TamR Breast Cancer Research Articles

Development of heterodimeric estrogen receptor alpha antagonists to target simultaneously the ligand and coactivator binding site.

One-third of breast cancer patients will develop recurrent cancer within 15 years of endocrine treatment. Notably, tumor growth in a hormone-refractory state still relies on the interaction between estrogen receptor alpha (ERα) and upregulated coactivators. Herein, we suggest that simultaneous targeting of the primary ligand binding site (LBS) and the coactivator binding site (CABS) at ERα represents a promising alternative therapeutic strategy to overcome mutation-driven resistance in breast cancer. We synthesized two series of compounds that connect the LBS-binder (E)-3-{4-[8-fluoro-4-(4-hydroxyphenyl)-2,3-dihydrobenzo[b]oxepin-5-yl]phenyl}acrylic acid 8 with the coactivator binding site inhibitors (CBIs) 4,6-bis(isobutyl(methyl)amino)pyrimidine or 3-(5-methoxy-1H-benzo[d]imidazol-2-yl)propanoic acid via covalent linkage. The most active benzoxepine-pyrimidine conjugate 31 showed strong inhibition of estradiol-induced transactivation (IC50 = 18.2 nM (ERα) and 61.7 nM (ERβ)) in a luciferase reporter gene assay as well as high antiproliferative effects in MCF-7 (IC50 = 65.9 nM) and tamoxifen-resistant MCF-7/TamR (IC50 = 88.9 nM) breast cancer cells. All heterodimers exhibited two- to sevenfold higher antagonism at ERα (compared with ERβ) and were superior to the acrylic acid precursor 8 in terms of ER antagonism and antiproliferative activity. It was demonstrated on the example of31 that the compounds did not influence the ERα content in MCF-7 cells and therefore act as pure antiestrogens without downregulating potency. Possible interactions of the CBI at the receptor surface, which enhanced the biological activities, were evaluated using molecular docking studies.

Matrix Metalloproteinase-1 (MMP1) Upregulation through Promoter Hypomethylation Enhances Tamoxifen Resistance in Breast Cancer.

Simple SummaryCancer recurrence caused by tamoxifen resistance hampers chemotherapy in breast cancer patients. The reasons behind the resistance were investigated by screening epigenetically regulated genes through analysis of methylation data from tamoxifen-resistant MCF-7 cells. MMP1 locus was found to be hypomethylated at a promoter CpG site and its expression was upregulated in the cell line, which was verified by the drug-resistant tumor tissues from breast cancer patients (n = 28). Downregulating MMP1 using a short hairpin RNA inhibited the growth of resistant cells and increased sensitivity to tamoxifen in vitro as well as in a xenografted mouse model in vivo. This study suggests that MMP1 is potentially a target gene to control tamoxifen resistance in breast cancer.Background: Tamoxifen (tam) is widely used to treat estrogen-positive breast cancer. However, cancer recurrence after chemotherapy remains a major obstacle to achieve good patient prognoses. In this study, we aimed to identify genes responsible for epigenetic regulation of tam resistance in breast cancer. Methods: Methylation microarray data were analyzed to screen highly hypomethylated genes in tam resistant (tamR) breast cancer cells. Quantitative RT-PCR, Western blot analysis, and immunohistochemical staining were used to quantify expression levels of genes in cultured cells and cancer tissues. Effects of matrix metalloproteinase-1 (MMP1) expression on cancer cell growth and drug resistance were examined through colony formation assays and flow cytometry. Xenografted mice were generated to investigate the effects of MMP1 on drug resistance in vivo. Results: MMP1 was found to be hypomethylated and overexpressed in tamR MCF-7 (MCF-7/tamR) cells and in tamR breast cancer tissues. Methylation was found to be inversely associated with MMP1 expression level in breast cancer tissues, and patients with lower MMP1 expression exhibited a better prognosis for survival. Downregulating MMP1 using shRNA induced tam sensitivity in MCF-7/tamR cells along with increased apoptosis. The xenografted MCF-7/tamR cells that stably expressed short hairpin RNA (shRNA) against MMP1 exhibited retarded tumor growth compared to that in cells expressing the control shRNA, which was further suppressed by tam. Conclusions: MMP1 can be upregulated through promoter hypomethylation in tamR breast cancer, functioning as a resistance driver gene. MMP1 can be a potential target to suppress tamR to achieve better prognoses of breast cancer patients.

HBXIP is a novel regulator of the unfolded protein response that sustains tamoxifen resistance in ER+ breast cancer

Endocrine-therapy-resistant estrogen receptor–positive (ER+) breast cancer cells often exhibit an augmented capacity to maintain endoplasmic reticulum (EnR) homeostasis under adverse conditions. Oncoprotein hepatitis B X-interacting protein (HBXIP) is a known transcriptional coactivator that promotes cancer development. However, it is unclear whether HBXIP participates in maintaining EnR homeostasis and promoting drug resistance in ER+ breast cancer. Here, we report that tamoxifen-resistant (TmaR) breast cancer cells exhibit increased expression of HBXIP, which acts as an inactivator of the unfolded protein response (UPR) to diminish tamoxifen-induced EnR stress. We show that HBXIP deficiency promotes EnR-associated degradation, enhances UPR-element reporter activity and cellular oxidative stress, and ultimately attenuates the growth of TmaR cells in vitro and in vivo. Mechanistically, we demonstrate that HBXIP acts as a chaperone of UPR transducer inositol-requiring enzyme 1a and diminishes production of reactive oxygen species (ROS) in TamR breast cancer cells. Upon loss of HBXIP expression, tamoxifen treatment hyperactivates IRE1α and its downstream proapoptotic pathways and simultaneously induces accumulation of intracellular ROS. This elevated ROS programmatically activates the other two branches of the UPR, mediated by PKR-like ER kinase and activating transcription factor 6α. Clinical investigations and Kaplan–Meier plotter analysis revealed that HBXIP is highly expressed in TamR breast cancer tissues. Furthermore, reinforced HBXIP expression is associated with a high recurrence and poor relapse-free survival rates in tamoxifen monotherapy ER+ breast cancer patients. These findings indicate that HBXIP is a regulator of EnR homeostasis and a potential target for TamR breast cancer therapy.

Low concentrations of 17β-estradiol exacerbate tamoxifen resistance in breast cancer treatment through membrane estrogen receptor-mediated signaling pathways.

The present study aims to discover the influences of tamoxifen and 17β-estradiol (E2) on tamoxifen-resistant (TamR) patients in vitro. Herein, we established a stabilized TamR MCF-7 cell line at 1μM via gradient concentrations of tamoxifen cultivation. The expression changes of four ER subtypes (ERα66, ERβ, ERα36 and GPR30) were found to bring about tamoxifen resistance. Moreover, the generation of tamoxifen resistance involved in apoptosis escape via a reactive oxygen species-regulated p53 signaling pathway. Interestingly, E2 at environmental concentrations (0.1-10nM) could activate the expression of both ERα36 and GPR30, and then stimulate the phosphorylation of ERK1/2 and Akt, resulting in cell growth promotion. Cell migration and invasion promotion, apoptosis inhibition, and cell cycle G1-S progression are involved in such proliferative effects. Conversely, the application of specific antagonists of ERα36 and GPR30 could restore tamoxifen's sensitivity as well as partially offset E2-mediated proliferation. In short, overexpression of ERα36 and GPR30 not only ablate tamoxifen responsiveness but also could promote tumor progression of TamR breast cancer under estrogen conditions. These results provided novel insights into underlying mechanisms of tamoxifen resistance and the negative effects of steroid estrogens at environmental concentrations on TamR MCF-7 cells, thus generating new thoughts for future management of ER-positive breast cancer.

Activating transcription factor-2 (ATF2) is a key determinant of resistance to endocrine treatment in an in vitro model of breast cancer

BackgroundActivating transcription factor-2 (ATF2), a member of the leucine zipper family of DNA binding proteins, has been implicated as a tumour suppressor in breast cancer. However, its exact role in breast cancer endocrine resistance is still unclear. We have previously shown that silencing of ATF2 leads to a loss in the growth-inhibitory effects of tamoxifen in the oestrogen receptor (ER)-positive, tamoxifen-sensitive MCF7 cell line and highlighted that this multi-faceted transcription factor is key to the effects of tamoxifen in an endocrine sensitive model. In this work, we explored further the in vitro role of ATF2 in defining the resistance to endocrine treatment.Materials and methodsWe knocked down ATF2 in TAMR, LCC2 and LCC9 tamoxifen-resistant breast cancer cell lines as well as the parental tamoxifen sensitive MCF7 cell line and investigated the effects on growth, colony formation and cell migration. We also performed a microarray gene expression profiling (Illumina Human HT12_v4) to explore alterations in gene expression between MCF7 and TAMRs after ATF2 silencing and confirmed gene expression changes by quantitative RT-PCR.ResultsBy silencing ATF2, we observed a significant growth reduction of TAMR, LCC2 and LCC9 with no such effect observed with the parental MCF7 cells. ATF2 silencing was also associated with a significant inhibition of TAMR, LCC2 and LCC9 cell migration and colony formation. Interestingly, knockdown of ATF2 enhanced the levels of ER and ER-regulated genes, TFF1, GREB1, NCOA3 and PGR, in TAMR cells both at RNA and protein levels. Microarray gene expression identified a number of genes known to mediate tamoxifen resistance, to be differentially regulated by ATF2 in TAMR in relation to the parental MCF7 cells. Moreover, differential pathway analysis confirmed enhanced ER activity after ATF2 knockdown in TAMR cells.ConclusionThese data demonstrate that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity.

IGFBP-1 Expression Promotes Tamoxifen Resistance in Breast Cancer Cells via Erk Pathway Activation.

Insulin-like growth factor (IGF) system plays a significant role in many cellular processes, including proliferation, and survival. In estrogen receptor positive breast cancer, the level of circulating IGF-1 is positively associated with the incidence and at least 50% of cases have elevated IGF-1R signaling. Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα-positive breast cancer. Unfortunately, tamoxifen resistance is a frequent occurrence in patients receiving treatment and the molecular mechanisms that underlie tamoxifen resistance not adequately defined. It has recently been reported that the inhibition of IGF-1R activation and the proliferation of breast cancer cells upon tamoxifen treatment is mediated by the accumulation of extracellular insulin-like growth factor binding protein 1 (IGFBP-1). Elevated IGFBP-1 expression was observed in tamoxifen-resistant (TamR) MCF-7 and T-47D cells lines suggesting that the tamoxifen-resistant state is associated with IGFBP-1 accumulation. MCF-7 and T-47D breast cancer cells stably transfected with and IGFBP-1 expression vector were generated (MCF7-BP1 and T47D-BP1) to determine the impact of breast cancer cell culture in the presence of increased IGFBP-1 expression. In these cells, the expression of IGF-1R was significantly reduced compared to controls and was similar to our observations in tamoxifen-resistant MCF-7 and T-47D cells. Also similar to TamR breast cancer cells, MCF7-BP1 and T47D-BP1 were resistant to tamoxifen treatment, had elevated epidermal growth factor receptor (EGFR) expression, increased phospho-EGFR (pEGFR), and phospho-Erk (pErk). Furthermore, tamoxifen sensitivity was restored in the MCF7-BP1 and T47D-BP1 upon inhibition of Erk phosphorylation. Lastly, the transient knockdown of IGFBP-1 in MCF7-BP1 and T47D-BP1 inhibited pErk accumulation and increased tamoxifen sensitivity. Taken together, these data support the conclusion that IGFBP-1 is a key component of the development of tamoxifen resistance in breast cancer cells.

Abstract 3893: Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice

Abstract Background: Melatonin-tamoxifen drug conjugate linked with a 5-carbon chain (C5) exhibited uterus protection in female mice, which did not occur when the same doses of unlinked melatonin and tamoxifen were co-administered. Anti-cancer actions of C5 and 4 others, with varying carbon lengths (C2, C4, C9, C15), were further tested in phenotypically diverse breast cancer (BC) cell lines—MCF-7(ER+/PR+), MMC(HER2+) and triple negatives(MDA-MB-231 and BT-549) in terms of cell viability and migration. In all BC lines, C4 and C5 exhibited superior potency and efficacy compared to C2, C9, C15 and unlinked melatonin and tamoxifen or 4-OH-tamoxifen controls. In this study, C4 and C5 were further tested for their anti-cancer effects in tamoxifen-resistant MCF-7 cells(TamR-MCF-7); their pharmacokinetic parameters were assessed in vitro and in vivo; and the mechanisms underlying C4- and C5-mediated anti-cancer actions were also determined by use of inhibitors against MEK1/2, MEK5 or PI3 kinase and western blot analysis. Results: C4 and C5 exhibited similar potency (TamR-MCF-7 IC50 = 4.22µM and 7.21µM; MCF-7 IC50 = 3.62nM and 440mM) and efficacy (TamR-MCF-7 = 83% and 81% inhibition; MCF-7= 71% and 90% inhibition) to inhibit TamR-MCF-7 cell viability as they did in MCF-7 cells. Regarding metabolism, C4 and C5 displayed similar CYP-mediated pharmacokinetic profiles as tamoxifen in both mouse and human liver microsomes where 40% loss of tamoxifen, C4 or C5 occurred by 10 min in mouse vs. 20 min in human. In vivo, the Tmax for C5 (oral) was 24h compared to 0.5h for C4 (oral) resulting in lower Cmax (8.9 ng/mL) and AUC0-24 (1.6 hr.ng/mL) for C5 compared to C4 (Cmax =102.6 ng/mL and AUC0-24 =143.7 hr.ng/mL) and lower oral bioavailability (0.11%) for C5 compared to C4 (4.52%). Regarding mechanisms, the effect of C4- or C5-mediated protein modulation was “context-specific” dependent upon BC phenotype (i.e., ER+, HER2+ or ER-/PR-/HER2-); the number of carbons linking melatonin to tamoxifen (i.e., 4 carbons for C4 or 5 carbons for C5); and the concentration (i.e., 1nM or 10µM) of C4 or C5 tested. For MCF-7, MMC and MDA- MB-231 cells, inhibition of MEK1/2, MEK5 and PI3K enhanced but did not block C4’s or C5’s anti-cancer actions; while for BT-549 cells, Bix02189 blocked C5’s effects. Also, in MCF-7 cells, pERK1/2, NF-κB and Runx2 were significantly modulated by C4 or C5; for MMCs, C4 and C5 modulated NF-κB and β1-integrin expression; for MDA-MB-231, MEK1/2 and MEK5 played a more central role possibly through co-modulation of NF-κB; and for BT-549, C5-mediated effects on pERK5 or PI3K-dependent regulation of pERK1/2 may underlie its anti-cancer actions. Conclusions: Melatonin-tamoxifen drug conjugates (C4 and C5) may be superior to tamoxifen therapy alone in a variety of cancers, including TamR BC. Citation Format: Mahmud Hasan, Thomas D. Wright, Saugat Adhikari, Mohamed A. Marzouk, Alaina Pericoloso, Kelsey Murgas, Miranda Burgman, Benton P. Miller, Ulrike Holzgrabe, Darius P. Zlotos, Robert E. Stratford, Paula A. Witt-Enderby. Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3893.

AhR ligand aminoflavone suppresses α6-integrin-Src-Akt signaling to attenuate tamoxifen resistance in breast cancer cells.

More than 40% of patients with luminal breast cancer treated with endocrine therapy agent tamoxifen demonstrate resistance. Emerging evidence suggests tumor initiating cells (TICs) and aberrant activation of Src and Akt signaling drive tamoxifen resistance and relapse. We previously demonstrated that aryl hydrocarbon receptor ligand aminoflavone (AF) inhibits the expression of TIC gene α6-integrin and disrupts mammospheres derived from tamoxifen-sensitive breast cancer cells. In the current study, we hypothesize that tamoxifen-resistant (TamR) cells exhibit higher levels of α6-integrin than tamoxifen-sensitive cells and that AF inhibits the growth of TamR cells by suppressing α6-integrin-Src-Akt signaling. In support of our hypothesis, TamR cells and associated mammospheres were found to exhibit elevated α6-integrin expression compared with their tamoxifen-sensitive counterparts. Furthermore, tumor sections from patients who relapsed on tamoxifen showed enhanced α6-integrin expression. Gene expression profiling from the TCGA database further revealed that basal-like breast cancer samples, known to be largely unresponsive to tamoxifen, demonstrated higher α6-integrin levels than luminal breast cancer samples. Importantly, AF reduced TamR cell viability and disrupted TamR mammospheres while concomitantly reducing α6-integrin messenger RNA and protein levels. In addition, AF and small interfering RNA against α6-integrin blocked tamoxifen-stimulated proliferation of TamR MCF-7 cells and further sensitized these cells to tamoxifen. Moreover, AF reduced Src and Akt signaling activation in TamR MCF-7 cells. Our findings suggest elevated α6-integrin expression is associated with tamoxifen resistance and AF suppresses α6-integrin-Src-Akt signaling activation to confer activity against TamR breast cancer.

Abstract 1804: Novel indole compound LX-039 as an oral selective estrogen receptor degrader (SERD) for treating estrogen receptor positive (ER+) breast cancer

Abstract Objective: Endocrine therapy (ET) now is the standard of care (SOC) for ER+ breast cancer treatment. The representative drug of ET is tamoxifen, a selective estrogen receptor modulator (SERM) that inhibits ER+ breast cancer growth by antagonizing ER function. Tamoxifen displays promising efficacy but also brings problems such as increased endometrial cancer risk and acquired resistance. SERD fulvestrant controls ER+ breast cancer by degrading in-cell ER. It shows improved efficacy in advanced or tamoxifen-resistant (TamR) breast cancer patients and is recently approved as first-line treatment for ER+ breast cancer. However, due to its poor PK profile, this drug could only be administrated intramuscularly, which restricts its applicable patient population. Thus, developing an oral SERD is still an unmet medical need. Method & Results: We have discovered an oral SERD, LX-039. LX-039 is a novel indole series compound. It robustly degraded ER in MCF-7 breast cancer cells (IC50 = 1.53 nM) and inhibited MCF-7 cell proliferation (IC50 = 2.56 nM). Besides, LX-039 exhibited good PK profile across species, F for mice, rat and dog were 32.2%, 44.5% and 48.1% respectively. Dose to p.o. AUC showed linear relationship, and very high p.o. AUCs could be obtained at high doses. For rat @500 mpk, p.o. AUC reached to 4.52M nM.h, and dog @300 mpk, p.o. AUC reached to 1.5M nM.h. LX-039 displayed promising in vivo antitumor efficacy. In naive MCF-7 breast cancer model, TGI was 87% @ 20 mpk; in TamR MCF-7 model, TGI was 70% @100 mpk. What makes LX-039 more advantageous is its safety profile. LX-039 showed purer ER antagonism. In rat uterine growth inhibition assay, the inhibition rate reached to 94% while inhibition rate of GDC-0810 and AZD-9496 were 65% and 89%, respectively. It had a clean CYP inhibition profile by various CYP assays, which indicates a low DDI risk. LX-039 selectively antagonizes ER without affecting other nuclear receptors such as AR, GR and PR. It showed a clean inhibition profile for a selected kinase/GPCR panel (IC50 >3 μM) and was negative in the GABA gated ion channel assay (IC50 > 10 μM) and the mini Ames test. In the 14 day rat non-GLP tox study, NOAEL of LX-039 was determined to be 300 mpk. Conclusion: The antitumor efficacy of LX-039 as demonstrated in the preclinical tumor models is highly encouraging and warrants testing the compound in the clinic as an oral SERD for treating ER+ breast cancer. The IND enabling studies of LX-039 are ongoing, and IND filing scheduled in the mid of 2018. Citation Format: Jianyu Lu, Lihong Hu, Ping Wang, Yusong Zhu, Peng Zou, Dongdong Wu, Charles Z. Ding, Shuhui Chen. Novel indole compound LX-039 as an oral selective estrogen receptor degrader (SERD) for treating estrogen receptor positive (ER+) 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 1804.

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

Abstract Luminal breast cancers account for ~75% of all cases and while adjuvant hormone therapy targeting ERα has significantly improved overall survival for patients with ER+ tumors, acquired resistance remains a major clinical problem. Tamoxifen-resistant (TamR) breast cancer models show loss of IGF1R concomitant with increased insulin-induced growth, underpinned by insulin receptor (InsR) compensation for IGF1R loss. PR (gene name PGR) is an estrogen-regulated target gene whose expression is used as a clinical marker of ER activity. However, its de novo relevance to breast cancer is unclear. We have shown previously that post-translational modifications create unique PR species whose altered behavior drives an endocrine-resistant gene signature, in part by crosstalk with the IGFR pathway. We propose that phospho-PR target gene selectivity is mediated by cooperation between PR-B and InsR/IGF1R pathway components. Herein we show that phospho-PR-expressing T47D cells lose expression of IGF1R compared to control cells expressing WT PR-B. Furthermore, expression of the adapter protein IRS-1 requires PRB expression and these IRS-1+ cells were more sensitive to insulin in anchorage-dependent growth assays. Phospho-PR T47D cells exhibit increased ALDH+ and CD24-/CD44+ tumorsphere formation compared to wt PRB-expressing cells. Inhibitors targeting IRS-1 and InsR were used to test their requirement for tumorsphere growth and, surprisingly, IRS-1 perturbation reduced phospho-PRB but not wt PRB tumorsphere growth. Interestingly, IRS-1 replaces IGF1R in phospho-PRB-containing transcriptional complexes that are recruited to the CTSD promoter, which we previously identified as a phospho-PR target gene. Finally, breast cancer cells expressing phospho-PR species exhibited tamoxifen-resistant growth. Collectively our data suggest that phospho-PRB cooperates with IRS-1 downstream of the Ins/IGF1R system to promote outgrowth of endocrine-resistant cells that include ALDH+ stem-like cells capable of forming secondary tumorspheres in vitro. Targeting phospho-PR species in addition to the signaling components of PR-complexes may provide a means to block emergence of endocrine-resistant cancer cells during breast cancer progression. Citation Format: Amy R. Dwyer, Deepali Sachdev, Carol A. Lange. Progesterone receptor/IRS-1 cooperation promotes stem cell outgrowth and endocrine resistance in estrogen receptor-positive luminal breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 949.

MiR-125a-3p inhibits ERα transactivation and overrides tamoxifen resistance by targeting CDK3 in estrogen receptor-positive breast cancer.

Tamoxifen (TAM) is a major adjuvant therapy for patients who are diagnosed with estrogen receptor-α (ER)-positive breast cancer; however, TAM resistance occurs often during treatment and the underlying mechanism is unclear. Here, we report that miR-125a-3p inhibits ERα transcriptional activity and, thus, ER+ breast cancer cell proliferation, which causes cell-cycle arrest at the G1/S stage, inducing apoptosis and suppressing tumor growth by targeting cyclin-dependent kinase 3 (CDK3) in vitro and in vivo. In addition, CDK3 and miR-125a-3p expression levels were measured in 37 cancerous tissues paired with noncancerous samples, and their expression levels were negatively associated with miR-125a-3p level. Of interest, miR-125a-3p level is down-regulated in MCF-7 TAM-resistant (TamR) cells. Of more importance, up-regulation of miR-125a-3p resensitizes MCF-7 TamR cells to TAM, which is dependent on CDK3 expression. These results suggest that miR-125a-3p can function as a novel tumor suppressor in ER+ breast cancer by targeting CDK3, which may be a potential therapeutic approach for TamR breast cancer therapy.-Zheng, L., Meng, X., Li, X., Zhang, Y., Li, C., Xiang, C., Xing, Y., Xia, Y., Xi, T. miR-125a-3p inhibits ERα transactivation and overrides tamoxifen resistance by targeting CDK3 in estrogen receptor-positive breast cancer.

Abstract P3-04-21: Androgen receptor in tamoxifen-resistant breast cancer is affected by SUMO

Abstract Resistance to tamoxifen is a major problem in treating women with estrogen receptor-positive (ERa+) breast cancer (BCa). Previous studies report elevated levels of the androgen receptor (AR) in tamoxifen-resistant (TamR) human mammary tumors. Inversely, in vitro overexpression of AR in ERa+-MCF-7 cells potentiates growth in the presence of tamoxifen. AR is a target for small ubiquitin like modifiers (SUMO) posttranslational modification (PTM). Prostate cancer (PCa) studies, show that SUMO-PTMs can have diverse effects on AR transcriptional activity, by modifying the AR itself or its co-regulators. SUMOylation regulates the AR's interaction with chromatin and modulates its function in a gene selective manner. Although SUMO-1 inhibited the AR transcriptional activity, SUMO-3 strongly enhanced the transactivation of AR in LNCaP cells but negatively affected the AR transcriptional activity in primary prostate epithelial cells. On the other hand, SUMO modifications and its subsequent effects on the AR in breast tumors and in particular TamR-BCa is unknown. Our objective is to understand how SUMO-PTMs affect AR activity in TamR-BCa. Others report that high Ubc9, a SUMO E2 conjugating enzyme, correlates with with aggressive phenotypes of BCa and that PIAS3, a SUMO E3 ligase, expression increases in breast tumors. Consistently, we now report that global SUMO2/3-conjugation is enhanced while the full-length SUMO protease SENP7 (SENP7L) is reduced in TamR-BCa cells. Collectively, these observations would suggest reduced SUMO dynamics in TamR-BCa as compared to tamoxifen-sensitive (TamS) BCa cells. Unlike PCa cells that favor unmodified over SUMOylated AR, we report that AR SUMOylation is potentiated in TamR-BCa cells. Functionally, SUMOylation of AR augments its chromatin binding and enhances its transcriptional activity. Our results suggest that the level of unmodified to SUMOylated AR is dramatically altered with onset of TamR and drives a functionally distinct population of AR in TamR versus TamS BCa. Consistently these biochemical reactions that define protein diversity could have a major impact on drug resistance in BCa. Citation Format: Bahnassy S, Kumar S, Ren J, Frutiz G, Karami S, Bawa-Khalfe T. Androgen receptor in tamoxifen-resistant breast cancer is affected by SUMO [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-21.

Abstract 1881: Insulin receptor targeting in breast cancer through yeast surface display

Abstract Selective estrogen receptor modulators (SERMs) such as tamoxifen are used to treat estrogen receptor (ER) positive breast cancers, the most common intrinsic subtype. The development of secondary resistance to SERMs is an unsolved clinical dilemma, leading to the exploration, and approval, of therapies targeting growth factor signaling pathways. The insulin-like growth factor (IGF) system is well documented to cooperate with ER and is implicated as a contributor to endocrine resistance. Unfortunately, antibodies directed against Type I IGF receptor (IGF1R) failed to demonstrate clinical efficacy in endocrine resistant breast cancer. We developed a tamoxifen resistant (TamR) model to further identify the role for this system in endocrine resistance. In these cells, IGF1R levels are low but the level of insulin receptor (InsR), a closely related receptor of IGF1R still remains functional. Thus, we hypothesize that InsR may serve as a compensatory pathway to the loss of IGF1R in TamR cells and InsR may be a target in the therapy of endocrine resistant breast cancer. Indeed, our results showed that TamR breast cancer cells are more sensitive to insulin stimulation than wild-type cells. As compared to the parental cells, insulin-stimulated TamR cells showed enhanced PI3K/AKT and MAPK/ERK activation, greater monolayer and anchorage-independent growth. Suppression of InsR expression with either lentiviral shRNA or pharmacological methods in TamR cells was able to attenuate their sensitivity towards insulin-regulated PI3K/MAPK activation and growth, suggesting InsR targeting may be necessary. To target InsR, we are developing InsR-selective small protein scaffolds - the 10th type III domain of human fibronectin (Fn3) and T7 phage gene 2 protein (Gp2) using yeast surface display. This technique has arisen as an alternative candidate to bind cells surface proteins. Compared to antibodies, protein scaffolds are smaller (<12kDa), thermally more stable, lack disulfide bonds, tolerance to mutations and have the ability to bind a large variety of proteins. Using error-prone polymerase mutagenesis, we have identified improved Fn3 and Gp2 binders with increased affinity, specificity, and stability for InsR. Yeast surface displayed (YSD) Gp2 and Fn3 libraries yield affinities for recombinant InsR in the high nanomolar range and some specificity with an ability to differentiate binding from IGF1R. Purified Gp2 binder was able to distinguish InsR expression levels between InsRlow and InsRhigh mammalian cells, verifying its specificity for cell surface InsR. Its affinity for cell surface InsR, however was much lower compared to YSD affinity. Preliminary analyses also indicated that Gp2 binders have minimal effect in InsR-regulated signaling and short-term biological functions in vitro. These InsR binding engineered protein scaffolds will further be improved and evaluated as a potential imaging, diagnostic, and therapeutic tools. Citation Format: Jie Ying Chan, Kelly LaPara, Benjamin Hackel, Douglas Yee. Insulin receptor targeting in breast cancer through yeast surface display. [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 1881.

Abstract 3737: Inhibition of O-GlcNAc transferase in tamoxifen resistant breast cancer cells

Abstract O-linked N-acetyl-glucosamine transferase (OGT) is an enzyme that catalyzes addition of the O-GlcNAc modification to a wide range of intracellular proteins. The O-GlcNAc modification is a product of the hexosamine biosynthetic pathway, which requires glucose and glutamine as substrates. Uptake of both of these nutrients is often up-regulated in cancer, which in turn leads to an increase in the total protein O-GlcNAcylation. Increased OGT expression has also been reported in most cancer types, including the most frequently diagnosed cancer in women, breast cancer. Many of the breast cancers rely on estrogen receptor alpha (ERα) for proliferation and have shown a strong response to the ERα inhibition, most commonly achieved by treatment with tamoxifen. However, while efficient, prolonged exposure to tamoxifen commonly causes resistance and relapse of the disease. It is therefore vital to uncover mechanisms which contribute to the resistance in order to develop adequate treatment strategy for these patients. Here, we have investigated the effect of targeting OGT in an isogenic pair of ERα-positive tamoxifen-sensitive MCF7 and tamoxifen-resistant TAMR breast cancer cell lines. OGT inhibition decreased viability and triggered cell death in both cell lines. These responses were associated with over 50% reduction in ERα expression in both MCF7 and TAMR cells. Reduced O-GlcNAcylation has previously been reported to induce endoplasmic reticulum stress and activation of transcription factor C/EBP homologous protein (CHOP), which promotes cell death. Targeting OGT resulted in a strong increase of CHOP expression, which appeared more prominent in the TAMR cells. Finally, targeting OGT induced a very pronounced cell cycle arrest in the G2/M phase in the TAMR cells, while the MCF7 cell lined showed a very modest response. Taken together, these results indicate that targeting OGT leads to a differential response in the tamoxifen-sensitive and resistant breast cancer cells. Currently, we are using an expanded panel of tamoxifen-resistant cell lines to perform expression microarrays, metabolic flux assays and DNA damage response analysis in order to uncover the cause of the differential response to OGT targeting. This may help us identify potential therapeutic combinations that might be suitable for treatment of tamoxifen-resistant cancers. Citation Format: Anna Barkovskaya, Lina Prasmickaite, Ian G. Mills, Gunhild M. Mælandsmo, Siver A. Moestue, Harri M. Itkonen. Inhibition of O-GlcNAc transferase in tamoxifen resistant breast cancer cells. [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 3737.

Abstract P2-04-03: SUMO wrestles breast cancer: SUMO posttranslational modification directs breast cancer cell epigenome

Abstract Epigenetic reprogramming allows breast cancer (BCa) cells to bypass normal growth checkpoints and acquire an aggressive morphology. Function of critical epigenetic proteins is directed by small ubiquitin-like modifiers (SUMO) posttranslational modification (PTM). Components of the SUMO system are dysregulated in BCa. Our objective is to delineate how changes to SUMO-PTM affect epigenetic programs that coordinate BCa pathology. We report that chromatin-bound SUMO protease SENP7L is upregulated in metastatic tamoxifen-resistant (TamR) and triple-negative BCa. SENP7L facilitates chromatin remodeling that potentiates the growth and invasiveness of these BCa subtypes. SENP7L interacts with and deSUMOylates heterochromatin protein 1 alpha (HP1-alpha/CBX5) to mediates these responses. Canonical models suggest that HP1-alpha reads the histone mark to localize predominantly at constitutive heterochromatin. Our studies challenge the old paradigm as we show that SUMOylated HP1-alpha is enriched outside heterochromatin at euchromatin, including E2F-responsive and mesenchymal gene loci. Recruitment of SUMOylated HP1-alpha supports de novo heterochromatin formation at these loci. However, SUMOylated HP1-alpha is lost in aggressive BCa due to SENP7L induction. Consistently, SENP7L upregulation prompts chromatin de-condensation and gene transcription. Nuclei acid and chemical targeting of SENP7L reduces self-renewal/differentiation properties of TamR mammospheres as well as tumor volume and secondary tumor formation of TamR cells. Hence, SENP7L inhibitors can serve as promising tools to modulate epigenetic abnormalities in TamR BCa. Citation Format: Bawa-Khalfe T, Kumar S, Lin F-M. SUMO wrestles breast cancer: SUMO posttranslational modification directs breast cancer cell epigenome. [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 P2-04-03.

Differential effect of EGFR inhibitors on tamoxifen-resistant breast cancer cells.

Although tamoxifen is the most common and effective therapy for treatment of estrogen receptor-α (ER-α) breast cancer patients, resistance of endocrine therapy occurs, either denovo or acquired during therapy. Here, we investigated the clinical value of epidermal growth factor receptor (EGFR) in tamoxifen-resistant (TamR) patients and the differential effect of EGFR inhibitors, neratinib and gefitinib, on TamR breast cancer cell model. The morphology of TamR MCF7 cells showed mesenchymal phenotypes and did not induce cell death by tamoxifen treatment compared with tamoxifen‑sensitive (TamS) MCF7 cells. In addition, mesenchymal marker proteins, including N-cadherin (N-cad), fibronectin (FN), and Slug, significantly increased in TamR cells. In contrast, ER-α and E-cadherin (E-cad) were greatly decreased. We also found that the levels of EGFR and HER2 expression were increased in TamR cells. Furthermore, we observed that EGFR expression was directly involved with poor prognosis of tamoxifen-treated breast cancer patients using the GSE1378 date set. Thus, we treated TamR and TamS cells with EGFR inhibitors, neratinib and gefitinib, respectively. Interestingly, neratinib induced apoptotic cell death of TamR but not gefitinib. Cleaved PARP-1 expression was also increased by neratinib treatment in TamR cells. Therefore, we suggest that neratinib may be a potential therapeutic drug for treating TamR breast cancer.

Abstract P4-06-02: Insulin receptor targeting in breast cancer through yeast surface display

Abstract Selective estrogen receptor modulators (SERMs) such as tamoxifen have been a conventional therapy for breast cancer patients expressing estrogen receptor, representing the most common breast cancer type. The development of secondary resistance to SERM is an unsolved clinical dilemma, leading to the exploration of more targeted therapies. The insulin-like growth factor (IGF) system is well documented and is implicated as a contributor to endocrine resistance. Unfortunately, antibodies directed against type I IGF receptor (IGF1R) failed to demonstrate efficacy in endocrine resistant breast cancer. We have previously shown that tamoxifen resistant (TamR) breast cancer cells lose IGF1R yet retain expression of insulin receptor (InsR), a closely related receptor to IGF1R. Thus, we hypothesized that InsR may serve as a compensatory pathway to the loss of IGF1R in TamR cells and InsR may be a target in the therapy of endocrine resistant breast cancer. Indeed, our preliminary results show that TamR breast cancer cells were more sensitive to insulin stimulation. As compared to the parental cells, TamR cells showed stronger PI3K/AKT and MAPK/ERK activation, greater MTT proliferation growth and anchorage-independent growth upon insulin treatment. Knocking down InsR in TamR cells with shRNA was able to attenuate their sensitivity towards insulin-mediated PI3K/MAPK activation and growth, suggesting InsR targeting may be necessary in endocrine resistant breast cancer. To develop new agents to target InsR, we used yeast surface display to develop an InsR-selective protein scaffold – the 10th type III domain of human fibronectin (Fn3). This technique has arisen as an alternative method for the development of specific binders of cell surface proteins. Compared to antibodies, the Fn3 proteins are smaller (∼10kDa), thermally more stable, lack disulfide bonds and have the ability to bind a large variety of proteins. Using error-prone polymerase mutagenesis, we identified improved Fn3 proteins with increased InsR affinity, specificity, and stability. Our current results show that InsR is an important target in endocrine resistant breast cancer cells. Development of engineered Fn3 peptides with increased InsR binding specificity and affinity for InsR compared to IGF1R could be used to disrupt signaling through this pathway. Engineered Fn3 will further be evaluated as a potential imaging and therapeutic tool. Citation Format: Jie Ying Chan, Benjamin Hackel, Douglas Yee. Insulin receptor targeting in breast cancer through yeast surface display [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 P4-06-02.

Abstract P4-06-06: Growth hormone induction of oncogenic signaling promotes survival of endocrine resistant breast cancer cells

Abstract Targeting endocrine resistant breast cancer cells has been successfully accomplished by the use of mTORC1 inhibitors in combination with either aromatase inhibitors or tamoxifen. While there was preclinical data suggesting that targeting of the type I IGF receptor (IGF1R) might also be a target for endocrine resistant cells, clinical trials using IGF1R monoclonal antibodies showed no advantage and even suggested potential harm for the combination. IGF1R inhibition results in the disruption of an endocrine feedback loop and results in elevation of the pituitary growth factor growth hormone (GH). GH is involved in normal human growth, development, metabolism, and longevity. Growth hormone released from the pituitary stimulates production of IGF-I from the liver. The GH/IGF-1 axis is known to promote mammary gland hyperplasia and breast cancer carcinogenesis. Previous studies have shown that GH promotes carcinogenesis independently of insulin-like growth factor. The role of GH in specific subtypes of breast carcinoma remains to be defined. Our laboratory has found that estrogen receptor positive breast cancer cell lines up regulated oncogenic STAT5, Akt, and MAPK signaling pathways in response to GH. Tamoxifen resistant (TamR) and long-term estrogen deprived (LTED) breast cancer cell lines derived from MCF-7 and T47D cells demonstrated increased oncogenic signaling in response to GH compared to the corresponding parental cell lines from which they were derived. In contrast, GH stimulation of a triple negative breast cancer cell line (MDA-MB-231) failed to induce signaling via these oncogenic pathways. Although GH treatment of parental, TamR, and LTED breast cancer cells had minimal effect on cell proliferation, cell cycle progression, or migration; GH signaling protected cells from cytotoxic chemotherapy induced apoptosis. This data shows that GH signaling is found in endocrine sensitive and resistant cells. Furthermore, GH may function in protecting cells from cell death induced by either endocrine or cytotoxic drugs. GH receptor blockade may be a valid treatment option for endocrine resistant breast cancer. Citation Format: Heather C Beckwith, Douglas Yee. Growth hormone induction of oncogenic signaling promotes survival of endocrine resistant breast cancer cells [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 P4-06-06.

An epigenomic approach to therapy for tamoxifen-resistant breast cancer.

Tamoxifen has been a frontline treatment for estrogen receptor alpha (ERα)-positive breast tumors in premenopausal women. However, resistance to tamoxifen occurs in many patients. ER still plays a critical role in the growth of breast cancer cells with acquired tamoxifen resistance, suggesting that ERα remains a valid target for treatment of tamoxifen-resistant (Tam-R) breast cancer. In an effort to identify novel regulators of ERα signaling, through a small-scale siRNA screen against histone methyl modifiers, we found WHSC1, a histone H3K36 methyltransferase, as a positive regulator of ERα signaling in breast cancer cells. We demonstrated that WHSC1 is recruited to the ERα gene by the BET protein BRD3/4, and facilitates ERα gene expression. The small-molecule BET protein inhibitor JQ1 potently suppressed the classic ERα signaling pathway and the growth of Tam-R breast cancer cells in culture. Using a Tam-R breast cancer xenograft mouse model, we demonstrated in vivo anti-breast cancer activity by JQ1 and a strong long-lasting effect of combination therapy with JQ1 and the ER degrader fulvestrant. Taken together, we provide evidence that the epigenomic proteins BRD3/4 and WHSC1 are essential regulators of estrogen receptor signaling and are novel therapeutic targets for treatment of Tam-R breast cancer.

Abstract A96: The role of the proto-oncogene AGR2 in endocrine resistant breast cancer.

Abstract Tamoxifen, a selective estrogen receptor modulator (SERM), is used as a first line treatment for estrogen receptor (ER) positive breast cancer. Though most ER-positive breast cancers initially respond to tamoxifen, the development of resistance impedes durable responses to this intervention. Further, predicting which tumors are most likely to develop resistance remains a challenge. In part, this is due to the complexity of the mechanisms underlying resistance and the difficulty in translating insights from studies of tamoxifen pharmacology in vitro to the clinical setting. However, the results of most studies support the idea that resistance represents a change that enables cells to recognize tamoxifen as an agonist instead of an antagonist. Thus, identifying biomarkers downstream of the ER pathway that read on the relative agonist/antagonist activity of tamoxifen would likely improve the clinical utility of tamoxifen and other endocrine therapies in breast cancer. Recent work by our lab and other groups has identified AGR2 as an ER target gene whose expression is induced by both 17β-estradiol (E2) and 4-hydroxytamoxifen (4OHT). AGR2 is a proto-oncogene whose elevated expression is associated with increased cell proliferation, migration and invasion in breast cancer. Additionally, in the clinical setting, AGR2 has been identified as playing an important role in tamoxifen resistance. In a cell line established within our lab from a human xenograft model of tamoxifen resistance (TamR), AGR2 is significantly overexpressed compared to the parental MCF7 cell line. Knockdown of AGR2 expression in the TamR cell line resulted in a dramatic inhibition of E2 induced cell growth and migratory potential, attesting to its importance in the pathobiology of advanced cancers. When overexpressed in the MCF7 cell line, AGR2 was secreted and induced migratory potential in the MCF7 and TamR breast cancer cell lines in vitro. Additionally, overexpressed AGR2 significantly increased tumor growth in vivo. In both the MCF7 and TamR cellular models of breast cancer, AGR2 expression is required for ER expression. However, in the TamR cell line, AGR2 expression is no longer controlled by ER, suggesting that under selective pressure, the TamR cell line has acquired an alternative mechanism to upregulate AGR2 expression. Additional work is currently underway to further understand the mechanism of AGR2 regulation as breast cancers progress to a tamoxifen resistant state. This work is supported by a DOD Breast Cancer Postdoctoral Fellowship BC103843 (TMW) and a National Institutes of Health Grant R37 DK048807 (DPM). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A96. Citation Format: Tricia M. Wright, Suzanne E. Wardell, Erik R. Nelson, Donald P. McDonnell. The role of the proto-oncogene AGR2 in endocrine resistant breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A96.