Endocrine Resistant Cell Lines Research Articles

A Regulatory Axis between Epithelial Splicing Regulatory Proteins and Estrogen Receptor α Modulates the Alternative Transcriptome of Luminal Breast Cancer

Epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the splicing pattern during epithelial to mesenchymal transition (EMT) in a physiological context and in cancer, including breast cancer (BC). Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs of patients with poor prognosis and correlates with estrogen receptor α (ERα) levels. Analysis of ERα genome-binding profiles in cell lines and primary breast tumors showed its binding in the proximity of ESRP1 and ESRP2 genes, whose expression is strongly decreased by ERα silencing in hormone-deprived conditions. The combined knock-down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs correlated with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein–protein interactions altered by ESRP1/2 silencing. The comparison of ERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected in primary BCs and endocrine-resistant cell lines. Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.

Collateral-resistance to estrogen and HER-activated growth is associated with modified AKT, ERα, and cell-cycle signaling in a breast cancer model.

Aim:A model of progressively endocrine-resistant breast cancer was investigated to identify changes that can occur in signaling pathways after endocrine manipulation.Methods:The MCF7 breast cancer model is sensitive to estrogens and anti-estrogens while variant lines previously derived from wild-type MCF7 are either relatively 17β-estradiol (E2 )-insensitive (LCC1) or fully resistant to estrogen and anti-estrogens (LCC9).Results:In LCC1 and LCC9 cell lines, loss of estrogen sensitivity was accompanied by loss of growth response to transforming growth factor alpha (TGFα), heregulin-beta and pertuzumab. LCC1 and LCC9 cells had enhanced AKT phosphorylation relative to MCF7 which was reflected in downstream activation of phospho-mechanistic target of rapamycin (mTOR), phospho-S6, and phospho-estrogen receptor alpha Ser167 [ERα(Ser167)]. Both AKT2 and AKT3 were phosphorylated in the resistant cell lines, but small interfering RNA (siRNA) knockdown suggested that all three AKT isoforms contributed to growth response. ERα(Ser118) phosphorylation was increased by E2 and TGFα in MCF7, by E2 only in LCC1, but by neither in LCC9 cells. Multiple alterations in E2-mediated cell cycle control were identified in the endocrine-resistant cell lines including increased expression of MYC, cyclin A1, cyclin D1, cyclin-dependent kinase 1 (CDK1), CDK2, and hyperphosphorylated retinoblastoma protein (ppRb), whereas p21 and p27 were reduced. Estrogen modulated expression of these regulators in MCF7 and LCC1 cells but not in LCC9 cells. Seliciclib inhibited CDK2 activation in MCF7 cells but not in resistant variants; in all lines, it reduced ppRb, increased p53 associated responses including p21, p53 up-regulated modulator of apoptosis (PUMA), and p53 apoptosis-inducing protein 1 (p53AIP1), inhibited growth, and produced G2/M block and apoptosis.Conclusions:Multiple changes occur with progression of endocrine resistance in this model with AKT activation contributing to E2 insensitivity and loss of ERα(Ser118) phosphorylation being associated with full resistance. Cell cycle regulation is modified in endocrine-resistant breast cancer cells, and seliciclib is effective in both endocrine-sensitive and resistant diseases.

PLK1 inhibition exhibits strong anti-tumoral activity in CCND1-driven breast cancer metastases with acquired palbociclib resistance

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.

Abstract P6-04-26: PI3K inhibitors restored the sensitivity to anti-estrogen drugs in endocrine therapy resistant cell lines

Abstract Background The Combination of endocrine therapy with molecular targeted drugs has become one of the standard treatment option for ER positive advanced or metastatic breast cancer. The therapeutic effects of PI3K-mTOR inhibitors, which inhibit intracellular phosphorylation of signaling pathways, have been clarified in clinical trials. However, the effects of these drugs on ER signal pathways in endocrine-resistant breast cancer have not been fully elucidated. We investigated the changes in the expression and functions of ER when endocrine-resistant cell lines, established in our laboratory, were treated with PI3K-mTOR inhibitors Methods Long-term estrogen deprivation-resistant (EDR) cell lines and fulvestrant-resistant cell lines (MFR) were established from MCF-7 cells in our previous studies. These cell lines exhibited different ER expression levels, including high (EDR-1), low (EDR-2), or no expression (MFR). All these cell lines had the same PIK3CA mutations as the parental cell lines. We used buparlisib (BUP, pan-class1 PI3K inhibitor) and alpelisib (ALP, α-specific PI3K inhibitor) as PI3K inhibitors and everolimus (EVE) as an mTOR inhibitor. Tamoxifen and fulvestrant were used in anti-estrogen therapy. In this study, the procedure to examine histone modifications was CHIP-qtPCR analysis. Results EDR-1, -2 and MFR were treated with the PI3K-TOR inhibitors for 24 h. There were no changes in ER expression and ER activity on EDR-1 and MFR before and after the addition of PI3K-mTOR inhibitors. Interestingly, ER expression and ER activity in EDR-2 increased after the addition of PI3K-mTOR inhibitors. In particular, a high re-expression of ER was observed when the PI3K inhibitors were added. Next, in order to evaluate the effects of endocrine therapy on these cell lines Using BUP, ALP and EVE, each drug resistant cell lines were established from EDR-2. When the cell proliferation assay was performed in the established resistant cell line, EDR-2 which showed re-expression of ER on adding the PI3K inhibitors, showed cell growth-inhibitory effects with the addition of anti-estrogen agents. Previously, we reported that epigenetic regulation contributes to the reduction of ER expression in EDR-2. (Tsuboi et al, J Steroid Biochem Mol Biol 2017). Therefore, we investigated the possibility that epigenetic regulation could contribute to the mechanism of ER re-expression when PI3K inhibitors were used in EDR-2. It was revealed that the change of H3K27me3, one of the histonemodifications, correlated with the expression of ER. Conclusion PI3K inhibitor could restore the expression of ER and sensitivity to anti-estrogen drugs in endocrine therapy resistant cell line in which ER expression was lost during acquired resistant period. This restoration might be due to a histone modification (H3K27me3). Citation Format: Emi Tokuda, Shunta Sasaki, Kouki Tsuboi, Masafumi Iida, Toshifumi Niwa, Shigehira Saji, Shin-Ichi Hayashi. PI3K inhibitors restored the sensitivity to anti-estrogen drugs in endocrine therapy resistant cell lines [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 P6-04-26.

Abstract PD2-04: Different mechanism of CDK4/6 inhibitor resistance between ribociclib and abemaciclib

Abstract Background: CDK4/6 inhibitors—palbociclib, ribociclib, and abemaciclib—result in prolonged progression free survival for advanced estrogen receptor positive breast cancer patients. However, the optimum CDK4/6 inhibitor for each patient remains unclear until the emergence of side effects, which are the key differentiators between the two treatments. Furthermore, an optimum treatment after acquisition of resistance is poorly understood. Therefore, we elucidated the difference in CDK4/6 inhibitor resistance mechanisms using ribociclib- and abemaciclib-resistant cell lines in vitro.Methods: We established hormone-resistant cell lines. Estrogen deprivation-resistant (EDR) cell lines (EDR1:ER-positive, EDR2:ER-negative) and fulvestrant-resistant (MFR) cell lines were established from the MCF-7 cell line. We established ribociclib-resistant cell lines (RIBR) and abemaciclib-resistant cell lines (ABER) from EDR1 by long-term culture (7 months for RIBR and 12 months for ABER) with ribociclib and abemaciclib, respectively. We also established CDK6 overexpressed cell lines (MCF7-C6) from MCF-7 cell line using the stably transfected CDK6 expression vector.Results: First, we identified the efficacies of ribociclib and abemaciclib in several cell lines. Luminal cell lines (MCF-7 and T-47D) exhibited high sensitivity to ribociclib and abemaciclib compared to non-luminal cell lines (MDA-MB-231, BT-20, and SKBR3). The hormone-resistant cell lines (EDR1, EDR2, and MFR) exhibited similar sensitivity to ribociclib and abemaciclib as that of MCF-7; this was independent of hormone resistant mechanism.Subsequently, we compared RIBR and ABER, which are the models of acquired ribociclib- and abemaciclib-resistant cell lines, respectively. We confirmed that RIBR and ABER decreased ribociclib and abemaciclib sensitivity, respectively. RIBR showed low sensitivity to abemaciclib. Similarly, ABER exhibited low sensitivity to ribociclib. Immunoblot analysis showed that compared to EDR1, RIBR and ABER had extremely low levels of total Retinoblastoma (RB). Interestingly, compared to EDR1, CDK6 levels were upregulated in ABER and remained unaltered in RIBR. On the other hand MCF7-C6 reduced sensitivity not only abemaciclib but also ribociclib compared to MCF-7 cell line. Furthermore, compared to EDR1, RIBR exhibited lower levels of p21 and p27 compared to EDR1 (P21 levels were extremely reduced, but p27 levels were slightly reduced). Additionally, ABER showed markedly lower levels of p27; however, the p21 level was slightly increased compared to EDR1. PI3K inhibitor and mTOR inhibitor suppressed cell growth in RIBR and ABER to the same extent as EDR1, indicating that resistance to CDK4/6 inhibitors was still dependent on the PI3K/Akt/mTOR pathway. Chemotherapeutic drugs were effective both in RIBR and ABER.Discussion & Conclusion: There are various mechanisms of CDK4/6 inhibitor resistance, such as loss of RB, CDK6 amplification, and high CDK2 activity. However, the mechanism of resistance between ribociclib and abemaciclib might be different because the CDK6 level was different, although RB level was decreased in both RIBR and ABER. Furthermore, both p21 and p27 are considered as the internal inhibitors of CDK2, although more factors in RIBR and ABER were different. CDK4/6 inhibitor resistant cell lines acquired cross resistance to another CDK4/6 inhibitor. Although the mechanism of resistance between ribociclib and abemaciclib might be different, use of drugs targeting the PI3K/Akt/mTOR pathway and chemotherapeutic drugs would be an effective strategy following the development of resistance. Our findings suggest that the mechanism of resistance differs depending on the type of CDK4/6 inhibitors used, and provide some insights into the treatment strategies after acquisition of CDK4/6 inhibitor resistance. Citation Format: Masafumi Iida, Daichi Toyosawa, Misato Nakamura, Emi Tokuda, Toshifumi Niwa, Ryuichi Yoshida, Takanori Ishida, Shin-ichi Hayashi. Different mechanism of CDK4/6 inhibitor resistance between ribociclib and abemaciclib [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 PD2-04.

NADPH: Quinone oxidoreductase 1 (NQO1) mediated anti-cancer effects of plumbagin in endocrine resistant MCF7 breast cancer cells

BackgroundPLB is a natural naphthoquinone compound isolated from the roots of Plumbago indica plant. Our previous study reported the inhibitory effect of Plumbagin (PLB) on human endocrine resistant breast cancer cell growth and cell invasion. Hypothesis/PurposeSince PLB is a naphthoquinone compound, it can be reduced by the cytosolic NADPH: quinone oxidoreductase 1 (NQO1) enzyme. NQO1 expression is upregulated in various types of aggressive cancer including breast cancer. This study investigated the impact of NQO1 on anti-cancer effects of PLB in endocrine-resistant breast cancer cells. Study DesignThis study was an in vitro study using ER-positive cell line (MCF7) and endocrine-resistant cell lines (MCF7/LCC2 and MCF7/LCC9 cells). MethodsThe roles of NQO1 in anti-cancer activity of PLB were investigated by using NQO1 knockdown cells, NQO1 inhibitor and NQO1 overexpressed cells. To study the impact of NQO1 on the effects of PLB on cell viability, apoptosis, invasion and generation of ROS, the following assays were used: MTT assays, annexin V-PE/7-ADD staining flow cytometry, matrigel invasion assays and DCFHDA assays. To study the mechanism of how NQO1 mediated PLB effects in tamoxifen response and apoptosis, we assessed the levels of mRNA expression by using qRT-PCR. Results1. In this study, NQO1 was upregulated in endocrine-resistant cells.2. PLB did not change the expression of NQO1 but it was able to increase NQO1 activity.3. The inhibitory effects of PLB on cell proliferation, cell invasion and expression of tamoxifen resistant gene were attenuated in NQO1 knockdown cells or in the presence of NQO1 inhibitor.4. The effects of PLB to induce apoptosis and generate ROS were also decreased when NQO1 activity was inhibited or when the NQO1 expression was reduced.5. The anti-cancer effects of PLB increased when NQO1 was upregulated. ConclusionThe effects of PLB in endocrine-resistant breast cancer cells is dependent on NQO1’s activity.

The Hedgehog Signaling Pathway: A Viable Target in Breast Cancer?

The hedgehog (Hh) pathway plays a key role in embryonic development and stem cell programs. Deregulation of the Hh pathway is a key driver of basal cell carcinoma, and therapeutic targeting led to approval of Hh inhibitor, vismodegib, in the management of this cancer. The Hh pathway is implicated in other malignancies including hormone receptor (HR+) positive and triple negative breast cancer (TNBC). Hh signaling, which is activated in human mammary stem cells, results in activation of glioma-associated oncogene (GLI) transcription factors. High GLI1 expression correlates with worse outcomes in breast cancer. Non-canonical GLI1 activation is one mechanism by which estrogen exposure promotes breast cancer stem cell proliferation and epithelial–mesenchymal transition. Tamoxifen resistant cell lines show aberrant activation of Hh signaling, and knockdown of Hh pathway inhibited growth of tamoxifen resistant cells. As in other cancers Hh signaling is activated by the PI3K/AKT pathway in these endocrine resistant cell lines. Hh pathway activation has also been reported to mediate chemotherapy resistance in TNBC via various mechanisms including paracrine signaling to tumor micro-environment and selective proliferation of cancer stem cells. Co-activation of Hh and Wnt signaling pathways is a poor prognostic marker in TNBC. Early phase clinical trials are evaluating the combination of smoothened (SMO) inhibitors and chemotherapy in TNBC. In addition to SMO inhibitors like vismodegib and sonidegib, which are in clinical use for basal cell carcinoma, GLI1 inhibitors like GANT58 and GANT61 are in preclinical drug development and might be an effective mechanism to overcome drug resistance in breast cancer. Gene signatures predictive of Hh pathway activation could enrich for patients likely to respond to these agents.

The p21 levels have the potential to be a monitoring marker for ribociclib in breast cancer.

Although cyclin-dependent kinase (CDK) 4/6 inhibitors have exhibited remarkable results for patients with estrogen receptor (ER)–positive breast cancer in clinical trials, the mechanism of CDK4/6 inhibitor resistance remains unclear. Thus, this study aimed to investigate the mechanism of CDK4/6 inhibitor resistance using two CDK4/6 inhibitor resistant breast cancer cell lines. We established CDK6 overexpressed cell lines (MCF7-C6) from MCF-7 cells using the stably transfected CDK6 expression vector. Additionally, acquired ribociclib-resistant (RIBR) cell lines were created using ER-positive hormone-resistant cell lines by long-term exposure to ribociclib. CDK6 overexpression and the knockdown of CDK4 experiments highlight the significance of high levels of CDK4 and low levels of CDK6 in CDK4/6 inhibitor sensitivity. Moreover, RIBR cell lines did not exhibit incremental CDK6 compared with ER-positive hormone-resistant cell lines. In MCF7-C6 and RIBR cell lines, p21 levels decreased, and p21 levels were proportional to CDK4/6 inhibitor sensitivity. This study suggests that overexpression of CDK6 is one of the many possible mechanisms of resistance to CDK4/6 inhibitors. Furthermore, p21 levels have the potential to serve as a marker for CDK4/6 inhibitors independent of the resistance mechanism.

Combined Targeting of Estrogen Receptor Alpha and XPO1 Prevent Akt Activation, Remodel Metabolic Pathways and Induce Autophagy to Overcome Tamoxifen Resistance.

A majority of breast cancer specific deaths in women with ERα (+) tumors occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to resensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and the nuclear transport protein XPO1 in overcoming endocrine resistance. Selinexor (SEL), an XPO1 antagonist, has been evaluated in multiple late stage clinical trials in patients with relapsed and/or refractory hematological and solid tumor malignancies. Our transcriptomics analysis showed that 4-Hydroxytamoxifen (4-OHT), SEL alone or their combination induced differential Akt signaling- and metabolism-associated gene expression profiles. Western blot analysis in endocrine resistant cell lines and xenograft models validated differential Akt phosphorylation. Using the Seahorse metabolic profiler, we showed that ERα-XPO1 targeting changed the metabolic phenotype of TAM-resistant breast cancer cells from an energetic to a quiescent profile. This finding demonstrated that combined targeting of XPO1 and ERα rewired the metabolic pathways and shut down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies to improve therapy response in relapsed ERα (+) tumors, our findings show great promise for uncovering the role that ERα-XPO1 crosstalk plays in reducing cancer recurrences.

Abstract P4-07-05: A novel, first in class Notch transcriptional inhibitor, CB-103 has activity on luminal breast cancer stem cells in combination with fulvestrant

Abstract The Notch signaling pathway plays a central role in cellular differentiation, growth and stem cell maintenance. Expression and activation of Notch pathway receptors and ligands have differential outcomes depending on the tissue, localization and cell type. When Notch pathway is aberrantly activated by genetic lesions, it can be a major driver for Notch-dependent cancers and can cause resistance to standard of care treatment. We and others have shown that in Estrogen Receptor (ER)-positive breast cancers, estrogen deprivation caused by endocrine therapy results in Notch1 and Notch4 activation. In turn, Notch1 stimulated ER-dependent transcription in the absence of estrogen, causing endocrine resistance. Combinations of Notch inhibitors and endocrine therapy are effective in preclinical models of ER-positive breast cancer and have shown promising signals in early clinical trials. Cellestia's lead development candidate CB-103 is a small molecule, first-in-class, oral pan-Notch inhibitor. CB-103 selectively blocks Notch pathway activation-related gene transcription through binding to a Notch specific protein in the transcription factor complex. The blockade occurs by protein-protein interaction inhibition with a binding site critical for the assembly of the Notch transcription complex. This is a unique mode of action, which allows blocking Notch signaling regardless of the genetic lesions which have activated the pathway. We have performed mammosphere assays to test the potency and efficacy of this compound on stem cell ability to form sphere. We used two different doses of CB-103; either alone or in combination with a fixed dose of Fulvestrant (30nM), a SERD, in our mammosphere assays. Two different ER+ luminal,endocrine resistant cell lines were tested and compared with their parental controls. From our data, it's apparent that there is a synergistic effect when using CB-103 in combination with Fulvestrant. It's also evident that the efficacy of CB-103 is maximal maximizes at the lowest concentration tested in our assays. The combination was effective in 3 out of 4 models. However, the effect of CB-103 on MCF7-TAMR either as a single agent or in combination was not statistically significant. Cellestia has received regulatory approval to start clinical development with CB-103 in a first-in-human study Phase l – lla study investigating safety (Ph l) and preliminary single agent efficacy (Ph lla) of CB-103 in patients with advanced solid cancers and haematological malignancies. Our data support the notion of testing this agent in ER-positive breast cancer in combination with SERDs. Citation Format: Majumder S, Crabtree J, Hossain F, Murone M, Lehal R, Miele L. A novel, first in class Notch transcriptional inhibitor, CB-103 has activity on luminal breast cancer stem cells in combination with fulvestrant [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-05.

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.

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.

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 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 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 P6-04-02: CDK6 might be a key factor for efficacy of CDK4/6 inhibitor and the hormone sensitivity following acquired resistance

Abstract Background: CDK4/6 inhibitors have received FDA breakthrough therapy designation as 1st line treatment for advanced estrogen receptor positive breast cancer patients. However, the benefit offered by CDK4/6 inhibitors is individually different and furthermore acquired resistance to the drugs is monumental challenges. It is urgent need to search for the biomarker and understand the drug sensitivity and its alteration after acquired resistance. Results: To identify the efficacy of CDK4/6 inhibitors, we assessed IC50 of ribociclib in several cell lines. Luminal cell lines (MCF-7, T-47D) exhibited lower ribociclib IC50 than HER-2 (SK-BR-3) and triple negative cell lines (MDA-MB-231, BT-20). Immunoblot analysis of Luminal cell lines showed extremely lower levels of CDK6 compared with others. CDK6-transfected MCF-7 by means of expression vector reduced the sensitivity equivalent to MDA-MB-231 not only to ribociclib but also to palbociclib and abemaciclib. Protein level of ERα in CDK6-transfected MCF-7 stayed unchanged and fulvestrant sensitivity was unaltered as well. Subsequently, we detect the efficacy of ribociclib in hormone resistant cell lines. Estrogen deprivation-resistant (EDR) cells (EDR1:ER-positive, EDR2:ER-negative) and fulvestrant resistance (MFR) cells (loss of ER expression) established from MCF-7 maintained ribocilib sensitivity to the same degree with MCF-7. No marked difference in IC50 was observed between EDR1/2 and MFR, and CDK6 expressions were comparable to MCF-7. These results suggest that high level of CDK6 expression weaken the sensitivity to CDK4/6 inhibitors. The inhibitors would provide more effective benefits to tumors expressing lower level of CDK6 than the higher, independent of hormone sensitivity. To understand the characteristics in acquired resistance, ribociclib resistant cell lines (RIBR1/2) were established from EDR1 by long-term culture with ribociclib. RIBR designed lower level of p21, p27 and ERα by immunoblot analysis. EDR1 were promoted cell growth by estrogen addition, while RIBR were not. Further, ER activity of RIBR was intensely decreased, and mRNA levels of the ER target genes, PgR and EGR3 were also decreased. Therefore, the responsiveness to tamoxifen and fulvestrant were lost. On the other hand, PI3K inhibitor and mTOR inhibitor suppressed cell growth to the same extent as EDR1, suggesting that RIBR were reduced ER dependence and remain reliant on PI3K/Akt/mTOR pathway. Conclusion: The possibility of CDK6 as a biomarker is corroborated by the finding that low level of CDK6 expression is positively correlated with the efficacy of CDK4/6 inhibitor. Further ER dependence had decreased after acquired CDK4/6 inhibitor resistance whereas the dependence on PI3K/Akt/mTOR pathway still remain, indicating the inhibition of PI3K/Akt/mTOR pathway would be amenable to therapeutic target. Citation Format: Iida M, Nakamura M, Tokuda E, Niwa T, Ishida T, Hayashi S-I. CDK6 might be a key factor for efficacy of CDK4/6 inhibitor and the hormone sensitivity following acquired 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 P6-04-02.

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.

The effect of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel and normal cell lines

BackgroundNullomer peptides are the smallest sequences absent from databases of natural proteins. We first began compiling a list of absent 5-amino acid strings in 2006 (1). We report here the effects of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel, derived from human cancers of 9 organs (kidney, ovary, skin melanoma, lung, brain, lung, colon, prostate and the hematopoietic system), and four normal cell lines (endothelial HUVEC, skin fibroblasts BJ, colon epithelial FHC and normal prostate RWPE-1).MethodsNCI-60 cancer cell panel and four normal cell lines were cultured in vitro in RPMI1640 supplemented with 10% Hyclone fetal bovine serum and exposed for 48 h to 5 μM, 25 μM and 50 μM of peptides 9R, 9S1R and 124R. Viability was assessed by CCK-8 assay. For peptide ATP depletion effects, one cell line representing each organ in the NCI-60 panel, and four normal cell lines were exposed to 50 μM of peptides 9R, 9S1R and 124R for 3 h. The ATP content was assessed in whole cells, and their supernatants.ResultsPeptides 9S1R and 9R are respectively lethal to 95 and 81.6% of the 60 cancer cell lines tested. Control peptide 124R has no effect on the growth of these cells. Especially interesting the fact that peptides 9R and 9S1R are capable of killing drug-resistant and hormone-resistant cell lines, and even cancer stem cells. Peptides 9R and 9S1R have a broader activity spectrum than many cancer drugs in current use, can completely deplete cellular ATP within 3 h, and are less toxic to 3 of the 4 normal cell lines tested than they are to several cancers.ConclusionsNullomer peptides 9R and 9S1R have a large broad lethal effect on cancer cell lines derived from nine organs represented in the NCI-60 panel. This broad activity crosses many of the categorical divisions used in the general classification of cancers: solid vs liquid cancers, drug sensitive vs drug resistant, hormone sensitive vs hormone resistant, cytokine sensitive vs cytokine non sensitive, slow growing vs rapid growing, differentiated vs dedifferentiated cancers. Furthermore peptides 9R and 9S1R are lethal to cancer stem cells and breast canrcinosarcoma.

Abstract 861: Global transcription factor repression by the coactivator SRC-1 mediates disease progression in endocrine-resistant breast cancer

Abstract Despite the clinical utility of endocrine therapies to treat ER positive breast cancer, 40% of patients eventually develop resistance, leading to disease progression. Steroid receptor co-activators have been described as “the powerhouses of transcription” with a role in mediating hormone receptor responsiveness. There is now substantial evidence that steroid receptor coactivator-1 (SRC-1) is central to the ability of endocrine tumours to adapt and overcome targeted therapy thereby facilitating metastatic disease progression. Recent work highlighted the potential ability of SRC-1 to also act as transcriptional repressor that can bi-directionally regulate genes that help it promote the resistant phenotype. This study employed a genome-wide multi-omics sequencing approach to determine the SRC-1 repression signature in endocrine resistance and elucidate the mechanism by which SRC-1 mediates this repression. RNA-sequencing was used to investigate the transcriptional network affected by SRC-1 in an endocrine resistant cell line model. This approach identified 1,495 genes significantly downregulated by SRC-1, with common functional pathways such as differentiation, cell morphogenesis and extracellular matrix enriched in the gene set. Parallel global methylation sequencing analysis revealed methylation as a possible mechanism by which SRC-1 was repressing target genes, which was confirmed with mechanistic studies utilising 5-AZA-2’-deoxycitidine. Through combined analysis of our global sequencing data we identified a SRC-1 regulated transcriptional hub of differentiation genes, that have protective roles in normal and treatment- sensitive breast cancer cells and which associate with poor disease-free survival in breast cancer patients. Furthermore, our work demonstrated that SRC-1 can re-programme cells to become poorly differentiated by supressing this normally protective transcriptional hub, and thereby promote aggressive endocrine resistant phenotype. Here we report a novel mechanism by which SRC-1 can drive endocrine resistant tumorigenesis. Our genome-wide discovery approach revealed an epigenetic re-programming pathway, whereby concerted differential DNA methylation is potentiated by the activation of SRC-1 in the endocrine resistant setting. This study suggests that therapeutic strategies of combined targeted epigenetic therapy with estrogen deprivation could be successful strategy to prevent acquired resistance to endocrine therapy. Citation Format: Damir Vareslija, Elspeth Ward, Ailis Fagan, William Gallagher, Arnold DK Hill, Leonie S. Young. Global transcription factor repression by the coactivator SRC-1 mediates disease progression in endocrine-resistant 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 861.

Src Is a Potential Therapeutic Target in Endocrine-Resistant Breast Cancer Exhibiting Low Estrogen Receptor-Mediated Transactivation.

Despite the effectiveness of endocrine therapies in estrogen receptor positive (ER+) breast cancer, approximately 40% of patients relapse. Previously, we identified the Focal-adhesion kinase canonical pathway as a major contributor of resistance to estrogen deprivation and cellular-sarcoma kinase (c-src) as a dominant gene in this pathway. Dasatinib, a pan-src inhibitor, has recently been used in clinical trials to treat ER+ patients but has shown mixed success. In the following study, using isogenic cell line models, we provide a potential explanation for these findings and suggest a sub-group that may benefit. A panel of isogenic cell lines modelling resistance to aromatase inhibitors (LTED) and tamoxifen (TAMR) were assessed for response to dasatinib ± endocrine therapy. Dasatinib caused a dose-dependent decrease in proliferation in MCF7-TAMR cells and resensitized them to tamoxifen and fulvestrant but not in HCC1428-TAMR. In contrast, in estrogen-deprived conditions, dasatinib increased the proliferation rate of parental-MCF7 cells and had no effect on MCF7-LTED or HCC1428-LTED. Treatment with dasatinib caused a decrease in src-phosphorylation and inhibition of downstream pathways, including AKT and ERK1/2 in all cell lines tested, but only the MCF7-TAMR showed a concomitant decrease in markers of cell cycle progression. Inhibition of src also caused a significant decrease in cell migration in both MCF7-LTED and MCF7-TAMR cells. Finally, we showed that, in MCF7-TAMR cells, in contrast to tamoxifen sensitive cell lines, ER is expressed throughout the cell rather than being restricted to the nucleus and that treatment with dasatinib resulted in nuclear shuttling of ER, which was associated with an increase in ER-mediated transcription. These data suggest that src has differential effects in endocrine-resistant cell lines, particularly in tamoxifen resistant models, with low ER genomic activity, providing further evidence of the importance of patient selection for clinical trials testing dasatinib utility in ER+ breast cancer.