Although last year our program was evaluated at the Site Visit of the laboratory, we succeeded to generate significant pool of new data withy relevance to initial stages of neoplastic development and with potential for use in therapeutic interventions. The molecular and cytogenetic evidence for cancer-specific translocations, amplification of oncogenes, deletion of tumor suppressor gene and viral integration at fragile sites (FSs) constitute the turning point in linking genomic regions of fragility and recombination to cancer development. Long time ago, we advanced the concept of FS as preferential target for integration of transforming viruses. This notion is currently generally accepted. A novel example of the role of specific viral integration at FS was provided showing Simian virus 40 (SV 40) integration at a FRA1F located on chromosome 1q21. SV40 insertion in the cells genome led to deregulation of genes involved in senescence and apotosis and thus plays a critical role in cellular immortalization. Subsequently a human genomic sequences flanking SV40 genome was isolated and identified a 150 KB BAC, corresponding to the viral integration site at 1q21. The BAC clone was retrofitted with a neo marker for selection and introduced into the parental immortal cell line. BAC transfer clones displayed growth characteristics of normal fibroblasts terminating in replicative senescence. These results show that ectopic expression of gene(s) disrupted by viral insertion restore normal growth pattern and senescence. A high-resolution map of 1q21 region, surrounding the BAC clone, identified genes that are either rearranged or show abnormal expression in many cancers and suggests a role for this region in the development of cancer. In the past few years we gained considerable experience in cytogenetics of embryonic stem cells. This expertise attracted the interest of several intra- and extramural scientists. One of the results is the generation of a KEPI knockout mouse in a collaboration with Dr. Uhls group from the Molecular Neurobiology Branch, NIDA, in Baltimore. KEPI as a morphine-regulated gene, which is a powerful inhibitor of protein phosphatase 1. The gene maps onto mouse chromosome 10 close to the locus that contains the -opioid receptor (Oprm1). After the final construct, pJD9 was linearized with Not I and electroporate into 129.3 mouse MC1 embryonic stem (ES), cells, primers vNEOf and J39 were used for a PCR screen of 376 colonies resistant to G418. Embryonic stem (ES) cell clones positive by PCR were further confirmed by Southern blot. Eight of the correctly targeted ES cell clones were extensively karyotyped and the two confirmed free of aneuploidy and structural deffects were microinjected into C57BL/6J E3.5 blastocysts. Heterozygous KEPI KO offspring of the resulting male chimeras were mated with each other, to amplify wild type (WT) and KO, (922 bp) alleles simultaneously. Recombinant, KEPIKO mice displayed set of characteristsics that support roles for KEPI gene action in adaptive responses Over past decade the human DLC-1 (Deleted in Liver Cancer 1) gene has emerged as an potent tumor suppressor gene and as one of the most frequently deregulated genes in cancer, being the 5th among top-50 genes implicated in multiple cancers. DLC1 loss of function is viewed as a driving event in the promotion and progression of liver and other cancers. A novel DLC1 isoform 4 (DLC1-i4) was identified and characterized. The DLC1-i4 encodes an 1125-aa protein with distinct N-terminus compared to the other isoforms and, as others, is expressed ubiquitously in normal tissues and immortalized normal epithelial cells, suggesting a role as a major DLC1 transcript. However, differential expression of the four DLC1 isoforms is found in tumor cell lines: Isoform 1 and 3 (probably nonfunctional) share a promoter and are silenced in almost all cancer and immortalized cell lines, while isoform 2 and 4 utilize different promoters and are frequently downregulated. DLC1-i4 is significantly down-regulated in a high number of nasopharyngeal, esophageal, gastric, breast, colorectal, cervical and lung carcinoma cell lines as well as in primary tumors. The functional DLC1-i4 promoter is within a CpG island and is activated by wild-type p53. Treatment with 5-aza-2-deoxycytidine led to demethylation of the promoter and reactivation of DLC1-i4 expression. Ectopic expression of isofor 4 in DLC1-i4-negative tumor cells strongly inhibited their growth and colony formation showing that this isoform has oncosuppressive role. The differential expression of various DLC1 isoforms suggests interplay in modulating the complex activities of DLC1 during carcinogenesis. Inactivation of tumor suppressor genes is a major contributing alteration in the initiation or progression of cancer. DLC1 is deleted in various common cancers more frequently than most other tumor suppressors genes, however down regulation and inactivation of DLC1 is mediated predominantly by promoter hypermethylation and histone deacetylation. Because DNA methyltransferase and histone deacetylase (HDAC) inhibitors can induce the restoration of DLC-1 expression, the DLC-1 protein may also represent a potential target for novel therapies. Given the considerable interest and progress in epigenetic therapy, a number of promising antineoplastic agents, particularly HDAC inhibitors, have been developed and used successfully in clinical trials. Both DLC1 and HDAC inhibitors exert antineoplastic functions, and their combined action could be exploited for a more effective cancer therapy. To evaluate the potential benefits of this approach, we examined the antineoplastic effects of adenoviral (Ad)-DLC1-mediated transduction, and the exposure to suberoylanilide hydroxamic acid (SAHA), a powerful HDAC inhibitor in two DLC1-negative human cancer cell lines - 22Rv1 (prostate cancer) and 7703K (human hepatocellular carcinoma) cells. Consistent with the oncosuppressive function of DLC1 in several cancers, transduction of prostate and liver cancer cells with an adenovirus AD-DLC1 expression vector resulted in alterations of cell morphology, induction of apoptosis, and inhibition of cell proliferation, migration, and anchorage-independent growth. A low concentration of SAHA efficiently restored the expression of DLC1 in prostate tumor cells that lack DLC1 expression due to histone deacetylation, but had a minimal effect in liver tumor cells in which silencing of the DLC1 gene is due mainly to promoter hypermethylation. Regardless of the epigenetic mechanism of DLC1 inactivation, SAHA treatment of DLC1-transduced cells had a synergistic inhibitory effect on tumor cell proliferation and tumorigenesis in both cell lines. In prostate tumor cells, this combination regimen virtually abolished the formation of colonies in semisolid media as a measure of tumorigenicity in vitro. Current in vitro results validate this protocol as a potentially new therapeutic option in certain cancers.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010038-15
Application #
8157252
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2010
Total Cost
$1,095,529
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Zhou, Xiaoling; Yang, Xu-Yu; Popescu, Nicholas C (2012) Preclinical evaluation of combined antineoplastic effect of DLC1 tumor suppressor protein and suberoylanilide hydroxamic acid on prostate cancer cells. Biochem Biophys Res Commun 420:325-30
Yang, Xuyu; Popescu, Nicholas C; Zimonjic, Drazen B (2011) DLC1 interaction with S100A10 mediates inhibition of in vitro cell invasion and tumorigenicity of lung cancer cells through a RhoGAP-independent mechanism. Cancer Res 71:2916-25
Low, J S W; Tao, Q; Ng, K M et al. (2011) A novel isoform of the 8p22 tumor suppressor gene DLC1 suppresses tumor growth and is frequently silenced in multiple common tumors. Oncogene 30:1923-35
Zhou, Xiaoling; Yang, Xu-Yu; Popescu, Nicholas C (2010) Synergistic antineoplastic effect of DLC1 tumor suppressor protein and histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), on prostate and liver cancer cells: perspectives for therapeutics. Int J Oncol 36:999-1005
Drgonova, J; Zimonjic, D B; Hall, F S et al. (2010) Effect of KEPI (Ppp1r14c) deletion on morphine analgesia and tolerance in mice of different genetic backgrounds: when a knockout is near a relevant quantitative trait locus. Neuroscience 165:882-95
Ullmannova-Benson, V; Guan, M; Zhou, X et al. (2009) DLC1 tumor suppressor gene inhibits migration and invasion of multiple myeloma cells through RhoA GTPase pathway. Leukemia 23:383-90
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Zimonjic, Drazen B; Zhou, Xiaoling; Lee, Ju-Seog et al. (2009) Acquired genetic and functional alterations associated with transforming growth factor beta type I resistance in Hep3B human hepatocellular carcinoma cell line. J Cell Mol Med 13:3985-92
Liu, Jinglan; Kaur, Gurpreet; Zhawar, Vikramjit K et al. (2009) Role of SV40 integration site at chromosomal interval 1q21.1 in immortalized CRL2504 cells. Cancer Res 69:7819-25
Zimonjic, Drazen B; Ullmannova-Benson, Veronika; Factor, Valentina M et al. (2009) Recurrent and nonrandom DNA copy number and chromosome alterations in Myc transgenic mouse model for hepatocellular carcinogenesis: implications for human disease. Cancer Genet Cytogenet 191:17-26