Fusobacterium nucleatum upregulates MMP7 to promote metastasis-related characteristics of colorectal cancer cell via activating MAPK(JNK)-AP1 axis.

BACKGROUND: Colorectal cancer (CRC) is the third most common malignant tumor. Fusobacterium nucleatum (F. nucleatum) is overabundant in CRC and associated with metastasis, but the role of F. nucleatum in CRC cell migration and metastasis has not been fully elucidated. METHODS: Differential gene analysis, protein-protein interaction, robust rank aggregation analysis, functional enrichment analysis, and gene set variation analysis were used to figure out the potential vital genes and biological functions affected by F. nucleatum infection. The 16S rDNA sequencing and q-PCR were used to detect the abundance of F. nucleatum in tissues and stools. Then, we assessed the effect of F. nucleatum on CRC cell migration by wound healing and transwell assays, and confirmed the role of Matrix metalloproteinase 7 (MMP7) induced by F. nucleatum in cell migration. Furthermore, we dissected the mechanisms involved in F. nucleatum induced MMP7 expression. We also investigated the MMP7 expression in clinical samples and its correlation with prognosis in CRC patients. Finally, we screened out potential small molecular drugs that targeted MMP7 using the HERB database and molecular docking. RESULTS: F. nucleatum infection altered the gene expression profile and affected immune response, inflammation, biosynthesis, metabolism, adhesion and motility related biological functions in CRC. F. nucleatum was enriched in CRC and promoted the migration of CRC cell by upregulating MMP7 in vitro. MMP7 expression induced by F. nucleatum infection was mediated by the MAPK(JNK)-AP1 axis. MMP7 was highly expressed in CRC and correlated with CMS4 and poor clinical prognosis. Small molecular drugs such as δ-tocotrienol, 3,4-benzopyrene, tea polyphenols, and gallic catechin served as potential targeted therapeutic drugs for F. nucleatum induced MMP7 in CRC. CONCLUSIONS: Our study showed that F. nucleatum promoted metastasis-related characteristics of CRC cell by upregulating MMP7 via MAPK(JNK)-AP1 axis. F. nucleatum and MMP7 may serve as potential therapeutic targets for repressing CRC advance and metastasis.

Long-Term Outcome Comparison Between Two Specimen Extraction Approaches for Middle Rectum Cancer: A Retrospective Study.

Objective. There are few studies comparing the long-term results of natural orifice specimen extraction surgery (NOSES) and conventional laparoscopic-assisted resection (LA) in the treatment of middle rectal cancer. This retrospective analysis aimed to evaluate the reliability of NOSES. Method. From January 2013 to December 2017, all patients diagnosed with median rectal cancer in our hospital who underwent NOSES and LA were enrolled. We used propensity-score matching (PSM) to balance baseline data between the NOSES group and the laparoscopic group. The primary endpoint was overall survival (OS) and disease-free survival (DFS). We used the Kaplan-Meier method to estimate OS and DFS. Student's t-test was used to analyze the difference of continuous data. Categorical data were compared using the Kruskal-Wallis test or Fisher's exact test. Results. After PSM, 38 patients were included in each group. We found that surgical bleeding volume in the NOSES group was considerably lower than that in the LA group (49.5 ± 47.5 mL vs. 86.3 ± 83.5 mL, P = .01). From the short-term results, the first flatus and regular diet time in the NOSES group were shorter than those in the LA group (41.3 ± 25.2 vs. 54.0 ± 19.2 hours, P < .01 and 63.9 ± 42.6 hours vs. 105.1 ± 66.8 hours, P < .01, respectively). Long-term OS and DFS were not different between the groups. Conclusion. Therefore, NOSES is a reliable technique for middle rectal cancer treatment. Short-term outcomes are pointedly better than LA, while the two surgical approaches did not differ in the long-term outcomes or complication rate.

Prognostic Significance of D3 Lymph Node for Survival in Patients With Colorectal Cancer.

BACKGROUND: The aim of this study was to investigate the prognostic value of D3 lymph node (TSLN) for the survival of patients with colorectal cancer. METHODS: A total of 156 patients with R0 resected colorectal cancer were selected from 2011 to 2015 to carry out a retrospective study. The survival rate according to the groups of positive lymph node number (N: 1-3, N2: 4-6, N3: ≥7) and TSLN (TSLN [-], TSLN [+]) was analyzed. The influences of covariates on the 5-year overall survival (OS) and 5-year disease-free survival (DFS) were determined by the Cox proportional risk model of backward stepwise analysis. Kaplan-Meier survival analysis was used to draw survival curves between and within groups. RESULTS: During the median follow-up period (44.0 months), the 5-year DFS rate and OS rate were 45.0% and 46.0%, respectively. Survival analysis of the TSLN group showed that the 5-year OS rate and 5-year DFS rate in the TSLN (+) group (20.0 and 16.2%, respectively) were significantly lower than those in the TSLN (-) group (68.3 and 51.6%, respectively) (P < 0.001). The 5-year OS rate and DFS rate of the TSLN (+) and TSLN (-) subgroups in the N1 group were 16.7%, 33.3%, 56.7%, and 55.7%, respectively (P < 0.001). Multivariate analysis showed that positive lymph node, TSLN, and Pathological T stage were independent prognostic factors of DFS and OS for 5 years. Patients in the TSLN (+) group had a poorer prognosis. CONCLUSIONS: TSLN metastasis is an independent factor influencing the prognosis of patients, and patients with TSLN (+) have a poor prognosis. As an independent prognostic factor, this factor should be considered when evaluating the prognosis of patients.

BMSC derived EVs inhibit colorectal Cancer progression by transporting MAGI2-AS3 or something similar.

In this study, we investigated the molecular mechanisms underlying the impact of extracellular vesicles (EVs) derived from bone marrow stromal cells (BMSCs) on colorectal cancer (CRC) development. The focus was on the role of MAGI2-AS3, delivered by BMSC-EVs, in regulating USP6NL DNA methylation-mediated MYC protein translation modification to promote CDK2 downregulation. Utilizing bioinformatics analysis, we identified significant enrichment of MAGI2-AS3 related to copper-induced cell death in CRC. In vitro experiments demonstrated the downregulation of MAGI2-AS3 in CRC cells, and BMSC-EVs were found to deliver MAGI2-AS3 to inhibit CRC cell proliferation, migration, and invasion. Further exploration revealed that MAGI2-AS3 suppressed MYC protein translation modification by regulating USP6NL DNA methylation, leading to CDK2 downregulation and prevention of colorectal cancer. Overexpression of MYC reversed the functional effects of BMSC-EVs-MAGI2-AS3. In vivo experiments validated the inhibitory impact of BMSC-EVs-MAGI2-AS3 on CRC tumorigenicity by promoting CDK2 downregulation through USP6NL DNA methylation-mediated MYC protein translation modification. Overall, BMSC-EVs-MAGI2-AS3 may serve as a potential intervention to prevent CRC occurrence by modulating key molecular pathways.

Minimally Invasive Concepts in Treating Synchronous Liver Metastases Rectal Cancer Patients: Report of Six Cases.

BACKGROUND: Rectal cancer patients with synchronous liver metastases (SLM) is common in clinical practice. However, the application of conventional natural orifice specimen extraction surgery (NOSES) and NOSES with specimen extraction via stoma/hepatectomy incision in the special population is rarely explored. CASE REPORT: Six SLM rectal cancer patients were treated with simultaneous surgical resection and the specimens were extracted via anal/stoma/hepatectomy incision. Respectively, intraoperative and postoperative data, anal function 3 months after surgery and long-term prognosis were reviewed. RESULTS: Intraoperative and postoperative data and anal function were reliable for the six cases. Only one patient died of brain and bone metastases at 84 months after surgery and the other five patients were alive at their last follow-up. CONCLUSIONS: Simultaneous surgical resection with the concept of conventional NOSES and NOSES with specimen extraction via stoma/hepatectomy incision is safety for SLM rectal patients.

A KRAS-Associated Signature for Prognostic, Immune and Chemical Anti-Cancer Drug-Response Prediction in Colon Cancer.

Background: KRAS mutation, one of the most important biological processes in colorectal cancer, leads to poor prognosis in patients. Although studies on KRAS have concentrated for a long time, there are currently no ideal drugs against KRAS mutations. Methods: Different expression analysis and weighted gene coexpression network analysis was conducted to select candidate genes. Log-rank tests and Cox regression picked out the prognostic genes to build a KRAS-related gene prognostic score (KRGPS). A nomogram based on KRGPS was built to predict survival of clinical patients. Comprehensive analysis showed the prognosis, immune microenvironment and response to immune therapy and chemotherapy in KRGPS subgroups. Results: We collected a KRGPS from the set of two genes GJB6 and NTNG1, with low-KRGSP patients having better progression-free survival (PFS). Low KRGPS is correlated with high infiltration of activated NK cells, plasma cells and activated memory CD4 T cells and that these cells benefit more from immune checkpoint inhibitor therapy. However, high KRGPS is associated with high infiltration of activated mast cells, pathways of immune dysregulation and a high ratio of TP53 and KRAS mutations. KRGPS subgroups are also sensitive to chemotherapy differently. A nomogram, established based on the KRGPS and pathological stage, predict 3- and 5-years PFS well. Conclusions: The KRAS-associated score acts as a promising signature to distinguish prognosis, molecular and immune characteristics, and benefits from immune and chemical therapy. These KRAS-associated genes could be promising targets for drug design.

Natural orifice specimen extraction surgery versus conventional laparoscopic-assisted resection for colorectal cancer in elderly patients: a propensity-score matching study.

Whether natural orifice specimen extraction surgery (NOSES) could provide beneficial effects in treating elderly patients is still under debate. The aim of the study was to compare the clinical outcomes of transanal NOSES with conventional laparoscopic-assisted resection (LA) in elderly colorectal cancer (CRC) patients. A retrospective analysis from the Second Affiliated Hospital of Harbin Medical University between 2013 and 2017 was performed. Outcomes related to surgery, body image, quality of life, anal function and long-term survival were compared between the two groups with the propensity-score matching (PSM) method. After PSM, 78 patients were successfully compared. Patients with NOSES had faster gastrointestinal function recovery (P = 0.028), less postoperative complications (P = 0.025), lower pain scores on days 1, 3 and 5 after surgery (P < 0.001). The body image score (P < 0.001) and cosmetic score (P < 0.001) were significantly higher in the NOSES group than the LA group at 1 month after surgery. Patients with NOSES posed better global health status (P < 0.001), role function (P = 0.009), emotional function (P = 0.011) and social function (P = 0.011) at 3 months after surgery. Moreover, NOSES showed non inferiority in anal function 6 months after surgery. No significant difference could be found regarding to overall survival (OS), disease-free survival (DFS), local recurrence (LR) and distant metastasis (DM). In elderly CRC patients, NOSES harbored favorable postoperative outcomes, excellent cosmetic properties and better quality of life. Besides, anal function and long-term outcomes of NOSES can be sure for elderly patients.

Identification of the Crucial Role of CCL22 in F. nucleatum-Related Colorectal Tumorigenesis that Correlates With Tumor Microenvironment and Immune Checkpoint Therapy.

Colorectal cancer (CRC) is the third most common malignant cancer worldwide with the second highest mortality. Gut microbiota can educate the tumor microenvironment (TME), consequently influencing the efficacy of immune checkpoint inhibitors (ICIs). Fusobacterium nucleatum is one of the most crucial bacteria contributing to colorectal tumorigenesis, but the molecular mechanisms between F. nucleatum and TME or ICIs are poorly investigated. In the present study, we firstly analyzed differentially expressed genes and the biological functions between F. nucleatum-infected and uninfected CRC cell lines, with the findings that CCL22 mRNA expression was markedly upregulated after F. nucleatum infection. Moreover, the survival analysis showed that CCL22 was significantly associated with the overall survival of CRC patients. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis suggested that CCL22 was related to immune-related terms. Furthermore, the ESTIMATE analysis indicated that the high-CCL22-expression subgroup had a higher immune/stromal/estimate score and lower tumor purity. The CIBERSORT analysis indicated that the high-CCL22-expression group had more immune-suppressive cells and less antitumor immune cells. In addition, immune checkpoint genes and cytotoxic genes were positively correlated with CCL22 expression. The immunophenoscore analysis suggested that CCL22 was associated with the IPS-CTLA4 and PD1/PD-L1/PD-L2 score. Interestingly, CCL22 expression in the KRAS and APC mutation groups was markedly reduced compared to that of the wild groups. In summary, our study provided evidence that CCL22 might play a crucial role in F. nucleatum-related colorectal tumorigenesis and correlate with TME and ICIs, which deserves further study.

Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway.

BACKGROUND: Epidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. METHODS: In this study, using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. RESULTS: Among the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. CONCLUSION: Our studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.

Comparison of natural orifice specimen extraction surgery and conventional laparoscopic-assisted resection in the treatment effects of low rectal cancer.

Natural orifice specimen extraction surgery (NOSES) is an intra-abdominal procedure that does not require an auxiliary incision to take a surgical sample from the abdominal wall through the natural orifice, but there are few systematic clinical studies on it. The aim of this study was to demonstrate the safety and feasibility of NOSES. We retrospectively analyzed the clinical data and follow-up of 165 patients with low rectal cancer who underwent NOSES or conventional laparoscopic surgery at our center from January 2013 to June 2015. From the perioperative data and postoperative follow-up results of both groups, patients in the NOSES group had less intraoperative bleeding (49.3 ± 55.8 ml vs. 75.1 ± 57.3 ml, p = 0.02), shorter postoperative gastrointestinal recovery (42.3 ± 15.5 h vs. 50.1 ± 17.0 h, p = 0.01), less postoperative analgesic use (35.6% vs. 57.6%, p = 0.02), lower postoperative pain scores, lower rate of postoperative complications (6.8% vs. 25.4%, p = 0.01), better satisfaction of the image and cosmesis of the abdominal wall postoperatively, and higher quality of life. Moreover, there was no significant difference in overall survival (OS) and disease-free survival (DFS) between two groups. Overall, NOSES is a safe and reliable minimally invasive surgical technique for patients with low rectal cancer.

Genomic Analysis Reveals Heterogeneity Between Lesions in Synchronous Primary Right-Sided and Left-Sided Colon Cancer.

Background: The synchronous primary right-sided and left-sided colon cancer (sRL-CC) is a peculiar subtype of colorectal cancer. However, the genomic landscape of sRL-CC remains elusive. Methods: Twenty-eight paired tumor samples and their corresponding normal mucosa samples from 14 patients were collected from the Second Affiliated Hospital of Harbin Medical University from 2011 to 2018. The clinical-pathological data were obtained, and whole-exome sequencing was performed based on formalin-fixed and paraffin-embedded samples of these patients, and then, comprehensive bioinformatic analyses were conducted. Results: Both the lesions of sRL-CC presented dissimilar histological grade and differentiation. Based on sequencing data, few overlapping SNV signatures, onco-driver gene mutations, and SMGs were identified. Moreover, the paired lesions harbored a different distribution of copy number variants (CNVs) and loss of heterozygosity. The clonal architecture analysis demonstrated the polyclonal origin of sRL-CC and inter-cancerous heterogeneity between two lesions. Conclusion: Our work provides evidence that lesions of sRL-CC share few overlapping mutational signatures and CNVs, and may originate from different clones.

Overcoming stemness and chemoresistance in colorectal cancer through miR-195-5p-modulated inhibition of notch signaling.

MiR-195-5p has been shown to have a regulatory role in a variety of cancers. Its influence on colorectal cancer (CRC), however, has never been evaluated. In the present study, we found that miR-195-5p expression was significantly decreased as compared to paired, tumor-adjacent normal colorectal tissues. We demonstrated that miR-195-5p inhibited the stem-like capacity of CRC cells. We established 5-FU-resistant SW620 and HT-29 cell lines and performed a variety of functional assays following exposure to miR-195-5p and anti-miR-195-5p. In 5-FU-resistant cells, expression of miR-195-5p, P-gp and ABCG2 was decreased. MiR-195-5p significantly increased cancer cell apoptosis and decreased tumor sphere formation. In order to determine the mechanism by which miR-195-5p reduced CRC cell stemness and chemoresistance, we first identified potential targets of miR-195-5p. The 3' UTR of Notch signaling proteins Notch2 and RBPJ, which are essential genes in CRC cell stemness and chemoresistance, possessed double putative binding sites of miR-195-5p. qRT-PCR and western blot assays demonstrated significant decreases in Notch2 and RBPJ when CRC cell lines were exposed to miR-195-5p and significant increases when exposed to anti-miR-195-5p. These findings indicate that miR-195-5p has the potential to improve standard therapeutic approaches to CRC.

Long non-coding RNA H19 confers 5-Fu resistance in colorectal cancer by promoting SIRT1-mediated autophagy.

Chemotherapy failure is the major cause of recurrence and poor prognosis in colorectal cancer (CRC) patients. The role of the differentially expressed lncRNAs in 5-Fluorouracil chemoresistance has not fully explained. Here, we observed lncRNA H19 was associated with the 5-Fu resistance in CRC. Quantitative analysis indicated that H19 was significantly increased in recurrent CRC patient samples. Kaplan-Meier survival analysis indicated that high H19 expression in CRC tissues was significantly associated with poor recurrent free survival. Our functional studies demonstrated that H19 promoted colorectal cells 5-Fu resistance. Mechanistically, H19 triggered autophagy via SIRT1 to induce cancer chemoresistance. Furthermore, bioinformatics analysis showed that miR-194-5p could directly bind to H19, suggesting H19 might work as a ceRNA to sponge miR-194-5p, which was confirmed by Dual-luciferase reporter assay and Immunoprecipitation assay. Extensively, our study also showed that SIRT1 is the novel direct target of miR-194-5p in CRC cells. Taken together, our study suggests that H19 mediates 5-Fu resistance in CRC via SIRT1 mediated autophagy. Our finding provides a novel mechanistic role of H19 in CRC chemoresistance, suggesting that H19 may function as a marker for prediction of chemotherapeutic response to 5-Fu.

miR-450b-5p Suppresses Stemness and the Development of Chemoresistance by Targeting SOX2 in Colorectal Cancer.

To investigate the role of miR-450b-5p, a newly identified microRNA, located in the Xq26 region, in development of chemoresistance in colorectal cancer (CRC), and to explore the underlying mechanism by which miR-450b-5p regulates this process. In this study, we demonstrated that expression of miR-450b-5p was downregulated in recurrent CRC tissues. We found that expression of miR-450b-5p was significantly inhibited in response to 5-fluorouracil (5-FU) treatment in HT-29 cells and HCT-116 cells. Importantly, overexpression of miR-450b-5p in 5-FU-resistant HT-29 cells reduced cell viability, but elevated DNA fragmentation levels and caspase-3 activity were induced by treatment with 5-FU. Conversely, inhibition of miR-450b-5p enhanced resistance to 5-FU, and promoted cell viability in HCT-116 cells. Mechanistically, we found that miR-450b-5p directly targeted SOX2, an essential factor in stem cells. Expression of miR-450b-5p was negatively correlated to the expression of SOX2, the percentages of CD133(+) cells present, and sphere-forming capacity in CRC cells. Finally, depletion of SOX2 abolished the effects of suppression of miR-450b-5p on stemness and chemoresistance in HT29 cells. We have demonstrated that miR-450b-5p inhibits stemness and development of chemoresistance to 5-FU in CRC cells. These results indicate that miR-450b-5p may be a key determinant of 5-FU sensitivity, and may represent a novel therapeutic target to facilitate chemotherapy for CRC.

The Incidence Characteristics of Second Primary Malignancy after Diagnosis of Primary Colon and Rectal Cancer: A Population Based Study.

BACKGROUND: With the expanding population of colorectal cancer (CRC) survivors in the United States, one concerning issue is the risk of developing second primary malignancies (SPMs) for these CRC survivors. The present study attempts to identify the incidence characteristics of SPMs after diagnosis of first primary colon cancer (CC) and rectal cancer (RC). METHODS: 189,890 CC and 83,802 RC cases were identified from Surveillance, Epidemiology and End Results Program (SEER) database. We performed rate analysis on incidence trend of SPMs in both CC and RC. Expected incidence rates were stratified by age, race and stage, calendar year of first CRC diagnosis and latency period since first CRC diagnosis. The standardized incidence ratios (SIRs), measure for estimating risk of SPMs, were calculated for CC and RC respectively. RESULTS: The trends of incidence of SPMs in both CC and RC were decreasing from 1992 to 2012. Both CC and RC survivors had higher risk of developing SPMs (SIRCC = 1.13; SIRRC = 1.05). For CC patients, the highest risks of SPM were cancers of small intestine (SIR = 4.03), colon (SIR = 1.87) and rectum (SIR = 1.80). For RC patients, the highest risks of SPMs were cancers of rectum (SIR = 2.88), small intestine (SIR = 2.16) and thyroid (SIR = 1.46). According to stratified analyses, we also identified incidence characteristics which were contributed to higher risk of developing SPMs, including the age between 20 and 40, American Indian/Alaska Native, localized stage, diagnosed at calendar year from 2002 to 2012 and the latency between 12 and 59 months. CONCLUSIONS: Both CC and RC survivors remain at higher risk of developing SPMs. The identification of incidence characteristics of SPMs is extremely essential for continuous cancer surveillance among CRC survivors.