Gengyin Zhou

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

Cancerous inhibitor of protein phosphatase 2A is overexpressed in cervical cancer and upregulated by human papillomavirus 16 E7 oncoprotein

OBJECTIVES Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein stabilizing c-Myc and promoting cell proliferation and transformation. Here we investigated the role of CIP2A in cervical cancer in vivo and in vitro. METHODS CIP2A expression was assessed in normal cervical, cervical intraepithelial neoplasia (CIN) I to III and cervical cancer tissues by immunohistochemistry and RT-PCR. Cell growth was explored by cell proliferation assay, colony formation assay and anchorage-independent growth in soft agar after inhibition of CIP2A by siRNA in HeLa, SiHa and Caski cells. Crosstalk of CIP2A and HPV16 E7 was investigated by immunohistochemistry in cervical cancer tissues and by real-time PCR and western blot analysis after HPV16 E7 inhibition by siRNA in SiHa cells. RESULTS CIP2A was transcribed in 73.3% of cervical cancer tissues (n=15) but not in normal cervical tissues (n=8). CIP2A protein was detected in 52.8% of cervical cancer (n=72) and 12.5% of CIN III tissues (n=24) but not in normal (n=15), CIN I (n=21) or CIN II samples (n=25). CIP2A protein level was positively associated with HPV16 E7 level in cervical cancer tissues. CIP2A expression was markedly reduced after E7 depletion. Moreover, CIP2A depletion reduced c-Myc protein level and impaired proliferation and growth of cervical cancer cells. CONCLUSIONS CIP2A is overexpressed in cervical cancer and promotes the malignant growth of cervical cancer cells. Its expression is upregulated by HPV16 E7. Therefore, CIP2A plays an important role in carcinogenesis of cervical cancer and shows promise for the diagnosis and treatment of cervical cancer.

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.

Targeting glucosylceramide synthase downregulates expression of the multidrug resistance gene MDR1 and sensitizes breast carcinoma cells to anticancer drugs

Drug resistance in breast cancer remains a major cause for the failure of chemotherapy. Glucosylceramide synthase (GCS) plays an important role in multidrug resistance (MDR) in breast cancer. P-glycoprotein (P-gp) also confers a cross-resistance of many unrelated drugs. In this study, we studied the MDR effect and potential mechanisms of breast cancer after constructing permanent breast cancer cell lines with GCS knockout by using recombinant vectors targeting GCS (pSUPER-GCSshRNAs). The GCSshRNA stably transfected cells were successfully established and significant lower levels of GCS mRNA and protein expression were confirmed. In in vitro experiments, the GCSshRNA stably transfected cells showed a significantly reduced level of MDR1 and P-gp expression and decreased drug efflux ability. Reduced level of GCS expression conveyed a significant reversal of drug resistance by MTT assay and increased caspase-3 activity. In in vivo experiments by using nude mice with xenograft tumors, a significant inhibition of tumor growth was observed after comparing with the control group. Furthermore, enhanced response of chemotherapy was acquired by reduced expression of GCS as well as MDR1 in vivo. In conclusion, GCSshRNA could efficiently suppress GCS and MDR1 expression in vitro and in vivo and these findings may be used as one of the methods to reverse MDR in breast cancer.

Doxorubicin Influences the Expression of Glucosylceramide Synthase in Invasive Ductal Breast Cancer

Introduction Glucosylceramide synthase (GCS) is one enzyme that provides a major route for ceramide clearance. Recent evidence has indicated an important role for GCS in multidrug resistance (MDR) tumors. Doxorubicin (DOX)can modulate the expression of GCS in leukemia and ovary cell lines. However, few studies have investigated their relationship in breast cancer; Methods We collected 84 excision biopsies from patients with invasive ductal breast cancer of whom 33 patients had undergone preoperative chemotherapy. Immunohistochemistry was used to analyze the expression of GCS protein and significantly showed that the expression of GCS was higher in the samples from patients treated with preoperative chemotherapy(p = 0.018). In order to investigate the underlying mechanism, breast cancer cell lines were cultured with different concentrations of DOX, and mRNA and protein levels of GCS were then detected; Results DOX significantly upregulated the expression of GCS at both the mRNA and protein level in ERα-positive MCF-7 cells.We then block down the Sp1 site of GCS promoter, which inhibited the DOX-mediated increase in GCS expression; and after Erα was inhibited in MCF-7 cells, the up-regulation of GCS by DOX also been inhibited. Conclusions In conclusion, our data demonstrated the novel finding that DOX could modulate the expression of GCS through the Sp1 site of GCS promoter in ERα-positive breast cancer cells.

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

MDR1 (multidrug resistence 1) can regulate GCS (glucosylceramide synthase) in breast cancer cells

Besides MDR1/P‐glycoprotein (MDR1/P‐gp), glucosylceramide synthase (GCS), an enzyme, which transfers UDP–glucose to ceramide to form glucosylceramide was also related with multidrug resistance (MDR) in breast cancer. Although many research showed that GCS could affect mdr1 in cancer cells, nobody knows that whether mdr1 can affect GCS in breast cancer. Our study aims to verify that.

Lysosomal-Associated Protein Transmembrane 4 Beta-35 Overexpression Is a Novel Independent Prognostic Marker for Gastric Carcinoma

Objective The purpose of this work was to analyze the relationships between the expression status of Lysosomal-associated protein transmembrane-4 beta 35 (LAPTM4B-35) in cancerous tissues and clinicopathological characteristics and prognosis of the patients with gastric carcinoma (GC). Methods The GC samples from 157 patients in a discovery cohort and 148 patients in a testing cohort with follow-up data were used to validate the feasibility of expression of LAPTM4B-35 protein in predicting GC prognosis. Immunohistochemical staining was used to determine the expression of LAPTM4B-35 protein in precancerous gastric lesions and gastric carcinomas. The correlation between the expression of LAPTM4B-35 and clinicopathologic characteristics of patients with gastric carcinoma was analyzed using chi-square test. Univariate and multivariate analyses were performed to determine the association between LAPTM4B-35 expression and prognosis. Results LAPTM4B-35 expression was increased steadily in sequential stages of precancerous gastric lesions. Positive LAPTM4B-35 expression was more frequently detected in patients with distant metastasis (P = 0.023) and III+IV TNM stages (P = 0.042) in the discovery cohort. Kaplan-Meier survival curves and univariate analysis showed that expression of LAPTM4B-35 had a significant impact on overall survival of patients with gastric carcinoma in discovery cohort (P<0.001) and testing cohort (P = 0.001). LAPTM4B-35 expression was an independent prognostic indicator for the overall survival of patients with gastric carcinoma in both cohorts. Conclusions The present research demonstrated that LAPTM4B-35 over-expression was an independent factor in gastric carcinoma prognosis. LAPTM4B gene may be a useful target of interventions slowing the progression of precancerous gastric lesions and a new therapy method to improve the prognosis of gastric carcinoma.