| Breast cancer is one of common malignant tumors around the world with a high incidence and mortality rate. Tumor metastasis is the major cause of breast cancer mortality rate. Thus, it is particularly important and urgent to discover new mechanism in breast cancer invasion and migration, thus to find new target for breast cancer clinical therapy.CUL4B is the scaffold protein of E3 ubiquitin ligase (Cullin4B-Ring E3 ligase complex, CRL4B). CRL4B involves in lots of important life processes by monoubiquitinating or multi-ubiquitylating specific substrate proteins. Our previous results have showed that CUL4B is highly expressed in various kinds of solid tumors (such as breast, colon, esophagus, lung, stomach, pancreas, etc.). CUL4B was reported to promote tumor metastasis, of which EMT (Epithelial and mesenchymal transition) is an important mechanism. Thus, we study the role of CUL4B in promoting EMT process and its mechanism using breast cancer cell lines.PART ONECUL4B promotes the EMT progression of breast cancer cellsOur previous results showed that CUL4B is highly expressed in malignant breast cancer. Thus, to explore the function of CUL4B in breast tumor metastasis and invasion, we firstly explored the function of CUL4B in EMT process. We got the following results:1. CUL4B expression is positively correlated with the EMT progression in breast cancer cell lines:We collected data from Cellminer NCI60 Analysis Tools database to analyze expression profile of CUL4B and EMT-related genes in six breast cancer cell lines. Meanwhile, we applied Western blotting to verify CUL4B expression in various breast cancer cell lines. The results showed that CUL4B expression is positively correlated with the capability of invasiveness of breast cancer cells, which is similar with expression profile of mesenchymal cells markers(TWIST1, ZEB1, ZEB2, FOXC2, CDH2 etc.) but opposite with epithelial cell markers (CDH1, KRT19, etc.). These results suggest CUL4B probably promotes EMT process in breast cancer cell.2. CUL4B overexpression led to upregulation of EMT promoters and down-regulation of CDH1 in normal mammary epithelial cells:We constructed CUL4B stable overexpressing and control cell lines of normal mammary epithelial cells (MCF 10A CUL4B and MCF 10A Con), and performed Western blotting and qRT-PCR to detect the expression of EMT-related markers in MCF 10A Con and MCF 10A CUL4B cell lines. We found that overexpression of CUL4B resulted in downregulation of CDH1 expression and upregulation of the expression of EMT transcription factor (ZEB1 and Snail), suggesting that CUL4B regulates EMT process in normal mammary epithelial cells.3. CUL4B promotes EMT process of breast cancer cells:To further explore the role of CUL4B in promoting EMT process, we constructed CUL4B stable overexpressing and control cell lines (MCF7 CUL4B and MCF7 Con) in MCF7 cells (low metastasis potential), and CUL4B stable interference and control cell lines (MDA-MB-231 shCUL4B and MDA-MB-231 shNC) in MDA-MB-231 cancer cells (high metastasis potential). We used Western blotting and qRT-PCR method to detect the expression of EMT-related markers in these stable cell lines. Overexpression of CUL4B decreased the expression of epithelial markers E-cadherin and increased the expression of mesenchymal cells markers (CDH2, VIM etc.) and EMT transcriptional factors (Snail, Twist etc.), while knockdown of CUL4B increased the expression of E-cadherin and decreased the expression of VIM and Snail. These results suggest CUL4B promotes EMT process in breast cancer cell lines.4. CUL4B promotes the migration and invasion ability of breast cancer cells:We performed Transwell migration/matrigel invasion assay to investigate migration and invasion ability of MDA-MB-231 shNC and MDA-MB-231 shCUL4B cell lines. The results showed down-regulated CUL4B decreased breast cancer cell migration and invasion ability in vitro.5. CUL4B promotes acquirement of cancer stem cell properties in breast cancer cells: EMT is one of the important mechanisms that helps tumor cells acquire cancer stem cell properties, so we performed sphere formation assay and flow cytometry analysis to study the function of CUL4B in acquirement of cancer stem cell characteristics. We found interfering CUL4B expression significantly reduced MDA-MB-231 cell microsphere formation ablity. Flow cytometry anlysis showed CUL4B overexpression increased the percentage of CD44HighCD24Low cells in MCF7 cells while interfering CUL4B expression significantly reduced the percentage of CD44HighCD24Low cells in MDA-MB-231 cells. The results show CUL4B promotes breast cancer cells to acquire cancer stem cell properties.In summary, CUL4B could promote EMT progression in breast cancer cell.PART TWOThe molecular mechanism of CUL4B-mediated EMT promotion in breast cancer cellsIn the first part we found CUL4B promotes the EMT process of breast cancer cells. CUL4B enhances the migration and invasion ability of breast cancer cells. So we further study the molecular mechanism of CUL4B in promoting EMT process in this section. We made the following findings:1. CUL4B inhibits CDH1 expression: It is the main feature during EMT progress that epithelial cell marker E-cadherin expression is reduced or loss. Therefore, we firstly confirmed the function of CUL4B in CDH1 repression in MCF7 cells, and we found CUL4B negatively regulates CDH1 expression.2. CRL4B coordinates with Snail/HDAC to inhibit CDH1 expression: CDH1 gene which encodes E-cadherin protein is regulated by Snail and other transcription factors. As CRL4B complex was reported to coordinate with PRC2, HDAC and DNMT complex to play a role in transcriptional repression, we wonder if CRL4B-mediated repression of CDH1 expression was mediated by Snail. Therefore, we first performed the immuneprecipitation to analyze whether CRL4B is physically associated with Snail. The result showed that CUL4B and DDB1 interact with Snail, suggesting the potential role of CRL4B to coordinate with Snail in CDH1 repression. Snail was reported as an important transcription inhibitor to inhibit CDH1 expression by recruiting PRC2 and HDAC complexes to CDH1 promoter. Our previous results showed CRL4B can coordinate with PRC2, HDAC and DNMT complexes in transcriptional repression. In order to define which complex involved in CRL4B-mediated CDH1 repression, we used 5-aza (DNMT inhibitor), DZNep (EZH2 inhibitor) and TSA (HDAC inhibitor) respectively to treat MCF7 CUL4B cell lines and detected CDH1 expression. We found TSA treatment obviously restored the reduced CDH1 expression in MCF7 cells caused by CUL4B overexpression. The results suggest that CRL4B probably coordinates with class â… HDACs to inhibit CDHl expression. Next, we performed co-immunoprecipitation to identify the association of CRL4B with HDACs. We also did ChIP assay and found CRL4B and Snail/HDAC bind to the same region of CDH1 promoter, indicating CRL4B coordinates with Snail/HDAC in CDH1 repression.3. CUL4B promotes H2AK119ub1 and histone deacetylation in CDH1 promoter region: To further indicate the function of CUL4B in CDHl regulation, we did qChIP assay in MDA-MB-231 shNC and MDA-MB-231 shCUL4B stable cell lines to detect changes in epigenetic modification status of CDH1 promoter caused by CUL4B interfering. We discovered that after CUL4B was downregulated, the ability of CUL4B and DDB1 bound to CDH1 promoter regions was significantly reduced, meanwhile, H2AK119ub1 and H3K27me3 in CDH1 promoter is declined while histone H3, H4 acetylation was significantly increased. These results suggest that CRL4B coordinates Snail/HDAC to repress CDH1 expression.4. Snail probably recruites CRL4B complex to CDHl promoter: Although many studies have shown that CRL4B plays an important role in transcription regulation, components of CRL4B complex do not have any DNA-binding domain. Thus, to test whether CRL4B is recruited to the CDH1 promoter by Snail, we used siRNA to specificly interfere Snail expression in MDA-MB-231 cells. We did qChIP assay and found less CRL4B associates with CDH1 promoter after Snail was reduced while Snail overexpression facilitates the bind of CRL4B to CDH1 promoter. These results suggest Snail promotes the association of CRL4B complex with CDH1 promoter5. CUL4B positively regulates Snail by repressing miRNA-34 expression: Our analysis found overexpression or interferencing CUL4B not only changed the CDH1 expression, but also affected the Snail expression. The rusults suggested that CRL4B complexes not only coordinated with Snail in CDH1 repression, but also affected Snail expression. There was a regulation loop between Snail and miRNA-34, and our previous studies had shown that CUL4B regulates miRNA expression. In order to know whether CUL4B regulates Snail through the miRNA-34, we examined the expression of miRNA-34a/b/c in the MDA-MB-231 shNC and MDA-MB-231 shCUL4B stable cell lines. Our results showed that after CUL4B interfering, miRNA-34a/b/c expression was significantly increased. Then we further confirmed that CRL4B associated with the promoter of microRNA-34b/c of Snail-binding domain by ChIP assay. We then treated MDA-MB-231 shCUL4B cells with microRNA-34 inhibitor and control, and found that Snail expression was restored. Transwell experiments further confirmed the miRNA-34 inhibitor can effectively restore the reduced migration ability because of interferencing CUL4B expression. These results indicated that except for coordinating with Snail to promote EMT process, CUL4B also promotes the expression of Snail by inhibiting miRNA-34.6. CUL4B promotes tumor metastasis by coordinating with Snail to inhibit CDH1 expression:The above results showed CRL4B complex regulates CDH1 directly and also by upregulating Snail, thus to accelerate the EMT process of breast cancer cells. In order to further corfirm their functional effect, we explored the rescue effect by overexpressing Snail or CDH1 interfering in MDA-MB-231 shCUL4B cells. Then we applied Transwell assay of these cell lines and found that the interference of CDH1 or overexpression of Snail was able to restore the reduced migration ability due to interference CUL4B.In summary, we explored the function and regulation mechanism of CUL4B in the EMT process of breast cancer cells. We found CUL4B effectively promotes the EMT process of breast cancer cells. On the one hand, CRL4B complex coordinates with Snail/HDAC to promote H2AK119ubl and histone deacetylation in the CDH1 promoter, thereby inhibits CDH1 transcription; on the other hand, CUL4B also positively regulates Snail by inhibiting the miRNA-34, thereby further promotes the EMT process. |
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