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Yichao Liang,1,* Xinling Cai,2,* Xu Zheng,2 Hongzhuan Yin1 1Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning, People’s Republic of China; 2Department of Clinical Oncology, Shengjing Hospital of China Medical University, Shenyang, Liaoning, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hongzhuan YinDepartment of General Surgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning, People’s Republic of ChinaTel +86 18940256969Fax +86 2496615-10094Email chad156@hotmail.comPurpose: To investigate the clinicopathological characteristics of stage I–III colorectal cancer (CRC) patients with deficient mismatch repair (dMMR) protein.Patients and Methods: A retrospective analysis of 61 patients with stage I–III CRC confirmed by immunohistochemistry as dMMR after radical resection at Shenjing Hospital of China Medical University from May 2017 to June 2019 was performed. A total of 183 stage I–III CRC patients with proficient mismatch repair (pMMR) protein from the same period were randomly selected as a control group. The clinicopathological data of the two groups were investigated.Results: There were significant differences between the two groups in age, sex, site of onset, maximum diameter of tumor, T stage, tumor differentiation, and histological type (P < 0.05). No significant difference was detected in nerve vessel invasion, cancer nodules, the N stage or the TNM stage. In the dMMR group, 41 patients (66.13%) showed PMS2/MLH1 deletion, and the number of MSH2/MSH6 deletion is 21 patients (33.87%). Among them, 34 patients (54.84%) had PMS2 and MLH1 deficiency. In total, 16 patients (25.81%) had MSH2 and MSH6 deficiency. A total of 5 patients (8.06%) showed simply PMS2 deletion and 5 patients (8.06%) showed simply MSH6 deletion. In total, 2 patients (3.23%) showed concurrent loss of PMS2, MLH1 and MSH2. No significant difference were found (P > 0.05) in the above factors among dMMR CRC patients with different MMR proteins deletions.Conclusion: Our results show that dMMR status may be more likely exist in female and younger (≤ 55 years) patients with a greater tumor burden (> 5cm), right colon, T4 stage disease, poor differentiation and mucinous adenocarcinoma. Loss of PMS2 and MLH1 is the most common pattern of MMR protein expression deficiency, followed by concurrent deletion of MSH2 and MSH6.Keywords: deficient mismatch repair, clinicopathological characteristics, colorectal cancer, microsatellite instability

Here, we investigated the clinicopathological and prognostic potential of the long noncoding RNA Colon Cancer-Associated Transcript 2 (CCAT2) in human colorectal cancer (CRC). We used qPCR to quantify CCAT2 levels in 44 pairs of CRC tissues and adjacent nontumor and healthy colon mucosa tissues, and in several CRC cell lines (SW620, SW480, HT-29, LOVO, HCT116 and DLD-1) and normal human colorectal epithelial cells (HFC). We assessed the effects of CCAT2 overexpression or knockdown on the proliferation, migration and invasion by SW620 and LOVO cells using CCK-8, transwell, and wound−healing assays, respectively. We also investigated the potential interaction between CCAT2 and TAF15 through RNA pull down and rescue experiments. Lastly, we evaluated the expression of the cell cycle progression markers and GSK3β signaling pathway proteins using Western blotting. Our results showed that CCAT2 was upregulated in CRC tissues and cell lines as com-pared to controls. Ectopic expression of CCAT2 promoted CRC cell proliferation, migration and invasion, likely through direct interaction with TAF15, transcriptional activation of RAB14, and activation of the AKT/GSK3β signaling pathway. In vivo, CCAT2 promoted CRC cell growth and metastasis in nude mice. Taken together, these results highlight the actions of CCAT2 as a CRC oncogene.

Ferroptosis, a newly discovered form of programmed cell death characterized by lipid peroxidation, crafts a new perspective on cancer treatment. Serine and arginine rich splicing factor 9 (SFRS9) is frequently described as a proto-oncogene in cervical and bladder cancer. However, the role of SFRS9 in colorectal cancer (CRC) and whether SFRS9 exerts its function associated with ferroptosis is largely unknown. Herein, we found that the expression of SFRS9 mRNA and protein in the CRC tissues was obviously higher than that in the paracancerous tissues. Function assays revealed that SFRS9 overexpression (SFRS9-OE) significantly promoted cell viability, cell cycle progression and colony formation of CRC cells. While SFRS9 knockdown by shRNAs transfection inhibited these progressions. Furthermore, cell death and lipid peroxidation induced by ferroptosis inducers erastin and sorafenib were suppressed by SFRS9-OE. Bioinformatics analysis indicated that SFRS9 can bind to peroxidase 4 (GPX4) mRNA which is a central regulator of ferroptosis. Western blot showed that GPX4 protein expression was clearly elevated upon SFRS9-OE, while it was decreased in SFRS9-inhibited CRC cells. RNA immunoprecipitation experiment was carried out in HCT116 cells to confirm the binding of SFRS9 and GPX4 mRNA specifically. SiGPX4 transfection reversed the inhibitory effects of SFRS9-OE on the erastin and sorafenib-induced ferroptosis. Consistent with our in vitro observations, SFRS9 promoted the growth of tumors while SFRS9 knockdown significantly inhibited tumor growth in nude mice. In conclusion, SFRS9 represents an obstructive factor to ferroptosis by upregulating GPX4 protein expression, and knocking down SFRS9 might be an effective treatment for CRC.

Purpose B-cell translocation gene 3 (BTG3) has been identified as a candidate driver gene for various cancers, but its specific role in colorectal cancer (CRC) is poorly understood. We aimed to investigate the relationship between expression of BTG3 and clinicopathological features and prognosis, as well as to explore the effects and the role of a possible BTG3 molecular mechanism on aggressive colorectal cancer behavior. Methods BTG3 expression was assessed by immunohistochemistry (IHC) on specimens from 140 patients with CRC. The association of BTG3 expression with clinicopathological features was examined. To confirm the biological role of BTG3 in CRC, two CRC cell lines expressing BTG3 were used and BTG3 expression was knocked down by shRNA. CCK-8, cell cycle, apoptosis, migration, and invasion assays were performed. The influence of BTG3 knockdown was further investigated by genomic microarray to uncover the potential molecular mechanisms underlying BTG3-mediated CRC development and progression. Results BTG3 was downregulated in colorectal cancer tissues and positively correlated with pathological classification (p = 0.037), depth of invasion (p = 0.016), distant metastasis (p = 0.024), TNM stage (p = 0.007), and overall survival (OS) and disease-free survival (DFS). BTG3 knockdown promoted cell proliferation, migration, invasion, relieved G2 arrest, and inhibited apoptosis in HCT116 and LoVo cells. A genomic microarray analysis showed that numerous tumor-associated signaling pathways and oncogenes were altered by BTG3 knockdown. At the mRNA level, nine genes referred to the extracellular-regulated kinase/mitogen-activated protein kinase pathway were differentially expressed. Western blotting revealed that BTG3 knockdown upregulated PAK2, RPS6KA5, YWHAB, and signal transducer and activator of transcription (STAT)3 protein levels, but downregulated RAP1A, DUSP6, and STAT1 protein expression, which was consistent with the genomic microarray data. Conclusions BTG3 expression might contribute to CRC carcinogenesis. BTG3 knockdown might strengthen the aggressive colorectal cancer behavior.

Previous studies on the expression of Sal-like 4 (SALL4), a zinc finger transcription factor, had conflicting results in colorectal cancer (CRC). The main aim of this study was to investigate the expression of SALL4 and its relationship with β-catenin in CRC. Immunohistochemistry was performed to examine the expression of SALL4 and β-catenin in a cohort of 149 patients with CRC, 12 with atypical hyperplasia, 25 with benign tumor and 38 with normal tissue. Expression patterns of SALL4 and β-catenin and correlation with clinicopathological features were investigated. The relationship between SALL4 and β-catenin was examined by immunofluorescence and co-immunoprecipitation using CRC cell lines, SW480, SW620, HCT116 and HT29. Immunohistochemical analysis revealed significantly lower expression of SALL4 in CRC (46.3 %) than atypical hyperplasia (68.0 %, p < 0.05) and normal tissue (78.9 %, p < 0.01). Well-differentiated CRC seemed to express more SALL4 (47.6 %) than moderately (45.8 %) and poorly-differentiated cancers (18.8 %) (p < 0.05). However, SALL4 expression positively correlated with lymph node metastasis and tumor-node-metastasis (TNM) and Dukes stages (all p < 0.05) suggesting a new mechanism involved in the function of SALL4 in CRC. β-catenin was expressed significantly higher in CRC (69.1 %) than normal tissue (21.1 %, p < 0.01), and positively correlated with CA19-9 level in serum (p < 0.05). SALL4 and β-catenin were positively correlated in CRC (Spearman correlation coefficient R = 0.536, p < 0.01). The group of co-expression of the two molecules showed advanced lymph node metastasis, TNM stage and Dukes stage (all p < 0.05). Double-labeling immunofluorescence and co-immunoprecipitation indicated that SALL4 and β-catenin co-localized in the nucleus and cytoplasm and interacted. Taken together, our results revealed that SALL4 and β-catenin were positively correlated in CRC. In CRC cells, SALL4 and β-catenin co-localized and interacted. The function of SALL4 in promoting lymph node metastasis and advanced clinical stage might partly be due to the interaction with β-catenin.

Aim: To evaluate the potential of tumor-targeted nanoprobe, N-(2-hydroxypropyl)methacrylamide copolymer-conjugated zinc protoporphyrin (PZP) for photodynamic therapy (PDT) and tumor imaging. Materials & Methods: Different tumor models including carcinogen-induced cancer were used, PZP was intravenously injected followed by irradiation with xenon or blue fluorescent light on tumor. Results: One PZP 20 mg/kg (ZnPP equivalent) dose with two or three treatments of light at an intensity of ≥20 J/cm2 caused necrosis and disappearance of most tumors (>70%) in different tumor models. We also confirmed PZP-based tumor imaging in carcinogen-induced breast tumor and colon cancer models. Conclusion: These findings support the potential application of PZP as a tumor-selective nanoprobe for PDT as well as tumor imaging, by virtue of the enhanced permeability and retention effect., To evaluate the potential of a tumor-targeted nanoprobe, PZP and normal xenon light source for photodynamic therapy and tumor imaging, different tumor models including cancer induced by carcinogen were used. In all models, a high accumulation of PZP in tumor was found after intravenous injection, resulting in remarkable therapeutic effect. These findings support further research to assess the potential application of PZP as a future nanomedicine for photodynamic cancer therapy and imaging in cancers of the esophagus, breast, lung, colon, rectum, urinary bladder and cervix.

Oxidative stress is associated with various pathological processes including inflammatory bowel disease, which is a major cause of colon cancer. Here, we examined the antioxidative and anti-inflammatory effects of 4-vinyl-2,6-dimethoxyphenol (canolol), a potent antioxidant compound obtained from crude canola oil. Oral administration of 2% dextran sulfate sodium (DSS) resulted in the progression of colitis with shortening of the large bowel length. Administering a diet containing canolol significantly suppressed pathogenesis; diarrhea markedly improved and the length of large bowel returned to almost normal. Pathological examination clearly revealed improvement of colonic ulcers. Production of inflammatory cytokines, i.e. interleukin-12 and tumor necrosis factor-α, was significantly increased during this pathological process; their production was markedly inhibited by canolol. In the azoxymethane/DSS-induced colon cancer model, mice receiving canolol had a reduced occurrence of cancer, to 60%, compared with control mice, 100% of which had colon cancer. The numbers of tumors in each mouse were also significantly reduced in mice receiving the canolol-containing diet (5.6±2.0) compared with azoxymethane/DSS control mice (10.8±4.2). No apparent toxicity of canolol was observed. Moreover, inflammatory cytokines (i.e. cyclooxygenase-2, inducible nitric oxide synthase and tumor necrosis factor-α) and oxidative responding molecules, i.e. heme oxygenase-1, in colon were suppressed during this treatment. In a mouse colon 26 solid tumor model, canolol significantly suppressed cyclooxygenase-2 expression; however, no significant tumor growth inhibition was observed, suggesting that canolol preferably shows chemopreventive effects during the stages of initiation/promotion. Canolol may, thus, be considered a potential cancer preventive agent or supplement.

Integrin-linked kinase (ILK), an intracellular serine-threonine kinase, has been reported to be overexpressed in multiple types of human malignancies, including colorectal cancer (CRC). The prognostic value of ILK in CRC, however, remains unknown. In the present study, expression of ILK in 25 paired primary CRC samples and adjacent noncancerous tissues were quantified using real-time PCR and Western blotting. ILK protein expression was analyzed in 102 archived, paraffin-embedded CRC samples using immunohistochemistry. The correlation between ILK expression and clinicopathological factors was evaluated by the χ(2) test. Patients' overall survival was analyzed by Kaplan-Meier method. We found that both ILK mRNA and protein expression levels were significantly up-regulated in primary CRC samples compared with their corresponding normal tissues. Immunohistochemical analysis revealed relative high expression of ILK in 43 of 102 (42.2 %) primary CRC samples. Statistical analysis showed a significant correlation of ILK expression with tumor differentiation, lymph node metastasis, tumor invasion, and tumor-node-metastasis stage. Patients with tumors displaying high-level ILK expression showed significantly shorter overall survival (P = 0.028, log-rank test). More importantly, multivariate analysis indicated that high ILK protein expression was an independent prognostic factor for CRC patients (P = 0.026). Taken together, our data suggest that ILK overexpression is associated with tumor progression and a poor prognosis in CRC patients and may represent a novel potential prognostic marker for patients with CRC.

Multi-exon genes may generate distinct isoforms in different conditions and exhibit versatile properties. Here we investigated the isoform-specific gene expression and the gene expression changes of without and with serum-induced human HCT-116 colon cancer cell lines. For these analyses, 4 transcriptome sequencing datasets were used and 2 replicates for each condition. We observed that ~73% of those expressed genes in four samples generated only one isoform, while ~27% encoded at least two isoforms. Our results show that human gene expression can exhibit great flexibility in alternative splicing. Those expressed genes generated ~1.4 isoforms for each gene across four samples on average. In addition, most of these expressed genes were expressed at moderate or low levels. We found that these four samples have similar patterns of their gene expression distributions. Furthermore, we also conducted the differential expression analysis between without and with serum-induced two conditions of these four samples. We detected that 123 genes were differentially expressed and 110 of them were up-regulated. Among those differentially expressed genes, we found intriguing phenomena that a portion of them could be clustered into different functional groups and some oncogenes and proto-oncogenes are differentially expressed.

Background Heme oxygenase-1 (HO-1) and its major product carbon monoxide (CO) are known to be involved in the development and progression of many tumors. The present study was to elucidate the expression and function of HO-1 in colorectal cancer (CRC), specially focusing on the circulation CO levels in CRC patients and the possible roles of HO-1 in chemoresistance of colon cancer cells. Methods One hundred and eighteen patients received resection for colorectal cancer and polyps at China Medical University Sheng Jing Hospital, were collected in this study. HO-1 expression in CRC tissues was analyzed by immnuohistochemical staining; circulation CO levels as carboxyhemoglobin (COHb) in CRC patients were analyzed by an ABL800 FLEX blood gas analyzer. HO-1 expression in murine colon cells C26 and human colon cancer cells HT29 and DLD1 under HO-1 inducer hemin and anticancer drug pirarubicin (THP) treatment was examined by RT-PCR, and the cell viability after each treatment was investigated by MTT assay. Data were analyzed by student’s t-test or one-way ANOVA followed by Bonferroni t-test or Fisher's exact test. Results HO-1 expression in tumor tissues of CRC (61.0%) was significantly higher than in normal colorectal tissues and polyps tissues (29.7%, P

Background Somatic variants, which occur in the genome of all cells, are well accepted to play a critical role in cancer development, as their accumulation in genes could affect cell proliferations and cell cycle. Methods In order to understand the role of somatic mutations in human colorectal cancers, we characterized the mutation spectrum in two colorectal tumor tissues and their matched normal tissues, by analyzing deep-sequenced transcriptome data. Results We found a higher mutation rate of somatic variants in tumor tissues in comparison with normal tissues, but no trend was observed for mutation properties. By applying a series of stringent filters, we identified 418 genes with tumor specific disruptive somatic variants. Of these genes, three genes in mucin protein family (MUC2, MUC4, and MU12) are of particular interests. It has been reported that the expression of mucin proteins was correlated with the progression of colorectal cancer therefore somatic variants within those genes can interrupt their normal expression and thus contribute to the tumorigenesis. Conclusions Our findings provide evidence of the utility of RNA-Seq in mutation screening in cancer studies, and suggest a list of candidate genes for future colorectal cancer diagnosis and treatment.