Xiaoling Zhou

DLC-1 gene inhibits human breast cancer cell growth and in vivo tumorigenicity

The human DLC-1 (deleted in liver cancer 1) gene was cloned from a primary human hepatocellular carcinoma (HCC) and mapped to the chromosome 8p21–22 region frequently deleted in common human cancers and suspected to harbor tumor suppressor genes. DLC-1 was found to be deleted or downregulated in a significant number of HCCs. We expanded our investigations to other cancers with recurrent deletions of 8p22, and in this study examined alterations of DLC-1 in primary human breast tumors, human breast, colon, and prostate tumor cell lines. Genomic deletion of DLC-1 was observed in 40% of primary breast tumors, whereas reduced or undetectable levels of DLC-1 mRNA were seen in 70% of breast, 70% of colon, and 50% of prostate tumor cell lines To see whether DLC-1 expression affects cell growth and tumorigenicity, two breast carcinoma cell lines lacking the expression of endogenous gene were transfected with the DLC-1 cDNA. In both cell lines, DLC-1 transfection caused significant growth inhibition and reduction of colony formation. Furthermore, introduction of the DLC-1 cDNA abolished the in vivo tumorigenicity in nude mice, suggesting that the DLC-1 gene plays a role in breast cancer by acting as a bona fide tumor suppressor gene.

DLC‐1:a Rho GTPase‐activating protein and tumour suppressor

•  Introduction ‐  The deleted in liver cancer family of RhoGAP domain proteins ‐  DLC‐1 ‐  DLC‐2 ‐  DLC‐3 ‐  Invertebrate DLC‐1‐like proteins •  Expression of DLC family proteins •  Features of DLC family protein domains ‐  SAM domain ‐  RhoGAP domain ‐  START domain ‐  Serine‐rich, unstructured middle region •  Biological functions of DLC‐1 ‐  Cytoskeletal organization ‐  DLC‐1 localizes to focal adhesions via binding to tensin family proteins ‐  Interaction of DLC‐1 with caveolin‐1 ‐  DLC‐1 and phosphoinositide signalling ‐  Biological activities of DLC‐2 and DLC‐3 •  Genetic analysis of DLC‐1 function ‐  Mouse DLC‐1 gene knockout ‐  RhoGAP88 C in Drosophila •  The DLC family proteins in cancer ‐  Decreased expression of DLC‐1 in human cancers ‐  Deletions of DLC1 in tumours ‐  Epigenetic inactivation of DLC‐1 expression ‐  DLC‐1 sequence variants in human cancers ‐  DLC‐1 suppresses tumour cell growth ‐  DLC‐1 as a metastasis suppressor gene ‐  Regulation of DLC‐1 by anti‐oncogenic factors ‐  Animal models of DLC‐1 in cancer ‐  Evidence for roles of DLC‐2 and DLC‐3 in neoplasia •  Conclusions and future directions

Restoration of DLC-1 gene expression induces apoptosis and inhibits both cell growth and tumorigenicity in human hepatocellular carcinoma cells.

The gene deleted in liver cancer-1 (DLC-1) is located on human chromosome 8p21–22, a region thought to harbor tumor suppressor genes on the basis of its frequent deletion or loss of heterozygosity in a variety of human cancers, including hepatocellular carcinoma (HCC). Deletion or altered expression of DLC-1 is common in HCC. In the current study, the subcellular localization of Dlc-1 protein was determined by immunostaining with antibody to DLC-1 and the possible tumor growth suppressor activity of DLC-1 was investigated by examining the effects of of DLC-1 cDNA transfection in two human HCC cell lines lacking expression of the endogenous gene. The results show that Dlc-1protein is localized in the cell cytoplasm, and the restoration of DLC-1 expression in HCC cells resulted in caspase-3-mediated apoptosis, inhibition of cell growth and invasiveness in vitro as well as in reduction of the ability of the cells to form tumors in athymic nude mice. These observations thus support the notion that Dlc-1 protein is involved in hepatocarcinogenesis and has oncosuppressive activity in HCC.

Aberrant methylation and deacetylation of Deleted in liver cancer-1 gene in prostate cancer : Potential clinical applications

Purpose: 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. Experimental Design: 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. Results: 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. Conclusions: 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.

Amplification and Overexpression of the EMS 1 Oncogene, a Possible Prognostic Marker, in Human Hepatocellular Carcinoma

DNA amplification in cancer cells frequently involves oncogenes whose increased expression confers a selective advantage on tumor cell growth. In an attempt to identify novel oncogenes involved in hepatocarcinogenesis, representational difference analysis (RDA) was performed using DNA from a primary human hepatocellular carcinoma (HCC) that showed high-level DNA amplifications on chromosomes 1p32 and 11q13 by comparative genomic hybridization. Ten amplification fragments were isolated by RDA, and when used to probe Southern blots of tumor DNA, there was a 5- to 50-fold increase in hybridization intensity relative to normal DNA. The sequence of one amplification product matched that of the EMS1 oncogene, which is located on chromosome 11q13 and is amplified in other cancers. We detected EMS1 amplification in 3 of 17 primary HCC. Overexpression of EMS1 mRNA was observed in 12 of 14 HCC cell lines in the absence of gene amplification or an increased copy-number of the gene. The EMS1 gene encodes cortactin, a cortical actin-associated protein that is a substrate for Src kinase and is involved in cytoskeleton organization. Alterations of the EMS1 gene that lead to overexpression of cortactin may be associated with tumor development in HCC. EMS1 amplification and overexpresion is indicative of unfavorable prognosis in several cancers and may have similar prognostic implications in liver cancer.

DLC1 suppresses distant dissemination of human hepatocellular carcinoma cells in nude mice through reduction of RhoA GTPase activity, actin cytoskeletal disruption and down-regulation of genes involved in metastasis.

The process of cell dissemination from the primary tumors to distant sites is the most harmful event during cancer progression, and the leading cause of cancer death. We have previously demonstrated that restoration of DLC1 tumor suppressor gene expression in the DLC1-negative Focus and 7703K human hepatocellular carcinoma (HCC) cell lines induced caspase-3 mediated apoptosis, reduced cell growth in vitro and tumorigenicity in vivo and diminished the ability to migrate through Matrigel, a property suggestive of metastatic potential in vivo. We now show that subcutaneous tumors developing after inoculation of Focus and 7703K cells into nude mice disseminate cells to liver and lung, and this process is markedly suppressed by restoration of DLC1 expression. Inhibition of tumor cell dissemination was associated with lower levels of RhoA activity, an increase in rounded cells and a reduction in actin stress fibers and focal adhesion molecules that are of critical importance in cancer cell invasion and metastasis. In addition, DLC1 down-regulated the expression of osteopontin and matrix metalloproteinase-9, which are highly up-regulated in most primary HCC with associated metastases. These observations implicate the DLC1 gene in suppression of HCC cell dissemination and identify novel cellular and genetic alterations that contribute to prevention of metastasis, a life-threatening event in cancer progression.

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.

Regulation of PI3K signaling in T-cell acute lymphoblastic leukemia: a novel PTEN/Ikaros/miR-26b mechanism reveals a critical targetable role for PIK3CD

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy, and T-ALL patients are prone to early disease relapse and suffer from poor outcomes. The PTEN, PI3K/AKT and Notch pathways are frequently altered in T-ALL. PTEN is a tumor suppressor that inactivates the PI3K pathway. We profiled miRNAs in Pten-deficient mouse T-ALL and identified miR-26b as a potentially dysregulated gene. We validated decreased expression levels of miR-26b in mouse and human T-ALL cells. In addition, expression of exogenous miR-26b reduced proliferation and promoted apoptosis of T-ALL cells in vitro, and hindered progression of T-ALL in vivo. Furthermore, miR-26b inhibited the PI3K/AKT pathway by directly targeting PIK3CD, the gene encoding PI3Kδ, in human T-ALL cell lines. ShRNA for PIK3CD and CAL-101, a PIK3CD inhibitor, reduced the growth and increased apoptosis of T-ALL cells. Finally, we showed that PTEN induced miR-26b expression by regulating the differential expression of Ikaros isoforms that are transcriptional regulators of miR-26b. These results suggest that miR-26b functions as a tumor suppressor in the development of T-ALL. Further characterization of targets and regulators of miR-26b may be promising for the development of novel therapies.