Plant homeodomain finger 2 (PHF2) has a role in epigenetic regulation of gene expression by demethylating H3K9-Me2. Several genome-wide studies have demonstrated that the chromosomal region including the PHF2 gene is often deleted in some cancers including colorectal cancer, and this finding encouraged us to investigate the tumor suppressive role of PHF2. As p53 is a critical tumor suppressor in colon cancer, we tested the possibility that PHF2 is an epigenetic regulator of p53. PHF2 was associated with p53, and thereby, promoted p53-driven gene expression in cancer cells under genotoxic stress. PHF2 converted the chromatin that is favorable for transcription by demethylating the repressive H3K9-Me2 mark. In an HCT116 xenograft model, PHF2 was found to be required for the anticancer effects of oxaliplatin and doxorubicin. In PHF2-deficient xenografts, p53 expression was profoundly induced by both drugs, but its downstream product p21 was not, suggesting that p53 cannot be activated in the absence of PHF2. To find clinical evidence about the role of PHF2, we analyzed the expressions of PHF2, p53 and p21 in human colon cancer tissues and adjacent normal tissues from patients. PHF2 was downregulated in cancer tissues and PHF2 correlated with p21 in cancers expressing functional p53. Colon and stomach cancer tissue arrays showed a positive correlation between PHF2 and p21 expressions. Informatics analyses using the Oncomine database also supported our notion that PHF2 is downregulated in colon and stomach cancers. On the basis of these findings, we propose that PHF2 acts as a tumor suppressor in association with p53 in cancer development and ensures p53-mediated cell death in response to chemotherapy.

BackgroundThe enhancement of cell motility is a critical event during tumor cell spreading. Since myosin light chain kinase (MLCK) regulates cell behavior, it is regarded as a promising target in terms of preventing tumor invasion and metastasis. Since MLCK was identified to be associated with human arrest defective-1 (hARD1) through yeast two-hybrid screening, we here tested the possibility that hARD1 acts as a regulator of MLCK and by so doing controls tumor cell motility.Methodology/Principal FindingsThe physical interaction between MLCK and hARD1 was confirmed both in vivo and in vitro by immunoprecipitation assay and affinity chromatography. hARD1, which is known to have the activity of protein lysine ε-acetylation, bound to and acetylated MLCK activated by Ca2+ signaling, and by so doing deactivated MLCK, which led to a reduction in the phosphorylation of MLC. Furthermore, hARD1 inhibited tumor cell migration and invasion MLCK-dependently. Our mutation study revealed that hARD1 associated with an IgG motif of MLCK and acetylated the Lys608 residue in this motif. The K608A-mutated MLCK was neither acetylated nor inactivated by hARD1, and its stimulatory effect on cell motility was not inhibited by hARD1.Conclusion/SignificanceThese results indicate that hARD1 is a bona fide regulator of MLCK, and that hARD1 plays a crucial role in the balance between tumor cell migration and stasis. Thus, hARD1 could be a therapeutic target in the context of preventing tumor invasion and metastasis.

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Chaetocin, an antibiotic produced by Chaetomium species fungi, was recently found to have antimyeloma activity. Here we examined whether chaetocin has anticancer activities against solid tumors. Chaetocin inhibited the growth of mouse and human hepatoma grafts in nude mice. Immunohistochemical analyses revealed that chaetocin inhibits hypoxia-inducible factor-1a (HIF-1a) expression and vessel formation in the tumors. Chaetocin also showed antiangiogenic anticancer activities in HIF-1a(1/1) fibrosarcoma grafted in mice, but not in HIF-1a(2/2) fibrosarcoma. Biochemical analyses showed that chaetocin down-regulated HIF-1a and the transcripts of HIF-1 target genes including vascular endothelial growth factor in hepatoma tissues and in various hepatoma cell lines. Based on the reported literature, unsuccessful efforts were made to determine the mechanism underlying the action of chaetocin. Unexpectedly, chaetocin was found to cause the accumulation of HIF-1a premessenger RNA (pre-mRNA) but to reduce mature mRNA levels in hepatoma cells and tissues. Such an effect of chaetocin was not observed in cell lines derived from normal cells, and was cell type-dependent even among cancer cell lines. Conclusions: Our results suggest that chaetocin could be developed as an anticancer agent to target HIF-1 in some cancers including hepatoma. It is also suggested that the HIF-1a premRNA splicing is a novel therapeutic target for controlling HIF-1-mediated pathological processes. (HEPATOLOGY 2011;53:171-180) H epatocellular carcinoma (also called malignant hepatoma) is one of the most common malignant tumors and the third leading cause of cancer mortality worldwide.1 Despite many efforts to develop various classes of agents, systemic chemotherapy and hormone therapy have failed to significantly increase the survival of patients with advanced hepatoma. However, recent advances in the understanding of hepatoma progression have led to the development of novel molecularly targeted therapies.2 Because angiogenesis is pivotal for the development and progression of hepatoma, key molecules regulating angiogenesis are regarded as promising targets for treating hepatoma. Hypoxia inevitably develops in rapidly growing tumors and is an important microenvironment that forces changes in tumor behavior. In particular, hypoxia activates hypoxia-inducible factor-1a (HIF-1a), which promotes the progression of malignancy by stimulating angiogenesis and by augmenting the ability of tumors to survive. 4,5 The roles of HIF-1a have been extensively investigated in cancer patients and in tumor-bearing mice. 6,7 Consequently, HIF-1a is believed to be a valid target for the treatment of aggressive tumors, and many efforts have been made to identify suitable HIF-1a inhibitors. 8 Chaetocin, which is produced by Chaetomium sp., is an antibiotic having the thiodioxopiperazine structure (a disulfide-bridged piperazine).9 Other thiodioxopiperazines are known to have antimicrobial, antiviral, Abbreviations: ATP, adenosine triphosphate; CA9, carbonic anhydrase 9...

Histone modifications on major transcription factor target genes are one of the major regulatory mechanisms controlling adipogenesis. Plant homeodomain finger 2 (PHF2) is a Jumonji domain-containing protein and is known to demethylate the histone H3K9, a repressive gene marker. To better understand the function of PHF2 in adipocyte differentiation, we constructed stable PHF2 knock-down cells by using the mouse pre-adipocyte cell line 3T3-L1. When induced with adipogenic media, PHF2 knock-down cells showed reduced lipid accumulation compared to control cells. Differential expression using a cDNA microarray revealed significant reduction of metabolic pathway genes in the PHF2 knock-down cell line after differentiation. The reduced expression of major transcription factors and adipokines was confirmed with reverse transcription- quantitative polymerase chain reaction and Western blotting. We further performed co-immunoprecipitation analysis of PHF2 with four major adipogenic transcription factors, and we found that CCATT/enhancer binding protein (C/EBP)α and C/EBPδ physically interact with PHF2. In addition, PHF2 binding to target gene promoters was confirmed with a chromatin immunoprecipitation experiment. Finally, histone H3K9 methylation markers on the PHF2-binding sequences were increased in PHF2 knock-down cells after differentiation. Together, these results demonstrate that PHF2 histone demethylase controls adipogenic gene expression during differentiation.