Survival Of CRC Patients Research Articles

A multi-omics machine learning framework in predicting the survival of colorectal cancer patients

BackgroundColorectal cancer (CRC), the 3rd most universal cancer globally, accounts for approximately 10% of newly diagnosed cancer incidences each year. Identifying biomarkers associated with CRC survival and predicting the survival of CRC patients are critical for personalized therapy. Existing studies on CRC survival are mainly based on single omics, studies using multi-omics to predict CRC survival are still vacant. To fill in this gap, we aim to identify biomarkers associated with CRC survival at mRNA, miRNA and tissue microbiome levels, and to evaluate the accuracy of potential biomarkers in predicting CRC survival. MethodsFirst, we collected 31 short-term survival (ST, less than 3 years) and 47 long-term survival (LT, longer than 3 years) CRC samples from the database, was named The Cancer Genome Atlas (TCGA). Then, we carried out bioinformatics analysis with collected multi-omics data: (1) comparing the bacterial community structures between ST and LT, (2) identifying differentially expressed mRNAs and miRNAs between ST and LT, and (3) exploring the relationship between bacteria and genes. Finally, we trained models based on multi-omics data to evaluate the performance of several omics data in predicting CRC survival. ResultsAmong the compared omics data, microbiome of CRC tissue had the best predictive power on the three-year survival of CRC patients, the area under the receiver operating characteristic curve (AUC) is 0.755 with 10-fold Cross-Validation (CV). In addition, we screened out 26 differential microbial communities and 13 differential expression genes (DEGs) between ST and LT. Thermoanaerobacterium, Parabacteroides, Oceanicaulis, and Acetonema were more abundant in the ST, while Methylotenera, Candidatus_Riesia and Aquamicrobium were enriched in the LT. We also found that up-regulated genes were significantly enriched in ST group, but the down-regulated genes were enriched in the LT group. ConclusionThe tissue bacterial communities of CRC patients with different survival periods show significant differences, and the bacteria in tumour tissue of CRC are potential biomarkers for predicting the three-year survival of CRC patients.

EPR22-117: Evaluation of the Influence of the BRAF V600E Mutation and of the Microsatellite Instability on the Overall Survival (OS) of Metastatic CRC Patients Based on Real World Data

"EPR22-117: Evaluation of the Influence of the BRAF V600E Mutation and of the Microsatellite Instability on the Overall Survival (OS) of Metastatic CRC Patients Based on Real World Data" published on 31 Mar 2022 by National Comprehensive Cancer Network.

Spatial transcriptome unveils a discontinuous inflammatory pattern in proficient mismatch repair colorectal adenocarcinoma

The preexistence of immune cells in the tumor microenvironment substantiates the efficacy of immunotherapy in cancer patients. Although the complex intratumoral immune heterogeneity has been extensively studied in single cell resolution, hi-res spatial investigations are limited. In this study, we performed a spatial transcriptome analysis of 4 colorectal adenocarcinoma specimens and 2 paired distant normal specimens to identify the molecular pattern involved in a discontinuous inflammatory response in pathologically annotated cancer regions. Based on the location of spatially varied gene expression, we unmasked the spatially-varied immune ecosystem and identified the locoregional “warmed-up” immune response in predefined “cold” tumor with substantial infiltration of immune components. This “warmed-up” immune profile was found to be associated with the in-situ copy number variance and the tissue remodeling process. Further, “warmed-up” signature genes indicated improved overall survival in CRC patients obtained from TCGA database.

TEAD4 nuclear localization and regulation by miR-4269 and miR-1343-3p in colorectal carcinoma

Background and aimsTEAD4 transcription factor belonging to TEAD-family, is a key downstream element of the Hippo Signalling pathway and is very important for YAPinduced tumor progression. YAP-TEAD interaction is required to promote tumor progression and metastasis in various cancers. This study aims to investigate the role of TEAD4 in CRC progression and to compare the TEAD4 expression with different clinicopathological parameters of the study population. We also aim to explore the expression pattern of miR-4269 and miR-1343–3p and their functional role in TEAD4 mediated CRC progression. Furthermore, we intend to evaluate the prognostic significance of TEAD4, miR-4269, and miR-1343–3p in colorectal carcinoma. MethodsReal-time PCR, Immunohistochemical Staining, and Western Blotting were performed on 71 human CRC tissue specimens and their adjacent normal tissues to evaluate the TEAD4 expression and the results were statistically analyzed against the clinicopathological variables of patient data and also with survival data using STATA software. miRNA expression was analyzed by quantitative real-time PCR. ResultsTEAD4 expression levels in tumor specimens were significantly higher than their paired normal specimens. The higher protein expression levels showed a significant association with TNM stage, Duke Stage, tumor grade, invasion depth, node status, necrosis of tumor tissue, lymphovascular and perineural invasion. As per the cox-regression model and classification tree analysis, TNM stage and perineural invasion were important predictors for TEAD4 expression and prognosis of CRC patients. Survival analysis indicated that TEAD4 overexpression was associated with poorer overall and disease-free survival. miR-4269 and miR-1343–3p were downregulated in CRC tumors and showed a negative correlation with TEAD4. The nuclear overexpressed TEAD4 and downregulated miR-4269 and miR-1343–3p evaluated for the first time in CRC, are believed to serve as important prognostic markers in CRC. ConclusionExpression of TEAD4 was increased in CRC and was negatively regulated by miR-4269 and miR-1343–3p. The overexpression of TEAD4 is linked with poor overall and disease-free survival of CRC patients. These findings support prior observations and thus TEAD4 may be a possible prognostic marker in CRC.

MicroRNA-based signature for diagnosis and prognosis of colorectal cancer using residuum of fecal immunochemical test

BackgroundColorectal cancer (CRC) is still among the most lethal and prevalent malignancies in the world. Despite continuous efforts, the diagnosis and prognosis of CRC have never been satisfying, especially the non-invasive assays. MethodsOur study comprised three independent cohorts of 835 qualified stool samples. From 46 literature-identified miRNA candidates, four miRNA ratios were selected and developed into a miRNA-based signature after applied to the training and test sets. The clinical performances of this signature were further evaluated in the prospective cohorts. ResultsFour miRNA ratios with significant alterations and the highest discriminating power between the CRC and control groups in the training set were successfully validated in the test set. In the training dataset, combining these four miRNA ratios using a logistic regression model improved the area under the curve value to 0.821 and obtained a sensitivity of 73.6% and specificity of 78.9%. This miRNA signature showed consistent performances in the other two sample cohorts, with the highest sensitivity of 85.7% in the prospective cohort. Additionally, the higher miRNA signature was associated with worse disease-free survival (hazard ratio = 2.27) and overall survival (hazard ratio = 1.83) of CRC patients. For fecal immunochemical test (FIT)-positive populations, the positive predictive value for CRC detection in miRNA-positive subjects was 3.43-fold higher in the prospective cohort, compared to FIT alone. ConclusionThis stool miRNA signature is highly associated with poor outcome of CRC and can be added to FIT tests to help identify the most at-risk group to receive prompt colonoscopy examination.

P057 Mucosal microbiota adapts to ATF6-induced alterations in host lipid metabolism with prognostic value in colorectal cancer

Abstract Background Colorectal cancer (CRC) is a leading cause of cancer deaths worldwide. The endoplasmic reticulum unfolded protein response (UPRER) signal transducer activating transcription factor 6 (ATF6) is a clinically relevant pre-cancerous marker in CRC and colitis-associated CRC. We established the interrelated role of the microbiota and ATF6 signalling as a novel tumor-promoting mechanism in our transgenic mouse model of spontaneous microbiota-dependent ATF6-driven CRC (nATF6IEC). Methods To elucidate the transcriptional program initiated by acute and chronic ATF6 signalling, mRNA sequencing analyses of murine colonic intestinal epithelial cells were performed 4 days or 5 weeks after induction of ATF6, respectively. Germfree (GF) mice served to dissect microbiota contribution to the transcriptional response. CRC patient datasets (TCGA) were used to estimate ATF6 activity and validate ATF6-driven signatures. To investigate the impact of ATF6 signalling on metabolites, untargeted metabolomics of faeces and targeted lipidomics of tissue were performed. Mucosal microbiota was spatially characterised by 16S rRNA profiling at mm resolution along the colonic longitudinal axis. Based on 16S rRNA data, Phylogenetic Investigation of Communities Reconstruction of Unobserved States (PICRUSt2) was used to infer microbiota lipid-specific functional content. Results We identified an ATF6-UPR core of 368 differentially expressed genes fully activated by acute ATF6 signalling. Functional analysis using KEGG pathways showed that chronic ATF6 signalling predominantly alters UPR-related and metabolic pathways, with 22% of metabolic pathway genes classified as lipid metabolism. GF mice confirmed that the microbiota enhances ATF6-induced metabolic changes. Kaplan-Meier analyses significantly associate our microbiota-dependent ATF6-driven and lipid-specific ATF6-driven gene signatures with decreased disease-free survival in CRC patients since primary therapy. Moreover, ATF6 activity correlates with the presence of CRC-associated bacteria in TCGA samples. Tumor-susceptible mice show alterations in lipid metabolites, particularly long-chain fatty acids (FA) and elongation of saturated FA. PICRUSt2 revealed bacterial lipid detoxification mechanisms, with an increased total abundance of oleate hydratase-positive species. Conclusion Chronic ATF6-signalling alters host lipid metabolism and the lipid milieu in tumor-developing nATF6IEC mice. ATF6-driven microbiota changes are concomitant with bacterial lipid detoxification mechanisms in the tumor niche. We postulate that chronic ATF6 signalling represents a clinically relevant pathologic response that alters the intestinal lipid milieu and thus selects for a tumor-promoting microbiota.

Targeted deep sequencing reveals APC mutations as predictors of overall survival in Chinese colorectal patients receiving adjuvant chemotherapy

Objective: Targeted deep sequencing was used to characterize the mutational spectrum of APC in Chinese colorectal tumors in comparison to that in Caucasians from The Cancer Genome Atlas (TCGA) and to investigate whether APC mutations can predict overall survival in CRC patients receiving adjuvant chemotherapy. Methods: A total of 315 Chinese CRC patients including 241 stage II/III patients receiving fluorouracil-based adjuvant chemotherapy were included in this study. Next generation sequencing was carried out to detect somatic mutations on all APC exons. The associations between APC mutations and overall survival were determined by the Cox proportional hazards model. Results: APC was mutated in 221 of 315 colorectal tumors (70.2%). Chinese CRC had a much higher frequency of missense mutations (16.2% vs. 2.4%), but a lower frequency of nonsense (41.0% vs. 54.2%) and frameshift mutations (10.5% vs. 18.4%) than Caucasian CRC. Among stage II/III patients receiving fluorouracil-based adjuvant chemotherapy, APC mutations showed a significant association with worse survival (HR = 1.69; 95% CI, 1.10-2.62; p = .0179). Of the mutation types, frameshift mutations conferred the highest risk of death (HR = 2.88; 95% CI, 1.54-5.37; p =.0009). Among individual mutation sites, Arg232Ter, the most frequent mutation in Chinese CRC, exhibited the strongest negative impact on survival (HR = 2.65; 95% CI, 1.16-6.03; p =.0202). Conclusion: APC overall mutation was an independent predictor for overall survival of stage II/III CRC patients receiving fluorouracil-based chemotherapy.

Bioinformatically Prediction of the miRNAs Targeting BCL9,FRMD6 Genes in Colorectal Cancer

CRC is a malignancy with no significant symptoms such as bleeding or abdominal pain, and the condition is usually diagnosed when the tumor growth makes the treatment difficult. The survival of CRC patients depends on the disease progression. Most of the CRCs begin with an unbalanced and noncancerous growth of a polyp. CRC may be prevented if the polyps are removed from the colon. Screening reduces the incidence and mortality of CRC. As the miRNAs play a significant role in the regulation of the cellular procedures, this study aims to predict the miRNAs, targeting the BCL9,FRMD6 genes , using bioinformatics tools. After getting the BCL9,FRMD6 protein chain from the NCBI database, the miRNAs targeting the genes were predicted using TargetScan, miRDB, DIANA and miRWalk databases using different algorithms. Based on the scoring system of the bioinformatics software and considering the best targeting scores, (hsa-miR-30e-5p , hsa-miR-30d-5p) and (hsa-miR-106b-5p , hsa-miR-106a-5p) are suggested as the potential miRNAs, targeting the BCL9,FRMD6 genes for future researches. Considering the significant role of the BCL9,FRMD6 genes on Colorectal cancer, the predicted miRNAs can be used as the molecular biomarkers for the early detection of Colorectal cancer patients.

Clinical characteristics and survival analysis of colorectal cancer in China: a retrospective cohort study with 13,328 patients from southern China.

BackgroundColorectal cancer (CRC) is one of the most common cancers in China. However, detailed clinical characteristics and survival information are limited. This study aimed to investigate the potential epidemiological and clinical risk factors affecting the survival of CRC patients in southern China.MethodsPatients with primary CRC between 1994 and 2019 at the First and the Sixth Affiliated Hospitals of Sun Yat-sen University (Guangzhou, China) were included. Clinical characteristics and survival outcomes were collected from medical records. The Kaplan–Meier method was used to estimate overall survival (OS) and progression-free survival (PFS), and Cox’s proportional-hazards regression model was used to estimate hazard ratios and 95% confidence intervals.ResultsOf all 13,328 patients, 60.1% were men; the mean age was 61.3 years; 53.5% had colon cancer. Among all patients, 1,864 (14.0%) were diagnosed with stage IV disease. The 3- and 5-year OS rates were 79.90% and 71.50%, respectively, whereas the 3- and 5-year PFS rates were 70.30% and 63.90%, respectively. The median OS and PFS times were 189 and 149 months, respectively. Among 13,328 patients, 428 (14.0%) patients with poor/undifferentiated cancer suffered recurrence. In patients with stage III and stage IV diseases, the median PFS times of the patients who received chemotherapy were significantly longer than those in patients who had not received chemotherapy (stage III: 147 vs 62 months, P < 0.001; stage IV: 14 vs 9.5 months, P < 0.001).ConclusionsThis retrospective cohort study illustrates the current status of the clinical characteristics of patients with CRC in southern China. Sex, age, family history, location of cancer occurrence, differentiation status, T status, N status, M status, clinical stage, operation, and surgical margin are independent factors associated with the OS of CRC patients.

Upregulation of SNTB1 correlates with poor prognosis and promotes cell growth by negative regulating PKN2 in colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most highly malignant tumors and has a complicated pathogenesis. A preliminary study identified syntrophin beta 1 (SNTB1) as a potential oncogene in CRC. However, the clinical significance, biological function, and underlying mechanisms of SNTB1 in CRC remain largely unknown. Thus, the present study aimed to investigate the role of SNTB1 in CRC.MethodsThe expression profile of SNTB1 in CRC samples was evaluated by database analysis, cDNA array, tissue microarray, quantitative real-time PCR (qPCR), and immunohistochemistry. SNTB1 expression in human CRC cells was silenced using short hairpin RNAs (shRNA)/small interfering RNAs (siRNA) and its mRNA and protein levels were assessed by qPCR and/or western blotting. Cell viability, survival, cell cycle, and apoptosis were determined by the CCK-8 assay, colony formation, and flow cytometry assays, respectively. A xenograft nude mouse model of CRC was established to validate the roles of SNTB1 in vivo. Immunohistochemistry and TUNEL staining were used to determine the expression of SNTB1, PCNA, and cell apoptosis in tissue samples. Isobaric tag for relative and absolute quantification (iTRAQ) was used to analyze the differentially expressed proteins after knockdown of SNTB1 in CRC cells. Silence of protein kinase N2 (PKN2) using si-PNK2 was performed for rescue experiments.ResultsSNTB1 expression was increased in CRC tissues compared with adjacent noncancerous tissues and the increased SNTB1 expression was associated with shorter overall survival of CRC patients. Silencing of SNTB1 suppressed cell viability and survival, induced cell cycle arrest and apoptosis in vitro, and inhibited the growth of CRC cells in vivo. Further elucidation of the regulation of STNB1 on CRC growth by iTRAQ analysis identified 210 up-regulated and 55 down-regulated proteins in CRC cells after SNTB knockdown. A PPI network analysis identified PKN2 as a hub protein and was up-regulated in CRC cells after SNTB1 knockdown. Western-blot analysis further confirmed that SNTB1 knockdown significantly up-regulated PKN2 protein expression in CRC cells and decreased the phosphorylation of both ERK1/2 and AKT. Moreover, rescue experiments indicated that PKN2 knockdown significantly rescued SNTB1 knockdown-mediated decrease in cell viability, survival, and increase of cell cycle arrest at G0/G1 phase and apoptosis of CRC cells.ConclusionsThese findings indicate that SNTB1 is overexpressed in CRC. Elevated SNTB1 levels are correlated with shorter patient survival. Importantly, SNTB1 promotes tumor growth and progression of CRC, possibly by reducing the expression of PKN2 and activating the ERK and AKT signaling pathway. Our study highlights the potential of SNTB1 as a new prognostic factor and therapeutic target for CRC.

Identification and verification of HCAR3 and INSL5 as new potential therapeutic targets of colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most common cancers of the gastrointestinal tract and ranks third in cancer-related deaths worldwide. This study was conducted to identify novel biomarkers related to the pathogenesis of CRC based upon a bioinformatics analysis, and further verify the biomarkers in clinical tumor samples and CRC cell lines.MethodsA series of bioinformatics analyses were performed using datasets from NCBI-GEO and constructed a protein–protein interaction (PPI) network. This analysis enabled the identification of Hub genes, for which the mRNA expression and overall survival of CRC patients data distribution was explored in The Cancer Genome Atlas (TCGA) colon cancer and rectal cancer (COADREAD) database. Furthermore, the differential expression of HCAR3 and INLS5 was validated in clinical tumor samples by Real-time quantitative PCR analysis, western blotting analysis, and immunohistochemistry analysis. Finally, CRC cells over-expressing INSL5 were constructed and used for CCK8, cell cycle, and cell apoptosis validation assays in vitro.ResultsA total of 286 differentially expressed genes (DEGs) were screened, including 64 genes with increased expression and 143 genes with decreased expression in 2 CRC database, from which 10 key genes were identified: CXCL1, HCAR3, CXCL6, CXCL8, CXCL2, CXCL5, PPY, SST, INSL5, and NPY1R. Among these genes, HCAR3 and INSL5 had not previously been explored and were further verified in vitro.ConclusionsHCAR3 expression was higher in CRC tissues and associated with better overall survival of CRC patients. INSL5 expression in normal tissue was higher than that in tumor tissue and its high expression was associated with a better prognosis for CRC. The overexpression of INSL5 significantly inhibited the proliferation and promoted the shearing of PARP of CRC cells. This integrated bioinformatics study presented 10 key hub genes associated with CRC. HCAR3 and INSL5 were expressed in tumor tissue and these were associated with poor survival and warrant further studies as potential therapeutic targets.

Colorectal Cancer Classification and Survival Analysis Based on an Integrated RNA and DNA Molecular Signature

Background: Colorectal cancer (CRC) is the third most common cancer among women and men in the USA, and recent studies have shown an increasing incidence in less developed regions, including Sub-Saharan Africa (SSA). We developed a hybrid (DNA mutation and RNA expression) signature and assessed its predictive properties for the mutation status and survival of CRC patients. Methods: Publicly-available microarray and RNASeq data from 54 matched formalin-fixed paraffin-embedded (FFPE) samples from the Affymetrix GeneChip and RNASeq platforms, were used to obtain differentially expressed genes between mutant and wild-type samples. We applied the support-vector machines, artificial neural networks, random forests, k-nearest neighbor, naïve Bayes, negative binomial linear discriminant analysis, and the Poisson linear discriminant analysis algorithms for classification. Cox proportional hazards model was used for survival analysis. Results: Compared to the genelist from each of the individual platforms, the hybrid genelist had the highest accuracy, sensitivity, specificity, and AUC for mutation status, across all the classifiers and is prognostic for survival in patients with CRC. NBLDA method was the best performer on the RNASeq data, while the SVM method was the most suitable classifier for CRC across the two data types. Nine genes were found to be predictive of survival. Conclusion: This signature could be useful in clinical practice, especially for colorectal cancer diagnosis and therapy. Future studies should determine the effectiveness of integration in cancer survival analysis and the application on unbalanced data, where the classes are of different sizes, as well as on data with multiple classes.

High PHD Finger Protein 19 (PHF19) expression predicts poor prognosis in colorectal cancer: a retrospective study.

BackgroundColorectal cancer (CRC) is the third most common cancer all around the world, and it seriously threats human health. PHF19 has been proved to be closely related to the prognosis of patients in a variety of malignant tumors, but the effect of PHF19 on the prognosis evaluation of CRC patients has not been confirmed.MethodsIn our study, we used GEO, TCGA database and IHC to verify the PHF19 expression in CRC samples. Survival analysis of PHF19 based on TCGA, GEO series, and our own CRC sample were performed. Cox regression was performed to reveal the relationship between PHF19 and prognosis. Co-expression was performed to find genes related to PHF19 expression. GO/KEGG enrichment analysis and GSEA analysis were used to confirm the most relevant signal pathway to PHF19. Next, cell experiments were performed to verify the effect of PHF19 on the proliferation, invasion and metastasis of CRC. Then, Western blot was used to verify the protein expression of the above two phenotypes. Finally, tumor formation experiments in nude mice were used to verify the role of PHF19 of tumor proliferation in vivo.ResultsWe found that PHF19 was significantly over-expressed in tumors compared with normal tissues. Kaplan–Meier (K–M) analysis indicated that high PHF19 in CRC associated with poor overall survival (OS) in CRC patients. Clinical correlation analysis showed that high expression of PHF19 was closely related to t umor progression in CRC patients, especially infiltration and metastasis. Bioinformatics revealed that PHF19 might affect tumor malignant phenotype by regulating the cell cycle in CRC. CCK-8 and clonal formation experiment showed that the proliferative ability of tumor cells was promoted. Flow cytometry showed that the cell cycle accelerated the transition from G1 to S phase. Western blot found that Cyclin D1, CDK4, and CDK6 expression were up-regulated. Transwell and wound-healing experiment found that invasive and migratory abilities was promoted after the over-expression of PHF19. Western blot showed that the expression of key proteins of Epithelial-Mesenchymal Transition (EMT) changed. Tumor formation experiments in nude mice showed that overexpression of PHF19 could promote tumor proliferation in vivo.ConclusionOur research proved that PHF19 could be an independent prognostic factor for CRC, PHF19 promoted the proliferative ability and the invasion and metastasis of CRC by up-regulating the expression of key molecules related to cell cycle and EMT pathway in vitro, promoting tumor proliferation in vivo.

LDL receptor related protein 2 to promote colorectal cancer metastasis via enhancing GSK3β/β-catenin signaling.

e15507 Background: LDL receptor related protein 2 (LRP2), a multi-ligand endocytic receptor, has been implicated in the tumourigenesis of various human cancers. However, the biological roles and underlying regulatory mechanism of LRP2 in CRC remain unclear. Methods: Real-time PCR (RT-PCR), western blot, and immunohistochemistry were used to examine LRP2 expression. Gene silencing and overexpressing efficiencies of LRP2 were confirmed by RT-PCR and western blot. Then wound-healing, migration assay and invasion assay were used to investigate the function of LRP2 in CRC cells. The protein expression and cellular localization of LRP2 and β-catenin were characterized by immunofluorescence staining. The nude mice tail vein metastasis model was established to observe the effect of LRP2 on the lung metastasis of CRC cells in vivo. In addition, gene set enrichment analysis was carried out to explore the potential mechanism of LRP2 in CRC. Results: LRP2 expression was highly upregulated in CRC compared with matched adjacent normal tissue. LRP2 overexpression was positively correlated with shorter overall survival in CRC patients. The biological function observation indicated that LRP2 promoted CRC metastasis in vitro and in vivo. Further molecular mechanism investigation demonstrated that LRP2 protein interacted with glycogen synthase kinase-3β (GSK-3β) and led to decreased expression of GSK3β at ser9, followed by enhanced β-catenin signaling pathway in CRC cells. Conclusions: Our study demonstrated that LRP2 could enhance the metastatic abilities of CRC cells in vitro and in vivo by inhibiting the expression of GSK3β and enhancing the activity of β-catenin signaling pathway, providing that LRP2 might be a novel therapeutic target for metastasis in CRC.

Activation of the clock gene TIMELESS by H3k27 acetylation promotes colorectal cancer tumorigenesis by binding to Myosin-9

BackgroundColorectal cancer (CRC) is a common tumor characterized by its high mortality. However, the underlying molecular mechanisms that drive CRC tumorigenesis are unclear. Clock genes have important roles in tumor development. In the present study, the expression and functions of clock gene TIMELESS (encoding the Timeless protein) in CRC were investigated.MethodsImmunohistochemistry, cell proliferation, migration, invasion, EMT and xenograft tumor experiments were used to prove the function of Timeless in the tumorigenesis of CRC. Immunoprecipitation, mass spectrometry, Immunofluorescence and Chromatin immunoprecipitation (ChIP) were utilized to clarify the mechanism of Timeless in regulating CRC tumorigenesis.ResultsWe found that Timeless was upregulated in CRC tissues compared with corresponding normal tissues and its expression was closely associated with the TNM stages and overall survival of CRC patients. Functional studies demonstrated that Timeless promoted the proliferation, invasion, and EMT of CRC cells in vitro and in vivo. Mechanistic investigations showed that Timeless activated the β-catenin signal pathway by binding to Myosin-9, which binds to β-catenin to induce its nuclear translocation. The upregulation of Timeless was attributed to CREB-binding protein (CBP)/p300-mediated H3K27 acetylation of the promoter region of Timeless.ConclusionTimeless regulates the tumorigenesis of CRC by binding to and regulating myosin-9, suggesting Timeless might be a potential prognostic biomarker and therapeutic target for CRC.

Cancer Susceptibility Candidate 9 (CASC9) Promotes Colorectal Cancer Carcinogenesis via mTOR-Dependent Autophagy and Epithelial-Mesenchymal Transition Pathways.

BackgroundColorectal cancer (CRC) is the third most common cancer worldwide. Many recent studies have demonstrated that different long non-coding RNAs (lncRNAs) are involved in the initiation, advancement, and metastasis of many cancers including CRC. Cancer susceptibility candidate 9 (CASC9) is an lncRNA that has been reported in many cancers, but its role in CRC is poorly understood. In this study, we aimed to examine the expression of CASC9 in CRC cell lines and to determine the mechanism of action of CASC9 in CRC carcinogenesis.MethodsThe expression of CASC9 in CRC tissues was compared with normal samples from publicly available datasets in The Cancer Genome Atlas (TCGA) and The Encyclopedia of RNA Interactomes (ENCORI). CASC9 expression was further verified in four CRC cell lines (DLD1, HT-29, SW480, and HCT-116) and normal colorectal cell line (CCD-112CoN) by real-time quantitative polymerase chain reaction (RT-qPCR). After gene silencing in HCT-116 and SW480, Cell Counting Kit-8 assay, clonogenic assay, and wound healing assay were performed to evaluate cell proliferation, viability, and migration index of cells. Western blotting was used to explore the key pathways involved.ResultsCASC9 was significantly upregulated as analyzed from both public datasets TCGA and ENCORI where its overexpression was associated with poor survival of CRC patients. Similarly, CASC9 was significantly overexpressed in the CRC cell lines compared with normal cells studied. The silencing of CASC9 in HCT-116 and SW480 attenuated cell proliferation and migration significantly. Furthermore, pathways investigations showed that silencing of CASC9 significantly induced autophagy, promoted AMP-activated protein kinase (AMPK) phosphorylation, inhibited mTOR and AKT signaling pathways, and altered epithelial–mesenchymal transition (EMT) marker protein expression.ConclusionWe demonstrated that silencing of CASC9 contributes to the reduced CRC cell proliferation and migration by regulating autophagy and AKT/mTOR/EMT signaling. Therefore, CASC9 plays an important role in carcinogenesis, and its expression may act as a prognostic biomarker and a potential therapeutic target of CRC management.

Hyperglycemia Promotes Liver Metastasis of Colorectal Cancer via Upregulation of Integrin αvβ6.

BackgroundDiabetes is related to higher risk of multiple cancers. This study aimed to explore the effect and mechanism of diabetes on liver metastasis of CRC.Material/MethodsOverall and liver metastasis-free survival in diabetic and non-diabetic CRC patients were compared by Kaplan-Meier analysis. Expression of αvβ6 was detected by immunohistochemistry in clinical specimens. Effects of hyperglycemia on αvβ6 expression in colon cancer cells were assessed by western blot, real-time PCR, and flowcytometry. Effects of hyperglycemia on migration and invasion were demonstrated by Transwell assay. Expression and activity of MMP-9 and MMP-2 were determined by real-time PCR and gelatin zymography. Liver metastatic nodules were counted and β6 expression was detected by western blot in a liver metastasis mouse model.ResultsCRC patients with diabetes had poorer overall and liver metastasis-free survival, and diabetes was associated with higher αvβ6 expression in CRC specimens. Hyperglycemia promoted the invasion and migration of colon cancer cells, and upregulated the expression and activity of MMP-9, which were attenuated by inhibition of αvβ6. Hyperglycemia upregulated the expression of β6 and cell surface expression of αvβ6, which was reduced by ERK inhibitor. The in vitro results were confirmed in vivo in the mouse model.ConclusionsOur study demonstrated the enhancing effect of hyperglycemia on liver metastasis of CRC, and showed that αvβ6 was involved in this process, suggesting that control of glucose levels and inhibition of αvβ6 can reduce the risk of liver metastasis in diabetic CRC patients.

A Prognostic Model Based on the Immune-Related lncRNAs in Colorectal Cancer

BackgroundColorectal cancer (CRC) is one of the most common malignant tumors with a poor prognosis. At present, the pathogenesis is not completely clear. Therefore, finding reliable prognostic indicators for CRC is of important clinical significance. In this study, bioinformatics methods were used to screen the prognostic immune-related lncRNAs of CRC, and a prognostic risk scoring model based on immune-related lncRNAs signatures were constructed to provide a basis for prognostic evaluation and immunotherapy of CRC patients.MethodsThe clinical information and RNA-seq data of CRC patients were obtained from The Cancer Genome Atlas (TCGA) database. The information of immune-related lncRNA was downloaded from the immunology database and analysis portal. The differentially expressed immune-related lncRNAs (IRLs) were screened by the edgeR package of R software. The prognostic value of IRLs was studied. Based on Cox regression analysis, a prognostic index (IRLPI) based on IRLs was established, and the relationship between the risk score and the clinicopathological characteristics of CRC was analyzed to determine the effectiveness of the risk score model as an independent prognostic factor.ResultsA total of 240 differentially expressed IRLs were identified between normal colorectal cancer tissues and normal colorectal cancer tissues, in which 8 were significantly associated with the survival of CRC patients (P < 0.05), including LINC00461, LINC01055, ELFN1-AS1, LMO7-AS1, CYP4A22-AS1, AC079612.1, LINC01351, and MIR31HG. And most of the lncRNAs related to survival were risk factors for the prognosis of CRC. The index established based on the 7 survival-related IRLs found to be highly accurate in monitoring CRC prognosis. Besides, IRLPI was significantly correlated with a variety of pathological factors and immune cell infiltration.ConclusionEight immune-related lncRNAs closely related to the prognosis of CRC patients were identified from the TCGA database. At the same time, an independent IRLPI was constructed, which may be helpful for clinicians to assess the prognosis of patients with CRC and to formulate individualized treatment plans.

Nomogram for predicting overall survival in colorectal cancer with distant metastasis

BackgroundColorectal cancer (CRC) is a major cancer burden, and prognosis is determined by many demographic and clinicopathologic factors. The present study aimed to construct a prognostic nomogram for colorectal cancer patients with distant metastasis.MethodsColorectal cancer patients with distant metastasis diagnosed between 2010 and 2016 were selected from the Surveillance, Epidemiology, and End Results database. Cox proportional hazards regression was used to identify independent prognostic factors. A nomogram was constructed to predict survival, and validation was performed.ResultsA total of 7099 stage IV colorectal cancer patients were enrolled in the construction cohort. The median overall survival was 20.0 (95% CI 19.3–20.7) months. Age at diagnosis, marital status, race, primary tumour site, tumour grade, CEA level, T stage, N stage, presence of bone, brain, liver and lung metastasis, surgery for primary site and performance of chemotherapy were independent prognostic factors. The nomogram was constructed and the calibration curve showed satisfactory agreement. The C-index was 0.742 (95% CI 0.726–0.758). In the validation cohort (7098 patients), the nomogram showed satisfactory discrimination and calibration with a C-index of 0.746 (95% CI 0.730–0.762).ConclusionA series of factors associated with the survival of CRC patients with distant metastasis were found. Based on the identified factors, a nomogram was generated to predict the survival of stage IV colorectal cancer patients. The predictive model showed satisfactory discrimination and calibration, which can provide a reference for survival estimation and individualized treatment decisions.

Mex3a promotes oncogenesis through the RAP1/MAPK signaling pathway in colorectal cancer and is inhibited by hsa-miR-6887-3p.

BackgroundAlthough Mex3 RNA‐binding family member A (Mex3a) has demonstrated an important role in multiple cancers, its role and regulatory mechanism in CRC is unclear. In this study, we aimed to investigate the role and clinical significance of Mex3a in CRC and to explore its underlying mechanism.MethodsWestern blotting and quantitative real‐time polymerase chain reaction (qRT‐PCR) were performed to detect the expression levels of genes. 5‐Ethynyl‐2'‐deoxyuridine (EDU) and transwell assays were utilized to examine CRC cell proliferation and metastatic ability. The R software was used to do hierarchical clustering analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Overexpression and rescue experiments which included U0126, a specific mitogen activated protein kinase kinase/extracellular regulated protein kinase (MEK/ERK) inhibitor, and PX‐478, a hypoxia‐inducible factor 1 subunit alpha (HIF‐1α) inhibitor, were used to study the molecular mechanisms of Mex3a in CRC cells. Co‐immunoprecipitation (Co‐IP) assay was performed to detect the interaction between two proteins. Bioinformatics analysis including available public database and Starbase software (starbase.sysu.edu.cn) were used to evaluate the expression and prognostic significance of genes. TargetScan (www.targetscan.org) and the miRDB (mirdb.org) website were used to predict the combination site between microRNA and target mRNA. BALB/c nude mice were used to study the function of Mex3a and hsa‐miR‐6887‐3p in vivo.ResultsClinicopathological and immunohistochemical (IHC) studies of 101 CRC tissues and 79 normal tissues demonstrated that Mex3a was a significant prognostic factor for overall survival (OS) in CRC patients. Mex3a knockdown substantially inhibited the migration, invasion, and proliferation of CRC cells. Transcriptome analysis and mechanism verification showed that Mex3a regulated the RAP1 GTPase activating protein (RAP1GAP)/MEK/ERK/HIF‐1α pathway. Furthermore, RAP1GAP was identified to interact with Mex3a in Co‐IP experiments. Bioinformatics and dual‐luciferase reporter experiments revealed that hsa‐miR‐6887‐3p could bind to the 3'‐untranslated regions (3'‐UTR) of the Mex3a mRNA. hsa‐miR‐6887‐3p downregulated Mex3a expression and inhibited the tumorigenesis of CRC both in vitro and in vivo.ConclusionsOur study demonstrated that the hsa‐miR‐6887‐3p/Mex3a/RAP1GAP signaling axis was a key regulator of CRC and Mex3a has the potential to be a new diagnostic marker and treatment target for CRC.