“Identification Card”: Sites on Histone Modification of Cancer Cell△

Chao Huang and Bin Wen*

Institute of Spleen and Stomach, Guangzhou University of Chinese Medicine, Guangzhou 510000, China

“Identification Card”: Sites on Histone Modification of Cancer Cell△

Chao Huang and Bin Wen*

Institute of Spleen and Stomach, Guangzhou University of Chinese Medicine, Guangzhou 510000, China

histone modification； epigenetics； oncogenesis； methylation； histone acetylation；cancer

Formation of malignant tumor originating from normal healthy cell is a multistep process including genetic and epigenetic lesions. Previous studies of cell line model systems displayed that early important epigenetic events happened in stepwise fashion prior to cell immortalization. Once these epigenetic alterations are integrated into chromatin， they will perform vertical propagation through cell subculture. Hence， status of epigenetics is dramatically important in maintaining of cell identity. Histone modification is another factor of epigenetic alterations during human oncogenesis. Histones， one of main components of chromatin， can be modified post-translationally. Histone tail modifications are regulated by corresponding modification enzymes. This review focuses on the description of relationship between the main sites of histone modification and oncogenesis.IMILAR to genetic lesions, epigenetic lesions can also alter structure and function of genome, which participates in the acquisition of limitless uncontrolled growth ability of cells and phenotypic hallmarks of cancer cell, as well as affects cell pluripotency and differentiation. Increasing evidence uncovers the vital significance of epigenetic dysfunction in oncogenesis and in maintaining of cancer cell identity,1,2the critical role of altered epigenetic modifications in early stage of cell reprogramming, and incompletely epigenetic inhibition of

Chin Med Sci J 2015； 30（4）:203-209

Simportant genes may be a bottleneck in the process of transformation of pluripotency state,3which suggest that the identities of cancer cells are different from normal cell in epigenetics.

Three post-translational histone modifications, such as methylation, acetylation, and phosphorylation are relatively important modifications. Histone modifications affect whole structure of chromatin and regulate gene expression by means of changing condensation of DNA or recruiting effector molecules which control downstream gene expression.4Histone modifications directly mediate gene activation and gene inhibition via individually specific modifications, as well as cell survival, cell identity maintaining and cell reprogramming. Hence, this review mainly discusses the sites of histone modification as

“identification card” of cancer cell involved in oncogenesis.

Histone Methylation

Histone specific methyltransferases (HKMTs), such as SMYD3 (H3K4 methyl-transferase) and EZH2 (H3K27 methyltransferase), can methylate histones. Furthermore, demethylases have influence on histones as well. Histone methylation defines an epigenetic entity,5and can accurately distinguish cancer from normal tissues. Popovic and Licht6uncover that both histone methyltransferases (HMTs) and demethylase are aberrant in human cancer. Therefore, the two kinds of enzymes may be related to oncogenesis. For instance, G9a, a H3K9 methylase in mammal, is overexpressed in hepatocellular carcinoma.7Study has found that G9a was associated with epigenetic silence of tumor suppressor genes (TSGs) and maintaining of malignant phenotypes.8Lysine-specific histone demethylase 1 (LSD1) can not only change histone modifications, but also interact with various proteins such as P53 and DNMTs and then contribute to malignant transformation.9Methylation patterns of H3K9 and H3K27 between cancer and normal tissues have an essential distinction. For example, H3K9me1 and H3K9me2 in penile squamous cell carcinoma have a transfer toward increased H3K9me3.5Compared with normal tissues, the expressions of H3K27me2 and H3K27me3 in penile carcinoma are down-regulated, which is likely related to over-weight of histone demethylases over activity of demethylase.5

Histone lysine residues which can be methylated by HMTs include H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20. Among these, H3K4, H3K36, and H3K79 are associated with gene activation of euchromatin, H3K9, H3K27, and H4K20 are involved in genome heterochromatin.10H3K4me2/3 frequently displays functional changes of promoters in the process of oncogenesis,11while H3K4me1 is associated with activation of enhancer.12H3K9me1 can be seen as “active gene”, but H3K9me3 takes part in gene suppression.10Study on colon cancer cell line CT26 revealed the increasing of inactive chromatin epigenetic marks H3K9me2 and H3K9me3 in promoter regions of low-expressed genes. Heterochromatin with increased EZH2 level indicates rising polycomb repressive compound 2 (PRC2) in silenced genes.13

Abnormal activation of oncogene and inactivation of tumor suppressor gene in neoplasia are caused by aberrant histone modifications, such as deregulation of H3K4me3 and H3K27me3, which cause chromosomal instability and DNA repair defects.14Overexpressed oncogene myelocytomatosis (MYC) preferentially combines to the promoter regions of active genes with the characteristic of H3K4me3 to improve the expression levels of these genes in pluripotent stem cells and cancer cells.15,16H3K4me2 mediates the status of MYC transcription, and its level is up-regulated in cancer.

The silence of TSGs caused by deficiency of H4K16 acetylation and H4K20me3 contributes to occurrence of lymphoma and colorectal cancer (CRC), hence, the deletion of H4K16 acetylation has become a general hallmark.17Some studies have noted deficiency of H4 lysine trimethylation or acetylation in hypomethylated repeat genome region,18as well as H3K27me3 loss in hypermethylated promotor region of cancer suppressor gene,19which elucidate that aberrant histone modifications leading to oncogenesis are in dynamic. In acute myelocytic leukemia (AML), H3K9 hypermethylation on promoter region of gene suppressor of cytokine signaling 1 (SOCS1）was found, furthermore, inhibition of H3K9 methylase can reactivate the expression of SOCS1.20Likewise, increasing H3K9 methylation rather than CpG methylation appears on the promoter region of the silent reticuloendotheliosis viral (v-rel) oncogene related B (RELB) gene.21Study from gastric carcinoma cell lines has uncovered that PRC1 member CBX7 launched H3K9me3 at the P16 through recruiting and/or activating HMT SUV39H2 to the target locus. This mechanism has two repressive epigenetic landmarks, H3K9me3 formation and PRC1 binding within silenced domains of euchromatin.22

Aberrant histone modification of H3K4 is also connected with occurrence of various cancers. For instance, in colon cancer HCT116 cell lines, arrested cell cycle and extensive apoptosis were caused by increased level of H3K4 and H3K9 methylation.23Other study has reported that LSD made H3K4 demethylation that inhibited apoptosis of breast cancer cell.24Therefore, we have reason to believe that oncogenesis of breast cancer refers to H3K4 demethylation. Today, we have successfully derived “induced pluripotent stem cells ” (iPSCs) from somatic differentiated cells through changing the epigenetic modifications of H3K4me2 in wide-genome associated with the maintenance of cellular identity,25,26implying that we can also transform aberrant epigenetic modifications of tumor cells in the same or similar way to induce the transformation of early malignant cells towards normal stem cells. The inhibition of LSD1 can guard against H3K4me1/me2 demethylation and marginally increase expression of calcium sensing receptor (CaSR). The result indicates that hypermethylation of CaSR promoter and H3K9 deacetylation instead of H3K4me2 demethylation are a significant mechanism that induces the silence of CaSR in CRC.27Interestingly, Wang et al28found that the level ofH3K4me2/me3 is parallel between gastric cancer and normal tissue. Consequently, on the basis of above data, we can draw a conclusion that the level of H3K4 methylation is increased in most tumors but not in gastric cancer. Nevertheless, it is vital to make further study for confirming whether H3K4 methylation may be used as a specific identity mark of tumor cells.

H3K27me3 is an inhibitory histone modification, and marks many domains of the genome, including promoter, gene bodies, and gene intergenic regions,29regulating development and differentiation. Abnormal H3K27 methylation is also observed in cancer. H3K27 demethylase, ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), also known as KDM6A, positively mediates gene expression related to cell proliferation. The down-regulation of UTX can significantly reduce cell proliferation of breast cancer in vitro and xenografted tumor model of rat. Because UTX is mutated in neoplasia,30resulting in aberrant H3K27 methylation. In consequence, H3K27me3 level is proposed by researcher as a key parameter for determining cell identity.31In a molecule, two kinds of histone modifications with opposite properties may appear at the same time. For example, the active H3K4me3 and repressive H3K27me3 marks called as“divalent marking” in gene promoter region.32The divalent marking which has been proved by CHIP-reCHIP appears frequently in embryonic stem cells (ESCs) and cancer cells, and is related to a large number of regulatory genes, like regulator of differentiation, TSGs and regulator of cell cycle.33Certainly, genes with bivalent histone marks in cancer also have increased propensity to be aberrantly hypermethylated.32Interestingly, the bivalent domains are found in normal tissues as well, and this signature is likewise maintained in adenoma, implying that the marks have not been decomposed during differentiation.13Study from CT26 cell lines has reported the decrease of H3K4me3 and H3K27me3, confirming this viewpoint that bivalence is maintained through mediating the transcriptional balance between active and inactive marks, which is considered to guard against aberrant expression of genes. Therefore, the divalent marks in promoter regions lose their balance in the process of carcinogenesis. For example, p16ink4alocus are in status of divalent marking in normal human mammary epithelial cells (HMECs), but the H3K27me3 of the marking is lost in variant HMECs (vHMECs),34leading to aberrant profiles of gene expression.

Long range epigenetic silencing (LRES) found in a good many cancers, such as colorectal, prostate, breast, gastric, bladder, and Wilms’ kidney tumors, is established within a silenced large region in chromosome 2q14.2, and this region involves lots of TSGs where large groups of CpG islands hypermethylation and abundant H3K9me2 of promoter regions are discovered.9In addition, transcription factor Ikaros controlling differentiation and a large region at 5q31 controlling many members of protocadherins tumor suppression gene family are under LRES.35,36On the basis of analysis of two regions undergoing LRES in human colon cancer and mouse cancer model, study has found that the genes embedded in these regions display a dynamic and autonomous regulation during differentiation of mouse intestine cells, suggesting that the coordinated regulation in LRES was limited to cancer in framework considered here.13

Histone Acetylation

Histone acetylation in dynamic is regulated by two kinds of competitive enzyme families, histone lysine acetyl transferase (HATs) and histone deacetylase (HDACs).37Deacetylation in histone N-acetyl lysine residues by HDACs closely integrates with the negative charge of DNA, resulting in dense crispation of chromatin and therefore suppression of gene transcription.38Histone acetylation makes compact nucleosome structure loosen and is in favor of gene transcription. However, HDACs is one of key enzymes that regulate dynamic equilibrium of histone acetylation and deacetylation in nucleosome. Moreover, HDACs target not only histones but also non-histone proteins, including nuclear transcription factors (NF-κB, STAT), cytoplasm protein (a-tubulin, heat shock protein 90), and so on.39For example, HDAC3 maintains functional deacetylation of DNA repair gene CtBP-interaction protein 1 (CtIP).40Class Ⅲ HDAC has an effect on cell survival through deacetylation of several important cell cycle and apoptosis regulator such as p53 and Rb.41

Changes of normal acetylation profiles, resulting from multiple mechanisms, will lead to oncogenesis. Reseachers have reported deregulation of HDACs activities during aberrant gene silencing and carcinogenesis. Heterozygosity loss and missense mutation of HATs, including p300, CREB-binding protein (CBP), and p300/CBP associated factor (PCAF) have been testified in gastric cancer,42consistent with the down-regulation of PCAF expression in gastric cancer. Previous study from ovarian cancer displays over-expression of CLDN3 and CLDN4 gene related to inhibitory histone modification.43In addition, silenced oncogene BCL6 can be reactivated by HDACs with histone deacetylation,44as well as reduced H3K9 acetylation and increased H3K9 methylation can result in silence of gene P16, MLH1, and MGMT.41In CRC, acetylation level of histone H3 is significantly reduced, as well as decreasedglobal and local histone acetylation levels on the promoter regions of gene P21 and bax are also observed.45,46Some HDACs inhibit cancer cell growth via deacetylation of certain signal pathways. Sirtuin-3 attenuates oxidative stress through mediating hypoxia inducible factor (HIF) and is served as a TSG.47Nucleosome remodeling and histone deacetylase (NuRD), a chromatin-remodeler, decides destiny of all kinds of signaling cataracts,48transcription factor activator protein-1 (AP-1) mediates intestinal proliferation and initiation of carcinogenesis.49On the contrary, NuRD at least partly suppresses the transcription activity of AP-1.

Histone Phosphorylation

Four histone tails of nucleosome include numerous acceptor sites which can be phosphorylated by protein kinase and dephosphorylated by phosphatase. Although phosphorylation of histone H3S10 and S32 on promoter regions of proto-oncogene c-fos, c-jun, and c-myc has been widely found,50,51which is in favor of activation of gene expression, the definite biological characteristics and functions of these phosphorylation sites and their roles in tumorigenesis remain unclear.

Histone modifications, especially phosphorylation and acetylation, affect structure and function of chromatin. Because dynamic chromatin remodelling is the basis of numerous biological processes, like gene transcription, DNA replication, and DNA damage repair, its chaos by dysregulation of histone phosphorylation is directly associated with initiation and development of cancer.41Histone phosphorylation is also regulated by some signal pathway. Extracellular signal-regulated kinase-mitogen activated protein kinase (ERK-MAPK) signal pathway can induce H3S10 phosphorylation to prompt chromatin concentration required for mitosis.52Caspase, a family of cysteine proteinase, controls histone modification enzymes and promotes post-translational modifications of histones, such as H2A deubiquitination, H3T45ph, and H2BS14ph. Phosphorylation of histone H2B at ser 14, catalyzed by Mst1 kinase, is deemed as an epigenetic mark of apoptosis induced by multiple death stimulation signals. Caspase cleavage of Mst1 facilitates its nuclear translocation and following chromatin condensation.53H2BS10 phosphorylation is directly catalyzed by Ste20 kinase translocated to cell nucleus but independent of caspase,54although the H2BS14ph relys on the caspase cleavage of Mst1 and its activation. Down-regulated Mst1 kinase activity leads to reduced H2BS14ph and then suppression of apoptosis. However, the combined treatment of a HDAC and glucocorticoid inhibits both H2BS14ph and internucleosomal DNA degradation without inhibition of apoptosis in thymocytes.55There is a possible explanation that adjacent H2BK15ac is characteristic of non-apoptosis cell and significantly decreased in apoptosis cell, H2BK15 deacetylation is required for S14 phosphorylation, inhibition of H2BK15ac can lead to suppression of apoptosis. That study discovers that both K15 deacetylation and S14ph have been involved in apoptosis. In consequence, apoptosis cannot be inhibited on account of K15 being likely to be deacetylated by endogenic HDACs in a more specific manner.55Therefore, it is not yet clear whether every histone modification mark participates in apoptotic programme.

H3S10ph is related to two completely opposite chromatin status, namely loosened chromatin of some kind of activated genes during interkinesis and highly condensed chromosomes during mitotic phase.56When cells are exposed to all kinds of death stimulation signals, H3S10 is phosphorylated as well, showing H3S10ph’s indicative role in controlling cell survival.57H3S10ph level is significantly higher in hepatoma carcinoma cell induced by diethylnitrosamine (DEN) than that of non-cancer cell, at the same time, inhibition of H3S10ph will bring about suppression of cell proliferation and cell transformation, as well as down-regulation of Brf1 and PolⅢ gene whose promoter region are occupied by H3S10ph.58Just as the relationship between H2BS14ph and H2BK15ac, H3K9 adjacent site is H3S10, studies have confirmed that there have been a crosstalk among H3K9, S10, and K14.59Unfortunately, at present, researches about related functions and mechanisms of these crosstalks in tumorigenesis are relatively less.

Perspectives

Considering the remarkable significance of histone modification, researchers have used “histone onco-modifications”, a new terminology, to state the covalent post-translational modifications of histones associated with oncogenesis.14In tumor, differential histone codes, H3, H4 acetylation and H3K4me2, have been discovered, and these codes are distinct active marks. Because of the crosstalk between the different histone modifications, a large number of aberrant histone modifications have been emerged during early carcinogenesis. Dysfunctional histone-modifying enzymes in cancer can target and modify some residues of histone, hence, abnormal HDACs may lead to many aberrant modifications of histone sites. The effects of histone modifications on cell functions, not only on gene expression, but also on chromatin remodeling, are diverse. Because chromatin remodeling is required for many biological processes, such as apoptosis, DNA damage repair, cellproliferation, cell self-renewal and differentiation, hence, its chaos can directly result in carcinogenesis. On the other hand, histone modifications affect cell signal pathway as well, for example, HATs may affect a lot of intricate cross-talk networks of signal pathways.

Because of the difference of histone modifications between cancer and normal cell and the active contribution of abnormal histone modifications in the maintaining of cancer cell identity, histone markers play a vital role in distinguishing cancer cell from normal cell, in this case, aberrant histone modifications in cancer can be used as a“identification card” with recognition function. Divalent domain in gene promoter is used as “on-off” of activation of gene transcription to mediate the profile of some gene expression. Therefore, the damage of this “on-off”resulting from the related aberrant enzyme or mutated gene will lead to aberrant gene expression, consequently, cancer is formed.

The sites of histone modifications and their roles in oncogenesis which are described in this review are only the tip of the iceberg. Others, such as modifications of histone variant, molecular chaperones of histones and other new patterns in the histone landscape, and their roles in oncogenesis in this review have not been described. Fortunately, with the further increasing knowledge about the complex interaction among histone modifications, DNA methylation and non-coding RNAs, we have reason for believing that we are able to create a complex epigenetic atlas of oncogenesis to contribute to early diagnosis and treatment of cancer and judgment of prognosis.

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for publication May 10, 2015.

Tel: 86-20-36585077, E-mail: wenbin@

gzucm.edu.cn

△Supported by the Natural Science Foundation of China (81173257).