Bao-Zhu Yuan

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

DLC-1 operates as a tumor suppressor gene in human non-small cell lung carcinomas

The deleted in liver cancer (DLC-1) gene at chromosome 8p21–22 is altered mainly by genomic deletion or aberrant promoter methylation in a large number of human cancers such as breast, liver, colon and prostate and is known to have an inhibitory effect on breast and liver tumor cell growth. Given the high frequency of deletion involving region 8p21–22 in human non-small cell lung carcinoma (NSCLC), we examined alterations of DLC-1 in a series of primary tumors and tumor cell lines and tested effects of DLC-1 on tumor cell growth. A significant decrease or absence of the DLC-1 mRNA expression was found in 95% of primary NSCLC (20/21) and 58% of NSCLC cell lines (11/19). Transcriptional silencing of DLC-1 was primarily associated with aberrant DNA methylation, rather than genomic deletion as 5-aza-2′-deoxycytidine induced reactivation of DLC-1 expression in 82% (9/11) NSCLC cell lines showing downregulated DLC-1. It was further evidenced by an aberrant DLC-1 promoter methylation pattern, which was detected by Southern blotting in 73% (8/11) of NSCLC cell lines with downregulation of the gene. The transfer of DLC-1 into three DLC-1 negative cell lines caused a significant inhibition in cell proliferation and/or a decrease in colony formation. Furthermore, stable transfer of DLC-1 abolished tumorigenicity in nude mice of two cell lines, suggesting that DLC-1 plays a role in NSCLC by acting as a bona fide new tumor suppressor gene.

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.

Nonrandom Breakpoints of Unbalanced Chromosome Translocations in Human Hepatocellular Carcinoma Cell Lines

In the search for specific chromosomal alterations in human hepatocellular carcinomas (HCC), we analyzed two new HCC cell lines and identified nonrandom changes by combined G-banding and fluorescence in situ hybridization (FISH). Cell line 7703 was established from an HCC deriving from a patient in the Qidong region of China, where the incidence of HCC is very high and is associated with hepatitis-B virus infection and exposure to aflatoxin. This line has a highly rearranged karyotype eliciting complex rearrangements involving the majority of chromosomes. The second line, SK-Hep-1, derived from a liver adenocarcinoma, is less heterogeneous, having few altered chromosomes. We have characterized the majority of structural and numerical alterations and identified in both lines unbalanced translocations with the breakpoints nonrandomly involving regions 1p36 and 3p14 and gain of chromosome 6p and 8q. While gain of 6p and 8q are recurrent in HCC, translocations of 1p and 3p are described for the first time. Damage and recombination at the breakpoint sites on chromosomes 1 and 3 might have resulted in activation of proto-oncogene, formation of new oncogenic chimeric genes, or loss of tumor suppressor genes.

Proteasome inhibitors induce apoptosis in human lung cancer cells through a positive feedback mechanism and the subsequent Mcl-1 protein cleavage.

Proteasome inhibitors (PIs) are promising new therapeutic agents for treating non-small cell lung carcinoma (NSCLC). To investigate the mechanisms of action of PIs, we analyzed the proapoptotic activities of PIs (MG132 or Bortezomib) in NSCLC cells. We found that both MG132 (>1 μM) and Bortezomib (>0.025 μM) induced a significant apoptosis in NCI-H1703, a PI-sensitive NSCLC cell line, through initially activating the intrinsic apoptosis pathway, leading to the activation of a positive feedback mechanism (PFM), which then conveyed apoptosis signaling from the intrinsic pathway to the extrinsic pathway with formation of a signaling loop for maximal caspase activation. Mcl-1 and Noxa were identified to be the major anti-apoptotic and proapoptotic proteins, respectively, in PI-induced apoptosis and mutually exclusive in protein stability. Although the Mcl-1 protein was upregulated by proteasome inhibition, it was also subjected to caspase 3-dependent cleavage governed by the PFM. Moreover, it was revealed that Mcl-1 protein cleavage contributed to PFM-governed apoptosis in following inter-related ways: reducing the anti-apoptotic Mcl-1; generating the truncated proapoptotic Mcl-1S; and inducing a shift of balance between Mcl-1 and Noxa. It was further manifested that tumor necrosis factor-related apoptosis-inducing ligand boosted MG132s proapoptotic activity through strengthening the PFM in both NCI-H1703 and NCI-H358, a PI-resistant NSCLC cell line. Therefore, this study provides a basis for enhancing the efficacy of PIs in treating NSCLC.

Abstract 3699: Multi-walled carbon nanotubes induce apoptosis in normal human small airway epithelial cells through proteasome-mediated Mcl-1 protein degradation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Engineered carbon nanotubes (CNTs) are emerging as the major building blocks in nanotechnology thanks to their unique physical and chemical properties. However, widespread application of CNTs, including single- and multi-walled (SWCNTs and MWCNT), has resulted in increased public concern regarding potential toxicities from occupational or environmental exposures to CNTs. Recently, both SWCNTs and MWCNTs have been observed to exhibit pulmonary toxicities. To better understand CNT-related pulmonary toxicities, the present in vitro study used normal human small airway epithelial cells (SAEC), which constitute a primary target of respiratory exposure to CNTs, as a model to investigate cell death effects of either SWCNTs or MWCNTs. We observed that, at 24 h after exposure, greater than 5 μg/cm2 MWCNT, but not SWCNT, induced a significant dose-dependent cell death in primary cultured SAEC cells as assayed by WST-1. The cell death was accompanied by DNA damage, as indicated by the appearance of sub G0/G1 cells in a flow cytometry assay, and protein cleavages for PARP, caspases 3, 7 and 9, as revealed by Western blotting. It was further observed that the pro-apotpotic activities of MWCNT were reduced by caspase specific inhibitors, demonstrating that MWCNT, but not SWCNT, can induce a caspase-dependent apoptosis in SAEC cells. To understand regulation of the MWCNT-induced apoptosis, we then examined expression of the anti-apoptotic Bcl-2 family proteins and found that only Mcl-1 was significantly reduced in protein level by MWCNT, but not SWCNT, and such reduction was blocked by proteasome inhibitor MG132. Furthermore, it was found that reduction of Mcl-1 protein expression through Mcl-1 siRNA enhanced sensitivity of SAEC cells to MWCNT-induced apoptosis, suggesting that proteasome-mediated Mcl-1 protein degradation contributes to MWCNT-induced apoptosis. This study reveals a difference in cell death effects on normal human SAEC cells between SWCNT and MWCNT. It also provides new insights into understanding of the MWCNT-induced pulmonary toxicity. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 3699.