The Wnt/β-catenin signaling cascade is highly conserved during evolution and plays a crucial role in the embryonic development of all animal species. Wnt signals regulate cellular processes such as cellular proliferation, survival and differentiation; misregulation of this signaling pathway is heavily implicated in the development of cancers, most notably colorectal cancers (3
). Uncontrolled Wnt signaling leads to constitutive activation of many oncogenes which are tightly regulated and only activated transiently during normal development. The pivotal mediator which responds to upstream Wnt signals and executes the downstream gene activation program is β-catenin. Stimulatory Wnt ligands induce elevated levels of β-catenin, which binds to transcription factors associated with the promoters of Wnt target genes; β-catenin recruits other transcriptional coactivators which activate transcription of the Wnt target genes (3
). Identification and functional investigation of proteins that bind β-catenin and cooperate in regulation of Wnt target genes will thus provide novel and exciting insights into the highly complicated mechanism of Wnt target gene activation. β-catenin-associated coactivators may also represent novel targets for therapeutic intervention against cancers with dysregulated Wnt signaling.
CARM1 is a member of the protein arginine methyltransferase family, and it methylates a large number of cellular substrates including histones as well as non-histone proteins involved in transcription, RNA processing, and other cellular functions. Deregulation of CARM1 expression is implicated in the pathogenesis of cancers, such that CARM1 expression has been correlated with tumor staging (20
). CARM1 functions as a transcriptional coactivator for several different types of DNA-binding transcriptional activator proteins, and thus altered CARM1 expression likely affects many transcriptional programs which target genes that control proliferation rate or other oncogenic properties. In fact, studies from the MCF7 breast cancer cell line showed that CARM1 is a positive regulator of estrogen receptor-responsive genes and is essential for estrogen-stimulated growth and proliferation of breast carcinomas (21
). Therefore, to further explore the mechanistic knowledge of how deregulated CARM1 might transform cells, we tested whether CARM1 is transcriptionally involved in oncogenic activation of the Wnt/β-catenin signaling cascade in human colorectal carcinomas.
We showed that CARM1 interacts with β-catenin () and cooperates with β-catenin at the transcriptional level in transient reporter gene assays (). In a more physiologically relevant model, CARM1 was essential for expression of several Wnt target genes (including known oncogenes) in the HT29 and HCT116 colorectal cancer cell lines ( and Supplementary Fig. S5
), which have aberrant activation of Wnt/β-catenin signaling and thus constitutive β-catenin regulated transcription. In HT29 cells the relative requirement for CARM1 versus β-catenin varied on the four different Wnt target genes tested: Expression of GPR49 and Axin2 was affected more strongly by depletion of β-catenin than by depletion of CARM1, while expression of S100A4 and c-Myc was affected to approximately equal extents by depletion of each of the two coactivators (). These results suggest that the Wnt/β-catenin pathway plays a more dominant role in controlling expression of Axin2 and GPR49 than c-Myc and S100A4. In fact, Wnt/β-catenin signaling is only one of several signaling cascades regulating expression of c-Myc and S100A4; c-Myc expression is known to be regulated by many transcription factors, including nuclear receptors and AP1 (22
), and the S100A4 gene is reported to be under the control of transcription factor NFAT5 (nuclear factor of activated T-cells 5) (24
Since CARM1 works as a coactivator for multiple transcription factors, and since Wnt target genes are controlled by Wnt/β-catenin signaling and by transcription factors regulated by other signaling cascades (13
), it is difficult to assess whether the requirement of CARM1 for constitutively high expression of Wnt target genes in cells with deregulated Wnt/β-catenin signaling is due to CARM1 actions through LEF1/β-catenin or through other transcription factors that bind to the same promoter. To address this problem we identified a cell line in which we could observe Wnt-inducible gene expression. After screening a number of colon cancer cell lines we identified RKO as a colon cancer cell line which has normal Wnt/β-catenin signaling. Importantly, in RKO cells the induction of Axin2 and GPR49 transcription by Wnt3a ligands was blocked by CARM1 depletion (), and Wnt3a ligand caused recruitment of CARM1 to the WRE of the Axin2 promoter, resulting in enhanced methylation of Arg 17 of histone H3 in nucleosomes associated with the WRE (). Together, these results indicate that CARM1 acts directly at the Axin2 promoter to mediate the actions of LEF1/β-catenin in response to Wnt signaling. Therefore, CARM1 is a bona fide coactivator for Wnt/β-catenin signaling.
Our results also have begun to dissect the mechanism of CARM1 action. Depletion of β-catenin from RKO cells diminished Wnt-induced occupancy of the Axin2 WRE by CARM1, thus demonstrating that CARM1 recruitment in response to Wnt3a depends upon the presence of β-catenin. In contrast, depletion of CARM1 levels did not diminish Wnt-induced accumulation of β-catenin in the cells or the recruitment of β-catenin to the Wnt target gene; instead, CARM1 depletion reduced methylation of Arg 17 on histone H3 at the WRE (). These findings indicate that mechanistically, CARM1 acts downstream from β-catenin and facilitates transcription complex formation by facilitating steps that are downstream from the recruitment of β-catenin and CARM1 to the promoter. In the LEF1/β-catenin reporter gene system, methyltransferase activity of CARM1 was not required for its transcriptional coactivator activity (Fig. S1
). The C-terminal activation domain of CARM1 may be responsible for the coactivator function of CARM1 in this case (26
). In addition, the importance of CARM1 enzymatic activity in the expression of endogenous Wnt target genes should be tested, given that 1) plasmid-based reporter gene studies may omit the contribution of core histone and its tail to the regulation of transcriptional activation, and 2) previous reports have indicated that arginine-specific histone methylation by CARM1 is a significant part of the transcriptional activation process for some genes.
Abnormal expression of CARM1 has been linked to human prostate (20
), breast (28
), and colorectal cancers (29
). In this report, we showed that CARM1 protein levels are higher in a panel of colorectal cancer cell lines than in a cell line that more closely resembles normal colon epithelial cells (). Moreover, CARM1 silencing adversely affected clonal survival and anchorage-independent growth of colorectal cancer cell lines with constitutively high levels of β-catenin ( and Supplementary Figure S5
). These CARM1-dependent phenotypes are presumably due to the fact that CARM1 depletion causes reduced expression of genes involved in Wnt-driven tumorigenesis (e.g. c-myc (5
)), metastasis (e.g. S100A4 (8
)) and prevention of apoptosis (e.g. GPR49 (10
)). Thus, CARM1 plays a key role in Wnt signaling through its role as a transcriptional coactivator that mediates the actions of β-catenin on Wnt target genes; some of the Wnt target genes that require CARM1 are critical for maintaining important aspects of the transformed phenotype of colon cancer cell lines. Therefore, selective inhibition of CARM1 methyltransferase activity or CARM1 binding to β-catenin may be a potential strategy for therapeutic treatment of abnormally activated Wnt/β-catenin signaling in colorectal cancers.