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    • This study was supported by the Finance

    2021-10-14

    This study was supported by the Finance Department Foundation of Jilin Province (3D5178963428).
    Introduction Breast cancer is one of the leading cause of death in women worldwide [1,2]. Due to recent advances of combined therapies, survival rate of breast has improved significantly. However, breast cancer still ranks second among cancer related deaths in women [3]. Therefore, it is of great importance to identify effective molecular targets influencing tumor proliferation and resistance to chemotherapy, which could be used as potential targets to overcome chemoresistance [[4], [5], [6], [7]]. RASSF6 is a member of RASSF family protein, which has been reported to play a part in the regulation of cancer development. Several RASSF family proteins are inactivated through mutation or promoter hyper-methylation. RASSF6 promoter hypermethylation was reported in metastatic melanoma and neuroblastoma [8]. Downregulation of RASSF6 was reported in children leukemia [9]. Loss of RASSF6 could serve as an independent prognostic indicator in gastric cancers [10]. To data, the expression pattern biological effects of RASSF6 in breast cancer and its molecular mechanism remains unclear.
    Materials and methods
    Results
    Discussion Breast cancer is a great threat to women's health and finding new cancer markers, especially those related to chemoresistance, is an important step towards development of targeted therapy. RASSF family proteins, including RASSF6, are frequently inactivated in various human cancers. Loss of RASSF6 has been observed in leukemia [9], gastric cancer [10] and neuroblastoma [8]. However, study concerning its role in breast cancer is missing. Here, we demonstrated RASSF6 expression is decreased in breast cancer tissues and cell lines, especially in TNBC tissues and cell lines. Our result was also supported by TCGA data showing downregulation of RASSF6 mRNA in breast cancer tissues. Loss of RASSF6 correlated with advanced clinical stage and nodal metastasis. Importantly, RASSF6 downregulation correlated with poor patient survival. This is the first report concerning its clinical significance in human breast cancers. CCK-8 and colony formation assay showed that RASSF6 overexpression inhibited growth while its depletion facilitated cell growth. Cell cycle and western blot analysis showed that RASSF6 overexpression inhibited a little of progression, with downregulation of cyclin D1. Western blot showed that RASSF6 downregulated cyclin D1 and upregulated p21. Cyclin D1 regulates G1-S progression and serves as an indicator of malignant proliferation [11,12]. p21 functions a cell cycle inhibitor. Downregulation of p21 often lead to growth of cancer cells [13,14]. In accord with our results, it is reported that, in renal cell carcinoma, RASSF6 leads to p21 dependent cell cycle arrest [15]. We also checked the role of RASSF6 on CDDP induced apoptosis. The results showed that RASSF6 significantly increased CDDP induced apoptosis rate, with upregulation of cleaved caspase 3 and cytochrome c. Cytochrome c is a component of the electron transport chain in mitochondria and its release indicates mitochondrial damage and induction of mitochondrial apoptosis [16]. Using JC-1 staining, we found RASSF6 decreased membrane potential. Loss of membrane potential triggers mitochondrial apoptosis pathway through elevated mitochondrial membrane permeability and release of cytochrome c. Together these results indicated that RASSF6 could facilitate the effect of CDDP on cancer cell apoptosis through regulation of mitochondrial function. Next we explored the potential mechanism of RASSF6 on chemosensitivity. Since there was a connection between RASSFs and Hippo pathway, we checked related proteins and found that RASSF6 induced p-MST1/2 and p-LATS1. RASSF6 also downregulated YAP expression. RASSF family proteins have been reported to interact with MST1/2 and activate Hippo signaling through SARAH domains. MST1/2 are orthologous to Drosophila Hippo, one of the core regulatory proteins in the Hippo signaling pathway. MST1/2 and SAV1 scaffold protein form a complex that leads to phosphorylation of LATS1, which phosphorylates YAP and promotes its cytoplasmic sequestration and degradation. YAP is an potent onco-protein which is overexpressed in various cancers, including breast cancer a little of [[17], [18], [19], [20]]. Our results showed that YAP plasmid was able to reduced cleaved caspase 3 and apoptosis induced by RASSF6, indicating YAP may serve as a mediator for the biological effects, especially reduction of chemoresistance, of RASSF6. YAP upregulation and inhibition of Hippo pathway have been reported to facilitate cell growth and repress apoptosis [21,22]. It has been reported that YAP was able to maintain mitochondrial structure and function too [23]. Thus it is possible YAP reduces apoptosis by maintaining mitochondrial membrane potential. The exact mechanism needs further investigation.