Inactivation of tumor suppressor gene HIC1 in gastric cancer is reversed via small activating RNAs

HIC1 is a tumor suppressor gene that is down-expressed in different malignancies, in part, because of promoter hypermethylation. However, the biological function of HIC1 in gastric cancer remains unclear. It is known that small double-stranded RNAs can induce gene expression by targeting promoter sequences. In the present study, we examined the expression levels of HIC1 in gastric cancer tissue. Several pieces of small double-stranded RNAs were used for the activation of HIC1. Tissue microarray analysis of gastric cancer indicated that down-regulation of HIC1 in gastric cancer tissue was dramatic compared with the adjacent gastric mucosa. Gastric cancer cell lines also showed down-regulated HIC1 expression compared with a human immortalized gastric mucosa cell line. One out of four dsRNAs produced activation of HIC1 as assessed by real-time PCR and Western blotting. Use of a cell counting kit 8 and clonogenicity assays indicated that dsRNA-mediated re-expression of HIC1 inhibited cell proliferation and clonogenicity in gastric cancer. Reactivation of HIC1 suppressed cell migration and induced cell cycle arrest in the G0/G1 phase, as well as induced apoptosis. These results suggest that HIC1 is a potential target of gene therapy against gastric cancer, and that dsRNAs could function as a therapeutic option for up-regulating tumor suppressor genes in gastric cancer and other malignancies.     

Expression of the class II tumor suppressor gene RIG1 is directly regulated by p53 tumor suppressor in cancer cell lines

Recent studies indicated that the RIG1 (RARRES3/TIG3) plays an important role in cell proliferation, differentiation, and apoptosis. However, the regulatory mechanism of RIG1 gene expression has not been clearly elucidated. In this study, we identified a functional p53 response element (p53RE) in the RIG1 gene promoter. Transfection studies revealed that the RIG1 promoter activity was greatly enhanced by wild type but not mutated p53 protein. Sequence specific mutation of the p53RE abolished p53-mediated transactivation. Specific binding of p53 protein to the rig-p53RE was demonstrated using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. Further studies confirmed that the expression of RIG1 mRNA and protein is enhanced through increased p53 protein in HepG2 or in H24-H1299 cells. In conclusion, our results indicated that RIG1 gene is a downstream target of p53 in cancer cell lines.     

Deletions in chromosome 4 differentially associated with the development of cervical cancer: evidence of slit2 as a candidate tumor suppressor gene

The aim of this study was to locate the candidate tumor suppressor genes (TSGs) loci in the chromosomal 4p15-16, 4q22-23 and 4q34-35 regions associated with the development of uterine cervical carcinoma (CA-CX). Deletion mapping of the regions by microsatellite markers identified six discrete areas with high frequency of deletions, viz. 4p16.2 (D1: 40%), 4p15.31 (D2: 35–38%), 4p15.2 (D3: 37–40%), 4q22.2 (D4: 34%), 4q34.2-34.3 (D5: 37–59%) and 4q35.1 (D6: 40–50%). Significant correlation was noted among the deleted regions D1, D2 and D3. The deletions in D1, D2, D5 and D6 regions are suggested to be associated with the cervical intraepithelial neoplasia (CIN), and deletions in the D2, D3, D5 and D6 regions seems to be associated with progression of CA-CX. The deletions in the D2 and D6 regions showed significant prognostic implications (P = 0.001; 0.02). The expression of the candidate TSG SLIT2 mapped to D2 region gradually reduced from normal cervix uteri →CIN → CA-CX. SLIT2 promoter hypermethylation was seen in 28% CIN samples and significantly increased with tumor progression (P = 0.04). Significant correlation was seen between SLIT2 deletion and its promoter methylation (P = 0.001), indicating that both these phenomena could occur simultaneously to inactivate this gene. Immunohistochemical analysis showed reduced expression of SLIT2 in cervical lesions and CA-CX cell lines. Although no mutation was detected in the SLIT2 promoter region (−432 to + 55 bp), CC and AA haplotypes were seen in −227 and −195 positions, respectively. Thus, it indicates that inactivation of SLIT2-ROBO1 signaling pathway may have an important role in CA-CX development.     

The cytoplasmic domain of C-CAM1 tumor suppressor is necessary and sufficient for suppressing the tumorigenicity of prostate cancer cells.

We have previously shown that C-CAM1 cell adhesion molecule can suppress the growth of prostate cancer cells in vivo. In this study, we determined the minimal domain of C-CAM1 that is required for its tumor-suppressive activity. DU145 prostate cancer cells were infected with recombinant adenoviruses containing various C-CAM1 mutant genes, and the effects of the mutant C-CAM1 proteins on the growth of DU145 cells were assessed in a nude-mice xenograft model. Deletion of C-CAM1's cytoplasmic domain, which is not required for its adhesion activity, abolished the growth-suppressive activity, whereas deletion of the adhesion domain did not. This observation suggests that C-CAM1's extracellular domain may be not essential for its tumor suppressive activity. Indeed, we found that expression of the C-CAM1 cytoplasmic domain alone led to growth suppression of DU145 cells. These results suggest that the cytoplasmic domain of C-CAM1 is necessary and sufficient for its growth-suppressive function.     

The Wilms' tumor suppressor Wt1 is associated with the differentiation of retinoblastoma cells.

We have demonstrated recently that Wilms' tumor suppressor 1 (Wt1),in addition to its role in genitourinary formation,is required for the differentiation of ganglion cells in the developing retina. Here we provide further evidence that Wt1 is associated with neuronal differentiation. Thus, the retinoblastoma-derived human cell line, Y-79, contained robust amounts of Wt1 mRNA and protein. Wt1 expression was down-regulated upon laminin-induced differentiation of Y-79 into neuron-like cells. Inhibition of Wt1 with antisense oligonucleotides dramatically reduced the capacity of undifferentiated Y-79 cells to undergo neuronal differentiation, whereas sense and missense oligonucleotides had no effect. Wt1 immunoreactivity was also detected in solid retinoblastomas, in which it resided mainly in areas with moderate proliferative activity. These findings suggest a role for Wt1 in the differentiation of retinoblastoma cells. Furthermore, Wt1 expression in retinoblastoma may reflect the potential of these tumors to initiate the early steps of neuronal differentiation.     

Metformin inhibition of colorectal cancer cell migration is associated with rebuilt adherens junctions and FAK downregulation

E-cadherin, a central component of the adherens junction (AJ), is a single-pass transmembrane protein that mediates cell–cell adhesion. The loss of E-cadherin surface expression, and therefore cell–cell adhesion, leads to increased cell migration and invasion. Treatment of colorectal cancer (CRC)-derived cells (SW-480 and HT-29) with 2.0 mM metformin promoted a redistribution of cytosolic E-cadherin to de novo formed puncta along the length of the contacting membranes of these cells. Metformin also promoted translocation from the cytosol to the plasma membrane of p120-catenin, another core component of the AJs. Furthermore, E-cadherin and p120-catenin colocalized with β-catenin at cell–cell contacts. Western blot analysis of lysates of CRC-derived cells revealed a substantial metformin-induced increase in the level of p120-catenin as well as E-cadherin phosphorylation on Ser^838/840, a modification associated with β-catenin/E-cadherin interaction. These modifications in E-cadherin, p120-catenin and β-catenin localization suggest that metformin induces rebuilding of AJs in CRC-derived cells. Those modifications were accompanied by the inhibition of focal adhesion kinase (FAK), as revealed by a significant decrease in the phosphorylation of FAK at Tyr³⁹⁷ and paxillin at Tyr¹¹⁸. These changes were associated with a reduction in the numbers, but an increase in the size, of focal adhesions and by the inhibition of cell migration. Overall, these observations indicate that metformin targets multiple pathways associated with CRC development and progression.     

Chromosome instability in colorectal tumor cells is associated with defects in microtubule plus-end attachments caused by a dominant mutation in APC

The attachment of microtubule plus ends to kinetochores and to the cell cortex is essential for the fidelity of chromosome segregation. Here, we characterize the causes underlying the high rates of chromosome instability (CIN+) observed in colorectal tumor cells. We show that CIN+ tumor cells exhibit inefficient microtubule plus-end attachments during mitosis, accompanied by impairment of chromosome alignment in metaphase. The mitotic abnormalities associated with CIN+ tumor cells correlated with status of adenomatous polyposis coli (APC). Importantly, we have shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and to cause a dramatic increase in mitotic abnormalities. We propose that APC functions to modulate microtubule plus-end attachments during mitosis, and that a single mutant APC allele predisposes cells to increased mitotic abnormalities, which may contribute to tumor progression.     

Inhibition of cellular proliferation by the Wilms' tumor suppressor WT1 is associated with suppression of insulin-like growth factor I receptor gene expression.

We have investigated the regulation of the insulin-like growth factor I receptor (IGF-I-R) gene promoter by the Wilms' tumor suppressor WT1 in intact cells. The levels of endogenous IGF-I-R mRNA and the activity of IGF-I-R gene promoter fragments in luciferase reporter constructs were found to be significantly higher in G401 cells (a Wilms' tumor-derived cell line lacking detectable WT1 mRNA) than in 293 cells (a human embryonic kidney cell line which expresses significant levels of WT1 mRNA). To study whether WT1 could suppress the expression of the endogenous IGF-I-R gene, WT1-negative G401 cells were stably transfected with a WT1 expression vector. Expression of WT1 mRNA in G401 cells resulted in a significant decrease in the rate of cellular proliferation, which was associated with a reduction in the levels of IGF-I-R mRNA, promoter activity, and ligand binding and with a reduction in IGF-I-stimulated cellular proliferation, thymidine incorporation, and anchorage-independent growth. These data suggest that a major aspect of the action of the WT1 tumor suppressor is the repression of IGF-I-R gene expression.     

Loss of circadian clock gene expression is associated with tumor progression in breast cancer

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2 and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P < 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2 and NFIL3 in the ER+/HER2- subgroup, CLOCK and NPAS2 in the ER-/HER2- subtype, and ARNTL2 in HER2+ breast cancer. In the multivariate Cox model, only PER3 (HR = 0.66; P = 0.016) and RORC (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.     

Overexpression of Rho GDP-dissociation inhibitor alpha is associated with tumor progression and poor prognosis of colorectal cancer

The GDP dissociation inhibitors (GDIs) are pivotal regulators of Rho GTPases, which are essential for tumor progression, particularly in the area of metastasis. One member of GDIs was identified as RhoGDI (Rho GDP-dissociation inhibitor alpha, or RhoGDIalpha), but little is known about this protein in tumors. In this study, we used comparative proteomic analysis to show that RhoGDI is markedly up-regulated in metastatic colorectal cancer (CRC). The elevated level of RhoGDI protein in metastatic CRC was confirmed by Western blot at the tissue ( n = 24) and cell ( n = 6) levels. Further, we analyzed RhoGDI protein expression in 126 clinicopathologically characterized CRC cases by immunohistochemistry. Statistical analysis showed that there were significant differences of RhoGDI overexpression in patients categorized according to tumor invasion ( p = 0.018), lymph node metastasis ( p = 0.001) and clinical stage ( p = 0.009). A trend was also identified between high expression of RhoGDI and shorter overall survival ( p = 0.013). In the present work, we also analyzed the effect of RhoGDI on CRC cell line. Gene transfection-mediated overexpression of RhoGDI in HT29 cells, containing a low detectable level of endogenous RhoGDI, resulted in a significant increase in cell proliferation and motility in vitro. These data suggest that RhoGDI may promote CRC progression and metastasis by stimulating tumor cell growth and migration.     

The cytostatic function of c-Abl is controlled by multiple nuclear localization signals and requires the p53 and Rb tumor suppressor gene products.

c-Abl is a non-receptor protein-tyrosine kinase lacking a clear physiological role. A clue to its normal function is suggested by overexpression of Abl in fibroblasts, which leads to inhibition of cell growth. This effect requires tyrosine kinase activity and the Abl C-terminus. c-Abl is localized to the cell nucleus, where it can bind DNA, and interacts with the retinoblastoma protein, a potential mediator of the growth-inhibitory effect. Nuclear localization of Abl can be directed by a pentalysine nuclear localization signal in the Abl C-terminus. Here, we have identified two additional basic motifs in the Abl C-terminus, either of which can function independently of the pentalysine signal to localize Abl to the nucleus. Using a quantitative transfection assay, we show that both c-Abl and transforming Abl proteins inhibit entry into S phase and this effect is absolutely dependent on nuclear localization. Further, we demonstrate that the Abl cytostatic effect requires both the Rb and p53 tumor suppressor gene products. These results indicate that Abl inhibits cell proliferation by interacting with central elements of the cell cycle control apparatus in the nucleus, and suggest a direct connection between p53 and Rb in this growth-inhibitory pathway.     

Identification of Fetuin-B as a member of a cystatin-like gene family on mouse chromosome 16 with tumor suppressor activity.

Studies of mouse models for multistage carcinogenesis have led to the identification of a susceptibility locus for skin tumor development (Skts9) in the proximal region of mouse chromosome 16. This chromosome region shows a loss of heterozygosity or an allelic imbalance in mouse skin and pancreatic islet carcinoma, and has been associated with angiogenesis. The microsatellite marker D16Mit2, which has the strongest linkage to skin tumor susceptibility, was used to screen a bacterial artificial chromosome (BAC) library, leading to the identification of the histidine-rich glycoprotein (Hrg) and Fetuin-B as the most tightly linked genes. These genes are members of a cystatin-like superfamily that includes the neighboring genes Kng and Ahsg/Fetuin. Overexpression of Fetuin-B in skin squamous carcinoma cells led to suppression of tumor growth in nude mice. The neighboring genes Kng and Ahsg also have potential roles in angiogenesis and (or) tumor development, and several genes in this locus may be candidates for the Skts9 gene.     

Regulatory T-cell-mediated inhibition of antitumor immune responses is associated with clinical outcome in patients with liver metastasis from colorectal cancer

Adaptive regulatory T cells (Tregs) contribute to an immunosuppressive microenvironment in colorectal cancer (CRC). Here, we examined whether the level of Treg-mediated inhibition of antitumor immune responses in patients with metastatic CRC (metCRC) selected for liver resection is associated with clinical outcome. Preoperatively and at follow-ups, we did flow-based phenotyping, examined antitumor immunity using peptides from carcinoembryonic antigen (CEA) protein in the presence or absence of CD4(+)CD25(+)CD127(dim/-) cells (Tregs) and determined cytokine and PGE(2) levels in patient blood samples. At 18 months post-surgery, 8 patients were disease free (7 alive and 1 dead of unrelated cause) and 10 had experienced disease recurrence (7 alive and 3 dead of metCRC). Prior to surgery, the patients demonstrated Treg-mediated suppression of TNFα and IFNγ expression that could be perturbed through the PGE(2)/cAMP pathway and the immune suppression was significantly higher in the group that later developed disease recurrence (P = 0.046). Furthermore, the post-surgery plasma PGE(2) levels were related to the clinical outcome (PGE(2) levels of 280 ± 47 vs. 704 ± 153 pg/ml (mean ± SEM) for disease free and recurrent disease, respectively). T-cell phenotyping revealed higher frequencies of COX-2(+) cells in the patients with recurrent disease. These findings support the notion that the level of Treg-mediated suppression of adaptive antitumor immune responses at the time of surgery may influence later clinical outcome of metCRC and provide valuable prognostic information.     

Successful inhibition of tumor development by specific class-3 semaphorins is associated with expression of appropriate semaphorin receptors by tumor cells

The class-3 semaphorins (sema3s) include seven family members. Six of them bind to neuropilin-1 (np1) or neuropilin-2 (np2) receptors or to both, while the seventh, sema3E, binds to the plexin-D1 receptor. Sema3B and sema3F were previously characterized as tumor suppressors and as inhibitors of tumor angiogenesis. To determine if additional class-3 semaphorins such as sema3A, sema3D, sema3E and sema3G possess anti-angiogenic and anti-tumorigenic properties, we expressed the recombinant full length semaphorins in four different tumorigenic cell lines expressing different combinations of class-3 semaphorin receptors. We show for the first time that sema3A, sema3D, sema3E and sema3G can function as potent anti-tumorigenic agents. All the semaphorins we examined were also able to reduce the concentration of tumor associated blood vessels although the potencies of the anti-angiogenic effects varied depending on the tumor cell type. Surprisingly, there was little correlation between the ability to inhibit tumor angiogenesis and their anti-tumorigenic activity. None of the semaphorins inhibited the adhesion of the tumor cells to plastic or fibronectin nor did they modulate the proliferation of tumor cells cultured in cell culture dishes. However, various semaphorins were able to inhibit the formation of soft agar colonies from tumor cells expressing appropriate semaphorin receptors, although in this case too the inhibitory effect was not always correlated with the anti-tumorigenic effect. In contrast, the anti-tumorigenic effect of each of the semaphorins correlated very well with tumor cell expression of specific signal transducing receptors for particular semaphorins. This correlation was not broken even in cases in which the tumor cells expressed significant concentrations of endogenous semaphorins. Our results suggest that combinations of different class-3 semaphorins may be more effective than single semaphorins in cases in which tumor cells express more than one type of semaphorin receptors.     

Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity.

Dopaminergic D2 receptor agonists, such as bromocriptine, are potent anti-proliferative agents in the treatment of human pituitary adenomas. We have reproduced the anti-proliferative effect of dopamine in an established pituitary cell line stably transfected with the rat D2 dopamine receptor cDNA. We found that dopaminergic inhibition of DNA synthesis parallels the stimulation of a phosphotyrosine phosphatase activity. Both actions are blocked by pertussis toxin and by the phosphotyrosine phosphatase inhibitor, vanadate. We suggest that the anti-proliferative action of dopamine is mediated, at least in part, by the dopaminergic stimulation of a phosphotyrosine phosphatase.     

Overexpression of integrin-linked kinase (ILK) is associated with tumor progression and an unfavorable prognosis in patients with colorectal cancer

Integrin-linked kinase (ILK), an intracellular serine-threonine kinase, has been reported to be overexpressed in multiple types of human malignancies, including colorectal cancer (CRC). The prognostic value of ILK in CRC, however, remains unknown. In the present study, expression of ILK in 25 paired primary CRC samples and adjacent noncancerous tissues were quantified using real-time PCR and Western blotting. ILK protein expression was analyzed in 102 archived, paraffin-embedded CRC samples using immunohistochemistry. The correlation between ILK expression and clinicopathological factors was evaluated by the χ² test. Patients’ overall survival was analyzed by Kaplan–Meier method. We found that both ILK mRNA and protein expression levels were significantly up-regulated in primary CRC samples compared with their corresponding normal tissues. Immunohistochemical analysis revealed relative high expression of ILK in 43 of 102 (42.2 %) primary CRC samples. Statistical analysis showed a significant correlation of ILK expression with tumor differentiation, lymph node metastasis, tumor invasion, and tumor-node-metastasis stage. Patients with tumors displaying high-level ILK expression showed significantly shorter overall survival (P = 0.028, log-rank test). More importantly, multivariate analysis indicated that high ILK protein expression was an independent prognostic factor for CRC patients (P = 0.026). Taken together, our data suggest that ILK overexpression is associated with tumor progression and a poor prognosis in CRC patients and may represent a novel potential prognostic marker for patients with CRC.     

IL-17 is associated with poor prognosis and promotes angiogenesis via stimulating VEGF production of cancer cells in colorectal carcinoma

IL-17, which exerts strong pro-inflammatory effects, has emerged as an important mediator in inflammation-associated cancer. However, the characteristics of IL-17-producing cells, the relevance of IL-17 to clinical parameters and its function in the development and progression of colorectal carcinoma still remain to be explored. In the present study, we first found the levels of IL-17 producing cells were significantly increased in the tumor regions of samples from colorectal carcinoma patients compared with non-tumor regions. Confocal microscopic analysis showed co-staining of IL-17 with CD4 and CD68, indicating IL-17 in colorectal carcinoma was expressed by macrophage and Th17. High expression of IL-17 was associated with high microvessel density. Univariate and multivariate analysis revealed that IL-17 was an independent prognostic factor for overall survival. To explore the underlying mechanisms of IL-17 in angiogenesis, we used PCR-array to find pro-angiogenic factor in cancer cells specifically induced by IL-17, then validated VEGF as one of factors in IL-17-mediated angiogenesis with the use of quantitative RT-PCR, ELISA and VEGF immunohistochemistry. Our results propose IL-17 as a novel indicator of prognosis in the patients with colorectal carcinoma and could serve as a novel therapeutic target for colorectal carcinoma, furthermore our results indicate that IL-17 producing cells may facilitate development of colorectal carcinoma by fostering angiogenesis via promote VEGF production from cancer cells.