1. Fudan University Shanghai Cancer Center & Institutes of Biomedical Sciences; Shanghai Medical College; Key Laboratory of Breast Cancer in Shanghai; Innovation Center for Cell Signaling Network: Cancer Institute, Fudan University, Shanghai 200032, China;
2. The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science & Technology of China, Hefei, Anhui 230027, China;
3. WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan;
4. State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing 100193, China
5. Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University, Shenzhen 518035, China;
*These authors contributed equally to this work.
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.
Keywords: NOTCH4, SLUG, GAS1, mesenchymal-like breast cancer stem cell (ML-BCSC), TNBC