Deleted In Liver Cancer 1 Promoter Research Articles

Curcumin inhibits growth of human breast cancer cells through demethylation of DLC1 promoter.

The heterogeneity of breast cancer makes it a challenging solid tumor to diagnose and treat. A tumor suppressor Deleted in Liver Cancer 1 (DLC1) has been reported to be down-regulated or even silenced in several kinds of cancer including breast cancer. Curcumin has been reported to modulate the growth of tumor cells through regulation of multiple cell signaling pathways and modulate epigenetic changes by CpG demethylation of many tumor suppressor genes. This study was designed to investigate the effect of curcumin on the expression of Deleted in Liver Cancer 1 (DLC1) in human breast cancer cell line MDA-MB-361 and the underlying mechanism in vitro and in vivo. Curcumin induced DLC1 expression in a dose-dependent manner. In curcumin-treated cells, methylation of DLC1 promoter was reduced and active forms of RhoA and Cdc42 were also decreased. DLC1 expression was closely related to tumor cell growth, demonstrated by Ki67 staining. Curcumin inhibited DNA methyltransferase 1 expression through down-regulation of transcription factor Sp1. Consistent with the in vitro data, in vivo administration of curcumin inhibited the growth of implanted MDA-MB-361 cells and induced DLC1 expression in tumor tissue. In MDA-MB-361 cells, curcumin down-regulates the expression of Sp1 to inhibit the expression of DNA methyltransferase 1, thus subsequently reducing hypermethylation of DLC1 promoter to induce DLC1 expression.

Identification of DLC-1 expression and methylation status in patients with non-small-cell lung cancer

In order to determine whether the deleted in liver cancer-1 (DLC-1) gene is deregulated in non-small-cell lung carcinoma (NSCLC) and to assess the contribution of molecular alterations in DLC-1 to lung carcinogenesis, a total of 84 tissue specimens (30 NSCLC and 30 corresponding adjacent normal tissues; 5 benign tumor and 5 corresponding adjacent normal tissues; and 10 pulmonary bullae and 4 corresponding adjacent normal tissues), were obtained from 45 patients who underwent curative surgical resection. DLC-1 mRNA expression was evaluated by reverse transcription-quantitative polymerase chain reaction (PCR) and its protein level was assessed by western blot analysis. A significant downregulation of DCL-1 at the mRNA and protein levels was observed in NSCLC tissues when compared to benign lung tumors and normal lung tissues (P<0.001). To further determine whether the decreased expression of DLC-1 at the mRNA and protein levels is associated with the methylation of its promoter, methylation-specific PCR was performed following extraction of genomic DNA from the samples. DLC-1 promoter methylation was identified in 7 of the 30 (23.3%) NSCLC tissue samples, but not in the corresponding adjacent normal tissues from NSCLC patients or in lung tissues from non-NSCLC patients. Our data indicated that DLC-1 hypermethylation may play a crucial role in lung carcinogenesis and may be a target for the treatment of NSCLC.

EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers

Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.

Abstract 5356: EZH2-mediated H3K27me3 is involved in epigenetic repression of DLC1 (Deleted in liver cancer 1) in human cancers.

Abstract Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive Complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers, including hepatocellular carcinoma (HCC). In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by short hairpin RNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation in the expression pattern between EZH2 and DLC1 was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors. Citation Format: Leung Kuen Au, Carmen Chak-Lui Wong, Joyce Man-Fong Lee, Chun-Ming Wong, Irene Oi-Lin Ng. EZH2-mediated H3K27me3 is involved in epigenetic repression of DLC1 (Deleted in liver cancer 1) in human cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5356. doi:10.1158/1538-7445.AM2013-5356

Correlation of DLC1 gene methylation with oncogenic PIK3CA mutations in extramammary Paget's disease

AbstractExtramammary Paget's disease is a rare cutaneous malignant neoplasm. The genetic and epigenetic mechanisms underlying its pathology remain unknown. In this study, we investigated the expression levels, and mutation and methylation status of a common tumor suppressor gene, deleted in liver cancer 1 (DLC1), and an oncogene, PIK3CA, in tumor (n=132) and normal tissues (n=20) from unrelated patients. The presence of epigenetic and genetic lesions was then correlated to the patient pathology data to determine the potential role of these genes in extramammary Paget's disease etiology and progression. The DLC1 gene was found to be downregulated in 43 (33%) tumors, as compared with immunohistochemistry results from normal tissues. Methylation-sensitive, high-resolution melting analysis indicated that the DLC1 promoter was hypermethylated in 51 (39%) extramammary Paget's disease tumors. This hypermethylation was associated with significantly decreased DLC1 levels (P=0.011), and had a strong positive correlation with advanced age (P=0.002). PIK3CA mutations were detected by direct sequencing in 32 (24%) tumors, the majority of which were invasive. Furthermore, PIK3CA mutations significantly correlated with DLC1 hypermethylation. Thus, aberrant DLC1 methylation and PIK3CA mutations may have important roles in extramammary Paget's disease pathogenesis, and may represent potential molecular targets for therapy.

Aberrant DNA Methylation and Transcriptional Silencing of DLC-1 in Waldenstrom's Macroglobulinemia.

Abstract 2954Poster Board II-930The deleted in liver cancer-1 (DLC-1) gene encodes a Rho GTPase activating protein (RhoGAP) with potential tumor-suppressor activity. Hypermethylation in the DLC-1 promoter is an aberrant epigenetic modification associated with transcriptional silencing of DLC-1 in various types of human cancers. To explore the epigenetic alteration of DLC-1 in Waldenström's macroglobulinemia (WM), we investigated the methylation status of the DLC-1 promoter and its correlation with DLC-1 mRNA expression in cell lines and primary WM patient samples. Initial analysis using methylation-specific PCR (MSP) showed that DLC-1 promoter was completely methylated in WM-WSU and partially methylated in BCWM.1. Using quantitative RT-PCR, DLC-1 mRNA expression was detectable in BCWM.1, but not WM-WSU. These results suggested a correlation between the DLC-1 methylation status and mRNA expression. Similarly, among multiple myeloma (MM) cell lines, we showed that RPMI and U266 exhibited complete methylation, whereas INA6 showed partial methylation, and no methylation was detectable in MM1S and MM1R cells. Similar in WM cells, DLC-1 methylation status was highly correlated with the mRNA expression in these MM cell lines. Of 37 WM patient samples examined, 24 (65%) exhibited methylation in the DLC-1 promoter. In contrast, no methylation of DLC-1 was observed in 4 healthy volunteers. The methylation status was further confirmed using bisulfite DNA sequencing in a subset of WM patients. Quantitative RT-PCR analysis showed that DLC-1 mRNA expression was significantly lower in WM patients compared to healthy volunteers (p=0.001). Treatment with demethylation agents azacytidine or 5-aza-deoxycytidine resulted in significant reactivation of DLC-1 transcription in the WM cell lines WM-WSU and BCWM.1. In addition, a synergistic induction of DLC-1 transcription was observed in the presence of azacytidine and the HDAC inhibitor Vorinostat in BCWM.1 and primary WM patient cells. Moreover, functional studies showed that overexpression of DLC-1 induced cell growth arrest and apoptosis in BCWM.1. DLC-1 methylation status was also correlated with serum sCD27 levels in WM patients (p=0.004), which is secreted by WM cells, serves as a marker of disease burden and facilitates CD40L directed paracrine stimulation by mast cells. Taken together, these results suggest that the down-regulation of DLC-1 via aberrant DNA methylation plays a role in the pathogenesis of WM, and represents a novel therapeutic target in the treatment of WM. Disclosures:No relevant conflicts of interest to declare.

DLC1 tumor suppressor gene inhibits migration and invasion of multiple myeloma cells through RhoA GTPase pathway

DLC1 (deleted in liver cancer 1), a tumor suppressor gene that encodes a RhoGTPase-activating protein, is recurrently downregulated or silenced in various solid tumors and hematological malignancies because of epigenetic modifications or genomic deletion. Here, we identified DLC1 promoter hypermethylation in 43 out of 44 multiple myeloma (MM) cell lines, which resulted in downregulation or silencing of DLC1 in 41 samples. High frequency of tumor-specific methylation and attenuation or silencing of DLC1 expression could serve as an independent diagnostic marker for MM. Combined treatment with demethylating and acetylating agents significantly elevated the expression of DLC1 and suppressed MM cell proliferation. Two cell lines exhibiting complete promoter methylation and the absence of DLC1 expression were transduced by an adenoviral vector containing DLC1 cDNA. In both cell lines, the reexpression of DLC1 inhibited myeloma cell invasion and migration, reduced RhoA activity and resulted in the reorganization of actin cytoskeleton. These results provide the first evidence for the antiproliferative effect of DLC1 in a hematological cancer and implicate RhoA pathway in suppression of MM migration and invasion. Given the myeloma cells sensitivity to the reactivation of DLC1 function, the potential for molecular targeted therapy of DLC1-mediated pathways as well as epigenetic therapies hold prospects.

IDENTIFICATION OF THE DELETED IN LIVER CANCER 1 GENE, DLC1, AS A CANDIDATE MENINGIOMA TUMOR SUPPRESSOR

Meningiomas are the second most common primary tumors of the central nervous system. Meningiomas at the cranial base pose technical challenges and result in increased morbidity. To investigate the molecular mechanisms of meningioma formation, the expression profiles of 12 000 genes from meningiomas and dural specimens were compared. Ribonucleic acid from 6 meningiomas (World Health Organization Grade I) and 4 dural specimens was profiled using U95A GeneChips (Affymetrix, Inc., Santa Clara, CA). Expression profiles of the 2 groups were compared using dChip and Data Mining Tool software packages (Affymetrix, Inc.) to identify differentially expressed genes. Down-regulation of a differentially expressed tumor suppressor gene, deleted in liver cancer 1 (DLC1), was verified by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Function and methylation of DLC1 were assessed by ectopic expression in 5 primary cultures, demethylation assay using 5-aza-2'-deoxycytidine, and methylation-specific polymerase chain reaction in 4 meningioma samples. Gene expression profiling revealed up-regulation of 5 genes (fibroblast growth factor 9, gibbon leukemia virus receptor 2, cyclin D1, eukaryotic translation initiation factor 5A, and 28S ribosomal ribonucleic acid) and down-regulation of 35 genes, including DLC1, in meningiomas. The down-regulation of DLC1 in meningiomas was confirmed by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Transfection of DLC1 complementary deoxyribonucleic acid into primary cultures of 5 meningiomas resulted in decreased replication. Although demethylation decreased meningioma cell growth rates in vitro, methylation-specific polymerase chain reaction did not detect DLC1 promoter methylation. The results suggest that DLC1 may function as a tumor suppressor gene in meningiomas. Furthermore, DLC1 promoter methylation does not appear to be responsible for the decreased DLC1 expression in these tumors.

Expression of the deleted in liver cancer-1 gene is regulated by DNA methylation and is a target for therapy in Waldenström’s Macroglobulinemia

19505 Background: The deleted in liver cancer-1 (DLC-1) gene encodes a Rho GTPase activating protein (RhoGAP) with tumor-suppressor activity. Hypermethylation of the DLC-1 promoter leads to transcriptional silencing of DLC-1, and its re-expression blocks proliferation and/or induces apoptosis. We therefore sought to determine the methylation status of the DLC-1 promoter and its impact on DLC-1 transcription and tumor growth in Waldenström’s macroglobulinemia (WM). Materials and Methods: The methylation status of the DLC-1 promoter was analyzed in WM (BCWM.1 and WM-WSU), multiple myeloma (INA6, MM1S, MM1R, RPMI 8226 and U266), and bone marrow CD19+ cells from 19 WM patients and 4 healthy individuals using methylation-specific PCR. The methylation pattern was subsequently determined by cloning and bisulfite DNA sequencing. DLC-1 mRNA expression was measured by quantitative RT-PCR. Results: The DLC- 1 promoter was completely methylated in WM-WSU and partially methylated in BCWM.1 WM cells. In myeloma cells, complete methylation was observed in RPMI 8226 and U266 cells; partial methylation in INA6 cells; and was undetectable in MM.1S and MM.1R cells. In WM and myeloma cells with complete methylation, DLC-1 transcripts were not expressed, whereas transcript expression was decreased in WM and myeloma cells with partial DLC-1 methylation. Treatment with the DNA methyltransferase inhibitor decitabine resulted in up-regulation of DLC-1 in BCWM.1 (7.4 fold) and re-expression of DLC-1 in WM-WSU WM cells. Importantly, decitabine abrogated cell growth in BCWM.1 and WM-WSU cells, and induced apoptosis of WM-WSU WM cells. Of the 19 WM patients examined, 13 (68%) had methylation in the DLC-1 promoter region. No methylation was seen in healthy individuals, and DLC-1 transcript expression was significantly lower in WM patients relative to the normal controls (p = 0.014). Conclusions: Methylation of CpG islands in the DLC-1 promoter region is a frequent epigenetic modification and contributes to down-regulation of DLC-1 in WM. Treatment with decitabine leads to up-regulation of DLC-1 expression and inhibits growth and/or induces apoptosis in WM cells setting the framework for the study of demethylating agents in WM. No significant financial relationships to disclose.

Aberrant methylation of the 8p22 tumor suppressor gene DLC1 in renal cell carcinoma

Epigenetic mechanisms involving DNA methylation and chromatin remodeling are important in silencing tumor suppressor genes (TSG) in various malignancies, including renal cell carcinoma (RCC). DLC1 ( deleted in liver cancer 1)/ ARHGAP7 is a recently identified 8p22 candidate TSG. Frequent methylation of the DLC1 promoter with resultant gene silencing has been reported in several tumors, but not in RCC yet. We examined DLC1 promoter methylation in 34 primary RCCs and the corresponding non-malignant tissues, and the correlation of DLC1 methylation with the clinicopathological characteristics of RCC patients. Although DLC1 methylation and downregulation were only detected in one of seven RCC cell lines using methylation-specific PCR (MSP) and semi-quantitative reverse-transcription PCR, we found that the DLC1 promoter was methylated in 35% (12/34) of primary RCC tumors, which was further confirmed by direct sequencing of MSP products and high-resolution bisulfite genomic sequencing. In contrast, only one of the 34 (3%) non-malignant renal tissues had weak methylation. Aberrant DLC1 methylation appeared to be a relatively early event during renal tumorigenesis since 33% of the RCC tumors with pT1 (TNM staging) showed methylation, which is similar to other late stage tumors. Thus, our results demonstrated that DLC1 methylation occurs in a subset of RCC tumors and may play a role in renal carcinogenesis.

The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal, esophageal, and cervical carcinomas, and inhibits tumor cell colony formation

Identification of tumor suppressor genes (TSG) silenced by methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic tumor markers for early cancer detection. Both nasopharyngeal carcinoma (NPC) and esophageal carcinoma are major tumors in Southern China and Southeast Asia. Through expression subtraction of NPC, we identified Deleted in Liver Cancer 1 (DLC1)/ARHGAP7 (NM_006094)--an 8p22 TSG as a major downregulated gene. Although expressed in all normal tissues, DLC1 was silenced or downregulated in 11/12 (91%) NPC, 6/15 (40%) esophageal, 5/8 (63%) cervical and 3/9 (33%) breast carcinoma cell lines. No genetic deletion of DLC1 was detected in NPC although a hemizygous deletion at 8p22-11 was found by 1-Mb array-CGH in some cell lines. We then located the functional DLC1 promoter by 5'-RACE and promoter activity assays. This promoter was frequently methylated in all downregulated cell lines and in a large collection of primary tumors including 89% (64/72) NPC (endemic and sporadic types), 51% (48/94) esophageal, 87% (7/8) cervical and 36% (5/14) breast carcinomas, but seldom in paired surgical marginal tissues and not in any normal epithelial tissue. The transcriptional silencing of DLC1 could be reversed by 5-aza-2'-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B. Furthermore, ectopic expression of DLC1 in NPC and esophageal carcinoma cells strongly inhibited their colony formation. We thus found frequent epigenetic silencing of DLC1 in NPC, esophageal and cervical carcinomas, and a high correlation of methylation with its downregulation, suggesting a predominant role of epigenetic inactivation. DLC1 appears to be a major TSG implicated in the pathogenesis of these tumors, and should be further tested as a molecular biomarker in patients with these cancers.

Aberrant Methylation and Deacetylation of Deleted in Liver Cancer-1 Gene in Prostate Cancer: Potential Clinical Applications

The deleted in liver cancer-1 (DLC-1) gene that encodes a Rho GTPase-activating protein with tumor suppressor function is located on chromosome 8p21-22, a region frequently deleted in prostate carcinomas. This study was designed to determine whether DLC-1 is deregulated in prostate carcinomas and to assess the contribution of DLC-1 alterations to prostate carcinogenesis. Primary prostate carcinomas, prostate carcinoma cell lines, benign prostatic hyperplasias, and normal prostatic tissues were examined for detection of functional and structural alterations of the DLC-1 gene by real-time PCR, methylation-specific PCR, and Southern and Western blots. Down-regulation or loss of DCL-1 mRNA expression was detected in 10 of 27 (37%) prostate carcinomas, 3 of 5 (60%) prostate carcinoma cell lines, and 5 of 21 (24%) benign prostatic hyperplasias. DLC-1 promoter methylation was identified in 13 of 27 (48%) prostate carcinomas and 2 matching normal tissues and in 15 of 21 (71%) benign prostatic hyperplasias but was absent in 10 normal prostatic tissues from noncancerous individuals. Genomic deletions were found in only 3 prostate carcinomas and 1 benign prostatic hyperplasia. DLC-1 protein was not detected in 8 of 27 (30%) prostate carcinomas and 11 of 21 (52%) benign prostatic hyperplasias. Methylation of DLC-1 correlated with age in prostate carcinoma patients (P = 0.006) and with prostate-specific antigen blood levels in benign prostatic hyperplasia patients (P = 0.029). Treatment of the three prostate carcinoma cell lines (PC-3, LNCaP, and 22Rv1) expressing a low level of DLC-1 transcripts with inhibitors of DNA methyltransferase or histone deacetylase increased DLC-1 expression. These results show that the transcriptional silencing of DLC-1 by two epigenetic mechanisms is common and may be involved in the pathogenesis of prostate carcinomas and benign prostatic hyperplasias and could have potential clinical application in the early detection and gene therapy of prostate cancer.

High-frequency promoter hypermethylation of the deleted in liver cancer-1 gene in multiple myeloma

Background: Deleted in liver cancer-1 (DLC-1) is a tumour suppressor gene that is inactive in liver carcinogenesis. It encodes a ρ-guanosine triphosphatase-activating protein (ρ-GAP) and maps to one of the...