Resistant Breast Cancer Cell Lines Research Articles

Abstract P3-14-07: Intra-cellular dsRNA receptor RIG-I: A ubiquitous novel target for treatment of chemotherapy drug resistant breast cancer

Abstract INTRODUCTION: Toll-like receptor 3 (TLR3), the cell surface receptor for double stranded RNA (dsRNA), expressed in ∼40% of advanced primary breast cancers (T2/T3/N+) has been shown to be an effective therapeutic target for synthetic dsRNA poly A:U in combination with radiation and adjuvant chemotherapy, producing a significant decrease in risk of metastatic relapse (HR 1.85-2.0; 1.03-3.89; Cancer Res;71:1607). AIM: The present pre-clinical study aimed to explore the therapeutic potential of a ubiquitously expressed intracellular dsRNA receptor, retinoic-acid-inducible gene-I (RIG-I) - a cytoplasmic pathogen recognition receptor directed at pathogen-associated molecular pattern (PAMP) motifs to differentiate viral from cellular dsRNAs. We have shown Ampligen, poly I:C12U (a synthetic dsRNA polymer designed to rapidly degrade in vivo to prevent the toxicity of long dsRNA polymers such as poly I:C) is capable of entering cells in its fragmented dsRNA oligomeric form (<1–2 kb) optimal for activation of intra-cellular RIG-I (J Exp Med 2008;205:1601–1610). METHODOLOGY & RESULTS: Preliminary experiments with Ampligen on several human breast cancer cell lines (MCF-7 & MDA-MB 453) and normal human mammary epithelial & fibroblast cell lines (HMEC & HFC) unexpectedly showed it to consistently cause a significant loss of cell viability (CellTiter-Glo Luminescent Cell Viability Assay) in p53-deficient drug resistant (5-FU/doxorubicin) MDA-MB 453 cell line in contrast to cell growth arrest (Guava cell Cycle Assay) in p53 wild type MCF-7 cancer and the two non-neoplastic cells lines. This selective effect was confirmed using syngeneic clones of MCF-7 breast cancer cells stably transfected with a dominant negative p53 construct or vector alone: p53-function blocked (DD1) vs p53-function active (EV1) MCF-7 cell lines, respectively (J Nucl Med 2006; 47:1525–1530), and shown to be associated with RIG-I specific mRNA induction (RT-PCR) and Type I interferon pathway activation both inhibited by BX795 (selective inhibitor of IRF3 activation and IFN-b production). Decitabine (DNA demethylating drug capable of intra-cellular generation of dsRNA through transcriptional activation of Alu retrotransposons - PNAS 2012; December 10: E89–E98 ) was next tested as an alternative source of intra-cellular dsRNA, and found to produce results similar to Ampligen on DD1 and EV1. Work is in progress to examine dose response and time-course relationships of the effects Ampligen or decitabine added singly or in combination with chemotherapeutic drug (e.g. doxorubicin) on DD1 and triple negative breast cancer cell lines such as MDA-MB 453 to explore potential therapeutically most effective protocols. PROVISIONAL CONCLUSIONS: Intra-cellular dsRNA receptor RIG-I constitutively expressed in all cells offers a more ubiquitous target compared to TLR3 for the treatment of breast cancer. Ampligen and decitabine with their selective growth inhibitory effect on drug resistant p53-deficient breast cancer cell lines, merit testing as novel drugs for treatment of p53-deficient drug resistant breast cancer e.g. triple negative breast cancer frequently (∼70%) associated with drug resistance and altered p53 status. Citation Format: Jasani B, Navabi N, Barrett-Lee P, Thompson A, Chester J, Mason M. Intra-cellular dsRNA receptor RIG-I: A ubiquitous novel target for treatment of chemotherapy drug resistant breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-07.

Molecular characterization of irinotecan (SN-38) resistant human breast cancer cell lines.

BackgroundStudies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30 % response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC.MethodsWe established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6 months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump.ResultsWe found that the resistant cell lines showed 7–100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance.ConclusionsBased on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2071-1) contains supplementary material, which is available to authorized users.

Essential oils from Inula japonica and Angelicae dahuricae enhance sensitivity of MCF-7/ADR breast cancer cells to doxorubicin via multiple mechanisms

Ethnopharmacological relevanceAngelicae dahurica (Hoffm.) Benth. & Hook.f.ex Franch. & Sav combined with Pueraria and Gastrodia elata Bl. combined with Inula japonica Thunb. are widely used in herb-pairs of traditional chinese medicine. Previous studies have shown that Angelicae dahuricae essential oil (ADO) enhanced puerarin internalization into ABCB1-overexpressed Caco-2 cells. These findings suggest the possibility that essential oils may enhance the absorption via certain mechanisms related to ABCB1 and reverse multidrug resistance (MDR). Aim of the studyADO and essential oils from Inula japonica (IJO) may reverse ABCB1-mediated MDR, but this ability has not been investigated in detail in the well-established cancer cell lines. In this study, the underlying molecular mechanisms were further investigated to examine how IJO and ADO reverse MDR in the resistant human breast cancer cell line of MCF-7/ADR. Also this work may help uncover the conceivable compatibility mechanisms of above herb-pairs involved in ABCB1. Materials and methodsThe MDR human breast cancer MCF-7/ADR cells were treated with IJO, its sesquiterpene component isoalantolactone (ISO) or ADOat non- cytotoxic concentrations. The MDR ability was examined by measuring the sensitivity to doxorubicin (DOX), DOX accumulation and efflux, ABCB1 ATPase activity, ABCB1 expression, membrane fluidity, and stability and localization of lipid rafts and caveolae. Finally, the molecular modeling was performed to postulate how ISO interacts with ABCB1. ResultsTreating MCF-7/ADR cells with IJ oil, ISO or AD oil reversed MDR 2- to 3-fold, without affecting the sensitivity of the non-MDR parental cell line. Mechanistic studies showed that these oils down-regulated mRNA and protein expression of ABCB1, and reduced the stability of lipid rafts in the cell membrane, which has previously been shown to reduce ABCB1-mediated transport. On the other hand, IJO, ISO and ADO did not inhibit ABCB1 ATPase activity, and fluorescence polarization experiments showed that low concentrations of the oils did not appear to alter membrane fluidity, unlike some MDR-reversing agents, ISO showed a higher docking score than verapamil but lower than dofequidar and tariquidar. ConclusionsOur results suggest that IJO, ISO and ADO could reverse MDR by down-regulating ABCB1 expression and reducing lipid raft stability. These findings may be useful for developing safer and effective MDR reversal agents and also help find out the compatibility mechanisms.

Abstract A02: The HER4 BH3-only protein and tumor suppressor is epigenetically silenced in endocrine refractory and triple negative breast cancers

Abstract Breast cancer is the most commonly diagnosed cancer in North American women and remains the second most common cause of cancer related deaths in women. In contrast to the mitogenic activities of the EGFR-family members EGFR itself, HER2, and HER3; HER4, the fourth and final member of the EGFR-family to be discovered, suppresses breast tumor cell growth. HER4 is also unique because it is the only member of the EGFR family to undergo proteolytic cleavage at the cell surface to generate an independently signaling intracellular domain (4ICD). Clinically, HER4 expression is extinguished during breast tumor progression and patients with tumors lacking HER4 expression have significantly worse prognosis. We investigated the apparent tumor suppressor activity of HER4 and mechanisms of HER4 suppression during breast tumor progression. We found that 4ICD harbors a pro-apoptotic BH3-only domain required for its tumor cell killing activity. To evade the potent tumor cell killing activity of 4ICD, tumor cells acquire mechanisms to suppress HER4 expression. Importantly, we identified a CpG island within the HER4 promoter and show by pyrosequencing of bisulfite treated DNA an inverse correlation between HER4 expression and the extent of promoter methylation. Clinically, increased levels of HER4 promoter methylation were significantly associated with the most aggressive breast cancer subtypes (hormone refractory, HER2 positive, and triple negative) and significantly worse patient prognosis. Consistent with these clinical findings, the HER4 promoter is hypermethylated and HER4 expression is suppressed in multiple endocrine resistant and triple negative breast cancer cell lines. Reestablishing, by ectopic expression, or reactivating, through 5-aza-2-deoxycytidine treatment, HER4 expression results in dramatic BH3-dependent apoptosis of both endocrine resistant and triple negative breast cancer cell lines. To identify alterations in epigenetic regulators common to multiple breast tumor cell lines with suppressed HER4 expression we used Qiagen RT-Profiler PCR Arrays to analyze expression of 168 key chromatin modifying enzymes and remodeling factors. The most dramatic alterations common to two endocrine resistant and three triple negative breast tumor cell lines were suppression of the chromatin remodeling protein CHD5 and suppression of the TET2 deoxygenase - which initiates a DNA demethylation pathway. We are currently investigating the impact of modulated CHD5 and TET2 expression on the reactivation of HER4 cell killing activity in endocrine resistant and triple negative breast tumors. In conclusion, our data supports a pro-apoptotic tumor suppressor function for HER4 which is epigenetically suppressed in endocrine refractory and triple negative breast tumors through promoter hypermethylation. Reactivation of epigenetically silenced HER4 results in dramatic tumor cell apoptosis, providing a therapeutic strategy for the treatment of the most aggressive forms of breast cancer. Note: This abstract was not presented at the conference. Citation Format: Wen Han, Eleanor Semmes, Frank E. Jones. The HER4 BH3-only protein and tumor suppressor is epigenetically silenced in endocrine refractory and triple negative breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A02.

Abstract B19: Quantitative proteomics analysis identifies MUC1 as an effect sensor of EGFR inhibition

Abstract Introduction The epidermal growth factor receptor (EGFR) is often hyperactivated through overexpression or somatic mutation in multiple cancer types, driving tumor growth and survival. Targeted therapies against EGFR show clinical benefit, but resistance to these agents invariably develops. Thus, there is need for markers of effective treatment, termed ‘effect sensors’, to monitor the effective treatment with EGFR therapeutics during therapy. Therefore, we investigated the cellular rewiring at the proteomic level in response to EGFR inhibition, with the aim of identifying proteins that can be used as effect sensors. These effect sensors potentially constitute powerful targets for the development of tracers to monitor EGFR-directed therapies. Materials and Methods Human breast cancer cell lines were tested for cell viability in presence of the EGFR inhibitor erlotinib and sensitive cell lines (SKBR3, SUM149, and BT474) were identified. These cell lines were labeled with stable (SILAC) isotopes and subsequently control-treated or treated with erlotinib for 48 hours. Membranes were then isolated and analyzed with quantitative mass-spectrometry (MS). Results were validated in erlotinib-sensitive (SKBR3, SUM149, and BT474) and resistant breast cancer cell lines (MB-231 and BT549), as well as erlotinib-sensitive human non-small cell lung cancer (NSCLC) cell lines (H292 and HCC827). Results In total, 1,485 overlapping proteins were quantitatively determined in all cell lines, containing 256 plasma membrane proteins as determined using Gene Ontology. Of those, 8 proteins showed at least 1.5 fold up-regulation and were selected for validation. We found MUC1 to be significantly upregulated in response to erlotinib treatment. Upregulation of MUC1 was detected both at protein and mRNA levels in all erlotinib-sensitive breast cancer cell lines, but not in erlotinib-resistant cell lines. In addition, MUC1 levels were elevated upon erlotinib treatment in NSCLC cell lines H292 (EGFR-WT) and HCC827 (EGFR-exon-19 deletion). MUC1 upregulation was observed as early as 48 hours after treatment, and persisted until 14 days with continuous erlotinib treatment. Upregulation of MUC1 was also observed in response to other clinical EGFR inhibitors, including lapatinib and afatinib, as well as in response to the monoclonal EGFR-directed antibody cetuximab. Erlotinib-induced MUC1 expression was dependent on PI3K/AKT/mTOR signaling, since MUC1 expression was completely blocked when EGFR inhibition was combined with either BEZ235, MK2206, or everolimus, which inhibit PI3K, AKT, and mTOR respectively. Importantly, MUC1 upregulation could be measured by flow cytometry, indicating that elevated MUC1 levels are expressed at plasma membranes and underscoring that MUC1 is a potential target for tracer development for medical imaging. Conclusion We identified MUC1 to be specifically upregulated in response to EGFR-directed treatments in breast and lung cancer cell lines. These results warrant in vivo assessment of MUC1 as an effect sensor, to ultimately test whether this strategy can be employed to stratify patients for treatment with EGFR therapeutics. Supported by ERC grant OnQview-2011-293445 to EGE de Vries. Citation Format: Harmen R. de Boer, Esméé Joosten, Fabrizia Fusetti, Rudolf S.N. Fehrmann, Elisabeth G.E. de Vries, Marcel A.T.M. van Vugt. Quantitative proteomics analysis identifies MUC1 as an effect sensor of EGFR inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B19.

Abstract A182: Targeting the topoisomerase I enzyme in cancer cells with acquired resistance to SN-38

Abstract De novo or acquired resistance to anti-cancer drugs represents a major obstacle to successful treatment of cancer patients. In order to improve future cancer drug development we have established the DEN-50R cell line panel which when fully developed will consist of isogenic pairs of drug sensitive and resistant human cancer cell lines representing the 5 most common cancer types. Based on the DEN-50R we have tested a number of novel anti-cancer drugs in this panel of cell lines. One class of drugs that includes the indenoisoquinolines LMP400 and LMP776, which have topoisomerase 1 inhibitory activity and recently passed phase 1 clinical studies, showed very interesting features in the cell line studies. When tested in SN-38 (the active metabolite of irinotecan) -sensitive and -resistant isogenic colorectal cancer cell lines and breast cancer cell lines, LMP400 showed significant dose related cytotoxicity independent of the state of SN-38-resistance (Table). As these resistant cell lines have significant up-regulation of either the BCRP and/or mdr-1 protein, it can be concluded that the indenoisoquinoline LMP400 can target topoisomerase 1 enzyme without being a substrate for these drug efflux pumps. We are now planning to initiate two clinical phase II studies (Simon's two stage design) with LMP400; one in irinotecan-failed metastatic colorectal cancer patients and one in late stage metastatic breast cancer patients. Patients for these studies will be preselected based on an LMP400-responsiveness profile we generated by gene expression data where associations between gene expression profiles and growth inhibition were compared in a panel of cell lines exposed to LMP400. A second step included filtering the identified gene expression profile against mRNA expression from a collection of 3200 human tumors. Only genes being differentially expressed in the clinical tumor material were retained in the model. Sensitivity to LMP776 and LMP400 of human colorectal and breast cancer cell lines with acquired resistance to SN-38 Cell lineCross-resistantSensitiveHCT116-SN-38*LMP400; LMP776HT29-SN-38LMP776LMP400LoVo-SN-38LMP776LMP400MDA-MB-231- SN-38LMP776LMP400MCF-7- SN-38LMP776; LMP400* HCT-116-SN38 resistant cells have gained a mutation in the binding site of SN38 and LMP400 during acquisition of SN-38 resistance. Citation Format: Jan Stenvang, Niels Frank Jensen, Haatisha Jandu, Steen Knudsen, Keli Agama, Thomas Jensen, Anker Hansen, Peter Buhl Jensen, Yves Pommier, Mark Cushman, Nils Brünner. Targeting the topoisomerase I enzyme in cancer cells with acquired resistance to SN-38. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A182.

E2F7 overexpression leads to tamoxifen resistance in breast cancer cells by competing with E2F1 at miR-15a/16 promoter.

About 50-70% of breast cancers are estrogen receptor α (ERα) positive and most of them are sensitive to endocrine therapy including tamoxifen. However, one third of these patients will eventually develop resistance and relapse. We found that the expression of miR-15a and miR-16 were significantly decreased in tamoxifen resistant ER positive breast cancer cell lines. Exogenous expression of miR-15a/16 mimics re-sensitized resistant cells to tamoxifen by inhibiting Cyclin E1 and B cell lymphoma-2 (Bcl-2) to induce cell growth arrest and apoptosis respectively. Further, we identified that a repressive member of E2F family, E2F7, was responsible for the suppression of miR-15a/16 cluster by competing with E2F1 for E2F binding site at the promoter of their host gene DLEU2. Moreover, high expression of E2F7 is correlated with high risk of relapse and poor prognosis in breast cancer patients receiving tamoxifen treatment. Together, our results suggest that overexpression of E2F7 represses miR-15a/16 and then increases Cyclin E1 and Bcl-2 that result in tamoxifen resistance. E2F7 may be a valuable prognostic marker and a therapeutic target of tamoxifen resistance in breast cancer.

Potential Therapeutic Effect of Natural Killer Cells on Doxorubicin-Resistant Breast Cancer Cells In Vitro.

ObjectiveThe aim of this study was to explore the therapeutic effect of natural killer (NK) cells on human doxorubicin-sensitive and resistant breast adenocarcinoma.MethodsHuman doxorubicin-sensitive and resistant breast cancer cell lines (MCF-7 and MCF-7/ADR) were tagged with renilla luciferase (Rluc) (MCF-7/RC and MCF-7/ADR/RC). NK cells were tagged with enhanced firefly luciferase (effluc) using a recombinant retrovirus transfection (NKF). Expression of Rluc, effluc, and NK cell surface markers CD16, CD56 as well as death receptors, DR4 and DR5, were assessed by using flow cytometry. In vitro cytotoxic effect of NK to MCF-7 and MCF-7/ADR was measured and in vivo bioluminescence imaging was also performed to visualize MCF-7/RC, MCF-7/ADR, and NKF in an animal model.ResultsNK92-MI, MCF-7, and MCF-7/ADR cells were successfully labeled with Rluc or effluc. Both the target breast cancer cells (with Rluc) and therapeutic NK cells (with effluc) were noninvasively visualized in nude mice. Doxorubicin-resistant breast cancer cells (MCF-7/ADR) presented a higher expression of DR5 and were more sensitive to NK cells compared with doxorubicin-sensitive breast cancer cells (MCF-7).ConclusionThe results of present study suggest that NK cell therapy has a therapeutic effect on doxorubicin-sensitive and resistant breast cancer cells.

Abstract 2703: Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106

Abstract Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that is overexpressed in about 20% of breast cancers and most often in the more aggressive inflammatory breast cancer (IBC). Lapatinib is one of the most potent small molecule inhibitors of HER2. Although Lapatinib has shown to be an effective targeted therapy, development of drug resistance stands as a major problem. Here we show that the anti-cancer activity of the novel fatty acid synthase inhibitor HS-106 can re-sensitize resistant cell lines to Lapatinib. Lapatinib resistant SKBR3 and BT474 breast cancer cell lines were treated with increasing concentrations of HS-106 either alone or in combination with Lapatinib and their effects on cell proliferation were assessed using the DNA dye Hoechst 33342. The ability of HS-106 and Lapatinib drug combination to induce apoptosis were measured in Caspase 3/7 activity assay using the fluorogenic Caspase substrate (DEVD)2 R110. Their effect on apoptosis was further confirmed in Lapatinib resistant BT474 cells using flow cytometry based Annexin V assay. In addition, the effect of HS-106 on the expression levels and phosphorylation of the kinases involved in Her2 pathway including Her2, AKT and ERK were determined. The fatty acid synthase inhibitor HS-106 significantly inhibits the proliferation of Lapatinib resistant cell lines with concentrations. Interestingly, when combined with Lapatinib, the drugs synergize to dramatically affect cell growth. This anti-proliferative activity of HS-106 was found to be the result of induction of apoptosis, Caspase 3/7 activity was found to increase in a dose dependent manner following HS-106 treatment. Low concentrations of Lapatinib and HS-106, which does not induce Caspase activity when administrated separately, were found to significantly induce Caspase activity when combined together. These results were confirmed with the Annexin V assay. By examining the expression levels of HER2 pathway proteins we found a decrease in HER2 levels which was reflected on the levels of the downstream kinases AKT and ERK when treating with high concentrations of HS-106. In a previous work, HS-106 showed a good tolerability and anti-cancer activity in the MMTV neu+ mouse model. By showing potent efficacy against Lapatinib resistant breast cancer cell lines, our data suggest that HS-106 is an excellent drug candidate to advance into translational studies for the treatment of Lapatinib resistant breast cancer. Citation Format: Yazan Alwarawrah, Philip Hughes, Rachid Safi, Donald P. McDonnell, Neil L. Spector, Timothy A. Haystead. Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2703. doi:10.1158/1538-7445.AM2015-2703

Abstract 4067: Pluronic micelle-encapsulated Disulfiram targets cancer stem-like cells and reverses pan-resistance in acquired resistant breast cancer cell lines

Abstract Background: Chemoresistance is the major obstacle for the success of cancer treatment. The advanced breast cancer (BC) is commonly pan-resistant to different anticancer drugs. BC contains a small population of cancer stem cells (CSCs). CSCs express stem cell markers (CD24low/CD44high, ALDH+, CD133+) and are resistant to a wide range of anticancer drugs. Therefore, CSCs may be responsible for the pan-chemoresistance in advanced BC. We have recently developed several acquired chemoresistant BC cell lines. In this study, we investigated CSC traits in these cell lines and examined the cross-resistance of these cell lines to a panel of anticancer drugs. Disulfiram (DS), an antialcoholism drug, shows high cytotoxicity in several different types of cancer cell lines in vitro. It also targets CSCs and reverses pan-chemoresistance. However, the clinical application of DS in cancer treatment is limited by its very short half-life in the bloodstream. In order to increase the bio-stability of DS in the bloodstream, we recently developed a pluronic micelle-encapsulated DS (PM-DS). In this study, we examined the cytotoxicity of a newly developed (PM-DS) in the resistant cell lines. The effect of PM-DS on CSC traits in the resistant BC cell lines was also examined. Cell lines and Methods: Cell lines: MDA-MB-231, MCF7 and T47D and acquired resistant BC cell lines [gemcitabine (MDA-MB-231GEM100), 5-fluouracil (MCF7FU10) and doxorubicin (T47DDOX100nM)]; Methodologies: MTT assay, Western blot, CSC markers detection (ALDH, CD24low/CD44high, CD133), embryonic stem cell markers detection (Nanog, Sox2, Oct 4). Results: The resistant cell lines are highly cross-resistant to 6 conventional anticancer drugs e.g doxorubicin, gemcitabine, docetaxel, cisplatin, 5-fluouracil and vincristine. In comparison with the parental cells, the resistant cell lines have significantly longer doubling time. Significantly higher population of resistant cells expresses high levels of CSC markers e.g. ALDH+, CD24low/CD44high, and CD133+ and embryonic stem cell markers (OCT4, Sox2 and Nanog). DS was successfully encapsulated in pluronic micelles with a high entrapment efficiency of about 90% and our in vitro release studies showed that DS was slowly released from the micelles. Our formulation PM-DS has a relatively small size of about 200nm with high drug loading content (90μgDS/1mgPM). PM-DS was highly toxic to chemoresistant cell lines. It also targeted CSC population and enhanced cytotoxicity of anticancer drugs in the resistant cell lines. Conclusions: The acquired resistant BC cell lines are highly cross-resistant to 6 different kinds of anticancer drugs. CSCs may be responsible for the cross-chemoresistance. PM-DS targets CSC population and reverses the acquired pan-chemoresistance in acquired resistant BC cell lines. Key words: Disulfiram, micelles, breast cancer, stem-like cells. Citation Format: EREBI P. TAWARI, Peng Liu, Zhipeng Wang, Vinodh Kannappan, Christopher Mcconville, Angel Armesilla, John Darling, Juan Irache, Krassimira Yoncheva, Weiguang Wang, Petar Petrov. Pluronic micelle-encapsulated Disulfiram targets cancer stem-like cells and reverses pan-resistance in acquired resistant breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4067. doi:10.1158/1538-7445.AM2015-4067

Abstract 726: Constitutive NF-kB-activation loop enhances resistance to trastuzumab in HER2 positive luminal B breast cancer

Abstract [Purpose] Luminal B breast cancer patients are significantly at the risk of relapse with in 5 years. Acquired resistance to the receptor tyrosine kinase HER2 targeting antibody trastuzumab is a major clinical problem in the treatment of recurrent HER2-positive breast cancer. The molecular mechanisms leading to trastuzumab resistance (TZR) remain unclear in luminal B breast cancer. Here, we investigated the mechanism of TZR in HER2-positive luminal B breast cancer cells with acquired resistance. [Methods and Results] We generated trastuzumab resistant breast cancer cell line (BT-474-R) by treating BT-474 cells with trastuzumab for over long-term. Initial validation showed that HER2 overexpression in the BT-474-R cells remained unaltered suggesting that probably HER2 independent signaling pathway is involved in the development of TZR. We demonstrated that NF-κB is constitutively activated in the BT-474-R cells by quantitative realtime RT-PCR, western blotting and immunofluorescent microscopy. Expression of pro-inflammatory cytokines were also demonstrated. Pharmacologic inhibition of NF-κB improved sensitivity to trastuzumab in BT-474-R cells. We identified both IKK-α and IKK-β as the major determinants of the activation of the NF-κB signaling in the resistant cells. [Conclusion] Our study suggests by activating NF-κB pathway, luminal B cells may facilitate acquiring basal-like, Her2+ phenotype a notion consistent with “progression through grade” hypothesis. Finally, our study suggests that blocking NF-κB will provide a new treatment option to limit relapse in HER2 positive breast cancer patients treated with trastuzumab. Citation Format: Hirotaka Kanzaki, Nishit Makhopadhya, Xiaojiang Cui, Krishnan V. Ramanujan, Tatsuaki Takeda, Yoshihisa Kitamura, Toshiaki Sendo, Ramachandran Murali. Constitutive NF-kB-activation loop enhances resistance to trastuzumab in HER2 positive luminal B breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 726. doi:10.1158/1538-7445.AM2015-726

Abstract LB-125: The identification of FOXA1/ERα pathway modulators for the treatment of hormone-based therapy resistant breast cancer

Abstract Estrogen receptor alpha (ERα) is expressed in ∼75% of breast cancers where it binds and regulates specific target genes which drive cancer cell proliferation. Hormone-based treatments such as selective estrogen receptor modulators (SERMs) and aromatase inhibitors prevent estrogen signalling through the ERα, but in many cases resistance to these therapies develops. FOXA1 is a pioneer factor for ERα that is required to facilitate ERα binding to its target genes and its expression is considered to be a defining gene that characterizes ERα positive luminal breast cancers. Here we show that FOXA1 is an essential gene in ERα positive, tamoxifen resistant and aromatase-resistant breast cancer cell lines suggesting inhibitors that target the FOXA1/ERα pathway could have broad utility in these settings. To this end we performed a cell-based screen to identify novel small molecule regulators of FOXA1/ERα pathway activity. Our novel cell-based assay allowed us to screen over 180, 000 discrete compounds in a high through-put manner. Counter screens and phenotypic profiling in FOXA1 positive and negative breast cancer cell lines were used to remove non-specific compounds and direct ERα modulators. Second batches of confirmed actives were profiled through the screening cascade and by using ChIP assays we have observed a reduction in FOXA1 binding at the chromatin that is commensurate with a similar loss of ERα binding, demonstrating for the first time pharmacological modulation of FOXA1/ERα occupancy at the chromatin level. Citation Format: jon roffey, Aurore Lejeune, Michelle Barnard, Julie Stock, Ai Ching Wong, Jenny McKelvie, Kelly Holmes, Jason Carroll, Stephen Myatt. The identification of FOXA1/ERα pathway modulators for the treatment of hormone-based therapy resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-125. doi:10.1158/1538-7445.AM2015-LB-125

Potentiation of Growth Inhibitory Responses of the mTOR Inhibitor Everolimus by Dual mTORC1/2 Inhibitors in Cultured Breast Cancer Cell Lines.

The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORC1, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORC1/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORC1 and mTORC2; and GDC-0941 is a pan-PI3K inhibitor. We have addressed the question of whether mTOR inhibitors may be more effective in combination than singly in inhibiting the proliferation of breast cancer cells. We selected a panel of 30 human breast cancer cell lines that included ER and PR positive, HER2 over-expressing, and “triple negative” variants, and determined whether signaling pathway utilization was related to drug-induced inhibition of proliferation. A significant correlation (p = 0.005) was found between everolimus IC50 values and p70S6K phosphorylation, but not with AKT or ERK phosphorylation, consistent with the mTOR pathway being a principal target. We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested. The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested. The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.

DMXL2 drives epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through Notch hyper-activation.

The acquisition of endocrine therapy resistance in estrogen receptor α (ERα) breast cancer patients represents a major clinical problem. Notch signalling has been extensively linked to breast cancer especially in patients who fail to respond to endocrine therapy. Following activation, Notch intracellular domain is released and enters the nucleus where activates transcription of target genes. The numerous steps that cascade after activation of the receptor complicate using Notch as biomarker. Hence, this warrants the development of reliable indicators of Notch activity. DMXL2 is a novel regulator of Notch signalling not yet investigated in breast cancer. Here, we demonstrate that DMXL2 is overexpressed in a subset of endocrine therapy resistant breast cancer cell lines where it promotes epithelial to mesenchymal transition through hyper-activation of Notch signalling via V-ATPase dependent acidification. Following DMXL2 depletion or treatment with Bafilomycin A1, both EMT targets and Notch signalling pathway significantly decrease. We show for the first time that DMXL2 protein levels are significantly increased in ERα positive breast cancer patients that progress after endocrine therapy. Finally, we demonstrate that DMXL2 is a transmembrane protein with a potential extra-cellular domain. These findings identify DMXL2 as a novel, functional biomarker for ERα positive breast cancer.

Synthesis and antitumor evaluation of novel hybrids of phenylsulfonylfuroxan and epiandrosterone/dehydroepiandrosterone derivatives

Thirteen novel furoxan-based nitric oxide (NO) releasing hybrids (14a–e, 15a–e, 17b–d) of 16,17-pyrazo-annulated steroidal derivatives were synthesized and evaluated against the MDA-MB-231, HCC1806, SKOV-3, DU145, and HUVEC cell lines for their in vitro anti-proliferative activity. Most of the compounds displayed potent anti-proliferative effects. Among them, 17c exhibited the best activity with IC50 values of 20–1.4nM against four cell lines (MDA-MB-231, SKOV-3, DU145, and HUVEC), and 1.03μM against a tamoxifen resistant breast cancer cell line (HCC1806). Furthermore, five compounds (14a, 15a, 17b–d) were selected to screen for VEGF inhibitory activity. Compounds 15a, 17b,c showed obviously better activity than 2-Methoxyestradiol (2-ME) on reducing levels of VEGF secreted by MDA-MB-231 cell line. In a Capillary-like Tube Formation Assay, compounds 17b,c exhibited a significant suppression of the tubule formation in the concentration of 1.75nM and 58nM, respectively. The preliminary SAR showed that steroidal scaffolds with a linker in 3-position were favorable moieties to evidently increase the bioactivities of these hybrids. Overall, these results implied that 17c merited to be further investigated as a promising anti-cancer candidate.

Abstract P2-07-06: Niclosamid overcomes epithelial-mesenchymal transition of lapatinib resistance through inhibiting Src activation in HER2 positive breast cancer

Abstract Background: The Src pathway is known to regulate tumor matastasis and plays a role in epithelial-mesenchymal transition(EMT). Lapatinib, although, has improved clinical outcome for HER2 positive breast cancer patients, acquired resistance to lapatinib remains an important reason influencing its clinical efficacy. Our previous research shows lapatinib acquired resistance HER2 positive breast cancer cell lines, SKBR3-R and BT474-R have EMT phenomenon and multiple pathway activated. Niclosamide is an FDA-approved antihelminthic agent, which is found has cytotoxicity effect on tumor stem cells recently. We wished to determine the effect of niclosamide on EMT and lapatinib resistance cells and investigate niclosamide as a potential therapeutic agent for HER2 positive breast cancer. Methods: SKBR3 and BT474, two HER2 positive breast cancer cell lines, were continuously exposed to increasing doses of lapatinib to establish two stable cell lines resistant to lapatinib, SKBR3-R and BT474-R. Cell proliferation was determined by CCK8 assay. Protein expression was determined by western-blotting. Invasion ability was analyzed by transwell assay. FITC staining flow cytometry (FCM) was conducted to observe the percentage of apoptosis. Results: Both two HER2 positive lapatinib resistant cell lines, SKBR3-R and BT474-R had EMT phenomenon. Niclosamide had a stronger inhibition effect on lapatinib resistant cell lines than parental cell lines, and induced more apoptosis by FCM. Western-blot showed niclosamide could reverse the EMT phenomenon of SKBR3-R and BT474-R with E-cadherin up-regulated and snail,vimentin down-regulated at the concentration of 0.5-1μM. A significant reduction of Src signaling was also confirmed, as well as the downstream Akt and Erk pathway. After adding niclosimide for 48 hours, SKBR3-R and BT474-R’s capability of invasion were inhibited. Conclusion: Our results suggested that niclosamide had a strong cytotoxic effect on HER2 positive lapatinib resistant breast cancer cell lines. The EMT induced by lapatinib resistance could be reversed by niclosamide, associated with the inhibition of Src pathway activation, as well as the downstream pathways. Niclosamide treatment produced reduced levels of invasion, serving as a novel therapeutic way for lapatinib-resistant breast cancer patients. Citation Format: Junjun Liu, Xiaosong Chen, Yan Mao, Kunwei Shen. Niclosamid overcomes epithelial-mesenchymal transition of lapatinib resistance through inhibiting Src activation in HER2 positive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-06.

Abstract P4-06-03: Targeting the insulin receptor with a small peptide (S961) in cancer

Abstract The insulin-like growth factor (IGF)/insulin receptors are members of the superfamily of growth factor receptor tyrosine kinases. The insulin and type I IGF-I receptors (IGF-1R) have very similar structure and share high homology (∼84%) in the kinase domain. Their dimeric structure allows for the formation of either holo or hybrid receptors. Thus, targeting of any individual receptor has been a challenge. We have previously shown that the insulin receptor is expressed in tamoxifen resistant breast cancer cell lines. Therefore, specific targeting might be a useful strategy to block this pathway. The antagonist S961 is a single chain peptide that binds insulin receptor and has been reported to have partial antagonist activity. The affinity of S961 for the insulin receptor is comparable to that of insulin and the selectivity of the insulin receptor versus IGF1 is higher than that of insulin itself (Sturis et al). To determine if S961 has activity in breast cancer cells, we tested its ability to disrupt insulin and IGF-I signaling and growth in a panel of cancer cell lines (MCF-7L, MCF-7L TamR, MDA-231, and HepG2 cells). MCF-7L cells express high levels of IGF1, while HepG2 express mostly insulin receptor. Pre-incubation with S961 significantly suppressed p-Akt and p-MAPK after insulin stimulation in the HepG2, but not in MCF-7L. In contrast, insulin receptor stimulation in MCF-7L TamR cells, which do not express IGF1R, was inhibited by S961. Even a 1000-fold increase in S961 was unable to suppress insulin receptor activation in cells expressing both insulin receptor and IGF1R. S961 inhibits insulin-stimulate cell cycle progression in MCF7L TamR cells. In these TamR cells, S961 also suppresses colony formation in anchorage independent growth assays. These data suggest S961 is an effective inhibitor of insulin receptor activation but only when little IGF1R is expressed. Thus, insulin receptor targeting might be useful in the management of endocrine resistant breast cancer. Citation Format: Kelly LaPara, Douglas Yee. Targeting the insulin receptor with a small peptide (S961) in cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-06-03.

Abstract OT2-1-10: RAD1901, a novel tissue-selective estrogen receptor degrader (SERD) demonstrates estrogen receptor engagement in a phase 1 clinical study

Abstract Despite advances in the treatment of metastatic breast cancer through modulation of estrogen receptor (ER) activity, after initial efficacy, the use of hormonal therapies is frequently followed by the development of de novo or acquired endocrine resistance. There therefore exists a potentially significant opportunity for new agents that can overcome endocrine resistance. One mechanism by which this could be achieved is through degradation of the estrogen receptor and thereby elimination of estrogen receptor mediated signaling. RAD1901 is a novel, non-steroidal small molecule that selectively binds and degrades the ER that is currently being evaluated for the treatment of metastatic breast cancer. RAD1901 has demonstrated good tissue selectivity in preclinical models, its does not stimulate the uterine endometrium, and it protects against bone loss in an ovariectomy-induced osteopenia rat model. In addition, we believe that RAD1901 has the ability to cross the blood-brain barrier. In vitro, treatment of tamoxifen-sensitive and resistant human breast cancer cell lines with RAD1901 resulted in a potent degradation of the ER (IC50 1.6nM in MCF7 cells) and inhibition of both basal and estradiol-stimulated proliferation. In a mouse xenograft tumor model with MCF7, oral dosing with RAD1901 resulted in a significant decrease in estradiol-stimulated tumor growth. An ongoing phase 1 clinical study in healthy volunteers is being conducted to evaluate the tolerability, safety and pharmacokinetics of RAD1901 at escalating doses. This study is also using 18F-estradiol positron emission tomography (FES-PET) to provide a pharmacodynamic assessment of estrogen receptor engagement/turnover. Following 6-days of daily treatment with RAD1901, at doses that were well tolerated, a complete suppression of FES-PET signal was observed, with standardized uptake values (SUV) comparable to background tissues. The relationship between PK and PD will be presented. To date, the maximum tolerated dose of RAD1901 has not been determined. In conclusion, RAD1901 is a novel SERD that we believe has the potential to be used for the treatment of hormone driven and hormone resistant metastatic breast cancers, including breast cancer brain metastasis. The proposed phase 1b study design in metastatic breast cancer will be outlined. Citation Format: Gary Hattersley, Frank David, Alan Harris, Marcie Clarkin, Kate Banks, Greg Williams, Andor Glaudemans, Janine Doorduin, Michel Koole, Erik de Vries, Rich Lyttle. RAD1901, a novel tissue-selective estrogen receptor degrader (SERD) demonstrates estrogen receptor engagement in a phase 1 clinical study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT2-1-10.

Aurora kinase B is important for antiestrogen resistant cell growth and a potential biomarker for tamoxifen resistant breast cancer.

BackgroundResistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer. The aim of the study was to explore the growth promoting pathways of antiestrogen resistant breast cancer cells to identify biomarkers and novel treatment targets.MethodsAntiestrogen sensitive and resistant T47D breast cancer cell lines were used as model systems. Parental and fulvestrant resistant cell lines were subjected to a kinase inhibitor library. Kinase inhibitors preferentially targeting growth of fulvestrant resistant cells were identified and the growth inhibitory effect verified by dose–response cell growth experiments. Protein expression and phosphorylation were investigated by western blot analysis. Cell cycle phase distribution and cell death were analyzed by flow cytometry. To evaluate Aurora kinase B as a biomarker for endocrine resistance, immunohistochemistry was performed on archival primary tumor tissue from breast cancer patients who have received adjuvant endocrine treatment with tamoxifen.ResultsThe selective Aurora kinase B inhibitor barasertib was identified to preferentially inhibit growth of fulvestrant resistant T47D breast cancer cell lines. Compared with parental cells, phosphorylation of Aurora kinase B was higher in the fulvestrant resistant T47D cells. Barasertib induced degradation of Aurora kinase B, caused mitotic errors, and induced apoptotic cell death as measured by accumulation of SubG1 cells and PARP cleavage in the fulvestrant resistant cells. Barasertib also exerted preferential growth inhibition of tamoxifen resistant T47D cell lines. Finally, high percentage of Aurora kinase B positive tumor cells was significantly associated with reduced disease-free and overall survival in 261 ER-positive breast cancer patients, who have received tamoxifen as first-line adjuvant endocrine treatment.ConclusionsOur results indicate that Aurora kinase B is a driving factor for growth of antiestrogen resistant T47D breast cancer cell lines, and a biomarker for reduced benefit of tamoxifen treatment. Thus, inhibition of Aurora kinase B, e.g. with the highly selective kinase inhibitor barasertib, could be a candidate new treatment for breast cancer patients with acquired resistance to antiestrogens.

The Role of Non‐coding RNAs in Breast Cancer Resistance to EGFR Therapy

To achieve the objective of characterizing the miRNA‐mediated mechanisms of resistance to current epidermal growth factor receptor (EGFR) targeted therapies, therapy resistant breast cancer (BC) cell lines were created by exposing therapy sensitive SKBR3 and BT‐474 human BC cells to varying concentrations of gefitinib or lapatinib. The fold resistance, as determined from the IC50s, was 2.5 and 21.5 respectively, for the SKBR3 gefitinib and lapatinib resistant cell lines and 1.9 for the BT‐474 gefitinib resistant cells. The therapy sensitive cells demonstrated higher levels of apoptosis in the presence of the therapeutics compared to the therapy resistant variants. Diferentially expressed miRNAs in therapy resistant and sensitive variants were identified from qRT‐PCR and their targets detected by Western blotting. The oncomiRs miR‐221/222 that promotes the epithelial to mesenchymal (EMT) transition, were upregulated, while miR‐200a, a miRNA that maintains the epithelial phenotype, was downregulated in the therapy resistant cell lines. Accordingly, the miR‐221/222 targets and tumor suppressors PTEN and p27 were downregulated, and the miR‐200 target β‐catenin, a pro‐cancer protein, was upregulated in the therapy resistant variants. In conclusion, this data suggest that downregulation of miR‐200 family, and upregulation of miR‐221/222 is a general mechanism of EGFR therapy resistance in BC.