In most cases, proliferation and differentiation are inversely coupled: repression of proliferation is a prerequisite for initiation of differentiation (11
). In many cell types, however, cell cycle arrest is necessary but not sufficient for differentiation. CARM1 appears to be a unique ER-coactivator regulating both processes. Over-expression of CARM1 in MCF7 cells results in inhibition of E2-dependent growth through inhibition of the G0/G1 transition to S-phase. This is in part due to up-regulation of key negative cell cycle regulators such as p21cip1
, and cyclin G2. Inhibition of E2 dependent cell growth by CARM1 is accompanied by morphological changes characteristic of a more differentiated phenotype and induction of multiple differentiation markers such as GATA-3 and MAZ. This finding is supported by previous reports that CARM1 can promote cell differentiation in other systems (19
). Nonetheless, regulation of cell differentiation by CARM1 appears to be cell-type and context dependent. In mouse embryo and embryonic stem cells, CARM1 was shown to elevate expression of key pluripotency genes and delay their response to differentiation signals (38
In contrast to growth inhibition by CARM1 overexpression, knocking down CARM1 in MCF7 did not alter E2-dependent cell growth in cell culture nor did it affect E2-induced S phase entry. This observation contradicts the conclusion by Frietze et al. that CARM1 increases growth of MCF7 cells. The discrepancies may be due to the transient transfection of CARM1 siRNA throughout the cell cycle study by Frietze et al. (16
). Moreover, the authors measured the percentage of cells in S+G2+M phase without distinguishing the percentage of cells in S phase. Also, in consistent with the observation of O’Brien et al (21
), we did not observe change of E2F1 with CARM1 knock down, in contrast to Frietze et al. (16
). Interestingly, and in contrast to cells grown in culture, knocking down CARM1 enhanced E2-induced xenograft tumors. This may be due to increased breast cancer cell interaction with the microenvironment which plays essential roles in promoting tumor growth in animals.
The growth inhibitory effect of CARM1 is unique from that of SRCs. Knocking down SRC2 and SRC3 but not SRC1 inhibits growth of MCF7 cells and decreases cyclin D1 expression (39
). Overexpression of SRC3 also increases breast cancer cell proliferation and invasiveness. Likewise, SRC-1 promotes breast tumor metastasis and inhibits tumor cell differentiation (40
). Thus, the ERα-dependent, growth inhibitory effect of CARM1 is unlikely to be mediated through SRC-1, 2 and -3.
Cell cycle genes that are regulated by E2 or loss of CARM1 include cyclin D1, c-myc, cyclin G2, cyclin L1, cyclin T2, p21cip1
, p130 and Rb (Table S1
). E2 treatment alone significantly represses cyclin G2 (33
), which is reversed by overexpressing CARM1. Cyclin D1 is a well-known E2-induced ERα target gene, however, its expression is not affected by overexpression of CARM1 in the presence of E2, yet knocking down CARM1 upregulates cyclin D1 in MCF7 cells (Table S1
). C-myc is upregulated by E2 alone or loss of CARM1 (Table S1
) but is not affected by depletion of any of the p160 coactivators in MCF7 cells (39
). Thus, the mechanism of CARM1 regulation of cell cycle regulators is complex and only partially depends on the p160 coactivators.
Microarray gene expression analyses reveal that approximately 16% of E2-activated genes were repressed by CARM1, consistent with the repressive effects of CARM1 on some ER-target genes (41
). The mechanism of CARM1-mediated repression is unclear. The major effect of CARM1 overexpression was to relieve E2-repressed genes. CARM1 methyltransferase activity may be responsible for the activation since we observed increased H3R17Me2 mark on p21cip1
promoter upon CARM1 induction in MCF7-tet-on-CARM1 (data not shown), consistent with a recent publication that CARM1 is recruited to p21cip1
). Whether CARM1 regulates ERα-target genes via an epigenetic mechanism remains to be determined. Nonetheless, global ERα transcriptional regulation by CARM1 leads to induction of many E2 repressed genes associated with differentiation.
Consistent with this finding, knocking-down CARM1 shares over 65% of the E2 gene signature. The majority of CARM1, E2-regulated genes are involved in gene expression, metabolism, cell cycle and differentiation. Knocking down CARM1 leads to up-regulation of positive cell cycle regulators (e.g. c-myc) and down-regulation of negative cell cycle regulators (e.g. cyclin G2). This result suggests that loss of CARM1 function may lead to the acquisition of a proliferative phenotype resembling estrogen stimulation of breast cancer. Further, knocking-down CARM1 also modulates genes involved in cell differentiation. For example, combination of CARM1 shRNA and E2 treatment significantly reduced the level of PPARγ, which induces terminal differentiation of breast cancer (43
). Loss of CARM1 also significantly decreases KRTAP10.12, an E2-repressed gene involved in keratin filament formation and potentially in cell differentiation processes (44
). Collectively, either loss of CARM1 or E2 treatment significantly inhibits expression of various differentiation markers (Table S1
). Overall, the gene expression data from CARM1 gain- and loss-of-function models suggest that CARM1 plays an important role in regulating ERα target genes in differentiation and proliferation.
Evidence for a functional interplay of ERα and CARM1 was explored in human breast cancer specimens. A direct correlation was observed between CARM1 and ERα in ER+ tumors. Higher ERα expression is associated with less aggressive and more differentiated tumors, and ER status is known to inversely correlate with histological grade (46
). Our observation contradicts an earlier report that CARM1 is overexpressed in grade III breast tumors (23
). The difference could result from analysis of RNA vs. protein and the sample size. In the study by El Messaoudi et al., CARM1 was only analyzed at the RNA level in 81 human breast tumors, while we analyzed CARM1 protein level in over 300 human breast tumors.
Histological grade using the Nottingham method, integrates scores from glandular differentiation, nuclear morphology and mitotic counts (47
) and higher grade is significantly associated with poor outcome and survival. The inverse correlation of CARM1 expression and tumor grade found in ER+ breast cancer cases together with enhanced tumor volume in CARM1 knock-down breast cancer xenografts in animal models support an association of low levels of CARM1 with less well differentiated, high grade breast cancers, and is consistent with the hypothesis that CARM1 inhibits breast cancer progression in ERα positive tumors. Our results suggest that co-expression of ERα and CARM1 together may serve as a better biomarker of well differentiated breast cancers.
ERα is believed to regulate growth and differentiation through balanced interaction with cofactors. This study reports an unexpected biological function of the ER-coactivator CARM1 in breast cancer. The hallmark of CARM1 action might be due to global modulation of E2-regulated genes, leading to re-programming of cell proliferation and differentiation. To our knowledge, CARM1 is the only ER coactivator that is able to simultaneously block cell proliferation and induce differentiation. Since CARM1 has histone modification activity, inducing differentiation of breast cancer cells by up-regulating CARM1 activity may be therapeutically effective in breast cancer.