Hyun Soo Cho

Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers

A number of histone demethylases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human disease like cancer have not been well understood. Here, we demonstrate important roles of lysine‐specific demethylase 1 (LSD1) in human carcinogenesis. Expression levels of LSD1 are significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001). cDNA microarray analysis also revealed its transactivation in lung and colorectal carcinomas. LSD1‐specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of various bladder and lung cancer cell lines. Concordantly, introduction of exogenous LSD1 expression promoted cell cycle progression of human embryonic kidney fibroblast cells. Expression profile analysis showed that LSD1 could affect the expression of genes involved in various chromatin‐modifying pathways such as chromatin remodeling at centromere, centromeric heterochromatin formation and chromatin assembly, indicating its essential roles in carcinogenesis through chromatin modification.

Demethylation of RB regulator MYPT1 by histone demethylase LSD1 promotes cell cycle progression in cancer cells.

Histone demethylase LSD1 (also known as KDM1 and AOF2) is active in various cancer cells, but its biological significance in human carcinogenesis is unexplored. In this study, we explored hypothesized interactions between LSD1 and MYPT1, a known regulator of RB1 phosphorylation. We found that MYPT1 was methylated in vitro and in vivo by histone lysine methyltransferase SETD7 and demethylated by LSD1, identifying Lys 442 of MYPT1 as a target for methylation/demethylation by these enzymes. LSD1 silencing increased MYPT1 protein levels, decreasing the steady state level of phosphorylated RB1 (Ser 807/811) and reducing E2F activity. MYPT1 methylation status influenced the affinity of MYPT1 for the ubiquitin-proteasome pathway of protein turnover. MYPT1 was unstable in murine cells deficient in SETD7, supporting the concept that MYPT1 protein stability is physiologically regulated by methylation status. LSD1 overexpression could activate RB1 phosphorylation by inducing a destabilization of MYPT1 protein. Taken together, our results comprise a novel cell cycle regulatory mechanism mediated by methylation/demethylation dynamics, and they reveal the significance of LSD1 overexpression in human carcinogenesis.

Histone Lysine Methyltransferase SETD8 Promotes Carcinogenesis by Deregulating PCNA Expression

Although the physiologic significance of lysine methylation of histones is well known, whether lysine methylation plays a role in the regulation of nonhistone proteins has not yet been examined. The histone lysine methyltransferase SETD8 is overexpressed in various types of cancer and seems to play a crucial role in S-phase progression. Here, we show that SETD8 regulates the function of proliferating cell nuclear antigen (PCNA) protein through lysine methylation. We found that SETD8 methylated PCNA on lysine 248, and either depletion of SETD8 or substitution of lysine 248 destabilized PCNA expression. Mechanistically, lysine methylation significantly enhanced the interaction between PCNA and the flap endonuclease FEN1. Loss of PCNA methylation retarded the maturation of Okazaki fragments, slowed DNA replication, and induced DNA damage, and cells expressing a methylation-inactive PCNA mutant were more susceptible to DNA damage. An increase of methylated PCNA was found in cancer cells, and the expression levels of SETD8 and PCNA were correlated in cancer tissue samples. Together, our findings reveal a function for lysine methylation on a nonhistone protein and suggest that aberrant lysine methylation of PCNA may play a role in human carcinogenesis.

Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B.

Although heat-shock protein 70 (HSP70), an evolutionarily highly conserved molecular chaperone, is known to be post-translationally modified in various ways such as phosphorylation, ubiquitination and glycosylation, physiological significance of lysine methylation has never been elucidated. Here we identify dimethylation of HSP70 at Lys-561 by SETD1A. Enhanced HSP70 methylation was detected in various types of human cancer by immunohistochemical analysis, although the methylation was barely detectable in corresponding non-neoplastic tissues. Interestingly, methylated HSP70 predominantly localizes to the nucleus of cancer cells, whereas most of the HSP70 protein locates to the cytoplasm. Nuclear HSP70 directly interacts with Aurora kinase B (AURKB) in a methylation-dependent manner and promotes AURKB activity in vitro and in vivo. We also find that methylated HSP70 has a growth-promoting effect in cancer cells. Our findings demonstrate a crucial role of HSP70 methylation in human carcinogenesis.

The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G1/S transition in cancer cells via transcriptional regulation of the HOXA1 gene.

A number of histone demethylases have been identified and biochemically characterized, yet their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. In this study, we describe important roles for the histone demethylase KDM3A, also known as JMJD1A, in human carcinogensis. Expression levels of KDM3A were significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001), when assessed by real‐time PCR. We confirmed that some other cancers including lung cancer also overexpressed KDM3A, using cDNA microarray analysis. Treatment of cancer cell lines with small interfering RNA targeting KDM3A significantly knocked down its expression and resulted in the suppression of proliferation. Importantly, we found that KDM3A activates transcription of the HOXA1 gene through demethylating histone H3 at lysine 9 di‐methylation by binding to its promoter region. Indeed, expression levels of KDM3A and HOXA1 in several types of cancer cell lines and bladder cancer samples were statistically correlated. We observed the down‐regulation of HOXA1 as well as CCND1 after treatment with KDM3A siRNA, indicating G1 arrest of cancer cells. Together, our results suggest that elevated expression of KDM3A plays a critical role in the growth of cancer cells, and further studies may reveal a cancer therapeutic potential in KDM3A inhibition.

Deregulation of the histone demethylase JMJD2A is involved in human carcinogenesis through regulation of the G1/S transition

Although a number of JmjC-containing histone demethylases have been identified and biochemically characterized, pathological roles of their dysfunction in human disease such as cancer have not been well elucidated. Here, we report the Jumonji domain containing 2A (JMJD2A) is integral to proliferation of cancer cells. Quantitative real-time PCR analysis revealed higher expression of JMJD2A in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P<0.0001). Immunohistochemical analysis also showed positive staining for JMJD2A in 288 out of 403 lung cancer cases, whereas no staining was observed in lung normal tissues. Suppression of JMJD2A expression in lung and bladder cancer cells overexpressing this gene, using specific siRNAs, inhibited incorporation of BrdU and resulted in significant suppression of cell growth. Furthermore, JMJD2A appears to directly transactivate the expression of some tumor associated proteins including ADAM12 through the regulation of histone H3K9 methylation. As expression levels of JMJD2A are low in normal tissues, it may be feasible to develop specific inhibitors targeting the enzyme as anti-tumor agents which should have a minimal risk of adverse reaction.

The histone methyltransferase Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) is involved in human carcinogenesis.

Histone lysine methylation plays a fundamental role in chromatin organization. Although a set of histone methyltransferases have been identified and biochemically characterized, the pathological roles of their dysfunction in human cancers are still not well understood. In this study, we demonstrate important roles of WHSC1L1 in human carcinogenesis. Expression levels of WHSC1L1 transcript were significantly elevated in various human cancers including bladder carcinoma. Immunohistochemical analysis of bladder, lung, and liver cancers confirmed overexpression of WHSC1L1. WHSC1L1‐specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of bladder and lung cancer cell lines. WHSC1L1 knockdown induced cell cycle arrest at the G2/M phase followed by multinucleation of cancer cells. Expression profile analysis using Affymetrix GeneChip® showed that WHSC1L1 affected the expression of a number of genes including CCNG1 and NEK7, which are known to play crucial roles in the cell cycle progression at mitosis. As WHSC1L1 expression is significantly low in various normal tissues including vital organs, WHSC1L1 could be a good candidate molecule for development of novel treatment for various types of cancer.

IFT46 plays an essential role in cilia development

Cilia are microtubule-based structures that project into the extracellular space. Ciliary defects are associated with several human diseases, including polycystic kidney disease, primary ciliary dyskinesia, left-right axis patterning, hydrocephalus and retinal degeneration. However, the genetic and cellular biological control of ciliogenesis remains poorly understood. The IFT46 is one of the highly conserved intraflagellar transport complex B proteins. In zebrafish, ift46 is expressed in various ciliated tissues such as Kupffer׳s vesicle, pronephric ducts, ears and spinal cord. We show that ift46 is localized to the basal body. Knockdown of ift46 gene results in multiple phenotypes associated with various ciliopathies including kidney cysts, pericardial edema and ventral axis curvature. In ift46 morphants, cilia in kidney and spinal canal are shortened and abnormal. Similar ciliary defects are observed in otic vesicles, lateral line hair cells, olfactory pits, but not in Kupffer׳s vesicle. To explore the functions of Ift46 during mouse development, we have generated Ift46 knock-out mice. The Ift46 mutants have developmental defects in brain, neural tube and heart. In particular Ift46(-/-) homozygotes displays randomization of the embryo heart looping, which is a hallmark of defective left-right (L/R) axis patterning. Taken together, our results demonstrated that IFT46 has an essential role in vertebrate ciliary development.

Functional interplay between Aurora B kinase and Ssu72 phosphatase regulates sister chromatid cohesion

Cohesins establish cohesion between replicated sister chromatids and are maintained as a multiprotein complex on chromosome arms until they are phosphorylated by mitotic kinases, such as Aurora B and Plk1. However, the mechanics of how the phosphorylation and dephosphorylation of cohesin subunits by kinases and phosphatases, respectively, leads to the dissociation of the cohesin complex from chromosomes remain unclear. Here we report that Aurora B kinase directly interacts with and phosphorylates Ssu72, a new cohesin-binding phosphatase, at Ser 19 in vitro and in vivo. The Aurora B-mediated phosphorylation of Ssu72 causes the structural modification of Ssu72 protein, downregulates phosphatase activity and triggers the ubiquitin-dependent degradation of Ssu72. Overexpression of the Aurora B-mediated phosphomimetic mutant of Ssu72 prevents maintainance chromosome arm cohesion. These results provide evidence that Aurora B kinase directly targets Ssu72 phosphatase for regulation of sister chromatid cohesion during early mitosis.

The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer

Molecular classifications of breast cancer (BRC), such as human epidermal growth factor receptor 2 (HER2), luminal A and luminal B, have been developed to reduce unnecessary treatment by dividing patients with BRC into low- and high-risk progression groups. However, these methods do not cover all of the pathological characteristics of BRC, and investigations into novel prognostic/therapeutic markers are thus continually required. In this study, we identified the overexpression of the histone methyltransferase, euchromatic histone-lysine N-methyltransferase 2 (EHMT2) in BRC samples (n=1,222) and normal samples (n=113) derived from the TCGA portal by performing a BRC tissue microarray. EHMT2 overexpression was clearly associated with a poor prognosis in multiple cohorts of patients with BRC (total, n=1,644). Furthermore, the knockdown of EHMT2 expression affected cell apoptosis via the downregulation and re-localization of heat shock protein family D (Hsp60) member 1 (HSPD1). In addition, a statistically significant positive correlation between EHMT2 and HSPD1 expression was revealed in the clinical cohorts. On the whole, the findings of this study may assist the development of novel therapeutic strategies and provide a prognostic marker (EHMT2) for patients with BRC.