TamR Cell Lines Research Articles

β‑catenin blockers enhance the effect of CDK4/6 inhibitors on stemness and proliferation suppression in endocrine‑resistant breast cancer cells.

Wnt/β‑catenin signaling is involved in endocrine resistance and stem cell‑like properties of hormone receptor‑positive breast cancer cells. Palbociclib is a well‑known inhibitor of cyclin‑dependent kinase 4 and 6 (CDK4/6 inhibitor) that downregulates the activation of retinoblastoma protein, thereby inhibiting the cell cycle in breast cancer cells. The inhibitory effects of a combination of palbociclib and ICG‑001, a β‑catenin small‑molecule inhibitor, were investigated in tamoxifen‑resistant breast cancer cell lines. Tamoxifen‑resistant MCF‑7 (TamR) cells were established by continuously exposing MCF‑7 cells to tamoxifen. The characteristics associated with the stem cell‑like property of cancer were assessed using western blotting, cell cycle analysis, and the mammosphere assay. The effects of the combination of palbociclib and ICG‑001 were evaluated in control MCF‑7 and TamR cell lines. Compared with control cells, TamR cells exhibited elevated levels of Nanog, Sox2, ALDH1, and p‑STAT3, indicating stem cell‑like characteristics, and elevated β‑catenin activity. TamR cells also showed significantly higher mammosphere‑forming efficiency. Several markers of stem cell‑like nature of TamR cells showed reduced levels upon treatment of cells with the drug combination; there was a greater reduction in the levels of these markers when the cells were treated with the combination than in the case where cells were treated with one of the drugs individually (combination index value for 25µM palbociclib and 50µM ICG‑001 was 1.1±0.02). TamR cells treated with the palbociclib and ICG‑001 combination demonstrated significantly reduced cell proliferation and mammosphere‑forming efficiency compared with the cells treated with one of these drugs. The combination of the drugs could additively inhibit proliferation and suppress stem cell‑like characteristics. These results suggest that β‑catenin plays a role in endocrine‑resistant breast cancer; the inhibition of β‑catenin and CDK4/6 together can overcome endocrine resistance in breast cancer cells.

Abstract P4-02-01: Targeting insulin receptor in estrogen receptor positive breast cancer

Abstract Obesity and metabolic dysfunction are on the rise in the United States. Hyperinsulinemia, frequently seen in these conditions, correlates with an increased risk of development and mortality from estrogen receptor (ER) positive breast cancer. Expression of components of the insulin and insulin-like growth factor (IGF) family of proteins promote cancer cell growth in vitro, link to shorter progression-free survival, and is associates with an increased risk of metastatic disease and death in breast cancer patients. Previously our lab found that development of resistance to the commonly used ER-targeting drug tamoxifen (Tam) in vitro is associated with a loss of the type I IGF receptor expression with an increased dependence on the homologous insulin receptor (IR). These data argue for targeting IR in breast cancer, but concern has been raised due to the potential of disrupting glucose homeostasis in patients with or without existing metabolic disease. To address this concern, a novel insulin-Fc fusion protein (AKS-130) was developed to lower blood glucose levels without causing clinical hypoglycemia with a potential to target tumor IR. AKS-130 was found to downregulate IR expression in models of colorectal cancer (HCT-116) and malignant melanoma (WM266.4). Further, AKS-130 suppressed xenograft growth in fed and fasted states. We examined if AKS-130 had similar effects on ER positive breast cancer cells. To test the hypothesis that resistance to endocrine therapy increases the reliance of ER positive breast cancer cells on IR signaling, we utilized the ER positive breast cancer cell lines (MCF-7L, T47D) and their Tam resistant derivatives (MCF-7L TamR, T47D TamR). Similar to in HCT-116 and WM266.4 cells, we found that AKS-130 is a modest agonist of IR in ER+ breast cancer cells. Exposure to AKS-130 led to a marked reduction in IR expression in both parental and TamR cell lines, and was associated with partially suppressed downstream signaling to Akt. Interestingly, AKS130 induced IR downregulation was inhibited by pretreatment of BMS 536924, a dual IGF1R/IR kinase inhibitor. In vivo, AKS-130 treatment by itself scaled down MCF7L parental xenograft growth as compared to vehicle group, and AKS-130 in combination with Tam trended toward extending time to a tumor size of 500mm3. In MCF7L TamR xenografts, addition of AKS-130 to Tam treatment also trended toward delaying xenograft growth, though it was not statistically significant. Importantly, prolonged treatment with AKS-130 did not lead to significant changes in weight or blood glucose. Taken together these data show that novel agents, such as AKS-130, may serve as way to disrupt IR signaling in cancer cells without affecting host glucose metabolism. Addition of AKS-130 to Tam treatment trended toward delaying xenograft growth though further studies are needed, especially animal model systems of insulin resistance. These data indicate that IR signaling may represent a target to overcome or delay endocrine therapy resistance in breast cancer. Citation Format: Jingran Cao, Lynsey M. Fettig, Kelly LaPara, Xihong Zhang, Sylaja Murikipudi, Andrea R. Delpero, Thomas M. Lancaster, Todd C. Zion, Douglas Yee. Targeting insulin receptor in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-01.

Establishment and transcriptome characterization of tamoxifen-resistant canine mammary gland tumor cells

Tamoxifen (TAM) currently is still the drug of choice for endocrine therapy in patients with estrogen receptor positive breast cancer. However, the development of drug resistance not only limits the drug utilization, but also greatly reduces the survival of patients. At the same time, TAM is poorly understood in canine mammary gland tumors. Therefore, it is crucial to find effective methods to reverse drug resistance and prevent the development of drug resistance so as to improve the efficacy of endocrine therapy for breast cancer.Firstly, we successfully established two TAM-resistant canine mammary gland tumor cells lines including TAMp,TAMm by drug concentration gradient plus drug maintenance, and then we confirmed that the resistant cells have stronger proliferation, migration, invasion and cloning ability by CCK8, Wound healing assay, Transwell invasion assay and Clone formation assay. Second, we performed sequencing analysis of TAMm and CHMm and detected a large number of different expression genes, including reported and novel drug-resistant genes, and genes involved in complex biological processes. Finally, we explored the role of the classical Wnt signaling pathway in drug-resistant cells, and immunofluorescence and western blot results showed increased expression of Wnt pathway related genes β-catenin and P-GSK3β in drug-resistant cells, indicating abnormal activation of the classical Wnt/β-catenin pathway This study successfully established two TamR cell lines and assayed its resistance generation in many aspects, which provides a good experimental model and theoretical support for a more comprehensive understanding of the endocrine drug resistance mechanism.

Cholesterol and Mevalonate: Two Metabolites Involved in Breast Cancer Progression and Drug Resistance through the ERRα Pathway.

Breast cancer is the second greatest cause of cancer-related death in women. Resistance to endocrine treatments or chemotherapy is a limiting drawback. In this context, this work aims to evaluate the effects of cholesterol and mevalonate during tumor progression and their contribution in the onset of resistance to clinical treatments in use today. In this study, we demonstrated that cholesterol and mevalonate treatments were able to activate the estrogen-related receptor alpha (ERRα) pathway, increasing the expression levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), ERbB2/human epithelial receptor (HER2), tumor protein D52 (TPD52), and NOTCH2 proteins in breast cancer cells. The activation of this pathway is shown to be responsible for intense metabolic switching, higher proliferation rates, sustained motility, the propagation of cancer stem-like cells (CSCs), and lipid droplet formation. All of these events are related to greater tumor propagation, aggressiveness, and drug resistance. Furthermore, the activation and expression of proteins induced by the treatment with cholesterol or mevalonate are consistent with those obtained from the MCF-7/TAMr cell line, which is largely used as a breast cancer model of acquired endocrine therapy resistance. Altogether, our data indicate that cholesterol and mevalonate are two metabolites implicated in breast cancer progression, aggressiveness, and drug resistance, through the activation of the ERRα pathway. Our findings enable us to identify the ERRα receptor as a poor prognostic marker in patients with breast carcinoma, suggesting the correlation between cholesterol/mevalonate and ERRα as a new possible target in breast cancer treatment.

Effect of artesunate on apoptosis and autophagy in tamoxifen resistant breast cancer cells (TAM-R).

BackgroundThe antitumor effect of artesunate (ART) is well-recognized. To investigate the effect of ART on tamoxifen-resistant breast cancer cells (TAM-R) proliferation, apoptosis, and autophagy with TAM-R cells of breast cancer as objects of study, and to investigate whether ART could re-sensitize TAM-R cells to TAM therapy.MethodsExperiments were performed using TAM-R cell lines. Cell Death Detection ELISA kit was used to detect the level of apoptosis. Western blot and immunofluorescent staining analysis were conducted to detect autophagy and apoptosis related proteins in TAM-R cells.ResultsAfter treated with ART, the proliferation activity of TAM-R cells was inhibited in a concentration-dependent manner. Increased apoptosis activity was detected in TAM-R cells when treated with ART. Compared with 10−6 M TAM monotherapy group, treatment group with ART and TAM in combination caused significant reduction in the levels of Bcl-2, XIAP, and Survivin proteins, and elevation of caspase-7. However, increased p53 proteins was not detected after ART treatment. No significant change was observed in autophagy proteins LC-3 and Beclin-1 among control, ART, TAM, and ART combined with TAM groups.ConclusionsThe intervention of ART could not inhibit protective autophagy in TAM-R cells, however, possess potential in inducing apoptosis. In addition, as ART inhibit TAM-R cells growth in a dose-dependent manner, co-administration of 1 or 3 µM of ART with 10−6 M TAM might not be enough to re-sensitize TAM-R cells to TAM therapy.

Abstract 2365: Dual inhibition of the PI3k/Akt and MEK5/ERK5 pathways in tamoxifen resistant breast cancer

Abstract Approximately 70% of breast cancers are considered estrogen receptor alpha (ER) and/or progesterone receptor (PR) positive, and their growth is primarily driven by the hormone estrogen. Initially these cancers are responsive to endocrine therapies such as tamoxifen. However, over time patients become resistant to conventional endocrine therapy and treatment becomes more difficult. Tamoxifen resistance occurs due to several mechanisms: loss of ER expression, gain of function mutations, pharmacological tolerance, alterations of co-activators, and ligand independent activation of ER by various kinase cascades. Aberrations in the Phosphoinositide-3-kinase (PI3K) and Mitogen Activated Protein Kinase (MAPK) pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. The crosstalk between PI3K/Akt and MEK1/2/ERK1/2 has been characterized in several systems. However, new evidence suggests that MEK5/ERK5, a member of the MAPK family, is a key component in the proliferation and survival of therapy resistant cancers. MEK5/ERK5 has been shown to promote ER alpha driven transcription in ER+ breast cancers and actin reorganization and metastasis in ER- breast cancers. Furthermore, MEK5/ERK5 promotes hormone independent tumorigenesis in breast cancer. Our lab has previously investigated these pathways in hormone independent breast cancers, TNBC (triple negative breast cancer). Our previous results indicate that combinations of PI3k/Akt and MEK5/ERK5 blockade are promising because they inhibit both the pro-proliferative and pro-metastatic pathways in TNBC. Additionally, PI3k/Akt and MEK5/ERK5 inhibition was shown synergistically reduce viability in a diverse panel of cell lines (TNBC, ER+, and Triple Positive). Based on these initial results we hypothesize that PI3k/Akt and MEK5/ERK5 inhibition decreases viability via a hormone independent mechanism: perhaps by reducing the cytosolic sequestration of Bad. In this study we investigate the utility PI3k/Akt and MEK5/ERK5 inhibition in a MCF-7 TamR cell line. The goal of our study is to elucidate the roles of PI3k/Akt/mTOR and MEK5/ERK5 in endocrine resistant breast cancer and broaden the scope of a dual inhibition strategy. Citation Format: Thomas D. Wright, Mahmud Hasan, Paula Witt-Enderby, Jane Cavanaugh. Dual inhibition of the PI3k/Akt and MEK5/ERK5 pathways in tamoxifen resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2365. doi:10.1158/1538-7445.AM2017-2365

Integrative analysis of miRNA and gene expression reveals regulatory networks in tamoxifen-resistant breast cancer.

Tamoxifen is an effective anti-estrogen treatment for patients with estrogen receptor-positive (ER+) breast cancer, however, tamoxifen resistance is frequently observed. To elucidate the underlying molecular mechanisms of tamoxifen resistance, we performed a systematic analysis of miRNA-mediated gene regulation in three clinically-relevant tamoxifen-resistant breast cancer cell lines (TamRs) compared to their parental tamoxifen-sensitive cell line. Alterations in the expression of 131 miRNAs in tamoxifen-resistant vs. parental cell lines were identified, 22 of which were common to all TamRs using both sequencing and LNA-based quantitative PCR technologies. Although the target genes affected by the altered miRNA in the three TamRs differed, good agreement in terms of affected molecular pathways was observed. Moreover, we found evidence of miRNA-mediated regulation of ESR1, PGR1, FOXM1 and 14-3-3 family genes. Integrating the inferred miRNA-target relationships, we investigated the functional importance of 2 central genes, SNAI2 and FYN, which showed increased expression in TamR cells, while their corresponding regulatory miRNA were downregulated. Using specific chemical inhibitors and siRNA-mediated gene knockdown, we showed that both SNAI2 and FYN significantly affect the growth of TamR cell lines. Finally, we show that a combination of 2 miRNAs (miR-190b and miR-516a-5p) exhibiting altered expression in TamR cell lines were predictive of treatment outcome in a cohort of ER+ breast cancer patients receiving adjuvant tamoxifen mono-therapy. Our results provide new insight into the molecular mechanisms of tamoxifen resistance and may form the basis for future medical intervention for the large number of women with tamoxifen-resistant ER+ breast cancer.

Abstract P3-05-04: AKT antagonist AZD5363 targets estrogen receptor (ER) function in endocrine resistant breast cancer (BC) and synergises with fulvestrant in vivo

Abstract AIM: To evaluate the efficacy and functional consquences of combining AZD5363 with endocrine therapy in pre-clinical models of endocrine-sensitive and resistant ER+ BC. BACKGROUND: The PI3K/AKT/mTOR signalling pathway plays an important role in BC. Its close interaction with ER signalling becomes more complex and inter-dependent with acquisition of endocrine resistance. Targeting the mTOR pathway in combination with endocrine therapy has shown clinical utility. However, a negative feedback loop exists downstream of the PI3K/AKT/mTOR pathway with mTOR inhibition leading to increased activation of IGFR1-dependent AKT activity potentially negating long-term benefit. Direct blockade of AKT in combination with endocrine therapy, may provide a better rationale for treatment of endocrine-resistant BC, impacting on both cell survival/apoptosis and ER ligand-independent signaling. In this investigation, we assessed the efficacy of AZD5363, a pan-AKT inhibitor with endocrine therapies in pre-clinical models of endocrine sensitive and resistant ER+ BC and its impact on molecular and cellular response. METHODS: Inhibition of AKT using AZD5363 was examined in 5 ER+ BC lines before and after adaptation to long-term estrogen deprivation (LTED) or tamoxifen (TAMR). The effects of AZD5363 on cell proliferation were determined alone and in combination with endocrine treatment and feedback upregulation and activation of receptor tyrosine kinases (RTKs) was examined by western blotting. ER-transactivation was measured with an estrogen-response element (ERE)-linked luciferase reporter construct and confirmed using chromatin-immunoprecipitation. Global gene expression analysis was used to identify pathways associated with response. Xenografts were treated with AZD5363 ± fulvestrant to determine in vivo effects. RESULTS: AZD5363 caused a dose-dependent decrease in proliferation in all cell lines tested (GI50<500nM) with the exception of HCC1428-wild-type and LTED. Of note both T47D-LTED and ZR75-LTED, which lose expression of ER were exquisitely sensitive (GI50∼100nM). AZD5363 re-sensitised the TAMR cell line to tamoxifen and acted synergistically with fulvestrant in MCF7-Wt and MCF7-LTED (CI<1). AZD5363 decreased phosphorylation of AKT/mTOR substrates PRAS40, p70S6 and S6 in all cell lines tested and caused a significant decrease in phosphorylation of ERser167 with an associated 50% reduction in ERα-mediated transcription and decrease in recruitment of ER and CBP to ERE on the TFF1 promoter. Furthermore, AZD5363 reduced Rb and cyclinD1. Inhibition of AKT with AZD5363 resulted in upregulation and activation of RTKs, including IGF-IR, EGFR, ERBB2 and ERBB3, which was cell line specific. Global gene expression and pathway analysis of MCF7 and MCF7-LTED treated with AZD5363 highlighted the relevance of ERBB2-ERBB3, ERK5 and IGF1 signalling as potential feedback loops. Combined treatment with AZD5363 and fulvestrant showed strong synergy in an MCF7 xenograft. CONCLUSION: These data suggest that AZD5363 plus fulvestrant may be effective in BC that is sensitive or resistant to E-deprivation or tamoxifen and that activated AKT is a determinant of response. These data strongly support the need for clinical evaluation. Citation Format: Lesley-Ann Martin, Stephanie K Guest, Sunil Pancholi, Ricardo Ribas, Qiong Gao, Nikiana Simigdala, Aradhana Rani, Barry Davies, Stephen Johnston, Mitch Dowsett. AKT antagonist AZD5363 targets estrogen receptor (ER) function in endocrine resistant breast cancer (BC) and synergises with fulvestrant in vivo [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 P3-05-04.

Gene expression profiling identifies FYN as an important molecule in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy.

To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer, we performed gene array analyses and identified 366 genes with altered expression in four unique tamoxifen-resistant (TamR) cell lines vs the parental tamoxifen-sensitive MCF-7/S0.5 cell line. Most of these genes were functionally linked to cell proliferation, death and control of gene expression, and include FYN, PRKCA, ITPR1, DPYD, DACH1, LYN, GBP1 and PRLR. Treatment with FYN-specific small interfering RNA or a SRC family kinase inhibitor reduced cell growth of TamR cell lines while exerting no significant effect on MCF-7/S0.5 cells. Moreover, overexpression of FYN in parental tamoxifen-sensitive MCF-7/S0.5 cells resulted in reduced sensitivity to tamoxifen treatment, whereas knockdown of FYN in the FYN-overexpressing MCF-7/S0.5 cells restored sensitivity to tamoxifen, demonstrating growth- and survival-promoting function of FYN in MCF-7 cells. FYN knockdown in TamR cells led to reduced phosphorylation of 14-3-3 and Cdc25A, suggesting that FYN, by activation of important cell cycle-associated proteins, may overcome the anti-proliferative effects of tamoxifen. Evaluation of the subcellular localization of FYN in primary breast tumors from two cohorts of endocrine-treated ER+ breast cancer patients, one with advanced disease (N=47) and the other with early disease (N=76), showed that in the former, plasma membrane-associated FYN expression strongly correlated with longer progression-free survival (P<0.0002). Similarly, in early breast cancer patients, membrane-associated expression of FYN in the primary breast tumor was significantly associated with increased metastasis-free (P<0.04) and overall (P<0.004) survival independent of tumor size, grade or lymph node status. Our results indicate that FYN has an important role in tamoxifen resistance, and its subcellular localization in breast tumor cells may be an important novel biomarker of response to endocrine therapy in 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.

The Wnt signalling pathway is upregulated in an in vitro model of acquired tamoxifen resistant breast cancer

BackgroundAcquired resistance to Tamoxifen remains a critical problem in breast cancer patient treatment, yet the underlying causes of resistance have not been fully elucidated. Abberations in the Wnt signalling pathway have been linked to many human cancers, including breast cancer, and appear to be associated with more metastatic and aggressive types of cancer. Here, our aim was to investigate if this key pathway was involved in acquired Tamoxifen resistance, and could be targeted therapeutically.MethodsAn in vitro model of acquired Tamoxifen resistance (named TamR) was generated by growing the estrogen receptor alpha (ER) positive MCF7 breast cancer cell line in increasing concentrations of Tamoxifen (up to 5 uM). Alterations in the Wnt signalling pathway and epithelial to mesenchymal transition (EMT) in response to Tamoxifen and treatment with the Wnt inhibitor, IWP-2 were measured via quantitative RT-PCR (qPCR) and TOP/FOP Wnt reporter assays. Resistance to Tamoxifen, and effects of IWP-2 treatment were determined by MTT proliferation assays.ResultsTamR cells exhibited increased Wnt signalling as measured via the TOP/FOP Wnt luciferase reporter assays. Genes associated with both the β-catenin dependent (AXIN2, MYC, CSNK1A1) and independent arms (ROR2, JUN), as well as general Wnt secretion (PORCN) of the Wnt signalling pathway were upregulated in the TamR cells compared to the parental MCF7 cell line. Treatment of the TamR cell line with human recombinant Wnt3a (rWnt3a) further increased the resistance of both MCF7 and TamR cells to the anti-proliferative effects of Tamoxifen treatment. TamR cells demonstrated increased expression of EMT markers (VIM, TWIST1, SNAI2) and decreased CDH1, which may contribute to their resistance to Tamoxifen. Treatment with the Wnt inhibitor, IWP-2 inhibited cell proliferation and markers of EMT.ConclusionsThese data support the role of the Wnt signalling pathway in acquired resistance to Tamoxifen. Further research into the mechanism by which activated Wnt signalling inhibits the effects of Tamoxifen should be undertaken. As a number of small molecules targeting the Wnt pathway are currently in pre-clinical development, combinatorial treatment with endocrine agents and Wnt pathway inhibitors may be a useful therapeutic option in the future for a subset of breast cancer patients.

Abstract 2214: COUP-TFII suppresses NFκB activation in endocrine-resistant breast cancer cells

Abstract COUP-TFII is an orphan nuclear receptor that can function as either a transcriptional activator or repressor. We have previously shown that COUP-TFII expression is reduced in tamoxifen-resistant LCC9 breast cancer cells and that overexpression of COUP-TFII restored the ability of tamoxifen to inhibit cell proliferation. An increase in NFκB activity and p65/RELA level has been reported in endocrine-resistant breast cancer cells. Because NFκB activation results in increased expression of anti-apoptotic and pro-proliferative genes, increased NFκB signaling promotes the survival of cancer cells. The goal of the current study was to further elucidate the mechanism by which COUP-TFII enables cells to maintain endocrine sensitivity and to determine if COUP-TFII modulates NFκB activity in tamoxifen-resistance. To examine whether COUP-TFII can suppress NFκB activity, MCF-7 (tamoxifen-sensitive, TAM-S) and LCC9 (tamoxifen-resistant, TAM-R) breast cancer cells were transfected with a NFκB luciferase reporter and treated with TNFα to activate NFκB. Cotransfection with COUP-TFII reduced NFκB activity in both TAM-S and TAM-R cell lines. Knockdown of COUP-TFII with siRNA led to an increase in p65 expression; conversely, COUP-TFII overexpression led to a suppression of p65 expression. COUP-TFII overexpression also reduced TNFα-induced endogenous NFκB target gene expression. Preliminary data also suggests that suppression of NFκB activation by COUP-TFII leads to a reduction in cell viability in MCF-7 and LCC9 cells in response to tamoxifen and TNFα treatment. This indicates a novel role for COUP-TFII in suppression of NFκB activity which may explain the ability of COUP-TFII to promote tamoxifen-sensitivity. COUP-TFII may be both a useful biomarker to predict tamoxifen-sensitivity as well as a target to restore endocrine sensitivity to resistant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2214. doi:1538-7445.AM2012-2214

The ERα coactivator, HER4/4ICD, regulates progesterone receptor expression in normal and malignant breast epithelium

The HER4 intracellular domain (4ICD) is a potent estrogen receptor (ERα) coactivator with activities in breast cancer and the developing mammary gland that appear to overlap with progesterone receptor (PgR). In fact, 4ICD has recently emerged as an important regulator and predictor of tamoxifen response, a role previously thought to be fulfilled by PgR. Here we investigated the possibility that the 4ICD coactivator regulates PgR expression thereby providing a mechanistic explanation for their partially overlapping activities in breast cancer. We show that 4ICD is both sufficient and necessary to potentiate estrogen stimulation of gene expression. Suppression of HER4/4ICD expression in the MCF-7 breast tumor cell line completely eliminated estrogen stimulated expression of PgR. In addition, the HER4/4ICD negative MCF-7 variant, TamR, failed to express PgR in response to estrogen. Reintroduction of wild-type HER4 but not the γ-secretase processing mutant HER4V673I into the TamR cell line restored PgR expression indicating that 4ICD is an essential PgR coactivator in breast tumor cells. These results were substantiated in vivo using two different physiologically relevant experimental systems. In the mouse mammary gland estrogen regulates expression of PgR-A whereas expression of PgR-B is estrogen independent. Consistent with a role for 4ICD in estrogen regulated PgR expression in vivo, PgR-A, but not PgR-B, expression was abolished in HER4-null mouse mammary glands during pregnancy. Coexpression of PgR and 4ICD is also commonly observed in ERα positive breast carcinomas. Using quantitative AQUA IHC technology we found that 4ICD potentiated PgR expression in primary breast tumors and the highest levels of PgR expression required coexpression of ERα and the 4ICD coactivator. In summary, our results provide compelling evidence that 4ICD is a physiologically important ERα coactivator and 4ICD cooperates with ERα to potentiate PgR expression in the normal and malignant breast. We propose that direct coupling of these signaling pathways may have important implications for mammary development, breast carcinogenesis, and patient response to endocrine therapy.