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Oncogenic MUC1-C promotes tamoxifen resistance in human breast cancer.

ABSTRACT: Tamoxifen resistance of estrogen receptor-positive (ER+) breast cancer cells has been linked in part to activation of receptor tyrosine kinases, such as HER2, and the PI3K-AKT pathway. Mucin 1 (MUC1) is aberrantly overexpressed in about 90% of human breast cancers, and the oncogenic MUC1-C subunit is associated with ER?. The present studies using HER2 overexpressing BT-474 breast cancer cells, which are constitutively resistant to tamoxifen, demonstrate that silencing MUC1-C is associated with (i) downregulation of p-HER2 and (ii) sensitivity to tamoxifen-induced growth inhibition and loss of clonogenic survival. In contrast, overexpression of MUC1-C in tamoxifen-sensitive MCF-7 breast cancer cells resulted in upregulation of p-AKT and tamoxifen resistance. We show that MUC1-C forms complexes with ER? on the estrogen-responsive promoter of Rab31 and that MUC1-C blocks tamoxifen-induced decreases in ER? occupancy. MUC1-C also attenuated tamoxifen-induced decreases in (i) recruitment of the coactivator CREB binding protein, (ii) Rab31 promoter activation, and (iii) Rab31 mRNA and protein levels. The importance of MUC1-C is further supported by the demonstration that targeting MUC1-C with the cell-penetrating peptide inhibitor, GO-203, sensitized tamoxifen-resistant cells to tamoxifen treatment. Moreover, we show that targeting MUC1-C in combination with tamoxifen is highly synergistic in the treatment of tamoxifen-resistant breast cancer cells. Combined, these findings indicate that MUC1-C contributes to tamoxifen resistance.

SUBMITTER: Kharbanda A

PROVIDER: S-EPMC3720729 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Oncogenic MUC1-C promotes tamoxifen resistance in human breast cancer.

Kharbanda Akriti A Rajabi Hasan H Jin Caining C Raina Deepak D Kufe Donald D

Molecular cancer research : MCR 20130328 7

Tamoxifen resistance of estrogen receptor-positive (ER+) breast cancer cells has been linked in part to activation of receptor tyrosine kinases, such as HER2, and the PI3K-AKT pathway. Mucin 1 (MUC1) is aberrantly overexpressed in about 90% of human breast cancers, and the oncogenic MUC1-C subunit is associated with ERα. The present studies using HER2 overexpressing BT-474 breast cancer cells, which are constitutively resistant to tamoxifen, demonstrate that silencing MUC1-C is associated with ( ...[more]

PMID: 23538857

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Project description:BackgroundEndocrine therapy plays a key role in estrogen receptor-positive breast cancer patients; but, tamoxifen resistance could be a real difficulty for these patients. Several attempts have been made to explore the mechanism and new therapies for these patients. We intend to clarify the expression change of SRC and SIRT1 in tamoxifen-resistant breast cancer cells and explore their functions on tamoxifen resistance.MethodsSRC and SIRT1 expressions were analyzed by RNA sequencing, qPCR and Western blotting. Loss and gain of function of SRC and SIRT1 were utilized to indicate their oncogenic roles in tamoxifen resistance in vitro and in vivo. Kaplan-Meier analysis and receiver operating characteristic curve were used to evaluate the survival and the predicted effects of SRC and SIRT1 on patients' prognosis.ResultsHigh expressions of SRC and/or SIRT1 were found in tamoxifen-resistant cells and related to poor overall survival (p<0.05 for SRC, p<0.001 for SIRT1, p<0.001 for SRC and SIRT1) and cancer-specific survival (p<0.05 for SRC, p<0.01 for SIRT1, p<0.01 for SRC and SIRT1) of tamoxifen-treated breast cancer patients. Down-regulation of SRC (p<0.01) or SIRT1 (p<0.05) separately reversed the resistance to tamoxifen and the minimal concentration of SRC inhibitor KX-01 (p<0.05) or SIRT1 inhibitor EX527 (p<0.001) could also suppress cell proliferation. The expression level of SIRT1 was positively correlated with that of SRC. Overexpression of SRC significantly promotes the cell resistance to tamoxifen inhibited by SIRT1 (p<0.01). In vivo experiments confirmed the effects of SRC on tumor growth by over- or down-regulating SRC expression (p<0.001 and p<0.001, respectively).ConclusionSRC and SIRT1 are both up-regulated in tamoxifen-resistant breast cancer cells and related to a poor prognosis in tamoxifen-treated breast cancer. Moreover, SRC could promote tamoxifen resistance by up-regulating SIRT1. SRC and SIRT1 might be novel therapeutic targets in tamoxifen-resistant breast cancer and the interaction between SRC and SIRT1 needs to be further explored.

Project description:BackgroundTamoxifen resistance remains a clinical challenge for hormone receptor-positive breast cancer. Recently, dysregulations in autophagy have been suggested as a potential mechanism for tamoxifen resistance. Although the long noncoding RNA H19 is involved in various stages of tumorigenesis, its role in tamoxifen resistance remains unknown. Here, we assessed the role of H19 in the development of tamoxifen-resistant breast cancer.MethodsQuantitative real-time PCR analyzed expression of H19 in tamoxifen-resistant breast cancer tissues. Knockdown of H19 was used to assess the sensitivity to tamoxifen in vitro and in vivo. Both knockdown and overexpression of H19 were used to analyze the status of autophagy. Real-time quantitative methylation-specific polymerase chain reaction, chromatin immunoprecipitation, immunofluorescence, and Western blot were used to explore the tamoxifen resistance mechanism of H19.ResultsIn this study, we observed that the expression of H19 was substantially upregulated in tamoxifen-resistant breast cancer cell line and tumor tissues, and knockdown of H19 enhanced the sensitivity to tamoxifen both in vitro and in vivo. Furthermore, knockdown of H19 significantly inhibited autophagy in MCF7 tamoxifen-resistant (MCF7/TAMR) cells. Conversely, overexpression of H19 promoted autophagy. Interestingly, overexpression of H19 in MCF7 tamoxifen-sensitive cells could recapitulate tamoxifen resistance. Moreover, an increase in methylation in the promoter region of Beclin1 was observed in MCF7/TAMR-shH19 cells. In the double knockdown groups, both shH19+shSAHH and shH19+shDNMT3B rescued the Beclin1 promoter region methylation levels and reactivated autophagy functions. A chromatin immunoprecipitation assay further validated that DNMT3B binds to the Beclin1 promoter region and the knockdown of H19 increases this binding.ConclusionsOur findings demonstrate that H19 induces autophagy activation via the H19/SAHH/DNMT3B axis, which could contribute to tamoxifen resistance in breast cancer.

Project description:BackgroundTamoxifen (TAM) is the eminent first-line drug for endocrine therapy of hormone receptor positive premenopausal breast cancer and reduces the risk of recurrence by ∼50%. However, many patients developed TAM resistance and their diseases recurred. Our previous study on transcriptome profile of TAM resistant breast cancer cells revealed that the TMEM47 is one of the most significantly differentially expressed genes. The mechanism of how TMEM47 is involved in TAM resistance was not known.MethodsWe constructed a mammal breast cancer cell line, in which TMEM47 was stably overexpressed (TMEM47-OE/MCF-7), to further verify the role of TMEM47 in TAM resistance. siRNA targeting TMEM47 was transfected into TAMR / MCF-7 cells by Liposome. TMEM47 expression was validated on mRNA and protein level by qRT-PCR and western blotting. We tested the cytotoxicity of TAM in the cells. Apoptosis was detected by flow cytometry.ResultsCompared to the MCF7 cells, TMEM47 mRNA was significantly up regulated more than 6 folds in the TAMR/MCF7 cells and so its protein. TMEM47 expression level in TMEM47-OE/MCF-7 was similar as in the TAMR/MCF-7 cells. The 50% inhibitory concentration (IC50) value (mean ± SD) of TAM in MCF-7, TAMR/MCF-7 and TMEM47-OE/MCF-7 cells was 1.58 ± 0.19, 2.74 ± 0.24 and 3.12 ± 0.32 µγ/mL, respectively. The apoptosis rates of TAMR/MCF-7 and TMEM47-OE/MCF-7 cell lines were significantly lower than that of MCF-7 cells. After 24 and 48 hours TAM treatments, cell viability was significantly inhibitied in TMEM47 knockdown TAMR/MCF7 cells (P < 0.01). Consistant with the decreased cell viability, the apoptosis rate in TMEM47 knockdown TAMR/MCF-7 cells was significantly increased.ConclusionsOur results suggest that overexpression of TMEM47 in MCF-7 cells acquired TAM resistance to those cells, and knockdown of TMEM47 in TAMR/MCF-7 cells reversed their resistance to TAM. TMEM47 might confer TAM resistance on MCF-7 cells through the inhibition of apoptosis.

Project description:BackgroundBreast cancer is the most prevalent cancer in women globally. Over-activated estrogen receptor (ER) α signaling is considered the main factor in luminal breast cancers, which can be effectively managed with selective estrogen receptor modulators (SERMs) like tamoxifen. However, approximately 30-40% of ER + breast cancer cases are recurrent after tamoxifen therapy. This implies that the treatment of breast cancer is still hindered by resistance to tamoxifen. Recent studies have suggested that post-translational modifications of ERα play a significant role in endocrine resistance. The stability of both ERα protein and its transcriptome is regulated by a balance between E3 ubiquitin ligases and deubiquitinases. According to the current knowledge, approximately 100 deubiquitinases are encoded in the human genome, but it remains unclear which deubiquitinases play a critical role in estrogen signaling and endocrine resistance. Thus, decoding the key deubiquitinases that significantly impact estrogen signaling, including the control of ERα expression and stability, is critical for the improvement of breast cancer therapeutics.MethodsWe used several ER positive breast cancer cell lines, DUB siRNA library screening, xenograft models, endocrine-resistant (ERα-Y537S) model and performed immunoblotting, real time PCR, RNA sequencing, immunofluorescence, and luciferase activity assay to investigate the function of USP36 in breast cancer progression and tamoxifen resistance.ResultsIn this study, we identify Ubiquitin-specific peptidase 36 (USP36) as a key deubiquitinase involved in ERα signaling and the advancement of breast cancer by deubiquitinases siRNA library screening. In vitro and in vivo studies showed that USP36, but not its catalytically inactive mutant (C131A), could promote breast cancer progression through ERα signaling. Conversely, silencing USP36 inhibited tumorigenesis. In models resistant to endocrine therapy, silencing USP36 destabilized the resistant form of ERα (Y537S) and restored sensitivity to tamoxifen. Molecular studies indicated that USP36 inhibited K48-linked polyubiquitination of ERα and enhanced the ERα transcriptome. It is interesting to note that our results suggest USP36 as a novel biomarker for treatment of breast cancer.ConclusionOur study revealed the possibility that inhibiting USP36 combined with tamoxifen could provide a potential therapy for breast cancer.

Project description:BackgroundTamoxifen is a frontline therapy for estrogen receptor (ER)-positive breast cancer in premenopausal women. However, many patients develop resistance to tamoxifen, and the mechanism underlying tamoxifen resistance is not well understood. Here we examined whether ER-c-Src-HER2 complex formation is involved in tamoxifen resistance.MethodsMTT and colony formation assays were used to measure cell viability and proliferation. Western blot was used to detect protein expression and protein complex formations were detected by immunoprecipitation and immunofluorescence. SiRNA was used to examine the function of HER2 in of BT474 cells. An in vivo xenograft animal model was established to examine the role of c-Cbl in tumor growth.ResultsMTT and colony formation assay showed that BT474 cells are resistant to tamoxifen and T47D cells are sensitive to tamoxifen. Immunoprecipitation experiments revealed ER-c-Src-HER2 complex formation in BT474 cells but not in T47D cells. However, ER-c-Src-HER2 complex formation was detected after overexpressing HER2 in T47D cells and these cells were more resistant to tamoxifen. HER2 knockdown by siRNA in BT474 cells reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was also disrupted and tamoxifen resistance was reversed in BT474 cells by the c-Src inhibitor PP2 and HER2 antibody trastuzumab. Nystatin, a lipid raft inhibitor, reduced ER-c-Src-HER2 complex formation and partially reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was disrupted by overexpression of c-Cbl but not by the c-Cbl ubiquitin ligase mutant. In addition, c-Cbl could reverse tamoxifen resistance in BT474 cells, but the ubiquitin ligase mutant had no effect. The effect of c-Cbl was validated in BT474 tumor-bearing nude mice in vivo. Immunofluorescence also revealed ER-c-Src-HER2 complex formation was reduced in tumor tissues of nude mice with c-Cbl overexpression.ConclusionsOur results suggested that c-Cbl can reverse tamoxifen resistance in HER2-overexpressing breast cancer cells by inhibiting the formation of the ER-c-Src-HER2 complex.

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Oncogenic MUC1-C promotes tamoxifen resistance in human breast cancer.

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Oncogenic MUC1-C promotes tamoxifen resistance in human breast cancer.

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