 nM in MCF7Ca and LTLTCa cells vs. 21 uM in palbociclib and letrozole-resistant cells. First line and second line palbociclib resistance correlated with changes in morphology, protein expression, and cancer stem cell characteristics. Under phase contrast microscopy, first line and second line palbociclib-resistant MCF7Ca and LTLTCa cells were larger in size, more irregular in shape, and tended not to grow in epithelial cell-like groups compared to palbociclib-sensitive MCF7Ca and letrozole cells, with second-line palbociclib resistant cells exhibiting these characteristics the most. Western blot showed that ER protein expression in ER+/HER2- MCF7Ca cells decreased with first line palbociclib and letrozole resistance, and that both ER and HER2 protein expression were decreased with second line palbociclib resistance in ERlow/HER2+ LTLTCa cells. Lastly, mammosphere assays demonstrated increasing percentage of cancer stem cells with letrozole resistance alone (4 per 1000 cells plated MCF7Ca vs. 58 per 1000 planted LTLTCa) and with palbociclib and palbociclib resistance (317 per 1000 cells plated MCF7Ca and 202 per 1000 cells plated LTLTCa cells). Overall, these results indicate that 1) resistance to palbociclib, whether as first line or second line treatment has significant effects on breast cancer cells that may be relevant to patient diagnosis and treatment. Citation Format: Armina A. Kazi, Antony Sare, Saranya Chumsri, Angela Brodie. Mechanisms of resistance to palbociclib and aromatase inhibitors in hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2053. doi:10.1158/1538-7445.AM2017-2053

Abstract P6-11-15: Pre-clinical investigation of estrogen receptor β agonists for the treatment of breast cancer

Abstract Breast Cancer is the primary cause of cancer-associated mortality worldwide, and in United States alone, more than 250,000 women are diagnosed every year. Current breast cancer treatment strategies focus on Estrogen Receptor α signaling, given that the majority of cases diagnosed are ERα positive. These treatment strategies include endocrine therapies; such as anti-estrogens or aromatase inhibitors. Although, endocrine therapy has been demonstrated to be successful and effective, therapy resistance commonly arises and results in relapse. While current endocrine therapies focus on ERα signaling, emerging studies highlight the importance of Estrogen Receptor β.Unlike ERα, ERβ has been shown to have tumor-suppressive function in various cancers, including breast cancer. Recent studies have identified, synthesized, and tested the clinical safety of ERβ-selective agonists. The objective of this study was to investigate the utility of using ERβ agonists in the treatment of breast cancer. To investigate the utility of ERβ agonists in the treatment of breast cancer, we used in-vitro and in-vivo pre-clinical models systems. Our results demonstrated that treatment with ERβ agonists, S-Equol and LY500307, was able to inhibit the short-term and long-term growth of both endocrine therapy sensitive and resistant breast cancer cells. Progression through the cell cycle, cell migration and cell invasion was also abrogated upon treatment. In-vivo, our syngeneic tumor mouse model demonstrates a decline in tumor growth rate after treating with a combination of letrozole and ERβ agonist. Gene expression array analysis reveal that treatment with ERβ agonist elicits changes in key signaling molecules involved in cell death and cell cycle pathways. In Letrozole resistant cells, Letrozole treatment had not effect on gene expression, while LY500307 treatment resulted in the modulation of 780 genes. Interestingly, combining Letrozole with LY500307 resulted in the modulation of 966 genes, of which 417 were unique to the combination treatment. Our studies suggest that activation of ERβ signaling is a valuable strategy in the treatment of breast cancer, even in cases which have developed resistance to current endocrine therapies. Citation Format: Samayoa C, Krishnegowda NK, Vadlamudi RK, Tekmal RR. Pre-clinical investigation of estrogen receptor β agonists for the treatment of breast cancer [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 P6-11-15.

Abstract PD1-04: CSMD1 SNPs selectively affect anastrozole response in postmenopausal breast cancer patients

Abstract BACKGROUND: Based on prospective clinical trials, there is no evidence for differences in efficacy between the 3 aromatase inhibitors (AIs) anastrozole, exemestane, and letrozole. The purpose of this study was to identify germline genetic variants associated with response to AIs and to help identify novel mechanisms associated with drug disease efficacy. METHODS: A genome-wide association study (GWAS) was performed for 624 patients (Steroids 2015;99:32-38) to identify SNPs associated with estrogen level change in women with estrogen receptor (ER) positive breast cancer treated with anastrozole. Replication of associated SNPs was performed in a GWAS from the MA.27 trial that compared adjuvant anastrozole and exemestane treatment of post-menopausal women with ER+ breast cancer. Functional studies were subsequently performed to determine SNP effects and underlying mechanisms. RESULTS: Our initial GWAS identified SNPs within CSMD1 that were associated with changes in estrogen levels during anastrozole therapy. An additional SNP in CSMD1 was also associated with breast cancer events in CCTG MA.27. Functionally, we showed that CSMD1 regulates CYP19 expression in a SNP-, and in an anastrozole- dependent fashion. These phenomena were not observed for either letrozole or exemestane. In MA.27, an anastrozole- specific effect was also seen with the minor allele having a protective effect on time to distant metastasis (HR=0.49, p=0.00259), but this was not the case for exemestane (HR=0.71, p=0.111). Our in vitro functional studies indicated that overexpression of CSMD1 sensitized anastrozole or letrozole resistant cells to anastrozole but not to the other two AIs. The SNP in CSMD1 that was associated with increased CSMD1 and CYP19 expression levels increased anastrozole sensitivity, but not letrozole or exemestane in lymphoblastoid cell lines (LCLs) homozygous for either WT or variant CSMD1 SNP genotypes. Based on these observations, we explored whether anastrozole has additional mechanisms beyond its function as a CYP19 inhibitor. Utilizing an estrogen response element (ERE) luciferase reporter assay in a CYP19 CRISPR knockout breast cancer T47D cell line and a surface plasmon resonance (SPR) assay, we found that anastrozole can also function as an ERα agonist, and can bind to, and result in, proteasome dependent ERα degradation, especially in the presence of E2. Treatment of these CYP19 CRISPR knockout cells with anastrozole in the presence of increasing concentrations of E2 results in greater sensitivity compared with anastrozole alone, while the addition of E2, as expected, does not improve letrozole or exemestane sensitivity. These same observations were also seen in letrozole and anastrazole resistant cells. CONCLUSIONS: Our findings suggest that anastrozole might be more effective than letrozole or exemestane in patients with the CSMD1 SNP. Furthermore, anastrozole can function as an ERα agonist, binding to ERα and resulting in its degradation, especially in the presence of E2. These findings should help to make it possible to develop precision endocrine therapies for women who are candidates for AIs. Citation Format: Cairns J, Ingle J, Dudenkov T, Kalari K, Buzdar A, Kubo M, Robson M, Ellis M, Goss P, Shepherd L, Goetz M, Weinshilboum R, Wang L. CSMD1 SNPs selectively affect anastrozole response in postmenopausal breast cancer patients [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 PD1-04.

Abstract 1820: Investigating the role of estrogen receptor β agonists in the prevention and treatment of breast cancer

Abstract Breast Cancer is the primary cause of cancer-associated mortality worldwide. In the United States alone, more than 250,000 women are diagnosed every year. Asian women have the lowest incidence rates. However, their rates increase to those of the US within one generation after migration. Suggesting that diet and lifestyle are major contributing factors in breast cancer risk. Since Asian women consume high amount of soy when compared to other populations, perhaps compounds found in soy may have a protective effect that is lost once they adopt a western diet. Recent studies have identified plant-derived compounds from soy and licorice that activate Estrogen Receptor β. Unlike previous ERβ agonists, these novel compounds are selective for ERβ and have been tested for safety in clinical trials. Current breast cancer treatment strategies focus on Estrogen Receptor α signaling, given that the majority of cases diagnosed are ERα positive. These treatment strategies include endocrine therapies; such as anti-estrogens or aromatase inhibitors. However, in the last decade, the importance of ERβ has emerged. Unlike ERα, ERβ has been shown to have tumor-suppressive function in various cancers, including breast cancer. The objective of this study was to investigate the utility of using ERβ agonists in the prevention and treatment on breast cancer. To investigate the significance of ERβ activation in the prevention of breast cancer, we used an immunocompetent transgenic mouse model of breast cancer. Specifically, HER2/neu overexpressing mice develop premalignant lesions at 4-5 months and tumors starting at month 7. HER2/neu mice were treated with ERβ agonists for 3 months and subsequently mammary glands were analyzed using whole mount and IHC. When compared to controls, ERβ agonist treated mice showed a decrease in branching and ductal hyperplasia, with no change in body weight. These results suggest that ER β agonist treatment is chemopreventive and provided protection against premalignant lesion in the mammary gland. To investigate the utility of ERβ agonists in the treatment of breast cancer, we used in-vitro and in-vivo models systems. Our results demonstrated that treatment with the agonists was able to inhibit the short-term and long-term growth of the breast cancer cell lines; MCF7aro and LTLT which represent post-menopausal breast cancer and letrozole resistant cells, respectively. In addition to growth inhibition, treatment with ERβ agonists also decreased cell migration and invasion. In addition to activating ERβ, we also found that the agonists are also able to increase the expression of ERβ without increasing ERα levels. Our studies also demonstrated that treatment with ERβ agonists modulated key signaling molecules involved in cell death and cell cycle. Our studies suggest that activation of ERβ signaling is a valuable strategy in the treatment and chemoprevention of breast cancer. Citation Format: Cathy Samayoa, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. Investigating the role of estrogen receptor β agonists in the prevention and treatment of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1820.

Abstract 1123: The role of proto-oncogene PELP1 in breast cancer stem cell maintenance and therapy resistance

Abstract BACKGROUND: Cancer stem cells (CSCs) are known to evade hormonal therapy and regrowth of tumor cells from cancer stem cells is a major clinical problem. Histone methyltransferase G9a/EHMT2 plays a critical role in stem cell maintenance. Proline, glutamic acid, and leucine rich protein (PELP1) is a proto-oncogene, functions as a coregulator of nuclear receptors, and is prognostically linked to shorter breast cancer survival. Recent studies from our lab discovered that PELP1 interacts with G9a/EHMT2. The objective of this study is to develop small molecular inhibitors that block PELP1 interactions with G9a/EHMT2. METHODS: We isolated CD44high/CD24low CSCs from three breast cancer cell lines (ZR75, MCF7, T47D) using FACS. Cells were analyzed for spheroid formation, morphological changes, immunofluorescence for differentiation markers, protein (Western) and RNA (RT-qPCR) analysis. Expression of differentiation markers K19 and K14 and stem cell markers CD133, CD44, Id1, Nestin, Musashi-1, SOX2, Notch2, and OCT1 was determined. Effect of inhibitors on the cell viability of breast cancer cells was determined using cell titer glow assays. Xenograft studies were used to determine the in vivo efficacy of the inhibitors. RESULTS: Using yeast based genetic screen, we identified a small peptide inhibitor (PIP1) that interferes PELP1 interaction with G9/EHMT2. Utilizing Hit-Ligand interaction site with the PELP1 hot spot residues based on 3D alignment and shape, we have identified 61 potential hits from Ligand-Based screening using a 10,000 Diverse Set. Screening of these 61 potential hits using MTT based cell viability assays identified three small organic molecule inhibitors (peptidomimetics) as leads. All three peptidomimetics showed activity similar to PELP1 peptide inhibitor 1 (PIP1) in MTT assays and in biochemical assays disrupted PELP1 interaction with G9a/EHMT2. Peptidomimetic treatment inhibited the proliferation of tamoxifen and letrozole resistant cells. In mechanistic studies, we found that knockdown of PELP1 inhibited stem cell maintenance. In FACS analysis of ZR75, ZR75-PELP1 and ZR75-PELP1KD cells, the percentage of CD44high/CD24low cells correlated with PELP1 status. In mammosphere assays, PELP1 targeting peptidomimetics significantly inhibited the formation of mammospheres. Further, in self-renewal assays, peptidomimetic-treated cells had decreased self-renewal capacity. In xenograft assays using ZR-75 cells, PELP1 inhibitor significantly reduced the tumor growth. CONCLUSIONS: Collectively, our studies have discovered an essential role for PELP1 in breast cancer stem cell maintenance and identified the PELP1-G9a/EHMT2 axis as a potential therapeutic target for reducing stemness. Further, the novel small molecule inhibitors of PELP1 could be used for therapeutic targeting of breast cancer stem cells and therapy resistance. Citation Format: Suryavathi Viswanadhapalli, Monica Mann, Gangadhara Reddy Sareddy, Xaionan Lix, Hariprasad Vankayalapati, Ratna K. Vadlamudi. The role of proto-oncogene PELP1 in breast cancer stem cell maintenance and therapy resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1123.

Contribution of Estrone Sulfate to Cell Proliferation in Aromatase Inhibitor (AI) -Resistant, Hormone Receptor-Positive Breast Cancer.

Aromatase inhibitors (AIs) effectively treat hormone receptor-positive postmenopausal breast cancer, but some patients do not respond to treatment or experience recurrence. Mechanisms of AI resistance include ligand-independent activation of the estrogen receptor (ER) and signaling via other growth factor receptors; however, these do not account for all forms of resistance. Here we present an alternative mechanism of AI resistance. We ectopically expressed aromatase in MCF-7 cells expressing green fluorescent protein as an index of ER activity. Aromatase-overexpressing MCF-7 cells were cultured in estrogen-depleted medium supplemented with testosterone and the AI, letrozole, to establish letrozole-resistant (LR) cell lines. Compared with parental cells, LR cells had higher mRNA levels of steroid sulfatase (STS), which converts estrone sulfate (E1S) to estrone, and the organic anion transporter peptides (OATPs), which mediate the uptake of E1S into cells. LR cells proliferated more in E1S-supplemented medium than did parental cells, and LR proliferation was effectively inhibited by an STS inhibitor in combination with letrozole and by ER-targeting drugs. Analysis of ER-positive primary breast cancer tissues showed a significant correlation between the increases in the mRNA levels of STS and the OATPs in the LR cell lines, which supports the validity of this AI-resistant model. This is the first study to demonstrate the contribution of STS and OATPs in E1S metabolism to the proliferation of AI-resistant breast cancer cells. We suggest that E1S metabolism represents a new target in AI-resistant breast cancer treatment.

Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer

IntroductionAlthough aromatase inhibitors (AIs; for example, letrozole) are highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Previous studies suggest that acquired resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as human epidermal growth factor receptor-2 (HER2). However, the role of HER2, and the identity of other relevant factors that may be used as biomarkers or therapeutic targets remain unknown. This study investigated the potential role of transcription factor hypoxia inducible factor 1 (HIF-1) in acquired AI resistance, and its regulation by HER2.MethodsIn vitro studies using AI (letrozole or exemestane)-resistant and AI-sensitive cells were conducted to investigate the regulation and role of HIF-1 in AI resistance. Western blot and RT-PCR analyses were conducted to compare protein and mRNA expression, respectively, of ERα, HER2, and HIF-1α (inducible HIF-1 subunit) in AI-resistant versus AI-sensitive cells. Similar expression analyses were also done, along with chromatin immunoprecipitation (ChIP), to identify previously known HIF-1 target genes, such as breast cancer resistance protein (BCRP), that may also play a role in AI resistance. Letrozole-resistant cells were treated with inhibitors to HER2, kinase pathways, and ERα to elucidate the regulation of HIF-1 and BCRP. Lastly, cells were treated with inhibitors or inducers of HIF-1α to determine its importance.ResultsBasal HIF-1α protein and BCRP mRNA and protein are higher in AI-resistant and HER2-transfected cells than in AI-sensitive, HER2- parental cells under nonhypoxic conditions. HIF-1α expression in AI-resistant cells is likely regulated by HER2 activated-phosphatidylinositide-3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, as its expression was inhibited by HER2 inhibitors and kinase pathway inhibitors. Inhibition or upregulation of HIF-1α affects breast cancer cell expression of BCRP; AI responsiveness; and expression of cancer stem cell characteristics, partially through BCRP.ConclusionsOne of the mechanisms of AI resistance may be through regulation of nonhypoxic HIF-1 target genes, such as BCRP, implicated in chemoresistance. Thus, HIF-1 should be explored further for its potential as a biomarker of and therapeutic target.

Abstract P5-09-05: Progranulin (GP88) expression and letrozole resistance in breast cancer

Abstract The 88 kDa glycoprotein GP88 (Progranulin, PCDGF, acrogranin) is the largest member of the granulin/epithelin family of growth modulators. GP88 was originally characterized in our laboratory through a biological screen to identify drivers of tumorigenesis. Published studies have established that GP88 represents an ideal therapeutic and diagnostic target in breast cancer (BC) leading to the development of validated tools to measure GP88 in tumor biopsies and biological fluids as well as blocking its action. It was shown that: 1) GP88 expression increases with tumorigenesis; 2) in ER+ breast cancer cells, GP88 stimulates proliferation and its overexpression confers estrogen independence and resistance to several anti-estrogens and aromatase inhibitor; 3) inhibition of GP88 expression by antisense transfection inhibited proliferation in vitro and in vivo; 4) In Her-2 overexpressing breast tumors, GP88 stimulated Her-2 phosphorylation and conferred trastuzumab resistance; 5) GP88 is expressed in 80% invasive ductal carcinoma (IDC) and 60% of ductal carcinoma whereas it is negative in lobular carcinoma, benign lesions and normal mammary tissues; 6) GP88 is secreted and can be detected in the serum of BC patients at an increased level when compared to healthy subjects; 7) Pathological studies with 530 cases of ER+ IDC with clinical outcomes showed that GP88 tumor expression was an independent prognostic indicator of recurrence in early stage BC patients. Training study followed by an independent validation study demonstrated that high GP88 tissue expression (GP88 3+) was associated with a 4-fold increase in risk of recurrence at 5 years. Since GP88 displays not only diagnostic but also therapeutic potentials, we developed a neutralizing anti-GP88 antibody AG1 that inhibited GP88 biological effect (proliferation and migration) in a dose-dependent fashion in vitro. AG1 was expressed in a high yield CHO cell line and formulated. We have shown that in tamoxifen resistant cells, treatment with AG1 would inhibit tumor growth and restore tamoxifen sensitivity. The present study examined the effect of AG1 in letrozole resistant cells. We have developed from a letrozole sensitive cell ER+ BC cell line, a letrozole-resistant cell line by long term selection in letrozole-supplemented medium. This cell line (LetR) shows also decreased letrozole responsiveness in vivo and therefore constitutes an excellent model for investigating letrozole resistance in vivo as well as in vivo. Here we investigated the effect of various doses of AG1 on LetR tumor development alone or in combination with letrozole. Treatment with AG1 (10 mg/kg i.p.) in combination with letrozole was efficient to maintain long term responsiveness to letrozole and inhibited tumor growth. In addition to the mouse xenografts study, an IRB approved clinical study examines changes in GP88 circulating levels in patients with resistance to aromatase inhibitors. Preliminary data will be presented. In conclusion, inhibiting GP88 could provide a novel and alternative therapeutic strategy for patients with resistance to anti-estrogen therapy, being tamoxifen or letrozole. This work is supported by 2R44CA124179 and HHSN 261201200060C from NCI and 02- 2013-018 from the Avon Foundation for Women. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-05.

Proteomic Signatures of Acquired Letrozole Resistance in Breast Cancer: Suppressed Estrogen Signaling and Increased Cell Motility and Invasiveness

Aromatase inhibitors, such as letrozole, have become the first-line treatment for postmenopausal women with estrogen-dependent breast cancer. However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated constitutive activation of the MAPK signaling, overexpression of HER2, and down-regulation of aromatase and ERα in letrozole-resistant breast cancer cells. Given the complex signaling network involved in letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of aromatase inhibitor resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. This study was undertaken to characterize for the first time global proteomic alterations occurring in a letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca (resistant) versus AC-1 cells (sensitive) was performed to identify significant protein expression changes. A total of 1743 proteins were identified and quantified, of which 411 were significantly up-regulated and 452 significantly down-regulated (p < 0.05, fold change > 1.20). Bioinformatics analysis revealed that acquired letrozole resistance is associated with a hormone-independent, more aggressive phenotype. LTLT-Ca cells exhibited 84% and 138% increase in migration and invasion compared with the control cells. The ROCK inhibitor partially abrogated the enhanced migration and invasion of the letrozole-resistant cells. Flow cytometric analyses also demonstrated an increase in vimentin and twist expression in letrozole-resistance cells, suggesting an onset of epithelial to mesenchymal transition (EMT). Moreover, targeted gene expression arrays confirmed a 28-fold and sixfold up-regulation of EGFR and HER2, respectively, whereas ERα and pS2 were dramatically reduced by 28-fold and 1100-fold, respectively. Taken together, our study revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence, enhanced cell motility, EMT and the potential values of several altered proteins as novel prognostic markers or therapeutic targets for letrozole resistant breast cancer.

Abstract P1-05-10: Targeting breast cancer metastasis through disruption of novel PELP1-G9a complex

Abstract Although there are several therapies available for the treatment of breast cancer, many patients have tumor recurrences that ultimately metastasize. There are several oncogenic estrogen receptor alpha (ERα) coregulators that promote breast cancer metastasis and targeting these coregulators could be a promising cancer therapeutic. One coactivator of ERα that is known to be upregulated in breast cancer and promote metastasis is proline-, glutamic acid-, and leucine-rich protein 1 (PELP1). PELP1 affects the migratory and invasive potential of breast cancer cells both in vitro and in vivo and modulates the expression of several genes involved in the epithelial to mesenchymal transition (EMT) including Snail, Twist and Zeb1. To elucidate the mechanism by which PELP1 promotes metastasis, we studied the proteins that interact with PELP1 and promote cancer metastasis. We identified a novel PELP1-interacting protein G9a (Bat8/KMT1C/EHMT2), a histone lysine methyltransferase known to overexpressed in aggressive breast cancer and promote invasion and metastasis. G9a contributes to the epigenetic silencing of tumor suppressor genes, interacts with Snail to promote EMT and its knockdown inhibits cell migration and invasion as well as lung metastasis. The objective of this study is to characterize the interaction of PELP1 and G9a in the promotion of breast cancer metastasis. The complex formation of PELP1 and G9a was confirmed by an in vivo immunoprecipitation and an in vitro binding assay. The region of PELP1 that binds to G9a was mapped further by PELP-GST deletions. To determine whether formation of the complex is necessary for PELP1's oncogenic functions including the promotion of metastasis, we developed a peptide to disrupt the complex formation. The sequence of the peptide has homology to both PELP1 and G9a with a TAT-sequence to enable cellular uptake. Cellular uptake was confirmed by fluorescent microscopy while biotin-avidin peptide pull-down assays confirmed PELP1-peptide binding. A peptide competition assay confirmed that the peptide could interrupt PELP1-G9a interaction. Treatment of two ER-positive, PELP1-overexpressing breast cancer cells (MCF7-PELP and ZR75-PELP) with the peptide significantly inhibited the PELP-1 mediated proliferation. Peptide treatment also substantially inhibited PELP1-mediated cell migration in Boyden chamber assays as well as decreased anchorage independence as seen by soft agar assay. Interestingly, disrupting PELP1-G9a interaction with the peptide also sensitized tamoxifen and letrozole resistant cells to the respective hormonal therapy. Mechanistic studies revealed that PELP1-G9a interactions play a critical role in epigenetic changes in therapy resistant cells. In conclusion, our studies identified a novel complex involved in breast cancer metastasis. The interaction of PELP1 and G9a is required for PELP1-mediated oncogenic properties and disruption of the complex by peptide treatment results in decreased metastatic potential and changes in epigenetic modifications. The novel peptide could be used for therapeutic targeting of breast cancer metastasis and therapy resistance. This work was supported by the NIH grant CA095681 and CPRIT pre-doctoral fellowship RP101491. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-10.

Efficacy and mechanism of action of Proellex, an antiprogestin in aromatase overexpressing and Letrozole resistant T47D breast cancer cells

Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the response of antiprogestin CDB-4124 (Proellex) on the aromatase overexpressing and Letrozole resistant cell lines and also studies its mechanism of action in inhibition of breast cancer cell proliferation. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10μM Letrozole. Cell proliferation was determined by MTT or crystal violet assays. Gene expressions were quantified by QRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's ‘t’ test or ANOVA followed by Bonferroni's post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80–90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50–60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex alone or in combination with other AIs as compared to AIs alone. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PR-B/EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients.

The importance of HER2 signaling in the tumor-initiating cell population in aromatase inhibitor-resistant breast cancer

Aromatase inhibitors (AIs) are an effective therapy in treating estrogen receptor-positive breast cancer. Nonetheless, a significant percentage of patients either do not respond or become resistant to AIs. Decreased dependence on ER-signaling and increased dependence on growth factor receptor signaling pathways, particularly human epidermal growth factor receptor 2 (EGFR2/HER2), have been implicated in AI resistance. However, the role of growth factor signaling remains unclear. This current study investigates the possibility that signaling either through HER2 alone or through interplay between epidermal growth factor receptor 1 (EGFR/HER1) and HER2 mediates AI resistance by increasing the tumor initiating cell (TIC) subpopulation in AI-resistant cells via regulation of stem cell markers, such as breast cancer resistance protein (BCRP). TICs and BCRP are both known to be involved in drug resistance. Results from in vitro analyses of AI-resistant versus AI-sensitive cells and HER2-versus HER2+ cells, as well as from in vivo xenograft tumors, indicate that (1) AI-resistant cells overexpress both HER2 and BCRP and exhibit increased TIC characteristics compared to AI-sensitive cells; (2) inhibition of HER2 and/or BCRP decrease TIC characteristics in letrozole-resistant cells; and (3) HER2 and its dimerization partner EGFR/HER1 are involved in the regulation of BCRP. Overall, these results suggest that reducing or eliminating the TIC subpopulation with agents that target BCRP, HER2, EGFR/HER1, and/or their downstream kinase pathways could be effective in preventing and/or treating acquired AI resistance.

Abstract LB-415: Global proteomic signature of letrozole-resistance in breast cancer cells

Abstract Postmenopausal women with early-stage metastatic estrogen-dependent breast cancer are generally treated with aromatase inhibitors (AIs) (e.g., anastrozole and letrozole). However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated letrozole-resistant breast cancer cells undergo adaptive changes resulting in constitutive activation of the MAPKinase signaling pathway and increased expression of HER2 and IGFR with decreased expression of aromatase and ERα. Given the complex signaling network involving letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of AI resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. The objective of this study was to characterize for the fist time global proteomic alterations occurring in the LTLT-Ca letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca cells vs. AC1 cells (letrozole-sensitive) was performed to identify significant protein expression changes. Protein extracts were digested with trypsin and the resulting peptides were labeled with tandem mass tags. Fractionation and nanoflow HPLC-MS/MS was used for peptide separation, identification and quantification. Proteomic data were analyzed by bioinformatics tools to gain insight into global adaptive signaling modulations as a result of acquired resistance to letrozole. A total of 1743 proteins were identified and quantified that were present in both cell lines, of which 411 were significantly upregulated and 452 were significantly downregulated (p&amp;lt;0.05, fold change &amp;gt; 1.20). Bioinformatics analysis of these altered proteins revealed that acquired letrozole resistance is associated with a hormone-independent, more motile phenotype. In particular, overexpression of multiple proteins was implicated in the enhanced migration and invasion in the resistant cell line, including ROCK, RAC1, S100 family proteins and the actin bundling protein, fascin. Subsequent migration and invasion assays demonstrated more than a 25% increase in the motility of the LTLT-Ca cells. Treatment with pharmacologic inhibitors of fascin, ROCK, and RAC1 partially abrogated both invasion and migration of the letrozole-resistant cells. Moreover, targeted gene expression arrays confirmed a 28-fold and 6-fold increase in the expression of EGFR and HER2, respectively, while ERα and pS2 were dramatically suppressed by 28-fold and 1100-fold, respectively. Taken together, our study for the first time revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence and enhanced cell motility and the potential values of several altered proteins as novel therapeutic targets for treatment of acquired letrozole resistance in postmenopausal breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-415. doi:1538-7445.AM2012-LB-415

P2-17-05: Antiprogestin Proellex Suppresses Proliferation of Aromatase Overexpressing and Letrozole Resistant T47D Breast Cancer Cells.

Abstract Background: Aromatase is a cytochrome P450 CYP19A1 enzyme responsible for the conversion of C19 androgens to C18 estrogen. Aromatase overexpression leads to increase in local estrogen concentration in post menopausal women diagnosed for breast cancer. Aromatase inhibitors (AI) are used as one of the first line therapies for the treatment of ER+PR+ breast cancers. However, many patients acquire resistance to AI treatment. Therefore alternative approaches are being sought for patients with AI resistance. Previous reports suggest that upregulation of progesterone receptor can lead to enhanced expression of EGFR/ERK/MAPK involved in acquiring resistance. In this study, we evaluated the effect of a selective progesterone receptor modulator, CDB4124 (Proellex) with low glucocorticoid activity on aromatase overexpressing and Letrozole resistant T47D cells. Methods: Aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines were generated by stable transfection with plasmid containing CYP19A1 coding region, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10μM Letrozole. Cell proliferation was determined by MTT or Crystal violet assays. Gene expressions were quantitated by qRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's ‘T’ test or ANOVA followed by Bonferroni's post hoc test. Results: T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA and protein) and as a result exhibited increased conversion of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone. In T47Darom cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex, Letrozole and other known AI (Anastrozole, Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. The inhibition of cell proliferation was significantly greater when cells were treated with Proellex in combination to other AIs. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex may be due to PR-B/EGFR modulation in ER+PR+, aromatase overexpressing cells. Conclusion: Overall these results suggest that antiprogestin, Proellex could be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for AI resistant breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-17-05.

GP88 (PC-Cell Derived Growth Factor, progranulin) stimulates proliferation and confers letrozole resistance to aromatase overexpressing breast cancer cells

BackgroundAromatase inhibitors (AI) that inhibit breast cancer cell growth by blocking estrogen synthesis have become the treatment of choice for post-menopausal women with estrogen receptor positive (ER+) breast cancer. However, some patients display de novo or acquired resistance to AI. Interactions between estrogen and growth factor signaling pathways have been identified in estrogen-responsive cells as one possible reason for acquisition of resistance. Our laboratory has characterized an autocrine growth factor overexpressed in invasive ductal carcinoma named PC-Cell Derived Growth Factor (GP88), also known as progranulin. In the present study, we investigated the role GP88 on the acquisition of resistance to letrozole in ER+ breast cancer cellsMethodsWe used two aromatase overexpressing human breast cancer cell lines MCF-7-CA cells and AC1 cells and their letrozole resistant counterparts as study models. Effect of stimulating or inhibiting GP88 expression on proliferation, anchorage-independent growth, survival and letrozole responsiveness was examined.ResultsGP88 induced cell proliferation and conferred letrozole resistance in a time- and dose-dependent fashion. Conversely, naturally letrozole resistant breast cancer cells displayed a 10-fold increase in GP88 expression when compared to letrozole sensitive cells. GP88 overexpression, or exogenous addition blocked the inhibitory effect of letrozole on proliferation, and stimulated survival and soft agar colony formation. In letrozole resistant cells, silencing GP88 by siRNA inhibited cell proliferation and restored their sensitivity to letrozole.ConclusionOur findings provide information on the role of an alternate growth and survival factor on the acquisition of aromatase inhibitor resistance in ER+ breast cancer.