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NOTCH4 maintains quiescent mesenchymal-like breast cancer stem cells via transcriptionally activating SLUG and GAS1 in triple-negative breast cancer.

ABSTRACT: Rationale: NOTCH4 receptor has been implicated in triple-negative breast cancer (TNBC) development and breast cancer stem cell (BCSC) regulation. However, the potential of NOTCH4 as a BCSC marker and the underlying mechanisms remain unclear. Methods: In this study, we determined the expression and activation of NOTCH4 in breast cancer cell lines and tumor samples by qRT-PCR, western blotting and immunohistochemistry. Subsequently, in vitro and in vivo serial dilution experiments were performed to demonstrate the application of NOTCH4 as an efficient mesenchymal-like (ML)-BCSC marker in TNBC. Stable overexpression of activated NOTCH4 and knockdown cell lines were established using lentivirus. RNA-seq and qRT-PCR were employed to reveal the downstream effectors of NOTCH4, followed by dual-luciferase reporter and chromatin immunoprecipitation assays to identify the genuine binding sites of NOTCH4 on SLUG and GAS1 promoters. Transwell assay, mammosphere formation and chemoresistance experiments were performed to determine the effects of SLUG, GAS1 and NOTCH4 on the mesenchymal-like characteristics of TNBC cells. Survival analysis was used to study the relation of NOTCH4, SLUG and GAS1 with prognosis of breast cancer. Results: NOTCH4 is aberrantly highly expressed and activated in TNBC, which contributes to the maintenance of ML-BCSCs. Furthermore, NOTCH4 shows significantly higher efficiency in labeling ML-BCSCs than the currently commonly used CD24-CD44+ marker. Mechanistically, NOTCH4 transcriptionally upregulates SLUG and GAS1 to promote EMT and quiescence in TNBC, respectively. The effects of NOTCH4 can be mimicked by simultaneous overexpression of SLUG and GAS1. Moreover, SLUG is also involved in harnessing GAS1, a known tumor suppressor gene, via its anti-apoptotic function. Conclusions: Our findings reveal that the NOTCH4-SLUG-GAS1 circuit serves as a potential target for tumor intervention by overcoming stemness of ML-BCSCs and by conquering the lethal chemoresistance and metastasis of TNBC.

SUBMITTER: Zhou L

PROVIDER: S-EPMC7019177 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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NOTCH4 maintains quiescent mesenchymal-like breast cancer stem cells via transcriptionally activating SLUG and GAS1 in triple-negative breast cancer.

Zhou Lei L Wang Dong D Sheng Dandan D Xu Jiahui J Chen Weilong W Qin Yuanyuan Y Du Ruikai R Yang Xiaoli X He Xueyan X Xie Ni N Liu Suling S Zhang Lixing L

Theranostics 20200119 5

Rationale: NOTCH4 receptor has been implicated in triple-negative breast cancer (TNBC) development and breast cancer stem cell (BCSC) regulation. However, the potential of NOTCH4 as a BCSC marker and the underlying mechanisms remain unclear. Methods: In this study, we determined the expression and activation of NOTCH4 in breast cancer cell lines and tumor samples by qRT-PCR, western blotting and immunohistochemistry. Subsequently, in vitro and in vivo serial dilution ...[more]

PMID: 32104513

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Project description:BackgroundDNA damage and DNA damage repair (DDR) are important therapeutic targets for triple-negative breast cancer (TNBC), a subtype with limited chemotherapy efficiency and poor outcome. However, the role of microRNAs in the therapy is emerging. In this study, we explored whether miR-26a-5p could act as BRCAness and enhance chemotherapy sensitivity in TNBC.MethodsQuantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-26a-5p in breast cancer tissues and cell lines. CCK-8 was used to measure drug sensitivity in concentration gradient and time gradient. Comet assay was used to detect DNA damage. Flow cytometry was performed to examine apoptosis. Moreover, we used western blot and immunofluorescence to detect biomarkers. Luciferase reporter assay was performed to verify the combination of miR-26a-5p and 3'UTR of target gene. Hormone deprivation and stimulation assay were used to validate the effect of hormone receptors on the expression of miR-26a-5p. Chromatin immunoprecipitation (ChIP) assays were used to verify the binding sites of ER-a or PR with the promoter of miR-26a-5p. Animal experiments were performed to the effect of miR-26a-5p on Cisplatin treatment.ResultsThe expression of miR-26a-5p was significantly downregulated in TNBC. Overexpressing miR-26a-5p enhanced the Cisplatin-induced DNA damage and following apoptosis. Interestingly, miR-26a-5p promoted the expression of Fas without Cisplatin stimulating. It suggested that miR-26a-5p provided a hypersensitivity state of death receptor apoptosis and promoted the Cisplatin sensitivity of TNBC cells in vitro and in vivo. Besides, miR-26a-5p negatively regulated the expression of BARD1 and NABP1 and resulted in homologous recombination repair defect (HRD). Notably, overexpressing miR-26a-5p not only facilitated the Olaparib sensitivity of TNBC cells but also the combination of Cisplatin and Olaparib. Furthermore, hormone receptors functioned as transcription factors in the expression of miR-26a-5p, which explained the reasons that miR-26a-5p expressed lowest in TNBC.ConclusionsTaken together, we reveal the important role of miR-26a-5p in Cisplatin sensitivity and highlight its new mechanism in DNA damage and synthetic lethal.

Dataset Information

NOTCH4 maintains quiescent mesenchymal-like breast cancer stem cells via transcriptionally activating SLUG and GAS1 in triple-negative breast cancer.

Publications

NOTCH4 maintains quiescent mesenchymal-like breast cancer stem cells via transcriptionally activating SLUG and GAS1 in triple-negative breast cancer.

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