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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. Cancer Res; 71(8); 2916–25. ©2011 AACR.

We have applied a functional gene transfer strategy to show the importance of viral integration site in cellular immortalization. The large tumor antigen of SV40 is capable of extending the cellular life span by sequestering tumor suppressor proteins pRB and p53 in virus-transformed human cells. Although SV40 large T antigen is essential, it is not sufficient for cellular immortalization, suggesting that additional alterations in cellular genes are required to attain infinite proliferation. We show here that the disruption of human chromosomal interval at 1q21.1 by SV40 integration can be an essential step for cellular immortalization. The transfer of a 150-kb bacterial artificial chromosome (BAC) clone, RP364B14, corresponding to viral integration site in CRL2504 cells, reverted their immortal phenotype. Interestingly, the BAC transfer clones of CRL2504 cells displayed characteristics of either senescence as shown by β-galactosidase activity or apoptosis as revealed by positive staining with M30 CytoDEATH antibody. The SV40 integration at 1q21.1, in the vicinity of epidermal differentiation complex (EDC) genes, resulted in the down-regulation of the filaggrin (FLG) gene that is part of the EDC. FLG gene expression was increased in BAC transfer senescent and apoptotic clones. Our results suggest that the disruption of native genomic sequence by SV40 may alter expression of genes involved in senescence and apoptosis by modulating chromatin structure. These studies imply that identification of genes located in the vicinity of viral integration sites in human cancers may be helpful in developing new diagnostic and therapeutic strategies. [Cancer Res 2009;69(19):7819–25]

The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1 small interfering RNA (siRNA) cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those clones, Top1 is reduced ∼5-fold and Top2α compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone γ-H2AX foci associated with replication defects. siTop1 cells also show rDNA and nucleolar alterations and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) expression was reduced in siTop1 cells and in cells with transient Top1 down-regulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacologic profiling showed l-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, indenoisoquinoline, aphidicolin, hydroxyurea, and staurosporine and hypersensitivity to etoposide and actinomycin D show that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis. Overall, our studies show the pleiotropic nature of human Top1 activities. In addition to its classic DNA nicking-closing functions, Top1 plays critical nonclassic roles in genomic stability, gene-specific transcription, and response to various anticancer agents. The reported cell lines and approaches described in this article provide new tools to perform detailed functional analyses related to Top1 function. [Cancer Res 2007;67(18):8752–61]

To identify new antigens that are targets for the immunotherapy of prostate and breast cancer, we used expressed sequence tag and genomic databases and discovered POTE, a new primate-specific gene family. Each POTE gene encodes a protein that contains three domains, although the proteins vary greatly in size. The NH2-terminal domain is novel and has properties of an extracellular domain but does not contain a signal sequence. The second and third domains are rich in ankyrin repeats and spectrin-like helices, respectively. The protein encoded by POTE-21, the first family member discovered, is localized on the plasma membrane of the cell. In humans, 13 highly homologous paralogs are dispersed among eight chromosomes. The expression of POTE genes in normal tissues is restricted to prostate, ovary, testis, and placenta. A survey of several cancer samples showed that POTE was expressed in 6 of 6 prostate, 12 of 13 breast, 5 of 5 colon, 5 of 6 lung, and 4 of 5 ovarian cancers. To determine the relative expression of each POTE paralog in cancer and normal samples, we employed a PCR-based cloning and analysis method. We found that POTE-2α, POTE-2β, POTE-2γ, and POTE-22 are predominantly expressed in cancers whereas POTE expression in normal tissues is somewhat more diverse. Because POTE is primate specific and is expressed in testis and many cancers but only in a few normal tissues, we conclude POTE is a new primate-specific member of the cancer-testis antigen family. It is likely that POTE has a unique role in primate biology. (Cancer Res 2006; 66(1): 52-6)

In various studies of sporadic breast cancers, 40-70% were strongly positive for fragile histidine triad (Fhit) protein expression, whereas only 18% of BRCA2 mutant breast cancers demonstrated strong Fhit expression, suggesting that the BRCA2 repair function may be necessary to retain intact fragile common chromosome fragile site 3B(FRA3B)/FHITloci. In the current study, 22 breast tumors with deleterious BRCA1 mutations were analyzed for Fhit expression by immunohistochemistry in a case-control matched pair analysis. Loss of Fhit expression was significantly more frequent in the BRCA1 cancers compared with sporadic breast tumors (9% Fhit positive versus 68% Fhit positive), suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To investigate the relationship between repair gene deficiencies and induction of chromosome fragile sites in vitro, we have analyzed the frequency of aphidicolin induction of chromosome gaps and breaks in PMS2-, BRCA1-, MSH2-, MLH1-, FHIT-, and TP53-deficient cell lines. Each of the repair-deficient cell lines showed elevated expression of chromosome gaps and breaks, consistent with the proposal that proteins involved in mismatch and double-strand break repair are important in maintaining the integrity of common fragile regions. Correspondingly, genes at common fragile sites may sustain elevated levels of DNA damage in cells with deficient DNA repair proteins such as those mutated in several familial cancer syndromes.

Deleted in liver cancer 1 (DLC1) is a multidomain Rho-GTPase-activating protein (RhoGAP) and a tumor suppressor in several human common cancers. DLC1's structure predicts its interaction with a number of other proteins with various cellular functions. Previously we have shown that S100A10 (also known as p11), a key plasminogen receptor on the surface of cell membrane, colocalizes and binds with DLC1 in cell cytoplasm. The binding sites were mapped to the central domain of the DLC1 and at the end of C-terminus of S100A10. The same terminus is also a binding site for Annexin A2, which is believed to regulate availability of extracellular S100A10 and, therefore, dynamics of plasmin formation. Now we demonstrate that DLC1 competes with Annexin A2 for interaction with S100A10. DLC1 binding to S100A10 did not affect DLC1's RhoGAP activity but did reduce availability of S100A10 for binding by Annexin A2. Furthermore, the expression of DLC1 also affected S100A10 expression in a dose dependent manner, and increased S100A10 ubiquitin-dependent degradation. All of the above interactions attenuated plasminogen activation and resulted in inhibition of in vitro cell migration, invasion, colony formation and anchorage independent growth of aggressive and tumorigenic lung tumor cells. Transduction of DLC1-negative breast tumor cells significantly reduced the ability of tumor cells to form pulmonary metastases as well as inhibited distant dissemination of liver tumor cells in athymic mice. Current observations provide a plausible mechanism for DLC1 antimetastatic function by negatively affecting plasminogen activation, which in turn lead to a significant reduction in invasiveness of tumor cells. 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 2349.