Hepatocellular carcinoma (HCC) is the third leading cause of cancer death, and its incidence is increasing worldwide in an alarming manner. The development of curative therapy for advanced and ...metastatic HCC is a high clinical priority. The HCC genome is complex and heterogeneous; therefore, the identification of recurrent genomic and related gene alterations is critical for developing clinical applications for diagnosis, prognosis and targeted therapy of the disease. This article focuses on recent research progress and our contribution in identifying and deciphering the role of defined genetic alterations in the pathogenesis of HCC. A significant number of genes that promote or suppress HCC cell growth have been identified at the sites of genomic reorganization. Notwithstanding the accumulation of multiple genetic alterations, highly recurrent changes on a single chromosome can alter the expression of oncogenes and tumor suppressor genes (TSGs) whose deregulation may be sufficient to drive the progression of normal hepatocytes to malignancy. A distinct and highly recurrent pattern of genomic imbalances in HCC includes the loss of DNA copy number (associated with loss of heterozygosity) of TSG-containing chromosome 8p and gain of DNA copy number or regional amplification of protooncogenes on chromosome 8q. Even though 8p is relatively small, it carries an unusually large number of TSGs, while, on the other side, several oncogenes are dispersed along 8q. Compelling evidence demonstrates that DLC1, a potent TSG on 8p, and MYC oncogene on 8q play a critical role in the pathogenesis of human HCC. Direct evidence for their role in the genesis of HCC has been obtained in a mosaic mouse model. Knockdown of DLC1 helps MYC in the induction of hepatoblast transformation in vitro, and in the development of HCC in vivo. Therapeutic interventions, which would simultaneously target signaling pathways governing both DLC1 and MYC functions in hepatocarcinogenesis, could result in progress in the treatment of liver cancer.
Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that γ-H2AX foci ...(γ-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic γ-foci remain after repair of radiation-induced γ-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.
The DLC1 gene encodes a Rho GTPase-activating protein (RhoGAP) that functions as a tumor suppressor in several common human cancers. The multidomain structure of DLC1 enables interaction with a ...number of other proteins. Here we report that the proinflammatory protein S100A10 (also known as p11), a key cell surface receptor for plasminogen which regulates pericellular proteolysis and tumor cell invasion, is a new binding partner of DLC1 in human cells. We determined that the 2 proteins colocalize in the cell cytoplasm and that their binding is mediated by central sequences in the central domain of DLC1 and the C-terminus of S100A10. Because the same S100A10 sequence also mediates binding to Annexin 2, we found that DLC1 competed with Annexin 2 for interaction with S100A10. DLC1 binding to S100A10 did not affect DLC1's RhoGAP activity, but it decreased the steady-state level of S100A10 expression in a dose-dependent manner by displacing it from Annexin 2 and making it accessible to ubiquitin-dependent degradation. This process attenuated plasminogen activation and resulted in inhibition of in vitro cell migration, invasion, colony formation, and anchorage-independent growth of aggressive lung cancer cells. These results suggest that a novel GAP-independent mechanism contributes to the tumor suppressive activity of DLC1, and highlight the importance and complexity of protein-protein interactions involving DLC1 in certain cancers.
Summary
Accumulation of DNA damage may play an essential role in both cellular senescence and organismal aging. The ability of cells to sense and repair DNA damage declines with age. However, the ...underlying molecular mechanism for this age‐dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, DNA damage‐induced foci of phosphorylated histone H2AX (γ‐H2AX), which occurs specifically at sites of DNA double‐strand breaks (DSBs) and eroded telomeres, were examined in human young and senescing fibroblasts, and in lymphocytes of peripheral blood. Here, we show that the incidence of endogenous γ‐H2AX foci increases with age. Fibroblasts taken from patients with Werner syndrome, a disorder associated with premature aging, genomic instability and increased incidence of cancer, exhibited considerably higher incidence of γ‐H2AX foci than those taken from normal donors of comparable age. Further increases in γ‐H2AX focal incidence occurred in culture as both normal and Werner syndrome fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to γ‐H2AX foci correlated inversely with age for both normal and Werner syndrome donors, perhaps due in part to the slower growth of γ‐H2AX foci in older donors. Because genomic stability may depend on the efficient processing of DSBs, and hence the rapid formation of γ‐H2AX foci and the rapid accumulation of DSB repair proteins on these foci at sites of nascent DSBs, our findings suggest that decreasing efficiency in these processes may contribute to genome instability associated with normal and pathological aging.
While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA ...nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.
Once immortalized, human cells are susceptible to transformation by introduction of an oncogene such as ras. Several lines of evidence now suggest that the maintenance of telomere length is a major ...determinant of replicative lifespan in human cells and thus of the immortalized state. The majority of human tumor cells acquire immortality through expression of the catalytic subunit of telomerase (hTERT), whereas others activate an alternative mechanism of telomere maintenance (ALT) that does not depend on the actions of telomerase. We have examined whether ALT could substitute for telomerase in the processes of transformation in vitro and tumorigenesis in vivo. Expression of oncogenic H-Ras in the immortal ALT cell line GM847 did not result in their transformation. However, subsequent ectopic expression of hTERT in these cells imparted a tumorigenic phenotype. Indeed, this outcome was also observed after introduction of a mutant hTERT that retained catalytic activity but was incapable of maintaining telomere length. These studies indicate that hTERT confers an additional function that is required for tumorigenesis but does not depend on its ability to maintain telomeres.
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Introduction
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The deleted in liver cancer family of RhoGAP domain proteins
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DLC‐1
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DLC‐2
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DLC‐3
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Invertebrate DLC‐1‐like proteins
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Expression of DLC family proteins
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Features of DLC ...family protein domains
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SAM domain
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RhoGAP domain
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START domain
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Serine‐rich, unstructured middle region
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Biological functions of DLC‐1
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Cytoskeletal organization
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DLC‐1 localizes to focal adhesions via binding to tensin family proteins
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Interaction of DLC‐1 with caveolin‐1
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DLC‐1 and phosphoinositide signalling
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Biological activities of DLC‐2 and DLC‐3
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Genetic analysis of DLC‐1 function
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Mouse DLC‐1 gene knockout
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RhoGAP88 C in Drosophila
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The DLC family proteins in cancer
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Decreased expression of DLC‐1 in human cancers
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Deletions of DLC1 in tumours
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Epigenetic inactivation of DLC‐1 expression
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DLC‐1 sequence variants in human cancers
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DLC‐1 suppresses tumour cell growth
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DLC‐1 as a metastasis suppressor gene
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Regulation of DLC‐1 by anti‐oncogenic factors
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Animal models of DLC‐1 in cancer
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Evidence for roles of DLC‐2 and DLC‐3 in neoplasia
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Conclusions and future directions
The deleted in liver cancer 1 (DLC‐1) gene encodes a GTPase activating protein that acts as a negative regulator of the Rho family of small GTPases. Rho proteins transduce signals that influence cell morphology and physiology, and their aberrant up‐regulation is a key factor in the neoplastic process, including metastasis. Since its discovery, compelling evidence has accumulated that demonstrates a role for DLC‐1 as a bona fide tumour suppressor gene in different types of human cancer. Loss of DLC‐1 expression mediated by genetic and epigenetic mechanisms has been associated with the development of many human cancers, and restoration of DLC‐1 expression inhibited the growth of tumour cells in vivo and in vitro. Two closely related genes, DLC‐2 and DLC‐3, may also be tumour suppressors. This review presents the current status of progress in understanding the biological functions of DLC‐1 and its relatives and their roles in neoplasia.
A number of genetic mutations have been identified in human breast cancers, yet the specific combinations of mutations required in concert to form breast carcinoma cells remain unknown. One approach ...to identifying the genetic and biochemical alterations required for this process involves the transformation of primary human mammary epithelial cells (HMECs) to carcinoma cells through the introduction of specific genes. Here we show that introduction of three genes encoding the SV40 large-T antigen, the telomerase catalytic subunit, and an H-Ras oncoprotein into primary HMECs results in cells that form tumors when transplanted subcutaneously or into the mammary glands of immunocompromised mice. The tumorigenicity of these transformed cells was dependent on the level of ras oncogene expression. Interestingly, transformation of HMECs but not two other human cell types was associated with amplifications of the c-myc oncogene, which occurred during the in vitro growth of the cells. Tumors derived from the transformed HMECs were poorly differentiated carcinomas that infiltrated through adjacent tissue. When these cells were injected subcutaneously, tumors formed in only half of the injections and with an average latency of 7.5 weeks. Mixing the epithelial tumor cells with Matrigel or primary human mammary fibroblasts substantially increased the efficiency of tumor formation and decreased the latency of tumor formation, demonstrating a significant influence of the stromal microenvironment on tumorigenicity. Thus, these observations establish an experimental system for elucidating both the genetic and cell biological requirements for the development of breast cancer.
The identification of mammary gland stem cells (MGSC) or progenitors is important for the study of normal breast development and tumorigenesis. Based on their immunophenotype, we have isolated a ...population of mouse mammary gland cells that are capable of forming "mammospheres" in vitro. Importantly, mammospheres are enriched for cells that regenerate an entire mammary gland on implantation into a mammary fat pad. We also undertook cytogenetic analyses of mammosphere-forming cells after prolonged culture, which provided preliminary insight into the genomic stability of these cells. Our identification of new cell surface markers for enriching mammosphere-initiating cells, including endoglin and prion protein, will facilitate the elucidation of the cell biology of MGSC.
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