Colorectal Cancer Tissue Samples Research Articles

Correlation of tumor-associated macrophage density and proportion of M2 subtypes with the pathological stage of colorectal cancer.

Colorectal cancer (CRC) is a prevalent global malignancy with complex prognostic factors. Tumor-associated macrophages (TAMs) have shown paradoxical associations with CRC survival, particularly concerning the M2 subset. We aimed to establish a simplified protocol for quantifying M2-like TAMs and explore their correlation with clinicopathological factors. A cross-sectional study included histopathological assessment of paraffin-embedded tissue blocks obtained from 43 CRC patients. Using CD68 and CD163 immunohistochemistry, we quantified TAMs in tumor stroma and front, focusing on M2 proportion. Demographic, histopathological, and clinical parameters were collected. TAM density was significantly higher at the tumor front, with the M2 proportion three times greater in both zones. The tumor front had a higher M2 proportion, which correlated significantly with advanced tumor stage (P = 0.04), pathological nodal involvement (P = 0.04), and lymphovascular invasion (LVI, P = 0.01). However, no significant association was found between the M2 proportion in the tumor stroma and clinicopathological factors. Our study introduces a simplified protocol for quantifying M2-like TAMs in CRC tissue samples. We demonstrated a significant correlation between an increased M2 proportion at the tumor front and advanced tumor stage, nodal involvement, and LVI. This suggests that M2-like TAMs might serve as potential indicators of disease progression in CRC, warranting further investigation and potential clinical application.

MYC and NCAPG2 as molecular targets of colorectal cancer and gastric cancer in nursing.

Colorectal cancer is a common malignant tumor in intestinal tract, the early symptoms are not obvious. Gastric cancer is a malignant tumor originating from the gastric mucosal epithelium. However, the role of MYC and non-SMC condensin II complex subunit G2 (NCAPG2) in colorectal cancer and gastric cancer remains unclear. The colorectal cancer datasets GSE49355 and gastric cancer datasets GSE19826 were downloaded from gene expression omnibus database. Differentially expressed genes (DEGs) were screened and weighted gene co-expression network analysis (WGCNA) was performed. Functional enrichment analysis, gene set enrichment analysis (GSEA) and immune infiltration analysis was performed. Construction and analysis of protein-protein interactions (PPI) network. Survival analysis and comparative toxicogenomics database (CTD) were performed. A heat map of gene expression was drawn. A total of 751 DEGs were obtained. According to the gene ontology (GO) analysis, in Biological process (BP) analysis, they are mainly enriched in cell differentiation, cartilage development, and skeletal development. In cellular component (CC) analysis, they are mainly enriched in the cytoskeleton of muscle cells and actin filaments. In molecular function (MF) analysis, they are mainly concentrated in Rho GTPase binding, DNA binding, and fibronectin binding. In Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, they are mainly enriched in the MAPK signaling pathway, apoptosis, and cancer pathways. The soft threshold power for WGCNA analysis was set to 9, resulting in the generation of 40 modules. Ultimately, 2 core genes (MYC and NCAPG2) were identified. The heatmap of core gene expression showed high expression of MYC and NCAPG2 in colorectal cancer tissue samples and low expression in normal tissue samples, while they were core molecules in gastric cancer. Survival analysis indicated that MYC and NCAPG2 were risk factors, showing an upregulation trend with increasing risk scores. CTD analysis revealed associations of MYC and NCAPG2 with colorectal cancer, gastric cancer, inflammation, and immune system diseases. MYC and NCAPG2 are highly expressed in colorectal cancer. The higher the expression of MYC and NCAPG2, the worse the prognosis. MYC and NCAPG2 are core molecules in gastric cancer.

Modified Unit-Mediated Strand Displacement Reactions for Direct Detection of Single Nucleotide Variants in Active Double-Stranded DNA.

Accurate identification of single nucleotide variants (SNVs) in key driver genes holds a significant value for disease diagnosis and treatment. Fluorescent probes exhibit tremendous potential in specific, high-resolution, and rapid detection of SNVs. However, additional steps are required in most post-PCR assays to convert double-stranded DNA (dsDNA) products into single-stranded DNA (ssDNA), enabling them to possess hybridization activity to trigger subsequent reactions. This process not only prolongs the complexity of the experiment but also introduces the risk of losing target information. In this study, we proposed two strategies for enriching active double-stranded DNA, involving PCR based on obstructive groups and cleavable units. Building upon this, we explored the impact of modified units on the strand displacement reaction (SDR) and assessed their discriminatory efficacy for mutations. The results showed that detection of low variant allele frequencies (VAF) as low as 0.1% can be achieved. The proposed strategy allowed orthogonal identification of 45 clinical colorectal cancer tissue samples with 100% specificity, and the results were generally consistent with sequencing results. Compared to existing methods for enriching active targets, our approach offers a more diverse set of enrichment strategies, characterized by the advantage of being simple and fast and preserving original information to the maximum extent. The objective of this study is to offer an effective solution for the swift and facile acquisition of active double-stranded DNA. We anticipate that our work will facilitate the practical applications of SDR based on dsDNA.

C6orf15 promotes liver metastasis via WNT/β-catenin signalling in colorectal cancer

BackgroundColon cancer ranks third among global tumours and second in cancer-related mortality, prompting an urgent need to explore new therapeutic targets. C6orf15 is a novel gene that has been reported only in Sjogren’s syndrome and systemic lupus erythematosus patients. We found a close correlation between increased C6orf15 expression and the occurrence of colon cancer. The aim of this study was to explore the potential of C6orf15 as a therapeutic target for colorectal cancer.MethodRNA-seq differential expression analysis of the TCGA database was performed using the R package ‘limma.’ The correlation between target genes and survival as well as tumour analysis was analysed using GEPIA. Western blot and PCR were used to assess C6orf15 expression in colorectal cancer tissue samples. Immunofluorescence and immunohistochemistry were used to assess C6orf15 subcellular localization and tissue expression. The role of C6orf15 in liver metastasis progression was investigated via a mouse spleen infection liver metastasis model. The association of C6orf15 with signalling pathways was assessed using the GSEA-Hallmark database. Immunohistochemistry (IHC), qPCR and western blotting were performed to assess the expression of related mRNAs or proteins. Biological characteristics were evaluated through cell migration assays, MTT assays, and Seahorse XF96 analysis to monitor fatty acid metabolism.ResultsC6orf15 was significantly associated with liver metastasis and survival in CRC patients as determined by the bioinformatic analysis and further verified by immunohistochemistry (IHC), qPCR and western blot results. The upregulation of C6orf15 expression in CRC cells can promote the nuclear translocation of β-catenin and cause an increase in downstream transcription. This leads to changes in the epithelial–mesenchymal transition (EMT) and alterations in fatty acid metabolism, which together promote liver metastasis of CRC.ConclusionOur study identified C6orf15 as a marker of liver metastasis in CRC. C6orf15 can activate the WNT/β-catenin signalling pathway to promote EMT and fatty acid metabolism in CRC.

Isthmin-1 promotes growth and progression of colorectal cancer through the interaction with EGFR and YBX-1

While previous studies have indicated the involvement of Isthmin 1 (ISM1), a secreted protein, in cancer development, the precise mechanisms have remained elusive. In this study, we unveiled that ISM1 is significantly overexpressed in both the blood and tissue samples of colorectal cancer (CRC) patients, correlating with their poor prognosis. Functional experiments demonstrated that enforced ISM1 expression significantly enhances CRC proliferation, migration, invasion and tumor growth. Notably, our investigation reveals an interaction of ISM1 with epidermal growth factor receptor (EGFR), a member of the receptor tyrosine kinase (RTK) family of CRC cells. The binding of ISM1 triggered EGFR activation and initiate downstream signaling pathways. Meanwhile, intracellular ISM1 interacted with Y-box binding protein 1 (YBX1), enhancing its transcriptional regulation on EGFR. Furthermore, our research uncovered the regulation of ISM1 expression by the hypoxia-inducible transcription factor HIF-1α in CRC cells. Mechanistically, we identified HIF-1α as a direct regulator of ISM1, binding to a hypoxia response element on its promoter. This novel mechanism illuminated potential therapeutic targets, offering insights into restraining HIF-1α/ISM1/EGFR-driven CRC progression and metastasis.

ZNF26-Associated Genes as Prognostic Signatures in Colorectal Cancer with Broad Therapeutic Implications.

The identification of biomarkers correlated with colorectal cancer (CRC) prognosis holds substantial importance from both clinical and scientific perspectives. Zinc finger protein 26 (ZNF26) has not been previously investigated or documented in solid tumors; thus, further research is necessary to ascertain its prognostic value in CRC. Gene expression profiles and clinicopathological data were acquired from The Cancer Genome Atlas (TCGA) database. Subsequently, expression correlation was assessed utilizing the TCGA CRC cohort. The prognostic value of ZNF26 was evaluated through Kaplan-Meier (KM) and ROC curve analyses. Following this, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to perform enrichment analysis between high- and low-ZNF26 expression groups. The association between immune cells, immune checkpoint genes, and ZNF26 expression levels was examined. Lastly, the research findings were further validated using CRC tissue samples. The results revealed that, in comparison to healthy controls, CRC significantly reduced ZNF26 expression. Elevated ZNF26 expression was associated with poorer overall survival in CRC patients. Additionally, high ZNF26 expression exhibited an inverse relationship with the immunological score and immune checkpoint gene expression in CRC patients. The findings from the TCGA data analysis were corroborated by the PCR results obtained from CRC tissue samples. ZNF26 is markedly upregulated in colorectal cancer tissues, potentially serving as a biomarker for CRC.

Extended Enrichment for Ultrasensitive Detection of Low-Frequency Mutations by Long Blocker Displacement Amplification.

Detecting low-frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele-specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14-44 nt enrichment regions which is 2-fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA-qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA-qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.

Blood miRNAs miR-549a, miR-552, and miR-592 serve as potential disease-specific panels to diagnose colorectal cancer

IntroductionmiRNAs originating from colorectal cancer (CRC) tissue receive significant focus in the early diagnosis of CRC due to their stability in body fluids. However, if these miRNAs originated from alternative organs, their prognostic value will diminish. Thus, in this study, we aim to identify disease-specific miRNAs for colorectal cancer (CRC) by employing bioinformatics and experimental methodologies. MethodTo identify CRC-specific miRNAs, we retrieved miRNA profiles of CRC and normal tissues from the Cancer Genome Atlas (TCGA) database. Subsequently, computational strategies were utilized to select potential candidate miRNAs. Following this, the expression levels of the potent miRNAs were assessed through RT-qPCR in both CRC tissue and serum samples from patients (N = 46), as well as healthy individuals (N = 46). Additionally, the associations between clinicopathological characteristics, survival outcomes, and diagnostic accuracy were evaluated. ResultsA total of 8893 RNA-seq expression data were acquired from TCGA, comprising 8250 data from 19 distinct cancer types and 643 corresponding healthy samples. Based on the computational methodology, miR-549a, miR-552, and miR-592 were identified as the principal expressed miRNAs in colorectal cancer (CRC). Within these miRNAs, miR-552 displayed a substantial association with tumors at the N and T stages. miR-549a and miR-592 were observed to be linked exclusively to the invasion of tumor depth and tumor stage (TNM), respectively. The receiver operating characteristic (ROC) analysis conducted on the miRNA expression in serum samples revealed that all miRNAs exhibited an area under the ROC curve (AUC) of up to 0.86, thereby indicating their high diagnostic accuracy. ConclusionConsidering the strong associations of these three identified miRNAs with CRC, they can collectively serve as a panel for specific discrimination of CRC from other types of cancer within the body. Although this study focused solely on CRC, this approach can potentially be applied to other cancer types as well.

Abstract 2444: Plasma levels of matrix metalloproteinases 8 are significantly increased in patients with colorectal cancer (CRC) and mRNA levels are increased in 71% of tumors vs normal tissue

Abstract Introduction: Matrix metalloproteinases 8 (MMP-8) is a member of the MMP protease family mainly involved in degrading extracellular matrix. A variety of cells express MMP-8 and during pathological inflammation neutrophils release IL-1, IL-8, TNF-α, and GM-CSF which triggers MMP-8 secretion. MMP-8 aids tumors in evading immune detection during the development of CRC and also regulates the role of endothelial cell in angiogenesis. Increased MMP-8 tumor expression and serum levels has been noted in hepatocellular cancer and melanoma patients and are associated with adverse outcomes. There is limited data regarding blood levels of MMP-8 in CRC patients. This study compared preoperative (PreOp) plasma MMP-8 levels in patients with CRC and benign colonic pathology (BCP) undergoing bowel resection. Method: Patients with CRC or BCP who had elective colorectal resection who were enrolled in an IRB approved tissue and data bank for whom preop plasma samples were available were eligible. Data analyzed included demographic, clinical, operative, and final pathology reports. Blood specimens were quickly processed and plasma stored at -80°C. MMP-8 levels in Preop plasma samples were measured via ELISA, in duplicate, and reported as median +95% CI (ng/ml). MMP8 expression was examined in paired CRC and normal mucosal tissue samples from a subset of the CRC patients (n=20) via QRT-PCR. Immunohistochemistry (IHC) was conducted on formalin-preserved tissue sections (tumor/normal pairs) following standard protocols. The concept of MMP8 as CRC diagnostic marker was evaluated by assessing the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC). The analysis was done with the Mann-Whitney test (significance, p<0.05). Results: 96 CRC patients (74% colon and 26% rectal cancer) and 96 BCP patients (32% adenoma, 64% diverticulitis, 4% other) met entry criteria. The CRC stage distribution was: Stage 1, 26%; Stage 2, 26% Stage 3, 40%; and stage 4, 8%. Median plasma MMP-8 levels were higher in CRC (8.21, CI: 6.92, 10.81) compared to BCP patients (5.59, CI: 4.68, 7.30; p<0.001). A non-significant increase in median MMP-8 levels were noted in STG IV vs STG I and STG II and STG III was found (38-89%). IHC validated the expression of MMP-8 in CRC tumors relative to normal colon. Increased expression of MMP-8 was seen in 71% of the tested CRC samples (12/17). The AUC value for the ROC curve was 0.653 (sensitivity 35%, specificity 0.95%). Conclusion: The median MMP-8 level in CRC patients was found to be 47% higher compared to BCP patients and 89% higher in stage IV vs stage II patients (p=0.06). The tumor is the most likely the cause of elevated plasma levels. Increased tumor expression of MMP-8 mRNA was noted in 71% of tumors. The low sensitivity of the ROC curve suggests MMP8 alone has no value as diagnostic factor. Larger studies are justified. Citation Format: Chandana S. K. Herath Mudiyanselage, Anuj R. Sharma, Neil Mitra, Aashutosh Sah, Xiaohong Yan, Pablo Palacios, Vesna Cekic, Richard L. Whelan. Plasma levels of matrix metalloproteinases 8 are significantly increased in patients with colorectal cancer (CRC) and mRNA levels are increased in 71% of tumors vs normal tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2444.

Abstract 4954: Improving microbial detection in cancer tissue samples with computational host depletion using the pangenome

Abstract Sensitive removal of host genetic information (i.e., host depletion) is a key first step towards accurate classification of potential microbial DNA sequences in low-biomass samples, such as human tumors. Inadequate filtration of human reads prior to microbial classification can cause false positives, biasing biological conclusions. Previous computational host depletion pipelines predominantly employed the human reference genome GRCh38 (hg38), which lacks genetic diversity of the human population and excludes substantial portions of the Y-chromosome. To address these deficiencies, we constructed a pipeline to maximally remove human reads that incorporates hg38, the first complete genome build T2T-CHM13v2.0 (T2T), and the 47 diploid genomes from the Human Pangenome Reference Consortium (HPRC) [1]. Reads passing maximal host depletion and quality control are considered sufficiently cleaned for downstream microbial analyses. We first evaluated the specificity of our pipeline by simulating Illumina whole genome sequencing (WGS) data from 10 heldout HPRC humans, whose genomes were excluded from the host depletion pipeline. We found significant improvement in the median number of human reads remaining across successive host depletion methods (hg38, 0.05369%; hg38+T2T, 0.00535%; hg38+T2T+HPRC, 0.00093%). For sensitivity, we applied our pipeline to simulated data from 962 microbes in the FDA-ARGOS dataset and found minimal removal of true microbial reads (median 99.9390% reads retained). As a real world example, we evaluated our pipeline on 583 metastatic colorectal cancer tissue (WGS) samples from the Hartwig Medical Foundation (HMF) database (median 106X coverage)[2] and found successive decreases in median number of reads retained (hg38, 185,135; hg38+T2T, 92,951; hg38+T2T+HPRC, 10,314). Using Woltka for taxonomic classification against database RefSeq210, we assessed biological sex similarity across the HMF samples using Robust Aitchison PCA (RPCA). Notably, microbial data following hg38-only host depletion showed significant microbial differences between male and female-labeled samples (p=0.00001), which was biologically unexpected. However, our updated pipeline (hg38+T2T+HPRC) removed the false sex-based separation in the microbial data (p=0.1837). Thus, utilization of diverse sets of complete human genomes for computational host depletion can mitigate artifactual bias, enabling more sensitive and specific host-microbe conclusions in cancer research. [1] Liao, et al. Nature 617, 312-324 (2023). [2] Priestley, et al. Nature 575, 210-216 (2019). Citation Format: Caitlin Guccione, Lucas Patel, Daniel McDonald, Cameron Martino, Antonio González, Gregory D. Sepich-Poore, Rob Knight, Kit Curtius. Improving microbial detection in cancer tissue samples with computational host depletion using the pangenome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4954.

Extended Enrichment for Ultrasensitive Detection of Low‐Frequency Mutations by Long Blocker Displacement Amplification

AbstractDetecting low‐frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele‐specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14–44 nt enrichment regions which is 2‐fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA‐qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA‐qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.

Decreased interleukin-17RA expression is associated with good prognosis in patients with colorectal cancer and inhibits tumor growth and vascularity in mice.

Interleukin-17 (IL-17) is a pro-inflammatory cytokine that plays a vital role in the promotion of tumorigenesis in various cancers, including colorectal cancer (CRC). Based on current evidence, IL-17 binds to interleukin-17 receptor A (IL-17RA); however, the role of IL-17RA has not been elucidated in previous studies on CRC. In this study, we explored the role of IL-17RA in human CRC tissues and the progression of CRC in humans and mice. The expressions of IL-17RA and epithelial-mesenchymal transition (EMT)-related genes were examined in CRC cells and tissue samples by quantitative real-time polymerase chain reaction. The role of IL-17RA in pathogenesis and prognosis was evaluated using a Chi-squared test, Kaplan-Meier analysis, univariate, and multivariate Cox regression analysis in 133 CRC patients. A tumor-bearing mice model was executed to evaluate the role of IL-17RA in tumor growth, vascularity and population of infiltrating immune cells. IL-17RA expression was found to be significantly higher in CRC tissues than in adjacent normal tissues. The expression of IL-17RA in Stage IV patients was significantly higher than that in Stages I and II patients. Patients with high IL-17RA expression exhibited significantly worse overall and CRC-specific survival than those with low IL-17RA expression. Functional assessment suggested that the knockdown of IL-17RA expression distinctly suppressed cellular proliferation, migration, invasion, and EMT-related gene expression. In a tumor-bearing mouse model, decreased IL-17RA expression significantly repressed tumor growth and vascularity and reduced the population of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Reduced IL-17RA expression also suppressed cellular proliferation, migration, and invasion, and the expression of EMT genes. Knockdown of IL-17RA inhibited tumor growth and vascularity and decreased the population of Tregs and MDSCs in mouse tumors. Overall, IL-17RA expression was identified to be independently associated with the prognosis of patients with CRC.

Comprehensive RNA-sequencing analysis of colorectal cancer in a Korean cohort

Considering the recent increase in the number of colorectal cancer (CRC) cases in South Korea, we aimed to clarify the molecular characteristics of CRC unique to the Korean population. To gain insights into the complexities of CRC and promote the exchange of critical data, RNA-sequencing (RNA-Seq) analysis was performed to reveal the molecular mechanisms that drive the development and progression of CRC; this analysis is critical for developing effective treatment strategies. We performed RNA-Seq analysis of CRC and adjacent normal tissue samples from 214 Korean participants (comprising a total of 381 including 169 normal and 212 tumor samples) to investigate differential gene expression between the groups. We identified 19,575 genes expressed in CRC and normal tissues, with 3,830 differentially expressed genes (DEGs) between the groups. Functional annotation analysis revealed that the upregulated DEGs were significantly enriched in pathways related to the cell cycle, DNA replication, and IL-17, whereas the downregulated DEGs were enriched in metabolic pathways. We also analyzed the relationship between clinical information and subtypes using the Consensus Molecular Subtype (CMS) classification. Furthermore, we compared groups clustered within our dataset to CMS groups and performed additional analysis of the methylation data between DEGs and CMS groups to provide comprehensive biological insights from various perspectives. Our study provides valuable insights into the molecular mechanisms underlying CRC in Korean patients and serves as a platform for identifying potential target genes for this disease. The raw data and processed results have been deposited in a public repository for further analysis and exploration. Data availability statementThe RNA-Seq data have been deposited in the Korean Nucleotide Archive under the accession number KAP230608 (https://www.kobic.re.kr/kona/search_bioproject?bioproject_id=KAP230608#none). Clinical data, preprocessed gene quantification (RSEM), normalized gene expression data (log2CPM), and downstream analysis results have been deposited in Zenodo (https://doi.org/10.5281/zenodo.8333650). Further information and requests for experimental resources should be directed to and will be fulfilled by the corresponding authors, Kil-yong Lee (cyboryee@hanmail.net), Jin Ok Yang (joy@kribb.re.kr), and Young-Joon Kim (yjkim@yonsei.ac.kr).

Association between Heavy Metals and Trace Elements in Cancerous and Non-cancerous Tissues with the Risk of Colorectal Cancer Progression in Northwest China.

Alterations in heavy metals and trace element levels may be associated with various cancers. However, the role of this interaction in colorectal cancer (CRC) progression is unclear. In recent years, Principal Component Analysis (PCA) and Bayesian Kernel Machine Regression (BKMR) models have provided new ideas for analyzing the effects of metal mixtures on CRC progression. Herein, we assessed the differences in the levels of arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), nickel (Ni), selenium (Se), and zinc (Zn) in tumors and adjacent healthy tissues, to investigate the relationship between heavy metals/trace elements and CRC progression. Surgical samples of CRC and noncancerous tissues were collected, and trace metal levels were analyzed using inductively coupled plasma mass spectrometry (ICP-MS). Logistic regression, PCA, and BKMR models were used to investigate the relationship between heavy metals and trace elements and the degree of tumor differentiation and lymph node metastasis in CRC. Cancer tissues showed lower As, Cd, Co, and Cr concentrations, and higher Se concentrations than healthy tissues (P < 0.05). In addition, CRC patients with poorly differentiated tumors and/or positive lymph node metastases had lower levels of Cd, Zn, Cu, and Se (P < 0.05). Logistic regression showed that single metal concentration was negatively correlated with CRC progression. PCA and BKMR models also showed that the metal mixture concentration was negatively correlated with CRC progression, with Cd contributing the most. Overall, changes in heavy metal and trace element levels may be related to the development of CRC; however, further mechanistic studies are required.

Activation of RhoA/ROCK2 signaling by hypoxia-inducible factor 1α in promoting tumor growth and metastasis in human colon cancer.

Recent evidence strongly suggests the profound role of the tumor microenvironment in cancer development and progression. A hypoxic microenvironment is widely acknowledged to be a typical feature of solid tumors, and altered hypoxia-inducible factor 1α (HIF-1α) expression has been associated with the formation and the progression of many solid tumors; however, the underlying mechanism of this relationship remains obscure. Clinical colorectal cancer tissue samples were collected to detect the differential expression of HIF-1α, Ras homolog family member A (RhoA), and Rho-associated, coiled-coil containing protein kinase 2 (ROCK2). With hypoxic stress, small interfering RNA (siRNA) targeting HIF-1α, lentivirus transfection of RhoA was used to study the mechanism of HIF-1α and RhoA/ROCK2 signaling pathways in the growth and metastasis of colon cancer. According to Cell Counting Kit 8, wound-healing, and Transwell assays, HIF-1α expression activated the RhoA/ROCK2 pathway within colon cancer cell lines, accelerating their growth and expansion. In cells transfected with LV-RhoA, inactivating the RhoA/ROCK2 pathway with the specific inhibitor Y-27632 decreased tumor growth and metastasis under hypoxic conditions, while activating the RhoA/ROCK2 pathway restored these biological properties. The Western blot results showed that the expression levels of pMYPT1, cyclin D1, and MMP2 in the siRNA + LV-RhoA group were also significantly increased compared with those in the siRNA group. For the first time, this study demonstrated that HIF-1α can promote the growth and metastasis of colon cancer via directly affecting RhoA/ROCK2 signaling and thus represents a novel therapeutic target for colon cancer.

The Role of High Mobility Group Box B-1 in the Prognosis of Colorectal Cancer Based on the Changes in the Intestinal Mucosal Barrier.

Background: To investigate the expression of high mobility group box B-1 (HMGB-1) in patients with colorectal cancer (CRC) and its association with clinicopathological features and prognosis in colorectal carcinoma by combining bioinformatics and clinical data analysis, and to clarify the role of HMGB-1. To examine whether HMGB-1 expression is related to the damage of the intestinal mucosal barrier, and then explore the potential HMGB-1-dependent mechanisms affecting the progression of CRC. Methods: CRC datasets of GSE12945, GSE17536, and GSE17537 from the public gene chip database were screened and downloaded. Clinical information and CRC tissue samples from patients with stage I-III CRC from the hospital were collected. Serum samples of patients were applied by enzyme-linked immunosorbent assay on HMGB-1, and were divided into high and low HMGB-1 expression, which was examined by 16S rDNA sequencing. Immunohistochemistry was performed to examine the relationship between the expression of HMGB-1 and tight junction protein, occludin, tumor necrosis factor-α, and interferon-γ. Results: Based on the Cutoff value of 10.24 ng/mL, the CRC patients were divided into high and low expression groups. In the HMGB-1H patient group, the TNM staging, overall survival, disease-free survival, recurrence, and metastasis were inferior to the HMGB-1L group. The results of 16S rDNA sequencing demonstrated that the Providencia genus was found to be enriched in the HMGB-1L group. Immunohistochemical results showed that HMGB-1 expression was negatively correlated with the expression of ZO-1 and occludin (R = 0.035, R = 0.003, P < .05), but was positively correlated with the expression of TNF-α and IFN-γ (R = 0.016, R = 0.001, P < .05). Conclusion: The survival of CRC patients with positive HMGB-1 expression was significantly shortened, which may be related to the decrease of Rovitensis content, the decreased expression of ZO-1 and occludin, and the increased levels of TNF-α and IFN-γ, which in turn damage the intestinal mucosal barrier, leading to the development of CRC.

Mối liên quan giữa mức độ sao chép gen KIAA1199 với một số đặc điểm lâm sàng, giải phẫu bệnh của người bệnh ung thư đại trực tràng tại Bệnh viện đa khoa Đức Giang năm 2022-2023

Background: Determine the level of KIAA1199 gene copy in FFPE tissue samples of colorectal cancer (CR) patients and evaluate of the relationship between KIAA1199 gene copy level and some clinical and pathological symptoms of CRC patients at Duc Giang General Hospital in year 2022-2023. Methods: A cross-sectional descriptive study on 40 FFPE samples and 40 corresponding medical records of CRC patients diagnosted and treated at Duc Giang General Hospital in year 2022-2023. Result: 26/40 FFPE tissue samples (65.0%) were processed to meet the requirements for RNA extraction quality to evaluate the KIAA1199 gene copy level. Furthermore, 20/26 samples (76.9%) had a higher level of KIAA1199 gene copy in tumor tissue than in normal tissue. There was a statistically significant association between increased KIAA1199 gene copy levels and level of tumor differentiations and types in CR patients (p&lt;0.05); among them, CR patients with neoplasms have increased 11.3 times the KIAA1199 gen copy level higher than CR patients with other types of tumors (p&lt;0.05). However, it was not possible to evaluate the statistically significant relationship between the KIAA1199 gene copy level and some histopathological characteristics such as tumor location, tumor number, histopathological type, level of serosal invasion, lymph node metastasis, and stages classified according to TNM (p&gt;0.05). Conclusion: In this study, the KIAA1199 gene copy levels in tumor tissue increased more strongly than in normal tissue and a statistically significant relationship between the increased KIAA1199 gene copy levels and the tumor differentiations and types in CR patients (p&lt;0.05).

Identification of potential key genes for colorectal cancer based on bioinformatics analysis.

This study aimed to explore key genes as potential biomarkers for colorectal cancer (CRC) diagnosis and prognosis in order to improve their clinical utility. To identify and screen candidate genes involved in CRC carcinogenesis and disease progression, we downloaded the microarray datasets GSE143939, GSE196006, and GSE200427 from the GEO database and applied the GEO2R tool to obtain differentially expressed genes (DEGs) between colorectal cancer tissue samples and normal tissue samples. Differentially expressed genes were analyzed using the DAVID online database for gene ontology and Kyoto encyclopedia of genes and genomes pathway enrichment analyses. Protein-protein interaction network was constructed and related module analysis was performed using STRING and Cytoscape. In total, 241 DEGs were identified, including 127 downregulated and 114 upregulated genes. DEGs enriched functions and pathways included cellular response to chemical stimulus, extracellular region, carbonate dehydratase activity, cell division, spindle, and cell division. The abundant functions and pathways of DEGs included cellular response to chemical stimulus, extracellular region, carbonate dehydratase activity, cell division, spindle, cell adhesion molecule binding, Aldosterone-regulated sodium reabsorption, and Cell cycle-related processes. Fifteen key genes were identified, and bioprocess analyses showed that these genes were mainly enriched in cell cycle, cell division, mitotic spindle, and tubulin binding processes. It was found that CDK1, CEP55, MKI67, and TOP2A may be involved in CRC cancer invasion and recurrence. The pivotal genes identified in this study contribute to our understanding of the molecular and pathogenic mechanisms of CRC carcinogenesis and progression, and provide possible biomarkers for the diagnosis and treatment of CRC.

BICDL1 Predicts Poor Prognosis and is Correlated with Methylation and Immune Infiltration in Colorectal Cancer.

Bicaudal-D (BICD) Family Like Cargo Adaptor 1 (BICDL1) is an essential component of the molecular mechanism during neuronal development. However, BICDL1 has not been reported in cancer. Using bioinformatics analysis, we systematically evaluated the potential role of BICDL1 in CRC. Colorectal cancer (CRC) and normal tissue samples were retrieved from the Gene Expression Omnibus (GEO), Genotype-Tissue Expression (GTEx), and Cancer Genome Atlas (TCGA) databases. Kaplan-Meier (K-M) analysis, nomogram, COX analysis, and receiver operating characteristic (ROC) curves were used to evaluate the prognostic power. Correlation analysis was also conducted to explore the correlation between mRNA expression and the methylation level of BICDL1 using cBioPortal, and the correlation between immune infiltration and BICDL1. RT-qPCR and Western blot assays were performed to analyze BICDL1 expression level between human colorectal cancer cell lines and normal colonic epithelial cells. BICDL1 had a higher expression in CRC tissues than in normal tissues (p < 0.001) in TCGA and GES 74602 datasets. Kaplan-Meier survival analysis revealed that patients with high BICDL1 expression had lower overall survival (OS) (1.53, 95% confidence interval: 1.07-2.17, p=0.019). The ROC curves demonstrated that BICDL1 has high specificity and efficiency in diagnosis (AUC=0.919, CI: 0.895-0.943). The expression level of BICDL1 was significantly correlated with the infiltrating levels of Treg (R=0.146, p <0.001), TFH (R=0.080, p=0.043), NK CD56bright cells (R=0.149, p <0.001), aDC (R=0.095, p=0.016), and T helper cell infiltration (R=-0.084, p=0.034). The correlation between BICDL1 expression and methylation levels was negative (R2=0.134, p <0.001), and CRC patients had lower methylation levels than normal people (p=0.036). BICDL1 mRNA and its protein expression levels in CRC cell lines (SW620) was markedly increased compared with that of normal colonic epithelial cells (NCM460) (p < 0.001). BICDL1 may be a potential biomarker for evaluating immune infiltration levels and prognosis of CRC.

Integration of single-cell transcriptomics and epigenetic analysis reveals enhancer-controlled TIMP1 as a regulator of ferroptosis in colorectal cancer.

Ferroptosis is an iron-dependent non-apoptotic programmed cell death. However, the regulatory mechanism of ferroptosis in colorectal cancer (CRC) is still unclear. The aim of this study was to investigate the role and mechanism of enhancer-controlled genes in ferroptosis in CRC. Dimensionality reduction and differentially expressed genes (DEGs) identification were conducted using Seurat algorithm based on single-cell RNA sequencing (scRNA-seq) data from the GSE200997 dataset. Ferroptosis-related pathway enrichment analysis was performed using the FerrDb V2 database. Enhancers were identified using HOMER algorithm based on H3K27ac ChIP-seq data from the GSE166254 dataset. Kaplan-Meier Plotter online tool was used to analyze prognosis and gene expression correlation. Transcription factors were predicted using the transcription factor affinity prediction web tool. The binding of enhancer to transcription factor and H3K27ac enrichment were detected by ChIP-qPCR. RSL3 was used to induce ferroptosis in CRC cells. Gene transcription was detected by qRT-PCR. Cell proliferation was detected by CCK8 assay. Nine cell clusters including T cells, natural killer cells, macrophages, mast cells, epithelial cells, fibroblasts, goblet cells, B cells and dendritic cells were identified in CRC and normal colonic tissue samples. Compared to normal colonic tissue-derived epithelial cells, 1075 DEGs were screened in CRC tissue-derived epithelial cells. Ferroptosis-related pathway enrichment suggested that DEGs were associated with the regulation of ferroptosis. DPEP1, ETV4, CEBPG, TIMP1, DUOX2 and LCN2 were identified as the significantly upregulated genes enriched in the "ferroptosis regulator" term, and their H3K27ac signals were significantly higher in CRC tissues than in normal colonic tissues. Of these, only the expression of TIMP1 predicted a poor prognosis of CRC patients. Transcription factor SPI1 drove TIMP1 transcription by binding to its enhancer. Overexpression of TIMP1 significantly promoted the resistance to ferroptosis induced by RSL3 in CRC cells, which was partially restored by SPI1 knockdown. Transcription of TIMP1 was driven by transcription factor SPI1 in combination with its enhancer, consequently promoting CRC cells against ferroptosis. The SPI1/TIMP1 axis confers ferroptosis resistance in CRC, and thus has the potential to be the molecular targets for CRC treatment.