Endocrine-resistant Cells Research Articles

Non-canonical AR activity facilitates endocrine resistance in breast cancer.

The role of androgen receptor (AR) in endocrine-resistant breast cancer is controversial and clinical trials targeting AR with an AR antagonist (e.g., enzalutamide) have been initiated. Here, we investigated the consequence of AR antagonism using in vitro and in vivo models of endocrine resistance. AR antagonism in MCF7-derived tamoxifen-resistant (TamR) and long-term estrogen-deprived breast cancer cell lines were achieved using siRNA-mediated knockdown or pharmacological inhibition with enzalutamide. The efficacy of enzalutamide was further assessed in vivo in an estrogen-independent endocrine-resistant patient-derived xenograft (PDX) model. Knockdown of AR inhibited the growth of the endocrine-resistant cell line models. Microarray gene expression profiling of the TamR cells following AR knockdown revealed perturbations in proliferative signaling pathways upregulated in endocrine resistance. AR loss also increased some canonical ER signaling events and restored sensitivity of TamR cells to tamoxifen. In contrast, enzalutamide did not recapitulate the effect of AR knockdown in vitro, even though it inhibited canonical AR signaling, which suggests that it is the non-canonical AR activity that facilitated endocrine resistance. Enzalutamide had demonstrable efficacy in inhibiting AR activity in vivo but did not affect the growth of the endocrine-resistant PDX model. Our findings implicate non-canonical AR activity in facilitating an endocrine-resistant phenotype in breast cancer. Unlike canonical AR signaling which is inhibited by enzalutamide, non-canonical AR activity is not effectively antagonized by enzalutamide, and this has important implications in the design of future AR-targeted clinical trials in endocrine-resistant breast cancer.

Epigenome-wide SRC-1–Mediated Gene Silencing Represses Cellular Differentiation in Advanced Breast Cancer

Purpose: Despite the clinical utility of endocrine therapies for estrogen receptor-positive (ER) breast cancer, up to 40% of patients eventually develop resistance, leading to disease progression. The molecular determinants that drive this adaptation to treatment remain poorly understood. Methylome aberrations drive cancer growth yet the functional role and mechanism of these epimutations in drug resistance are poorly elucidated.Experimental Design: Genome-wide multi-omics sequencing approach identified a differentially methylated hub of prodifferentiation genes in endocrine resistant breast cancer patients and cell models. Clinical relevance of the functionally validated methyl-targets was assessed in a cohort of endocrine-treated human breast cancers and patient-derived ex vivo metastatic tumors.Results: Enhanced global hypermethylation was observed in endocrine treatment resistant cells and patient metastasis relative to sensitive parent cells and matched primary breast tumor, respectively. Using paired methylation and transcriptional profiles, we found that SRC-1-dependent alterations in endocrine resistance lead to aberrant hypermethylation that resulted in reduced expression of a set of differentiation genes. Analysis of ER-positive endocrine-treated human breast tumors (n = 669) demonstrated that low expression of this prodifferentiation gene set significantly associated with poor clinical outcome (P = 0.00009). We demonstrate that the reactivation of these genes in vitro and ex vivo reverses the aggressive phenotype.Conclusions: Our work demonstrates that SRC-1-dependent epigenetic remodeling is a 'high level' regulator of the poorly differentiated state in ER-positive breast cancer. Collectively these data revealed an epigenetic reprograming pathway, whereby concerted differential DNA methylation is potentiated by SRC-1 in the endocrine resistant setting. Clin Cancer Res; 24(15); 3692-703. ©2018 AACR.

Abstract 3744: Multiple endocrine-resistant breast cancer cell lines retain ER and sensitivity to endocrine therapy

Abstract Endocrine therapy is the standard of care for breast cancer expressing estrogen receptor (ER), which occurs in 70% of patients. Unfortunately, acquired or de novo resistance to endocrine therapy is observed in up to 50% of patients, leaving a significant portion of patients with insufficient treatment options. Endocrine-resistance, usually defined as resistance to tamoxifen and aromatase inhibitor (AI) therapy, can also include resistance to selective estrogen receptor degraders (SERDs), since these also target ER. Since multiple mechanisms contribute to resistance, development of multiple resistant cell lines is needed for drug discovery and to identify characteristics that may suggest susceptibility to alternative and combination therapies. We have developed 5 stable, endocrine-resistant cell lines from a parent MCF-7 cell line, which all retain ER: one of these cell lines, MCF-7:CFR is resistant to the SERD fulvestrant. Clinical metastatic breast cancers that have gained endocrine resistance are overwhelmingly ER+. In addition to ER, progesterone receptor (PR) and glucocorticoid receptor (GR) status of these lines was assessed, and correlated with the response of these cells in culture to four classes of endocrine therapeutics: SERDs, selective ER modulators (SERMs), and selective human ER partial agonists (ShERPAs). Growth of all 5 cell lines was endocrine independent, indicating resistance to AI therapy. Two of the ER+, PR- cell lines were most resistant to the spectrum of endocrine therapies, but these cell lines both showed sensitivity to ShERPAs, especially in combination with non-endocrine targeted therapies, such as the PI3K inhibitor, alpelisib. Paradoxically, all endocrine-resistant cell lines responded to at least one of the endocrine therapies tested, demonstrating that if ER is not lost in the metastatic state, it remains a vulnerability suitable for therapeutic targeting. Citation Format: Lauren M. Gutgesell, Rui Xiong, Jiong Zhao, Huiping Zhao, Debra A. Tonetti, Gregory R. Thatcher. Multiple endocrine-resistant breast cancer cell lines retain ER and sensitivity to endocrine therapy [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 3744.

Abstract 5839: Growth hormone receptor (GHR) expression confers resistance to ruxolitinib in endocrine-resistant breast cancer cells

Abstract More than 30% of patients with early-stage estrogen receptor-positive breast cancer (ER+) treated with adjuvant endocrine therapy will relapse within fifteen years and all patients with metastatic breast cancer expressing ER eventually acquire resistance to antiestrogen therapy. The insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) are involved in the development of resistance to endocrine therapies such as tamoxifen. The IGF-1/growth hormone (GH) signaling axis has been implicated as a mitogenic pathway in the development and progression of breast carcinogenesis. Preclinical data demonstrate that blockade of the IGF-1R inhibits breast cancer growth, progression, and drug resistance. Unfortunately, IGF-1R targeted therapies have failed to show a benefit in prolonging either disease-free or overall survival in clinical trials. IGF-1R inhibition results in upregulation of GH due to disruption of a negative feedback pathway. We propose GH is able to stimulate mitogenic pathways independently of IGF-IR and the GHR is a potential therapeutic target in endocrine-resistant breast cancers. Tamoxifen-resistant (TamR) and long-term estrogen deprived (LTED) cells were derived from parental MCF-7L and T47D estrogen receptor-positive breast cancer cells. The endocrine-resistant cell lines express increased levels of growth hormone receptor (GHR) mRNA compared to the parental cell lines from which they were derived when measured by RT-PCR. TamR and LTED cell lines have reduced IGF-1R expression, but continue to phosphorylate insulin receptor substrate 1(IRS-1) downstream of IGF-1R. In these endocrine-resistant cells, GH treatment activates protein kinase B (Akt), phosphoinositide 3-kinase (PI3K), and mitogen activated protein kinase (MAPK) pathways in the absence of IGF-1. To determine if GHR signals through janus kinase-2 (JAK-2) in these cells, we treated MCF-7L and T47D TamR cells with 500 nM GH, which resulted in JAK-2 phosphorylation. To examine growth effects, we treated cells with the JAK-2 inhibitor ruxolitinib. Both MCF-7L and T47D endocrine-resistant cell lines treated with ruxolitinib have increased IC50 values compared to their parental counterparts. The MCF-7L parental cells had an IC50 value of 6.7 µM compared to 20 µM and 21.7 µM in TamR and LTED cells, respectively. The T47D parental cells had an IC50 value of 28.6 µM whereas the IC50 of T47D TamR cells was 45 µM. These data indicate endocrine-resistant breast cancer cell lines with elevated GHR expression have decreased sensitivity to JAK-2 inhibition. Based on these results, GHR and its downstream signaling should be considered as a target in the development of novel therapeutics to treat breast cancers that are resistant to traditional endocrine therapies. Citation Format: Anja N. Holtz, Douglas Yee, Heather Beckwith. Growth hormone receptor (GHR) expression confers resistance to ruxolitinib in endocrine-resistant breast cancer cells [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 5839.

Abstract 1926: Npy1r as a prognostic marker and a novel drug target in estrogen receptor-positive breast cancer

Abstract Despite the crucial role of some G protein-coupled receptors (GPCRs) in cancer progression and metastases reported in recent studies, the function of majority of GPCRs, as a class, remains largely unexplored in breast cancer. In this study, we evaluated transcriptome, proteome and phosphoproteome data for 398 non-sensory GPCRs (IUPHAR v1.0) using the landmark TCGA breast cancer proteogenomics dataset and Illumina HiSeq dataset. Neuropeptide Y Receptor Y1 (NPY1R) was the top most hypo-phosphorylated (at S368) GPCR in 92 out of 99 evaluable samples. Furthermore, NPY1R gene expression was found to be significantly lower in tumor samples than tumor-matched normal (n=112). Among tumor samples, the LumA subtype had significantly higher NPY1R expression than LumB, Basal and Her2 subtype (p<0.05, One-way ANOVA, Tukey test). Interestingly, the trend of NPY1R gene expression, protein expression and phosphosite expression was decreasing in the order of LumA, LumB, Basal and Her2 subtype (p<0.01, Jonckheere trend test). Using publicly available dataset of 22,277 genes on survival in 2,422 patients, we found that higher NPY1R expression predicted better overall survival and recurrence-free survival in estrogen receptor-positive (ER+) breast cancer patients. In light of this, we interrogated NPY1R gene expression in endocrine sensitive and resistant cell line models and the impact of NPY1R antagonist in ER+ breast cancer cell lines. NPY1R expression was increased in response to estradiol in various ER+ breast cancer cell line models (MCF7, T47D, and BT474) in publicly available datasets. Conversely, we found that tamoxifen or estrogen deprivation (ED) treatment reduced NPY1R expression in MCF7 and T47D cells. In our endocrine-resistant (tamoxifen-resistant, estrogen deprivation-resistant, and fulvestrant-resistant) cell line models, NPY1R expression remained significantly lower compared to the parental cells in vitro and in vivo (p<0.05, One-way ANOVA, Tukey test). Treatment of MCF7 parental cells with 100 nM NPY significantly reduced estrogen-stimulated cell growth (p<0.05, One-way ANOVA, Tukey test). Chronic treatment of cells with 1 μM BIBP3226, an NPY1R antagonist, for 2 weeks reversed the effect of NPY on the estradiol-stimulated cell growth. Estradiol-stimulated cell growth was modest (~40%) in ED-resistant MCF7 cells compared to that (~250%) in parental MCF7 cells. While 100 nM NPY treatment did not affect the estrogen-stimulated growth in ED-resistant MCF7 cells, which can be explained by the undetectable expression of NPY1R, it caused significant inhibition of the estrogen-stimulated growth in the same cells treated chronically with BIBP3226. Molecular mechanisms to explain these effects of chronic NPY1R antagonism are underway. Our ongoing studies will further elucidate the role of NPY1R as a novel drug target in ER+ breast cancer. Citation Format: Raksha Bhat, Suhas Vasaikar, Leon Bae, De Angelis Carmine, Maria Letizia Cataldo, Sarmistha Nanda, Bing Zhang, Rachel Schiff, Meghana V. Trivedi. Npy1r as a prognostic marker and a novel drug target in estrogen receptor-positive 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 1926.

PO-507 ADAM22 as a therapeutic target for endocrine resistant metastatic breast cancer

IntroductionThe development of endocrine resistance is a major hurdle for the treatment of oestrogen receptor positive (ER+) breast cancer. One of the key contributors to this resistant phenotype is overexpression of the nuclear receptor coactivator SRC-1, which in turn has been shown to promote metastatic disease. Using CHIP-seq and microarray studies, the neuronal protein ADAM22 (A Disintegrin And Metalloproteinase) was identified as a potential pro-metastatic SRC-1 regulated gene in endocrine resistant cell lines. ADAM22 expression promotes both migration and de-differentiation, key hallmarks of metastasis. While clinically, elevated ADAM22 expression predicts poor disease free survival. Here, both the role of ADAM22 in metastatic development and the clinical potential of ADAM22 as a biomarker and therapeutic target were explored.Material and methodsThe functional effect of ADAM22 modulation on anchorage independent growth and mammosphere formation was investigated using CRISPR/Cas9 knockout and lentiviral overexpression in the endocrine resistant LY2 cell line. To examine the clinical relevance of ADAM22 as a biomarker, ADAM22 expression was examined in matched primary breast and metastatic cancer tissues. A small peptide mimetic of the ADAM22 endogenous ligand LGI1 (LGIMIM) was designed and explored as a potential anti-ADAM22 therapeutic both in vitro and in vivo.Results and discussionsADAM22 expression promotes anchorage independent growth and mammosphere formation, suggesting ADAM22 contributes to both the survival of metastatic cells and their ability to colonise distant sites. ADAM22 expression was significantly higher in primary breast tumours which went on to metastasise versus primaries which did not. A significant increase in ADAM22 expression was found in brain metastatic tissue compared to matched primary tissue, suggesting ADAM22 may prime endocrine resistant cancer cells to colonise the brain. LGIMIM treatment was sufficient to inhibit ADAM22 mediated phenotypes in vitro and to reduce metastatic burden in a xenograft model of endocrine resistance in vivo.ConclusionADAM22 is a promising biomarker and therapeutic target for endocrine resistant breast cancer.

Abstract 949: Progesterone receptor/IRS-1 cooperation promotes stem cell outgrowth and endocrine resistance in estrogen receptor-positive luminal breast cancer

Abstract Luminal breast cancers account for ~75% of all cases and while adjuvant hormone therapy targeting ERα has significantly improved overall survival for patients with ER+ tumors, acquired resistance remains a major clinical problem. Tamoxifen-resistant (TamR) breast cancer models show loss of IGF1R concomitant with increased insulin-induced growth, underpinned by insulin receptor (InsR) compensation for IGF1R loss. PR (gene name PGR) is an estrogen-regulated target gene whose expression is used as a clinical marker of ER activity. However, its de novo relevance to breast cancer is unclear. We have shown previously that post-translational modifications create unique PR species whose altered behavior drives an endocrine-resistant gene signature, in part by crosstalk with the IGFR pathway. We propose that phospho-PR target gene selectivity is mediated by cooperation between PR-B and InsR/IGF1R pathway components. Herein we show that phospho-PR-expressing T47D cells lose expression of IGF1R compared to control cells expressing WT PR-B. Furthermore, expression of the adapter protein IRS-1 requires PRB expression and these IRS-1+ cells were more sensitive to insulin in anchorage-dependent growth assays. Phospho-PR T47D cells exhibit increased ALDH+ and CD24-/CD44+ tumorsphere formation compared to wt PRB-expressing cells. Inhibitors targeting IRS-1 and InsR were used to test their requirement for tumorsphere growth and, surprisingly, IRS-1 perturbation reduced phospho-PRB but not wt PRB tumorsphere growth. Interestingly, IRS-1 replaces IGF1R in phospho-PRB-containing transcriptional complexes that are recruited to the CTSD promoter, which we previously identified as a phospho-PR target gene. Finally, breast cancer cells expressing phospho-PR species exhibited tamoxifen-resistant growth. Collectively our data suggest that phospho-PRB cooperates with IRS-1 downstream of the Ins/IGF1R system to promote outgrowth of endocrine-resistant cells that include ALDH+ stem-like cells capable of forming secondary tumorspheres in vitro. Targeting phospho-PR species in addition to the signaling components of PR-complexes may provide a means to block emergence of endocrine-resistant cancer cells during breast cancer progression. Citation Format: Amy R. Dwyer, Deepali Sachdev, Carol A. Lange. Progesterone receptor/IRS-1 cooperation promotes stem cell outgrowth and endocrine resistance in estrogen receptor-positive luminal 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 949.

Abstract 1435: Investigation of phosphoserine aminotransferase 1: Its role in breast cancer progression

Abstract Metastasis and endocrine resistance are two factors that complicate therapeutic intervention in breast cancer patients and lead to poorer overall survival. Metastasis is known to be responsible for 90% of cancer related deaths, and is especially prevalent in triple negative breast cancer (TNBC); while endocrine resistance can affect up to 50% of patients diagnosed with estrogen receptor positive breast cancer (ER+BC). Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second step within de novo serine biosynthesis and increased expression of enzymes in this pathway have been linked to progression of breast cancer and poor clinical outcomes. Within our preliminary retrospective analysis of human breast cancer patients, we identified an inverse association with elevated transcript levels and poorer distant metastasis free survival, which, coupled to a previously reported correlation of PSAT1 with response to endocrine therapy in patients with ER+BC, we postulate that PSAT1 contributes to breast cancer progression through promotion of metastasis and/or endocrine resistance. To initially determine relevance for PSAT1, immunohistochemistry was performed to determine PSAT1 expression in human breast cancer patients. We found that PSAT1 expression is increased through breast cancer progression, with highest levels observed within metastatic conditions. To investigate the metastatic contribution of PSAT1, we silenced PSAT1 expression within the triple negative breast cancer cell line (TNBC), MDA-MB-231. While suppression of PSAT1 had no effect on proliferation, there was a significant decrease in the motility and invasion of these cells. In addition, decreased PSAT1 substantially inhibited tumor nodule formation following tail-vein injections of MDA-MB-231 cells in vivo. To investigate PSAT1's role in endocrine resistance, we used parental MCF-7 cells and an endocrine-resistant derivative cell lines and found that resistant cells exhibited higher PSAT1 expression compared to parental MCF-7 cells. Lastly, suppression of PSAT1 trended to sensitize the resistant cells to 4-hydroxytamoxifen treatment. Taken together, these data indicate that PSAT1 may contribute to the progression of human breast cancer via either metastasis or endocrine resistance or both and could potentially serve as a viable target for new therapies. Citation Format: Stephanie Metcalf, Traci Kruer, James Wittliff, Carolyn Klinge, Brian F. Clem. Investigation of phosphoserine aminotransferase 1: Its role in breast cancer progression [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 1435.

Abstract SY01-01: Endocrine resistance in metastatic breast cancer: Mechanisms and new therapeutic strategies

Abstract SY01-01: Endocrine resistance in metastatic breast cancer: Mechanisms and new therapeutic strategies

SNAI2 upregulation is associated with an aggressive phenotype in fulvestrant-resistant breast cancer cells and is an indicator of poor response to endocrine therapy in estrogen receptor-positive metastatic breast cancer

BackgroundEndocrine resistance in estrogen receptor-positive (ER+) breast cancer is a major clinical problem and is associated with accelerated cancer cell growth, increased motility and acquisition of mesenchymal characteristics. However, the specific molecules and pathways involved in these altered features remain to be detailed, and may be promising therapeutic targets to overcome endocrine resistance.MethodsIn the present study, we evaluated altered expression of epithelial-mesenchymal transition (EMT) regulators in ER+ breast cancer cell models of tamoxifen or fulvestrant resistance, by gene expression profiling. We investigated the specific role of increased SNAI2 expression in fulvestrant-resistant cells by gene knockdown and treatment with a SNAIL-p53 binding inhibitor, and evaluated the effect on cell growth, migration and expression of EMT markers. Furthermore, we evaluated SNAI2 expression by immunohistochemical analysis in metastatic samples from two cohorts of patients with breast cancer treated with endocrine therapy in the advanced setting.ResultsSNAI2 was found to be significantly upregulated in all endocrine-resistant cells compared to parental cell lines, while no changes were observed in the expression of other EMT-associated transcription factors. SNAI2 knockdown with specific small interfering RNA (siRNA) converted the mesenchymal-like fulvestrant-resistant cells into an epithelial-like phenotype and reduced cell motility. Furthermore, inhibition of SNAI2 with specific siRNA or a SNAIL-p53 binding inhibitor reduced growth of cells resistant to fulvestrant treatment. Clinical evaluation of SNAI2 expression in two independent cohorts of patients with ER+ metastatic breast cancer treated with endocrine therapy in the advanced setting (N = 86 and N = 67) showed that high SNAI2 expression in the metastasis correlated significantly with shorter progression-free survival on endocrine treatment (p = 0.0003 and p = 0.004).ConclusionsOur results suggest that SNAI2 is a key regulator of the aggressive phenotype observed in endocrine-resistant breast cancer cells, an independent prognostic biomarker in ER+ advanced breast cancer treated with endocrine therapy, and may be a promising therapeutic target in combination with endocrine therapies in ER+ metastatic breast cancer exhibiting high SNAI2 levels.

ERβ alters the chemosensitivity of luminal breast cancer cells by regulating p53 function.

Estrogen receptor α (ERα)-positive breast cancers tend to develop resistance to both endocrine therapy and chemotherapy. Despite recent progress in defining molecular pathways that confer endocrine resistance, the mechanisms that regulate chemotherapy response in luminal tumors remain largely elusive. Luminal tumors often express wild-type p53 that is a major determinant of the cellular DNA damage response. Similar to p53, the second ER subtype, ERβ, has been reported to inhibit breast tumorigenesis by acting alone or in collaboration with p53. However, a synergistic mechanism of action has not been described. Here, we suggest that ERβ relies on p53 to elicit its tumor repressive actions in ERα-positive breast cancer cells. Upregulation of ERβ and treatment with ERβ agonists potentiates the tumor suppressor function of p53 resulting in decreased survival. This effect requires molecular interaction between the two proteins that disrupts the inhibitory action of ERα on p53 leading to increased transcriptional activity of p53. In addition, we show that the same interaction alters the chemosensitivity of endocrine-resistant cells including their response to tamoxifen therapy. Our results suggest a collaboration of ERβ and p53 tumor suppressor activity in breast cancer cells that indicates the importance of ligand-regulated ERβ as a tool to target p53 activity and improve the clinical management of resistant disease.

A bi-stable feedback loop between GDNF, EGR1, and ERα contribute to endocrine resistant breast cancer.

Discovering regulatory interactions between genes that specify the behavioral properties of cells remains an important challenge. We used the dynamics of transcriptional changes resolved by PRO-seq to identify a regulatory network responsible for endocrine resistance in breast cancer. We show that GDNF leads to endocrine resistance by switching the active state in a bi-stable feedback loop between GDNF, EGR1, and the master transcription factor ERα. GDNF stimulates MAP kinase, activating the transcription factors SRF and AP-1. SRF initiates an immediate transcriptional response, activating EGR1 and suppressing ERα. Newly translated EGR1 protein activates endogenous GDNF, leading to constitutive GDNF and EGR1 up-regulation, and the sustained down-regulation of ERα. Endocrine resistant MCF-7 cells are constitutively in the GDNF-high/ ERα-low state, suggesting that the state in the bi-stable feedback loop may provide a ‘memory’ of endocrine resistance. Thus, we identified a regulatory network switch that contributes to drug resistance in breast cancer.

ER-positive breast cancer cells are poised for RET-mediated endocrine resistance.

The RET tyrosine kinase signaling pathway is involved in the development of endocrine resistant ER+ breast cancer. However, we know little about how ER+ cells activate RET signaling and initiate an endocrine resistant phenotype. Here we show that both ER+ endocrine resistant and sensitive breast cancers have a functional RET tyrosine kinase signaling pathway, but that endocrine sensitive breast cancer cells lack RET ligands that are necessary to drive endocrine resistance. Transcription of one RET ligand, GDNF, is necessary and sufficient to confer resistance in the ER+ MCF-7 cell line. Endogenous GDNF produced by endocrine resistant cells is translated, secreted into the media, and activates RET signaling in nearby cells. In patients, RET ligand expression predicts responsiveness to endocrine therapies and correlates with survival. Collectively, our findings show that ER+ tumor cells are “poised” for RET mediated endocrine resistance, expressing all components of the RET signaling pathway, but endocrine sensitive cells lack high expression of RET ligands that are necessary to initiate the resistance phenotype.

Investigation Of Phosphoserine Aminotransferase 1 And Its Role In Breast Cancer Progression

Metastasis and endocrine resistance are two factors that complicate therapeutic intervention in breast cancer patients and lead to poorer overall survival. Metastasis is known to be responsible for 90% of cancer related deaths, and is especially prevalent in triple negative breast cancer (TNBC); while endocrine resistance can affect up to 50% of patients diagnosed with estrogen receptor positive breast cancer (ER+BC). Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second step within de novo serine biosynthesis and increased expression of enzymes in this pathway have been linked to progression of breast cancer and poor clinical outcomes. Within our preliminary retrospective analysis of human breast cancer patients, we identified an inverse association with elevated transcript levels and poorer distant metastasis free survival, which, coupled to a previously reported correlation of PSAT1 with response to endocrine therapy in patients with ER+BC, we postulate that PSAT1 contributes to breast cancer progression through promotion of metastasis and/or endocrine resistance. To initially determine relevance for PSAT1, immunohistochemistry was performed to determine PSAT1 expression in human breast cancer patients. We found that PSAT1 expression is increased through breast cancer progression, with highest levels observed within metastatic conditions. To investigate the metastatic contribution of PSAT1, we silenced PSAT1 expression within the triple negative breast cancer cell line (TNBC), MDA‐MB‐231. While suppression of PSAT1 had no effect on proliferation, there was a significant decrease in the motility and invasion of these cells. In addition, decreased PSAT1 substantially inhibited tumor nodule formation following tail‐vein injections of MDA‐MB‐231 cells in vivo. To investigate PSAT1's role in endocrine resistance, we used parental MCF‐7 cells and an endocrine‐resistant derivative cell lines and found that resistant cells exhibited higher PSAT1 expression compared to parental MCF‐7 cells. Lastly, suppression of PSAT1 trended to sensitize the resistant cells to 4‐hydroxytamoxifen treatment. Taken together, these data indicate that PSAT1 may contribute to the progression of human breast cancer via either metastasis or endocrine resistance or both and could potentially serve as a viable target for new therapies.Support or Funding InformationSupported by a Research Scholar Grant, RSG‐13‐139‐01 from the American Cancer SocietyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P4-03-12: Effects of PI3K inhibitors on endocrine-resistant cell lines and differences in the characteristics of ER positive breast cancer cells after acquired resistance to the inhibitors

Abstract Background Mutations in phosphatidylinositol 3-kinase (PI3K), which encodes the catalytic subunit of PI3Kα, is one of the most frequent genomic alterations and is found in about 40% of estrogen receptor (ER)-positive, HER2 negative breast cancers. PIK3CA mutations promote the growth and proliferation of cancers via activation of the PI3K/mammalian target of rapamycin pathway and can mediate resistance to endocrine therapies in breast cancer. Although several clinical trials for PI3K inhibitors (PI3Kis) in ER-positive metastatic breast cancers are ongoing, the effects of PI3Kis on endocrine-resistant breast cancers with various characteristics and definitive biomarkers of PI3Kis are unclear. Using endocrine-resistant cells established in our laboratory, we evaluated the efficacy of PI3Ki in these cell lines and identified the characteristics associated with acquired resistance to PI3Kis in endocrine-resistant cells. Results Long-term estrogen deprivation-resistant (EDR) cell lines and fulvestrant-resistant cell lines (MFR and TFR) were established from MCF-7 and T-47D cells in our previous studies. These cell lines showed different ER expression levels, including high expression (EDR-1), low expression (EDR-2, -3), or no expression (MFR, TFR); all of these cell lines had the same PIK3CA mutations as the parental cell lines. The pan-class1 PI3Ki buparlisib (BKM120) and α-specific PI3K inhibitor alpelisib (BYL719) inhibited the proliferation of endocrine-resistant cell lines when compared with their parental cells. Among endocrine-resistant cells, MFR cells were dramatically inhibited by PI3Kis. Colony formation assays indicated that MFR cells were more sensitive to PI3Kis than other cells lines. Next, we established PI3Ki- and everolimus-resistant cell lines from EDR-1, EDR-2, and MFR cells using BKM120, BYL719, and everolimus. Analysis of the time required to generate resistant cells showed that MFR cells required twice as long to acquire resistance compared with EDR cells. Furthermore, the time required to acquire resistance to BYL719 was shorter than that for BKM120. BYL719-resistant (BYL-R) cells were effectively inhibited by BKM120 to a degree similar to that of parental cells; however, BYL-R cells lost sensitivity to BYL719 and everolimus. Evelolimus-resistant (EVE-R) cells were also the same. In contrast, BKM120-resistant (BKM-R) cells showed less sensitivity to BKM120, BYL719, and everolimus. In other words, the pan-PI3Ki BKM120 was able to inhibit the growth of BYL-R and EVE-R cells, whereas BYL719 and everolimus were not able to inhibit BKM-R cells sufficiently. In addition, there were no changes in ER expression in EDR-1, EDR-2, and MFR cells exposed to PI3Kis for 1 h. Interestingly, ER expression on EDR-2 cells with acquired resistance to PI3Kis was increased compared with that in parental cells. Conclusion Our findings showed that PI3Kis exhibited remarkable efficacy in all types of EDR cells, particularly in fulvestrant-resistant cell lines. In PI3Ki- and everolimus-resistant cell lines, BKM120 and BYL719 showed different effects, and BYL719 and everolimus may exhibit cross-resistance. Furthermore, PI3Kis were likely to change the expression of ER. Citation Format: Tokuda E, Komatsu T, Sasaki S, Tsuboi K, Nakamura M, Iida M, Niwa T, Saito M, Hayashi S-I. Effects of PI3K inhibitors on endocrine-resistant cell lines and differences in the characteristics of ER positive breast cancer cells after acquired resistance to the inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-12.

Abstract P1-09-09: Efficacy of estrogen receptor β agonists in the prevention of breast cancer progression to therapy resistance

Abstract Estrogen plays an important role in the initiation and progression of breast cancer (BCa). Approximately, 70% of breast tumors are estrogen receptor (ER) positive at the time of presentation. Endocrine therapy using aromatase inhibitors (AI), or anti-estrogen (AE) molecules are widely used for treating ER+ve BCa. However, their efficacy is limited by intrinsic and acquired therapy resistance and most patients develop resistance to these drugs. The transcriptional effects of estrogen are mediated by two ERs (ERα and ERβ) and both are expressed in normal breast tissue. Unlike ERα, ERβ functions as tumor suppressor. However, role of ERβ specific agonists in the prevention of BCa progression remains elusive. In this study, we investigated the effectiveness of two ERβ agonists (S-Equol and LY500307) in the prevention of BCa progression using endocrine therapy sensitive (MCF7-aro) and letrozole resistant (MCF7-aro-LTLT) cells. Our results demonstrated that treatment with ERβ agonists inhibit short-and long-term growth of both endocrine therapy sensitive and resistant BCa cells. In addition, ERβ agonists treatment inhibited invasion and migration of both MCF7-aro and MCF7-aro-LTLT cells. Importantly, cell cycle analysis revealed that ERβ agonists induced cell cycle arrest. Our gene microarray analysis demonstrated that both ERβ agonists significantly modulated genes involved in the cell cycle progression, DNA replication and cell death pathways. Further, gene enrichment analysis of differentially expressed genes revealed that genes involved in the cell cycle checkpoints emerged as significant pathway modulated by ERβ agonists treatment in MCF7-aro cells. Interestingly, in letrozole-resistant MCF7-aro cells, DNA replication was significantly affected by ERβ agonists treatment. Pathway analysis also identified enrichment for chemokine signaling pathways. We confirmed pathway analysis by qRT-PCR and western blot analysis. Accordingly, treatment of in vivo syngeneic xenografts with ERβ agonists significantly inhibited BCa progression. Collectively, these results from this study suggest that ERβ agonists have potential to prevent the progression of BCa progression. Citation Format: Ramasamy K, Samayoa C, Krishnegowda NK, Vadlamudi RK, Tekmal RR. Efficacy of estrogen receptor β agonists in the prevention of breast cancer progression to therapy resistance [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-09.

Abstract P1-01-15: Investigation of phosphoserine aminotransferase 1 and its role in breast cancer progression

Abstract Metastasis and endocrine resistance are two factors that complicate therapeutic intervention in breast cancer patients and lead to poorer overall survival. Metastasis is known to be responsible for 90% of cancer related deaths, and is especially prevalent in triple negative breast cancer (TNBC); while endocrine resistance can affect up to 50% of patients diagnosed with estrogen receptor positive breast cancer (ER+BC). Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second step within de novo serine biosynthesis and increased expression of enzymes in this pathway have been linked to progression of breast cancer and poor clinical outcomes. Within our preliminary retrospective analysis of human breast cancer patients, we identified an inverse association with elevated transcript levels and poorer distant metastasis free survival, which, coupled to a previously reported correlation of PSAT1 with response to endocrine therapy in patients with ER+BC, we postulate that PSAT1 contributes to breast cancer progression through promotion of metastasis and/or endocrine resistance. To initially determine relevance for PSAT1, immunohistochemistry was performed to determine PSAT1 expression in human breast cancer patients. We found that PSAT1 expression is increased through breast cancer progression, with highest levels observed within metastatic conditions. To investigate the metastatic contribution of PSAT1, we silenced PSAT1 expression within the triple negative breast cancer cell line (TNBC), MDA-MB-231. While suppression of PSAT1 had no effect on proliferation, there was a significant decrease in the motility and invasion of these cells. In addition, decreased PSAT1 substantially inhibited lung metastasis following tail-vein injections of MDA-MB-231 cells in vivo. To investigate PSAT1's role in endocrine resistance, we used parental MCF-7 cells and an endocrine-resistant derivative cell line (LY2) and found that LY2-resistant cells exhibited higher PSAT1 expression compared to parental MCF-7 cells. Lastly, suppression of PSAT1 was able to sensitize the LY2 cells to 4-hydroxytamoxifen treatment. Taken together, these data indicate that PSAT1 may contribute to the progression of human breast cancer via either metastasis or endocrine resistance or both and could potentially serve as a viable target for new therapies. Citation Format: Metcalf S, Kruer T, Klinge C, Clem B. Investigation of phosphoserine aminotransferase 1 and its role in breast cancer progression [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-15.

Endocrine Therapy of Estrogen Receptor-Positive Breast Cancer Cells: Early Differential Effects on Stem Cell Markers.

Endocrine therapy of breast cancer, which either deprives cancer tissue of estrogen or prevents estrogen pathway signaling, is the most common treatment after surgery and radiotherapy. We have previously shown for the estrogen-responsive MCF-7 cell line that exposure to tamoxifen, or deprivation of estrogen, leads initially to inhibition of cell proliferation, followed after several months by the emergence of resistant sub-lines that are phenotypically different from the parental line. We examined the early responses of MCF-7 cells following either exposure to 4-hydroxytamoxifen or deprivation of estrogen for periods of 2 days-4 weeks. Endocrine-sensitive or -resistant breast cancer cell lines were used to examine the expression of the stem cell gene SOX2, and the Wnt effector genes AXIN2 and DKK1 using quantitative PCR analysis. Breast cancer cell lines were used to assess the anti-proliferative effects (as determined by IC50 values) of Wnt pathway inhibitors LGK974 and IWP-2. Hormone therapy led to time-dependent increases of up to 10-fold in SOX2 expression, up to threefold in expression of the Wnt target genes AXIN2 and DKK1, and variable changes in NANOG and OCT4 expression. The cells also showed increased mammosphere formation and increased CD24 surface protein expression. Some but not all hormone-resistant MCF-7 sub-lines, emerging after long-term hormonal stress, showed up to 50-fold increases in SOX2 expression and smaller increases in AXIN2 and DKK1 expression. However, the increase in Wnt target gene expression was not accompanied by an increase in sensitivity to Wnt pathway inhibitors LGK974 and IWP-2. A general trend of lower IC50 values was observed in 3-dimensional spheroid culture conditions (which allowed enrichment of cells with cancer stem cell phenotype) relative to monolayer cultures. The endocrine-resistant cell lines showed no significant increase in sensitivity to Wnt inhibitors. Hormone treatment of cultured MCF-7 cells leads within 2 days to increased expression of components of the SOX2 and Wnt pathways and to increased potential for mammosphere formation. We suggest that these responses are indicative of early adaptation to endocrine stress with features of stem cell character and that this facilitates the survival of emerging hormone-resistant cell populations.

Salinomycin overcomes acquired tamoxifen resistance through AIB1 and inhibits cancer cell invasion in endocrine resistant breast cancer.

Salinomycin is a monocarboxylic polyether ionophore isolated from Streptomyces albus. It has been widely used as an antibiotic in veterinary medicine in poultry. A recent study demonstrated that salinomycin selectively inhibits human breast cancer stem cells; one possible mechanism of tamoxifen resistance. Our results show that salinomycin is effective in inhibiting MCF-7/LCC2 and MCF-7/LCC9 cell lines which are well-established endocrine resistant cells and has a synergistic effect in combination with tamoxifen using MTT proliferation assay. The inhibitory effect of salinomycin on the reduction of critical ER co-activator; amplified breast 1 (AIB1) mRNA and protein expression is overcoming tamoxifen resistance. Moreover, salinomycin significantly inhibits cell invasion in Matrigel invasion assay. The effect was mediated at least in part by the decrease of matrix metalopeptidase 9 (MMP-9) which is one critical enzyme facilitated in the cell invasion process. In conclusion, salinomycin should be developed as a novel agent used alone or in combination for endocrine-resistant breast cancer.

Abstract 430: Combination of CB-839 and everolimus is effective in inhibiting growth of endocrine resistant breast cancer in vivo

Abstract About 70% of all breast cancers are estrogen receptor alpha positive (ER+). Anti-hormone therapy such as antiestrogens are often used to treat ER+ breast cancer but breast cancer cells can develop resistance to these drugs (endocrine resistance). Unfortunately, ~50% percent of all antiestrogen treated tumors eventually develop endocrine resistance, and therefore, there is an urgent need to find ways to treat this incurable disease. We have shown that endocrine resistant breast cancer cells show an increased dependence on the amino acid glutamine and this process is regulated by MYC activation via the unfolded protein response (UPR). Metabolites of glutamine such as glutamate and proline are significantly elevated in endocrine resistant cells. Cellular changes in glutamine are sensed by the mammalian target of rapamycin (mTOR) complex, mTORC1, which is known to be deregulated in endocrine resistant breast cancer. In this study, we used human antiestrogen (Fulvestrant/Faslodex or Tamoxifen) sensitive and resistant ER+ breast cancer cells and xenografts to test the efficacy of CB-839, an anti-glutaminase, and everolimus (Afinitor), a mTORC1 inhibitor. Combination of CB-839 and everolimus, but not each drug alone, inhibited growth of antiestrogen resistant tumors compared to vehicle alone at 4 weeks post-treatment. The combination treatment did not significantly inhibit growth of antiestrogen sensitive tumors compared with its respective vehicle alone treatment. Thus, a combination strategy that targets glutamine dependence and increased mTOR activation may be a novel strategy in treating endocrine resistant breast cancer. Citation Format: Yassi Fallah, Diane M. Demas, Susan Demo, Ayesha N. Shajahan-Haq. Combination of CB-839 and everolimus is effective in inhibiting growth of endocrine resistant breast cancer in vivo [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 430. doi:10.1158/1538-7445.AM2017-430