Colorectal Cancer Patient Samples Research Articles

TET2–BCLAF1 transcription repression complex epigenetically regulates the expression of colorectal cancer gene Ascl2 via methylation of its promoter

Ascl2 has been shown to be involved in tumorigenesis in colorectal cancer (CRC), although its epigenetic regulatory mechanism is largely unknown. Here, we found that methylation of the Ascl2 promoter (bp -1670 ∼ -1139) was significantly increased compared to the other regions of the Ascl2 locus in CRC cells and was associated with elevated Ascl2 mRNA expression. Furthermore, we found that promoter methylation was predictive of CRC patient survival after analyzing DNA methylation data, RNA-Seq data, and clinical data of 410 CRC patient samples from the MethHC database, the MEXPRESS database, and the Cbioportal website. Using the established TET methylcytosine dioxygenase 2 (TET2) knockdown and ectopic TET2 catalytic domain–expression cell models, we performed glucosylated hydroxymethyl–sensitive quatitative PCR (qPCR), real-time PCR, and Western blot assays to further confirm that hypermethylation of the Ascl2 promoter, and elevated Ascl2 expression in CRC cells was partly due to the decreased expression of TET2. Furthermore, BCLAF1 was identified as a TET2 interactor in CRC cells by LC-MS/MS, coimmunoprecipitation, immunofluorescence colocalization, and proximity ligation assays. Subsequently, we found the TET2–BCLAF1 complex bound to multiple elements around CCGG sites at the Ascl2 promoter and further restrained its hypermethylation by inducing its hydroxymethylation using chromatin immunoprecipitation-qPCR and glucosylated hydroxymethyl-qPCR assays. Finally, we demonstrate that TET2-modulated Ascl2-targeted stem gene expression in CRC cells was independent of Wnt signaling. Taken together, our data suggest an additional option for inhibiting Ascl2 expression in CRC cells through TET2–BCLAF1–mediated promoter methylation, Ascl2-dependent self-renewal of CRC progenitor cells, and TET2–BCLAF1–related CRC progression.

Landscape of endocytosis pathway in colorectal cancer (CRC).

3148 Background: Recent proteogenomic analyses of CRC revealed that driver gene alterations are enriched in the endocytosis pathway (Vasaikar S, et al. Cell 2019;177:1035-49). Endocytosis is a cellular system involving post-translational modification of plasma membrane proteins through internalization, intracellular trafficking, degradation, and recycling. Clathrin-mediated endocytosis (CME) is the main endocytic portal, and endosomal sorting complexes required for transport (ESCRT) play a critical role in the lysosomal degradation pathway. Besides the well-known function of endocytosis attenuating signaling pathways through receptor clearance from the cell surface, the opposite function contributing to signal maintenance has also been reported. However, the clinical implications of the endocytosis pathway alterations in CRC are largely unclear. Methods: We retrospectively reviewed CRC patient samples (n = 15025) submitted to a commercial CLIA-certified laboratory (Caris Life Sciences, Phoenix AZ). Next-generation sequencing of DNA and RNA (whole-transcriptome sequencing) and immunohistochemistry (IHC) were performed. CME-related (47 genes) and ESCRT-related (35 genes) expression signatures were calculated as composite z-scores and compared between subgroups stratified by RAS/ BRAF mutation status, MSS/MSI status, tumor sidedness, and consensus molecular subtype (CMS). VPS4A/ VPS4B expression correlation with major oncogenic pathway signatures (composite z-scores) and CMTM6/ CMTM4/ HIP1R expression association with PD-L1+ IHC were also assessed. Results: Among 17 endocytosis-related genes, no pathogenic/likely pathogenic mutations were identified. The CME-related signature was increased in RAS/ BRAF wild type vs. mutant (0.93 z-score difference, p= 0.04) and MSS vs. MSI-high (6.0 z-score difference, p< 0.01), while the ESCRT-related signature was higher in MSS compared to MSI-high (2.7 z-score difference; p< 0.01). No differences between tumor sidedness were observed in both CME- and ESCRT-related signatures (0.81 and 1.17 z-score differences, respectively). CMS4 had the highest expression of both signatures, while CMS3 had the lowest, of both CME- and ESCRT-related genes (each > 20 z-score difference, p< 0.01). VPS4A and VPS4B expression had a strong positive correlation with WNT, EGFR/MAPK, TGF-beta, and Notch pathway signatures (0.65-0.83 Spearman, all p< 0.01). CMTM6 expression was positively associated with PD-L1+ IHC (1.2-fold increase vs PD-L1-negative, p< 0.01), while CMTM4 and HIP1R expression showed a negative association (0.7- and 0.9-fold decrease, respectively, p< 0.01). Conclusions: This large study indicates endocytosis pathway expression is positively associated with oncogenic pathway signaling in CRC. Further analysis of RAS/ BRAF wild type, MSS, and CMS4 patient subgroups are warranted to determine the efficacy of targeting endocytosis pathways in CRC.

Identification of potential therapeutic targets associated with diagnosis and prognosis of colorectal cancer patients based on integrated bioinformatics analysis

Colorectal cancer (CRC) is the most common malignancy of digestive system with significant mortality rate. CRC patients with comparable clinical symptoms or at similar stages of the disease have different outcomes. This underlying clinical result is almost inevitably due to genetic heterogeneity. Therefore, the current study aimed to highlight gene signatures during CRC and unveil their potential mechanisms through bioinformatic analysis. The gene expression profiles (GSE28000, GSE33113, GSE44861, and GSE37182) were downloaded from the Gene Expression Omnibus database, and the differential expressed genes (DEGs) were identified in normal tissues and tumor tissue samples of CRC patients. In total, 8931 DEGs were identified in CRC, including 411 up-regulated genes and 166 down-regulated genes. Further, a protein-protein interaction network was constructed and the highly related genes were clustered using the Molecular Complex Detection algorithm (MCODE) to retrieve the core interaction in different genes’ crosstalk. The screened hub genes were subjected to functional enrichment analysis. GO analysis results showed that up-regulated DEGs were significantly enriched in biological processes (BP), including cell division, cell cycle, and cell proliferation; the down-regulated DEGs were significantly enriched in BP, including cellular homeostasis, detoxification, defense response, intracellular signaling cascade. Additionally, KEGG pathway analysis displayed the up-regulated DEGs were enriched in the cell cycle, TNF signaling, chemokine signaling pathway, while the down-regulated DEGs were enriched in NF-kB signaling, mineral reabsorption. Furthermore, the overall survival and expression levels of hub genes were detected by the UALCAN database and were further validated using Human Protein Atlas database. Taken together the identified DEGs (MT2A, CCNB1, DLGAP5, CCNA2, CXCL2, and RACGAP1) enhance our understanding of the molecular pathways that underpin CRC pathogenesis and could be exploited as molecular targets and diagnostic biomarkers for CRC therapy.

Cell line models for drug discovery in PIK3CA-mutated colorectal cancers.

Colorectal cancer remains a major cause of cancer-related morbidity and mortality. Metastatic disease is still incurable in most cases. New therapies based on a better understanding of the pathogenesis are needed to improve outcomes. Mutations in the catalytic sub-unit of kinase PI3K encoded by gene PIK3CA are common in colorectal cancer cell lines and patient samples. The characteristics of colorectal cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE), with and without PIK3CA mutations, were evaluated and compared. A panel of colorectal cancer cell lines with and without PIK3CA mutations were compared for their sensitivity to PIK3 inhibitors. Concomitant molecular abnormalities of sensitive versus resistant cell lines were identified. Colorectal cancer cell lines with PIK3CA mutations are commonly diploid and have microsatellite instability (MSI) and a high tumor mutation burden (TMB), compared with cell lines without PIK3CA mutations. Cell lines with PIK3CA mutations tend to have higher sensitivity to some but not all PI3K inhibitors tested and display variability in sensitivity. Both cell lines with MSI and microsatellite stable (MSS) are among the most sensitive to PI3K inhibitors. Multiple concomitant mutations in the PI3K/AKT and KRAS/BRAF/MEK/ERK pathways are often observed in sensitive cell lines. In concordance with patient samples, colorectal cancer cell lines with PIK3CA mutations display more commonly MSI and tend to be more sensitive to PI3K inhibitors. Variability in sensitivity of PIK3CA-mutated cell lines suggests that additional molecular abnormalities contribute to sensitivity.

Alterations of the Gut Microbiome and Fecal Metabolome in Colorectal Cancer: Implication of Intestinal Metabolism for Tumorigenesis.

Objective: The gut microbiota and its metabolites are important for host physiological homeostasis, while dysbiosis is related to diseases including the development of cancers such as colorectal cancer (CRC). In this study, we characterized the relationship of an altered gut microbiome with the fecal metabolome in CRC patients in comparison with volunteers having a normal colorectal mucous membrane (NC). Methods: The richness and composition of the microbiota in fecal samples of 30 CRC patients and 36 NC controls were analyzed through 16S rRNA gene sequencing, and the metabolome was determined by ultra-performance liquid chromatography coupled to tandem mass spectrometry. Spearman correlation analysis was to determine the correlation between the gut microbiome and fecal metabolome in CRC patients. Results: There were significant alterations in the gut microbiome and fecal metabolome in CRC patients compared with NC controls. Bacteroidetes, Firmicutes, Actinobacteriota, and Proteobacteria dominated the gut microbial communities at the phylum level in both groups. Compared with NC controls, CRC patients had a lower frequency of Blautia and Lachnospiracaea but a higher abundance of Bacteroides fragilis and Prevotella. Regarding the fecal metabolome, twenty-nine metabolites were identified as having significantly changed, showing increased levels of adrenic acid, decanoic acid, arachidonic acid, and tryptophan but a reduction in various monosaccharides in the fecal samples of CRC patients. Moreover, increased abundance of Bacteroides fragilis was strongly associated with decreased levels of monosaccharides, while Blautia was positively associated with the production of monosaccharides in the fecal samples. Conclusion: These results highlight alterations of gut microbiota in association with certain metabolites in CRC progression, implying potential diagnostic and intervention potential for CRC.

Expression of specific microRNAs in tissue and plasma in colorectal cancer.

MicroRNAs (miRNA/miR) play significant roles in the regulation of cell differentiation, cell cycle progression, and apoptosis. They become dysregulated during carcinogenesis and are eventually released into the circulation, enabling their detection in body fluids. Thus, this study compared the miRNA expression in tissue and plasma samples of colorectal cancer (CRC) patients and clinically healthy controls and determined miRNA expression as a potential CRC biomarker. Using quantitative reverse transcription polymerase chain reaction (RT-qPCR), miR-21-5p, miR-29a-3p, miR-92a-3p, miR-135b-5p, miR-196b-5p, and miR-197-3p, expression was analyzed and compared between the malignant (n = 41) and the adjacent neoplasm free mucosal tissues (n = 41) of CRC patients. The findings were validated in plasma samples (n = 36) collected from the same CRC patients prior to surgery or any form of treatment and compared to plasma from their age and sex-matched controls (n = 36). MiR-21-5p, miR-29a-3p, miR-92a-3p, and miR- 196b-5p were upregulated and miR-135b-5p was downregulated in CRC malignant tissues compared to their expression in adjacent neoplasm-free tissue. This was further observed in the plasma of the same CRC cases compared to controls. MiR-92a-3p showed itself the most sensitive (0.93; p < .001) and most specific (0.95; p < .001) in detecting CRC in tissue. In plasma, miR-196b-5p was the most sensitive (0.97; p < .001) and specific (0.94; p < .001) in detecting CRC. Plasma miR-92a-3p and miR-196b-5p were the most sensitive (0.95; p < .001) and specific (0.94; p < .001) in the early detection of CRC. Results show that specific miRNAs dysregulated in malignant tissues are released and can be detected in the circulation, supporting their potential as non-invasive biomarkers of CRC.

Health-related quality of life of breast and colorectal cancer patients undergoing active chemotherapy treatment: Patient-reported outcomes.

We sought to explore the Health-Related Quality of Life (HRQoL) of Breast Cancer (BC) and Colorectal Cancer (CRC) patients receiving active chemotherapy. A cross-sectional study was conducted among a convenient sample of BC and CRC patients between May 2018 and June 2019. HRQoL was measured with the Functional Assessment of Cancer Therapy-Breast (FACT-B; 36 items, score range 0-144)) and the Functional Assessment of Cancer Therapy-Colon (FACT-C; 34 items, score range 0-136) scales. Both scales measured Physical Well-Being (PWB), Social Well-Being (SWB), emotional well-being, Functional Well-Being (FWB), and an additional disease-specific HRQoL items. A total of 209 BC and 159 CRC patients were included, with a mean age 49.73±10.41 and 55.38±11.35years, respectively. 110 (52.6%) of BC and 86 (54.1%) CRC patients were dependent on caregivers, and 115 (55%) of BC and 92 (57.9%) CRC patients slept > 7h/night. Reported HRQoL mean scores of BC (FACT-B) and CRC (FACT-C) were 85.53±14.81 and 87.69±20.21, respectively. For BC, the PWB score of patients aged >49years (postmenopausal) was statistically significantly (p=0.013) worse than those aged ≤49years (premenopausal). Patients dependent on caregivers had statistically significant better PWB and worse EWB (p=0.041; p=0.027, respectively). CRC patients' dependent on caregivers had better statistically significant differences scoring in FACT-C (p=<0.001), PWB (p=0.001), EWB (p=<0.001), and FWB (p=0.001). In this study, BC and CRC patients who received active chemotherapy were more likely to have poor HRQoL. BC and CRC HRQoL should be addressed early and continuously, to limit their effects on treatment plan.

Clusterin mRNA silencing reduces cell proliferation, inhibits cell migration, and increases CCL5 expression in SW480, SW620, and Caco2 cells

Abstract Objectives This study aimed to investigate the effects of specific gene silencing in colorectal cancer cells. Clusterin protein was found in the serum samples of colorectal cancer patients infected with Schistosoma mansoni previously. Methods For this reason, silencing clusterin mRNA in colorectal cancer cells was first performed to study the cytotoxic effect by lactate dehydrogenase assay. Next, propidium iodide staining and flow cytometry were performed to investigate the cell cycle profile in clusterin-silenced cells. A wound-healing assay was also used to examine the migration rate of clusterin-silenced cells. The mRNA expression of cell proliferation- and migration-related genes was then assessed by real-time PCR. Results Clusterin mRNA silencing caused a significant reduction in cell growth but induced no cell cycle arrest or potential apoptosis in all cells. It was found in this study that cell migration rate was inhibited in clusterin-silenced cells. Surprisingly, significantly induced chemokine (C–C motif) ligand 5 (CCL5) mRNA expression was detected in clusterin-silenced Caco2, which indicated that the cell proliferation and migration of clusterin-silenced Caco2 were likely associated with CCL5 mRNA expression. Conclusions Clusterin may be a potential target for regulation, staging, surveillance, and developing a cost-effective therapeutic agent for treating parasite-infected Caco2 type of colorectal cancer patients.

Vanillin Derivatives Reverse Fusobacterium nucleatum-Induced Proliferation and Migration of Colorectal Cancer Through E-Cadherin/β-Catenin Pathway.

Colorectal cancer (CRC) is a common clinical malignant tumor and closely related to intestinal microbiome disorders. Especially, Fusobacterium nucleatum (F. nucleatum) is one of the most prevalent pathogens in CRC. However, its change in CRC patients of Northwest China, an area with a high incidence of gastrointestinal tumors, is unclear, and therapeutic strategies targeting F. nucleatum remain unresolved. Here, fecal samples of healthy people and CRC patients were studied using 16S rRNA sequencing to explore microbial community alterations. Additionally, vanillin derivate (IPM711 and IPM712) intervention by coculture with CRC cells and potential mechanism were investigated. Results showed that intestinal microbial homeostasis was gradually dysregulated, and the abundance of Fusobacterium was higher in CRC patients. Moreover, IPM711 and IPM712 showed better anti-F. nucleatum activity than vanillin by increasing cell membrane permeability and destroying bacterial integrity. In addition, IPM711 and IPM712 could downregulate the expression of E-cadherin and β-catenin, thus, suppressing the migration of HCT116. Collectively, IPM711 and IPM712 have both anticolorectal cancer and anti-F. nucleatum activities, providing potential natural product drug candidates for microbe-targeted strategies for the treatment of CRC.

Evaluation of the Prognostic Relevance of Differential Claudin Gene Expression Highlights Claudin-4 as Being Suppressed by TGFβ1 Inhibitor in Colorectal Cancer.

Background: Claudins (CLDNs) are a family of closely related transmembrane proteins that have been linked to oncogenic transformation and metastasis across a range of cancers, suggesting that they may be valuable diagnostic and/or prognostic biomarkers that can be used to evaluate patient outcomes. However, CLDN expression patterns associated with colorectal cancer (CRC) remain to be defined. Methods: The mRNA levels of 21 different CLDN family genes were assessed across 20 tumor types using the Oncomine database. Correlations between these genes and patient clinical outcomes, immune cell infiltration, clinicopathological staging, lymph node metastasis, and mutational status were analyzed using the GEPIA, UALCAN, Human Protein Atlas, Tumor Immune Estimation Resource, STRING, Genenetwork, cBioportal, and DAVID databases in an effort to clarify the potential functional roles of different CLDN protein in CRC. Molecular docking analyses were used to probe potential interactions between CLDN4 and TGFβ1. Levels of CLDN4 and CLDN11 mRNA expression in clinical CRC patient samples and in the HT29 and HCT116 cell lines were assessed via qPCR. CLDN4 expression levels in these 2 cell lines were additionally assessed following TGFβ1 inhibitor treatment. Results: These analyses revealed that COAD and READ tissues exhibited the upregulation of CLDN1, CLDN2, CLDN3, CLDN4, CLDN7, and CLDN12 as well as the downregulation of CLDN5 and CLDN11 relative to control tissues. Higher CLDN11 and CLDN14 expression as well as lower CLDN23 mRNA levels were associated with poorer overall survival (OS) outcomes. Moreover, CLDN2 and CLDN3 or CLDN11 mRNA levels were significantly associated with lymph node metastatic progression in COAD or READ lower in COAD and READ tissues. A positive correlation between the expression of CLDN11 and predicted macrophage, dendritic cell, and CD4+ T cell infiltration was identified in CRC, with CLDN12 expression further being positively correlated with CD4+ T cell infiltration whereas a negative correlation was observed between such infiltration and the expression of CLDN3 and CLDN15. A positive correlation between CLDN1, CLDN16, and neutrophil infiltration was additionally detected, whereas neutrophil levels were negatively correlated with the expression of CLDN3 and CLDN15. Molecular docking suggested that CLDN4 was able to directly bind via hydrogen bond with TGFβ1. Relative to paracancerous tissues, clinical CRC tumor tissue samples exhibited CLDN4 and CLDN11 upregulation and downregulation, respectively. LY364947 was able to suppress the expression of CLDN4 in both the HT29 and HCT116 cell lines. Conclusion: Together, these results suggest that the expression of different CLDN family genes is closely associated with CRC tumor clinicopathological staging and immune cell infiltration. Moreover, CLDN4 expression is closely associated with TGFβ1 in CRC, suggesting that it and other CLDN family members may represent viable targets for antitumor therapeutic intervention.

Identification and Comprehensive Analysis of FREM2 Mutation as a Potential Prognostic Biomarker in Colorectal Cancer.

Gene mutations play an important role in tumor progression. This study aimed to identify genes that were mutated in colorectal cancer (CRC) and to explore their biological effects and prognostic value in CRC patients. We performed somatic mutation analysis using data sets from The Cancer Genome Atlas and International Cancer Genome Consortium, and identified that FREM2 had the highest mutation frequency in patients with colon adenocarcinoma (COAD). COAD patients were divided into FREM2-mutated type (n = 36) and FREM2-wild type (n = 278), and a Kaplan-Meier survival curve was generated to perform prognostic analysis. A FREM2-mutation prognosis model was constructed using random forest method, and the performance of the model was evaluated using receiver operating characteristic curve. Next, the random forest method and Cox regression analysis were used to construct a prognostic model based on the gene expression data of 36 FREM2-mutant COAD patients. The model showed a high prediction accuracy (83.9%), and 13 prognostic model characteristic genes related to overall survival were identified. Then, the results of tumor mutation burden (TMB) and microsatellite instability (MSI) analyses revealed significant differences in TMB and MSI among the risk scores of different prognostic models. Differentially expressed genes were identified and analyzed for functional enrichment and immune infiltration. Finally, 30 samples of CRC patients were collected for immunohistochemical staining to analyze the FREM2 expression levels, which showed that FREM2 was highly expressed in tumor tissues. In conclusion, CRC patients had a high level of FREM2 mutations associated with a worse prognosis, which indicated that FREM2 mutations may be potential prognostic markers in CRC.

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications.

In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.

Zebrafish xenograft model for studying the function of lncRNA SNHG4 in the proliferation and migration of colorectal cancer.

The zebrafish xenograft model has become a reliable in vivo model for human cancer research. Compared to a mouse model, the zebrafish xenograft has many advantages, including optical transparency, intuitive in vivo observation, and speed. Long noncoding RNAs (lncRNAs) have been identified as crucial regulatory factors in the progression of colorectal cancer (CRC). The biological function of lncRNA small nucleolar RNA host gene 4 (SNHG4) in CRC is still unclear. We analyzed the expression of SNHG4 in CRC patient samples by the Gene Expression Profiling Interactive Analysis (GEPIA) software. The quantitative real time-polymerase chain reaction (qRT-PCR) was used to verify in CRC cell lines. The colony formation assay was used to study the cell proliferation, and we used the transwell assay to detect the migration ability. Then the zebrafish xenograft models were used to confirm these roles of SNHG4 in vivo. Moreover, we detected epithelial mesenchymal transition (EMT) related genes by qRT-PCR. We found the expression of SNHG4 was upregulated in CRC patient samples by analyzing GEPIA software, which was also verified in CRC cell lines. We also found that silencing SNHG4 inhibited the proliferation and migration of CRC cells, and its roles were verified in zebrafish xenografts in vivo. Further, we found that the expression of E-cadherin was significantly upregulated and N-cadherin was downregulated when knocking-down SNHG4 in CRC cells. Our findings demonstrated that SNHG4 played oncogenic roles in CRC, which could be a potential target for treatment of CRC patients, and the results strongly revealed that zebrafish xenograft could be used for functional research of lncRNAs in human cancer.

Analysis of 16S rRNA sequencing in advanced colorectal cancer tissue samples.

163 Background: Changes in the colon’s microbiota composition are contributors to the pathogenesis of colorectal cancer (CRC). Features of this microbiome may have prognostic significance. For this study, we analyzed 16S rRNA sequencing data from tumor tissue samples of advanced CRC patients and determined if there were potential correlations between microbiome composition and clinical outcomes. Methods: One hundred and thirty three advanced CRC patients in St. Vincent’s Hospital in Korea were enrolled. DNA was extracted from collected tissue samples, the V3-V4 regions were amplified, and a 16S rRNA gene amplicon library was prepared using an Illumina protocol. DNA was sequenced on an Illumina MiSeq instrument. We used three different bioinformatic packages to process the sequence data and evaluate the microbiome composition of each tumor. Results: The classification performances of three different analytic pipelines (Kraken2, QIIME2, and DADA2) were compared in a microbiome control sample. Among these, DADA2 and QIIME2 were chosen for use in subsequent analysis, due to their lower Chi-square (χ2) test statistic values on the control data. After excluding samples that retained less than 5% of total reads after merging, 120 samples were analyzed. The median age of the cohort was 63 years, 63.3% were male, and all were Korean. The distribution over primary sites was 27.5% from the right-side colon, 30.8% from the left-side colon, and 41.7% from the rectum. All subjects received the first line of systemic treatment. The median progression-free survival time was 8.9 months. Twenty-nine patients (24.2%) survived more than 24 months. When examining the results from the two bioinformatic packages, pairwise comparisons showed positive correlations in the relative abundances of the top 20 genera. Fusobacterium, a microbe known to relate to pathogenesis and prognosis of CRC, was not detected by QIIME2. Stratifying by primary site, rectal cancers showed higher alpha diversity than left- or right-side colon cancers. Separately, a higher serum CEA level (≥8) at diagnosis, and the presence of lung metastasis were both found to be related to higher alpha-diversity, a global indicator of microbiome composition. When excluding minimally abundant (&lt; 1% per patient) genera, beta-diversity was found to be differentiable by T stage, the presence of lung metastasis, and the presence of liver metastasis. Most notably, beta-diversity differed between patients who survived more than two years and patients who died within 2 years. Using the DADA2 results, we confirmed that the presence of Fusobacterium nucleatum in CRC tissue was found to be a strong predictor of poor overall survival (OS), along with old age and liver metastasis. Conclusions: This study suggests potential associations between microbiome composition and clinical parameters of advanced CRC. Microbial biomarkers may be a valuable prognostic tool in this population.

Differential expression of miRNAs related to autophagy pathway in tissue and serum samples of colorectal cancer patients.

This study was aimed to investigate the relationship of miR-17-5p, miR-30b, miR-30d, miR-216a and miR-216b associated with autophagy gene beclin 1, and beclin 1 gene with colorectal cancer (CRC). Forty-seven patients with CRC and 50 healthy individuals with no cancer history were included in this study. In the serum, tumor and non-tumoral tissue samples of the CRC patients, and in the serum samples of the healthy subjects, expression levels of miRNAs were detected by qRT-PCR. The beclin 1 gene expression levels were determined by qRT-PCR, and protein levels were determined by Western blot method in tumor and non-tumor tissue samples of the patients. The miR-17-5p and miR-30d expressions were found to be higher in tumor tissue as compared to patient non-tumor tissues, while expressions of beclin-1, miR-30b and miR-216a were found to be lower. In addition, the beclin-1 protein levels were significantly decreased in the tumor tissue as compared to those in the patient non-tumor tissues. The miR-30d expression was significantly reduced in the serum of the patients when the serum samples of CRC patients and healthy controls were compared. The beclin 1 gene may play a role as a tumor suppressor in CRC. Moreover, these miRNAs cannot be used as highly reliable biomarkers in serum for CRC diagnosis (Tab. 2, Fig. 6, Ref. 46).

MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma

Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein−protein interactions are critical in MCL1’s nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53−/− CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53−/− CRC via exploitation of this unique MCL1-based chemoresistance mechanism.

Evaluation of plasma circ_0006282 as a novel diagnostic biomarker in colorectal cancer.

BackgroundNowadays, non‐invasive and rapid detection of cancers through molecular biomarkers has received much attention. Therefore, this study investigated the non‐invasive and rapid diagnosis of colorectal cancer through one of the newest biomarkers (circular RNA).MethodsFor this purpose, we collected tumoral, adjacent normal tissue, and plasma samples from 100 colorectal cancer (CRC) patients, 25 postoperative CRC patients, 28 colitis patients, and 108 healthy donors. First Illumina high‐throughput (Hi Seq 2000) sequencing was performed to identify known and novel differentially expressed circRNAs in the cancerous and adjacent normal tissues (n = 3). We used quantitative real‐time fluorescent polymerase chain reaction (qRT‐PCR) to detect the expression level of hsa_circ_0006282 among the different samples. Moreover, inter‐ and intra‐assays were performed to evaluate the potential of hsa_circ_0006282 as being a biomarker. The receiver operating characteristic curve (ROC) was drawn to appraise its diagnostic efficacy, and the sensitivity of this circ RNA was evaluated.ResultsBased on RNA‐sequencing results circ_0006282, cirs7, circ‐0001313, circ_0055625, circ_000984, circ_0055625, circ_0001178, circ_0071589, circ‐001569 were upregulated, and circ‐ITGA7, circ‐CDYL, circITCH, circ_0026344, circ_0000038, circ_0002220, circ_0067480, circIGHV3‐20‐1, circ_104916, circ_0009361 were downregulated circRNA. The hsa_circ_0006282 was the highest upregulated differentially expressed circRNA. Expression evaluation of this circRNA on different samples showed upregulation in CRC tissues (p < 0.0001) and plasma samples of CRC patients in comparison to healthy controls (p < 0.0001), while the area under the curve (AUC) was 0.831 (95% CI: 0.779–0.883). Expression of hsa_circ_0006282 in CRC patients decreased to normal after surgery (p < 0.0001). Our results showed high specificity and sensitivity of CRC detection when hsa_circ_0006282, carcinoembryonic antigen (CEA), and carbohydrate antigen199 (CA199) are combined.ConclusionPlasma hsa_circ_0006282 can be used as a novel diagnostic and dynamic monitoring biomarker in CRC patients.

High frequency of enterotoxigenic Bacteroides fragilis and Enterococcus faecalis in the paraffin-embedded tissues of Iranian colorectal cancer patients

BackgroundThe association between specific bacteria and colorectal cancer (CRC) has been proposed. Only a few studies have, however, investigated this relationship directly in colorectal tissue with conflicting results. So, we aimed to quantitate Streptococcus gallolyticus, Fusobacterium spp, Enterococcus faecalis and enterotoxigenic Bacteroides fragilis (ETBF) in formalin-fixed and paraffin-embedded (FFPE) colorectal tissue samples of Iranian CRC patients and healthy controls.MethodsA total of 80 FFPE colorectal tissue samples of CRC patients (n = 40) and healthy controls (n = 40) were investigated for the presence and copy number of above bacterial species using quantitative PCR. Relative quantification was determined using ΔΔCT method and expressed as relative fold difference compared to reference gene.ResultsRelative abundance and copy number of E. faecalis and ETBF were significantly higher in CRC samples compared to control group. E. faecalis was more prevalent than ETBF in tumor samples. Frequency of ETBF and E. faecalis in late stages (III/IV) of cancer was significantly higher than early stages (I/II). We did not detect a significant difference in abundance of S. gallolyticus and Fusobacterium spp between two groups.ConclusionOur study revealed the higher concentration of E. faecalis and ETBF in FFPE samples of CRC patients than controls. However, additional investigations on fecal and fresh colorectal cancer tissue samples are required to substantiate this correlation.

Application of droplet digital polymerase chain reaction of plasma methylated septin 9 on detection and early monitoring of colorectal cancer

Methylated septin 9 (SEPT9) has been approved for non-invasive screening of colorectal cancer (CRC), but data on monitoring of CRC is sparse. Droplet digital polymerase chain reaction (ddPCR), with higher detection precision and simpler quantification than conventional PCR, has not been applied in SEPT9 detection. We explored the role of SEPT9 ddPCR for CRC detection and to measure serial SEPT9 levels in blood samples of CRC patients before and 3-month after surgery. SEPT9 methylated ratio, methylated abundance, and CEA levels were all higher in CRC patients than normal controls (all P < 0.05). The area under the curve (AUC) for methylated ratio and abundance to detect CRC was 0.707 and 0.710, respectively. There was an increasing trend for SEPT9 methylated abundance from proximal to distal cancers (P = 0.017). At 3-month after surgery, both methylated abundance and ratio decreased (P = 0.005 and 0.053, respectively), especially methylated abundance in stage III and distal cancer (both P < 0.01). We have developed a ddPCR platform for the quantitative detection of plasma SEPT9 in CRC patients. SEPT9 methylated abundance had an early post-operative decline, which may be useful in monitoring of treatment response.

ATPase Activity of the Subcellular Fractions of Colorectal Cancer Samples under the Action of Nicotinic Acid Adenine Dinucleotide Phosphate.

In tumor cells with defects in apoptosis, autophagy allows prolonged survival. Autophagy leads to an accumulation of damaged mitochondria by autophagosomes. An acidic environment is maintained in compartments of cells, such as autophagosomes, late endosomes, and lysosomes; these organelles belong to the “acid store” of the cells. Nicotinic acid adenine dinucleotide phosphate (NAADP) may affect the release of Ca2+ from these organelles and affect the activity of Ca2+ ATPases and other ion transport proteins. Recently, a growing amount of evidence has shown that the variations in the expression of calcium channels or pumps are associated with the occurrence, disease-presentation, and the prognosis of colorectal cancer. We hypothesized that activity of ATPases in cancer tissue is higher because of intensive energy metabolism of tumor cells. The aim of our study was to ascertain the effect of NAADP on ATPase activity on tissue samples of colorectal cancer patients’ and healthy individuals. We tested the effect of NAADP on the activity of Na+/K+ ATPase; Ca2+ ATPase of endoplasmic reticulum (EPR) and plasma membrane (PM) and basal ATPase activity. Patients’ colon mucus cancer samples were obtained during endoscopy from cancer and healthy areas (control) of colorectal mucosa of the same patients. Results. The mean activity of Na+/K+ pump in samples of colorectal cancer patients (n = 5) was 4.66 ± 1.20 μmol Pi/mg of protein per hour, while in control samples from healthy tissues of the same patient (n = 5) this value was 3.88 ± 2.03 μmol Pi/mg of protein per hour. The activity of Ca2+ ATPase PM in control samples was 6.42 ± 0.63 μmol Pi/mg of protein per hour and in cancer −8.50 ± 1.40 μmol Pi/mg of protein per hour (n = 5 pts). The mean activity of Ca2+ ATPase of EPR in control samples was 7.59 ± 1.21 μmol Pi/mg versus 7.76 ± 0.24 μmol Pi/mg in cancer (n = 5 pts). Basal ATPase activity was 3.19 ± 0.87 in control samples versus 4.79 ± 1.86 μmol Pi/mg in cancer (n = 5 pts). In cancer samples, NAADP reduced the activity of Na+/K+ ATPase by 9-times (p < 0.01) and the activity of Ca2+ ATPase EPR about 2-times (p < 0.05). NAADP caused a tendency to decrease the activity of Ca2+ ATPase of PM, but increased basal ATPase activity by 2-fold vs. the mean of this index in cancer samples without the addition of NAADP. In control samples NAADP caused only a tendency to decrease the activities of Na+/K+ ATPase and Ca2+ ATPase EPR, but statistically decreased the activity of Ca2+ ATPase of PM (p < 0.05). In addition, NAADP caused a strong increase in basal ATPase activity in control samples (p < 0.01). Conclusions: We found that the activity of Na+/K+ pump, Ca2+ ATPase of PM and basal ATPase activity in cancer tissues had a strong tendency to be higher than in the controls. NAADP caused a decrease in the activities of Na+/K+ ATPase and Ca2+ ATPase EPR in cancer samples and increased basal ATPase activity. In control samples, NAADP decreased Ca2+ ATPase of PM and increased basal ATPase activity. These data confirmed different roles of NAADP-sensitive “acidic store” (autophagosomes, late endosomes, and lysosomes) in control and cancer tissue, which hypothetically may be connected with autophagy role in cancer development. The effect of NAADP on decreasing the activity of Na+/K+ pump in cancer samples was the most pronounced, both numerically and statistically. Our data shows promising possibilities for the modulation of ion-transport through the membrane of cancer cells by influence on the “acidic store” (autophagosomes, late endosomes and lysosomes) as a new approach to the treatment of colorectal cancer.