Enping Xu

IGFBP7 plays a potential tumor suppressor role in colorectal carcinogenesis

Insulin-like growth factor binding protein-7 (IGFBP7) is a gene identified as being low expressed in colorectal adenocarcinoma (CRC) cell lines. In the current study, we investigated the function of IGFBP7 in CRC by transfection studies. We found that IGFBP7 could inhibit cell growth, decrease soft agar colony formation activity and induce apoptosis in RKO and SW620 cells. Correlation analysis between the expression of IGFBP7 in CRC tissue and the prognosis in 218 patients showed that high expression of IGFBP7 was associated with favourable prognosis. Based on above results, we conclude that IGFBP7 plays a potential tumor suppressor role in colorectal carcinogenesis.

Analysis of SOX9 Expression in Colorectal Cancer

Our purpose was to investigate the role of SOX9, a novel downstream molecule of beta-catenin, in colorectal cancer. Expression of SOX9 and beta-catenin was detected by immunostaining, quantitative real-time reverse transcription-polymerase chain reaction (Q-PCR), and Western blot in colorectal cancer. The correlation between SOX9 or beta-catenin expression and clinicopathologic parameters was also analyzed. Immunostaining, Q-PCR, and Western blot consistently confirmed SOX9 up-regulation in colorectal cancer compared with normal mucosa (P < .05). Immunostaining showed more SOX9+ cells in the lower zone of colonic crypts than in the upper zone (P < .05). Cancers with strong SOX9 immunostaining were significantly associated with a lower 5-year overall survival (40% [17/43] vs low expression, 69% [66/95]; P < .01). The Cox proportional hazards model showed that strong SOX9 expression was an independent adverse prognosticator in colorectal cancer (P < .05). The detection of SOX9 expression might contribute to predicting clinical outcomes for patients with colorectal cancer.

Differential Expression of Mimecan and Thioredoxin Domain–Containing Protein 5 in Colorectal Adenoma and Cancer: A Proteomic Study

Adenoma is the major precursor lesion of colorectal cancer, one of the most common cancers worldwide. The elucidation of the molecular mechanism underlying adenoma is essential for early detection, prevention, and intervention of colorectal cancer. Using a combination of two-dimensional gel electrophoresis and mass spectrometry, we identified 27 differentially expressed proteins in adenoma, compared with matched normal mucosa and cancer tissue. Seventeen proteins were upregulated and six downregulated in adenoma when compared with the same proteins in individual-matched normal mucosa. Four were downregulated, but none upregulated in adenoma when compared with the same proteins in matched cancer tissue. Two novel proteins, mimecan and thioredoxin domain–containing protein 5 (TXNDC5), were further validated by Western blot in 8 colorectal adenomas and 19 cancers that were matched with normal mucosa. All adenoma and cancer tissues did not express mimecan, but all normal mucosa did (P < 0.01). In contrast, TXNDC5 was significantly upregulated in colorectal adenoma and cancer tissues as compared with that in normal mucosa (P < 0.05). This study clearly demonstrated that absence of mimecan and upregulation of TXNDC5 are involved in the early development of colorectal cancer. Thus, the differentially expressed proteins might serve as potential biomarkers for colorectal cancer detection and intervention.

Association of matrix metalloproteinase-2 and -9 promoter polymorphisms with colorectal cancer in Chinese.

Matrix metalloproteinase‐2 (MMP‐2) and ‐9 (MMP‐9) play an important role in cancer initiation, invasion, and metastasis. The aim of this study was to investigate whether common genetic variants in these two key MMPs are associated with the development and progression of colorectal cancer in a Chinese population. We detected the MMP‐2‐790T/G, ‐955A/C, ‐1575G/A, and MMP‐9‐1562 C/T polymorphisms in 126 colorectal cancer patients and matched normal controls by PCR‐denaturing high‐performance liquid chromatography (DHPLC) or PCR‐restriction fragment length polymorphism (RFLP), respectively. We found that the G/G genotype in the MMP‐2‐1575G/A polymorphism was significantly increased in colorectal cancer patients than in the controls (odds ratio (OR), 1.96; 95% CI, 1.06–3.64). Colorectal cancers with G/G genotype were more common with serosa/adventitia layer involvement than those with G/A + A/A genotypes (P < 0.05). However, no significant differences were observed in distribution of the MMP‐2‐790T/G, ‐955A/C, and MMP‐9‐1562 C/T polymorphisms or haplotype of MMP‐2 SNPs between patients and controls. Our results suggest that the presence of ‐1575G allele in the MMP‐2 promoter region may be of significance in the assessment of colorectal cancer risk and invasive potential.

Secretagogin, a novel neuroendocrine marker, has a distinct expression pattern from chromogranin A

Preliminary data suggest that secretagogin (SCGN), a calcium-binding protein identified from our previous proteomics study of colorectal cancers, is a potentially useful neuroendocrine marker. In this study, we further analyzed SCGN expression in normal and tumor tissues from various organ sites compared with three other conventional neuroendocrine markers [chromogranin A (CgA), neuron specific enolase, and synaptophysin]. We found strong SCGN staining in most normal neuroendocrine tissues except in the adrenal cortex. SCGN expression was identified in 140 out of 213 neuroendocrine tumors and 12 conventional carcinomas with neuroendocrine differentiation. In a subset of neuroendocrine tumors, such as gastric neuroendocrine cancers and typical carcinoid tumors of rectum and ovary, SCGN showed strong staining while CgA expression was often negative. It is intriguing to note that SCGN staining was positive in 26 out of 31 small cell lung cancers, more frequently than the other three markers. We conclude that SCGN is a novel neuroendocrine marker that may be useful in routine surgical pathology practice.

TaqMan low density array is roughly right for gene expression quantification in colorectal cancer

BACKGROUND TaqMan low density array (LDA) is promising for high throughput screening in functional genomics by simultaneously measuring mRNA expression of multiple genes. However, the reproducibility and reliability remain to be explored. METHODS We applied LDA to detect mRNA expression of 95 gastrointestinal differentiation associated genes in 27 colorectal cancers with individual-matched normal mucosa. Conventional Q-PCR assay was done to detect 18 differentially expressed genes in additional 22 colorectal cancers. RESULTS A total of 97.2% (11,520/11,844) gene samples were successfully amplified by LDA. There was a perfect agreement between intra-LDA assays in all gene samples (CCC=0.952, p<0.0001). Seventy-nine genes showed perfect or substantial agreement between intra-LDA tests (CCC>0.713). Genes with low Ct values (<30 cycles) had more genes showing perfect agreement, less showing moderate agreement, and lower DeltaCt variances between intra-plate assays than that with high Ct values (>30 cycles) (p<0.01). All 18 genes showed the same directional changes in colorectal cancers versus normal mucosa by both SYBR Green and LDA approaches. CONCLUSIONS LDA is a roughly robust method for gene quantification in colorectal cancer, but its reproducibility decreased in low copy genes. Hence, we strongly recommend caution when analyzing LDA results of those low copy genes.

Erratum to: RegIV expression showing specificity to gastrointestinal tract and its potential role in diagnosing digestive tract neuroendocrine tumor.

Regenerating gene IV (RegIV), a member of the regenerating gene family discovered in 2001, has been found to be involved in malignancy in several different organs including the stomach, colorectum, pancreas and prostate, but the overall expression profile of RegIV has not been reported. To learn more about RegIV, we evaluated its distribution by immunohistochemistry (IHC) in a total of 360 samples including 24 types of normal tissue, 40 benign and malignant lesions, and 18 neuroendocrine tumors. We found that in normal tissues, in addition to its relative specificity for the gastrointestinal tract, RegIV was detected in the adrenal gland and mammary gland. Among all the malignancies of various histological types under evaluation, RegIV was found mostly in adenocarcinomas. Studies on additional sets of colorectal tumor samples showed that RegIV expression was predominant in colorectal adenoma (87.5%) and peritumoral tissue (100%) but not in cancer tissue (30.8%). Among neuroendocrine tumors, RegIV had a relatively restricted expression to those of digestive system.

Mutations of key driver genes in colorectal cancer progression and metastasis

The association between mutations of key driver genes and colorectal cancer (CRC) metastasis has been investigated by many studies. However, the results of these studies have been contradictory. Here, we perform a comprehensive analysis to screen key driver genes from the TCGA database and validate the roles of these mutations in CRC metastasis. Using bioinformatics analysis, we identified six key driver genes, namely APC, KRAS, BRAF, PIK3CA, SMAD4 and p53. Through a systematic search, 120 articles published by November 30, 2017, were included, which all showed roles for these gene mutations in CRC metastasis. A meta-analysis showed that KRAS mutations (combined OR 1.18, 95% CI 1.05–1.33) and p53 mutations (combined OR 1.49, 95% CI 1.23–1.80) were associated with CRC metastasis, including lymphatic and distant metastases. Moreover, CRC patients with a KRAS mutation (combined OR 1.29, 95% CI 1.13–1.47), p53 mutation (combined OR 1.35, 95% CI 1.06–1.72) or SMAD4 mutation (combined OR 2.04, 95% CI 1.41–2.95) were at a higher risk of distant metastasis. Subgroup analysis stratified by ethnic populations indicated that the BRAF mutation was related to CRC metastasis (combined OR 1.42, 95% CI 1.18–1.71) and distant metastasis (combined OR 1.51, 95% CI 1.20–1.91) in an Asian population. No significant association was found between mutations of APC or PIK3CA and CRC metastasis. In conclusion, mutations of KRAS, p53, SMAD4 and BRAF play significant roles in CRC metastasis and may be both potential biomarkers of CRC metastasis as well as therapeutic targets.

No association between the polymorphisms in CDX2 coding regions and colorectal cancer in Chinese

CDX2 has been shown to play an important role in the pathogenesis of colorectal cancer. The aim of this study was to investigate whether genetic variants in CDX2 contributed to the development and progression of colorectal cancer in a Chinese population. We detected the polymorphisms in the CDX2 coding regions in 126 patients with colorectal cancer and matched tumor-free subjects by PCR-based DHPLC. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was also investigated. Three SNPs were identified in the coding region of the CDX2 gene. Neither the genotype frequencies nor allele frequencies of CDX2 polymorphisms showed significant difference from those in healthy controls. There were also no significant association between genotypes and clinicopathological features. When we examined the linkage disequilibrium between three SNPs using expectation-maximization algorithm, we found that there is strong linkage disequilibrium among these SNPs, but no significant difference was found in haplotypes distribution. Our present data suggest that the CDX2 polymorphisms may not be used as a useful marker to predicate susceptibility of colorectal cancer in Chinese.

SRSF6-regulated alternative splicing that promotes tumour progression offers a therapy target for colorectal cancer

Objective To investigate the molecular function of splicing factor SRSF6 in colorectal cancer (CRC) progression and discover candidate chemicals for cancer therapy through targeting SRSF6. Design We performed comprehensive analysis for the expression of SRSF6 in 311 CRC samples, The Cancer Genome Atlas and Gene Expression Omnibus (GEO) database. Functional analysis of SRSF6 in CRC was performed in vitro and in vivo. SRSF6-regulated alternative splicing (AS) and its binding motif were identified by next-generation RNA-sequencing and RNA immunoprecipitation sequencing (RIP-seq), which was validated by gel shift and minigene reporter assay. ZO-1 exon23 AS was investigated to mediate the function of SRSF6 in vitro and in vivo. Based on the analysis of domain-specific role, SRSF6-targeted inhibitor was discovered de novoby virtual screening in 4855 FDA-approved drugs and its antitumour effects were evaluated in vitroand in vivo. Results SRSF6 was frequently upregulated in CRC samples and associated with poor prognosis, which promoted proliferation and metastasis in vitro and in vivo. We identified SRSF6-regulated AS targets and discovered the SRSF6 binding motif. Particularly, SRSF6 regulates ZO-1 aberrant splicing to function as an oncogene by binding directly to its motif in the exon23. Based on the result that SRSF6 RRM2 domain plays key roles in regulating AS and biological function, indacaterol, a β2-adrenergic receptor agonist approved for chronic obstructive pulmonary disease treatment, is identified as the inhibitor of SRSF6 to suppress CRC tumourigenicity. Conclusions SRSF6 functions the important roles in mediating CRC progression through regulating AS, and indacaterol is repositioned as an antitumour drug through targeting SRSF6. Accession numbers The accession numbers for sequencing data are SRP111763 and SRP111797.