Clic4, a novel protein that sensitizes ß-cells to apoptosis.

OBJECTIVES: Chloride intracellular channel protein 4 (Clic4) is a ubiquitously expressed protein involved in multiple cellular processes including cell-cycle control, cell differentiation, and apoptosis. Here, we investigated the role of Clic4 in pancreatic ß-cell apoptosis. METHODS: We used ßTC-tet cells and islets from ß-cell specific Clic4 knockout mice (ßClic4KO) and assessed cytokine-induced apoptosis, Bcl2 family protein expression and stability, and identified Clic4-interacting proteins by co-immunoprecipitation and mass spectrometry analysis. RESULTS: We show that cytokines increased Clic4 expression in ßTC-tet cells and in mouse islets and siRNA-mediated silencing of Clic4 expression in ßTC-tet cells or its genetic inactivation in islets ß-cells, reduced cytokine-induced apoptosis. This was associated with increased expression of Bcl-2 and increased expression and phosphorylation of Bad. Measurement of Bcl-2 and Bad half-lives in ßTC-tet cells showed that Clic4 silencing increased the stability of these proteins. In primary islets ß-cells, absence of Clic4 expression increased Bcl-2 and Bcl-xL expression as well as expression and phosphorylation of Bad. Mass-spectrometry analysis of proteins co-immunoprecipitated with Clic4 from ßTC-tet cells showed no association of Clic4 with Bcl-2 family proteins. However, Clic4 co-purified with proteins from the proteasome suggesting a possible role for Clic4 in regulating protein degradation. CONCLUSIONS: Collectively, our data show that Clic4 is a cytokine-induced gene that sensitizes ß-cells to apoptosis by reducing the steady state levels of Bcl-2, Bad and phosphorylated Bad.

ING2 controls the G1 to S-phase transition by regulating p21 expression.

ING2 (Inhibitor of Growth 2) is a candidate tumor suppressive protein frequently lost in human tumors. Recently, we have reported that ING2 downregulation impairs DNA replication forks progression and leads to genome instability. To better understand the tumor suppressive functions of ING2 and its role in the cell cycle, we downregulated its expression in cells and studied the consequences of this downregulation on the G(1)/S transition. We observed that the inhibition of ING2 expression accelerated the progression of cells from G(1) to S-phase, and was accompanied by a decrease of p21 expression. Moreover, we show that the regulation of p21 by ING2 is independent of the tumor suppressive protein p53. Interestingly, this function seems to be unique for ING2 since its closest homolog ING1 does not regulate the G(1)/S transition. It has been suggested previously that ING2 may modulate the trimethylation of H3K4 at the promoter of p21. Accordingly, our results suggest that there may be a link between the regulation of the G(1)/S transition by ING2 and the level of H3K4Me3. All together, these results bring new information concerning the role of ING2 in the regulation of the cell cycle and suggest that it may play important roles in controlling several S-phase checkpoints.

Expression of candidate tumor suppressor gene ING2 is lost in non-small cell lung carcinoma.

ING2 is a candidate tumor suppressor gene involved in cell cycle control, apoptosis and senescence. Furthermore, we have recently shown that loss of ING2 expression is associated with increased genome instability. We investigated its status in a series of 120 non-small cell lung cancer (NSCLC) by using immunohistochemistry (IHC). The results showed that ING2 protein expression is downregulated in more than 50% of NSCLC, with a higher frequency in adenocarcinoma (ADK) as compared to squamous cell carcinoma (SCC) (68% versus 45%, P=0.021). Loss of ING2 expression occurs in a high proportion of tumors from stage I and was not associated with patient's gender, age and 5-year survival. When investigating the possible mechanisms responsible for the decrease of ING2 expression, we did not observe any loss of heterozygosity or mutation in the ING2 gene. However, in 95% of the cases examined, we identified a silent single nucleotide polymorphism (SNP). By using quantitative RT-PCR, we found that ING2 loss of expression may be due to the decrease of its mRNA level. Analysis of CpG islands present in the promoter region of the ING2 gene did not allow for the detection of methylation. Mechanistically, although p53 can regulate ING2 transcription and ING2 enhances p53 activity, no correlation between ING2 and p53 IHC status was observed. Overall, these results indicate that loss of ING2 expression could contribute to lung tumorigenesis independently of p53.

WNT16B is a new marker of cellular senescence that regulates p53 activity and the phosphoinositide 3-kinase/AKT pathway.

Senescence is a tumor suppression mechanism that is induced by several stimuli, including oncogenic signaling and telomere shortening, and controlled by the p53/p21(WAF1) signaling pathway. Recently, a critical role for secreted factors has emerged, suggesting that extracellular signals are necessary for the onset and maintenance of senescence. Conversely, factors secreted by senescent cells may promote tumor growth. By using expression profiling techniques, we searched for secreted factors that were overexpressed in fibroblasts undergoing replicative senescence. We identified WNT16B, a member of the WNT family of secreted proteins. We found that WNT16B is overexpressed in cells undergoing stress-induced premature senescence and oncogene-induced senescence in both MRC5 cell line and the in vivo murine model of K-Ras(V12)-induced senescence. By small interfering RNA experiments, we observed that both p53 and WNT16B are necessary for the onset of replicative senescence. WNT16B expression is required for the full transcriptional activation of p21(WAF1). Moreover, WNT16B regulates activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overall, we identified WNT16B as a new marker of senescence that regulates p53 activity and the PI3K/AKT pathway and is necessary for the onset of replicative senescence.

ING2 controls the progression of DNA replication forks to maintain genome stability.

Inhibitor of growth 2 (ING2) is a candidate tumour suppressor gene the expression of which is frequently lost in tumours. Here, we identified a new function for ING2 in the control of DNA replication and in the maintenance of genome stability. Global replication rate was markedly reduced during normal S-phase in small interfering RNA (siRNA) ING2 cells, as seen in a DNA fibre spreading experiment. Accordingly, we found that ING2 interacts with proliferating cell nuclear antigen and regulates its amount to the chromatin fraction, allowing normal replication progression and normal cell proliferation. Deregulation of DNA replication has been previously associated with genome instability. Hence, a high proportion of siRNA ING2 cells presented endoreduplication of their genome as well as an increased frequency of sister chromatid exchange. Thus, we propose for the first time that ING2 might function as a tumour suppressor gene by directly maintaining DNA integrity.

The new tumor suppressor genes ING: genomic structure and status in cancer.

The Inhibitor of Growth 1 (ING1) gene has been identified and characterized as a Type-II tumor suppressor gene (TSG). Subsequently, 4 additional members of the family were identified by homology search. ING proteins contain a nuclear localization sequence (NLS) and a plant homeo domain (PHD) finger motif in their C-terminus. These proteins are involved in numerous signaling pathways especially in 2 tumor suppressor pathways: apoptosis and senescence. In human tumors, several studies have shown that the expression of ING1 is frequently lost or downregulated. It occurs most frequently at the RNA level, and thus epigenetics mechanism could be involved. We summarize the current knowledge on ING proteins functions and their involvement in various signaling pathways. We also review the studies that have investigated the ING protein status in human tumors. The interest of ING proteins as biomarkers and their role in tumor initiation and progression is discussed.

Inhibitor of growth 4 suppresses cell spreading and cell migration by interacting with a novel binding partner, liprin alpha1.

Inhibitor of growth 4 (ING4) is a candidate tumor suppressor that plays a major role in gene regulation, cell cycle control, apoptosis, and angiogenesis. ING4 expression is down-regulated in glioblastoma cells and head and neck squamous cell carcinoma. Here, we identified liprin alpha1/PPFIA1, a cytoplasmic protein necessary for focal adhesion formation and axon guidance, as a novel interacting protein with ING4. ING4 and liprin alpha1 colocalized at lamellipodia in the vicinity of vinculin. Overexpressed ING4 suppressed cell spreading and cell migration. In contrast, overexpressed liprin alpha1 enhanced cell spreading and cell migration. Knockdown of endogenous ING4 with RNA interference induced cell motility, whereas knockdown of endogenous liprin alpha1 suppressed cell motility. ING4 also suppressed cell motility that was enhanced by liprin alpha1. However, ING4 did not further suppress cell motility when liprin alpha1 was suppressed with RNA interference, suggesting a functional and mechanistic interdependence between these proteins. In addition to its nuclear functions, cytoplasmic ING4 interacts with liprin alpha1 to regulate cell migration and, with its known antiangiogenic function, may prevent invasion and metastasis.