Guohao Wu

Prolyl Hydroxylase-3 Is Down-regulated in Colorectal Cancer Cells and Inhibits IKKβ Independent of Hydroxylase Activity

BACKGROUND & AIMS Prolyl hydroxylase (PHD) hydroxylates hypoxia inducible factor (HIF) alpha, leading to HIFalpha degradation. The PHD family comprises PHD1, PHD2, and PHD3. The enzymatic activity of PHDs is O(2)-dependent, so PHDs are believed to be oxygen sensors as well as tumor suppressors. However, the expression pattern of PHDs in colorectal cancer and the correlation between their expression level and tumorigenesis is unclear. METHODS We determined the expression of PHDs in 60 human primary colorectal carcinoma tissues, paired with normal colorectal tissues. PHD3 expression levels were knocked down using small interfering RNA (siRNA); cells were analyzed by immunoblotting, immunoprecipitation, and histochemical analyses. In vivo tumor growth was analyzed in nu/nu mice. RESULTS Expression of PHD3 is decreased in colorectal cancer tissues. Decreased expression of PHD3 is associated with higher tumor grade and metastasis. PHD3 inhibits phosphorylation of inhibitor of kappaB (IkappaB) kinase (IKK) beta and activation of (NF) kappaB, independent of its hydroxylase activity. PHD3 associates with IKKbeta but does not target it for destruction; instead, PHD3 blocks the interaction between IKKbeta and Hsp90 that is required for phosphorylation of IKKbeta. Knockdown of PHD3 increased resistance of colorectal cancer cells to the effects of tumor necrosis factor-alpha and tumorigenesis. CONCLUSIONS PHD3 appears to be a tumor suppressor in colorectal cancer cells that inhibits IKKbeta/NF-kappaB signaling, independent of its hydroxylase activity. Activation of NF-kappaB has been observed in colon cancer. Determination of PHD3 status could aid targeted therapy selection for patients with colorectal tumors that have increased NF-kappaB activity.

Pyruvate kinase type M2 is upregulated in colorectal cancer and promotes proliferation and migration of colon cancer cells.

Pyruvate kinase type M2 (PKM2) has been reported to be involved in aerobic glycolysis and cell growth in various tumors. However, the expression pattern of PKM2 in colorectal cancer (CRC) and the correlation between PKM2 expression and CRC remains unclear. The aim of this study is to investigate PKM2 expression and its possible role in CRC. We found that expression of PKM2 was increased in CRC and the increased PKM2 expression was associated with later stage and lymph metastasis of the tumors. Knockdown of PKM2 suppressed the aerobic glycolysis and decreased lactate production of colon cancer RKO cells. Knockdown of PKM2 repressed proliferation and migration of the cells. Inhibition of PKM2 suppressed xenograft tumor growth of RKO cells in vivo. These results suggest that the expression of PKM2 plays a critical role in development of CRC, and it may provide a growth advantage for colon cancer cells. Thus, PKM2 might be a potential therapeutic target for CRC.

BCLAF1 and its splicing regulator SRSF10 regulate the tumorigenic potential of colon cancer cells

Bcl-2-associated transcription factor 1 (BCLAF1) is known to be involved in multiple biological processes. Although several splice variants of BCLAF1 have been identified, little is known about how BCLAF1 splicing is regulated or the contribution of alternative splicing to its developmental functions. Here we find that inclusion of alternative exon5a was significantly increased in colorectal cancer (CRC) samples. Knockdown of the BCLAF1 protein isoform resulting from exon5a inclusion inhibited growth and that its overexpression increased tumorigenic potential. We also found that the splicing factor SRSF10 stimulates inclusion of exon5a and has growth-inducing activity. Importantly, the upregulation of SRSF10 expression observed in clinical CRC samples parallels the increased inclusion of BCLAF1 exon5a, both of which are associated with higher tumour grade. These findings identify SRSF10 as a key regulator of BCLAF1 pre-mRNA splicing and the maintenance of oncogenic features in human colon cancer cells.

PAQR3 Plays a Suppressive Role in the Tumorigenesis of Colorectal Cancers

PAQR3 is a member of the progestin and adipoQ receptor (PAQR) family and was recently characterized as a spatial regulator that negatively modulates Ras/Raf/MEK/ERK signaling cascade. However, little is known about the physiological functions of PAQR3 in the tumorigenesis of colorectal cancers. The function of PAQR3 in colorectal cancer development in mice was analyzed by crossing Paqr3-depleted mice with Apc(Min/+) mice that have a germline mutation of the gene-encoding tumor suppressor adenomatous polyposis coli (APC). The survival time and tumor area in the small intestine of the Apc(Min/+) mice was significantly aggravated by Paqr3 deletion. The cell proliferation rate, anchorage-independent growth, EGF-stimulated ERK phosphorylation and EGF-induced nuclear accumulation of β-catenin were inhibited by PAQR3 overexpression and enhanced by PAQR3 knockdown in SW-480 colorectal cancer cells. In humans, the expression level of PAQR3 was significantly decreased in colorectal cancer samples in comparison with adjacent normal tissues. In addition, the expression level of PAQR3 was inversely associated with tumor grade in the colorectal cancer samples. Collectively, our data reveal for the first time that PAQR3 has a tumor suppressor activity in the development of colorectal cancers.

The orphan nuclear receptor EAR2 is overexpressed in colorectal cancer and it regulates survivability of colon cancer cells

EAR2 is a member of the chick ovalbumin upstream promoter-transcription factors (COUP-TFs). COUP-TFs belong to orphan nuclear receptors and regulate many biological processes. Little is known regarding EAR2 in cancer, though much progress has been made in understanding the function of other COUP-TF members. The aim of this study is to investigate the expression and possible function of EAR2 in colorectal cancer. We determined expression of EAR2 in human primary colorectal malignant tumors and their paired adjacent normal colorectal tissues. We found that expression of EAR2 was upregulated in colorectal tumors. Knockdown of EAR2 induced apoptosis of colon cancer cells, suggesting that EAR2 may function to regulate survivability of colon cancer cells. In vivo tumor study demonstrated that knockdown of EAR2 inhibited the xenograft growth of colon cancer cells. We found that knockdown of EAR2 inhibited the expression of X-linked inhibitor of apoptosis protein (XIAP), suggesting that EAR2 regulates cell survivability, at least partly, through XIAP. In this manuscript, we demonstrated that expression of EAR2 was elevated in colorectal cancer and knockdown of EAR2 reduced survivability and tumor growth of colon cancer cells. Our results suggest that EAR2 plays an important role in development of colorectal cancer. The findings also suggest that EAR2 may serve as a potential therapeutic target of colorectal cancer.

PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function.

Background: Prolyl hydroxylases (PHDs) are linked to inflammatory bowel diseases (IBD); however, the exact role of PHD3, a member of PHD family, in IBD is unknown. Results: Deletion of Phd3 in mice intestinal epithelial cells causes spontaneous colitis. Conclusion: PHD3 protects the intestinal epithelial barrier function. Significance: The results are helpful for the development of therapeutic strategies for IBD. Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.

A non-secretory form of FAM3B promotes invasion and metastasis of human colon cancer cells by upregulating Slug expression

FAM3B mRNA has been predicted to have multiple splicing forms. Its secretory form PANDER is decreased in gastric cancers with high invasiveness and metastasis. Here we found that its non-secretory form FAM3B-258 was highly expressed in most colon cancer cell lines and colorectal adenocarcinoma tissues but not hepatocellular carcinoma, lung carcinoma and pancreatic adenocarcinoma cell lines. Elevation of FAM3B-258 was associated with poor cancer cell differentiation. Stable overexpression of FAM3B-258 in colon cancer cells downregulated adhesion proteins, upregulated Slug and Cdc42, promoted cell migration and invasion in vitro and metastasis in nude mice. Slug mediated FAM3B-258-induced downregulation of adhesion molecules, upregulation of Cdc42, and invasion of colon cancer cells. The expression of FAM3B-258 in human colorectal adenocarcinomas was positively correlated with Slug. These results suggest that FAM3B-258 promotes colon cancer cell invasion and metastasis through upregulation of Slug.

PAX2 Protein Induces Expression of Cyclin D1 through Activating AP-1 Protein and Promotes Proliferation of Colon Cancer Cells

Background: PAX2, a transcription factor, plays a role in embryogenesis. Results: Knockdown of PAX2 inhibits colon cancer cells proliferation and xenograft growth. PAX2 activates AP-1, leading to increased expression of cyclin D1. Conclusion: PAX2 promotes proliferation of colon cancer cells via AP-1. Significance: PAX2 exhibits oncogenic properties in colon cancer cells and may serve as a target for cancer therapy. Paired box (PAX) 2, a transcription factor, plays a critical role in embryogenesis. When aberrantly expressed in adult tissues, it generally exhibits oncogenic properties. However, the underlying mechanisms remain unclear. We reported previously that the expression of PAX2 was up-regulated in human colon cancers. However, the role of PAX2 in colon cancer cells has yet to be determined. The aim of this study is to determine the function of PAX2 in colon cancer cells and to investigate the possible mechanisms underlain. We find that knockdown of PAX2 inhibits proliferation and xenograft growth of colon cancer cells. Inhibition of PAX2 results in a decreased expression of cyclin D1. Expression of cyclin D1 is found increased in human primary colon malignant tumors, and its expression is associated with that of PAX2. These data indicate that PAX2 is a positive regulator of expression of cyclin D1. We find that knockdown of PAX2 inhibits the activity of AP-1, a transcription factor that induces cyclin D1 expression, implying that PAX2 induces cyclin D1 through AP-1. PAX2 has little effect on expression of AP-1 members including c-Jun, c-Fos, and JunB. Our data show that PAX2 prevents JunB from binding c-Jun and enhances phosphorylation of c-Jun, which may elevate the activity of AP-1. Taken together, these results suggest that PAX2 promotes proliferation of colon cancer cells through AP-1.

Dietary Fat Intake and Risk of Gastric Cancer: A Meta-Analysis of Observational Studies

Background and Objectives Consumption of dietary fat has been reported to be associated with gastric cancer risk, but the results of epidemiologic studies remain inconsistent. We conducted a meta-analysis to summarize the evidence regarding the association between dietary fat intake and gastric cancer risk. Methods A comprehensive search of PubMed and EMBASE was performed to identify observational studies providing quantitative estimates between dietary fat and gastric cancer risk. Random effects model was used to calculate the summary relative risk(SRR) in the highest versus lowest analysis. Categorical dose-response analysis was conducted to quantify the association between dietary fat intake and gastric cancer risk. Heterogeneity among studies was evaluated using I2 and tau2(between study variance)statistics. Subgroup analysis and publication bias analysis were also performed. Results Twenty-two articles were included in the meta-analysis. The SRR for gastric cancer was 1.18 for individuals with highest intake versus lowest intake of total fat (95% confidence interval [CI]: 0.999–1.39; n = 28; P< 0.001; tau2 = 0.12; I2 = 69.5%, 95% CI: 55%-79%) and 1.08 with a daily increase in total fat intake (20 g/d) (95%CI: 1.02–1.14; n = 6; P = 0.09; tau2 = 0.002; I2 = 46.8%, 95% CI: 0%-79%). Positive association between saturated fat intake (SRR = 1.31; 95%CI: 1.09–1.58;n = 18;P<0.001; tau2 = 0.08; I2 = 60.6%, 95% CI: 34%-76%), inverse association between polyunsaturated fat intake (SRR = 0.77; 95%CI: 0.65–0.92; n = 16; P = 0.003; tau2 = 0.06; I2 = 56.2%, 95% CI: 23%-75%) and vegetable fat intake (SRR = 0.55; 95%CI: 0.41–0.74; n = 4;P = 0.12; tau2 = 0.04; I2 = 48.6%, 95% CI: 0%-83%), and no association between monounsaturated fat intake (SRR = 1.00; 95%CI: 0.79–1.25; n = 14; P< 0.001; tau2 = 0.10; I2 = 63.0%, 95% CI: 34%-79%) and animal fat intake (SRR = 1.10; 95%CI: 0.90–1.33; n = 6; P = 0.13;tau2 = 0.02; I2 = 42.0%, 95% CI: 0%-70%) and gastric cancer risk were observed. Conclusions Our results suggest that intake of total fat is potentially positively associated with gastric cancer risk, and specific subtypes of fats account for different effects. However, these findings should be confirmed by further well-designed cohort studieswith detailed dietary assessments and strict control of confounders.

Prolyl hydroxylase domain protein 3 targets Pax2 for destruction

Prolyl hydroxylase domain proteins (PHDs) hydroxylate HIFα in the presence of oxygen, leading to HIFα proteasomal destruction. The PHDs family comprises PHD1, 2, and 3. Recent studies indicate that, in addition to HIFα, PHDs have other substrates. Paired box (Pax) 2, a transcription factor, was found aberrantly expressed in a variety of cancers. However, the underlying mechanisms remain unknown. Here we demonstrate that PHD3 is a negative regulator of expression of Pax2. We found that PHD3 bound to Pax2 and mediated Pax2 destruction directly. Inhibition of PHD3 hydroxylase activity led to upregulation of Pax2 protein but not mRNA level. We found that Pax2 protein was increased and PHD3 protein was decreased in colorectal cancer, and the increased Pax2 was associated with decreased PHD3. Our results suggest that PHD3 targets Pax2 for destruction. The findings may disclose a mechanism for the regulation of Pax2 expression in cancer cells.