Acquired Tamoxifen Resistance Research Articles

Downregulation of 15-hydroxyprostaglandin dehydrogenase during acquired tamoxifen resistance and association with poor prognosis in ERα-positive breast cancer.

Aim:Tamoxifen (TAM) resistance remains a clinical issue in breast cancer. The authors previously reported that 15-hydroxyprostaglandin dehydrogenase (HPGD) was significantly downregulated in tamoxifen-resistant (TAMr) breast cancer cell lines. Here, the authors investigated the relationship between HPGD expression, TAM resistance and prediction of outcome in breast cancer.Methods:HPGD overexpression and silencing studies were performed in isogenic TAMr and parental human breast cancer cell lines to establish the impact of HPGD expression on TAM resistance. HPGD expression and clinical outcome relationships were explored using immunohistochemistry and in silico analysis.Results:Restoration of HPGD expression and activity sensitised TAMr MCF-7 cells to TAM and 17β-oestradiol, whilst HPGD silencing in parental MCF-7 cells reduced TAM sensitivity. TAMr cells released more prostaglandin E2 (PGE2) than controls, which was reduced in TAMr cells stably transfected with HPGD. Exogenous PGE2 signalled through the EP4 receptor to reduce breast cancer cell sensitivity to TAM. Decreased HPGD expression was associated with decreased overall survival in ERα-positive breast cancer patients.Conclusions:HPGD downregulation in breast cancer is associated with reduced response to TAM therapy via PGE2-EP4 signalling and decreases patient survival. The data offer a potential target to develop combination therapies that may overcome acquired tamoxifen resistance.

Computational prediction methods to simulate structure and binding sites of coumarin with olfactory receptor 5P3

ABSTRACTOlfactory receptor 5P3 is coded by OR5P3 gene in human OR5P3 and recognized as the receptor for coumarin present in 30% of fragrances and might adversely affect human health. OR5P3 are also associated with coumarin-related breast cancer development and acquired tamoxifen resistance in breast cancer cells. Previously various investigators noted that coumarin acts as a ligand for OR5P3 and binding of this molecule to OR5P3 leads to downstream changes in the olfactory bulb and central nervous system. However, the interaction between OR5P3 and its ligands at molecular level is not well understood. The aim of this study was to develop a computerized approach to simulate the binding process of coumarin to OR5P3 and determine the binding sites at a molecular level. It is conceivable that understanding this binding mechanism to a specific OR may aid in identification of receptors responsible olfaction and non-olfaction disorders such as breast cancer. The approach employed in this study may thus be utilized in future studies to simulate bind of specific chemicals to ORs and potentially have therapeutic applications.

Activated zinc transporter ZIP7 as an indicator of anti-hormone resistance in breast cancer.

ZIP7, a member of the ZIP family of zinc importers, resides on the endoplasmic reticulum membrane and transports zinc from intracellular stores to the cytoplasm after activation by CK2 phosphorylation on two serine residues (S275 and S276). ZIP7 is known to be required for the growth of anti-hormone resistant breast cancer models, especially those with acquired tamoxifen resistance developed from MCF-7. Using our new pS275S276ZIP7 antibody which only recognises activated ZIP7 (pZIP7), we have demonstrated that the hyperactivation of ZIP7 is prevalent in tamoxifen-resistant breast cancer cells. This evidence suggests that pZIP7 might have potential as a biomarker of acquired resistance to such anti-hormones in breast cancer, a current unmet clinical need. In this regard, we have also developed a new immunohistochemical assay for pZIP7 which allowed pZIP7 to be tested on a small clinical series of breast cancer tissues confirming its prevalence in such tumours and relationship to a variety of clinicopathological parameters and biomarkers previously associated with endocrine resistant phenotypes, notably increased activated MAPK signalling, expression of ErbB2, CD71 and the proto-oncogene c-Fos, as well as with increased tumour grade.

The COOH-Terminal Proline-Rich Region of GRP78 Is a Key Regulator of Its Cell Surface Expression and Viability of Tamoxifen-Resistant Breast Cancer Cells

Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.

Time resolved gene expression analysis during tamoxifen adaption of MCF-7 cells identifies long non-coding RNAs with prognostic impact

ABSTRACT Acquired tamoxifen resistance is a persistent problem for the treatment of estrogen receptor positive, premenopausal breast cancer patients and predictive biomarkers are still elusive. We here analyzed gene expression changes in a cellular model to identify early and late changes upon tamoxifen exposure and thereby novel prognostic biomarkers. Estrogen receptor positive MCF-7 cells were incubated with 4OH-tamoxifen (10 nM) and gene expression analyzed by array hybridization during 12 weeks. Array results were confirmed by nCounter- and qRT-PCR technique. Pathway enrichment analysis revealed that early responses concerned mainly amine synthesis and NRF2-related signaling and evolved into a stable gene expression pattern within 4 weeks characterized by changes in glucuronidation-, estrogen metabolism-, nuclear receptor- and interferon signaling pathways. As a large number of long non coding RNAs was subject to regulation, we investigated 5 of these (linc01213, linc00632 linc0992, LOC101929547 and XR_133213) in more detail. From these, only linc01213 was upregulated but all were less abundant in estrogen-receptor negative cell lines (MDA-MB 231, SKBR-3 and UACC3199). In a web-based survival analysis linc01213 and linc00632 turned out to have prognostic impact. Linc01213 was investigated further by plasmid-mediated over-expression as well as siRNA down-regulation in MCF-7 cells. Nevertheless, this had no effect on proliferation or expression of tamoxifen regulated genes, but migration was increased. In conclusion, the cellular model identified a set of lincRNAs with prognostic relevance for breast cancer. One of these, linc01213 although regulated by 4OH-tamoxifen, is not a central regulator of tamoxifen adaption, but interferes with the regulation of migration.

ACT001 can prevent and reverse tamoxifen resistance in human breast cancer cell lines by inhibiting NF-κB activation.

Endocrine therapy is one of the main treatments for estrogen receptor-positive breast cancers. Tamoxifen is the most commonly used drug for endocrine therapy. However, primary or acquired tamoxifen resistance occurs in a large proportion of breast cancer patients, leading to therapeutic failure. We found that the combination of tamoxifen and ACT001, a nuclear factor-κB (NF-κB) signaling pathway inhibitor, effectively inhibited the proliferation of both tamoxifen-sensitive and tamoxifen-resistant cells. The tamoxifen-resistant cell line MCF7R/LCC9 showed active NF-κB signaling and high apoptosis-related gene transcription, especially for antiapoptotic genes, which could be diminished by treatment with ACT001. These results demonstrate that ACT001 can prevent and reverse tamoxifen resistance by inhibiting NF-κB activation.

Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF-7 Cells Revealed by Targeted, Quantitative Proteomic Analysis.

Development of tamoxifen resistance remains a tremendous challenge for the treatment of estrogen-receptor (ER)-positive breast cancer. Small GTPases of the Ras superfamily play crucial roles in intracellular trafficking and cell signaling, and aberrant small-GTPase signaling is implicated in many types of cancer. In this study, we employed a targeted, quantitative proteomic approach that relies on stable-isotope labeling by amino acids in cell culture (SILAC), gel fractionation, and scheduled multiple-reaction-monitoring (MRM) analysis, to assess the differential expression of small GTPases in MCF-7 and the pairedtamoxifen-resistant breast cancer cells. The method displayed superior sensitivity and reproducibility over the shotgun-proteomic approach, and it facilitated the quantification of 96 small GTPases. Among them, 13 and 10 proteins were significantly down- and up-regulated (with >1.5-fold change), respectively, in the tamoxifen-resistant line relative to in the parental line. In particular, we observed a significant down-regulation of RAB31 in tamoxifen-resistant cells, which, in combination with bioinformatic analysis and downstream validation experiments, supported a role for RAB31 in tamoxifen resistance in ER-positive breast-cancer cells. Together, our results demonstrated that the targeted proteomic method constituted a powerful approach for revealing the role of small GTPases in therapeutic resistance.

Loss of Estrogen-Regulated MIR135A1 at 3p21.1 Promotes Tamoxifen Resistance in Breast Cancer

The dysregulation of miRNAs has been increasingly recognized as a critical mediator of cancer development and progression. Here, we show that frequent deletion of the MIR135A1 locus is associated with poor prognosis in primary breast cancer. Forced expression of miR-135a decreased breast cancer progression, while inhibition of miR-135a with a specific miRNA sponge elicited opposing effects, suggestive of a tumor suppressive role of miR-135a in breast cancer. Estrogen receptor alpha (ERα) bound the promoter of MIR135A1 for its transcriptional activation, whereas tamoxifen treatment inhibited expression of miR-135a in ERα+ breast cancer cells. miR-135a directly targeted ESR1, ESRRA, and NCOA1, forming a negative feedback loop to inhibit ERα signaling. This regulatory feedback between miR-135a and ERα demonstrated that miR-135a regulated the response to tamoxifen. The tamoxifen-mediated decrease in miR-135a expression increased the expression of miR-135a targets to reduce tamoxifen sensitivity. Consistently, miR-135a expression was downregulated in ERα+ breast cancer cells with acquired tamoxifen resistance, while forced expression of miR-135a partially resensitized these cells to tamoxifen. Tamoxifen resistance mediated by the loss of miR-135a was shown to be partially dependent on the activation of the ERK1/2 and AKT pathways by miR-135a-targeted genes. Taken together, these results indicate that deletion of the MIR135A1 locus and decreased miR-135a expression promote ERα+ breast cancer progression and tamoxifen resistance.Significance: Loss of miR-135a in breast cancer disrupts an estrogen receptor-induced negative feedback loop, perpetuating disease progression and resistance to therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4915/F1.large.jpg Cancer Res; 78(17); 4915-28. ©2018 AACR.

Abstract 1930: Targeting the c-Cbl tumor suppressor protein as a novel therapeutic approach in overcoming acquired tamoxifen resistance in luminal breast cancer

Abstract Background and aims: Treatment of luminal breast cancer (LBC) with tamoxifen (TMX) is associated with the emergence of resistance in ~40% of patients. Such resistance is associated with emergence of tumor initiating cells (TICs, also known as cancer stem cells), resistance to other therapeutic agents and multiple other obstacles to successful treatment. This study aims to investigate the role of tumor suppressor gene c-Cbl in TMX resistance and identify novel therapeutic approaches in treatment of acquired TMX resistant LBC that address unmet clinical needs. Methods: TMX resistant MCF7 and T47D cell lines were generated by chronic exposure to TMX in vitro. Unbiased drug screening using the NINDS-II library revealed previously approved drugs with unexpected activities to restore c-Cbl activity. Assays, including dose-response studies, western blot, co-immunoprecipitation analyses and tumorsphere assays, were used to determine the mechanisms of action and utility of our lead agent (CRA-1). Finally, response to treatment was verified in xenograft mouse models. Findings: In TMX-resistant LBC cells, c-Cbl was inhibited due to complex formation with the Cool-1/ßpix (C1ßp) protein. Restoration of c-Cbl function by C1ßp knockdown or by treatment with our lead c-Cbl restorative agent (CRA-1, which is approved for other purposes and is suitable for prolonged use in patients) restored sensitivity to TMX and decreased TIC function. Treatment with CRA-1+TMX increased c-Cbl activation via the non-canonical redox/Fyn/c-Cbl (RFC) pathway by harnessing TMX's estrogen receptor-independent pro-oxidative activity. This treatment also decreased levels of the c-Cbl/C1ßp inhibitory complex, and of levels and/or activity of multiple direct and indirect c-Cbl targets important in TMX resistance (e.g. EGFR, HER2, Notch-1, sox2, ß-catenin). Treatment with CRA-1+TMX also increased sensitivity to cyclophosphamide (CPP), one of the standard chemotherapeutic agents to treat TMX resistant LBCs. In vivo, treatment with MPT+TMX + ultra-low-dose of CPP completely suppressed tumor growth in a xenograft animal model for 20 weeks without any obvious toxicity. Tumors thus far have not recurred (15 weeks) after treatment was stopped. Conclusions: Our study offers a new approach to overcoming acquired TMX resistance that also: (i) has the potential for rapid clinical translation due to the use of drugs already approved for other purposes; (ii) eliminates TICs; (iii) suppresses tumor growth in vivo with only a small fraction of clinically relevant CPP dosages; and (iv) exhibits greatly reduced toxicity effects and applicability for extended treatment periods. Citation Format: Xiaowen Wang, Hsing-Yu Chen, Jennifer Stripay, Mark Noble. Targeting the c-Cbl tumor suppressor protein as a novel therapeutic approach in overcoming acquired tamoxifen resistance in 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 1930.

Reactivation of cAMP Pathway by PDE4D Inhibition Represents a Novel Druggable Axis for Overcoming Tamoxifen Resistance in ER-positive Breast Cancer.

Purpose: Tamoxifen remains an important hormonal therapy for ER-positive breast cancer; however, development of resistance is a major obstacle in clinics. Here, we aimed to identify novel mechanisms of tamoxifen resistance and provide actionable drug targets overcoming resistance.Experimental Design: Whole-transcriptome sequencing, downstream pathway analysis, and drug repositioning approaches were used to identify novel modulators [here: phosphodiesterase 4D (PDE4D)] of tamoxifen resistance. Clinical data involving tamoxifen-treated patients with ER-positive breast cancer were used to assess the impact of PDE4D in tamoxifen resistance. Tamoxifen sensitization role of PDE4D was tested in vitro and in vivo Cytobiology, biochemistry, and functional genomics tools were used to elucidate the mechanisms of PDE4D-mediated tamoxifen resistance.Results: PDE4D, which hydrolyzes cyclic AMP (cAMP), was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. Higher PDE4D expression predicted worse survival in tamoxifen-treated patients with breast cancer (n = 469, P = 0.0036 for DMFS; n = 561, P = 0.0229 for RFS) and remained an independent prognostic factor for RFS in multivariate analysis (n = 132, P = 0.049). Inhibition of PDE4D by either siRNAs or pharmacologic inhibitors (dipyridamole and Gebr-7b) restored tamoxifen sensitivity. Sensitization to tamoxifen is achieved via cAMP-mediated induction of unfolded protein response/ER stress pathway leading to activation of p38/JNK signaling and apoptosis. Remarkably, acetylsalicylic acid (aspirin) was predicted to be a tamoxifen sensitizer using a drug repositioning approach and was shown to reverse resistance by targeting PDE4D/cAMP/ER stress axis. Finally, combining PDE4D inhibitors and tamoxifen suppressed tumor growth better than individual groups in vivoConclusions: PDE4D plays a pivotal role in acquired tamoxifen resistance via blocking cAMP/ER stress/p38-JNK signaling and apoptosis. Clin Cancer Res; 24(8); 1987-2001. ©2018 AACR.

Abstract P3-06-03: Dual p38/NLK kinase inhibitor as potential novel therapeutic agent for tamoxifen-resistant luminal breast cancer

Abstract Background: Tamoxifen is the most commonly used endocrine agent for estrogen receptor (ER) positive breast cancer (also known as luminal breast cancer). However, approximately half of the patients develop resistance after initial response to tamoxifen. To date, no effective targeted therapy exists to overcome it. We previously identified the role of nemo-like kinase (NLK), a serine-threonine kinase that functions in stress response and neurite outgrowth, in breast endocrine resistance. In addition, activation of p38 MAPK has been reported to modulate ER signaling and promote endocrine resistance. We identified a highly selective dual p38 and NLK kinase inhibitor (PNKI) through analysis of public kinase profiling datasets, and evaluated its therapeutic effect in endocrine-resistant breast cancers using in vitro and in preclinical mouse models. Experimental design and methods: To determine the in vitro therapeutic window of PNKI, we treated an acquired tamoxifen-resistant cell line (MCF7-TamR) and a benign breast epithelial cell line (MCF10A) with gradually increasing doses of PNKI. To determine the effect of PNKI on tamoxifen-resistant breast cancer cells, we treated primary tamoxifen-resistant breast cancer cell line BT483, and MDAMB415, together with acquired tamoxifen-resistant line MCF7 TamR, T47D TamR, and ZR-75-B TamR, with 0.5 uM PNKI in the presence of different doses of Tamoxifen. To evaluate the therapeutic effect of PNKI in a T47D-derived xenograft tumor model with acquired tamoxifen resistance, we administered PNKI alone or in combination with Fulvestrant, the second-line endocrine therapy agent, or with Everolimus, the mTOR inhibitor that could improve patient outcomes in several clinical trials. Mice bearing xenografts were randomized into six treatment groups (Vehicle, PNKI, Fulvestrant, Fulvestrant+PNKI, Everolimus, Everolimus+PNKI). Tumor growth was tracked closely. The tumors harvested two weeks after treatments started were profiled with Reverse Phase Protein Array (RPPA) to assess the early signaling changes after treatments. The therapeutic effect of PNKI were also evaluated in a patient-derived xenograft (PDX) model of de novo endocrine resistant breast cancer. Mice bearing the PDX tumors were randomized to four treatment groups (Vehicle, PNKI, Everolimus, Everolimus+PNKI) and tumor growth curve was measured timely. Results: PNKI showed an in vitro therapeutic window at 0.1-1μM for MCF7-TamR cells. Breast cancer cell lines with either de novo or acquired Tamoxifen resistance became more sensitive to tamoxifen when treated with 0.5uM PNKI. The concomitant treatment of PNKI and Everolimus results in significant decreased tumor burden and prolonged progression free survival in the both T47D-TamR xenograft tumors and re-transplanted de novo endocrine-resistant PDX tumors compared to other treatments. RPPA data of T47D-TamR tumors harvested following 2-week treatments revealed that several key survival signaling in breast cancer are repressed only when PNKI are combined with Everolimus. Conclusion: The dual p38 and NLK inhibitor (PNKI) exhibited potential therapeutic value as adjuvant agent to the mTOR inhibitor everolimus for acquired or de novo tamoxifen-resistant luminal breast cancers. Citation Format: Wang X, Cao X, Veeraraghavan J, Qin L, Kim J-A, Tan Y, Hilsenbeck SG, Schiff R, Wang X. Dual p38/NLK kinase inhibitor as potential novel therapeutic agent for tamoxifen-resistant luminal breast cancer [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 P3-06-03.

Abstract OT3-05-10: A single arm phase II study of palbociclib in combination with tamoxifen as first line therapy for metastatic hormone receptor positive breast cancer

Abstract Background: Hormone receptor positive breast cancer is the most commonly diagnosed subset of breast cancer (60-65%). Endocrine therapy is effective for this subset of breast cancer, in both the adjuvant and metastatic settings. Despite advances in endocrine therapy, many patients relapse during or after completing adjuvant therapy and metastatic breast cancer remains incurable. Palbociclib is a reversible, oral, small molecule inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6). CDK4 and CDK6 together with cyclin D have important roles in regulation of the G1/S transition via regulation of the phosphorylation state of retinoblastomaprotein (Rb). Palbociclib showed significantly improved progression-free survival taken together with endocrine agents in treatment of metastatic breast cancer. Preclinical data showed that in combination with tamoxifen, palbociclib had synergistic growth inhibitory activity as well as efficacy in a model of acquired tamoxifen resistance. Combining palbociclib with tamoxifen in first line treatment of metastatic hormone receptor positive breast cancer may offers an appealing alternative to other endocrine combinations. Methods: This is a non-randomized, open-label, single-arm, multicenter, phase II study of palbociclib in combination with tamoxifen in patients with hormone receptor positive/HER2 negative advanced breast cancer. The primary objective is to determine the objective response rate (complete or partial response) based on RECIST 1.1 or MDA Criteria (for patients with bone only disease). Secondary objectives are: safety and tolerability, progression-free survival, clinical benefit rate, 2-year overall survival. Correlative objectives will explore alterations in circulating tumor DNA and changes in gene expression pattern at the time of progression. Eligibility criteria: women or men with diagnosis of hormone receptor positive/ HER2 negative locally advanced or metastatic breast cancer, not amenable to curative surgery; no prior systemic anti-cancer therapy for advanced hormone receptor positive breast cancer; adequate organ function; pre and post menopausal women are allowed. Drug administration: palbociclib dose will be 125 mg orally once daily on days 1-21 of each 28-day cycle; tamoxifen dose will be 20 mg orally once daily for every day of the 28-day cycle. As of June 2017, the study enrolled 10/71 patients and it is still open to enrollment. NCT 02668666; ocdanciu@uic.edu Citation Format: Danciu OC, Hoskins K, Tamkus D, Truica C, Blaes A, Green L, Liu L, Toppmeyer D, Wisinski K. A single arm phase II study of palbociclib in combination with tamoxifen as first line therapy for metastatic hormone receptor positive breast cancer [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 OT3-05-10.

Acquired Tamoxifen Resistance Is Returned by GW8510 Through RRM2 Downregulation-Mediated Autophagy Induction

Acquired Tamoxifen Resistance Is Returned by GW8510 Through RRM2 Downregulation-Mediated Autophagy Induction

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.

Potential role of solid lipid nanoparticles in overcoming the acquired tamoxifen resistance of breast cancer

Potential role of solid lipid nanoparticles in overcoming the acquired tamoxifen resistance of breast cancer

Abstract 1223: Evaluation of the dual p38/NLK kinase inhibitor as potential new therapeutic agent for tamoxifen-resistant breast cancer

Abstract Background: Endocrine therapy has been considered an effective initial treatment for ER positive breast cancer and tamoxifen is the most commonly used endocrine agent. However, about half of the patients develop resistance or relapse eventually. No effective targeted therapy exists to overcome it. In our previous study, we have identified the role of nemo-like kinase (NLK) in breast cancer endocrine resistance – a serine-threonine kinase that functions in stress response and neurite outgrowth. In addition to NLK, activation of the other stress kinases such as p38 MAPK has been reported to modulate ER signaling and promote endocrine resistance. In this study, we have identified a highly selective dual p38 and NLK kinase inhibitor (PNKI). This study aimed to evaluate the therapeutic effect of the PNKI inhibitor in tamoxifen-resistant breast cancers using in vitro and preclinical mouse models. Experimental design and methods: To determine the effect of PNKI on tamoxifen-resistant breast cancer cells, we treated a primary tamoxifen-resistant breast cancer cell line MDAMB415 and an acquired-resistant line MCF7 TamR, with 0.5 uM PNKI in the presence of different doses of Tamoxifen. To evaluate the therapeutic effect of PNKI in a T47D-derived xenograft tumor model with acquired tamoxifen resistance, we administered PNKI alone or in combination with Fulvestrant, or with the mTOR inhibitor Everolimus. Mice bearing T47D-TamR xenografts were randomized into six treatment groups (Vehicle, PNKI, Fulvestrant, Fulvestrant + PNKI, Everolimus, Everolimus + PNKI). Tumor growth was tracked closely. The tumors harvested 2 weeks following treatments were profiled with Reverse Phase Protein Array (RPPA) to assess the early signaling changes after treatments. Furthermore, the therapeutic effect of PNKI were also evaluated in a patient-derived xenograft (PDX) model of de novo endocrine resistant breast cancer. Mice bearing the PDX tumors were randomized to one of four treatment groups (Vehicle, PNKI, Everolimus, Everolimus + PNKI) and tumor growth curve was measured. Summary of the Results: Breast cancer cell lines with either de novo or acquired Tamoxifen resistance became more sensitive to tamoxifen when treated with 0.5 uM PNKI. The concomitant treatment of PNKI and Everolimus results in significant decrease of tumor growth in the T47D-TamR xenograft tumor compared to Fulvestrant, Fulvestrant + PNKI, PNKI, or Everolimus treatments. RPPA data revealed that a majority of key survival signaling in breast cancer are repressed only when PNKI are combined with Everolimus. The de novo endocrine-resistant PDX tumors showed diverse response to PNKI mono-treatment, whereas the combination of PNKI and Everolimus resulted in significantly decreased tumor growth. Conclusion: The PNKI exhibited potential therapeutic value as adjuvant agent to the mTOR inhibitor everolimus for acquired or de novo tamoxifen-resistant breast cancer. Citation Format: XIAN WANG, Xixi Cao, Jamunarani Veeraraghavan, Lanfang Qin, Jin-Ah Kim, Ying Tan, Susan G. Hilsenbeck, Rachel Schiff, Xiaosong Wang. Evaluation of the dual p38/NLK kinase inhibitor as potential new therapeutic agent for tamoxifen-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1223. doi:10.1158/1538-7445.AM2017-1223

Molecular Analysis of Acquired Tamoxifen Resistance in Breast Cancer Cell Line

Background: Tamoxifen is an FDA approved drug for the prevention and the treatment of breast cancer, but its therapeutic benefit is limited by the development of drug resistance. Many Studies suggest that a basic biological difference exists between tumors with acquired tamoxifen resistance and those with intrinsic resistance to the drug. However the reasons why human mammary tumors become resistant to tamoxifen therapy are mainly unknown. Changes in gene expression may occur as cells acquire resistance to tamoxifen. A better understanding of gene expression alterations associated with tamoxifen resistance will facilitate circumventing this problem.Methods: We undertook a comparative gene expression analysis of tamoxifen-sensitive (MCF7 and T47D) and acquired tamoxifen-resistant human breast cancer cell lines (T47D tamoxifen resistant) in vitro models using Real-time PCR, to analyze differential gene expression. These genes were functionally linked to 4 major groups named as ER signaling/cell cycling, EGFR signaling, cancer stem cell (CSCs) and apoptosis.Results: Our results have been widely demonstrated that the altered expression of some genes involved in apoptosis, EGFR signaling, CSCs and Cell cycle-independent expression of estrogen receptor such as ESR1, TP53, CDKN1B, Casp3, CD44, CD24, BAX, Bcl2, Her2, and PTEN in T47D tamoxifen resistant cell line can be resulted in drug resistance in long-term treatment.Conclusions: Our findings correspond with the results of many earlier studies indicated tamoxifen resistance can be a result from combination of molecular mechanisms including Her2 activation, cell cycle progression out of ESR1 control, increased ratio of CSCs and inhibition of mitochondrial apoptosis.

Acquired Tamoxifen Resistance in MCF-7 Breast Cancer Cells Requires Hyperactivation of eIF4F-Mediated Translation.

While selective estrogen receptor modulators, such as tamoxifen, have contributed to increased survival in patients with hormone receptor-positive breast cancer, the development of resistance to these therapies has led to the need to investigate other targetable pathways involved in oncogenic signaling. Approval of the mTOR inhibitor everolimus in the therapy of secondary endocrine resistance demonstrates the validity of this approach. Importantly, mTOR activation regulates eukaryotic messenger RNA translation. Eukaryotic translation initiation factor 4E (eIF4E), a component of the cap-dependent translation complex eIF4F, confers resistance to drug-induced apoptosis when overexpressed in multiple cell types. The eIF4F complex is downstream of multiple oncogenic pathways, including mTOR, making it an appealing drug target. Here, we show that the eIF4F translation pathway was hyperactive in tamoxifen-resistant (TamR) MCF-7L breast cancer cells. While overexpression of eIF4E was not sufficient to confer resistance to tamoxifen in MCF-7L cells, its function was necessary to maintain resistance in TamR cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of TamR cells and partially restored sensitivity to tamoxifen. Further, the use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, the use of a pharmacologic agent which inhibited the eIF4E-eIF4G interaction also decreased the proliferation and anchorage-dependent colony formation in TamR cells. These results highlight the eIF4F complex as a promising target for patients with acquired resistance to tamoxifen and, potentially, other endocrine therapies.

Simvastatin suppresses the DNA replication licensing factor MCM7 and inhibits the growth of tamoxifen-resistant breast cancer cells

Acquired tamoxifen resistance (TamR) remains a major challenge in breast cancer endocrine therapy. The mechanism of acquiring tamoxifen resistance remains elusive, and no effective drugs are available. In this investigation, we determined that the expression of the DNA damage marker γH2AX is upregulated under minichromosome maintenance protein 7 (MCM7) knockdown in phospho Ser807/811-retinoblastoma protein (p-Rb) defect cells. In addition, the expression of p-Rb was lower in TamR cells than in parental cells, and the expression of γH2AX was significantly upregulated when MCM7 was knocked down in TamR cells. Simvastatin, an agent for hypercholesterolemia treatment, activated the MCM7/p-RB/γH2AX axis and induced DNA damage in TamR cells, especially when combined with tamoxifen. Finally, in vitro and in vivo experiments demonstrated that simvastatin combined with tamoxifen increased TamR cell apoptosis and inhibited xenograft growth. In conclusion, simvastatin may suppress TamR cell growth by inhibiting MCM7 and Rb and subsequently inducing DNA damage.

SH003 enhances paclitaxel chemosensitivity in MCF-7/PAX breast cancer cells through inhibition of MDR1 activity.

Paclitaxel is an anti-cancer drug for treating cancer, but paclitaxel resistance is reported in cancer cells. Multidrug resistance (MDR) is related with the epithelial-to-mesenchymal transition (EMT) mechanism, which plays a key role in cancer metastasis. Moreover, EMT mechanism is connected to tamoxifen resistance in breast cancer cells. Consequently, oncologists are interested in finding new MDR1 inhibitors originating from herbal medicines to have less side-effect. Here, we investigated an inhibition effect of SH003 on MDR1 activity in paclitaxel-resistant MCF-7/PAX breast cancer cells. Our results showed that paclitaxel did not inhibit a proliferation in paclitaxel-resistant MCF-7 breast cancer cells. Paclitaxel-resistant MCF-7 cells showed an increase of MDR1 activity, which was confirmed by measuring an amount of accumulated rhodamine 123 in the cells. Also, qRT-PCR and Western blot assays confirmed that paclitaxel-resistant MCF-7 cells exhibited high MDR1 expression level. Furthermore, paclitaxel-resistant MCF-7 cells showed mesenchymal morphology with alterations of EMT markers, and acquired tamoxifen resistance with a decrease of ERα expression. We also found that a combinatorial treatment of SH003 and paclitaxel in paclitaxel-resistant MCF-7 cells caused apoptosis in synergistic manner, which was due to SH003 inhibition of MDR1 expression. Therefore, SH003 could be a potential agent for overcoming MDR in drug-resistant cancer cells.