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Supplementary Figure 1 from KLF4-Mediated Negative Regulation of IFITM3 Expression Plays a Critical Role in Colon Cancer Pathogenesis

Purpose: To use gene transcript profiling to identify cancer-associated gene expression.Experimental Design: Methods included (i) marker discovery using laser capture microdissection (LCM)-assisted specimen preparation and antisense RNA-long serial analysis of gene expression (aRNA-LongSAGE) on matched colon cancer and uninvolved colon tissue specimens (n = 5). Candidate tumor-associated genes were selected by combining the LongSAGE libraries reported herein with our previous colon cancer LCM-microarray transcript profiling data; (ii) marker selection and validation by quantitative real-time PCR (n = 15) and immunohistochemistry (n = 31); and (iii) independent validation on multiple tissue microarray (n = 203).Results: Among 30 upregulated and 73 downregulated genes, upregulation of fermitin family member 1 (FERMT1), adenosylhomocysteinase (AHCY), secernin 1 (SCRN1), and SAC3 domain-containing protein 1 (SAC3D1) expression and downregulation of IgJ and MALL expression in colon cancer were confirmed by quantitative PCR. FERMT1 and AHCY protein expression was also upregulated in colon cancer compared with uninvolved colon mucosa, and FERMT1 expression showed upregulation in colon adenoma. Patients with moderate/strong tumor FERMT1 protein expression (n = 122) showed significantly poorer overall survival (OS; P = 0.011) and disease-free survival (DFS; P = 0.005) than patients with negative/weak tumor FERMT1 protein expression (n = 81). Multivariate Cox regression analysis showed that FERMT1 protein expression was also an independent prognostic factor for OS (P = 0.018) and DFS (P = 0.009). In addition, upregulated FERMT1 protein expression appeared to have some specificity among alimentary tract tumors.Conclusions: FERMT1 is a novel prognostic factor for colon carcinoma. Clin Cancer Res; 17(9); 2908–18. ©2011 AACR.

Supplementary Figure legends

Supplementary Tables 1-9 from KLF4-Mediated Negative Regulation of IFITM3 Expression Plays a Critical Role in Colon Cancer Pathogenesis

Supplementary Figures S1-S3; Supplementary Tables S1-S4.

Supplementary Fig. S1. KLF4 protein expression in human HCC specimens and its association with tumor grade. Supplementary Fig. S2. KLF4 protein expression in human HCC and paired nontumor tissue specimens. Supplementary Fig. S3. Expression of differentiation-associated markers in microdissected well-differentiated (grade 1) and poorly differentiated (grade 3) HCC cells in frozen tissue sesctions. Supplementary Fig. S4. Characterization of ZFD-deleted KLF4 protein (KLF4 ZFD). Supplementary Fig. S5. The influence of treatment with sodium butyrate on the expression of LETFs and KLF4. Supplementary Fig. S6. HNF-6 and KLF4 protein expression in human HCC specimens and their association with tumor grade. Supplementary Fig. S7. Alb-Cremediated KLF4 deletion in the murine liver. Supplementary Fig. S8. Basic activity of HNF-6 promoter reporters in HCC cells. Supplementary Fig. S9. Endogenous binding of KLF4 to the HNF-6 promoter. Supplementary Fig. S10. Prognostic significance of KLF4 and HNF-6 expression in HCC patients after OLT.

Supplementary Table S1. Primer Information for and the Genes Studied Using Real-time PCR and RT-PCR Analysis Supplementary Table S2. KLF4 Protein Expression in HCC and Matched Nontumor Tissue Specimens (n= 98*) Supplementary Table S3. Associations Between KLF4 Expression and Clinicopathological Parameters in HCC Patients Who Underwent OLT (n = 98*) Supplementary Table S4. Differences in HNF-6 Protein Expression in HCC and Matched Nontumor Tissue Specimens (n = 99) Supplementary Table S5. Association Between HNF-6 Expression and Clinicopathological Parameters in HCC Patients Who Underwent OLT (n = 99) Supplementary Table S6. Correlation Between KLF4 and HNF-6 Protein Expression in Human HCC Specimens (n = 98†) Supplementary Table S7. Univrariate and Multivariate Analysis of Parameters Related to OS and RFS in HCC Patients After OLT

Immunohistochemistry and TMA Analysis Western Blot Analysis Cell Lines and Culture Conditions Plasmids and Small Interfering RNAs Transient Transfection Conditional Deletion of Klf4 in Murine Hepatocytes RNA Extraction, RT-PCR and Real-time PCR LCM Genotyping and Gene Expression Analyses Cell Immunofluorescence Construction of Mutant KLF4 ZFD-Expressing Vectors Construction of HNF-6 Promoter Reporter Plasmids and Mutagenesis Promoter Reporter and Dual Luciferase Assay ChIP Assay Cell Proliferation Assay Flow Cytometric Cell-Cycle Analysis Scratch Assay Cell Invasion Assay Mouse Xenograft Tumor Study

Purpose: Tumor differentiation is a behavioral index for hepatocellular carcinoma (HCC) and a prognostic factor for patients with HCC who undergo orthotopic liver transplantation (OLT). However, the molecular basis for HCC differentiation and prognostic value of the underlying molecules that regulate HCC differentiation are unclear. In this study, we defined a potential driver pathway for HCC differentiation and prognostication.Experimental Design: The regulation and function of Krüppel-like factor 4 (KLF4) and hepatocyte nuclear factor-6 (HNF-6) in HCC differentiation was evaluated using human tissues, molecular and cell biology, and animal models, and its prognostic significance was determined according to its impact on patient survival.Results: There was a direct relationship between the expression levels of KLF4 and HNF6 in HCC. Reduced KLF4 or HNF6 expression correlated with high HCC grade. Poorly differentiated HCC cells had lower expression of KLF4 or HNF6 and differentiation-associated markers than did well-differentiated cells. Elevated KLF4 of HNF6 expression induced differentiation of poorly differentiated hepatoma cells. Mechanistically, KLF4 trans-activated HNF-6 expression. Restored HNF-6 expression upregulated expression of differentiation-associated markers and inhibited HCC cell migration and invasion, whereas HNF-6 knockdown did the opposite. Loss of KLF4 expression in primary HCC correlated with reduced overall survival and shortened relapse-free survival durations after OLT. Combination of KLF4 expression and the Milan criteria improved prognostication for HCC after OLT.Conclusions: The dysregulated KLF4/HNF-6 pathway drives dedifferentition and progression of HCC, and KLF4 is a biomarker for accurate prognostication of patients with HCC treated by OLT when integrated with the Milan Criteria. Clin Cancer Res; 22(2); 502–12. ©2015 AACR.

Purpose:IFITM3, an IFN-inducible gene, is overexpressed in human colorectal cancer. In this study, we sought to determine the clinical significance and underlying mechanisms of its dysregulated expression in human colon tumor specimens and murine models of this disease.Experimental Design: IFITM3 expression in a tissue microarray of tumor and matched normal colon tissue specimens and lymph node metastasis specimens obtained from 203 patients with colon cancer was measured immunohistochemically.Results: IFITM3 was expressed at higher levels in colon tumors and, particularly, nodal metastases than in normal colon tissue. A Cox proportional hazards model showed that IFITM3 expression was an independent prognostic factor for disease-free survival in patients with colon cancer. Knockdown of IFITM3 expression by a specific siRNA significantly suppressed the proliferation, colony formation, migration, and invasion of colon cancer cells in vitro and tumor growth and metastasis in a xenograft model. Restored expression of KLF4, a putative tumor suppressor, downregulated IFITM3 expression in colon cancer cells in vitro. Two KLF4-binding sites in the IFITM3 promoter bound specifically to KLF4 protein in a chromatin immunoprecipitation assay and promoter mutagenesis analyses. Specific deletion of KLF4 led to IFITM3 overexpression in colon mucosa in Villin-Cre+;Klf4fl/fl mice. An inverse correlation between loss of KLF4 expression and IFITM3 overexpression was evident in human colon tumors.Conclusion: These clinical and mechanistic findings indicate that IFITM3 is a direct transcriptional target of KLF4 and that dysregulated KLF4 expression leads to aberrant IFITM3 expression, thus contributing to colon cancer progression and metastasis. Clin Cancer Res; 17(11); 3558–68. ©2011 AACR.

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Purpose: The mammalian Forkhead Box (Fox) transcription factor FOXM1 is implicated in tumorigenesis including mouse intestinal cancer. However, the clinical significance of FOXM1 signaling in human colorectal cancer pathogenesis remains unknown.Experimental Design: We investigated FOXM1 expression in 203 cases of primary colon cancer and matched normal colon tissue specimens and explored the underlying mechanisms of altered FOXM1 expression and the impact of this altered expression on colon cancer growth and metastasis using in vitro and animal models of colon cancer.Results: We found weak expression of FOXM1 protein in the colon mucosa, whereas we observed strong FOXM1 expression in tumor-cell nuclei of colon cancer and lymph node metastases. A Cox proportional hazards model revealed that FOXM1 expression was an independent prognostic factor in multivariate analysis. Experimentally, overexpression of FOXM1 by gene transfer significantly promoted the growth and metastasis of colon cancer cells in orthotopic mouse models, whereas knockdown of FOXM1 expression by siRNA did the opposite. Promotion of colon tumorigenesis by FOXM1 directly and significantly correlated with activation of urokinase-type plasminogen activator receptor (PLAUR) expression and elevation of invasion and metastasis.Conclusions: Given the importance of FOXM1 in regulation of the expression of genes key to cancer biology, dysregulated expression and activation of FOXM1 may play important roles in colon cancer progression and metastasis. Clin Cancer Res; 19(1); 62–72. ©2012 AACR.

PDF file - 113K, Table S1. Different FOXM1 protein expressions in primary colon cancer and matched lymph node metastasis; Table S2. FOXM1 expression and clinicopathologic characteristics; Table S3. Correlation of FOXM1 expression with metastasis of colon cancer patients after radical colectomy (n = 185); Table S4. Multivariate analyses of overall survival and metastasis free survival of colon cancer patients; Table S5. Different PLAUR protein expressions in primary colon cancer and matched lymph node metastasis; Table S6. PLAUR expression and clinicopathologic characteristics; Table S7. Multivariate analyses of overall survival and metastasis free survival of colon cancer patients; Table S8. Correlation of PLAUR expression with metastasis of colon cancer patients after radical colectomy (n = 185); Table S9. Correlation between FOXM1 and PLAUR protein expressions in primary colon cancer (n = 203)

PDF file - 502K, S1. FOXM1 and PLAUR expression in colon cancer specimens; S2. The effect of FOXM1 expression on colon cancer cell migration and invasion; S3. Transcriptional activation of PLAUR expression in colon cancer cells by FOXM1

Background Copy number variation (CNV) is a type of structural variation, which is a gain or loss event with abnormal changes in copy number. Methods to predict the pathogenicity of CNVs are required to realize the relationship between these variants and clinical phenotypes. ClassifyCNV, X-CNV, StrVCTVRE, etc. have been trained to predict the pathogenicity of CNVs, but few studies have been reported based on the deleterious significance of features. Results From single nucleotide polymorphism (SNP), gene and region dimensions, we collected 79 informative features that quantitatively describe the characteristics of CNV, such as CNV length, the number of protein genes, the number of three prime untranslated region. Then, according to the deleterious significance, we formulated quantitative methods for features, which fall into two categories: the first is variable type, including maximum, minimum and mean; the second is attribute type, which is measured by numerical sum. We used Gradient Boosted Trees (GBT) algorithm to construct dbCNV, which can be used to predict pathogenicity for five-tier classification and binary classification of CNVs. We demonstrated that the distribution of most feature values was consistent with the deleterious significance. The five-tier classification model accuracy for 0.85 and 0.79 in loss and gain CNVs, which proved that it has high discrimination power in predicting the pathogenicity of five-tier classification CNVs. The binary model achieved area under curve (AUC) values of 0.96 and 0.81 in the validation set, respectively, in gain and loss CNVs. Conclusion The performance of the dbCNV suggest that functional deleteriousness-based model of CNV is a promising approach to support the classification prediction and to further understand the pathogenic mechanism.

5-Fluorouracil (5-FU)-based chemotherapy is the first-line treatment for colorectal cancer (CRC) but is hampered by chemoresistance. Despite its impact on patient survival, the mechanism underlying chemoresistance against 5-FU remains poorly understood. Here, we identified serine hydroxymethyltransferase-2 (SHMT2) as a critical regulator of 5-FU chemoresistance in CRC. SHMT2 inhibits autophagy by binding cytosolic p53 instead of metabolism. SHMT2 prevents cytosolic p53 degradation by inhibiting the binding of p53 and HDM2. Under 5-FU treatment, SHMT2 depletion promotes autophagy and inhibits apoptosis. Autophagy inhibitors decrease low SHMT2-induced 5-FU resistance in vitro and in vivo. Finally, the lethality of 5-FU treatment to CRC cells was enhanced by treatment with the autophagy inhibitor chloroquine in patient-derived and CRC cell xenograft models. Taken together, our findings indicate that autophagy induced by low SHMT2 levels mediates 5-FU resistance in CRC. These results reveal the SHMT2–p53 interaction as a novel therapeutic target and provide a potential opportunity to reduce chemoresistance.

Acute rejection of the transplanted heart is mediated by oxidative programmed cell death through the synergistic effects of the innate and adaptive immune systems. However, the role of ferroptosis, a newly discovered form of oxidative cell death, has not been widely evaluated. Tumor necrosis factor-α-induced protein-8 like 2 (TNFAIP8L2), also known as TIPE2, is required for maintaining immune homeostasis. To characterize the role of TIPE2 in mediating heart allografts, BALB/c hearts were transplanted into C57BL/6 wild-type (WT) and TIPE2

Metastatic GIST (gastrointestinal stromal tumor) is most commonly seen in the liver. Surgical resection and Imatinib administration are the preferred treatment for localized and potentially resectable GIST. However, it is still a matter of debate about the optimal therapeutic management for unresectable, liver-confined, metastatic GIST even after the administration of imatinib. The present case illustrates the possibility of LT surgery maybe for unresectable GIST.A 56-year-old man revealed clinical symptoms such as abdominal pain, eating little, fullness of abdomen, and fatigue. A 6.0 cm tumor in the fundus of the stomach was found by gastroscopy, which was confirmed to be GIST by pathological biopsy and molecular testing. Abdominal enhanced CT scanning showed multiple hepatic mass occupying synchronous. Then the patient initiated on targeted drug therapy of Imatinib (400 mg daily). A year later, a follow-up abdominal enhanced CT scanning showed that no tumor was found in the gastric fundus except the thickened gastric wall with poor dilatation, and the liver tumors were significantly smaller than before. When the patient showed symptoms of drug resistance, he was refered to our hospital for LT. The surgery was very successful, and the patient is disease-free and there is no evidence of recurrence until the paper was finished.Metastatic GIST to the whole liver is a rare clinical entity requiring unique considerations for treatment. Treatment based on LT might be the last resort therapy for unresectable, liver-confined, metastatic GIST in transplant oncology.

BACKGROUND Tumor recurrence after orthotopic liver transplantation (OLT) remains a serious threat for long-term survival of the recipients with hepatocellular carcinoma (HCC), since very few factors or measures have shown impact on overcoming HCC recurrence after OLT. Postoperative infection suppresses tumor recurrence and improves patient survival in lung cancer and malignant glioma probably via stimulating the immune system. Post-transplant infection (PTI), a common complication, is deemed to be harmful for the liver transplant recipients from a short-term perspective. Nevertheless, whether PTI inhibits HCC recurrence after OLT and prolongs the long-term survival of HCC patients needs to be clarified. AIM To investigate the potential influence of PTI on the survival and tumor recurrence of patients with HCC after OLT. METHODS A total of 238 patients with HCC who underwent OLT between August 2002 and July 2016 at our center were retrospectively included and accordingly subdivided into a PTI group (53 patients) and a non-PTI group (185 patients). Univariate analyses, including the differences of overall survival (OS), recurrence-free survival (RFS), and post-recurrence survival (PRS), between the PTI and non-PTI subgroups as well as survival curve analysis were performed by the Kaplan-Meier method, and the differences were compared using the log rank test. The variables with a P-value < 0.1 in univariate analyses were included in the multivariate survival analysis by using a Cox proportional-hazards model. RESULTS The 1-, 3-, and 5-year OS and RFS rates of the whole cohort were 86.6%, 69.0%, and 63.6%, and 75.7%, 60.0%, and 57.3%, respectively. The 1-, 3-, and 5-year OS rates for the PTI patient group (96.0%, 89.3%, and 74.0%) were significantly higher than those for the non-PTI group (84.0%, 63.4%, and 60.2%) (P = 0.033). The absence of PTI was an independent risk factor for dismal OS (relative risk [RR] = 2.584, 95%CI: 1.226-5.449) and unfavorable RFS (RR = 2.683, 95%CI: 1.335-5.390). Subgroup analyses revealed that PTI remarkably improved OS (P = 0.003) and RFS (P = 0.003) rates of HCC patients with vascular invasion (IV), but did not impact on OS (P = 0.404) and RFS (P = 0.304) of patients without VI. Among the patients who suffered post-transplant tumor recurrence, patients with PTI showed significantly better OS (P = 0.026) and PRS (P = 0.042) rates than those without PTI. CONCLUSION PTI improves OS and RFS of the transplant HCC patients at a high risk for post-transplant death and tumor recurrence, which is attributed to suppressive effect of PTI on HCC recurrence.

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXY

Colorectal cancer (CRC) is a common tumor that harms human health with a high recurrence rate. It has been reported that the expression of microRNA-539 (miR-539) is low in several types of cancer, including CRC. Tumor necrosis factor (TNF)-α-induced protein 8 (TNFAIP8/TIPE) is highly expressed in CRC and promotes the proliferation, migration and angiogenesis of CRC. However, the relationship between miR-539 and TIPE and the mechanisms by which they regulate the proliferation of CRC remain to be explored. We aimed to investigate the functions and mechanisms of miR-539 in CRC proliferation. Functionally, miR-539 can bind to and regulate the expression of TIPE, and miR-539 activates SAPK/JNK to downregulate the expression of glutathione peroxidase 4 (GPX4) and promote ferroptosis. Our data reveal the novel role of miR-539 in regulating ferroptosis in CRC via activation of the SAPK/JNK axis, providing new insight into the mechanism of abnormal proliferation in CRC and a novel potential therapeutic target for advanced CRC.

Bacterial antibiotic resistance is a serious global problem; the underlying regulatory mechanisms are largely elusive. The earlier reports states that the vital role of transcriptional regulators (TRs) in bacterial antibiotic resistance. Therefore, we have investigated the role of TRs on enoxacin (ENX) resistance in Aeromonas hydrophila in this study. A label-free quantitative proteomics method was utilized to compare the protein profiles of the ahslyA knockout and wild-type A. hydrophila strains under ENX stress. Bioinformatics analysis showed that the deletion of ahslyA triggers the up-regulated expression of some vital antibiotic resistance proteins in A. hydrophila upon ENX stress and thereby reduce the pressure by preventing the activation of SOS repair system. Moreover, ahslyA directly or indirectly induced at least 11 TRs, which indicates a complicated regulatory network under ENX stress. We also deleted six selected genes in A. hydrophila that altered in proteomics data in order to evaluate their roles in ENX stress. Our results showed that genes such as AHA_0655, narQ, AHA_3721, AHA_2114, and AHA_1239 are regulated by ahslyA and may be involved in ENX resistance. Overall, our data demonstrated the important role of ahslyA in ENX resistance and provided novel insights into the effects of transcriptional regulation on antibiotic resistance in bacteria.

SlyA is a well-known transcription factor that plays important roles in the regulation of diverse physiological functions including virulence and stress response in various bacterial species. The biological effects of slyA have species-specific characteristics. In this study, a phenotype assay showed that slyA gene deletion in Aeromonas hydrophila (ahslyA) decreased biofilm formation capability but did not affect bacterial hemolytic activity or acid stress response. The differentially expressed proteins between ΔahslyA and wild-type strains were compared by label-free quantitative proteomics to further understand the effects of AhSlyA on biological functions. Bioinformatics assays showed that ΔahslyA may be involved in the regulation of several intracellular metabolic pathways such as galactose metabolism, arginine biosynthesis, and sulfur metabolism. A further phenotypic assay confirmed that AhSlyA plays an important role in the regulation of sulfur and phosphate metabolism. Moreover, ahslyA also directly or indirectly regulated at least eight outer membrane proteins involved in the maintenance of cell permeability. Overall, the results provide insights into the functions of ahslyA and demonstrate its importance in A. hydrophila. BIOLOGICAL SIGNIFICANCE: In this study, we compared the DEPs between the transcriptional regulator slyA-deleted and the wild-type A. hydrophila strains using a label-free quantitative proteomics method. The bioinformatics analysis showed that slyA may be involved in the regulation of several metabolic pathways. Subsequent phenotype and growth assays confirmed that ΔahslyA affected sulfur and phosphate metabolism, and OM permeability. Finally, a ChIP-PCR assay further confirmed that AhSlyA directly binds to the promoters of several candidate genes, including sulfur metabolism-related genes. These results indicated that slyA plays an important regulatory role in pleiotropic physiological functions of A. hydrophila, and these functions may be different from those identified in previous reports from other bacterial species.

Background We aimed to evaluate the analytical performance of five commercial RT‐PCR kits (Genekey, Daan, BioGerm, Liferiver, and Yaneng) commonly used in China, since such comparison data are lacking. Methods A total of 20 COVID‐19 confirmed patients and 30 negative nasopharyngeal swab specimens were analyzed by five kits. The detection ability of five RT‐PCR kits was evaluated with 5 concentration gradients diluted by a single positive sample. The limit of detection was evaluated by N gene fragment solid standard. Two positive clinical specimens were used to evaluate the repeatability and imprecision. Finally, we used six human coronaviruses plasmid and four respiratory pathogens plasmid to check for cross‐reactivity. Results The positive detection rate was 100% for Genekey, Daan, and BioGerm,and 90% for Liferiver and Yaneng in 20 clinical SARS‐CoV‐2 infection. The coincidence rate of five kits in 10 negative samples was 100%. The detection rate of target genes for Daan, BioGerm, Liferiver, and Yaneng was 100% from Level 1 to Level 3. In Level 4, only Daan detection rate was 100%. In Level 5, five kits presented poor positive rate. The limit of detection declared by each manufacturer was verified. The repeatability for target genes was less than 5% and so did the total imprecision. There is no cross‐reactivity of five kits with six human coronaviruses and four respiratory pathogens for ORF1ab and N gene. Conclusions Five RT‐PCR kits assessed in this study showed acceptable analytical performance characteristics and are useful tools for the routine diagnosis of SARS‐CoV‐2., In the work, we presented the analytical performance evaluations of five RT‐PCR kits using nasopharyngeal swabs samples from patients with confirmed SARS‐CoV‐2 infection, and negative nasopharyngeal swabs samples (Figure 1). A total of 20 COVID‐19 confirmed patients and 30 negative nasopharyngeal swab specimens were analyzed by five kits. The detection ability of five RT‐PCR kits was evaluated with 5 concentration gradients diluted by a single positive sample. The limit of detection was evaluated by N gene fragment solid standard. Two positive clinical specimens were used to evaluate the repeatability and imprecision. Finally, we used six human coronaviruses plasmid and four respiratory pathogens plasmid to check for cross‐reactivity

The bifacial p-type silicon (p-Si) passivated emitter and rear cells (PERCs) have dominated the industrial bifacial solar cells. In this paper, we have investigated the impact of hydrogenated amorphous silicon oxynitride (SiOxNy) films on the optical and electrical performance of bifacial p-Si PERCs. Simulation and characterization are carried out to show the improvement of bifacial p-Si PERCs, yielding an absolute front-side efficiency improvement of 0.14% and 016% with SiOxNy films introduced to the front-side and rear-side, respectively. When SiOxNy films are employed to both sides of bifacial p-Si PERCs, the front-side and rear-side efficiency separately increase by 0.27% and 0.26% compared to the baseline without SiOxNy films. We have achieved on the bifacial p-Si PERCs production line an averaged front-side efficiency of 23.23% (champion efficiency of 23.42%), together with a 17.31% averaged rear-side efficiency. Furthermore, the introduction of SiOxNy films has been demonstrated to reduce the rear-side potential induced degradation (PID) effect through both single-cell modules and commercial glass-glass modules, due to the SiOxNy films protecting AlOx from corrosion and maximizing the field-effect passivation of AlOx layers.

// Zehua Wu 1, * , Xiangying Zhu 2, 3, * , Wendi Xu 4 , Yu Zhang 5 , Lin Chen 3 , Fabo Qiu 1 , Binyuan Zhang 1 , Liqun Wu 1 , Zhihai Peng 2 and Huamei Tang 5 1 Department of Hepatobiliary Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266003, People’s Republic of China 2 Department of General Surgery, Shanghai Jiaotong University Affiliated First People’s Hospital, Shanghai 200080, People’s Republic of China 3 R & D Department, Shanghai GeneChem Limited Company, Shanghai 201203, People’s Republic of China 4 Department of Continuing Medical Education, The Affiliated Hospital of Qingdao University, Qingdao 266003, People’s Republic of China 5 Department of Pathology, Shanghai Jiaotong University Affiliated First People’s Hospital, Shanghai 200080, People’s Republic of China * These authors have contributed equally to this work Correspondence to: Huamei Tang, email: huamei-tang@hotmail.com Zhihai Peng, email: zhihai.peng@hotmail.com Keywords: CIT, colon cancer, cell growth, apoptosis, p53 Received: July 18, 2016 Accepted: May 06, 2017 Published: June 27, 2017 ABSTRACT Colon cancer is one of the major causes of cancer mortality worldwide. However, the underlying mechanism and therapeutic targets of colon cancer have not yet been fully elucidated. In the present study, we demonstrate that citron rho-interacting, serine/threonine kinase 21 (CIT) promotes the growth of human colon cancer cells. CIT is overexpressed in human colon cancer tissues and cell lines. High expression of CIT predicts poor survival for patients with colon cancer. In colon cancer cells, CIT knockdown represses cellular proliferation and colony formation. Our in vivo xenograft experiments showed that CIT knockdown reduces the growth rate of colon cancer cells and the final tumor weight. We found that CIT knockdown induces cell cycle arrest and apoptosis in colon cancer cells. Further microarray and bioinformatics analyses indicated that CIT regulates the p53 signaling pathway, which may account for the effects of CIT on colon cancer cells. Taken together, our findings provide evidence that CIT may promote the development of colon cancer, at least in part, through the p53 signaling pathway. Therefore, CIT may be a potential therapeutic target for colon cancer treatment.

We previously discovered that Ras association domain family member 6 (RASSF6) was downregulated and predicted poor prognosis in GC patients. However, the mechanisms of the down regulation of RASSF6 in GC remained unclear. Increasing evidence indicates that dysregulation of microRNAs promotes the progression of cancer through the repression of tumour suppressors. Here, we identified miR-181a-5p as a novel regulator of RASSF6 in GC. Functionally, ectopic expression or silencing of miR-181a-5p, respectively, promoted or inhibited GC cell proliferation, colony formation and cell cycle transition, as well as enhanced or prevented the invasion, metastasis of GC cells and epithelial to mesenchymal transition of GC cells in vitro and in vivo. Molecularly, miR-181a-5p functioned as an onco-miRNA by activating the RASSF6-regulated MAKP pathway. Overexpression or silencing of RASSF6 could partially reverse the effects of the overexpression or repression of miR-181a-5p on GC progress caused by activation of the MAKP pathway in vitro and in vivo. Clinically, high miR-181a-5p expression predicted poor survival in GC patients, especially combined with low RASSF6 expression. Collectively, we identified miR-181a-5p as an onco-miRNA, which acts by directly repressing RASSF6 in GC. (C) 2017 The Authors. Published by Elsevier Ireland Ltd. Funding Agencies|National High Technology Research and Development Program of China [SS2014AA020803]; National Natural Science Foundation of China [81272750, 81302083]

Hyperactivation of transforming growth factor-β (TGF-β) signaling pathway is a common feature of hepatocellular carcinoma (HCC) progression. However, the driver factors leading to enhanced TGF-β activity are not well characterized. Here, we explore the mechanisms that loss of Krüppel-like factor 4 (KLF4) exacerbates oncogenic TGF-β signaling in human HCC. The expression of KLF4 and TGF-β signaling components in primary HCC and their clinicopathologic relevance and significance was evaluated by using tissue microarray and immunohistochemistry. Cellular and molecular impacts of altered KLF4 expression and TGF-β signaling were determined using immunofluorescence, western blot, reverse-transcriptase PCR, chromatin immunoprecipitation and promoter reporter assays. Loss of KLF4 expression in primary HCC closely correlated with decreased Smad7 expression, increased p-Smad2/3 expression and independently predicts reduced overall and relapse-free survival after surgery. TGF-β signaling components were expressed in most HCC cells, and activation of TGF-β signaling promoted cell migration and invasion. Enforced KLF4 expression blocked TGF-β signal transduction and inhibited cell migration and invasion via activation of Smad7 transcription, whereas deletion of its C-terminal zinc-finger domain diminished this effect. KLF4 protein physically interacts with the Smad7 promoter. Promoter deletion and point mutation analyses revealed that a region between nucleotides -15 bp and -9 bp of the Smad7 promoter was required for the induction of Smad7 promoter activity by KLF4. Our data indicate that KLF4 suppresses oncogenic TGF-β signaling by activation of Smad7 transcription, and that loss of KLF4 expression in primary HCC may contribute to activation of oncogenic TGF-β signaling and subsequent tumor progression.

// Shengtao Lin 1, * , Tao Jiang 2, * , Ling Ye 1 , Zhongbo Han 3 , Yuan Liu 1 , Chenchen Liu 1 , Chenwei Yuan 1 , Senlin Zhao 1 , Jian Chen 1 , Jingtao Wang 1 , Huamei Tang 4 , Su Lu 4 , Liguang Yang 5 , Xiaoliang Wang 1 , Dongwang Yan 1 , Zhihai Peng 1 , Junwei Fan 1 1 Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080,China 2 Department of Anal-Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan 750004, China 3 Department of General Surgery, Central Hospital of Zi Bo, Zi Bo, Shandong 255000, China 4 Department of Pathology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China 5 Key Lab of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China * These authors contributed equally to this work Correspondence to: Junwei Fan, email: drjunweifan@163.com Zhihai Peng, email: profzhihai@163.com Keywords: BRG1, colon cancer, progression, WNT3A Received: June 15, 2016 Accepted: November 07, 2016 Published: November 12, 2016 ABSTRACT In this study, we aimed to elucidate the clinical significance and underlying mechanisms of BRG1 in colon cancer. In the clinical analysis, overexpression of BRG1 correlates with colon cancer progression in two cohorts ( n = 191 and n = 75). Kaplan-Meier survival analysis revealed that BRG1 is a prognosis predictor for overall survival ( P < 0.001) and disease-free survival ( P = 0.001). Knocking down BRG1 expression significantly suppressed the proliferation and invasion in colon cancer cells. The expression pattern of WNT3A is consistent with BRG1 in colon cancer tissues and WNT3A expression was inhibited in BRG1 knockdown cells. In addition, restoring WNT3A expression rescues the inhibition of cell proliferation and invasion induced by BRG1. In this study, we demonstrate that BRG1 may contribute to colon cancer progression through upregulating WNT3A expression.

Human BarH-like homeobox 2 (Barx2), a homeodomain factor of the Bar family, has an important role in controlling the expression of cell adhesion molecules and has been reported in an increasing array of tumor types except colorectal cancer (CRC). The purpose of the current study was to characterize the expression of Barx2 and assess the clinical significance of Barx2 in CRC. First, we analyzed the expression of Barx2 in two independent public datasets from Oncomine. Subsequently, we evaluated Barx2 mRNA and protein expression by quantitative real-time PCR and western blotting, respectively. It was determined that Barx2 expression was lower in tumor tissues than in adjacent non-tumorous colorectal tissues of CRC patients, consistent with results from the public datasets. Subsequently, a tissue microarray containing 196 CRC specimens was evaluated for Barx2 expression by immunohistochemical staining. It was found that low expression of Barx2 significantly correlated with TNM stage, AJCC stage, differentiation, and relapse in patients with CRC. Patients with lower levels of Barx2 expression showed reduced disease-free survival and overall survival. Furthermore, a trend toward shorter overall survival in the patient group with Barx2-negative tumors independent of advanced AJCC stage and poor differentiation was determined by Kaplan-Meier survival analysis. Based on univariate and multivariate analyses, Barx2 expression was an independent prognostic factor for determining CRC prognosis. Taken together, low Barx2 expression was associated with the progression of CRC and could serve as a potential independent prognostic biomarker for patients with CRC. (C) 2016 The Authors. Published by Elsevier Inc. Funding Agencies|National Natural Science Foundation of China [81272750]

// Chao Li 1, * , Jian Chen 1, * , Yupeng Wang 1, * , Guohe Song 1 , Chao Xiao 1 , Dongwang Yan 1 , Lin Zhong 1 , Xing Sun 1 , Xiaoliang Wang 1 , Fudong Yu 1 , Yang Yu 1 , Huamei Tang 2 , Zhihai Peng 1 1 Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, People’s Republic of China 2 Department of Pathology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, People’s Republic of China * There authors have contributed equally to this work Correspondence to: Zhihai Peng, email: pengzh@hotmail.com Keywords: PAK7, colon cancer, EMT, metastasis, prognosis Received: November 18, 2015 Accepted: May 08, 2016 Published: June 14, 2016 ABSTRACT P21 protein (Cdc42/Rac)-activated kinase 7 (PAK7) can promote neurite outgrowth, induce microtubule stabilization, and activate cell survival signaling pathways. PAK7 expression was found to increase with colon carcinoma progression, but the prognostic value, clinical significance, and underlying mechanisms have not been explored. In my study, the expression of PAK7 was up-related at both the transcriptional and the translational levels in colon tumors compared to that in adjacent normal colon tissue. Patients with PAK7-positive tumors had a lower rate of overall survival (OS) and metastasis-free survival (MFS) (log-rank test, P < 0.001). A Cox proportional hazards model showed that PAK7 expression was an independent prognostic factor for OS (hazard ration [HR], 2.08; 95% confidence interval [CI], 1.16-3.73; P = 0.004) and MFS (HR, 2.88; 95% CI, 1.53-5.42; P < 0.001) in patients with colon cancer. Patients with tumors that were over-expressing PAK7 experienced metastasis, and died within a significantly shorter time after surgery ( P < 0.001). Knockdown of PAK7 by a specific short hairpin RNA (shRNA) significantly suppressed the progression of epithelial to mesechymal transition (EMT), migration, and invasion of colon cancer cells in vitro and tumor growth in vivo . However, overexpression of PAK7 significantly promoted these processes. These findings indicate that aberrant PAK7 expression is associated with the occurrence of metastasis and poor clinical outcomes of human colon cancer by promoting the EMT, and the assessment of PAK7 expression might be helpful in predicting metastasis and prognostication for patients with colon cancer.

// Dantong Cheng 1, * , Senlin Zhao 1, 5, * , Huamei Tang 2, * , Dongyuan Zhang 1 , Hongcheng Sun 1 , Fudong Yu 1 , Weiliang Jiang 4, * , Ben Yue 1 , Jingtao Wang 1 , Meng Zhang 3 , Yang Yu 1 , Xisheng Liu 1 , Xiaofeng Sun 5 , Zongguang Zhou 6 , Xuebin Qin 7 , Xin Zhang 8 , Dongwang Yan 1 , Yugang Wen 1, 5 , Zhihai Peng 1 1 Department of General Surgery, Shanghai General Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China 2 Department of Pathology, Shanghai General Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China 3 Department of Pathology, Fudan University Affiliated Shanghai Cancer Center, Shanghai, China 4 Department of Gastroenterology, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China 5 Department of Oncology and Department of Clinical and Experimental Medicine, Linkoping University, Linkoping, Sweden 6 Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu, Sichuan, China 7 Department of Neuroscience, School of Medicine, Temple University, Philadelphia, PA, USA 8 Department of Pathology, Zhejiang Provincial People’s Hospital, Hangzhou, Zhejiang, China * These authors contributed equally to this work Correspondence to: Zhihai Peng, email: pengpzhh@hotmail.com Yugang Wen, email: wenyg1502@hotmail.com Dongwang Yan, email: yandw70@163.com Keywords: miR-20a-5p, colorectal cancer, metastasis, prognosis, Smad4 Received: February 09, 2016 Accepted: May 28, 2016 Published: June 07, 2016 ABSTRACT Background: Tumor metastasis is one of the leading causes of poor prognosis for colorectal cancer (CRC) patients. Loss of Smad4 contributes to aggression process in many human cancers. However, the underlying precise mechanism of aberrant Smad4 expression in CRC development is still little known. Results: miR-20a-5p negatively regulated Smad4 by directly targeting its 3′UTR in human colorectal cancer cells. miR-20a-5p not only promoted CRC cells aggression capacity in vitro and liver metastasis in vivo , but also promoted the epithelial-to-mesenchymal transition process by downregulating Smad4 expression. In addition, tissue microarray analysis obtained from 544 CRC patients’ clinical characters showed that miR-20a-5p was upregulated in human CRC tissues, especially in the tissues with metastasis. High level of miR-20a-5p predicted poor prognosis in CRC patients. Methods: Five miRNA target prediction programs were applied to identify potential miRNA(s) that target(s) Smad4 in CRC. Luciferase reporter assay and transfection technique were used to validate the correlation between miR-20a-5p and Smad4 in CRC. Wound healing, transwell and tumorigenesis assays were used to explore the function of miR-20a-5p and Smad4 in CRC progression in vitro and in vivo . The association between miR-20a-5p expression and the prognosis of CRC patients was evaluated by Kaplan–Meier analysis and multivariate cox proportional hazard analyses based on tissue microarray data. Conclusions: miR-20a-5p, as an onco-miRNA, promoted the invasion and metastasis ability by suppressing Smad4 expression in CRC cells, and high miR-20a-5p predicted poor prognosis for CRC patients, providing a novel and promising therapeutic target in human colorectal cancer.

// Senlin Zhao 1, * , Huamei Tang 2, * , Dongwang Yan 1, * , Junwei Fan 1, * , Hongcheng Sun 1 , Yugang Wen 1 , Fudong Yu 1 , Feifei Cui 1 , Dongyuan Zhang 1 , Yingming Xue 1 , Chenchen Liu 1 , Ben Yue 1 , Jian Chen 1 , Jingtao Wang 1 , Xiao Wang 1 , Meng Zhang 3 , Yang Yu 1 , Weiliang Jiang 4 , Xisheng Liu 1 , Yushuai Mi 1 , Zongguang Zhou 5 , Xuebin Qin 6 , Zhihai Peng 1 1 Department of General Surgery, Shanghai First People’s Hospital, Affiliated to Shanghai Jiao Tong University, Shanghai, China 2 Department of Pathology, Shanghai First People’s Hospital, Affiliated to Shanghai Jiao Tong University, Shanghai, China 3 Department of Pathology, Fudan University Affiliated Shanghai Cancer Center, Shanghai, China 4 Department of Gastroenterology, Shanghai First People’s Hospital, Affiliated to Shanghai Jiaotong University, Shanghai, China 5 Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu, China 6 Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, USA * These authors contributed equally to this work Correspondence to: Zhihai Peng, e-mail: pengpzhh@hotmail.com Xuebin Qin, e-mail: xuebin.qin@temple.edu Zongguang Zhou, e-mail: zhouzzghuaxi@163.com Keywords: DDA1, colon cancer, tumor recurrence, prognosis, NFκB Received: September 01, 2015 Accepted: February 06, 2016 Published: March 02, 2016 ABSTRACT Conventional high-recurrence risk factors are not sufficient to predict post-operative risk of tumor recurrence or sensitivity to 5-fluorouracil (5-FU)-based chemotherapy for stage II colon cancer. DDA1, an evolutionarily conserved gene located at 19p13.11, may be involved in the activation of nuclear factor kappaB (NFκB). This study aimed to investigate whether DDA1 contributes to tumorigenesis and progression of stage II colon cancer via activation of the NFκB pathway. We found that positive expression of DDA1 alone or in combination with p65 nuclear translocation correlated with increased risk of tumor recurrence in patients with stage IIB–IIC colon cancer. DDA1 overexpression in colon cancer lines promoted cell proliferation, facilitated cell cycle progression, inhibited 5-FU-induced apoptosis, enhanced invasion, and induced the epithelial-mesenchymal transition. Suppression of DDA1 inhibited tumor progression, and reduced tumor growth in vivo . We also demonstrated that DDA1-mediated tumor progression is associated with the activation of the NFκB/COP9 signalosome 2(CSN2)/glycogen synthase kinase3β (GSK3β) pathway. These results indicate that DDA1 promotes colon cancer progression through activation of NFκB/CSN2/GSK3β signaling. DDA1, together with NFκB activation status, may serve as a sensitive biomarker for tumor recurrence risk and prognosis in patients with stage IIB–IIC colon cancers.

The bifacial p-type silicon (p-Si) passivated emitter and rear cells (PERCs) are predicted to dominate the industrial bifacial solar cells. In this work, we have investigated the impact of different rear surface morphologies on the electrical performance of bifacial PERCs with both five-busbar (5BB) and nine-busbar (9BB) grid design. The passivation and optical properties with differing rear surfaces are evaluated on semi-device structures. The depth of local aluminum back surface field is hardly affected by the rear surface morphology. The calculated efficiency loss analysis indicates that the negative electrical impact with enlarged rear surface area is more serious for rear side than that of front side. The batch conversion efficiency of 9BB bifacial PERCs increases by 0.2%–0.3% comparing to 5BB ones depending on the rear surface roughness. Consequently, a highest front-side average efficiency of 22.57%, with a champion efficiency of 22.75%, has been achieved for 9BB bifacial p-Si PERCs with a nearly planar rear surface. A highest bifaciality of 78.7% is realized for both 5BB and 9BB bifacial PERCs with the roughest rear surface. We have further simulated the relative enhancement of electric generation to compare the performance of bifacial PERCs in practical application.

Purpose: Tumor differentiation is a behavioral index for hepatocellular carcinoma (HCC) and a prognostic factor for patients with HCC who undergo orthotopic liver transplantation (OLT). However, the molecular basis for HCC differentiation and prognostic value of the underlying molecules that regulate HCC differentiation are unclear. In this study, we defined a potential driver pathway for HCC differentiation and prognostication. Experimental Design: The regulation and function of Krüppel-like factor 4 (KLF4) and hepatocyte nuclear factor-6 (HNF-6) in HCC differentiation was evaluated using human tissues, molecular and cell biology, and animal models, and its prognostic significance was determined according to its impact on patient survival. Results: There was a direct relationship between the expression levels of KLF4 and HNF6 in HCC. Reduced KLF4 or HNF6 expression correlated with high HCC grade. Poorly differentiated HCC cells had lower expression of KLF4 or HNF6 and differentiation-associated markers than did well-differentiated cells. Elevated KLF4 of HNF6 expression induced differentiation of poorly differentiated hepatoma cells. Mechanistically, KLF4 trans-activated HNF-6 expression. Restored HNF-6 expression upregulated expression of differentiation-associated markers and inhibited HCC cell migration and invasion, whereas HNF-6 knockdown did the opposite. Loss of KLF4 expression in primary HCC correlated with reduced overall survival and shortened relapse-free survival durations after OLT. Combination of KLF4 expression and the Milan criteria improved prognostication for HCC after OLT. Conclusions: The dysregulated KLF4/HNF-6 pathway drives dedifferentition and progression of HCC, and KLF4 is a biomarker for accurate prognostication of patients with HCC treated by OLT when integrated with the Milan Criteria. Clin Cancer Res; 22(2); 502–12. ©2015 AACR.

Background Postoperative 5-fluoruracil (5-FU)-based adjuvant chemotherapy is recommended for stage II colon cancer patients with high conventional risk factors; however, some of these patients still experience tumor recurrence. Identifying novel biomarkers to distinguish the risk of tumor recurrence after surgery is vital for improving their prognoses. We previously showed that ubiquitin D (UBD) can predict the prognosis of colon cancer; however, there are limited data on whether UBD is an independent prognostic factor for stage II patients treated with 5-FU-based adjuvant chemotherapy. Methods Quantitative real-time PCR and Western blot analyses were used to examine UBD expression in randomly selected stage II patients' tumor tissues. UBD expression and p65 distribution were assessed using immunohistochemistry in paraffin-embedded specimens from the 101 tumor recurrence patients and 178 nonrelapse patients who received postoperative 5-FU-based adjuvant chemotherapy. Results UBD expression, both at transcriptional and posttranscriptional levels, was higher in relapse tumors (P

Recent studies have determined that cartilage oligomeric matrix protein (COMP) plays a vital role in carcinogenesis. We sought to clarify the role of COMP in colon cancer. We investigated gene expression data from The Cancer Genome Atlas (TCGA) dataset. Tissue microarrays (TMA) containing paired samples from 253 patients with colon cancer were subjected to immunostaining. COMP levels in serum of colon cancer patients and healthy donors were measured with ELISA. We established COMP-knockout cells using the CRISPR/Cas9 system and COMP-overexpressing cells using lentiviral vectors to detect the effects of COMP on colon cancer cells using Cell Counting Kit-8 (CCK8), colony formation, apoptosis detection kit, and tumorigenesis assays in nude mice. The analysis of TCGA dataset and the results of the TMA suggested that COMP expression levels were significantly higher in cancer tissues than in adjacent normal tissues. Moreover, high COMP expression was correlated with the poor outcome of colon cancer patients. COMP levels in the sera of preoperative patients with colon cancer were much higher than those in healthy donors and were significantly reduced after colectomy. Colon cancer cells without COMP were defective with respect to the ability to proliferate, colony formation, the ability to resist 5-Fluorouracil-induced apoptosis and the growth of xenograft tumors in mice. Contrasting results were observed in COMP overexpressed cells. COMP promoted colon cancer cell proliferation partially through the activation of PI3K/ Akt/ mTOR/ p70S6K pathway. COMP may be a novel prognostic indicator and biomarker and also a potential therapeutic target for colon cancer.

Signal peptide-CUB-epidermal growth factor-like domain-containing protein 2 (SCUBE2), a member of the SCUBE protein family, is a secreted and membrane-associated multi-domain glycoprotein. SCUBE2 is known as a novel tumor suppressor and a useful prognostic marker in breast cancer. In the present study, we investigated the expression (including mRNA and protein levels) of SCUBE2 in colorectal cancer (CRC) and adjacent normal tissues, using quantitative real-time PCR, western blot analysis and immunohistochemistry on a tissue microarray. Upregulation of SCUBE2 was achieved by transient transfection in RKO cell lines, and the effects of SCUBE2 on tumor proliferation, invasion, migration and apoptosis were evaluated by a series of functional experiments. The results indicated that SCUBE2 expression was decreased at the transcriptional and translational levels in CRC tissues and significantly associated with clinical stage, the depth of tumor invasion, lymph‑node metastasis, distant metastasis and histological grade. Patients with SCUBE2‑positive tumors had a lower recurrence rate and better survival than patients with SCUBE2-negative tumors. Moreover, upregulation of SCUBE2 had a limited effect on cell apoptosis but significantly inhibited tumor cell proliferation, migration and invasion in vitro. In conclusion, SCUBE2 plays an important role in suppressing CRC progression and prognosis. Our findings suggested that SCUBE2 may serve as a novel tumor suppressor and a potential therapeutic target for CRC patients.

Zinc-α-2-glycoprotein (AZGP1) is a 41-kDa secreted glycoprotein, which has been detected in several malignancies. The diagnostic value of AZGP1 in serum of prostate and breast cancer patients has been reported. Analyzing “The Cancer Genome Atlas” data, we found that in colon cancer AZGP1 gene expression was upregulated at transcriptional level. We hypothesized that AZGP1 could be used as a diagnostic marker of colon cancer. First, we confirmed AZGP1 expression was higher in a set of 28 tumor tissues than in normal colonic mucosa tissues by real-time quantitative PCR and western blot in a Chinese population. We verified that serum concentration of AZGP1 was higher in 120 colon cancer patients compared with 40 healthy controls by ELISA (p &lt, 0.001). Then receiver-operating characteristic (ROC) curve analysis was used to evaluate the predictive diagnostic value of AZGP1 in serum. The area under the curve (AUC) of AZGP1 was 0.742 (p &lt, 0.001, 95% confidence interval (CI) = 0.656–0.827) in between the AUC of carcinoembryonic antigen (CEA) and the AUC of CA19-9, suggesting that predictive diagnostic value of AZGP1 is between CEA and Carbohydrate 19-9 (CA19-9). The combination of AZGP1 with traditional serum biomarkers, CEA and CA19-9, could result in better diagnostic results. To further validate the diagnostic value of AZGP1, a tissue microarray containing 190 samples of primary colon cancer tissue paired with normal colonic tissue was analysed and the result showed that AZGP1 was significantly upregulated in 68.4% (130 of 190) of the primary cancer lesions. In contrast, there was a weakly positive staining in 29.5% (56 of 190) of the normal colonic tissue samples (p &lt, 0.001). Leave-one-out cross-validation was performed on the serum data, and showed that the diagnostic value of AZGP1 had 63.3% sensitivity and 65.0% specificity. Combination of AZGP1, CEA and CA19-9 had improved diagnosis value accuracy with 74.2% sensitivity and 72.5% specificity. These results suggest that AZGP1 is a useful diagnostic biomarker in tissues and serum from a Chinese population.

// Jian Chen 1,* , Huijun Lu 2,* , Dongwang Yan 1 , Feifei Cui 1 , Xiaoliang Wang 1 , Fudong Yu 1 , Yingming Xue 1 , Xiaodong Feng 3 , Jingtao Wang 1 , Xiao Wang 1 , Tao Jiang 4 , Meng Zhang 2 , Senlin Zhao 1 , Yang Yu 1 , Huamei Tang 2 and Zhihai Peng 1 1 Department of General Surgery, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China 2 Department of Pathology, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China 3 Basic Medical College, Taishan Medical University, Tai’an, People’s Republic of China 4 Department of Anal-Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, People‘s Republic of China * These authors contributed equally to this work Correspondence: Zhihai Peng, email: // Huamei Tang, email: // Keywords : p21-activated kinase 6, Colon cancer, 5-fluorouracil, Chemoresistance Received : June 09, 2014 Accepted : November 07, 2014 Published : November 07, 2014 Abstract p21-Activated kinase 6 (PAK6) has been implicated in radiotherapy and docetaxel resistance. We have further evaluated PAK6 as a predictor of 5-fluorouracil (5-FU) treatment response in colon cancer. Here we report that in colon cancer PAK6 promotes tumor progression and chemoresistance both in vitro and in vivo . In the clinical analysis, PAK6 was overexpressed in 104 of 147 (70.75%) stage II and III patients who received 5-FU based chemotherapy after surgery. Multivariate Cox regression analysis indicated that PAK6 was an independent prognostic factor for overall survival ( P < 0.001) and disease-free survival ( P < 0.001). Colon cancer cell lines showed increased PAK6 expression upon 5-FU treatment. In PAK6-knockdown cells treated with 5-FU, cell viability and phosphorylation of BAD decreased, and the number of apoptotic cells, levels of cleaved caspase 3 and PARP increased compared to control cells. The opposite was observed in PAK6 overexpressing cells. Short hairpin RNA knockdown of PAK6 blocked cells in G2-M phase. Furthermore, Animal experiments results in vivo are consistent with outcomes in vitro. This study demonstrates that PAK6 is an independent prognostic factor for adjuvant 5-FU-based chemotherapy in patients with stage II and stage III colon cancer.

Deregulation of chromatin assembly factor 1, p150 subunit A (CHAF1A) has recently been reported to be involved in the development of some cancer types. In this study, we identified that the frequency of positive CHAF1A staining in primary tumor mucosa (45.8%, 93 of 203 samples) was significantly elevated compared to that in paired normal mucosa (18.7%, 38 of 203 samples). The increased expression was strongly associated with cancer stage, tumor invasion, and histological grade. The five-year survival rate of patients with CHAF1A-positive tumors was remarkably lower than that of patients with CHAF1A-negative tumors. Colon cancer cells with CHAF1A knockdown exhibited decreased cell growth index, reduction in colony formation ability, elevated cell apoptosis rate as well as impaired colon tumorigenicity in nude mice. Hence, CHAF1A upregulation functions as a poor prognostic indicator of colon cancer, potentially contributing to its progression by mediating cancer cell proliferation.

Background Although the rat orthotopic liver transplantation (OLT) model has existed for many years, only a few models can be applied for dynamic bile collection. The aim of this study was to introduce a dependent rat OLT model with hepatic rearterialization and an expediently dynamic bile collection system. Methods Forty-five male Sprague–Dawley rats were divided into the following three groups ( n = 15 each): group A, OLT without hepatic rearterialization; group B, OLT with hepatic rearterialization; group C, OLT with hepatic rearterialization and a biliary extradrainage system. In groups B and C, a modified sleeve anastomosis between the donor common hepatic artery and the recipient proper hepatic artery was performed to restore the hepatic artery blood flow. In group C, after hepatic rearterialization, biliary extradrainage and jejunum stoma were performed to reestablish the bile flow, and a waistcoat-like external fixator was introduced to protect this system. Results The surgical success rates in groups A, B, and C were 100% (15/15), 93% (14/15), and 93% (14/15), respectively. In groups B and C, the hepatic artery patency rates were 93% and 86% on postoperative day 3 and postoperative day 21, respectively. Also, the liver function and bile duct integrity were preserved better than that in group A. In group C, the biliary extradrainage system was well preserved and bile collection was easily performed. Conclusions The rat OLT model with hepatic rearterialization and a convenient biliary extradrainage system was satisfactory in maintaining the survival rate, hepatic artery patency rate, and recovery of graft function, so it can be applied in various studies after transplantation.

Objective A new working model of heterotopic heart transplantation in rats was established using a simplified technique. Materials and methods Sprague-Dawley rats were used as donors and recipients. The donor left common carotid artery and left pulmonary artery were anastomosed to the recipient left renal artery and vein by a “sleeve and cuff” method, respectively. The donor left ventricle was blood volume loaded by anastomosing the left atrium to the recipient's abdominal aorta in end-to-side fashion. The characteristics of the donor heart were evaluated by palpating the abdominal wall of the rats. We examined the surgical success rate, changes in heart weight, and histology at 1 month after transplantation. Results The model was attempted in 32 rats with the success rate of 93.7% (30/32); the 2 failed cases died due to postoperative bleeding. There was no significant difference in mean weight changes between the donor and native hearts at 1 month after transplantation (1.13 ± 0.13 g vs 1.09 ± 0.12 g, P = .244). The donor heart myocardium showed regularly shaped, unidirectional, healthy muscle similar to the native heart. Conclusions The technique was easily learned, allowing less recipient surgical stress. The hemodynamic performance appeared to be similar to the normal cardiac physiological situation, and thus may be more suitable for pre-clinical studies.

5-Fluorouracil (5FU)-based adjuvant therapy is the first-line therapy for treating stage II and III colon cancer after surgery. However, its therapeutic efficacy is limited because of chemoresistance, especially in deficient mismatch repair (dMMR) colon cancer. Here, we first used laser capture microdissection to obtain purified cells from four dMMR and four proficient mismatch repair (pMMR) colon cancer tissues. Second, microRNA (miRNA) microarray chips were used to identify miRNAs that are differentially expressed between these two classes of tumors. Third, we analyzed their differential expression by qRT-PCR in a panel of 5-FU-resistant colon cancer cell lines. We identified that miR-1290 was one of the most upregulated miRNAs in both dMMR colon cancer tissues and 5-FU-resistant cells. We also found that miR-1290 was positively correlated with dMMR status and predicted poor prognosis in stage II and III colon cancer patients who received 5-FU-based chemotherapy. Furthermore, we demonstrated that inhibition of the expression of miR-1290 enhanced sensitivity to 5-FU treatment in vitro and in tumor xenografts in vivo by direct targeting hMSH2. Our study indicates that miR-1290 may become a promising biomarker of dMMR colon cancer and predicts the prognosis of stage II and III patients who receive 5-FU-based adjuvant therapy.

Background: Despite advancements in the diagnosis and treatment of colorectal cancer (CRC), many patients die because of tumor metastasis or recurrence. Therefore, identifying new prognostic markers and elucidating the mechanisms of CRC metastasis and recurrence will help to improve the prognosis of the disease. As dysregulation of microRNAs is strongly related to cancer progression, the aim of this study was to identify the role of miR-4775 in the prognosis of CRC patients and the underling mechanisms involved in CRC progression. Methods: qPCR and in situ hybridization were used to evaluate the expression of miR-4775 in 544 pairs of paraffin-embedded normal and CRC tissues. Kaplan-Meier analysis with the log-rank test was used for survival analyses. Immunohistochemical staining was applied to investigate the expression of miR-4775-regulated Smad7/TGF beta pathway-associated markers. In vitro and in vivo invasion and metastasis assays were used to explore the function of miR-4775 in the progression of CRC. Results: miR-4775 was identified as a high-risk factor for CRC metastasis and recurrence, with high levels predicting poor survival among the 544 studied CRC patients. Furthermore, high miR-4775 expression promoted the invasion of CRC cells as well as metastasis and the epithelial to mesenchymal transition (EMT) via Smad7-mediated activation of TGF beta signaling both in vitro and in vivo. Downregulating miR-4775 or overexpressing Smad7 reversed the tumor-promoting roles of miR-4775/ Smad7/TGF beta in vitro and in vivo. Conclusion: miR-4775 promotes CRC metastasis and recurrence in a Smad7/TGF beta signaling-dependent manner, providing a new therapeutic target for inhibiting the metastasis or recurrence of the disease. Funding Agencies|National High Technology Research and Development Program of China [SS2014AA020803]; National Natural Science Foundation of China [81220108021, 81302083, 81272750]; Natural Science Foundation of Shanghai [16ZR1427700]; Project of Shanghai Science and Technology Commission [124119a1700, 14411950502]; Project of Songjiang District [0702 N14001]

Multivariate Cox proportional hazard analyses of miR-4775 expression, T stage, Lymph node metastasis and distant metastasis in association with DFS and OS in 544 CRC patients. (DOCX 16 kb)

miR-4775 expression with relation to metastasis in 544 CRC patients. (DOCX 18 kb)

Correlations between miR-4775 expression and Smad7, p-Smad2, p-Smad3 staining in tumor tissues from 544 CRC patients. (DOCX 16 kb)

Correlations between miR-4775 expression and E-cadherin, N-cadherin and vimentin staining in tumor tissues from 544 CRC patients. (DOCX 16 kb)