In the past year novel and highly recurrent genomic alterations harboring critical cancer-related genes have been identified in human and mouse model for liver cancer and significant progress has been made in the understanding of the Deleted in Liver Cancer 1, (DLC1), a bona fide tumor suppressor gene function and its potential use for therapeutic interventions. During the neoplastic process certain tumor cells acquire resistance to the antiproliferative signaling of TGFβ. We examined a human hepatocellular carcinoma (HCC) cell line sensitive to TGFβ1 (Hep3B-TS) and its derivative (Hep3B-TR) rendered resistant to TGFβ1 by stepwise exposure to the agent by spectral karyotyping (SKY) and array comparative genomic hybridization (aCGH) analysis. We discovered that the acquisition of TGFβresistance was caused by an interstitial microdeletion encompassing the site of TGFβreceptor II (TGFβRII) gene, which occurred in the only apparently normal chromosome 3. Comparative differential gene expression analysis of two cell lines using an oligonucleotide microarray of 21397 genes revealed that 197 and 110 genes were up- and down-regulated, respectively in Hep3BTR. Among these, 6 genes were downstream targets of tumor necrosis factor (TNF) gene, suggesting that loss of TGFβRII triggered the activation of TNF network, shown to be regulated by TGFβ1 pathway. Functionally, loss of the TGFβRII in cells resistant to TGFβ1 significantly enhanced cell migration and anchorage independent growth in vitro and in vivo tumorigenicity compared with parental sensitive cells. Mouse models for HCC provide an experimental ground to dissect the genetic and biological complexities of human liver cancer and enhance our ability to gain insights into the relevance of candidate cancer genes. We used mouse SKY and aCGH to examine several cell lines derived from HCC developed in c-Myc transgenic animals, and four cell lines established from the tumors induced in nude mice by s.c. inoculation of the original HCC cell lines. All cell lines exhibited gain and loss of material from several chromosomes, and also the recurrent rearrangements whose breakpoints clustered in the regions previously identified as important for the early stages of c-Myc induced hepatocarcinogenesis. The results underscore the importance of recurrent genomic reorganizations, loss of chromosomes 4, 9 and 14, and gain of chromosomes 15 and 19 in mouse liver neoplasia. The identification of those five genomic imbalances represent an unique example, as identical imbalances have been found in HCC developed in other transgenic mouse models, in spontaneous mouse HCC and derivative cell lines, as well as in preneoplastic liver lesions induced with chemical carcinogens. These results demonstrate selective, non-random genomic changes involving chromosomal regions homologous to those implicated in human HCC. DLC1, that encodes a Rho GTPase-activating protein, is an authentic tumor suppressor gene in several common cancers. Loss of DLC-1 expression represents a frequent mechanism for activation of Rho GTPases, key mediators of human oncogenesis. The predominant mechanisms of silencing or down regulation of DLC-1 in solid tumors and hematological malignancies are promoter hypermethylation and/or histone deacetylation. We screened 44 multiple myeloma (MM) cell lines for abnormal DLC1 promoter methylation and mRNA expression and found that the majority of them exhibited various degree of promoter methylation that correlated with downregulation or silencing of DLC1 expression. Recently, others provided conclusive evidence that an asymptomatic monoclonal gammopathy of unknown significance (MGUS) preceded the diagnosis of MM in all cases and emphasized that novel markers are necessary to predict progression to MM in individuals with MGUS. Thus, high frequency of DLC1 methylation and downregulation may provide such a useful molecular marker for MGUS and MM. We also demonstrated that treatment of MM cell lines lacking DLC1 expression due to full promoter hypermethylation with demethylating and acetylating agents significantly augmented the expression of DLC1 and inhibited cell proliferation. In addition and most importantly, when cells exhibiting complete promoter methylation and absence of DLC1 expression were transduced by an adenoviral vector containing DLC1 cDNA, resulted in restoration of DLC1 expression. Reexpression of DLC1 inhibited myeloma cell invasion and migration, reduced RhoA activity and resulted in reorganization of actin cytoskeleton. These results provide the first evidence for antiproliferative effect of DLC1 in a hematological cancer and implicate RhoA pathway in suppression of MM migration and invasion. DLC1-mediated suppression of cell migration and invasion is of significant importance as the two processes are instrumental in the myeloma cell movement within the bone morrow and metastasis to secondary sites. Given the myeloma cells sensitivity to reactivation of DLC-1 function, the potential for molecular targeted therapy of DLC-1 mediated pathways as well as epigenetic therapies hold prospects. A yeast two-hybrid screen first identified 14 proteins that interact of DLC1 protein and several interactions were confirmed in human cells. Identification of such interacting partners could enhance our understanding of the mechanisms that regulate the function of DLC1. We demonstrated DLC1 protein interaction with p120Ras-GAP protein. Endogenous DLC1 forms a stable complex with endogenous Ras-GAP, and the two proteins co-localize in focal adhesions. The interaction was mapped to the Rho-GAP catalytic domain of DLC1 and the SH3 domain of Ras-GAP. This interaction induced a dramatic reduction of DLC1Rho-GAP activity in vitro. Moreover, overexpression of Ras-GAP, in colon carcinoma cells that harbored mutant Ras and thus were resistant to the negative regulation of Ras by Ras-GAP, increased the level of endogenous active Rho in a DLC1-dependent manner and antagonized the growth suppressive effects of DLC1. Our data showing that Ras-GAP can promote the growth of cells containing mutant Ras add to a growing body of evidence suggesting that Ras-GAP might represent a valid therapeutic target. Another significant achievement was the generation of mice with a conditional knockout allele of the DLC1 gene by using gene targeting in ES cells to introduce loxP sites on either side of exon 4. Expression of Cre recombinase deleted the loxP-flanked DNA segment containing exon 4, leading to a reading frame shift that should result in premature translation termination of the DLC1 polypeptide. Mice homozygous for the floxed allele (Dlc1fl/fl) are viable and fertile and can be crossed with transgenic strains that express Cre recombinase under the control of various tissue-specific and inducible promoters. This conditional knockout mouse that was generated in collaboration with Drs. Snorri Thorgeirsson and Douglas Lowy will provide a valuable model system for analyzing the effects of DLC1 deficiency in normal developing and adult tissues and in cancer and other pathological conditions.
