Project description:Tamoxifen is an effective anti-estrogen treatment for patients with estrogen receptor-positive (ER+) breast cancer. However, about 30% of such patients receiving tamoxifen as an adjuvant therapy experience recurrence within 15 years, and most patients with advanced disease eventually develop resistance to tamoxifen. 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 human breast cancer cell lines (TamRs) compared to their parental tamoxifen-sensitive MCF-7/S0.5 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. ER+ and tamoxifen sensitive breast cancer cell line (MCF-7/S0.5) and its derived tamoxifen resistant clones: TAMR-1, TAMR-4 and TAMR-8 were miRNA expression profiled in triplicates of each using Exiqon's miRCURY LNA based microRNA Ready-to-use PCR, Human panel I+II, V2.R (Exiqon, product number 203608).
Project description:Tamoxifen, an antagonist to estrogen receptor (ER), is a first line drug used in breast cancer treatment. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying the resistance to tamoxifen, we established a tamoxifen-resistant cell line by treating the MCF7 breast cancer cell line with tamoxifen for over 6 months. We showed that this cell line exhibited resistance to tamoxifen both in vitro and in vivo. In order to quantify the phosphorylation alterations associated with tamoxifen resistance, we performed SILAC-based quantitative phosphoproteomic profiling on the resistant and vehicle-treated sensitive cell lines where we identified >5,600 unique phosphopeptides. We found phosphorylation levels of 1,529 peptides were increased (>2 fold) and 409 peptides were decreased (<0.5-fold) in tamoxifen resistant cells compared to tamoxifen sensitive cells. Gene set enrichment analysis revealed that focal adhesion pathway was the top enriched signaling pathway activated in tamoxifen resistant cells. We observed hyperphosphorylation of the focal adhesion kinases FAK1 and FAK2 in the tamoxifen resistant cells. Of note, FAK2 was not only hyperphosphorylated but also transcriptionally upregulated in tamoxifen resistant cells. Suppression of FAK2 by specific siRNA knockdown could sensitize the resistant cells to the treatment of tamoxifen. We further showed that inhibiting FAK activity using the small molecule inhibitor PF562271 repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 significantly associated with short metastasis-free survival of ER-positive breast cancer patients treated with tamoxifen-based hormone therapy. Our studies suggest that FAK2 is a great potential target for the development of therapy for the treatment of hormone refractory breast cancers.
Project description:Tamoxifen is an effective anti-estrogen treatment for patients with estrogen receptor-positive (ER+) breast cancer. However, about 30% of such patients receiving tamoxifen as an adjuvant therapy experience recurrence within 15 years, and most patients with advanced disease eventually develop resistance to tamoxifen. 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 human breast cancer cell lines (TamRs) compared to their parental tamoxifen-sensitive MCF-7/S0.5 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.
Project description:MCM3 is one of several genes whose expression profile is markedly altered in tamoxifen-resistant breast cancer cell lines. We observed that increased MCM3 expression is associated with tamoxifen resistance. Knockdown of MCM3 resulted in increased susceptibility of tamoxifen-resistant breast cancer cell lines. Moreover, MCM3 expression is significantly associated with clinical outcome of endocrine treated receptor positive breast cancer. To understand the effect of MCM3 on the mechanism of endocrine resistance, we performed gene expression array on tamoxifen-resistant breast cancer cell lines. Here we show that MCM3 knockdown affects the expression of hundreds of genes. Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer is a major clinical problem with poorly understood mechanisms. There is an unmet need for prognostic and predictive biomarkers to allow appropriate therapeutic targeting. We evaluated the mechanism by which minichromosome maintenance protein 3 (MCM3) influences endocrine resistance and its predictive/prognostic potential in ER+ breast cancer. We discovered that ER+ breast cancer cells survive tamoxifen and letrozole treatments through upregulation of minichromosome maintenance proteins (MCMs), including MCM3, which are key molecules in cell cycle and DNA replication. Lowering MCM3 expression in endocrine-resistant cells restored drug sensitivity and altered phosphorylation of cell cycle regulators, including p53(Ser315,33), CHK1(Ser317) and cdc25b(Ser323), suggesting that the interaction of MCM3 with cell cycle proteins is an important mechanism of overcoming replicative stress and anti-proliferative effects of endocrine treatments. Evaluation of MCM3 levels in primary tumors from four independent cohorts of breast cancer patients receiving adjuvant tamoxifen mono-therapy or no adjuvant treatment, including the Stockholm tamoxifen (STO-3) trial, showed MCM3 to be an independent prognostic adding information beyond Ki67. In addition, MCM3 was shown to be a predictive marker of response to endocrine treatment. Our study reveals a coordinated signaling network centered around MCM3 that limits response to endocrine therapy in ER+ breast cancer and identifies MCM3 as a clinically useful prognostic and predictive biomarker that allows personalized treatment of ER+ breast cancer patients.
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.
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.