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Kindlin 2, as a focal adhesion protein, controls integrin activation and regulates Wnt signaling in an integrin-binding independent manner. However, the association of Kindlin 2 with cancer-related microRNAs is unknown. Here, we report that Kindlin 2 markedly downregulates the expression of miR-200 family by inducing CpG island hypermethylation. Mechanistically, Kindlin 2 forms a complex with DNMT3A in the cell nucleus and the two proteins co-occupy the promoter of miRNA-200b. Functionally, repression of miR-200b is required for Kindlin 2-induced breast cancer cell invasion and tumor formation. Our data indicate that Kindlin 2 plays a novel role in epigenetic repression of miR-200 family, a mechanism that promotes breast cancer invasion.
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by inhibiting protein translation or degrading target mRNAs. Hundreds of miRNAs so far have been identified to play key roles in a variety of physiological pathways, including development, cell differentiation, proliferation and cell death.[2, 3] Moreover, aberrant expression of miRNA has been found in most cancers. Some miRNAs are oncogenic and some function as tumor suppressors.[5, 6] Loss of miR-200 family (miR-200a, -200b, -200c, -141 and -429) is believed to be crucial in tumor progression. MiR-200 family significantly inhibits the process of epithelial to mesenchymal transition (EMT), in which it directly targets to transcriptional factors ZEB1/ZEB2 in epithelial cells.[7, 8] Interestingly, ZEB1 also regulates the transcription of miR-200 family by a double negative feedback loop.[9, 10] Although the importance of miR-200 family in tumor progression has been established, the underlying mechanism of miR-200 repression is not completely clear. Recent reports showed that epigenetic regulation, especially DNA methylation, mediates the silencing of miR-200 family in human tumorigenesis.[11, 12] However, the mechanism accounting for the epigenetic regulation of miR-200 remained unknown.
Fermitin family homolog 2 (FERMT2, Kindlin 2) is a member of Fermitin family, originally characterized in the regulation of integrin activation and cell–matrix adhesion. Beside its essential role in the activation of integrin, recent studies revealed that Kindlin 2 is related to tumor progression. Kindlin 2 was found highly expressed in human uterine leiomyomas and human malignant mesothelioma.[13, 14] Functionally Kindlin 2 was reported to promote tumor cell proliferation, adhesion, migration, and invasion.[14-17] Recently, Kindlin 2 was found to induce EMT by activating Wnt signaling. However, association of Kindlin 2 with cancer-related miRNAs in the EMT process remains largely unknown.
Since both Kindlin 2 and miR-200 family function in the regulation of EMT, it is warranted to explore whether Kindlin 2 involves in the regulation of miR-200 family. Here, we report that Kindlin 2 regulates EMT via an epigenetic silencing of miR-200 family through interaction with DNA methyltransferase 3A at the miR-200b promoter.
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Kindlin 2 has been reported to associate with integrin, migfilin, integrin-linked kinase (ILK) and β-catenin for regulating cell adhesion, migration and invasion.[15, 25-27] Here, we reported that DNMT3A is a newly identified molecule of potential importance in associating with Kindlin 2 (Figs. 4g–4k). This complex of Kindlin 2 and DNMT3A co-occupied the miR-200b promoter (Fig. 4f) and resulted in the repression of miR-200b expression. Thus, knocking down endogenous DNMT3A in Kindlin 2 overexpressing cells rescued the expression of miR-200b (Fig. 4c). However, knockdown of DNMT3A or treatment of 5-aza-CdR only partially reversed the expression of miR-200b. In this scenario, we do not exclude other players that may also involve in the regulation on miR-200b expression. In fact, DNA methylation and histone modification often collaborate to lead to tumor suppressor gene silencing. For example, some histone methyltransferases, such as EZH2 and SETDB1, interact with DNMT3A to form a complex, co-occupying the CpG islands of tumor suppressors and regulating their expression.[28, 29] Therefore, EZH2, SETDB1 or other histone methyltransferases may be recruited by complex of Kindlin 2 and DNMT3A at the promoter of miR-200b, together silencing the expression of miR-200b. These possibilities deserve further investigation. Besides DNMT3A, knocking down endogenous DNMT1 in Kindlin 2 overexpressing cells also partially rescued miR-200b expression (Fig. 4c). However, the absence of DNMT1 binding on miR-200b promoter (Fig. 4e) suggested that DNMT1 may indirectly mediate the regulation of Kindlin 2 on miR-200b through other factors.
Since the mRNA level of DNMT3A was constant in Kindlin 2-overexpressed cells compared with the control cells (data not shown), the interaction between Kindlin 2 and DNMT3A may be beneficial for maintaining the stability of DNMT3A. It is reported that SUMOylation, a post-translational modification, mediates the stability of DNMT3. Cbx4 (chromobox 4), a member of Polycomb group proteins, functions as an E3 ligase to promote the SUMOylation of DNMT3A. Our data from a PCR array indicated that Cbx4 was downregulated by approximately 2.6-fold in Kindlin 2-overexpressed stable cells, compared with the control cells (data not shown). This suggests that Kindlin 2 may be involved in the Cbx4-mediated SUMOylation of DNMT3A and overexpression of Kindlin 2 may inhibit the degradation of DNMT3A dependent on the SUMOylation, which leaves room for future investigation.
Previously findings have revealed the CpG methylation of both clusters of miR-200 family. Davalos et al. found that MCF7 cells were unmethylated in miR-200b/200a/429 promoter, whereas Wee et al. indicated that MCF7 cells were methylated in miR-200b promoter. In this study, bisulfite sequencing analyses showed that the CpG methylation rate of miR-200b is 56 % in MCF7 cells (Fig. 3c). To clarify this ambiguity, methylation-specific PCR analyses were performed and both methylated and unmethylated bands were observed in MCF7 cells (Supporting Information Fig. 3). When Kindlin 2 was overexpressed in MCF7 cells, the unmethylated band was totally disappeared, and only the methylated band was observed. Considering the inconsistency in CpG methylation of miR-200b in MCF7, it is possible that different CpG dinucleotides were detected. Thus, multiple CpG regions in miR-200b promoter remain to be investigated in future research. In addition, we observed that MCF7 is totally unmethylated in miR-200c/141 promoter, whereas Hs578T is highly methylated in the same region, which is consistent with the observation of Vrba et al.
Since knockdown of Kindlin 2 could not markedly remove the methylation of miR-200b/200a/429 promoter in Hs578T cells (Fig. 3d), we considered the following possible reasons. Previous publication has indicated that some miRNAs have multiple promoters.[33, 34] For example, two independent promoters (P1 and P2) have been confirmed in the miR-200b/200a/429 cluster. As the significant re-expression of miR-200b/200a/429 was observed in Kindlin 2 shRNA-treated Hs578T cells compared with control cells (Fig. 2c); therefore, we do not exclude other promoters rather than the one we detected may involve in the epigenetic regulation of miR-200b/200a/429 in Hs578T cells. In addition, other mesenchymal phenotypes of breast cancer cell lines, such as MDA-MB-231 and BT549 might be used for future investigation to uncover the methylation of miR-200b/200a/429 promoter.
We and other researchers have identified that Kindlin 2 plays a positive role in promoting the invasion of breast cancer cells. However, an opposite role of Kindlin 2 was indicated in mesenchymal cancer cell invasion. These evidences suggested that the biological function of Kindlin 2 was complex in different types of tumors, which may provide an explanation for the different expression levels of Kindlin 2 in different tumors.[13, 14, 17]
It is reported that miR-200b inhibits the invasion of tumor cells by directly targeting transcriptional factor ZEB1. Consistent with this, our results demonstrated that Kindlin 2 induces the downregulation of miR-200b (Fig. 2b), which leads to the accumulation of ZEB1, in turn triggering EMT and promoting cell invasion (Fig. 6). In addition to ZEB1, miR-200 also inhibits tumor cell invasion by targeting VEGFR1. Our data from a PCR array indicated that VEGFR1 was increased by approximately 10-fold in Kindlin 2 overexpressing cells, compared with the control cells (data not shown). This suggests that the regulation of Kindlin 2 on the targets of miR-200b may be not only restricted to ZEB1, but also includes VEGFR1.
In conclusion, this study identified that Kindlin 2 is a negative regulator for miR-200 family both in vitro and in vivo. We demonstrated that Kindlin 2 interacts with DNMT3A in the nucleus, and Kindlin 2 and DNMT3A co-occupies the promoter of miR-200b that leads to the epigenetic repression of miR-200b. Finally, disruption of the interaction between Kindlin 2 and DNMT3A may hold promise for blocking breast cancer invasion.