Tingguo 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.

Immunohistochemical evaluation of solid pseudopapillary tumors of the pancreas: The expression pattern of CD99 is highly unique

The aim of this study was to investigate CD99 as a new marker to characterize solid pseudopapillary tumors (SPTs), and to determine a specific panel of markers to identify the disease. We analyzed the clinicopathological characteristics and immunohistochemical features of 37 patients with SPT. All 37 tumors displayed intracytoplasmic dot-like immunoreactivity of CD99 in contrast to membranous staining in all pancreatic endocrine tumors and most of acinar cell carcinomas, along with negative immunostaining in ductal carcinomas. In addition, we observed a loss of expression of E-cadherin in all SPTs as well as in some other pancreatic tumors, and aberrant nuclear expression of β-catenin in most SPTs. Our findings demonstrated for the first time that the pattern of CD99 expression was highly specific for distinguishing SPTs from other pancreatic tumors. CD99 combined with E-cadherin/β-catenin and CD10 can be used as a relatively specific expression profile of SPTs.

The different expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and possible roles in gastric carcinomas

The purpose of this study was to investigate the expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its effects on promoting angiogenesis in gastric adenocarcinomas. Paraffin wax sections of 222 patients with gastric adenocarcinomas having undergone surgery between 2001 and 2006 were classified into three histotypes: intestinal, diffuse, and mixed carcinomas following the Laurén classification. Immunohistochemistry (IHC) was used to study the distribution of CEACAM1, and double-labeling immunohistochemistry was used to observe the relationship between CEACAM1 expression and neovascularization in carcinoma areas. No CEACAM1 expression was found in normal non-metaplastic mucosa adjacent to the tumors; but in metaplastic mucosa, CEACAM1 was expressed on the apical surface. However, all of the collected gastric carcinomas expressed CEACAM1 with cytoplasmic or membranous staining. CEACAM1 was expressed mainly with a membranous pattern in the intestinal carcinomas, and with a cytoplasmic pattern in the diffuse carcinomas. There was a significant difference between the expression patterns and the histotypes (P<0.0001). CEACAM1 expression was classified as high (> or =66% positive cells) and low (<66% positive cells), and high CEACAM1 expression was associated with lymph nodes metastasis (P<0.05). High microvessel density (MVD) was observed more frequently in the tumors with membranous expression, and low MVD in the tumors with cytoplasmic staining (P<0.0001). The transformation of CEACAM1 distribution from membrane to cytoplasm is an important incident for the reverse effects on the tumorous angiogenesis, and high expression of CEACAM1 facilitates the metastasis of carcinoma cells to lymph nodes. Moreover, the different distribution of CEACAM1 in the intestinal and diffuse carcinomas indicates a different tumorigenic pathway.

CyclinD1, a prominent prognostic marker for endometrial diseases

PurposeAlteration of CyclinD1 was suggested to relate with development of endometrial carcinogenesis before, however CyclinD1 expression is not well defined in endometrial hyperplasia lesions. We checked the relationship between its expression and clinic-pathological variables of endometrial lesions to explore the possibility for CyclinD1 as a potential diagnostic and prognostic marker.MethodsCyclin D1 immunohistochemical analysis (IHC) was used to evaluate 201 fixed, paraffin-embedded endometrial samples which included simple hyperplasia (n = 27), atypical complex hyperplasia (ACH) (n = 41), endometrioid carcinoma (n = 103), endometrial serous carcinoma (ESC) (n = 21) and clear cell carcinoma (CCC) (n = 9). A breast cancer with known CyclinD1 expression was selected as a positive control in each immunohistochemistry run. We also performed follow-up study to estimate patients’ prognosis.ResultsCyclinD1 was significantly overexpressed in atypical complex hyperplasia (ACH), endometrioid carcinoma and clear cell carcinoma (CCC). The positive signaling of CyclinD1 was showed less than 40% in simple hyperplasia and endometrial serous carcinoma (ESC). The high expression of CyclinD1 was observed in metastasis carcinoma group more significantly than non-metastasis carcinoma group. Kaplan Meier analysis demonstrated that patients with high CyclinD1 expression had an obviously poor prognosis than patients without CyclinD1 staining (p < 0.05). Moreover, according to multivariate Cox regression analysis, CyclinD1 expression, as crucial as metastasis, was a risk marker for overall survival rate.ConclusionCyclinD1 exhibited a promising potential to predict the prognosis of patients with endometrial carcinoma. However, the statistical analysis demonstrated that CyclinD1 exhibited a poor ability to differentiate neoplastic lesions from non-neoplastic lesions; thus, the application of CyclinD1 only is not so credible for differentiation between benign and malignant lesions.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1871063048950173.

Detection of CHK1 and CCND1 gene copy number changes in breast cancer with dual-colour fluorescence in-situ hybridization

Mu K, Li L, Yang Q, Zhang T, Gao P, Meng B, Liu Z, Wang Y & Zhou G
(2011) Histopathology 58, 601–607
Detection of CHK1 and CCND1 gene copy number changes in breast cancer with dual‐colour fluorescence in‐situ hybridization

The evaluation of SOX9 expression and its relationship with carcinoembryonic antigen-related cell adhesion molecule 1 in gastric neoplastic and nonneoplastic lesions.

The aims of this study were to investigate the expression of SOX9 (sex determining region Y [SRY]-related high-mobility group box 9) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in benign, premalignant, and malignant gastric lesions and to explore the association between SOX9 and CEACAM1 in gastric carcinogenesis. SOX9 and CEACAM1 expression was detected in normal gastric mucosa, hyperplastic polyp, intestinal metaplasia, gastric intraepithelial neoplasia, and adenocarcinoma by immunohistochemistry. There was low expression of SOX9 and no CEACAM1 expression in normal gastric mucosa and hyperplastic polyps. Intestinal metaplasia began to express CEACAM1 and showed more membranous staining of CEACAM1 than normal mucosa and hyperplastic polyps (P = .000), but SOX9 expression had no significant difference, and the coexpression of SOX9 and CEACAM1 ascended; therefore, the difference was significant (P = .000). Gastric intraepithelial neoplasia showed more SOX9 expression, coexpression of SOX9, and CEACAM1 than in intestinal metaplasia (P = .014 and P = .026, respectively). Carcinoma showed more cytoplasmic CEACAM1 (P = .010), more SOX9 expression (P = .001), and more their coexpression (P = .023) than gastric intraepithelial neoplasia. As to the histologic classification, poorly differentiated carcinoma showed more cytoplasmic CEACAM1 than well and moderately differentiated carcinoma (P = .006 and P = .024, respectively). In the Laurén classification, diffuse carcinoma showed more cytoplasmic CEACAM1 than intestinal carcinoma (P = .0035), but the SOX9 expression and their coexpresison showed no difference (P = .065 and P = .074, respectively). With the elevation of SOX9 expression and the changing of CEACAM1 expression patterns, the coexpressions of SOX9 and CEACAM1 were highly elevated from benign proliferative lesions to malignant lesions. Moreover, the SOX9 expression and the coexpression with CEACAM1 were correlated positively (r = 0.310; P = .015). In addition, SOX9 expression was positively correlated with CEACAM1 expression patterns (r = 0.124; P = .032). In addition, CEACAM1 expression patterns and coexpression of SOX9 and CEACAM1 show significant difference between T1 and T2 and T3 and T4 (P = .021 and P = .011, respectively). Accordingly, compared with N0, N2 and N3 showed significant difference in SOX9 expression (P = .018), CEACAM1 expression patterns (P = .010), and their coexpression (P = .010). SOX9 expression significantly increased from nonneoplastic lesions to neoplastic lesions, and CEACAM1 expression patterns markedly changed; their coexpression also showed signally elevated suggesting that SOX9, as a transcriptional regulator, play important roles in the changing of CEACAM1 expression patterns, which might promote the tumor progression.

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.

Up-regulated expression of scavenger receptor class B type 1 (SR-B1) is associated with malignant behaviors and poor prognosis of breast cancer.

Scavenger receptor class B type 1 (SR-B1) is an integral membrane protein that is expressed in numerous cells and tissue types. The primary role of SR-B1 is to facilitate uptake of cholesteryl esters from high-density lipoproteins (HDL) in the liver. Altered SR-B1 expression contributes to human diseases. This study assessed association of SR-B1 expression in breast tissue specimens with breast cancer development and prognosis. Tissue specimens from 30 cases of adjacent normal breast tissues, ductal carcinoma in situ (DCIS) and invasive ductal breast cancer (IDCA) were subjected to Western blot analysis, and 135 cases of DCIS and IDCA were used for quantitative immunohistochemical analysis of SR-B1 expression. The data showed that SR-B1 was significantly overexpressed in IDCA tissues compared to normal breast and DCIS tissues. SR-B1 expression was associated with pre-menopausal status, tumor size, and worse overall survival of patients. The data from this ex vivo study suggests that up-regulated SR-B1 protein expression is associated with malignant behaviors of breast cancer and that SR-B1 is an independent predictor for poor survival in breast cancer patients.

CIP2A is overexpressed in human endometrioid adenocarcinoma and regulates cell proliferation,invasion and apoptosis

OBJECTIVE Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that stabilizes c-Myc and promotes cell proliferation and transformation. Here, we investigated the clinical significance and biological function of CIP2A in endometrial cancer. METHOD CIP2A expression was assessed in normal endometrium, endometrial hyperplasia, endometrial atypical hyperplasia, and endometrioid adenocarcinoma tissues using immunohistochemistry, western blot, and RT-PCR. The effect of reduced CIP2A expression was assessed by siRNA knockdown in Ishikawa and An3ca endometrial cell lines. The roles of CIP2A in proliferation, apoptosis, and the cell cycle were assessed using CCK-8 assays, colony formation assays, and flow cytometry, respectively. RESULTS Our results show that CIP2A expression was higher in endometrioid adenocarcinoma tissues and cell lines. Furthermore, CIP2A siRNA significantly reduced the proliferation rate and invasion of Ishikawa and An3ca cells, and induced a significant level of apoptosis in Ishikawa cells. Moreover, CIP2A depletion resulted in reduced c-Myc and cyclin D1 protein levels, and increased caspase-3 expression. CONCLUSIONS CIP2A is overexpressed in endometrioid adenocarcinoma and CIP2A promotes the malignant growth and invasion,decrease apoptosis in entometrioid adenocarcinoma cell lines. These results validate that CIP2A plays an important role in the carcinogenesis of endometrioid adenocarcinoma and establishes CIP2A as a clinically relevant oncoprotein and may presents a promising therapeutic target for cancer treatment.

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.