Deleted In Liver Cancer 1 Protein Research Articles

Proteasomal turnover of the RhoGAP tumor suppressor DLC1 is regulated by HECTD1 and USP7

The Rho GTPase activating protein Deleted in Liver Cancer 1 (DLC1) is frequently downregulated through genetic and epigenetic mechanisms in various malignancies, leading to aberrant Rho GTPase signaling and thus facilitating cancer progression. Here we show that in breast cancer cells, dysregulation of DLC1 expression occurs at the protein level through rapid degradation via the ubiquitin–proteasome system. Using mass spectrometry, we identify two novel DLC1 interaction partners, the ubiquitin-ligase HECTD1 and the deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7). While DLC1 protein expression was rapidly downregulated upon pharmacological inhibition of USP7, siRNA-mediated knockdown of HECTD1 increased DLC1 protein levels and impaired its degradation. Immunofluorescence microscopy analyses revealed that the modulation of HECTD1 levels and USP7 activity altered DLC1 abundance at focal adhesions, its primary site of action. Thus, we propose opposing regulatory mechanisms of DLC1 protein homeostasis by USP7 and HECTD1, which could open up strategies to counteract downregulation and restore DLC1 expression in cancer.

Effects of DLC1 Deficiency on Endothelial Cell Contact Growth Inhibition and Angiosarcoma Progression.

Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined. We investigated the effects of shRNA-induced DLC1 depletion in endothelial cells. Cell growth was measured by 3H thymidine incorporation, IncuCyte imaging, and population doublings; cell death by cell cycle analysis; gene expression by Affimetrix arrays and quantitative polymerase chain reaction; NF-κB activity by reporter assays; and protein levels by immunoblotting and immunofluorescence staining. We tested Tanespimycin/17-AAG and Fasudil treatment in groups of nine to 10 mice bearing ISOS-1 angiosarcoma. All statistical tests were two-sided. We discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in endothelial cells, promoting statistically significant (P < .001) cell death when cells are confluent (mean [SD] % viability: control DLC1 = 15.6 [19.3]; shDLC1 = 73.4 [13.1]). This prosurvival phenotype of DLC1-depleted confluent endothelial cells is attributable to a statistically significant and sustained increase of NF-κB activity (day 5, P = .001; day 8, P = .03) associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistently, we found that DLC1 is statistically significantly reduced (P < .001 in 5 of 6) and TNFAIP3/A20 is statistically significantly increased (P < .001 in 2 of 3 and P = 0.02 in 1 of 3) in human angiosarcoma compared with normal adjacent endothelium. Treatment with the NF-κB inhibitor Tanespimycin/17-AAG statistically significantly reduced angiosarcoma tumor growth in mice (treatment tumor weight vs control, 0.50 [0.19] g vs 0.91 [0.21] g, P = .001 experiment 1; 0.66 [0.26] g vs 1.10 [0.31] g, P = .01 experiment 2). These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma and support NF-κB targeting for the treatment of angiosarcoma where DLC1 is lost.

MiR-429 promotes the proliferation of non-small cell lung cancer cells via targeting DLC-1.

The microRNA (miR)-200 family has been demonstrated to be associated with the tumorigenesis and progression of multiple types of human cancer, including non-small cell lung cancer (NSCLC). As a member of the miR-200 family, miR-429 was recently identified to have an oncogenic role in NSCLC. However, the role of miR-429 in NSCLC growth as well as the underlying mechanism remains to be fully elucidated. In the present study, NSCLC cell line H1229 was transfected with miR-429 mimic or inhibitor, respectively. It was observed that overexpression of miR-429 led to a significant increase in NSCLC cell proliferation, while knockdown of miR-429 suppressed the proliferation of H1229 cells. Bioinformatic prediction suggested that deleted in liver cancer 1 (DLC-1), a tumor suppressor in NSCLC, was a putative target gene of miR-429. Therefore, a luciferase reporter assay was performed and confirmed that miR-429 was able to bind the 3'-untranslated region of DLC-1 mRNA in H1229 cells. Furthermore, overexpression of miR-429 inhibited the protein expression of DLC-1, while knockdown of miR-429 promoted the protein expression of DLC-1 in NSCLC H1229 cells. In addition, overexpression of DLC-1 not only inhibited H1229 cell proliferation, but also additionally reversed the promoting effect of miR-429 overexpression on H1229 cell proliferation. Based on these findings, the present study suggests that miR-429 may have an oncogenic role in the regulation of cell proliferation via direct inhibition of DLC-1 protein expression in NSCLC cells. Therefore, miR-429 may present a putative therapeutic target for the treatment of NSCLC growth.

Flavone inhibits migration through DLC1/RhoA pathway by decreasing ROS generation in breast cancer cells

Tumor suppressor protein deleted in liver cancer 1 (DLC1) is a RhoGTPase-activating protein (RhoGAP) and inhibits cancer cell migration by inactivating downstream target protein RhoA. A few studies have reported the regulations of reactive oxygen species (ROS) on RhoGAP. In this study, we investigated flavone (the core structure of flavonoids)-induced regulation on ROS generation and DLC1/RhoA pathway in MCF-7 and MDA-MB-231 breast cancer cells and explored whether flavone-induced upregulation of DLC1 is mediated by ROS. Our results showed that flavone decreased ROS production and inhibited cell migration through DLC1/RhoA pathway. To further investigate the role of ROS in flavone-induced regulation on DLC1/RhoA pathway, hydrogen peroxide was added to restore the ROS levels. Flavone-induced upregulation of DLC1 expression, downregulation of RhoA activity, and inhibition of cell migration were all restrained by hydrogen peroxide. We also found that flavone increased DLC1 stability by inhibiting DLC1 protein degradation in breast cancer cells. In summary, our study demonstrated that flavone inhibited cell migration through DLC1/RhoA pathway by decreasing ROS generation and suppressed DLC1 degradation in MCF-7 and MDA-MB-231 breast cancer cells.

Tumor suppressor DLC-1 induces apoptosis and inhibits the growth and invasion of colon cancer cells through the Wnt/β-catenin signaling pathway

The aim of the present study was to investigate the biological role and molecular mechanism of the deleted in liver cancer-1 (DLC-1) gene in human colon cancer growth and invasion. Recombinant lentiviral vectors encoding the DLC-1 gene were constructed for transfection into the human colon cancer cell line SW480. Real-time quantitative polymerase chain reaction (real-time qPCR) and western blot analysis were employed to evaluate the expression of DLC-1, β-catenin, GSK-3β and c-myc in DLC-1-transfected cells. Moreover, cell proliferation assay, cell colony formation assay, cell cycle analysis, apoptosis analysis and cell migration and invasion assays were performed in order to elucidate the role of DLC-1 in colorectal cancer development and progression. Both real-time qPCR and western blot analyses showed that the DLC-1 gene and protein were overexpressed in the DLC-1-transfected SW480 cells. In addition, the expression of β-catenin and GSK-3β was upregulated and the expression of the c-myc gene was downregulated in the DLC-1-transfected SW480 cells. Furthermore, DLC-1 overexpression inhibited cell proliferation, colony formation, migration and invasion, and induced cell cycle arrest at the G1 phase with subsequent apoptosis. DLC-1 inhibits cell growth and invasion in human colon cancer, functioning as a tumor-suppressor gene, possibly through the regulation of the Wnt/β-catenin signaling pathway.

Protein expression and promoter methylation of the deleted in liver cancer-1 gene in papillary thyroid carcinoma

Objective To investigate the protein expression and promoter methylation of deleted in liver cancer-1 (DLC1) gene in papillary thyroid carcinoma (PTC) and pericarcinomatous thyroid tissue (PCTT),and its clinical significance.Methods Methylation-specific polymerase chain reaction (MSP),immunohistochemistry (IHC) and Western blotting were empolyed to detect the methylation status of DLC1 gene promoter and its protein expression in 40 PTC and PCTT samples.The location of DLC1 protein expression was examined by IHC,and the differential expression of DLC1 protein by Western blotting,respectively.Results By immunohistochemical technique,DLCl was expressed in the cytoplasm of thyrocytes.The positive expression rate of DLC1 protein in PCTT and PTC was 90％ (36/40) and 47.5％ ％(19/40),respectively (x2 =26.36,P ＜ 0.05).The absorbance (A) value of DLC1 protein confirmed was 0.18 ±0.12 in PTC versus 0.33 ±0.14 in PCTT (P ＜0.05).In PTC,the positive expression rate of DLC1 protein was correlated with the tumor size,lymph node metastasis and the TNM stage (x2 =18.050,15.132,7.020,P ＜ 0.05).The methylation rate of DLC 1 gene in PCTT and PTC was 12.5 ％ (5/40) and 40.0％ (16/40) respectively (x2 =7.813,P ＜0.05).The methylation of DLC1 gene in PTC was correlated with tumor size (x2 =12.857,P ＜0.05).Futhermore,there was distinct correlation between methylation of DLC1 gene promoter and its protein expression (r =0.526,P ＜ 0.05).Conclusion Methylation of promoter may be one of the important inactivating factors of DLC1 gene,and it plays an important role in the carcinogenesis and progression of PTC. Key words: Papillary thyroid carcinoma; Pericarcinomatous thyreoid tissue; Deleted in liver cancer-1 gene ; Methylation

Role of microRNA181-b and its target gene oncogene deleted in liver cancer-1 in hepatocellular carcinoma pathogenesis

Objective To investigate the regulatory effect ofmicroRNA-181b (miR-181b) on deleted in liver cancer-1 (DLC-1) and its role in hepatocarcinogenesis.Methods miR-181b and DLC-1 mRNA expression levels in normal tissue,paraneoplastic tissue,liver cancer tissue and liver cancer cells were detected by using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).DLC-1 protein expression was detected by using Western blotting.miR-181b inhibitor was transfected into SMMC7721 cells and the expression changes of DLC-1 mRNA and protein were examined by using FQ-PCR and Western blotting respectively.Results miR-181b was up-regulated in hepatocellular carcinoma and adjacent tissues as compared to normal liver tissues and the expression of miR-181 b was also increased in SMMC7721 cells as compared to fetal liver cells LO2,whereas the expression levels of DLC-1 mRNA and protein were decreased in paraneoplastic and HCC tissues compared with normal liver tissues.The expression of miR-181b and DLC-1 was negatively correlated.No obvious difference was found in the DLC-1 mRNA expression after miR-181b inhibitor transfection.Conclusion The DLC-1 abnormal low-expression may play a dynamic role in hepatocarcinogenesis due to the miR-181 b up-regulation. Key words: Carcinoma, hepatocellular; MicroRNA-181b; Deleted in liver cancer-1

Expression of deleted in liver cancer 1 and plasminogen activator inhibitor 1 protein in ovarian carcinoma and their clinical significance

BackgroundThe deleted in liver cancer 1 (DLC1) and plasminogen activator inhibitor 1 (PAI-1) are known to be closely associated with tumor growth and metastasis in several kinds of human tumors. The aim of this study was to investigate the expression of DLC1 and PAI-1 in ovarian carcinoma, and evaluate their relations with the prognosis of ovarian carcinoma.MethodsImmunohistochemical staining and Western blot were used to examine the expressions of DLC1 and PAI-1 protein in 25 specimens normal ovarian tissues, 52 specimens of serous cystadenocarcinoma tissues and 23 specimens of mucinous cystadenocarcinoma tissues. Chi-square test, Logistic regression and Partial Correlate analysis were performed to evaluate the association between DLC1 and PAI-1 with clinicopathological characteristics. Overall survival was estimated by Kaplan-Meier curves and multivariate Cox analysis. The relationships between DLC1 and PAI-1 protein expression were analyzed by Pearson’s correlation coefficient.ResultsThe expression of DLC1 protein in ovarian carcinoma tissues was significantly lower than that in normal ovarian tissues, but it was converse for PAI-1. In ovarian carcinoma, the expression of DLC1 was significantly associated with advanced FIGO stage, ascites and positive lymph node metastasis, whereas PAI-1 protein was closely related with advanced FIGO stage, poor histological differentiation and lymph node metastasis. The expression of DLC1 was negatively correlated with PAI-1 in ovarian carcinoma. Ovarian cancer patients with negative expression of DLC1 and positive expression of PAI-1 had the worst overall survival time compared to other patients.ConclusionsThe expression of DLC1 and PAI-1 were closely related with the metastasis and invasion of ovarian carcinoma, only the combination of DLC1 and PAI-1 could serve as an independent prognostic factor of ovarian carcinoma.

Aberrant deleted in liver cancer‐1 expression is associated with tumor metastasis and poor prognosis in urothelial carcinoma

This study explored the potential role of deleted in liver cancer-1 (DLC-1) as a prognostic indicator of cancer metastasis and survival in urothelial carcinoma (UC). Tissue microarrays were constructed from paraffin-embedded specimens from 88 UC patients, and immunohistochemical staining was performed to investigate the association of DLC-1 with clinicopathologic characteristics and clinical outcome. The DLC-1 expression showed a significant positive correlation with tumor location (p = 0.041) and a significant negative correlation with advanced histological grade (p = 0.013). In tumors with low DLC-1 expression, Bcl-2 positivity was observed in 24.4% of cases. The DLC-1 expression had significant negative associations with Bcl-2 expression (p = 0.032) and with highly metastatic UC (p = 0.032). Kaplan-Meier analysis showed that DLC-1 protein expression was negatively associated with both overall survival (OS) (p = 0.035) and with distant metastasis-free survival (DMFS) (p = 0.041), but not with disease-free survival. Multivariate analyses indicated that tumor size was the significant independent predictors of OS (p = 0.048); however, only DLC-1 expression was a significant independent predictor of DMFS (p = 0.019). In conclusion, reduced DLC-1 protein expression may be an important factor in tumor progression and a useful prognostic molecular marker in UC.

Nuclear-Targeted Deleted in Liver Cancer 1 (DLC1) Is Less Efficient in Exerting Its Tumor Suppressive Activity Both In Vitro and In Vivo

BackgroundDeleted in liver cancer 1 (DLC1) serves as an important RhoGTPase activating protein (RhoGAP) protein that terminates active RhoA signaling in human cancers. Increasing evidence has demonstrated that the tumor suppressive activity of DLC1 depends not only on RhoGAP activity, but also relies on proper focal adhesion localization through its interaction with tensin family proteins. Recently, there are reports showing that DLC1 can also be found in the nucleus; however, the existence and the relative tumor suppressive activity of nuclear DLC1 have never been clearly addressed.Methodology and Principal FindingsWe herein provide new evidence that DLC1 protein, which predominantly associated with focal adhesions and localized in cytosol, dynamically shuttled between cytoplasm and nucleus. Treatment of cells with nuclear export blocker, Leptomycin B (LMB), retained DLC1 in the nucleus. To understand the nuclear entry of DLC1, we identified amino acids 600–700 of DLC1 as a novel region that is important for its nuclear localization. The tumor suppressive activity of nuclear DLC1 was directly assessed by employing a nuclear localization signal (NLS) fusion variant of DLC1 (NLS-DLC1) with preferential nuclear localization. In SMMC-7721 HCC cells, expression of NLS-DLC1 failed to suppress colony formation and actin stress fiber formation in vitro. The abrogated tumor suppressive activity of nuclear DLC1 was demonstrated for the first time in vivo by subcutaneously injecting p53−/− RasV12 hepatoblasts with stable NLS-DLC1 expression in nude mice. The injected hepatoblasts with NLS-DLC1 expression effectively formed tumors when compared with the non-nuclear targeted DLC1.Conclusions/SignificanceOur study identified a novel region responsible for the nuclear entry of DLC1 and demonstrated the functional difference of DLC1 in different cellular compartments both in vitro and in vivo.

Abstract 4017: DLC1 and α-catenin protein interaction enhances DLC1 antioncogenic activity by stabilizing adherens junctions and suppressing NFκB signaling

Abstract Adherens junctions (AJs), which are cell-cell adhesion complexes closely associated with actin cytoskeleton, play an important role in maintaining epithelial tissue homeostasis. Both malignant transformation, and tumor progression to metastasis are often associated with major changes in cytoskeletal organization, the cell-cell adhesion, and aberrant adhesion-mediated signaling. The tumor suppressor gene DLC1 (Deleted in Liver Cancer 1) encodes a RhoGTPase activating protein (GAP) that acts as a negative regulator of the Rho family of small GTPases, which are implicated in cytoskeleton organization and regulation of AJs. A yeast two-hybrid screening identified α-catenin, a component of AJs, as a potential DLC1's binding partner. We relied on this finding and investigated the role of DLC1 in the regulation of AJs and in adhesion-mediated signaling. Using human embryonic kidney (HEK 293) cells we demonstrated that DLC1 interacted with α-catenin and associated with E-cadherin and β-catenin, both constitutive AJs proteins. Co-immunoprecipitation using various deletion constructs of DLC1 and of α-catenin established that the N-terminal (340-435 aa) of DLC1 interacted with the N-terminal (39-161 aa) of α-catenin. Immuno-fluorescence staining and western blot of cellular membrane and cytoplasmic fractions, demonstrated that binding of DLC1 to α-catenin recruited α-catenin to the plasma membrane and required DLC1 GAP activity. In contrast, the DLC1 GAP mutant (R718E) failed to produce the same effect. Fluorescence Recovery After Photobleaching (FRAP) analysis demonstrated that DLC1-α-catenin interaction increased the GFP-E-cadherin mobility at cell-cell junctions, whereas phalloidin staining showed intact actin cable formation around the cell periphery, resulting in the stabilization of AJs. The DLC1-α-catenin interaction is instrumental for a maximal oncosuppressive effects of DLC1 (i.e., by inhibiting proliferation and abolishing colony formation of prostate carcinoma cells); DLC1 mutant defective for α-catenin binding failed to reproduce such an effect. Loss of α-catenin is usually accompanied by an increase in the activation of NFkB, which is associated with increased cell proliferation and resistance to apoptosis. We demonstrated that, by immobilizing NFkB to the AJ complex, DLC1-α-catenin interaction effectively inhibited NFkB phosphorylation and, therefore, its activation. Together, these results unravel a new mechanism through which DLC1 exerts its oncosuppressive function by stabilizing AJs and suppressing NFkB signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4017. doi:10.1158/1538-7445.AM2011-4017

Abstract 5007: Nuclear targeted Deleted in liver cancer 1 exhibited reduced tumor suppressive function both in vitro and in vivo

Abstract The tumor suppressor Deleted in liver cancer 1 (DLC1) protects cell from transformation by catalyzing the GTP hydrolysis of active RhoA. Besides the RhoGAP activity, accumulating evidences have supported that localization at focal adhesions and interaction with tensin protein are key factors guiding the tumor suppressive activity of DLC1. In this study, we provided new evidence that DLC1 was a dynamic protein which shuttles between cytoplasm and nucleus instead of statically staying in the cytoplasm. Subcellular fractionation assay showed that DLC1 protein can be detected in cytoplasmic and nuclear fractions. Treatment of Leptomycin B (LMB), a nuclear export blocker, could lead to the nuclear retention of exogenous and endogenous DLC1 in different human cancer cell lines. Detail examination of relevant DLC1 mutants had shown that the center region of DLC1 was necessary and sufficient for its nuclear localization. We transiently expressed a NLS fusion DLC1 (NLS-DLC1) with preferential nuclear localization in SMMC HCC cells and found that NLS-DLC1 was less potent in suppressing colony formation and actin stress fiber formation. To study the tumor suppressive function of nuclear DLC1 both in vitro and in vivo, we employed retroviral transduction to stably express NLS-DLC1 in a p53−/− RasV12 hepatoblast model. We found that NLS-DLC1 expressing cells proliferated faster in vitro and exhibited increased tumorigenicity upon nude mice injection when compared with the non-nuclear targeted DLC1. Although the function of nuclear DLC1 remains to be answered, our results clearly demonstrated that nuclear localization of DLC1 may serve as a potential mechanism in negatively regulating its tumor suppressive activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5007.

Abstract B33: A novel isoform of the major 8p22 tumor suppressor Deleted in Liver Cancer 1 (DLC1) gene with growth suppression function is frequently silenced epigenetically in multiple tumors

Abstract Aberrant DNA methylation of Tumor Suppressor Gene (TSG) promoter regions, in addition to being a hallmark of cellular transformation, could provide potential tumor markers for early detection and monitoring of treatment effect. In a previous study to identify candidate TSGs relevant in Nasopharyngeal Carcinoma (NPC), our group identified the Deleted in Liver Cancer 1 (DLC1) gene to be epigenetically silenced not just in NPC, but in esophageal, breast and cervical carcinomas also. In this study, we report the cloning and characterization of a novel isoform of the human DLC1 gene with growth suppression function transcribing from a functional alternative promoter and silenced epigenetically in multiple tumors. Designated as DLC1 isoform 4 (DLC1-i4), this novel isoform encodes a protein of 1125 amino acids with a distinct N-terminus as compared to the other known isoforms. DLC1-i4 was expressed ubiquitously in normal tissues and immortalized normal epithelial cells, but significantly down-regulated in multiple tumor cell lines. Promoter DNA methylation was further found in 6/16 (38%) esophageal, 3/4 (75%) cervical, 2/2 (100%) colorectal, 4/4 (100%) NPC cell lines, and in 37/39 (95%) and 12/31 (39%) of primary NPC and esophageal tumors respectively. In contrast, no DNA methylation was found in normal cell lines and tissues. Pharmacological treatment with 5-aza-2′-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B could demethylate the DLC1-i4 promoter and activate transcription, indicating a direct epigenetic mechanism. Ectopic expression of DLC1-i4 had a strong inhibitory effect on the growth and colony formation ability of silenced tumor cell lines. As a whole, our results suggest that transcription of multiple DLC1 isoforms driven off alternative promoters could modulate the activity of the candidate tumor suppressor DLC1 protein in the pathogenesis of these tumors, and should be further tested as a tumor specific molecular biomarker. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B33

DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma

Deletions on chromosome 8p are common in human tumors, suggesting that one or more tumor suppressor genes reside in this region. Deleted in Liver Cancer 1 (DLC1) encodes a Rho-GTPase activating protein and is a candidate 8p tumor suppressor. We show that DLC1 knockdown cooperates with Myc to promote hepatocellular carcinoma in mice, and that reintroduction of wild-type DLC1 into hepatoma cells with low DLC1 levels suppresses tumor growth in situ. Cells with reduced DLC1 protein contain increased GTP-bound RhoA, and enforced expression a constitutively activated RhoA allele mimics DLC1 loss in promoting hepatocellular carcinogenesis. Conversely, down-regulation of RhoA selectively inhibits tumor growth of hepatoma cells with disabled DLC1. Our data validate DLC1 as a potent tumor suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically.

DLC‐1 suppresses non‐small cell lung cancer growth and invasion by RhoGAP‐dependent and independent mechanisms

Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multi-domain protein that includes a Rho GTPase activating protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full-length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC, and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-delta1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.

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.