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lncRNA can regulate tumorigenesis development and distant metastasis of colorectal cancer (CRC). However, the detailed molecular mechanisms are still largely unknown. Using RNA-sequencing data, RT-qPCR, and FISH assay, we found that HIF1A-AS2 was upregulated in CRC tissues and associated with poor prognosis. Functional experiments were performed to determine the roles of HIF1A-AS2 in tumor progression and we found that HIF1A-AS2 can promote the proliferation, metastasis, and aerobic glycolysis of CRC cells. Mechanistically, HIF1A-AS2 can promote FOXC1 expression by sponging miR-141-3p. SP1 can transcriptionally activate HIF1A-AS2. Further, HIF1A-AS2 can be packaged into exosomes and promote the malignant phenotype of recipient tumor cells. Taken together, we discovered that SP1-induced HIF1A-AS2 can promote the metabolic reprogramming and progression of CRC via miR-141-3p/FOXC1 axis. HIF1A-AS2 is a promising diagnostic marker and treatment target in CRC., (© 2024. The Author(s).)

The enteric nervous system (ENS) functions largely independently of the central nervous system (CNS). Glutamate, the dominant neurotransmitter in the CNS and sensory afferents, is not a primary neurotransmitter in the ENS. Only a fraction (∼2%) of myenteric neurons in the mouse distal colon and rectum (colorectum) are positive for vesicular glutamate transporter type 2 (VGLUT2), the structure and function of which remain undetermined. Here, we systematically characterized VGLUT2-positive enteric neurons (VGLUT2-ENs) through sparse labeling with adeno-associated virus, single-cell mRNA sequencing (scRNA-seq), and GCaMP6f calcium imaging. Our results reveal that the majority of VGLUT2-ENs (29 of 31, 93.5%) exhibited Dogiel type I morphology with a single aborally projecting axon; most axons (26 of 29, 89.7%) are between 4 and 10 mm long, each traversing 19 to 34 myenteric ganglia. These anatomical features exclude the VGLUT2-ENs from being intrinsic primary afferent or motor neurons. The scRNA-seq conducted on 52 VGLUT2-ENs suggests different expression profiles from conventional descending interneurons. Ex vivo GCaMP6f recordings from flattened colorectum indicate that almost all VGLUT2-EN (181 of 215, 84.2%) are indirectly activated by colorectal stretch via nicotinic cholinergic neural transmission. In conclusion, VGLUT2-ENs are a functionally unique group of enteric neurons with single aborally projecting long axons that traverse multiple myenteric ganglia and are activated indirectly by colorectal mechanical stretch. This knowledge will provide a solid foundation for subsequent studies on the potential interactions of VGLUT2-EN with extrinsic colorectal afferents via glutamatergic neurotransmission. NEW & NOTEWORTHY We reveal that VGLUT2-positive enteric neurons (EN), although constituting a small fraction of total EN, are homogeneously expressed in the myenteric ganglia, with a slight concentration at the intermediate region between the colon and rectum. Through anatomic, molecular, and functional analyses, we demonstrated that VGLUT2-ENs are activated indirectly by noxious circumferential colorectal stretch via nicotinic cholinergic transmission, suggesting their participation in mechanical visceral nociception.

Sex differences in visceral nociception have been reported in clinical and preclinical studies, but the potential differences in sensory neural encoding of the colorectum between males and females are not well understood. In this study, we systematically assessed sex differences in colorectal neural encoding by conducting high-throughput optical recordings in intact dorsal root ganglia (DRGs) from control and visceral hypersensitive mice. We found an apparent sex difference in zymosan-induced behavioral visceral hypersensitivity: enhanced visceromotor responses to colorectal distension were observed only in male mice, not in female mice. In addition, a higher number of mechanosensitive colorectal afferents were identified per mouse in the zymosan-treated male group than in the saline-treated male group, whereas the mechanosensitive afferents identified per mouse were comparable between the zymosan- and saline-treated female groups. The increased number of identified afferents in zymosan-treated male mice was predominantly from thoracolumbar (TL) innervation, which agrees with the significant increase in the TL afferent proportion in the zymosan group as compared with the control group in male mice. In contrast, female mice showed no difference in the proportion of colorectal neurons between saline- and zymosan-treated groups. Our results revealed a significant sex difference in colorectal afferent innervation and sensitization in the context of behavioral visceral hypersensitivity, which could drive differential clinical symptoms in male and female patients. NEW & NOTEWORTHY We used high-throughput GCaMP6f recordings to study 2,275 mechanosensitive colorectal afferents in mice. Our results revealed significant sex differences in the zymosan-induced behavioral visceral hypersensitivity, which were present in male but not female mice. Male mice also showed sensitization of colorectal afferents in the thoracolumbar pathway, whereas female mice did not. These findings highlight sex differences in sensory neural anatomy and function of the colorectum, with implications for sex-specific therapies for treating visceral pain.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting reaction in the de novo synthesis pathway of guanine nucleotides that is highly required for cancer cell outgrowth. Herein, we found that IMPDH isoform 2 (IMPDH2) is highly expressed in colorectal cancer (CRC) and is correlated with poor patient prognosis. Via structure-based virtual screening, we identified berberrubine, a critical ingredient of the medical plant Coptis chinensis, as a novel, selective, and competitive inhibitor of IMPDH2, which demonstrated over 15-fold selectivity to IMPDH2 than IMPDH1. Besides, we also confirmed the interaction between berberrubine and IMPDH2. Of note, berberrubine treatment significantly impairs the growth of human CRC cells in a dose-dependent manner, which can be rescued by supplementing with guanosine. Furthermore, oral administration of berberrubine remarkably reduced tumor volume and weight in a human cell line-derived xenograft model. Importantly, the anti-cancer activity of berberrubine was also confirmed by using the azoxymethane (AOM) / dextran sulfate sodium (DSS)-induced spontaneous CRC mouse model. Taken together, our study highlights that berberrubine acts as a novel IMPDH2 inhibitor, suppressing the growth of CRC in vitro and in vivo, providing a fresh perspective for its potential application in the treatment of CRC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dectin-1 (gene Clec7a), a receptor for β-glucans, plays important roles in the host defense against fungi and immune homeostasis of the intestine. Although this molecule is also suggested to be involved in the regulation of tumorigenesis, the role in intestinal tumor development remains to be elucidated. In this study, we find that azoxymethane-dextran-sodium-sulfate-induced and Apc Min -induced intestinal tumorigenesis are suppressed in Clec7a -/- mice independently from commensal microbiota. Dectin-1 is preferentially expressed on myeloid-derived suppressor cells (MDSCs). In the Clec7a -/- mouse colon, the proportion of MDSCs and MDSC-derived prostaglandin E 2 (PGE 2 ) levels are reduced, while the expression of IL-22 binding protein (IL-22BP; gene Il22ra2) is upregulated. Dectin-1 signaling induces PGE 2 -synthesizing enzymes and PGE 2 suppresses Il22ra2 expression in vitro and in vivo. Administration of short chain β-glucan laminarin, an antagonist of Dectin-1, suppresses the development of mouse colorectal tumors. Furthermore, in patients with colorectal cancer (CRC), the expression of CLEC7A is also observed in MDSCs and correlated with the death rate and tumor severity. Dectin-1 signaling upregulates PGE 2 -synthesizing enzyme expression and PGE 2 suppresses IL22RA2 expression in human CRC-infiltrating cells. These observations indicate a role of the Dectin-1-PGE 2 -IL-22BP axis in regulating intestinal tumorigenesis, suggesting Dectin-1 as a potential target for CRC therapy., (© 2023. The Author(s).)

Background: Cuprotosis is a novel form of programmed cell death that involves direct targeting of key enzymes in the tricarboxylic acid (TCA) cycle by excess copper and may result in mitochondrial metabolic dysfunction. However, whether cuprotosis may mediate the tumor microenvironment (TME) and immune regulation in colorectal cancer (CRC) remains unclear., Methods: Ten cuprotosis-related genes were selected and unsupervised consensus clustering was performed to identify the cuprotosis patterns and the correlated TME characteristics. Using principal component analysis, a COPsig score was established to quantify cuprotosis patterns in individual patients. The top 9 most important cuprotosis signature genes were analyzed using single-cell transcriptome data., Results: Three distinct cuprotosis patterns were identified. The TME cell infiltration characteristics of three patterns were associated with immune-excluded, immune-desert, and immune-inflamed phenotype, respectively. Based on individual cuprotosis patterns, patients were assigned into high and low COPsig score groups. Patients with a higher COPsig score were characterized by longer overall survival time, lower immune cell as well as stromal infiltration, and greater tumor mutational burden. Moreover, further analysis demonstrated that CRC patients with a higher COPsig score were more likely to respond to immune checkpoint inhibitors and 5-fluorouracil chemotherapy. Single-cell transcriptome analysis indicated that cuprotosis signature genes recruited tumor-associated macrophages to TME through the regulation of TCA and the metabolism of glutamine and fatty acid, thus influencing the prognosis of CRC patients., Conclusion: This study indicated that distinct cuprotosis patterns laid a solid foundation to the explanation of heterogeneity and complexity of individual TME, thus guiding more effective immunotherapy as well as adjuvant chemotherapy strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xu, Ding, Zhong, Qin, Shu, Yu, Abuduaini, Zhang, Yang and Feng.)

Mucin 3A (MUC3A) is overexpressed in colorectal cancer (CRC) and associated with poor prognosis, but the related mechanism remains unclear. Our study found that MUC3A promotes the progression of CRC by activating the PI3K/Akt/mTOR signaling pathway. Knockout of MUC3A significantly inhibited the proliferation of CRC cells and induced G1 phase arrest by upregulating p21 protein, an important cell cycle regulator. Moreover, knockout of MUC3A significantly inhibited invasion ability and enhanced the sensitivity to the chemotherapeutic agent 5-FU. Furthermore, we found that knockout of MUC3A repressed the PI3K/Akt/mTOR pathway through RNA-seq. Treatment with the PI3K/Akt/mTOR pathway inhibitor rapamycin successfully eliminated the difference in proliferation, invasion and chemoresistance between MUC3A knockout cells and control cells. Our study suggests that MUC3A is a potential oncogene that promotes the proliferation, invasion, and chemotherapy resistance of CRC. Moreover, CRC patients with high expression of MUC3A may benefit from rapamycin treatment., (© 2022. The Author(s).)

Abstract: Clinical evidence indicates dorsal root ganglion (DRG) stimulation effectively reduces pain without the need to evoke paresthesia. This paresthesia-free anesthesia by DRG stimulation can be promising to treat pain from the viscera, where paresthesia usually cannot be produced. Here, we explored the mechanisms and parameters for DRG stimulation using an ex vivo preparation with mouse distal colon and rectum (colorectum), pelvic nerve, L6 DRG, and dorsal root in continuity. We conducted single-fiber recordings from split dorsal root filaments and assessed the effect of DRG stimulation on afferent neural transmission. We determined the optimal stimulus pulse width by measuring the chronaxies of DRG stimulation to be below 216 µs, indicating spike initiation likely at attached axons rather than somata. Subkilohertz DRG stimulation significantly attenuates colorectal afferent transmission (10, 50, 100, 500, and 1000 Hz), of which 50 and 100 Hz show superior blocking effects. Synchronized spinal nerve and DRG stimulation reveals a progressive increase in conduction delay by DRG stimulation, suggesting activity-dependent slowing in blocked fibers. Afferents blocked by DRG stimulation show a greater increase in conduction delay than the unblocked counterparts. Midrange frequencies (50-500 Hz) are more efficient at blocking transmission than lower or higher frequencies. In addition, DRG stimulation at 50 and 100 Hz significantly attenuates in vivo visceromotor responses to noxious colorectal balloon distension. This reversible conduction block in C-type and Aδ-type afferents by subkilohertz DRG stimulation likely underlies the paresthesia-free anesthesia by DRG stimulation, thereby offering a promising new approach for managing chronic visceral pain., (Copyright © 2021 International Association for the Study of Pain.)

Background and Objectives: To investigate the effects of different suture reinforcement methods for anastomotic leakage and other postoperative complications after the use of a laparoscopic double stapling technique (DST)., Methods: We collected the data of 124 patients who underwent laparoscopic radical resection of colorectal cancer from July 2017 to September 2018 at our institution. Patients were divided into three groups according to the suture reinforcement methods: intermittent, continuous suture reinforcement, and non-reinforcement (n = 41, 41, and 42, respectively). One-way analysis of variance, χ 2 , Fisher's exact, and nonparametric tests were used for statistical analysis., Results: Among the 124 patients, there were no statistically significant differences in operation times, intraoperative blood loss, postoperative hospital stays and recovery of bowel movement. Nine patients were diagnosed with anastomotic leakage (AL). The incidences of serious AL in the intermittent and continuous suture reinforcement groups were lower than that in the control group, with lower reoperation rate, shorter average lengths of stay and lower treatment costs of two experimental groups., Conclusion: Intermittent and continuous sutures after laparoscopic DST is effective, safe, and feasible on anastomotic leakage prevention. These procedures could be popularized in rectal surgery on patients with high risk of AL., (© 2021 Wiley Periodicals LLC.)

Colorectal cancer (CRC) is the leading cause of cancer-related mortality in the world. Accumulating evidence indicate that tumour infiltrating immune cells participated in cancer progression. Among them, tumour infiltrating neutrophils (TINs) are reported to play crucial role in various cancers. In this study, we used CIBERSORTx, a digital cytometry tool to evaluate the neutrophils infiltration in CRC based on gene expression data of CRC tissues from GSE39582 data set and The Cancer Genome Atlas data set (TCGA-COAD and TCGA-READ). Weighted gene co-expression network analysis (WGCNA) was conducted in GSE39582 data set to identify hub genes associated with neutrophil infiltration. The association of hub gene and neutrophils was then validated in TCGA cohorts and an independent RJ cohort. Functional analysis was performed to investigate the molecular mechanisms of the interested hub gene. We found that neutrophil infiltration is elevated in CRC tissues, and it is related to a poorer prognosis. A total of 18 gene modules are identified by WGCNA in GSE39582 data set, among which lightcyan module is significantly correlated with neutrophils infiltration. Furthermore, Superoxide Dismutase 2 (SOD2) in lightcyan module was proved to correlated with neutrophils infiltration in various cancer types. In addition, SOD2 expression is highly associated with several chemokines, including CXCL8, a neutrophils-related attractant, and functional analysis revealed that SOD2 is involved in neutrophils recruitment biological process. These results indicate that an 'SOD2-CXCL8-neutrophil recruitment' axis plays a potential role in colorectal cancer progression., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: N6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulation in eukaryotic cells. However, understanding of m6A in colorectal cancer (CRC) is very limited. We designed this study to investigate the role of m6A in CRC., Methods: Expression level of METTL14 was extracted from public database and tissue array to investigate the clinical relevance of METTL14 in CRC. Next, gain/loss of function experiment was used to define the role of METTL14 in the progression of CRC. Moreover, transcriptomic sequencing (RNA-seq) was applied to screen the potential targets of METTL14. The specific binding between METTL14 and presumed target was verified by RNA pull-down and RNA immunoprecipitation (RIP) assay. Furthermore, rescue experiment and methylated RNA immunoprecipitation (Me-RIP) were performed to uncover the mechanism., Results: Clinically, loss of METTL14 correlated with unfavorable prognosis of CRC patients. Functionally, knockdown of METTL14 drastically enhanced proliferative and invasive ability of CRC cells in vitro and promoted tumorigenicity and metastasis in vivo. Mechanically, RNA-seq and Me-RIP identified lncRNA XIST as the downstream target of METTL14. Knockdown of METTL14 substantially abolished m6A level of XIST and augmented XIST expression. Moreover, we found that m6A-methylated XIST was recognized by YTHDF2, a m6A reader protein, to mediate the degradation of XIST. Consistently, XIST expression negatively correlated with METTL14 and YTHDF2 in CRC tissues., Conclusion: Our findings highlight the function and prognostic value of METTL14 in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.

Background: N-myc downstream-regulated gene 1 (NDRG1) has been shown to play a key role in tumor metastasis. Recent studies demonstrate that NDRG1 can suppress tumor growth and is related to tumor proliferation; however, the mechanisms underlying these effects remain obscure., Methods: Immunohistochemistry (IHC) was used to detect NDRG1 and p21 protein expression in colorectal cancer tissue, and clinical significance of NDRG1 was also analyzed. CCK-8 assay, colony formation assay, flow cytometry, and xenograft model were used to assess the effect of NDRG1 on tumor proliferation in vivo and in vitro. The mechanisms underlying the effect of NDRG1 were investigated using western blotting, immunofluorescence, immunoprecipitation, and ubiquitylation assay., Results: NDRG1 was down-regulated in CRC tissues and correlated with tumor size and patient survival. NDRG1 inhibited tumor proliferation through increasing p21 expression via suppressing p21 ubiquitylation. NDRG1 and p21 had a positive correlation both in vivo and in vitro. Mechanistically, E3 ligase NEDD4 could directly interact with and target p21 for degradation. Moreover, NDRG1 could emulatively antagonize NEDD4-mediated ubiquitylation of p21, increasing p21 expression and inhibit tumor proliferation., Conclusion: Our study could fulfill potential mechanisms of the NDRG1 during tumorigenesis and metastasis, which may serve as a tumor suppressor and potential target for new therapies in human colorectal cancer.

Mechanical distension beyond a particular threshold evokes visceral pain from distal colon and rectum (colorectum), and thus biomechanics plays a central role in visceral nociception. In this study we focused on the layered structure of the colorectum through the wall thickness and determined the biomechanical properties of layer-separated colorectal tissue. We harvested the distal 30 mm of mouse colorectum and dissected this tissue into inner and outer composite layers. The inner composite consists of the mucosa and submucosa, whereas the outer composite includes the muscular layers and serosa. We divided each composite axially into three 10-mm-long segments and conducted biaxial mechanical extension tests and opening-angle measurements for each tissue segment. In addition, we quantified the thickness of the rich collagen network in the submucosa by nonlinear imaging via second-harmonic generation (SHG). Our results reveal that the inner composite is slightly stiffer in the axial direction, whereas the outer composite is stiffer circumferentially. The stiffness of the inner composite in the axial direction is about twice that in the circumferential direction, consistent with the orientations of collagen fibers in the submucosa approximately ±30° to the axial direction. Submucosal thickness measured by SHG showed no difference from proximal to distal colorectum under the load-free condition, which likely contributes to the comparable tension stiffness of the inner composite along the colorectum. This, in turn, strongly indicates the submucosa as the load-bearing structure of the colorectum. This further implies nociceptive roles for the colorectal afferent endings in the submucosa, which likely encode tissue-injurious mechanical distension. NEW & NOTEWORTHY Visceral pain from distal colon and rectum (colorectum) is usually elicited from mechanical distension/stretch, rather than from heating, cutting, or pinching, which usually evoke pain from the skin. We conducted layer-separated biomechanical tests on mouse colorectum and identified an unexpected role of submucosa as the load-bearing structure of the colorectum. Outcomes of this study will focus attention on sensory nerve endings in the submucosa that likely encode tissue-injurious distension/stretch to cause visceral pain.

Objective: To analyze the status of domestic surgical treatment of synchronous peritoneal carcinomatosis from colorectal cancer in China. Methods: Clinicopathological data of patients who underwent surgery from October 2003 to October 2018 in 16 domestic medical centers was retrospectively analyzed. Excel database was created which covered 77 fields of 7 parts: baseline information of patients, laboratory tests, imaging tests, chemoradiotherapy information, intra-operative findings, postoperative pathology and follow-up data. The Wilcoxon rank-sum test was used for comparison of the measurement data between groups. The χ(2) test was used for comparison of the categorical data between groups. The survival curve was calculated by the Kaplan-Meier method. Results: Of the 1 003 patients, there were 575 male and 428 female patients with the age of (58.5±14.1) years (range: 18 to 92 years). In a total of 920 patients, the carcinoma of sigmoid colon was performed in 292 cases (31.8%) with the highest ratio. The proportion of patients with liver metastasis and lung metastasis were 27.9% (219/784) and 8.3% (64/769). Preoperative detection of carcino-embryonic antigen level was the most common method in China (87.74%, 880/1 003), and the positive rate was 64.5% (568/880). The correct rate of preoperative imaging tests was 40.7% (280/688). The ratio of peritoneal carcinomatosis index (PCI) scores between 0 and 10 was the highest (59.6%, 170/285). Two hundred and sixty-two (27.0%) patients were performed by totally laparoscopic operation in 971 patients. The resection of primary tumor was performed in 588 of the 817 patients (72.0%). In a total of 457 cases, 253 (55.4%) patients were performed cytoreduction which group scored completeness of cytoreduction (CCR) 0. The postoperative hyperthermic intraperitoneal chemotherapy was implemented in 70 of the 334 cases (21.0%). Among 1 003 cases, 562 cases (56.03%) had complete follow-up data and the median overall survival was 15 months. The primary tumor resection and the CCR scores were affected by the PCI scores. The patients underwent primary tumor resection (187/205 vs . 26/80, χ(2)=105.085, P= 0.000) and the patients were performed cytoreduction which scored CCR 0 or CCR 1 (162/204 vs . 8/78, Z= -10.465, P= 0.000) had significant difference between the groups of PCI<20 and ≥20. There was a close correlation between the surgical method and the CCR scores ( Z= -3.246, P= 0.001).When the maximum degree of tumor reduction was planned, most surgeons would choose laparotomy. The overall survival time was longer in patients with primary tumor resection ( P= 0.000). The median survival time was 18.6 months in the group of primary tumor resection. Conclusions: It is difficult to diagnose the synchronous peritoneal carcinomatosis from colorectal cancer before the operation. Primary tumor resection has an obvious effect to prolong the survival time. It is necessary to standardize the treatment of peritoneal metastasis.

Background and Aim: Removal of neoplastic polyps is important for colorectal cancer prevention. The primary aim was to compare the complete resection rate of diminutive (≤ 5 mm) or small colorectal polyps (6-10 mm) using cold snare polypectomy (CSP) versus hot snare polypectomy (HSP)., Methods: To April 2018, databases of Medline, EMBASE, Cochrane Central Register of Controlled Trials, and Web of Science were searched for randomized controlled trials comparing CSP and HSP for diminutive or small colorectal polypectomy. Two authors independently assessed trial quality and extracted data. We contacted study authors for additional information. We used the Mantel-Haenszel method for binary endpoints and inverse variance method for continuous outcomes. Subgroup analysis was conducted to explore sources of heterogeneity., Results: Twelve trials involving 2481 patients and 4535 polyps were analyzed. Regarding complete resection rate, there was no statistical significance between CSP and HSP (odds ratio [OR] 0.86, 95% confidence interval [CI] 0.60-1.24). CSP shows more immediate bleeding cases than HSP in per-lesion analysis while no statistical significance in bleeding needing treatment (OR 1.99, 95% CI 0.59-6.75). In terms of all adverse events, both per-lesion and per-patient analysis revealed no difference (OR 1.49, 95% CI 0.87-2.56 and 0.57, 0.11-2.97, respectively). As far as post-polypectomy bleeding is concerned, there was also no statistical significance between CSP and HSP. Regarding procedure time, CSP was superior to HSP (standard mean difference -1.04, 95% CI -1.22 to -0.87)., Conclusions: Cold snare polypectomy is a safe, efficient, and effective polypectomy technique for diminutive or small colorectal polyps., (© 2018 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

The aberrant expression of mucin-type O-glycosylation plays important roles in cancer malignancy. The polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) are a family of conserved enzymes that initiate the mucin-type O-glycosylation in cells. In human, consistent up- or down-regulation of ppGalNAc-Ts expression during cancer development has been frequently reported. Here, we provide evidence that ppGalNAc-T4 shows a stage-dependent expression at the different stages of colorectal cancer (CRC) in the 62 pair-matched tumor/normal tissues. In detail, ppGalNAc-T4 expression is significantly induced at stage I and II but not at stage III and IV. Overexpression of ppGalNAc-T4 in CRC cells enhances colony formation and sphere formation suggesting an important role of ppGalNAc-T4 in tumorigenesis. Conversely, knockdown of ppGalNAc-T4 in CRC cells increases the cell migration and invasion, and leads to an epithelial-mesenchymal transition-like transition. Further analysis suggests that loss of ppGalNAc-T4 contributes to the dedifferentiation of CRC and high expression of ppGalNAc-T4 correlates to a good prognosis of patients. Taken together, our results not only demonstrate a stage-dependent expression of ppGalNAc-T4 in CRC progression, but also suggest that such stage-dependent expression may contribute to the tumorigenesis at the early stage and promote cell migration and invasion at the advanced stage., (© 2018 Federation of European Biochemical Societies.)

Background: It remains unknown whether blockade of c-Met signaling and epidermal growth factor receptor signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. In this study, we investigated the effects of the c-Met inhibitor PHA-665752 alone and in combination with cetuximab on the growth of human CRC cells in vitro and in mouse xenografts., Methods: Human CRC cell lines (Caco2, HCT-116, and HT-29) and mice bearing HCT-116 xenografts were treated with cetuximab in the absence or presence of PHA-665752. Cell viability and apoptosis were examined using the MTT and TUNEL assays, respectively. Vimentin was measured by immunohistochemistry as a marker for epithelial-to-mesenchymal transition. Western blotting was used to determine signaling protein expression levels., Results: The MTT assay showed that the growth of Caco2, HCT-116, and HT-29 cells was inhibited by PHA-665752 in a dose-dependent manner, but only Caco2 cell growth was suppressed by cetuximab. Combination treatment with PHA-665752 and cetuximab inhibited the proliferation of Caco2 cells and RAS mutant CRC cell lines. However, relative to the PHA-665752-alone treatment group, HT-29 cells with a BRAF mutation showed no noticeable effect. The mean tumor volume in mice treated with cetuximab in combination with PHA-665752 was significantly smaller than that in the mice treated with only cetuximab (P = 0.033) or PHA-665752 (P < 0.01). Similarly, the expression of vimentin in the mice treated with PHA-665752 in combination with cetuximab was significantly lower than that in the mice treated with cetuximab or PHA-665752 alone (P < 0.05 in each case). TUNEL assays revealed that treatment with PHA-665752 in combination with cetuximab markedly increased CRC cell apoptosis. Western blotting analysis of signaling protein expression showed that PHA- 665752 inhibited Met phosphorylation (P < 0.05). In addition, treatment with cetuximab alone or in combination with PHA-665752 effectively inhibited EGFR phosphorylation (P < 0.05)., Conclusion: Combination treatment with PHA-665752 and cetuximab suppressed in vitro and in vivo CRC cell growth more than treatment with either agent alone did., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Cetuximab has been regularly added to the treatments for metastatic colorectal cancer worldwide. However, due to its therapeutic insensitivity and underlying mechanisms being largely unknown, the clinical implementation of cetuximab in colorectal cancer remains limited., Methods: The gene expression profile GSE56386 was retrieved from the Gene Expression Omnibus database. Differentially expressed genes were identified between cetuximab-responsive patients and nonresponders, annotated by gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and further analyzed by protein-protein interaction networks. The integrative prognostic analysis was based on The Cancer Genome Atlas and PrognoScan., Results: 1350 differentially expressed genes were identified with 298 upregulated and 1052 downregulated. Epidermis development, the cornified envelope, calcium ion binding, and amoebiasis were enriched in upregulated genes while digestion, the apical part of the cell, the 3',5'-cyclic-adenosine monophosphate phosphodiesterase activity and pancreatic secretion were found enriched in downregulated genes. The top 10 hub genes were identified, including epithermal growth factor, G-protein subunit β 5, G-protein subunit γ 4, fibroblast growth factor 2, B-cell lymphoma protein 2, acetyl-coenzyme A carboxylase β, KIT proto-oncogene receptor tyrosine kinase, adenylate cyclase 4, neuropeptide Y, and neurotensin. The hub genes exhibited distinct correlations in cetuximab-treated and untreated genomic profiles (GSE56386, GSE5851 and GSE82236). The highest correlation was found between B-cell lymphoma protein 2 and acetyl-coenzyme A carboxylase β in GSE56386. The mRNA expression of hub genes was further validated in the genomic profile GSE65021. Furthermore, B-cell lymphoma protein 2 and acetyl-coenzyme A carboxylase β also exhibited highest degrees among the hub genes correlation networks based on The Cancer Genome Atlas. Both B-cell lymphoma and acetyl-coenzyme A carboxylase β were not independent prognostic factors for colorectal cancer in univariate and multivariate Cox analysis. However, integrative survival analysis indicated that B-cell lymphoma protein 2 was associated with favorable prognosis (hazard ratio = 0.62, 95% confidence interval, 0.30-0.95, P = .024)., Discussion: This in silico analysis provided a feasible and reliable strategy for systematic exploration of insightful target genes, pathways and mechanisms underlying the cetuximab insensitivity in colorectal cancer. B-cell lymphoma protein 2 was associated with favorable prognosis.

Elevated expression of β-galactoside α2,6-sialyltranferase 1 (ST6GAL1) has been observed in colorectal cancer (CRC) and demonstrated to be important for its tumorigenesis. Here, we found that ST6GAL1 expression was significantly higher in non-metastatic tumors (stage I and II) than that in metastatic tumors (stage III and IV) using 62 pair-matched tumor/normal tissues. To elucidate the molecular mechanisms of how ST6GAL1 affected the CRC progression, we performed a global identification of the substrates of ST6GAL1 in the colon adenocarcinoma cell line SW480. A total of 318 membrane proteins were identified differentially affected by ST6GAL1 overexpression using metabolic labeling and proteomic analysis. Subsequent bioinformatic analysis revealed a list of potential substrates that might mediate the different functions of ST6GAL1 in CRC including cell movement, cell death and survival. Taken together, these results indicate a dynamic change in the expression of ST6GAL1 during the CRC progression and provide a list of sialylated proteins potentially relevant to the different functions of ST6GAL1 in CRC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. While surgery remains the mainstay of treatment in early stage CRC, chemotherapy is usually given to prolong the overall survival and improve the quality of life for metastatic colorectal cancer (mCRC). But drug resistance is one of the major hurdles of mCRC treatment, and the underlying mechanisms are still largely unknown. In this study, we show that, compared with parental cells, RhoA is up-regulated in irinotecan (CPT-11)-resistant CRC cells. Furthermore, inhibition of RhoA in drug resistant cells, at least partially, rescues the resistance against irinotecan and increases the sensitivity to other chemotherapeutic drug by inhibiting expression of MDR1, MRP1and GSTP1, promotes apoptosis by suppressing the expression of BCL-XL and Bcl-2 and increasing Bax expression, and significantly decreases side population cells. Our results suggest that, in addition to survival, proliferation, migration, adhesion, cell cycle and gene transcription, RhoA is also involved in chemoresistance by regulating the expression of membrane transporter and apoptosis protein in colorectal cancer. They raise an interesting possibility that the expression of RhoA may indicate a poor prognosis due to the high probability to therapy resistance and, on the other hand, inhibition of RhoA activity and function may overcome chemoresistance and improve the effectiveness of clinical treatment of CRC.

Prefoldin (PFDN) subunits have been reported upregulated in various tumor types, while the expression and functions of PFDN1 (PFDN subunit 1) in colorectal cancer (CRC) are not well elucidated. The aim of this study was to investigate the use of PFDN1 as a poor prognosis indicator for CRC and explore the functions of PFDN1 in CRC. The relationship between PFDN1 expression and CRC clinical-pathological statistics was detected on the tissue microarray containing 145 cases of CRC. ShRNA was used to silence PFDN1 expression in SW480 and RKO CRC cells, and these transfected cells were analyzed for changes in proliferation, colony formation, cell cycle, migration, and invasion. Immunofluorescence and immunoblot were used to determine the remodeling of the F-actin and α-tubulin. Finally, tumor growth on nude mice was observed and measured. In this study, we found PFDN1 was upregulated in CRC tissues compared with adjacent normal tissues. Also, PFDN1 expression positively correlated with tumor size and tumor invasion. Moreover, after silencing PFDN1 in SW480 and RKO cells, the proliferation and motility of CRC cells were significantly suppressed. The inhibitory effect of PFDN1 on tumor cell growth and motility was partially due to G2/M cell cycle blockage and cytoskeletal deficiency. Finally, in vivo assay showed that downregulation of PFDN1 inhibited tumor growth on nude mice and PFDN1 expression correlated with higher levels of Ki-67 staining. These findings indicate that PFDN1 was involved in the progression of CRC, and provide new insights into PFDN1 as a potential therapeutic target for CRC treatment.

STIM1 (stromal interaction molecule 1), an endoplasmic reticulum Ca(2+) sensor that triggers the store-operated Ca(2+) entry activation, has recently been implicated in cancer progression. However, the role of STIM1 in the progression and metastasis of colorectal cancer (CRC) has not been addressed. In this study, we confirmed increased expression of STIM1 in highly invasive CRC cell lines. Enhanced expression of STIM1 promoted CRC cell metastasis in vitro and in vivo, whereas silencing of STIM1 with small interfering RNA resulted in reduced metastasis. Ectopic expression of STIM1 in CRC cells induced epithelial-to-mesenchymal transition (EMT), whereas silencing of STIM1 had the opposite effect. Furthermore, STIM1 expression was markedly higher in CRC tissues than in adjacent noncancerous tissues. STIM1 overexpression correlated with poor differentiation and higher tumor node metastasis stage. CRC patients with positive STIM1 expression had poorer prognoses than those with negative STIM1 expression. Moreover, STIM1 was found to be a direct target of miR-185, a microRNA (miRNA) that has not previously been reported to be involved in EMT, in both CRC tissues and cell lines. Taken together, these findings demonstrate for the first time that STIM1 promotes metastasis and is associated with cancer progression and poor prognosis in patients with CRC. In addition, we show that expression of STIM1 is regulated by a posttranscriptional regulatory mechanism mediated by a new EMT-related miRNA. This novel miR-185-STIM1 axis promotes CRC metastasis and may be a candidate biomarker for prognosis and a target for new therapies.

Background: MicroRNAs (miRNAs) have been reported to play crucial roles in regulating a variety of genes pivotal for tumor metastasis. MicroRNA-301a (miR-301a) is overexpressed and displays oncogenic activity in many cancers. However, little is known about the potential roles of miR-301a in colorectal cancer (CRC)., Methods: Taqman probe stem-loop real-time PCR was used to quantitatively measure the expression level of miR-301a in 48 cases of CRC tissues and the matched adjacent non-tumor mucosa as well as in CRC cell lines. miR-301a mimics and inhibitors were used to up-regulate and down-regulate miR-301a in CRC cells, respectively; lentivirus was used to construct miR-301a stably up- and down-regulated CRC cell lines. Metastasis ability was evaluated by transwell and wound healing assays while invasion was measured by transwell coated with matrix gel in vitro; in vivo metastasis was performed on nude mice model. The target of miR-301a was predicted by TargetScan software and validated by qRT-PCR, immunohistochemistry, western blot and luciferase reporter gene assay., Results: The expression of miR-301a was significantly higher in lymph node metastasis positive CRC samples compared with negative ones. Downregulation of miR-301a significantly inhibited the migration and invasion both in vitro and in vivo while forced up-regulation of miR-301a promoted migration and invasion. TGFBR2 was identified to be the downstream target of miR-301a. Knockdown of TGFBR2 in cells treated by miR-301a inhibitor elevated the previously abrogated migration and invasion., Conclusions: Our data indicated that miR-301a correlated with the metastatic and invasive ability in human colorectal cancers and miR-301a exerted its role as oncogene by targeting TGFBR2.

Previous reports have associated GRHL2 with tumor progression. However, the biological role of GRHL2 in human colorectal cancer (CRC) has not been explored. We examined the expression of GRHL2 in 75 CRC samples, as well as the paired non-tumor tissues, by immunohistochemistry, qRT-PCR, and western blot analysis. The association between GRHL2 expression and various clinicopathological parameters including Ki-67, a marker of proliferative activity, was also evaluated. We performed lentivirus-mediated shRNA transfection to knock down GRHL2 gene expression in HT29 and HCT116 CRC cells. Cell proliferation was examined by the CCK-8 (Cell Counting Kit-8) assay, colony formation, and cell cycle assay in vitro. Tumorigenesis in vivo was assessed using a mouse xenograft model. Moreover, we transiently silenced ZEB1 expression in GRHL2-knockdown CRC cells using specific shRNA, and then examined the effects on GRHL2 and E-cadherin expression, as well as cell proliferation. Herein, we demonstrated that enhanced GRHL2 expression was detected in CRC, and correlated with higher levels of Ki-67 staining, larger tumor size, and advanced clinical stage. Knocking down GRHL2 in HT29 and HCT116 CRC cells significantly inhibited cell proliferation by decreasing the number of cells in S phase and increasing that in the G 0/G 1 phaseof the cell cycle. This resulted in inhibition of tumorigenesis in vivo, as well as increased expression of ZEB1. Furthermore, transient ZEB1 knockdown dramatically enhanced cell proliferation and increased GRHL2 and E-cadherin expression. Collectively, our study has identified ZEB1 as a target of GRHL2 and suggested a reciprocal GRHL2-ZEB1 repressive relationship, providing a novel mechanism through which proliferation may be modulated in CRC cells.

Aim: To evaluate the value of positron emission tomography (PET)/computerized tomography (CT) in surveillance of colorectal cancer (CRC) patients with different carcinoembryonic antigen (CEA) concentrations., Methods: One hundred and six postoperative CRC patients who had suspected recurrence or metastasis and received fluorodeoxyglucose (FDG) PET/CT within one week were included in this study. The final diagnosis was confirmed by histological examination or clinical follow-up over at least six months., Results: The sensitivity, specificity, and accuracy of FDG PET/CT were 95.2%, 82.6%, and 92.5%, and 94.8%, 81.4% and 92.8%, respectively, in the case- and lesion-based analyses. The sensitivity and accuracy of FDG PET/CT significantly differed from CT in both analyses (χ(2) = 8.186, P = 0.004; χ(2) =6.201, P = 0.013; χ(2) =13.445, P = 0.000; χ(2) =11.194, P = 0.001). In the lesion-based analysis, the sensitivity, specificity, and accuracy of FDG PET/CT in the abnormal CEA group were 97.8%, 82.6%, and 95.6%, compared with 81.3%, 80%, and 80.6% for patients with normal CEA levels. In case-based analysis, the sensitivity, specificity, and accuracy of FDG PET/CT were 97.2%, 77.8%, and 95% in abnormal CEA group. Only in lesion-based analysis, the sensitivity and accuracy of FDG PET/CT in the abnormal CEA group were significantly superior to those in the normal CEA group (χ(2) =6.432, P = 0.011; χ(2) =7.837, P = 0.005). FDG PET/CT changed the management in 45.8% of patients with positive scans., Conclusion: FDG PET/CT showed superior diagnostic value and is an advisable option in surveillance of postoperative CRC patients with a vague diagnosis.

MicroRNAs (miRNAs) have emerged as critical epigenetic regulators involved in cancer progression. miR-320a has been identified to be a novel tumour suppressive miRNA in colorectal cancer (CRC). However, the detailed molecular mechanisms are not fully understood. Here, we reported that miR-320a inversely associated with CRC aggressiveness in both cell lines and clinical specimens. Functional studies demonstrated that miR-320a significantly decreased the capability of cell migration/invasion and induced G0/G1 growth arrest in vitro and in vivo. Furthermore, Rac1 was identified as one of the direct downstream targets of miR-320a and miR-320a specifically binds to the conserved 8-mer at position 1140-1147 of Rac1 3'-untranslated region to regulate Rac1 protein expression. Over-expression of miR-320a in SW620 cells inhibited Rac1 expression, whereas reduction of miR-320a by anti-miR-320a in SW480 cells enhanced Rac1 expression. Re-expression of Rac1 in the SW620/miR-320a cells restored the cell migration/invasion inhibited by miR-320a, whereas knockdown of Rac1 in the SW480/anti-miR-320a cells repressed these cellular functions elevated by anti-miR-320a. Conclusively, our results demonstrate that miR-320a functions as a tumour-suppressive miRNA through targeting Rac1 in CRC.

Transmembrane protease/serine 4 (TMPRSS4) is a member of the type II transmembrane serine protease (TTSP) family and it was found highly expressed in several cancers. This study aims to evaluate the expression of TMPRSS4 in colorectal cancer (CRC) and investigate its role in proliferation and self-renewal of colon cancer cells. qRT-PCR and immunohistochemistry were used to detect the mRNA and protein expression level of TMRPSS4 in CRC samples respectively. Loss of function assay was conducted with RNAi technique. Cell proliferation was done with WST-8 assay; cell apoptosis and cell cycle analysis were performed with flow cytometry; invasion and migration were done with transwell assay. Plate and soft agarose clonogenic assays were used to detect clone-formation ability. CD44 and CD133 expressions were analyzed by flow cytometry and western blot. We found that TMPRSS4 was highly expressed in CRC tissues both at mRNA and protein level and correlated with pathological stage. Knockdown of TMPRSS4 in highly expressed colon cancer cell line HCT116 resulted in inhibition of cell proliferation, induction of cell apoptosis and suppression of invasion and migration; moreover, knockdown of TMPRSS4 suppressed the in vitro clone-formation ability of HCT116 and reduced the expressions of CD44 and CD133. The findings in this research showed that TMPRSS4 was associated with CRC stage and regulated the proliferation and self-renewal ability of colon cancer cells; TMRPSS4 was involved in the development and progression of CRC.

Background and Objective: Transcriptional factor E2A is crucial for the normal development and differentiation of B and T lymphocytes. Dysregulation of E2A leads to leukemia and tumorigenesis of some solid tumors. The expression and clinical significance of E2A as well as its role in colorectal cancer (CRC) are still unknown. This study aims to assess E2A expression in CRC tissues, evaluate its prognosis value, and investigate its role in colon cancer cell growth., Methods: E2A expression in CRC tissues and normal mucosa was detected by immunohistochemical staining; Kaplan-Meier survival curve and Cox regression model were used to evaluate the prognostic value of E2A. Lentivirus was used to construct E2A stably knocked-down cells. MTT assay was employed to detect cell proliferation change; cell cycle was analyzed by flow cytometry; and chromatin immunoprecipitation (ChIP) assay was used to validate the predicted binding target of E2A., Results: Expression of E2A was lower in CRC tissues than normal mucosa; low E2A expression correlated with advanced TNM stage and larger tumor size, and predicted poor prognosis of CRC patients. E2A knockdown resulted in increased cell proliferation rate and cell cycle acceleration. ChIP assay showed miR-320a was a direct target of E2A and upregulation of miR-320a in E2A downregulated cells could reverse cell proliferation and cell cycle changes caused by E2A deficiency., Conclusions: E2A is an independent prognostic factor for CRC patients and targets miR-320a to regulate cell proliferation of colon cancer cells.

Background: E2A gene, which encodes two basic helix-loop-helix (bHLH) transcription factors E12 and E47, has been identified as regulator of B lymphoid hematopoiesis and suppressor of lymphoma. E47 protein was found to decrease E-cadherin expression and induce epithelial-mesenchymal transition (EMT). However, the role of E2A in colorectal cancer (CRC) metastasis is still elusive., Methods: qRT-PCR and semi-qRT-PCR were performed to determine mRNA level of E2A in CRC specimens and colorectal cancer cells. RNAi was employed to downregulate E2A expression and subsequent protein level change was evaluated by immunoblot. Cell invasion and migration capacity were detected by transwell assay using cell culture inserts with or without basement membrane matrix, respectively., Results: E2A expression was decreased in metastatic CRC tissues. Invasion and migration assays showed downregulation of E2A increased metastatic capacity of CRC cells while forced expression of E12 or E47 could offset this effect. Both E12 and E47 suppressed EMT induced by E2A downregulation. Moreover, Yes-Associated Protein (YAP) was a downstream target of E2A and suppression of YAP inhibited the pro-migration/invasion of E2A deficiency., Conclusion: Our results suggest that E2A suppresses CRC cell metastasis, at least partially if not all, by inhibiting YAP expression.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. The discovery of non-invasive biomarker candidates for diagnosis and prognosis is important for the management of CRC. In this study, we performed proteomic profiling of serum from patients with different stages of CRC using a 2D-LC-MS/MS based approach combined with the APEX quantitative method. 917 proteins were identified and 93 were differentially expressed in normal and three patient groups (stages I, II and III). These proteins were predominantly involved in cell adhesion, immune response, coagulation process and metabolism. Importantly, we found collagen I dynamically changed from stages I to IV, with maximum expression in stage II, as detected in serum by MS analysis. Expression of collagen I was also validated in tumor tissues from the same group of CRC patients by real-time PCR and western blotting. Furthermore, we demonstrate that serum levels of collagen I degradation telopeptide (CTx) are correlated with staging and poor disease-free survival of CRC patients by ELISA analysis. These results suggest (1) serum proteomics may reflect biological changes in colorectal tumor tissues and (2) altered collagen I expression may be an early event in CRC tumorigenesis and CTx may provide additional information for prognosis of CRC., Biological Significance: In this work, we performed a systematic characterization of serum proteomic alterations in colorectal cancer (CRC) with different stages using a LC-MS based approach combined with the APEX quantitative method, attempting to gain overview of relevant proteins in tumorigenesis and discover non-invasive CRC-derived markers. We found a significant up-regulation of collagen I based on the proteomics data, and confirmed its expression in tissue and serum of the same group of patients. In addition, we also demonstrated that serum levels of collagen I degradation telopeptide (CTx) are correlated with the staging and poor survival rate of CRC. Those findings imply that alternation of collagen I might be an early event during tumorigenesis of CRC, and might contribute to the metastasis of CRC under the degradation regulated by some specific proteases. This work provides evidence for the clinical application of serum proteomics, and would aid the understanding of the role of the ECM in the clinical progression of CRC., (© 2013.)

Transmembrane protease/serine 4 (TMPRSS4), a member of the type II transmembrane serine protease family, is highly expressed in some human cancers and involved in the EMT regulation of cancer cells. The prognostic value of TMPRSS4 in colorectal cancer (CRC) has not been discussed. This study aims to evaluate the association between TMPRSS4 expressions and survival in CRC patients. Immunohistochemistry revealed high expression of TMPRSS4 in 69/122 CRC samples, compared with 14/47 in normal tissues (P < 0.01). Correlation analysis showed high expression of TMPRSS4 was significantly associated with advanced TNM stage (P = 0.011), pT (P = 0.019), pN (P = 0.035), and pM status (P = 0.004). Higher TMPRSS4 predicted shorter overall survival (OS) and disease-free survival (DFS) in CRC patients (P < 0.01, both). Moreover, both TMPRSS4 expression and TNM stage were independent predictive factors of OS and DFS in Cox regression analysis. The findings in our study demonstrated the potential value of TMPRSS4 expression level as a prognostic biomarker for CRC patients.

Metastasis remains to be one of the most prevalent causes leading to poor long-term survival of colorectal cancer (CRC) patients. The clinical significances of tumor metastatic suppressor, N-myc downregulated gene 1 (NDRG1), have been inconsistently reported in a variety of cancerous diseases. In this study with 240 CRC clinical specimens, we showed that NDRG1 expression was significantly decreased in most of CRC tissues compared to the paired non-tumor counterparts. Statistical analysis revealed a significant inverse correlation of NDRG1 expression with tumor stage, differentiation status and metastasis. Compared with NDRG1-negative group, NDRG1-positve group had better disease-free/overall survival (p = 0.000) over 5 years' follow-up. Furthermore, NDRG1 was considered to be an independent prognostic factor for overall survival (p = 0.001) and recurrence (p = 0.003). Our study concludes that NDRG1 is a novel favorable predictor for the prognosis in CRC patients.

Recent studies suggest that biofluid-based metabonomics may identify metabolite markers promising for colorectal cancer (CRC) diagnosis. We report here a follow-up replication study, after a previous CRC metabonomics study, aiming to identify a distinct serum metabolic signature of CRC with diagnostic potential. Serum metabolites from newly diagnosed CRC patients (N = 101) and healthy subjects (N = 102) were profiled using gas chromatography time-of-flight mass spectrometry (GC-TOFMS) and ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS). Differential metabolites were identified with statistical tests of orthogonal partial least-squares-discriminant analysis (VIP > 1) and the Mann-Whitney U test (p < 0.05). With a total of 249 annotated serum metabolites, we were able to differentiate CRC patients from the healthy controls using an orthogonal partial least-squares-discriminant analysis (OPLS-DA) in a learning sample set of 62 CRC patients and 62 matched healthy controls. This established model was able to correctly assign the rest of the samples to the CRC or control groups in a validation set of 39 CRC patients and 40 healthy controls. Consistent with our findings from the previous study, we observed a distinct metabolic signature in CRC patients including tricarboxylic acid (TCA) cycle, urea cycle, glutamine, fatty acids, and gut flora metabolism. Our results demonstrated that a panel of serum metabolite markers is of great potential as a noninvasive diagnostic method for the detection of CRC.

Background: RhoE, an atypical Rho protein, is differently deregulated in some solid tumors, and there are conflicting data describing the role of RhoE in tumor cell migration and invasion. This study aimed to investigate RhoE expression in human colorectal cancer and its relationship with clinicopathological features and prognosis., Methods: Colorectal cancer and adjacent normal tissues from 202 patients were examined by immunohistochemistry. Staining evaluation results were analyzed statistically in relation to various clinicopathological parameters, disease-free survival, and overall survival. RhoE expression was also investigated by immunohistochemistry in 80 node metastases and the corresponding primary lesions, and by Western blot test in six cancer and adjacent normal tissues. The relationship between RhoE and invasion was examined by transwell assay and Western blot test., Results: The positive rate for RhoE in colorectal cancer was significantly higher than that of normal colorectal tissues. In colorectal cancer, RhoE expression was significantly correlated with depth of invasion, lymph node metastasis, and distant metastasis. Consistently, overexpression of RhoE in SW620 cells up-regulated vimentin, down-regulated E-cadherin, increased the expression of matrix metalloproteinase (MMP) 9, and enhanced cell invasion in vitro; in contrast, silencing of RhoE by a specific siRNA caused opposite effects. Most importantly, disease-free and overall survivals were significantly poorer for patients with RhoE-positive tumors than for those with RhoE-negative tumors., Conclusions: These findings emphasize the positive role of RhoE in invasion and metastasis in human colorectal cancer. It could also serve as an independent prognostic marker in addition to the tumor, node, metastasis staging system.

Aim: To evaluate the effect of nigericin on colorectal cancer and to explore its possible mechanism., Methods: The human colorectal cancer (CRC) cell lines HT29 and SW480 were treated with nigericin or oxaliplatin under the conditions specified. Cell viability assay and invasion and metastasis assay were performed to evaluate the effect of nigericin on CRC cells. Sphere-forming assay and soft agar colony-forming assay were implemented to assess the action of nigericin on the cancer stem cell properties of CRC cells undergone epithelial-mesenchymal transition (EMT)., Results: Compared with oxaliplatin, nigericin showed more toxicity for the HT29 cell line (IC50, 12.92 ± 0.25 μmol vs 37.68 ± 0.34 μmol). A similar result was also obtained with the SW116 cell line (IC50, 15.86 ± 0.18 μmol vs 41.02 ± 0.23 μmol). A Boyden chamber assay indicated that a significant decrease in the number of HT29 cells migrating through polyvinylidene fluoride membrane was observed in the nigericin-treated group, relative to the vehicle-treated group [11 ± 2 cells per high-power field (HPF) vs 19.33 ± 1.52 cells per HPF, P < 0.05]. Compared to the control group, the numbers of HT29 cells invading through the Matrigel-coated membrane also decreased in the nigericin-treated group (6.66 ± 1.52 cells per HPF vs 14.66 ± 1.52 cells per HPF, P < 0.05). Nigericin also reduced the proportion of CD133+ cells from 83.57% to 63.93%, relative to the control group (P < 0.05). Nigericin decreased the number of spheres relative to the control group (0.14 ± 0.01 vs 0.35 ± 0.01, P < 0.05), while oxaliplatin increased the number of spheres relative to the control group (0.75 ± 0.02 vs 0.35 ± 0.01; P < 0.05). Nigericin also showed a decreased ability to form colonies under anchorage-independent conditions in a standard soft agar assay after 14 d in culture, relative to the control group (1.66 ± 0.57 vs 7 ± 1.15, P < 0.05), whereas the colony numbers were higher in the oxaliplatin group relative to the vehicle-treated controls (14.33 ± 0.57 vs 7 ± 1.15, P < 0.05). We further detected the expression of E-cadherin and vimentin in cells treated with nigericin and oxaliplatin. The results showed that HT29 cells treated with nigericin induced an increase in E-cadherin expression and a decrease in the vimentin expression relative to vehicle controls. In contrast, oxaliplatin downregulated the expression of E-cadherin and upregulated the expression of vimentin in HT29 cells relative to vehicle controls., Conclusion: This study demonstrated that nigericin could partly reverse the EMT process during cell invasion and metastasis.

The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor., (2012 AACR)

Background: Fish consumption may protect against colorectal cancer, but results from observational studies are inconsistent; therefore, a systematic review with a meta-analysis was conducted., Methods: Relevant studies were identified by a search of MEDLINE and EMBASE databases to May 2011, with no restrictions. Reference lists from retrieved articles also were reviewed. Studies that reported odds ratio (OR) or relative risk estimates with 95% confidence intervals (CIs) for the association between the consumption of fish and the risk of colorectal, colon, or rectal cancer were included. Two authors independently extracted data and assessed study quality. The risk estimate (hazard ratio, relative risk, or OR) of the highest and lowest reported categories of fish intake were extracted from each study and analyzed using a random-effects model., Results: Twenty-two prospective cohort and 19 case-control studies on fish consumption and colorectal cancer risk met the inclusion criteria and were included in the meta-analysis. Our analysis found that fish consumption decreased the risk of colorectal cancer by 12% (summary OR, 0.88; 95% CI, 0.80-0.95). The pooled ORs of colorectal cancer for the highest versus lowest fish consumption in case-control studies and cohort studies were 0.83 (95% CI, 0.72-0.95) and 0.93 (95% CI, 0.86-1.01), respectively. There was heterogeneity among case-control studies (P<.001) but not among cohort studies. A significant inverse association was found between fish intake and rectal cancer (summary OR, 0.79; 95% CI, 0.65-0.97), and there was a modest trend seen between fish consumption and colon cancer (summary OR, 0.96; 95% CI, 0.81-1.14). This study had no publication bias., Conclusion: Our findings from this meta-analysis suggest that fish consumption is inversely associated with colorectal cancer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Polo-like kinase 1 (PLK1) is an important molecule in proliferation of many human cancers. The aim of study is to clarify the expression patterns and potential function of PLK1 in colorectal cancers., Material/methods: Fifty-six colorectal cancers samples were collected and arranged onto a tissue array and the expression of PLK1 were detected by immunohistochemistry and correlated with clinico-pathological characteristics and expression of PCNA. Expression of PLK1 in 9 colorectal cancer cells lines was investigated by RT-PCR and Western blot, then SW1116 cells lines were treated with PLK1 siRNA and the efficiency was examined by Western blot. Transwell test was applied to detect the migration and invasion capability of cancer cells by counting the number of cells passing through the membranes. Cell proliferation and apoptosis were examined by Cell Counting Kit-8 (CCK-8) and Annexin-V Kit., Results: PLK1 was positively expressed in 73.2% (41/56) of colorectal cancers tissues, but in only 3.6% (2/56) of normal tissues, and was associated with Duke's stage (P<0.01), tumor size (P<0.01), invasion extent (P<0.05) and lymphatic metastasis (P<0.01). The expression of PLK1 was correlated with expression of PCNA (R=0.553, P<0.01). PLK1 was inhibited in SW1116 cells by treating with PLK1 siRNA oligos, which resulted in a decreased number of cells passing through the membrane as compared with control groups (P<0.01) at 24 hours after transfection. Cell proliferation was inhibited from 48 hours after transfection, while cells apoptosis was induced from 72 hours after transfection., Conclusions: PLK1 could be a progression marker for colorectal cancer patients and PLK1 depletion can inhibit migration and invasion capability of colorectal cancer cells SW1116, suggesting that PLK1 might be involved in metastasis and invasion of colorectal cancer. Therapeutic strategies targeting PLK1 may be a new approach to colorectal cancer.

A full spectrum of metabolic aberrations that are directly linked to colorectal cancer (CRC) at early curable stages is critical for developing and deploying molecular diagnostic and therapeutic approaches that will significantly improve patient survival. We have recently reported a urinary metabonomic profiling study on CRC subjects (n = 60) and health controls (n = 63), in which a panel of urinary metabolite markers was identified. Here, we report a second urinary metabonomic study on a larger cohort of CRC (n = 101) and healthy subjects (n = 103), using gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry. Consistent with our previous findings, we observed a number of dysregulated metabolic pathways, such as glycolysis, TCA cycle, urea cycle, pyrimidine metabolism, tryptophan metabolism, polyamine metabolism, as well as gut microbial-host co-metabolism in CRC subjects. Our findings confirm distinct urinary metabolic footprints of CRC patients characterized by altered levels of metabolites derived from gut microbial-host co-metabolism. A panel of metabolite markers composed of citrate, hippurate, p-cresol, 2-aminobutyrate, myristate, putrescine, and kynurenate was selected, which was able to discriminate CRC subjects from their healthy counterparts. A receiver operating characteristic curve (ROC) analysis of these markers resulted in an area under the receiver operating characteristic curve (AUC) of 0.993 and 0.998 for the training set and the testing set, respectively. These potential metabolite markers provide a novel and promising molecular diagnostic approach for the early detection of CRC.

MicroRNAs have been implicated in the regulation of several cellular signaling pathways of colorectal cancer (CRC) cells. Although emerging evidence proves that microRNA (miR)-106a is expressed highly in primary tumor and stool samples of CRC patients; whether or not miR-106a mediates cancer metastasis is unknown. We show here that miR-106a is highly expressed in metastatic CRC cells, and regulates cancer cell migration and invasion positively in vitro and in vivo. These phenotypes do not involve confounding influences on cancer cell proliferation. MiR-106a inhibits the expression of transforming growth factor-β receptor 2 (TGFBR2), leading to increased CRC cell migration and invasion. Importantly, miR-106a expression levels in primary CRCs are correlated with clinical cancer progression. These observations indicate that miR-106a inhibits the anti-metastatic target directly and results in CRC cell migration and invasion.

Background: Cancer stem-like cells (CSCs) in colorectal cancers (CRC) may account for the failure of treatments because they are resistant to many current anticancer therapies. Salinomycin, a potassium ionophore, was recently identified as a selective inhibitor of breast CSCs., Methods: The human CRC cell lines HT29 and SW480 were treated with salinomycin and oxaliplatin. Cell viability was determined with cell counting kit 8. Fraction of CD133+ cell subpopulations was assessed by Flow Cytometric analysis. Clonogenecity and migration were determined with soft agar and Boyden chamber assays. Molecular changes were assessed by immunofluorescence staining, RT-PCR, and Western blot analysis., Results: We report that salinomycin reduces the proportion of CD133+ subpopulations in human CRC HT29 and SW480 cells. Furthermore, salinomycin treatment decreases colony-forming ability and cell motility in HT29 cells. Moreover, salinomycin downregulates the expression of vimentin and induces the E-cadherin expression in HT29 cells., Conclusions: This study demonstrates the ability of salinomycin to selectively target "CD133+" cell subpopulations and decrease the malignant traits in colorectal cancer lines.

Objective: To examine the role of Polo-like kinase 1(PLK1) in the migration and invasiveness of human colorectal cancer cells., Methods: Nine colorectal cancer cell lines were cultured. Cell lines with the highest level of PLK1 expression was selected by PCR and Western blot. Three siRNA oligo segments targeting PLK1 were designed and selected cell lines transfected. Successful transfection was confirmed using real-time PCR and Western blot. Changes in migration and invasiveness of the selected cell line were evaluated by Transwell test., Results: Colorectal cancer cell line SW1116 was selected with the highest expression of PLK1 at both mRNA level and protein level. The expression of PLK1 in SW1116 was reduced by the three siRNA oligo segments to varying degrees, and the No.1 siRNA oligo segment was the most efficient. In migration test, the number of cells crossing through chambers in PLK1-siRNA group was 44 ± 14, which was lower than that in the negative control group (242 ± 40) and in blank control group(240 ± 38). In invasion test, the number of cells crossing through chambers in PLK1-siRNA group was 62 ± 3, which was lower than that in negative control group (207 ± 12) and in blank control group (211 ± 15). These differences were statistically significant(P<0.01)., Conclusion: PLK1 silencing by siRNA may inhibit the migration and invasiveness of colorectal cancer cells, suggesting that PLK1 might play an important role in the infiltration and metastasis of colorectal cancer.

Colorectal carcinoma (CRC) is the third most commonly encountered cancer and fourth cause of cancer-associated death worldwide. Abundant studies have demonstrated that one of the best effective therapies for enhancing the 5-year survival rate of patients is to diagnose the disease at an early stage. Urine metabonomics is widely being utilized as an efficient platform to investigate the metabolic changes and discover the potential biomarkers of malignant diseases. In this study both ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and online affinity solid phase extraction-high performance liquid chromatography (SPE-HPLC) were used to analyze the urinary metabolites from 34 healthy volunteers, 34 benign colorectal tumor and 50 colorectal carcinoma patients to produce comprehensive metabolic profiling data. A reliable separation between the control and disease groups as well as significantly changed metabolites were obtained from orthogonal signal correction partial least squares models which were built based on the two separate data sets from UPLC-MS and affinity SPE-HPLC, respectively. 15 metabolites, showing the metabolic disorders of CRC, were identified finally. These metabolites were found to be related to glutamine metabolism, fatty acid oxidation, nucleotide biosynthesis and protein metabolism.

Pak1 has been reported to be overexpressed in colorectal cancer, but the role of Pak1 in colorectal cancer remains unclear. In this study, Pak1 expression and activity were associated with aggressive behavior of colorectal cancer. Overexpression of Pak1 increased colorectal cancer cell motility and invasion, whereas down-regulation of Pak1 expression or activity reduced colorectal cancer cell migration and invasion. In addition, activated Pak1 inhibited stress fiber and focal adhesion complex formation in colorectal cancer cells and led to formation of motile phenotypes. Importantly, activated Pak1 elicited phosphorylation of FAK at Ser-910 via an ERK-dependent pathway in colorectal cancer cell lines and clinical samples. In conclusion, our results suggest that activated Pak1 regulates colorectal cancer metastasis requiring an ERK-dependent phosphorylation of FAK at Ser-910.

Objective: To explore the relationship between gamma-synuclein gene expression and CpG island demethylation in colorectal cancer(CRC), and the relationship between the demethylation and clinicopathological factors of CRC., Methods: The expression of gamma-synuclein mRNA was examined in 30 pairs of tumor tissues and tumor-matched non-neoplastic adjacent tissues(NNAT) by RT-PCR. CRC cell lines including COLO205, LoVo, and SW480 were used and treated with a demethylating agent, 5-aza-2'-deoxycytidine(5-aza-C). Before and after the treatment, the expression of gamma-synuclein mRNA in the cells was determined by RT-PCR, and bisulfite sequencing PCR was also used to analyze methylation status of CpG island. The methylation status of gamma-synuclein was then examined in 67 CRC samples and 30 NNAT samples by nested methylation-specific PCR (NMSP) and real time methylation-specific PCR(real-time MSP). The relationship between the demethylation of gamma-synuclein in CRC and clinicopathological factors was analyzed., Results: The mean gamma-synuclein mRNA expression was 0.66+/-0.34 in CRC samples, which was much higher than 0.45+/-0.26 in NNAT samples(P=0.011). 5-aza-C could induce expression and demethylation of gamma-synuclein in COLO205, LoVo and SW480 cells. gamma-Synuclein gene was demethylated in 80.0%(24/30) of the CRC samples and 50.0%(15/30) of the NNAT samples. The demethylated status of gamma-synuclein was much higher in CRC samples than that in NNAT samples(P=0.030), and was significantly correlated with clinical stage, lymph node involvement, and distant metastasis of CRC(P<0.05)., Conclusion: The upregulation of gamma-synuclein expression in CRC is primarily attributed to the demethylation of CpG island, which may be used as a marker for prognosis.

The synucleins (alpha-, beta- and gamma-synuclein) are a small, soluble, highly conserved group of neuronal proteins that attracted considerable attention due to their involvement in both neurodegenerative diseases and cancer. In this study, we examined the synuclein exprsssion in colorectal cancer (CRC) tissues, tumor-matched non-neoplastic adjacent tissues (NNAT), and CRC cell lines, and then investigated clinical significance of synucleins. By using semi-quantitative RT-PCR, synuclein mRNA expression was detected in eight CRC cell lines. It was much higher in CRC samples than in NNAT samples (P<0.05). The results of western blotting showed that the levels of synucleins protein expression in CRC cells approximately corresponded to the levels of synuclein mRNA expression. Immunohistochemical staining revealed that gamma-synuclein protein expression was up-regulated in CRC samples compared to NNAT samples (P=0.022), and was significantly correlated with clinical stage and lymph node involvement of CRC (P<0.05). Although, there was no significant difference in either alpha- or beta-synuclein protein expression between tumor and normal samples (P>0.05), often more than one form of synuclein was expressed in a tumor sample. More ratios of later stage and lymph node-positive tumors expressed a least one type of synuclein protein, and more ratios showed positive for either alpha or gamma-synuclein expression, as well as positive either for beta or gamma-synuclein in more ratios of lymph node-positive tumors. These results show that alpha-, beta- and gamma-synuclein are expressed in a high percentage of CRC. gamma-synuclein protein is valuable for evaluation of progression of CRC, and it is more sensitive to predict advanced stage and lymph node invasion by detection of gamma-synuclein protein combined with either alpha- or beta-synuclein protein or both than by detection of gamma-synuclein only.

Aim: To investigate the expression pattern of gamma-synuclein in colorectal cancer (CRC) tissues, and to study the effects of gamma-synuclein on CRC cell line HCT116 biological features in vitro., Methods: The expression pattern of gamma-synuclein was determined in 54 CRC tissues and 30 tumor-matched nonneoplastic adjacent tissues (NNAT) 5 cm away from the tumor via real-time quantitative reverse transcription PCR (RT-PCR) and immunohistochemistry. The relationship between gamma-synuclein protein expression and clinicopathological factors of CRC tissues was analyzed. Three small interfering RNA (siRNA) targeting gamma-synuclein mRNA plasmids were constructed and transfected into the CRC cell line HCT116. The stable cell lines were selected with G-418 for 28 d, and the biological features of these cells were examined by cell growth curve, soft agar assay, and cell migration and invasion assays in vitro., Results: The expression of gamma-synuclein mRNA and protein was much higher in CRC tissue samples than in NNAT samples (P = 0.02, P = 0.036). There was a significant correlation between the gamma-synuclein protein expression and clinical stage and lymph node involvement of CRC (P = 0.02, P = 0.033). In functional analysis we found that down-regulation of gamma-synuclein expression in HCT116 cells could inhibit the growth, colony formation rate, and migration and invasion ability of HCT116 cells., Conclusion: Increased expression of gamma-synuclein in CRC tissues and the biological effects of reduced gamma-synuclein expression on HCT116 cells suggest that gamma-synuclein may play a positive role in the progression of CRC.

Colorectal cancer is one of the most commonly diagnosed cancers and cause of cancer-related deaths worldwide. Studies have demonstrated that patient outcome is substantially influenced by cancer stage at the time of diagnosis. For example, patients with early stage colorectal have a significant higher 5-year survival rates compared to patients diagnosed at late stage. Thus, it is important to develop effective methods for early diagnosis as well as for precise staging of this disease process. Although traditional colonoscopy remains the most effective means to diagnose colorectal cancer, this approach generally suffers from poor patient compliance. As such, it is imperative to develop accurate and specific tests that utilize a more convenient approaches including urine examination. Urine collection is noninvasive, requires no presampling preparation, and substantially improves compliance. Recent advances in metabolomics, analytical techniques, and data analysis have tremendous potential for application in diagnostic pathology including identification of novel urinary markers in colorectal cancer.

Recent evidence suggests that gamma-synuclein is abnormally expressed in a high percentage of tumor tissues of diversified cancer types, but rarely expressed in tumor-matched non-neoplastic adjacent tissues (NNAT). The molecular mechanism of CpG island demethylation may underlie aberrant gamma-synuclein expression. To fully understand the roles of aberrant gamma-synuclein expression and demethylation in the development of colorectal cancer (CRC), we examined the expression and methylation status of gamma-synuclein in 67 CRC samples, 30 NNAT samples, and five CRC cell lines as well. By using reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry analyses, gamma-synuclein expression was detected in both HT-29 and HCT116 cells, and was much higher in CRC samples than in NNAT samples (P < 0.05). The demethylating agent, 5-aza-2 cent-deoxycytidine, can induce re-expression of gamma-synuclein in COLO205, LoVo, and SW480 cells. Unmethylated gamma-synuclein alleles were detected in HT-29, HCT116, and LoVo cells by nested methylation-specific PCR, and the demethylated status of gamma-synuclein was much higher in CRC samples than in NNAT samples by real-time quantitative methylation-specific PCR (P < 0.05). The results of genomic bisulfite DNA sequencing further confirmed that the aberrant gamma-synuclein expression in CRC was primarily attributed to the demethylation of CpG island. The protein expression and demethylation status of gamma-synuclein in 67 CRC samples correlated with clinical stage, lymph node involvement, and distant metastasis. These findings suggest an involvement of aberrant gamma-synuclein expression and demethylation in progression of CRC, especially in advanced stages.

Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality, and elucidation of its underlying genetics has advanced diagnostic screening, early detection, and treatment. Because CRC genomes are characterized by numerous non-random chromosomal structural alterations, we sought to delimit regions of recurrent amplifications and deletions in a collection of 42 primary specimens and 37 tumor cell lines derived from chromosomal instability neoplasia and microsatellite instability neoplasia CRC subtypes and to compare the pattern of genomic aberrations in CRC with those in other cancers. Application of oligomer-based array-comparative genome hybridization and custom analytic tools identified 50 minimal common regions (MCRs) of copy number alterations, 28 amplifications, and 22 deletions. Fifteen were highly recurrent and focal (<12 genes) MCRs, five of them harboring known CRC genes including EGFR and MYC with the remaining 10 containing a total of 65 resident genes with established links to cancer. Furthermore, comparisons of these delimited genomic profiles revealed that 22 of the 50 CRC MCRs are also present in lung cancer, glioblastoma, and/or multiple myeloma. Among 22 shared MCRs, nine do not contain genes previously shown genetically altered in cancer, whereas the remaining 13 harbor 35 known cancer genes, of which only 14 have been linked to CRC pathogenesis. Together, these observations point to the existence of many yet-to-be discovered cancer genes driving CRC development, as well as other human cancers, and show the utility of high-resolution copy number analysis in the identification of genetic events common and specific to the development of various tumor types.