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RNA-Seq analysis of long-term estrogen-deprived (LTED) MDA-MB-134VI (MM134) and SUM44PE (SUM44) ILC cell lines

ABSTRACT: Background: Invasive lobular breast carcinoma (ILC) is a histological subtype of breast cancer that is characterized by loss of E-cadherin, and high expression of estrogen receptor alpha (ER). Many patients with ILC are effectively treated with adjuvant aromatase inhibitors (AIs), however, acquired AI resistance remains a significant problem. Methods: To identify underlying mechanisms of acquired antiestrogen resistance in ILC, we developed a total of 6 long-term estrogen-deprived (LTED) variant cell lines of the human ILC cell lines SUM44PE (SUM44; 2 lines) and MDA-MB-134VI (MM134; 4 lines). To better understand mechanisms of AI resistance in these models, we performed transcriptional profiling analysis by RNA-sequencing. Results: MM134 LTED cells expressed ER at decreased level and lost growth response to estradiol, while SUM44 LTED cells retained partial ER activity. Our transcriptional profiling analysis identified shared activation of lipid metabolism across all 6 independent models. However, the underlying basis of this signature was distinct between models. Oxysterols were able to promote the proliferation of SUM44 LTED cells, but not MM134 LTED. In contrast, MM134 LTED cells displayed high expression of the Sterol regulatory element-binding protein 1 (SREBP1), a regulator of fatty acid and cholesterol synthesis, and were hypersensitive to genetic or pharmacological inhibition of SREBPs. Several SREBP1 downstream targets involved in fatty acid synthesis, including FASN, were induced, and MM134 LTED cells were more sensitive to etomoxir, an inhibitor of the rate-limiting enzyme in β-oxidation, than their respective parental control cells. Conclusions: Our characterization of a unique series of AI-resistant ILC models identifies a lipogenic phenotype, including overexpression of SREBP1. This novel metabolic target deserves further study for the prevention and treatment of AI-resistance for patients with ILC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE116744 | GEO | 2018/08/30

REPOSITORIES: GEO

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Project description:Postmenopausal breast cancer patients benefit from aromatase inhibitors (AIs) that reduce the levels of estrogens critical for the growth of estrogen receptor (ER)-positive tumors. Unfortunately, many tumors are resistant to AI, and we are only beginning to understand the complex mechanisms underlying treatment resistance. Here we set out to determine whether epigenetic changes could contribute to therapy resistance. For AI-resistance models, we used previously established MCF-7 cell clones, termed C4-12 and long-term estrogen deprivation (LTED), that were isolated after being cultured in estrogen-free media for 9 months and 18M-bM-^@M-^S24 months, respectively. Methyl CpG Binding Domain (MBD) - pull down followed by affymetrix promoter array was used to detect promoter methylation patterns in these cell lines. These studies identified widespread genomic hyper- and hypomethylation events, with a significant enrichment of promoter hypermethylation of development-associated genes in both cell lines. A developmentally regulated gene that was heavily methylated and lost expression in both cell-line systems was HOXC10. Moreover, we found that HOXC10 is an estrogen-regulated gene and lack of ER regulation is associated with progressive epigenetic silencing through EZH2/H3K27me3 and DNA hypermethylation. Stable knockdown of HOXC10 in MCF-7 cells resulted in increased cell growth, reduced cell apoptosis, and increased cell motility. A preliminary study using AI-treated breast tumors did not show significant associations, however, the numbers were small. We identified HOXC10 methylation as a novel determinant of endocrine resistance. Also, we revealed that epigenetic reprogramming of genes involved in development may be a fundamental phenomenon in hormone resistance. Therefore, our study might provide the basis for the expansion of clinical markers for endocrine resistance and future clinical trials such as combination of endocrine and epigenetic therapies. Long-term estrogen deprived previously established MCF-7 cell clones termed C4-12 and LTED genomic DNA subjected to Methyl CpG Binding Domain (MBD) - pull down followed by affymetrix promoter array to detect promoter methylation patterns

Project description:Despite the success of the aromatase inhibitors (AIs) in treating estrogen receptor positive breast cancer, 15-20% of patients receiving adjuvant AIs will relapse within 5-10 years of treatment initiation. Long term estrogen deprivation (LTED) of breast cancer cells in culture has been successfully used to mimic AI-induced estrogen depletion to dissect mechanisms of AI resistance. However, we hypothesized that a subset of patients receiving AI therapy may maintain low circulating concentrations of estrogens that influence the development of endocrine resistance. We sought to expand established LTED models to account for these observations. MCF-7 cells were grown in medium with charcoal stripped serum supplemented with defined concentrations of E2 or the estrogenic androgen metabolite 3βAdiol. Cells were selected in the EC10 and EC90 of E2 or 3βAdiol, or estrogen deprived. Estrogen-independence was evaluated during selection by assessing cell growth in the absence or presence of E2 or 3βAdiol. Following >7 months of selection, estrogen-independence developed in estrogen-deprived cells and cells maintained in low concentrations of steroid hormone. Functional analyses demonstrated that estrogen-deprived and low-steroid selected cells developed estrogen-independence via unique mechanisms, independent and dependent of ERα, respectively. Estrogen-independent growth in low-steroid selected cells could be blocked by kinase inhibitors. However, these cells were completely resistant to kinase inhibition in the presence of low steroid hormone concentrations. These data offer a novel perspective on the development of resistance to AI therapy, and may yield novel approaches to treat AI-resistant tumors. MCF-7 cells were selected for >7months in IMEM+10% Charcoal Stripped FBS, supplmented with defined steroid hormone conditions. Following outgrowth of estrogen-independent cells, cell lines were grown in estrogen-free conditions and gene expression analysis was performed to identify drivers of estrogen-independent growth. Cells were assessed in biological triplicates.

Project description:Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer that is frequently associated with favorable outcomes, as ~90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that ILC patients receiving adjuvant endocrine therapy may not benefit from improved outcomes versus other breast cancer patients. Based on these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome in vitro via gene expression microarray analyses and ER ChIP-Seq. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. Response to endocrine therapy was also examined in ILC cell lines. We identified 915 genes that were uniquely E2-regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also regulated in vivo in HCI-013. We observed that MM134 were de novo tamoxifen resistant, and were induced to grow by 4-hydroxytamoxifen, as well as other anti-estrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required FGFR1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance. Cells were hormone deprived by replacing growth medium with IMEM+2% charcoal stripped serum for 3 days. Following deprivation, cells were treated for 3 or 24 hours with 1nM E2 or vehicle (0.01% EtOH) in biological quadruplicate. Following treatment, cells were lysed and RNA was harvested using the Illustra RNAspin Mini kit (GE Health). cRNA synthesis and labeling was performed using the Ambion MessageAmp Premier Kit (Life Technologies), and cRNA was hybridized to U133A 2.0 arrays (Affymetrix, Santa Clara, CA). cRNA synthesis and labeling, hybridization, and scanning were performed by the University of Pittsburgh Cancer Biomarkers Facility.

Project description:Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer that is frequently associated with favorable outcomes, as ~90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that ILC patients receiving adjuvant endocrine therapy may not benefit from improved outcomes versus other breast cancer patients. Based on these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome in vitro via gene expression microarray analyses and ER ChIP-Seq. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. Response to endocrine therapy was also examined in ILC cell lines. We identified 915 genes that were uniquely E2-regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also regulated in vivo in HCI-013. We observed that MM134 were de novo tamoxifen resistant, and were induced to grow by 4-hydroxytamoxifen, as well as other anti-estrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required FGFR1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance. Cells were hormone deprived by replacing growth medium with IMEM+10% charcoal stripped serum for 3 days. Following deprivation, cells were treated for 3 or 24 hours with 1nM E2 or vehicle (0.01% EtOH) in biological quadruplicate. Following treatment, cells were lysed and RNA was harvested using the Illustra RNAspin Mini kit (GE Health). cRNA synthesis and labeling was performed using the Ambion MessageAmp Premier Kit (Life Technologies), and cRNA was hybridized to U133A 2.0 arrays (Affymetrix, Santa Clara, CA). cRNA synthesis and labeling, hybridization, and scanning were performed by the University of Pittsburgh Cancer Biomarkers Facility.

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RNA-Seq analysis of long-term estrogen-deprived (LTED) MDA-MB-134VI (MM134) and SUM44PE (SUM44) ILC cell lines

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