Youyong Lu

Combination of hsa-miR-375 and hsa-miR-142-5p as a predictor for recurrence risk in gastric cancer patients following surgical resection

BACKGROUND Recurrence is a major factor leading to treatment failure and death in gastric cancer (GC) patients following surgical resection. Importantly, the prediction of recurrence is critical in improving clinical outcomes. We isolated a group of microRNAs (miRNAs) and evaluated their usefulness as prognostic markers for the recurrence of GC. PATIENTS AND METHODS A total of 65 GC patients were selected for systematic analysis, 29 patients with recurrence and 36 patients without recurrence. Firstly, miRNAs microarray and bioinformatics methods were used to characterize classifiers from primary tumor samples (n = 8). Following, we validated these predictors both in frozen fresh and paraffin-embedded tissue samples (n = 57) using quantitative PCR. RESULTS We have identified 17 differential miRNAs including 10 up-regulated and 7 down-regulated miRNAs in recurrence group. Using k-top scoring pairs (k-TSP) method, we further ascertained hsa-miR-375 and hsa-miR-142-5p as a classifier to recognize recurrence and nonrecurrence cases both in the training and test samples. Moreover, we validated this classifier in 34 frozen fresh tissues and 38 paraffin-embedded tissues with consistent sensitivity and specificity with training set; among them, 15 cases were matched. A high frequency recurrence and poor survival were observed in GC cases with high level of hsa-miR-375 and low level of hsa-miR-142-5p (P < 0.001). In addition, we evaluated that hsa-miR-375 and hsa-miR-142-5p were involved in regulating target genes in several oncogenic signal pathways, such as TP53, MAPK, Wnt and vascular endothelial growth factor. CONCLUSION Our results indicate that the combination of hsa-miR-375 and hsa-miR-142-5p as a predictor of disease progression has the potential to predict recurrence risk for GC patients.

Novel recurrently mutated genes and a prognostic mutation signature in colorectal cancer

Background Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication. Objective To identify recurrent somatic mutations with prognostic significance in patients with CRC. Method Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases. Results Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6–14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study. Conclusions The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging.

Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing

Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.

Expression of programmed cell death 5 gene involves in regulation of apoptosis in gastric tumor cells

The protein of programmed cell death 5 (PDCD5) is believed to participate in regulation of apoptosis. Although PDCD5 is reducibly expressed in various human tumors, it is not clear which expression level of PDCD5 is in gastric cancer (GC). In this study, we have systematically employed the approaches of RT-PCR, Real- time PCR, Immunohistochemistry (IHC), Immunofluorescence staining (IFS) and Western blot to determine the PDCD5 expression in GC cells and primary tumors, at mRNA and protein level, respectively. Our data revealed that the positive rate of PDCD5 expression in the gastric tumor tissues was significantly less than that of the normal tissues (14 out of 102 vs 36 out of 51), whereas, the decreased expression of PDCD5 protein was well correlated with the up-regulated expression of Bcl-2 in these tissues, and the up-regulated expression and nuclear translocation of PDCD5 protein were verified in the apoptotic GC cells induced by Diallyl trisulfide (DATS). Furthermore, the survival curve has suggested that the more PDCD5 expressions were found in the patients, the longer the survival periods were. Therefore, our observations lay down a reasonable postulation that PDCD5 may play a key role to regulate the apoptotic processes in the GC cells and gastric tumors.

Assessment of Immunoreactive Synthetic Peptides from the Structural Proteins of Severe Acute Respiratory Syndrome Coronavirus

Abstract Background: The widespread threat of severe acute respiratory syndrome (SARS) to human life has spawned challenges to develop fast and accurate analytical methods for its early diagnosis and to create a safe antiviral vaccine for preventive use. Consequently, we thoroughly investigated the immunoreactivities with patient sera of a series of synthesized peptides from SARS-coronavirus structural proteins. Methods: We synthesized 41 peptides ranging in size from 16 to 25 amino acid residues of relatively high hydrophilicity. The immunoreactivities of the peptides with SARS patient sera were determined by ELISA. Results: Four epitopic sites, S599, M137, N66, and N371-404, located in the SARS-coronavirus S, M, and N proteins, respectively, were detected by screening synthesized peptides. Notably, N371 and N385, located at the COOH terminus of the N protein, inhibited binding of antibodies to SARS-coronavirus lysate and bound to antibodies in >94% of samples from SARS study patients. N385 had the highest affinity for forming peptide-antibody complexes with SARS serum. Conclusions: Five peptides from SARS structural proteins, especially two from the COOH terminus of the N protein, appear to be highly immunogenic and may be useful for serologic assays. The identification of these antigenic peptides contributes to the understanding of the immunogenicity and persistence of SARS coronavirus.

Gastrokine 1 induces senescence through p16/Rb pathway activation in gastric cancer cells

Background and aims Gastrokine 1 (GKN1) is a stomach-specific protein that is normally expressed in gastric mucosa but not in primary tumours and cell lines. Based on this evidence, it was presumed that GKN1 might play a role in gastric cancer development; however, its function and molecular mechanism are not clear. A systematic study was initiated that combined multiple approaches to define the molecular mechanism of GKN1 in gastric cancer cells. Method Proteomics, western blotting and immunohistochemistry were used to measure the expression level of GKN1. Western blotting combined with immunofluorescence was used to monitor the secretory process of this protein. Subsequently, the function and molecular mechanism of GKN1 was explored in vitro and in vivo. Results It was shown that GKN1 is an autocrine/paracrine protein and inhibits cell growth due to senescence, which resulted from activation of p16/Rb and p21waf pathways. Furthermore, sustained activation of Ras/Raf/MEK/ERK signalling was characterised in gastric cancer cells and a xenograft nude mouse model following GKN1 treatment. Conclusion These results provide comprehensive molecular evidence of GKN1 in inducing senescence of gastric cancer cells, and indicate that GKN1 might be a potential novel target for gastric cancer therapeutics.

Integrative Identification of Epstein–Barr Virus–Associated Mutations and Epigenetic Alterations in Gastric Cancer

BACKGROUND & AIMS The mechanisms by which Epstein-Barr virus (EBV) contributes to the development of gastric cancer are unclear. We investigated EBV-associated genomic and epigenomic variations in gastric cancer cells and tumors. METHODS We performed whole-genome, transcriptome, and epigenome sequence analyses of a gastric adenocarcinoma cell line (AGS cells), before and after EBV infection. We then looked for alterations in gastric tumor samples, with (n = 34) or without (n = 100) EBV infection, collected from patients at the Prince of Wales Hospital, Chinese University of Hong Kong (from 1998 through 2004), or the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (from 1999 through 2006). RESULTS Transcriptome analysis showed that infected cells expressed 9 EBV genes previously detected in EBV-associated gastric tumors and 71 EBV genes not previously reported in gastric tumors. Ten viral genes that had not been reported previously in gastric cancer but were expressed most highly in EBV-infected cells also were expressed in primary EBV-positive gastric tumors. Whole-genome sequence analysis identified 45 EBV-associated nonsynonymous mutations. These mutations, in genes such as AKT2, CCNA1, MAP3K4, and TGFBR1, were associated significantly with EBV-positive gastric tumors, compared with EBV-negative tumors. An activating mutation in AKT2 was associated with reduced survival times of patients with EBV-positive gastric cancer (P = .006); this mutation was found to dysregulate mitogen-activated protein kinase signaling. Integrated epigenome and transcriptome analyses identified 216 genes transcriptionally down-regulated by EBV-associated hypermethylation; methylation of ACSS1, FAM3B, IHH, and TRABD increased significantly in EBV-positive tumors. Overexpression of Indian hedgehog (IHH) and TraB domain containing (TRABD) increased proliferation and colony formation of gastric cancer cells, whereas knockdown of these genes reduced these activities. We found 5 signaling pathways (axon guidance, focal adhesion formation, interactions among cytokines and receptors, mitogen-activated protein kinase signaling, and actin cytoskeleton regulation) to be affected commonly by EBV-associated genomic and epigenomic alterations. CONCLUSIONS By using genomic, transcriptome, and epigenomic comparisons of EBV infected vs noninfected gastric cancer cells and tumor samples, we identified alterations in genes, gene expression, and methylation that affect different signaling networks. These might be involved in EBV-associated gastric carcinogenesis.

CD44-positive cancer stem cells expressing cellular prion protein contribute to metastatic capacity in colorectal cancer

Cancer stem cells are implicated in tumor progression, metastasis, and recurrence, although the exact mechanisms remain poorly understood. Here, we show that the expression of cellular prion protein (PrPc, PRNP) is positively correlated with an increased risk of metastasis in colorectal cancer. PrPc defines a subpopulation of CD44-positive cancer stem cells that contributes to metastatic capacity. PrPc(+)CD44(+) colorectal cancer stem cells displayed high liver metastatic capability, unlike PrPc(-)CD44(+) stem cells, that was inhibited by RNAi-mediated attenuation of PrPc. Notably, administration of PrPc monoclonal antibodies significantly inhibited tumorigenicity and metastasis of colorectal cancer stem cells in mouse models of orthotopic metastasis. PrPc promoted epithelial to mesenchymal transition (EMT) via the ERK2 (MAPK1) pathway, thereby conferring high metastatic capacity. Our findings reveal the function of PrPc in regulating EMT in cancer stem cells, and they identify PrPc as candidate therapeutic target in metastatic colorectal cancer.

Reciprocal Interactions between Tumor-Associated Macrophages and CD44-Positive Cancer Cells via Osteopontin/CD44 Promote Tumorigenicity in Colorectal Cancer

Purpose: CD44 is of functional importance for tumor initiation and progression in colorectal cancer, but how this molecule benefits cancer cells from the tumor microenvironment, especially tumor-associated macrophages (TAM), remains poorly defined. Experimental Design: In vivo tumorigenic assays were conducted to assess the role of murine TAMs in the tumorigenesis of human colorectal cancer cells. Both in vitro and in vivo osteopontin (OPN) expression levels in TAMs were examined by immunohistochemistry, quantitative PCR, and Western blotting. Soft agar colony formation assays were used to estimate the clonogenicity of colorectal cancer cells that had received different treatments. The relationships between the expression levels of OPN, CD44v6, and CD68 and clinical prognosis were evaluated by tissue microarray analysis. Results: We found that macrophages, when coinjected or cocultured with CD44-positive colorectal cancer cells, were able to produce higher levels of OPN, which in turn facilitated the tumorigenicity and clonogenicity of the colorectal cancer cells. The knockdown of CD44 or treatment with blocking antibodies to CD44 attenuated OPN secretion. OPN, through binding to its receptor CD44, activated c-jun-NH2-kinase signaling and promoted the clonogenicity of colorectal cancer cells. Moreover, tissue microarray data have shown that OPN expression, in combination with CD44v6, has a negative correlation with colorectal cancer patient survival. Conclusions: These results suggest that the OPN–CD44 interaction is important for colorectal cancer progression and could serve as a potential therapeutic target for the treatment of colorectal cancer. Clin Cancer Res; 19(4); 785–97. ©2012 AACR.

MiR‐23a in amplified 19p13.13 loci targets metallothionein 2A and promotes growth in gastric cancer cells

Copy number variation (CNV) and abnormal expression of microRNAs (miRNAs) always lead to deregulation of genes in cancer, including gastric cancer (GC). However, little is known about how CNVs affect the expression of miRNAs. By integrating CNV and miRNA profiles in the same samples, we identified eight miRNAs (miR‐1274a, miR‐196b, miR‐4298, miR‐181c, miR‐181d, miR‐23a, miR‐27a and miR‐24‐2) that were located in the amplified regions and were upregulated in GC. In particular, amplification of miR‐23a‐27a‐24‐2 cluster and miR‐181c‐181d cluster frequently occurred at 19p13.13 and were confirmed by genomic real‐time PCR in another 25 paired GC samples. Moreover, in situ hybridization (ISH) experiments represented that mature miR‐23a was increased in GCs (75.5%, 40/53) compared with matched normal tissues (28.6%, 14/49, P = 0.001). Knocking down of miR‐23a expression inhibited BGC823 cell growth in vitro and in vivo. In addition, the potential target genes of miR‐23a were investigated by integration of mRNA profile and miRNA TargetScan predictions, we found that upregulation of miR‐23a and downregulation of metallothionein 2A (MT2A) were detected simultaneously in 70% (7/10) of the miRNA and mRNA profiles. Furthermore, an inverse correlation between miR‐23a and MT2A expression was detected in GCs and normal tissues. Through combining luciferase assay, we confirmed that MT2A is a potential target of miR‐23a. In conclusion, these results suggest that integration of CNV‐miRNA‐mRNA profiling is a powerful tool for identifying molecular signatures, and that miR‐23a might play a role in regulating MT2A expression in GC. J. Cell. Biochem. 114: 2160–2169, 2013.