CPSF4 promotes triple negative breast cancer metastasis by upregulating MDM4

Abstract

Dear Editor, Breast cancer (BrC) is the most common cancer in women. Triple negative BrC (TNBC) is the subtype with highly aggressive clinical behaviors and heterogeneity. Metastasis is the leading cause of TNBC-related deaths, but its mechanism is not well-understood. Apart from PIK3CA, TP53, and PTEN, few recurrent mutations have been identified in TNBC so far, suggesting that TNBC phenotype could be driven by nongenetic alterations such as aberrant expression of oncogenes. Cleavage and polyadenylation-specific factor complexes (CPSFs) participate in the processes of transcription initiation, cleavage, and formation of the poly(A) tail, as well as RNA splicing. Among the CPSF proteins, CPSF4 has been reported to cause the malignant phenotypes in lung cancer by upregulating the transcription of telomerase reverse transcriptase. However, the role of CPSF4 in TNBC remains unclear. Here we aimed to investigate the potential role of CPSF4 in TNBC metastasis and the underlying mechanism. First, we analyzed the clinical significance of CPSF4 using GenExMiner and The Cancer Genome Atlas (TCGA). Elevated mRNA level of CPSF4 was associated with shorter overall survival (OS) in BrC patients, and the expression of CPSF4 was significantly higher in basal-like BrC than that in normal breast tissues and other BrC subtypes (Supplementary Fig. S1a, b). Consistently, analysis of TCGA data showed that CPSF4 mRNA level was higher in basal-like and HR+ BrCs than normal breast tissues (Supplementary Fig. S1c–e). These results indicate that CPSF4 may play a critical role in TNBC. We then detected the protein abundance of CPSF4 in BrC tissues and cell lines. The expression of CPSF4 was higher in BrC compared to that in normal breast tissues and TNBC cells had higher expression of CPSF4 than cells in other subtypes (Supplementary Fig. S1f, g). Accordingly, we performed subsequent experiments in MDAMB-231 and SUM-159PT TNBC cell lines. Transwell migration and matrigel invasion assays were performed after transfecting cells with small interfering RNA/short hairpin RNA to suppress the expression of CPSF4. As shown in Fig. 1a and Supplementary Fig. S2a, knockdown of CPSF4 significantly decreased the capacity of cell invasion and migration. In contrast, transwell migration and matrigel invasion assays performed in cells with lentiviral vector-mediated overexpression of CPSF4 showed that increased expression of CPSF4 significantly enhanced the invasion and migration in both cell lines (Fig. 1b and Supplementary Fig. S2b). Epithelial–mesenchymal transition (EMT) is one of the most important steps in metastasis. To verify whether CPSF4 induces EMT, we detected the expression of Snail, ZEB1, and vimentin in response to changes of CPSF4 expression. Our results demonstrated that knockdown of CPSF4 inhibited EMT, whereas the expressions of EMT-related markers were increased after overexpression of CPSF4 (Supplementary Fig. S2c, d), proving that CPSF4 promoted TNBC cells metastasis through inducing EMT. Moreover, we observed that cell proliferation was repressed by CPSF4 knockdown in TNBC cells (Supplementary Fig. S2e, f). Next, we performed chromatin immunoprecipitation (ChIP)sequencing to identify the target genes of CPSF4, which was found to correlate with the promoter regions of 65 genes out of 162 candidates. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed the target genes were related to genome nucleotide-excision repair, epidermal growth factor receptor signaling, and metastasis (Supplementary Fig. S3a, b). We examined the expression of the metastasis-related genes by quantitative reverse transcription PCR (qRT-PCR) and discovered that the expression of Mouse double minute 4 (MDM4), a suppressor of p53, decreased markedly after CPSF4 knockdown (Supplementary Fig. S3c, d). We then confirmed the binding of CPSF4 at the MDM4 gene promoter by ChIPquantitative PCR (Fig. 1c). In addition, we constructed MDM4 promoter-driven luciferase reporter plasmids, which contained the full-length MDM4 promoter or its different deletion fragments. As shown in Fig. 1d, the +178 ~+215 region was crucial to the transcription activation of MDM4 and the +188 ~ +197 region contained the essential elements for promoter activity, whereas the +178 ~+187 region was indispensable to CPSF4-dependent transcription. In line with the observations above, western blotting showed that CPSF4 promoted the expression of MDM4 (Fig. 1e and Supplementary Fig. S3f). Given that CPSF4 had no DNA-binding domain, we surmised that CPSF4 was recruited to the MDM4 promoter by other proteins to enhance MDM4 transcription. It is known that transcriptional regulation can be coupled to RNA splicing and CPSF4 has been reported to participate in the alternative splicing of TP53 mRNA. Therefore, we performed qRT-PCR to determine whether CPSF4 functions in the alternative splicing of MDM4-S, the variant that lacks exon 6 of the full-length transcript and is abundant in proliferating cell. As shown in Supplementary Fig. S4a, b, the level of MDM4-S remained unaffected despite changed CPSF4 expression. Moreover, we tested the stability of MDM4 mRNA and protein, and found CPSF4 had no effect on it (Supplementary Fig. S4c, d). Also, neither knockdown nor overexpression of CPSF4 influenced the expression of p53 and MDM2 (Supplementary Fig. S4e), the interaction partners of MDM4. Collectively, these findings suggest that CPSF4 transcriptionally regulated the expression of MDM4. Furthermore, we performed rescue assays to determine whether CPSF4 promoted metastasis of TNBC cells through MDM4. Transwell migration, matrigel invasion, and wound scratch assays unanimously showed that knockdown of CPSF4 inhibited TNBC cell migration and invasion, whereas overexpression of MDM4 partially reversed the effects caused by CPSF4 silencing (Fig. 1f and Supplementary Fig. S5a, c, d). Consistently, knockdown of CPSF4 decreased the expression of Snail and vimentin, which was rescued partly by MDM4 overexpression (Fig. 1g, h and