Selectins mediate leukocyte rolling and prime leukocytes for integrin-mediated leukocyte adhesion. However, neither the in vivo importance of nor the signaling pathway by which selectin-mediated integrin activation occurs has been determined. We report here that P-selectin-deficient mice manifested impaired leukocyte adhesion, which was 'rescued' by soluble P-selectin. Mechanistically, the cytoplasmic domain of P-selectin glycoprotein ligand 1 formed a constitutive complex with Nef-associated factor 1. After binding of P-selectin, Src kinases phosphorylated Nef-associated factor 1, which recruited the phosphoinositide-3-OH kinase p85-p110delta heterodimer and resulted in activation of leukocyte integrins. Inhibition of this signal-transduction pathway diminished the adhesion of leukocytes to capillary venules and suppressed peritoneal infiltration of leukocytes. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by P-selectin glycoprotein ligand 1.

In cancer cells, glucose is often converted into lactic acid, which is known as the 'Warburg effect'. The reason that cancer cells have a higher rate of aerobic glycolysis, but not oxidative phosphorylation, remains largely unclear. Herein, we proposed an epigenetic mechanism of the Warburg effect. Fructose-1,6-bisphosphatase-1 (FBP1), which functions to antagonize glycolysis was downregulated through NF-kappaB pathway in Ras-transformed NIH3T3 cells. Restoration of FBP1 expression suppressed anchorage-independent growth, indicating the relevance of FBP1 downregulation in carcinogenesis. Indeed, FBP1 was downregulated in gastric carcinomas (Po0.01, n ¼ 22) and gastric cancer cell lines (57%, 4/7). Restoration of FBP1 expression reduced growth and glycolysis in gastric cancer cells. Moreover, FBP1 downregulation was reversed by pharmacological demethylation. Its promoter was hypermethylated in gastric cancer cell lines (57%, 4/7) and gastric carcinomas (33%, 33/ 101). Inhibition of NF-kappaB restored FBP1 expression, partially through demethylation of FBP1 promoter. Notably, Cox regression analysis revealed FBP1 promoter methylation as an independent prognosis predicator for gastric cancer (hazard ratio: 3.60, P ¼ 0.010). In summary, we found that NF-kappaB functions downstream of Ras to promote epigenetic downregulation of FBP1. Promoter methylation of FBP1 can be used as a new biomarker for prognosis prediction of gastric cancer. Such an important epigenetic link between glycolysis and carcinogenesis partly explains the Warburg effect.

Purpose: Nasopharyngeal carcinoma is prevalent in southern China and Southeast Asia, with distinct geographic and ethnic distribution. One candidate susceptibility locus has been identified at 4p11-14, with the associated candidate gene(s) not identified yet. This study investigated the role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in nasopharyngeal carcinoma pathogenesis.Experimental Design: UCHL1 expression and methylation were examined in nasopharyngeal carcinoma. Furthermore, the mechanism of its tumor-suppressive function was elucidated in nasopharyngeal carcinoma cells.Results: Through genomewide expression profiling, we identified UCHL1, a 4p14 gene normally expressed in normal upper respiratory tract tissues, being silenced in all nasopharyngeal carcinoma cell lines. Its silencing is mediated by CpG methylation because UCHL1 promoter methylation was detected in all silenced cell lines, and pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. UCHL1 methylation was also frequently detected in primary tumors but only weakly detected in few normal nasopharyngeal tissues, indicating that the methylation-mediated silencing of UCHL1 is important in nasopharyngeal carcinoma pathogenesis. Ectopic UCHL1 expression dramatically inhibited the growth of nasopharyngeal carcinoma cells through promoting tumor cell apoptosis. We further found that UCHL1 formed a complex with p53/p14 ARF /Mdm2 p53 binding protein homolog (mouse), MDM2 and activated the p53 signaling pathway. UCHL1 expression extended p53 and p14ARF protein half-life and shortened MDM2 protein half-life. Conclusions:These results indicate that UCHL1 could deubiquitinate p53 and p14 ARF and ubiquitinate MDM2 for p53 stabilization to promote p53 signaling, thus involved in nasopharyngeal carcinoma pathogenesis, whereas it is frequently silenced in this tumor.

The ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a carboxyl-terminal ubiquitin hydrolase regulating cellular ubiquitin levels, recently suggested as a tumor suppressor. However, the role of UCHL1 in hepatocellular carcinoma (HCC) is not clear. We investigated the expression and DNA methylation of the UCHL1 in primary HCC, liver metastases from digestive carcinomas, and primary digestive cancers. UCHL1 is expressed in all normal tissues and immortalized normal epithelial cell lines, but was low or silenced in 77% (10/13) of HCC cell lines, which is well correlated with its promoter methylation status. Methylation was further detected in 44% (12/27) of HCCs, but less in metastatic tumors generated from colorectal and stomach in the liver (19%, 3/16; P < 0.05). Methylation was also detected in primary digestive tumors, including 71% (

Tumor Suppressor genes (TSGs) often locate at chromosomal regions with frequent deletions in tumors. Loss of 16q23 occurs frequently in multiple tumors, indicating the presence of critical TSGs at this locus, such as the well-studied WWOX. Herein we found that ADAMTS18, located next to WWOX, was significantly downregulated in multiple carcinoma cell lines. No deletion of ADAMTS18 was detected with multiplex differential DNA-PCR or high resolution 1-Mb array-based CGH analysis. Instead, methylation of the ADAMTS18 promoter CpG Island was frequently detected with methylation-specific PCR and bisulfite genome sequencing in multiple carcinoma cell lines and primary carcinomas, but not in any non-tumor cell line and normal epithelial tissue. Both pharmacological and genetic demethylation dramatically induced ADAMTS18 expression, indicating that CpG methylation directly contributes to the tumor-specific silencing of ADAMTS18. Ectopic ADAMTS18 expression leads to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells lacking the expression. Thus, through functional epigenetics, we identified ADAMTS18 as a novel functional tumor suppressor, being frequently inactivated epigenetically in multiple carcinomas.

BackgroundNonhistone chromosomal proteins in concert with histones play important roles in the replication and repair of DNA and in the regulation of gene expression. The deregulation of these proteins can contribute to the development of a variety of diseases such as cancer. As a nonhistone chromosomal protein, chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as the product of a novel tumor suppressor gene (TSG), promoting the transcription of p19ink4a and p16arf. The inactivation of CHD5 was achieved partly through genetic deletion since it is located in 1p36, a region frequently deleted in human tumors. In this study, we aim to study the involvement of CHD5 in gastric cancer, the second most common cancer worldwide.MethodsCHD5 expression in a panel of gastric cancer cells were determined by quantitative RT-PCR. The methylation of CHD5 was evaluated by methylation specific PCR and bisulfite genome sequencing. The effect of CHD5 on growth of gastric cancer cells was tested by colony formation assay.ResultsCHD5 expression was down-regulated in all of gastric cancer cell lines used (100%, 7/7) and significantly restored after pharmacological demethylation. Methylation of CHD5 promoter was detected in all of seven gastric cancer cell lines and in the majority of primary gastric carcinoma tissues examined (73%, 11/15). Finally, ectopic expression of CHD5 in gastric cancer cells led to a significant growth inhibition.ConclusionCHD5 was a TSG epigenetically down-regulated in gastric cancer.

The enrichment of Fusobacterium nucleatum (Fn) has been identified in CRC patients and associated with worse outcomes. However, whether Fn was involved in the metastasis of CRC was not well determined. Here, we found that the abundance of Fn was significantly increased in CRC patients with lymph nodes metastasis. To further clarify the role of Fn in CRC metastasis, we performed transwell and wound healing assays after incubating CRC cell lines with or without Fn and injected Fn-treated or untreated CRC cells into nude mice via tail vein. The results indicated that Fn infection promoted CRC cells migration in vitro, as well as lung metastasis in vivo. Interestingly, colonization of Fn was detected in metastatic lung lesions of nude mice by fluorescence in situ hybridization. Mechanistically, RNA sequencing and validation study revealed that Fn significantly upregulated the expression of long non-coding RNA Keratin7-antisense (KRT7-AS) and Keratin7 (KRT7) in CRC cells. Importantly, Fn-induced CRC lung metastasis was attenuated by the depletion of KRT7-AS. In addition, KRT7-AS facilitated CRC cells migration by upregulating KRT7. Subsequently, we found that NF-κB signaling pathway was involved in the upregulation of KRT7-AS upon Fn infection. In conclusion, Fn infection upregulated KRT7-AS/KRT7 by activating NF-κB pathway, which promoted CRC cell migration in vitro and metastasis in vivo.

Ras has achieved notoriety as an oncogene aberrantly activated in multiple human tumors. Approximately 30% of all human tumors express an oncogenic form of this GTPase that is locked in an active conformation as a result of being insensitive to Ras GTPaseactivating proteins (GAPs), proteins that normally regulate the inactivation of Ras by enhancing its intrinsic GTPase activity. Besides oncogenic mutations in Ras, signaling by wild-type Ras is also frequently deregulated in tumors through aberrant coupling to activated cell surface receptors. This indicates that alternative mechanisms of aberrant wild-type Ras activation may be involved in tumorigenesis. Here, we describe another mechanism through which aberrant Ras activation is achieved in human cancers. We have established that Ras GTPase-activating-like protein (RASAL), a Ca 2؉ -regulated Ras GAP that decodes the frequency of Ca 2؉ oscillations, is silenced through CpG methylation in multiple tumors. With the finding that ectopic expression of catalytically active RASAL leads to growth inhibition of these tumor cells by Ras inactivation, we have provided evidence that epigenetically silencing of this Ras GAP represents a mechanism of aberrant Ras activation in certain cancers. Our demonstration that RASAL constitutes a tumor suppressor gene has therefore further emphasized the importance of Ca 2؉ in the regulation of Ras signaling and has established that deregulation of this pathway is an important step in Ras-mediated tumorigenesis.calcium ͉ Ras GTPase-activating-like protein ͉ tumorigenesis