Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer

Aihara, Masaki; Yamamoto, Shigeru; Nishioka, Hiroko; Inoue, Yuichiro; Hamano, Kimikazu; Oka, M.; Mizukami, Yoichi

doi:10.1016/j.gene.2012.04.029

Cited by 8 publications

(4 citation statements)

References 48 publications

(45 reference statements)

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“…The expression vector for the somatic mutation SIN3A c.2830 C>T; p.Gln944* was produced by introduction into the SIN3A -WT by site-directed mutagenesis as described previously 44 . Briefly, the expression vector of halo -tagged SIN3A was methylated with CpG methyltransferase (NewEngland Biolabs) in the presence of S-adenosylmethionine (NewEngland Biolabs), and then amplified by PCR using Taq polymerase (KOD Plus Neo, Toyobo, Osaka, Japan) with primers for mutagenesis as shown below at 95 °C for 2 min, 20 cycles of 98 °C for 10 sec and 68 °C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

A novel somatic mutation of SIN3A detected in breast cancer by whole-exome sequencing enhances cell proliferation through ERα expression

Watanabe

Yamamoto

Sakaguti

et al. 2018

Sci Rep

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Breast cancer is the most frequent tumor in women, and in nearly two-thirds of cases, the tumors express estrogen receptor α (ERα, encoded by ESR1). Here, we performed whole-exome sequencing of 16 breast cancer tissues classified according to ESR1 expression and 12 samples of whole blood, and detected 310 somatic mutations in cancer tissues with high levels of ESR1 expression. Of the somatic mutations validated by a different deep sequencer, a novel nonsense somatic mutation, c.2830 C>T; p.Gln944*, in transcriptional regulator switch-independent 3 family member A (SIN3A) was detected in breast cancer of a patient. Part of the mutant protein localized in the cytoplasm in contrast to the nuclear localization of ERα, and induced a significant increase in ESR1 mRNA. The SIN3A mutation obviously enhanced MCF7 cell proliferation. In tissue sections from the breast cancer patient with the SIN3A c.2830 C>T mutation, cytoplasmic SIN3A localization was detected within the tumor regions where nuclear enlargement was observed. The reduction in SIN3A mRNA correlates with the recurrence of ER-positive breast cancers on Kaplan-Meier plots. These observations reveal that the SIN3A mutation has lost its transcriptional repression function due to its cytoplasmic localization, and that this repression may contribute to the progression of breast cancer.

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Section: Methodsmentioning

confidence: 99%

A novel somatic mutation of SIN3A detected in breast cancer by whole-exome sequencing enhances cell proliferation through ERα expression

Watanabe

Yamamoto

Sakaguti

et al. 2018

Sci Rep

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“…G protein-coupled estrogen receptor-1 (GPER-1 or GPR30) is a G protein-coupled receptor (GPCR) sited in the cell membrane that triggers a broad range of biological activities in response to stimulation by endogenous estrogens or dietary phytoestrogens ( 2 , 7 ). Its gene is located on chromosome 7p22.3 and encodes a protein of 375 amino acids with a theoretical molecular mass of 41 kDa that is ubiquitously expressed in a large number of tissues ( 8 – 11 ).…”

Section: Introductionmentioning

confidence: 99%

Continuous Exposure of Breast Cancer Cells to Tamoxifen Upregulates GPER-1 and Increases Cell Proliferation

et al. 2020

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GPER-1 is a novel membrane sited G protein-coupled estrogen receptor. Clinical studies have shown that patients suffering an estrogen receptor a (ERa)/GPER-1 positive, breast cancer have a lower survival rate than those who have developed ERa-positive/GPER-1 negative tumors. Moreover, absence of GPER-1 improves the prognosis of patients treated with tamoxifen, the most used selective estrogen receptor modulator to treat ERapositive breast cancer. MCF-7 breast cancer cells were continuously treated with 1,000 nM tamoxifen for 7 days to investigate its effect on GPER-1 protein expression, cell proliferation and intracellular [Ca 2+ ]i mobilization, a key signaling pathway. Breast cancer cells continuously treated with tamoxifen, exhibited a robust [Ca 2+ ]i mobilization after stimulation with 1,000 nM tamoxifen, a response that was blunted by preincubation of cells with G15, a commercial GPER-1 antagonist. Continuously treated cells also displayed a high [Ca 2+ ]i mobilization in response to a commercial GPER-1 agonist (G1) and to estrogen, in a magnitude that doubled the response observed in untreated cells and was almost completely abolished by G15. Proliferation of cells continuously treated with tamoxifen and stimulated with 2,000 nM tamoxifen, was also higher than that observed in untreated cells in a degree that was approximately 90% attributable to GPER-1. Finally, prolonged tamoxifen treatment did not increase ERa expression, but did overexpress the kinin B1 receptor, another GPCR, which we have previously shown is highly expressed in breast tumors and increases proliferation of breast cancer cells. Although we cannot fully extrapolate the results obtained in vitro to the patients, our results shed some light on the occurrence of drug resistance in breast cancer patients who are ERa/GPER-1 positive, have been treated with tamoxifen and display low survival rate. Overexpression of kinin B1 receptor may explain the increased proliferative response observed in breast tumors under continuous treatment with tamoxifen.

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“…We have shown that GPER induced by oxidative stress leads to apoptotic cell death through p53 activation [11]. On the other hand, GPER enhances cell proliferation in breast cancer cells [12,13]. These observations indicate that GPER has different physiological effects depending on distinct cell types.…”

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confidence: 88%

GPER negatively regulates TNFα-induced IL-6 production in human breast cancer cells <i>via</i> NF-κB pathway

Okamoto

Mizukami

2016

Endocr J

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Estrogen is known to have anti-inflammatory effects, that are thought to be mediated by the classical estrogen receptors (ERs), ERα and ERβ. G protein coupled estrogen receptor1 (GPER) is a novel membrane-type estrogen receptor that can mediate non-genomic estrogenic responses. Although there have been several reports asserting that the participation of GPER in anti-inflammatory effects is induced by estrogen, the role of GPER remains poorly understood. In this study, we investigated the involvement of GPER in the regulation of a representative inflammatory cytokine, IL-6. We first examined the expression of IL-6 mRNA by TNFα stimulation in the transfection of GPER-expression plasmid into HeLa cells. Exogenous GPER significantly inhibited TNFα-induced IL-6 expression, and blocked NF-κB promoter activity inducing the expression of IL-6 in a dose-dependent manner. The promoter activity was restored almost to control level by transfection with the C-terminal deletion mutant of GPER. Similar results have been observed in endogenous GPER using SKBR3 cells which do not express the classical ERs. The data have been validated by treatment of GPER with siRNA. These findings indicate that GPER negatively regulates TNFα-induced IL-6 expression, probably through inhibition of NF-κB promoter activity by a signal(s) derived from the C-terminal region of GPER.

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Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer

Cited by 8 publications

References 48 publications

A novel somatic mutation of SIN3A detected in breast cancer by whole-exome sequencing enhances cell proliferation through ERα expression

A novel somatic mutation of SIN3A detected in breast cancer by whole-exome sequencing enhances cell proliferation through ERα expression

Continuous Exposure of Breast Cancer Cells to Tamoxifen Upregulates GPER-1 and Increases Cell Proliferation

GPER negatively regulates TNFα-induced IL-6 production in human breast cancer cells <i>via</i> NF-κB pathway

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Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer

Cited by 8 publications

References 48 publications

A novel somatic mutation of SIN3A detected in breast cancer by whole-exome sequencing enhances cell proliferation through ERα expression

A novel somatic mutation of SIN3A detected in breast cancer by whole-exome sequencing enhances cell proliferation through ERα expression

Continuous Exposure of Breast Cancer Cells to Tamoxifen Upregulates GPER-1 and Increases Cell Proliferation

GPER negatively regulates TNF&alpha;-induced IL-6 production in human breast cancer cells <i>via</i> NF-&kappa;B pathway

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GPER negatively regulates TNFα-induced IL-6 production in human breast cancer cells <i>via</i> NF-κB pathway