In an attempt to isolate cofactors capable of influencing estrogen receptor ␣ (ER␣) transcriptional activity, we used yeast two-hybrid screening and identified protein arginine methyltransferase 2 (PRMT2) as a new ER␣-binding protein. PRMT2 interacted directly with three ER␣ regions including AF-1, DNA binding domain, and hormone binding domain in a ligand-independent fashion. The ER␣-interacting region on PRMT2 has been mapped to a region encompassing amino acids 133-275. PRMT2 also binds to ER␤, PR, TR␤, RAR␣, PPAR␥, and RXR␣ in a ligand-independent manner. PRMT2 enhanced both ER␣ AF-1 and AF-2 transcriptional activity, and the potential methyltransferase activity of PRMT2 appeared pivotal for its coactivator function. In addition, PRMT2 enhanced PR, PPAR␥, and RAR␣-mediated transactivation. Although PRMT2 was found to interact with two other coactivators, the steroid receptor coactivator-1 (SRC-1) and the peroxisome proliferator-activated receptor-interacting protein (PRIP), no synergistic enhancement of ER␣ transcriptional activity was observed when PRMT2 was coexpressed with either PRIP or SRC-1. In this respect PRMT2 differs from coactivators PRMT1 and CARM1 (coactivator-associated arginine methyltransferase). These results suggest that PRMT2 is a novel ER␣ coactivator.

Fat, a candidate tumor suppressor in Drosophila, is a component of Hippo signaling pathway involved in controlling organ size. We found that a ∼3 Mbp deletion in mouse chromosome 3 caused tumorigenesis of a non‐tumorigenic mammary epithelial cell line. The expression of Fat4 gene, one member of the Fat family, in the deleted region was inactivated, which resulted from promoter methylation of another Fat4 allele following the deletion of one Fat4 allele. Re‐expression of Fat4 in Fat4‐deficient tumor cells suppressed the tumorigenecity whereas suppression of Fat4 expression in the non‐tumorigenic mammary epithelial cell line induced tumorigenesis. We also found that Fat4 expression was lost in a large fraction of human breast tumor cell lines and primary tumors. Loss of Fat4 expression in breast tumors was associated with human Fat4 promoter methylation. Together, these findings suggest that Fat4 is a strong candidate for a breast tumor suppressor gene. © 2008 Wiley‐Liss, Inc.

To identify genes involved in breast tumorigenesis, we applied the retroviral LoxP-Cre system to a nontumorigenic mouse mammary epithelial cell line NOG8 to create random chromosome deletion/translocation. We found that the disruption of one allele of Smyd4 (SET and MYND domain containing 4) gene through chromosome translocation led to tumorigenesis. The expression of Smyd4 was markedly decreased in tumor cells. Re-expression of Smyd4 resulted in growth suppression of tumor cells and inhibition of tumor formation in nude mice. Furthermore, the RNA interferencemediated suppression of Smyd4 expression in human MCF10A mammary epithelial cells caused their growth in soft agar. Microarray studies revealed that platelet-derived growth factor receptor A polypeptide (Pdgfr-A) was highly expressed in tumor cells compared with NOG8 cells. Reexpression of Smyd4 significantly reduced the expression of Pdgfr-A in tumor cells. In human breast cancers, reverse transcription-PCR results revealed that Smyd4 expression was totally silenced in 2 of 10 specimens. These findings indicate that Smyd4, as a potential tumor suppressor, plays a critical role in breast carcinogenesis at least partly through inhibiting the expression of Pdgfr-A, and could be a novel target for improving treatment of breast cancer. [Cancer Res 2009;69(9):4067-72]

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Nuclear receptor coactivator [peroxisome proliferator-activated receptor-binding protein (PBP)/mediator subunit 1 (MED1)] is a critical component of the mediator transcription complex. Disruption of this gene in the mouse results in embryonic lethality. Using the PBP/MED1 liver conditional null (PBP/MED1ΔLiv) mice, we reported that PBP/MED1 is essential for liver regeneration and the peroxisome proliferator-activated receptor α ligand Wy-14,643-induced receptor-mediated hepatocarcinogenesis. We now examined the role of PBP/MED1 in genotoxic chemical carcinogen diethylnitrosamine (DEN)-induced and phenobarbital-promoted hepatocarcinogenesis. The carcinogenic process was initiated by a single intraperitoneal injection of DEN at 14 days of age and initiated cells were promoted with phenobarbital (PB) (0.05%) in drinking water. PBP/MED1ΔLiv mice, killed at 1, 4 and 12 weeks, revealed a striking proliferative response of few residual PBP/MED1-positive hepatocytes that escaped Cre-mediated deletion of PBP/MED1 gene. No proliferative expansion of PBP/MED1 null hepatocytes was noted in the PBP/MED1ΔLiv mouse livers. Multiple hepatocellular carcinomas (HCCs) developed in the DEN-initiated PBP/MED1fl/fl and PBP/MED1ΔLiv mice, 1 year after the PB promotion. Of interest is that all HCC developing in PBP/MED1ΔLiv mice were PBP/MED1 positive. None of the tumors was PBP/MED1 negative implying that hepatocytes deficient in PBP/MED1 are not susceptible to neoplastic conversion. HCC that developed in PBP/MED1ΔLiv mouse livers were transplantable in athymic nude mice and these maintained PBP/MED1fl/fl genotype. PBP/MED1fl/fl HCC cell line derived from these tumors expressed PBP/MED1 and deletion of PBP/MED1fl/fl allele by adeno-Cre injection into tumors caused necrosis of tumor cells. These results indicate that PBP/MED1 is essential for the development of HCC in the mouse.