Qing-hui Zhang

Epigenetic inactivation of the tumor suppressor gene RIZ1 in hepatocellular carcinoma involves both DNA methylation and histone modifications

BACKGROUND & AIMS The retinoblastoma-interacting zinc finger gene RIZ1 is inactivated in many cancers, but the underlying mechanisms remain unknown. This study aimed to investigate the epigenetic mechanisms of RIZ1 inactivation by analyzing the relationship between DNA methylation and histone modifications during regulation of RIZ1 expression. METHODS Methylation-specific PCR, RT-PCR, and immunohistochemistry were performed to examine RIZ1 methylation and expression. Dynamic changes in histone H3 lysine 9 (H3K9) modifications and histone deacetylases (HDACs) associated with the promoter were analyzed by chromatin immunoprecipitation (ChIP). RESULTS RIZ1 methylation was detected in 66.7% (32/48) HCC tissues, 6.3% (3/48) corresponding non-cancerous tissues, and 66.7% (4/6) HCC cell lines. All 32 HCC tissues with promoter methylation showed complete loss of RIZ1 protein, whereas RIZ1 protein was present in all the corresponding non-cancerous tissues. Neither 5-aza-2-deoxycitidine (5-Aza-dC) nor Trichostatin A (TSA) reversed promoter methylation, but did restore RIZ1 mRNA and resulted in the downregulation of HDAC1 but not HDAC3. However, 5-Aza-dC+TSA induced a partial reversal of promoter methylation and a markedly synergistic reactivation of RIZ1. Moreover, both HDAC1 and HDAC3 were downregulated. The ChIP assays showed 5-Aza-dC and/or TSA also contributed to the dynamic conversion of trimethylated to acetylated H3K9 at the promoter. Furthermore, a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation. CONCLUSIONS Our results suggest that promoter methylation and H3K9 modifications work together to silence the RIZ1 gene in HCC. 5-Aza-dC can restore the expression of RIZ1, as reflected by its effects on histone modification levels. This finding indicates that cooperative effects between these epigenetic modifications exist.

Transcriptional silencing of the TMS1/ASC tumour suppressor gene by an epigenetic mechanism in hepatocellular carcinoma cells

DNA methylation and histone modifications have emerged as key mechanisms in transcriptional regulation. The target of methylation‐induced silencing 1 (TMS1) is a bipartite protein. Recent studies have indicated that methylation‐associated silencing of TMS1 occurs in many cancers. However, whether and how TMS1 is regulated by epigenetic mechanisms in cancers remains unknown. In this study we showed that methylation of the TMS1 promoter occurred in five of six hepatocellular carcinoma (HCC) cell lines. TMS1 expression was reduced in four HCC cell lines and correlated with methylation status. Furthermore, the TMS1 promoter was completely methylated and mRNA expression was undetectable. TMS1 expression could be restored by 5‐aza‐2′‐deoxycitidine (5‐Aza‐dC) (a DNA methyltransferase inhibitor) or trichostatin A (TSA) (a histone deacetylase inhibitor) alone and the promoter methylation was partially reversible. TSA was more efficient than 5‐Aza‐dC in inducing TMS1 expression, and the combination of 5‐Aza‐dC and TSA resulted in markedly synergistic reactivation of the gene and completely reversed promoter methylation. Interestingly, TMS1 promoter methylation‐associated gene silencing was accompanied by histone H3 Lysine 9 (H3K9) hypoacetylation and trimethylation. 5‐Aza‐dC and/or TSA also had some effect on conversion of methylated to acetylated H3K9 in restoring TMS1. This conversion was dynamic at the TMS1 promoter and a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation after 5‐Aza‐dC and/or TSA treatment. Our results thus suggest that epigenetic inactivation of TMS1 expression is regulated by promoter hypermethylation and H3K9 modifications in a coordinated way. Copyright

Lymphangiogenesis in gastric carcinoma correlates with prognosis

Lymphatic metastasis is an important way that gastric carcinomas can spread. However, little is known about the mechanisms of lymphangiogenesis and its clinical significance in gastric carcinomas. In the present study, lymphatic vessel density (LVD), VEGF‐C expression, and proliferative activity of lymphatic endothelium were determined in human gastric carcinomas and xenografts of gastric cancer cells in nude mice. The development of lymphangiogenesis and its correlation with patient prognosis were investigated. The results showed that lymphatic vessels were observed mainly in peripheral tumour tissue with significantly (p < 0.05) higher P‐LVD (peri‐tumoural‐LVD) than I‐LVD (intra‐tumoural‐LVD). The expression of VEGF‐C was heterogeneous within tumours, with a significantly higher expression (immunostaining score) at the margin than at the tumour centre (p < 0.05). A significant correlation was found between VEGF‐C expression at the margin (but not at the centre) and P‐LVD (r = 0.72, p < 0.01). High proliferative activity of lymphatic endothelium was also observed in the peripheral tissues, with a significant correlation between proliferative activity of lymphatic endothelium and VEGF‐C expression (p < 0.05). These data imply that the increased lymphatics may have been newly formed following stimulation by VEGF‐C. High VEGF‐C expression at the margin of gastric carcinomas could induce lymphangiogenesis in the peri‐tumoural stroma and contribute to the increased P‐LVD. The data from mice tumour xenografts also suggested that VEGF‐C produced from the transplanted gastric carcinoma cells could induce lymphangiogenesis around them. In patients, VEGF‐C expression at tumour margins was associated with nodal metastasis, lymphatic vessel invasion, poor recurrence‐free survival, and poor overall survival, and could serve as an independent predictor for patients with gastric carcinoma. Copyright

Clinicopathological significance of peritumoral lymphatic vessel density in gastric carcinoma

Lymphangiogenesis has recently been considered important for spread of malignant tumors. In the present study, lymphatic vessel density (LVD) including peritumoral LVD (P-LVD) and intratumoral LVD (I-LVD) was determined, respectively, by immunohistochemical staining with the antibody to LYVE-1 in 63 cases of early gastric carcinoma and 105 cases of advanced gastric carcinoma. The aim of the study is to investigate whether or not increased LVD could be a risk factor for nodal metastasis and survival. We conclude that increased P-LVD, but not I-LVD, could serve as an independent risk factor for nodal metastasis, recurrence and overall survival in gastric carcinoma.

Expression of ER, Ki-67 and CylinD1 in the Pre-cancerous Breast of Chinese Patients

To investigate the expression and association of ER, Ki-67 and cyclinD1 in usual ductal hyperplasia(UDH), atypical ductal hyperplasia (ADH) and ductal carcinoma in situ(DCIS) in the breast. The study included 56 cases of pre-cancerous lesions which were surgically excised at Qi Lu Hospital of Shangdong University. Immunohistochemistry was used to determine the expression of ER, Ki-67 and cyclinD1 and double-labelling immunofluorescence technique was used to observe the coexpression of ER and Ki-67. The expression and distribution of ER-positive cells were significantly different in UDH, ADH and DCIS. The ER-positive cells were much more in UDH than in normal TDLUs (terminal duct lobular units). The distribution of ER-positive cells interspersed amid ER-negative cells within UDH. However , the ER positive cells showed marked increases in ADH and low grade nuclear DCIS (P < 0.05), distributing in almost all constituent cells. The expression of ki-67 and cyclinD1 were significantly different between UDH and DCIS (P < 0.05) , and a positive correlation was found between expression of Ki-67 and morphological classification of pre-cancerous lesions (r = 0.3522, P < 0.05) as well as cyclinD1 (r = 0.3901, P < 0.05). Double-labelling immunofluorescence showed that there was no coexpression of ER and Ki-67 in normal breast tissue. The coexpression of the two markers was found in ADH and increased in DCIS. Overexpression of ER, Ki-67 and cyclinD1 significantly accompanies the transition of normal cells and UDH to ADH and DCIS. The coexpression of ER and ki-67 may present the early change in carcinogenesis of breast cancer.

Inhibitor of differentiation is overexpressed with progression of benign to malignant lesions and related with carcinoembryonic antigen–related cell adhesion molecule 1 distribution in mammary glands

The purpose of the study was to investigate the expression and association of inhibitor of differentiation (Id-1) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in benign, premalignant, and malignant lesions of human mammary glands. The study included 97 cases of benign, premalignant, and malignant lesions of human mammary glands including normal terminal duct lobular units, usual ductal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma that were surgically excised at the Second Hospital of Shangdong University. Immunohistochemistry was used to determine the expression of Id-1 and CEACAM1. The Id-1 expression was increased with the progression of benign to malignant transformation (P < .05) and positively related with CEACAM1 different expression patterns (r = 0.279, P < .01) in benign, premalignant, and malignant lesions: apical membranous staining in benign, and cytoplasmic and uniform membranous staining in premalignant and malignant lesions. A positive correlation was found between Id-1 expression and morphologic classification of benign to premalignant and malignant lesions (r = 0.641, P < .0001). The CEACAM1 expression patterns showed a significance between benign, premalignant, and malignant lesions (P < .05). The Id-1 expression is increased with the progression of benign to malignant transformation and promotes the CEACAM1 expression; the CEACAM1 expression patterns are changed by movement from apical membrane to bilateral membrane and cytoplasm. That the Id-1 overexpression promotes the transformation of CEACAM1 expression patterns may occur at the early stage in the breast carcinogenesis; and the Id-1 should be regarded as the transforming factor, which may regulate the transformation of CEACAM1 expression patterns.

Nuclear and cytoplasmic Id-1 expression patterns play different roles in angiogenesis and lymphangiogenesis in gastric carcinoma

The purpose of the study was to investigate the expression and impact of Id-1 (inhibitor of differentiation) on tumor progression, angiogenesis, and lymphangiogenesis in gastric adenocarcinoma. The study included 97 cases of gastric adenocarcinoma, which were surgically excised at the Second Hospital of Shandong University. Immunohistochemistry was used to detect the Id-1 expression, and dual-labeling immunohistochemistry was used to evaluate the microvessel density (MVD) and lymphatic vessel density (LVD). The Id-1 protein was mainly expressed with nuclear staining in well-differentiated carcinoma, but with cytoplasmic staining in moderately and poorly differentiated carcinoma, which showed a significant difference (P < .0001). Moreover, the expression patterns had different and crucial effects on angiogenesis and lymphangiogenesis. Nuclear staining of Id-1 inhibited angiogenesis, but cytoplasmic staining promoted angiogenesis (MVD, 110.57 ± 32.32 vs 141.45 ± 55.60) (P < .05). Consistent with their roles in angiogenesis, the nuclear and cytoplasmic expressions of Id-1 had similar effects on lymphangiogenesis: nuclear expression inhibited and cytoplasmic expression promoted lymphangiogenesis (LVD, 2.62 ± 1.03 vs 4.05 ± 2.04) (P < .05). Microvessel density and LVD showed no significant difference in low-and high-Id-1 expression groups (P > .05). Aberrant expression of Id-1 from nuclear to cytoplasm is accompanied with tumor malignant progression, which promotes angiogenesis and lymphangiogenesis; and Id-1 should be developed as a target for gastric carcinoma therapy.