Fulvestrant-resistant Breast Cancer Cells Research Articles

Induction of the TEAD Co-activator VGLL1 by Estrogen Receptor-Targeted Therapy Drives Resistance in Breast Cancer.

Resistance to endocrine therapies (ET) is common in estrogen receptor (ER) positive breast cancer, and most relapsed patients die with ET-resistant disease. While genetic mutations provide explanations for some relapses, mechanisms of resistance remain undefined in many cases. Drug-induced epigenetic reprogramming has been shown to provide possible routes to resistance. By analyzing histone H3 lysine 27 acetylation (H3K27ac) profiles and transcriptional reprogramming in models of ET resistance, we discovered that selective ER degraders (SERDs), such as fulvestrant, promote expression of VGLL1, a co-activator for TEAD transcription factors. VGLL1, acting via TEADs, promoted expression of genes that drive growth of fulvestrant-resistant breast cancer cells. Pharmacological disruption of VGLL1/TEAD4 interaction inhibited VGLL1/TEAD-induced transcriptional programs to prevent growth of resistant cells. EGFR was among the VGLL1/TEAD-regulated genes, and VGLL1-directed EGFR upregulation sensitized fulvestrant-resistant breast cancer cells to EGFR inhibitors. Taken together, these findings identify VGLL1 as a transcriptional driver in ET resistance and advance therapeutic possibilities for relapsed ER+ breast cancer patients.

Anticancer effect of zoledronic acid in endocrine-resistant breast cancer cells via HER-2 signaling

HER-2 overexpression is a major mechanism involved in endocrine-resistant breast cancer, which has very limited treatment options. Zoledronic acid (ZA) is a drug in the bisphosphonate group used to treat osteoporosis. ZA was reported to exhibit activity in various cancers, with higher efficacy associated with estrogen-deprivation states. ZA inhibits cell proliferation in lung cancer through the epidermal growth factor receptor signaling pathway. Because endocrine-resistant breast cancer cells overexpress HER-2 and grow independently without estrogen, ZA may exert anticancer effects in these cell types. The inhibitory effects and mechanisms of ZA in endocrine-resistant cells through HER-2 signaling were investigated. The efficacy of ZA was higher in the endocrine-resistant breast cancer cells when compared with the wild-type cells. ZA also exhibited a synergistic effect with fulvestrant and may circumvent fulvestrant resistance. ZA decreased phosphorylated ERK (pERK) levels in resistant cell lines and attenuated HER-2 signaling in tamoxifen- and fulvestrant-resistant cells. ZA significantly decreased HER-2 levels and its downstream signaling molecules, including pAKT and pNF-κB in fulvestrant-resistant breast cancer cells. This inhibitory effect may explain the lower IC50 values of ZA in fulvestrant-resistant cells compared with tamoxifen-resistant cells. Moreover, ZA inhibited the migration and invasion in the resistant cell lines, suggesting an ability to inhibit tumor metastasis. The results indicate that ZA has potential for repurposing as an adjuvant treatment for patients with endocrine-resistant breast cancer.

Comprehensive Transcriptomic Analysis Reveals Dysregulated Competing Endogenous RNA Network in Endocrine Resistant Breast Cancer Cells.

BackgroundTamoxifen and fulvestrant, both approved for endocrine therapy, have remarkably increased the prognosis of hormone receptor-positive breast cancer patients. However, acquired resistance to endocrine therapy greatly reduces its clinical efficacy. Accumulating evidence suggests a pivotal role of non-coding RNAs (ncRNAs) in breast cancer endocrine resistance, but the specific functions of ncRNAs in tamoxifen and fulvestrant resistance remain largely unknown.MethodsMicroarray analysis was performed for endocrine therapy sensitive (MCF-7), tamoxifen-resistant (LCC2), and dual tamoxifen and fulvestrant-resistant (LCC9) breast cancer cells. Gene ontology and pathway analysis were conducted for functional prediction of the unannotated differentially expressed ncRNAs. Competing endogenous RNA regulatory networks were constructed.ResultsWe discovered a total of 3,129 long non-coding RNAs (lncRNAs), 13,556 circular RNAs (circRNAs), 132 microRNAs, and 3358 mRNAs that were significantly differentially expressed. We constructed co-expression networks for lncRNA-mRNA, circRNA-mRNA, and microRNA-mRNA. In addition, we established lncRNA-microRNA-mRNA and circRNA-microRNA-mRNA regulatory networks to depict ncRNA crosstalk and transcriptomic regulation of endocrine resistance.ConclusionsOur study delineates a comprehensive profiling of ncRNAs in tamoxifen and fulvestrant resistant breast cancer cells, which enriches our understanding of endocrine resistance and sheds new light on identifying novel endocrine resistance biomarkers and potential therapeutic targets to overcome endocrine resistance.

A dual role of miR-22 modulated by RelA/p65 in resensitizing fulvestrant-resistant breast cancer cells to fulvestrant by targeting FOXP1 and HDAC4 and constitutive acetylation of p53 at Lys382

Antiestrogen resistance is a major challenge encountered during the treatment of estrogen receptor alpha positive (ERα+) breast cancer. A better understanding of signaling pathways and downstream transcription factors and their targets may identify key molecules that can overcome antiestrogen resistance in breast cancer. An aberrant expression of miR-22 has been demonstrated in breast cancer; however, its contribution to breast cancer resistance to fulvestrant, an antiestrogen drug, remains unknown. In this study, we demonstrated a moderate elevation in miR-22 expression in the 182R-6 fulvestrant-resistant breast cancer line we used as a model system, and this elevation was positively correlated with the expression of the miRNA biogenesis enzymes AGO2 and Dicer. The level of phosphorylated HER2/neu at Tyr877 was also upregulated in these cells, whereas the level of RelA/p65 phosphorylated at Ser536 (p-p65) was downregulated. Knockdown of HER2/neu led to an induction of p-p65 and a reduction in miR-22 levels. Luciferase assays identified two NF-κB binding motifs in the miR-22 promoter that contributed to transcriptional repression of miR-22. Activation of RelA/p65, triggered by LPS, attenuated miR-22 expression, but this expression was restored by sc-514, a selective IKKβ inhibitor. Inhibition of miR-22 suppressed cell proliferation, induced apoptosis and caused cell cycle S-phase arrest, whereas enhancing expression of p21Cip1/Waf1 and p27Kip1. Surprisingly, ectopic expression of miR-22 also suppressed cell proliferation, induced apoptosis, caused S-phase arrest, and promoted the expression of p21Cip1/Waf1 and p27Kip1. Ectopic overexpression of miR-22 repressed the expression of FOXP1 and HDAC4, leading to a marked induction of acetylation of HDAC4 target histones. Conversely, inhibition of miR-22 promoted the expression of both FOXP1 and HDAC4, without the expected attenuation of histone acetylation. Instead, p53 acetylation at lysine 382 was unexpectedly upregulated. Taken together, our findings demonstrated, for the first time, that HER2 activation dephosphorylates RelA/p65 at Ser536. This dephosphoryalted p65 may be pivotal in transactivation of miR-22. Both increased and decreased miR-22 expression cause resensitization of fulvestrant-resistant breast cancer cells to fulvestrant. HER2/NF-κB (p65)/miR-22/HDAC4/p21 and HER2/NF-κB (p65)/miR-22/Ac-p53/p21 signaling circuits may therefore confer this dual role on miR-22 through constitutive transactivation of p21.

SNAI2 upregulation is associated with an aggressive phenotype in fulvestrant-resistant breast cancer cells and is an indicator of poor response to endocrine therapy in estrogen receptor-positive metastatic breast cancer

BackgroundEndocrine resistance in estrogen receptor-positive (ER+) breast cancer is a major clinical problem and is associated with accelerated cancer cell growth, increased motility and acquisition of mesenchymal characteristics. However, the specific molecules and pathways involved in these altered features remain to be detailed, and may be promising therapeutic targets to overcome endocrine resistance.MethodsIn the present study, we evaluated altered expression of epithelial-mesenchymal transition (EMT) regulators in ER+ breast cancer cell models of tamoxifen or fulvestrant resistance, by gene expression profiling. We investigated the specific role of increased SNAI2 expression in fulvestrant-resistant cells by gene knockdown and treatment with a SNAIL-p53 binding inhibitor, and evaluated the effect on cell growth, migration and expression of EMT markers. Furthermore, we evaluated SNAI2 expression by immunohistochemical analysis in metastatic samples from two cohorts of patients with breast cancer treated with endocrine therapy in the advanced setting.ResultsSNAI2 was found to be significantly upregulated in all endocrine-resistant cells compared to parental cell lines, while no changes were observed in the expression of other EMT-associated transcription factors. SNAI2 knockdown with specific small interfering RNA (siRNA) converted the mesenchymal-like fulvestrant-resistant cells into an epithelial-like phenotype and reduced cell motility. Furthermore, inhibition of SNAI2 with specific siRNA or a SNAIL-p53 binding inhibitor reduced growth of cells resistant to fulvestrant treatment. Clinical evaluation of SNAI2 expression in two independent cohorts of patients with ER+ metastatic breast cancer treated with endocrine therapy in the advanced setting (N = 86 and N = 67) showed that high SNAI2 expression in the metastasis correlated significantly with shorter progression-free survival on endocrine treatment (p = 0.0003 and p = 0.004).ConclusionsOur results suggest that SNAI2 is a key regulator of the aggressive phenotype observed in endocrine-resistant breast cancer cells, an independent prognostic biomarker in ER+ advanced breast cancer treated with endocrine therapy, and may be a promising therapeutic target in combination with endocrine therapies in ER+ metastatic breast cancer exhibiting high SNAI2 levels.

Combined inhibition of EGFR and c-ABL suppresses the growth of fulvestrant-resistant breast cancer cells through miR-375-autophagy axis

Fulvestrant is the FDA-approved “pure anti-estrogen” agent for malignant breast cancer therapy. But endocrine resistance causes drug failure. A new approach is desired for fulvestrant-resistant breast cancer (FRBC) therapy. This study aims to find an effective approach to inhibit FRBC for patients with advanced breast cancer. MTT assay was first performed to detect the effect of inhibitors of c-ABL (imatinib) and EGFR (lapatinib) on FRBC cells. Microarray analysis was carried out to identify microRNA which is significantly changed between parental and FRBC cells. The related mechanisms were analyzed by qRT-PCR, MTT, AO staining and western blotting. Dual treatment significantly inhibited cell growth of FRBC and upregulated microRNA-375 (miR-375). Overexpression of miR-375 inhibited growth of FRBC cells, reduced autophagy, and decreased expression of ATG7 and LC3-II. Dual treatment elevated expression of miR-375 more than any single one of these two inhibitors. Overexpression of miR-375 increased cell growth inhibition induced by dual treatment, and the effect was attenuated when miR-375 was inhibited.In conclusion, we identified that combined inhibition of EGFR and c-ABL can suppress the growth of FRBC cells and elucidated a mechanism within FRBC cells involving regulation of miR-375 and autophagy. Dual treatment may be useful for inhibiting fulvestrant-resistant breast cancer.

Poster session 6: signalling pathways and apoptosis

Introduction: Acquired resistance to anti-hormonal agents in breast cancer is frequently associated with altered growth factor expression and the development of an aggressive cell phenotype in vitro. Chronic exposure of MCF-7 cells to the pure antioestrogen Fulvestrant results in acquired Fulvestrant resistance. Significantly, these resistant cells also overexpress the c-Met receptor, a protein that clinically has been linked to invasive breast carcinoma and metastasis. Subsequent exposure of Fulvestrant-resistant (Fas-R) cells to the c-Met ligand, hepatocyte growth factor/scatter factor (HGF/SF), results in a profound increase in their motile and invasive capacities. In other cancer cells, HGF/SF is known to induce invasive behaviour by matrix metalloproteinase (MMP) overproduction via the phosphoinositide-3 kinase (PI3K)/AKT pathway. In order to investigate whether a similar mechanism might operate in our resistant cell line, we first established whether HGF/SF stimulated MMP expression and subsequently determined the effects of PI3K pathway inhibition on both MMP expression and the aggressive phenotype. Methods: MCF-7 and Fas-R cells were exposed to HGF/SF (20ng/ml). MMP mRNA and protein expression were assessed using RT-PCR and zymography respectively. In vitro migratory and invasive capacities of both cell types were measured by migration and invasion assays. Pharmacological inhibition of the PI3K/AKT pathway was achieved using LY294002; changes in PI3K signalling pathway activity were assessed by Western Blotting and phosphospecific antibodies. Results: HGF/SF significantly promoted the migration and invasion of Fas-R cells whilst having only a modest effect in MCF-7 cells. Moreover, in Fas-R cells HGF/SF induced MMP-1, -9 and -10 expression and increased the level of AKT phosphorylation. Inclusion of LY294002 inhibited HGF/SF-enhanced Fas-R cell migration and invasion. AKT inhibition furthermore downregulated MMP-9 mRNA levels and abolished MMP-9 protein expression. Conclusion: These data suggest that HGF/SF-induced stimulation of c-Met, overexpressed in Fulvestrant-resistant breast cancer cells, promotes an aggressive, invasive phenotype through a mechanism involving PI3K/AKT-mediated MMP-9 expression.

Cooperative effect of gefitinib and fumitremorgin c on cell growth and chemosensitivity in estrogen receptor α negative fulvestrant-resistant MCF-7 cells

The selective ER downregulator, fulvestrant, is currently approved as a second line endocrine therapy after onset of resistance to prior antiestrogen therapy in postmenopausal breast cancer patients. Resistance to antihormonal therapies is common and, therefore, we anticipate that fulvestrant-resistance will occur as well. The current study was undertaken to investigate the underlying molecular changes after fulvestrant-resistance and find new therapeutic targets and agents for fulvestrant-resistant breast cancer cells. We developed a unique fulvestrant-resistant cell line (MCF-7/F), derived from MCF-7 estrogen receptor alpha (ERalpha)-positive human breast cancer cells, by culturing them in 1 microM fulvestrant containing medium for approximately 18 months. MCF-7/F cells became irreversibly ERalpha negative as withdrawal of fulvestrant did not alter the ERalpha-negative phenotype, determined by real-time PCR, Western blot analysis, and ERE-luciferase transfection assays. MCF-7/F cells grew in a hormone-independent manner. Interestingly, MCF-7/F cells overexpressed both epidermal growth factor receptor (EGFR) and breast cancer resistant protein (BCRP). Gefitinib, a specific EGFR tyrosine kinase inhibitor, preferentially inhibited the growth of MCF-7/F cells relative to MCF-7 cells by inhibiting both MAPK44/42 and Akt phosphorylation. MCF-7/F cells became less sensitive to chemotherapeutic agents such as mitoxantrone. Moreover, fumitremorgin C, a specific BCRP inhibitor, significantly increased the efficacy of mitoxantrone in MCF-7/F cells. Gefitinib increased the inhibitory effect of mitoxantrone on cell growth. Similarly, fumitremorgin C increased the inhibitory effect of gefitinib on cell growth, suggesting that there is a bidirectional crosstalk between EGFR and BCRP. More importantly, these results provide a molecular basis for using gefitinib, BCRP inhibitors, and chemotherapeutic agents as combination therapy approaches in fulvestrant-resistant breast cancer.