Colorectal Cancer Tumor Samples Research Articles

Abstract 2439: Potential target genes for early stage detection in Hispanic colorectal cancer

Abstract Background: Colorectal Cancer (CRC) has become the second leading cause of death related to cancer worldwide and the third most common malignancy. CRC tumors are classified into stages to describe the cancer; early (stages I and II) and advanced (stages III and IV). Despite the available screening methods and the scientific efforts in the development of a universal biomarker panel for CRC early detection, there still exists a gap in a reliable non-invasive screening method for the Hispanic community. We aimed to elucidate the most important pathways along with the differential gene expression in early and advanced CRC stages in a Hispanic population. Methods: We examined restrospectively 7 tissue samples from a Puerto Rican Hispanic population recruited throughout the Puerto Rico Familial Colorectal Cancer Registry (PURIFICAR) IRB #A2210207. We evaluated transcriptomic changes in advance tumors (n=4) vs. early tumors (n=3) with their respective normal mucosal tissue as negative control. Extracted RNA was sequenced using the Illumina NextSeq (2 × 75bp). The higher-quality reads were aligned, annotated, and quantified to the human genome (hg38) using STAR and RSEM softwares. Gene expression analysis was performed in R statistical software (packages tximport and DESeq2). We compared significantly expressed genes (adjusted p-value <0.05) between study groups (Tumor Advance [TA] vs. Mucosal Advance [MA] and Tumor Early [TE] vs. Mucosal Early [ME]) using Venny, and we performed enrichment analyses and protein-protein interaction networks using Metascape and STRING database, respectively. Results: There were 2284 differentially expressed genes (TA vs. MA) and 984 (TE vs. ME), in which 1736 were unique to advance stages, 436 were unique to early stages and 548 were shared in both tumor stages. Significantly impacted biological processes and pathways include: granulocyte chemotaxis, regulation of inflammatory response, positive regulation of apoptotic process, tissue morphogenesis, G alpha signaling events, interleukin-10 signaling, signaling by interleukins. Additionally, physical and functional interaction networks showed 11 major clusters (≥ 5 genes), which contained several gene hubs including LYN (proto-oncogene), IL-1β, IL-1α, TNF, CCL3, CCL4, CXCR1 and CXCR2 (cytokines/chemokines) among others genes previously reported as CRC biomarkers (DKK1, AQP8, AQP9, CTNNB1, and GNAQ). Discussion/Conclusions: Differential analysis of early CRC tumor samples shows a deregulation of genes related to the inflammatory response which plays an important role in CRC. Modulation of these genes suggests significant potential targets for early detection of CRC in a Hispanic population. Future studies will be focus on the evaluation of selected target genes as a non-invasive early CRC screening method. Citation Format: Elba V. Caraballo, Camille Zenón-Meléndez, Josué Pérez-Santiago, Marcia Cruz-Correa. Potential target genes for early stage detection in Hispanic colorectal cancer [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 2439.

Circular RNA as a source of neoantigens for cancer vaccines

BackgroundThe effectiveness of somatic neoantigen-based immunotherapy is often hindered by the limited number of mutations in tumors with low to moderate mutation burden. Focusing on microsatellite-stable colorectal cancer (CRC), this...

The integrative multi-omics approach identifies the novel competing endogenous RNA (ceRNA) network in colorectal cancer

Circular RNAs (circRNA) are known to function as competing endogenous RNA (ceRNA) in various cancers by regulating microRNAs (miRNA). However, in colorectal cancer (CRC), the precise pathological role of circ000240/miRNA/mRNA remains indeterminate. The expression level of hsa_circ_000240 was evaluated using qRT-PCR in matching pairs of CRC tumor and adjacent normal tissue samples in our laboratory. Then, to determine whether hsa_circ_000240 acted as a ceRNA in CRC, the linked miRNAs and gene targets were retrieved. Topological analysis of candidate genes using a network approach identified the most critical hub genes and subnetworks related to CRC disease. Microarray and bulk RNA sequencing analyses were utilized to comprehensively evaluate the expression levels of both miRNA and mRNA in CRC. Single-cell RNA-seq analysis was also used to evaluate the significant overall survival (OS) genes at the cellular level. ATAC-seq data provided insights into candidate genes' accessible chromatin regions. The research uncovered a considerable upregulation of hsa_circ_000240 in CRC tissues. Three miRNAs interacted with the target circRNA. One thousand six hundred eighty intersected genes regulated by three miRNAs were further identified, and the relevant functionality of identified neighbor genes highlighted their relevance to cancer. The topological analysis of the constructed network has identified 33 hub genes with notably high expression in CRC. Among these genes, eight, including CHEK1, CDC6, FANCI, GINS2, MAD2L1, ORC1, RACGAP1, and SMC4, have demonstrated a significant impact on overall survival. The utilization of single-cell RNA sequencing unequivocally corroborated the augmented expression levels of CDC6 and ORC1 in individuals with CRC, alongside their noteworthy connection with the infiltration of immune cells. ATAC-seq analyses revealed altered accessibility regions in Chr2, 4, and 12 for CDC6 and ORC1 high-expression. Correlation analysis of CDC6 and ORC1 further highlighted the association of candidate gene expression with exhaustion markers such as CTLA4, CD247, TIGIT, and CD244. The candidate genes exhibit a positive correlation with chromatin remodeling and histone acetylation. These epigenetic modifications play a significant role in influencing the cancer progression following expression of CDC6 and ORC1 in CRC. Additionally, results showed that the methylation rate of the promoter region of CDC6 was elevated in CRC disease, confirming the functional importance of CDC6 and their interaction with hsa_circ_000240 and associated ceRNA in CRC. In conclusion, this study highlights hsa_circ_000240's role as a ceRNA in CRC. It opens new avenues for further dissection of CDC6, ORC1, and underlying novel epigenetics and immunotherapy targets for CRC therapy.

Cell-free circulating tumor RNAs in plasma as the potential prognostic biomarkers in colorectal cancer.

Cell free RNA (cfRNA) contains transcript fragments from multiple cell types, making it useful for cancer detection in clinical settings. However, the pathophysiological origins of cfRNAs in plasma from colorectal cancer (CRC) patients remain unclear. To identify the tissue-specific contributions of cfRNAs transcriptomic profile, we used a published single-cell transcriptomics profile to deconvolute cell type abundance among paired plasma samples from CRC patients who underwent tumor-ablative surgery. We further validated the differentially expressed cfRNAs in 5 pairs of CRC tumor samples and adjacent tissue samples as well as 3 additional CRC tumor samples using RNA-sequencing. The transcriptomic component from intestinal secretory cells was significantly decreased in the in-house post-surgical cfRNA. The HPGD, PACS1, and TDP2 expression was consistent across cfRNA and tissue samples. Using the Cancer Genome Atlas (TCGA) CRC datasets, we were able to classify the patients into two groups with significantly different survival outcomes. The three-gene signature holds promise in applying minimal residual disease (MRD) testing, which involves profiling remnants of cancer cells after or during treatment. Biomarkers identified in the present study need to be validated in a larger cohort of samples in order to ascertain their possible use in early diagnosis of CRC.

Circ_RNF13 Regulates the Stemness and Chemosensitivity of Colorectal Cancer by Transcriptional Regulation of DDX27 Mediated by TRIM24 Stabilization.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers with high incidence and poor prognosis worldwide. Circ_RNF13 is upregulated in CRC; however, the biological roles and downstream signaling of circ_RNF13 remain undefined. The characterization of circ_RNF13 was determined by Sanger sequencing, qRT-PCR, subcellular fractionation assay, and RNA FISH. Western blot analysis and qRT-PCR were employed to detect the expression of the key molecules and stemness markers in CRC tumor samples and cells. The stem-like activities of CRC cells were assessed by sphere formation assay, flow cytometry, and immunofluorescence (IF). Cell viability was monitored by CCK-8 assay. The chemosensitivity of CRC cells was assessed by colony formation and cell apoptosis assays. Bioinformatics analysis, RIP assay, RNA pull-down assay, and FISH/IF staining were used to detect the association between circ_RNF13 and TRIM24. The transcriptional regulation of DDX27 was investigated by ChIP assay, and the post-translational regulation of TRIM24 was detected by Co-IP. The in vitro findings were verified in a xenograft model. circ_RNF13 and DDX27 were elevated in CRC tumor samples and cells. Knockdown of circ_RNF13 or DDX27 inhibited stemness and increased chemosensitivity in CRC cells. Mechanistically, circ_RNF13 regulated DDX27 expression via TRIM24-mediated transcriptional regulation, and circ_RNF13 stabilized TRIM24 via suppressing FBXW7-mediated TRIM24 degradation. In vivo studies revealed that the knockdown of circ_RNF13 impaired stemness and enhanced the chemosensitivity of CRC in the xenograft model. circ_RNF13 regulated the stemness and chemosensitivity of CRC by transcriptional regulation of DDX27 mediated by TRIM24 stabilization.

Up regulation of long non-coding RNAs BACE1 and down regulation of LINC-PINT are associated with CRC clinicopathological characteristics

BackgroundLong non-coding RNAs (LncRNAs) are known to have regulatory consequences for aberrant gene expression in cancers. The aim of this study was to evaluate the expression levels of long non-encoding RNAs, BACE1 (β-secretase1) and LINC-PINT (Long Intergenic Non-Protein Coding RNA, P53 Induced Transcript), in colorectal cancer (CRC) with clinicopathological parameters.Methods and resultsBioinformatics analysis defining effectual signalling pathways Wnt. A total of 130 tissue samples (50 fresh CRC tissues with parallel adjacent normal tissues (ADJ) accompanied with 30 normal healthy control tissue samples) were collected from the Iranian population. mRNA expression analysis was performed via Real Time Q-PCR. Statistical analysis for comparing CRC expression levels with ADJ and normal healthy tissues were carried out using Kruskal–Wallis tests. The Receiver Operating Characteristic (ROC) curve was plotted for each LNC, separately. We discovered that PINT and BACE1 expression levels were decreased and increased respectively in CRC tumour samples compared with ADJ normal and healthy tissues. Clinicopathological parameter assessment revealed a significant relationship between PINT expression, tumour location, staging and distant metastasis (p < 0.009, p < 0.014, p < 0.008, respectively). Also, BACE1 over expression was significantly associated with tumour site (p < 0.009), metastasis (p < 0.017) and histological differentiation (p < 0.028) and staging (p < 0.017). Furthermore, ROC curve plotting showed LINC-PINT LNC-BACE1 may distinguish between early and late-stage of CRC, highlighting the value of both BACE1 and PINT as CRC progression biomarkers.ConclusionWe investigated two LNCRNAs (PINT and BACE1) as potential CRC prognostic biomarkers, which are imperative for early and effective medical intervention in CRC. Expression levels of PINT and BACE1 in CRC tissue samples may serve to identify metastasis earlier, increasing patient survival rates and expediating clinical treatment options.

Dysregulated expression of suppressor loop of circadian rhythm genes in colorectal cancer pathogenesis.

Colorectal cancer (CRC) is a heterogeneous disease and activation of WNT and TGFβ mediated oncogenic pathways is frequently observed in this pathology. However, to date, limited reports have been published addressing the association of circadian clock with CRC pathogenesis and stratification. The current study aims at assessing the expression of important circadian markers, PER2, PER3 and NR1D1, in independent CRC cohorts and their associations with CRC-related pathways. Gene expression analysis was performed using available GEO (GSE39582) and TCGA datasets. Quantitative real time polymerase chain reaction was used to quantify the expression levels of PER2, PER3 and NRID1 in FFPE (formalin fixed paraffin embedded) CRC tissue samples. Furthermore, enrichment of circadian markers in WNT and TGFβ pathways-activated tumors was assessed. Statistically significant downregulation of PER3 was found in tumor versus control samples in GEO (P<0.0001) and TCGA colon and rectal adenocarcinoma datasets (P<0.05). Analysis of GEO dataset revealed a statistically significant upregulation of PER2 (P<0.01), and NR1D1 in colon adenocarcinoma, which was confirmed by qRT-PCR in CRC tumor samples versus controls in FFPE validation cohort. Higher expression of NR1D1 was associated with poor prognosis in colon adenocarcinoma. Contrastingly, PER3 was significantly downregulated in tumors (P<0.001) compared to controls and was associated with high-grade CRC tumors versus low-grade tumors. Tumors with WNT pathway activation had significantly low PER3 and slightly upregulated PER2 (<0.0001) expression. Interestingly, differential expression of PER3 and NR1D1 was significantly correlated with TGFβ1-expressing tumors (P<0.0001). Moreover, MYC- amplified tumors exhibited decreased PER3 levels. Thus, low PER3 expression in CRC and poor survival of patients with NR1D1-high tumors reveal that genes in the suppressor loop of circadian rhythm are dysregulated in CRC, hence pointing out to the importance of dissecting the circadian pathway in cancer.

The Long Non-Coding RNA FAM222A-AS1 Negatively Modulates MiR-Let-7f to Promote Colorectal Cancer Progression.

Accumulating evidence indicates that lncRNAs are potential biomarkers and key regulators of tumor development and progression. The present study aimed to screen abnormal expression lncRNAs and investigate the mechanisms underlying the function in the progression of colorectal cancer (CRC). Potential CRC prognosis-associated dysregulated lncRNAs were screened and identified using bioinformatics analysis. Loss/gain-of-function experiments were performed to detect the biological roles of FAM222A-AS1 in CRC cell phenotypes in vitro and in vivo. The potential microRNAs that interact with FAM222A-AS1 were identified using online tools and were verified using qRT-PCR and luciferase reporter assay. The expression of FAM222A-AS1 is significantly upregulated in CRC tumor samples and cell lines. CRC patients with elevated FAM222A-AS1 expression in the tumor samples had unfavorable overall survival and disease-free survival. Silencing FAM222A-AS1 expression significantly inhibited CRC cell proliferation, migration, and invasion both in vitro and in vivo. Furthermore, FAM222A-AS1 was mainly distributed in the cytoplasm. It may directly bound to miR-let-7f and inhibit its expression and upregulate MYH9. In summary, FAM222A-AS1, as a novel oncogene in CRC, may promote the CRC progression by inhibiting miR-let-7f/MYH9 axis. The FAM222A-AS1/miR-let-7f/MYH9 signaling pathway may be a novel valuable target for inhibiting CRC.

CircCDC6 restrains tumor growth and glycolysis energy metabolism in colorectal cancer via regulating miR-3187-3p and downstream PRKAA2.

The aberrant downregulation of circCDC6 in colorectal cancer (CRC) was previously identified by circRNA microarray analysis. However, the detailed role of circCDC6 in CRC is still lacking. We thus investigated the function of circCDC6 in CRC. The expression of circCDC6, miR-3187-3p and PRKAA2 mRNA was checked by real-time quantitative PCR (RT-qPCR). Cell growth was evaluated by MTT, EdU and colony formation assays. Cell apoptosis was evaluated by flow cytometry. Glycolysis was evaluated by glycolysis stress test and lactic acid level. The expression of PRKAA2, HK2 and LDHA proteins was checked by western blotting. The potential binding between miR-3187-3p and circCDC6 or PRKAA2 was confirmed by dual-luciferase reporter assay, RIP assay and pull-down assay. Xenograft model was established in nude mice. CircCDC6 showed poor expression in CRC tumor samples and cells. CircCDC6 ectopic expression repressed CRC cell proliferation, survival and glycolysis energy metabolism. MiR-3187-3p was targeted by circCDC6, and miR-3187-3p depletion also repressed CRC cell growth and glycolysis. PRKAA2 was a downstream target of circCDC6/miR-3187-3p pathway, and circCDC6 upregulated PRKAA2 expression via targeting miR-3187-3p. PRKAA2 knockdown rescued the functional effects of circCDC6 ectopic expression. CircCDC6 overexpression in vivo impeded tumor development in animal models. CircCDC6, acting as a tumor inhibitor, repressed tumor growth and glycolysis metabolism in CRC via targeting the miR-3187-3p/PRKAA2 axis, which partly clarified the role of circCDC6 in CRC.

Expression of TMED3 is independently associated with colorectal cancer prognosis.

Colorectal cancer (CRC) is the third most common cancer type and one of the deadliest cancers worldwide. Transmembrane p24 trafficking protein 3 (TMED3) has previously been indicated to suppress CRC metastasis, but its clinical significance has remained undetermined. In the present study, the expression of TMED3 was indicated to be elevated at the mRNA and protein levels in CRC tumor samples relative to that in para-cancerous healthy tissue samples (P<0.05). Furthermore, Kaplan-Meier survival analysis revealed a significant negative association between elevated TMED3 protein levels and overall survival of patients with CRC (P<0.001, log-rank test). Multivariate Cox regression analysis additionally determined that elevated TMED3 expression in primary CRC tumors was an independent predictor of poor prognosis (P<0.05). These results revealed that elevated TMED3 expression in CRC was associated with patient survival outcomes, suggesting that TMED3 may be a potential prognostic biomarker for this cancer type.

Identifying Differentially Expressed tRNA-Derived Small Fragments as a Biomarker for the Progression and Metastasis of Colorectal Cancer.

Objectives The epithelial-to-mesenchymal transition (EMT) is one key step for the invasion and metastasis of colorectal cancer (CRC). Up until now, the underlying mechanism of EMT in CRC is still unpromising. Thus, it is essential to have a better understanding of its carcinogenesis. The transfer RNA-derived small fragments (tsRNAs) are a new group of small noncoding RNAs (sncRNAs), including tRNA-derived stress-induced RNAs (tiRNAs) and tRNA-derived fragments (tRFs), which have been observed to play an important role in many cancers. However, the relationship between tRFs and EMT in CRC is still unknown. Herein, we aimed to investigate the involvement of tRFs in EMT and its contribution to CRC development. Methods We identified the differentially expressed tsRNAs in colorectal cancer cell line HT29 treated with TGF-β compared with control cells by using high-throughput sequencing and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). QRT-PCR was conducted to validate the differentially expressed fragments in 68 CRC tumor samples (22 women and 46 men) and adjacent nontumor samples. The association of the expression of tRFs with CRC metastasis and clinical stage was analyzed. Meanwhile, the correlation between tRF expression and overall survival (OS) was also analyzed. TargetScan and miRanda and multiple bioinformatic approaches were used to predict the possible target genes of tsRNAs and analyze possible functions of the tRFs. Results A series of differentially expressed tsRNAs were identified in TGF-β-treated HT29 cells compared with control cells. tRF-phe-GAA-031 and tRF-VAL-TCA-002 were found to be significantly upregulated in CRC tissues compared to adjacent nontumor tissues. They were significantly correlated with distant metastasis and clinical stage. We compared the differences between tumor samples and nontumor tissues from the ROC curves. The area under the ROC curve (AUC) was up to 0.7554 (95% confidence interval: 0.6739 to 0.8369, p < 0.0001) for tRF-Phe-GAA-031 and up to 0.7313 (95% confidence interval: 0.6474 to 0.8151, p < 0.0001) for tRF-VAL-TCA-002. For OS analysis, higher tRF-phe-GAA-031 and tRF-VAL-TCA-002 expressions were associated with shorter survival for CRC patients. Conclusion A series of differentially expressed tsRNAs are identified in the EMT process of CRC. And tRF-phe-GAA-031 and tRF-VAL-TCA-002 are higher expressed in CRC tissues, and they might play an important role in the metastasis of CRC. Meanwhile, they may be potential biomarkers and intervention targets in the clinical treatment of CRC.

Genome-wide circular RNA (circRNA) and mRNA profiling identify a circMET-miR-410-3p regulatory motif for cell growth in colorectal cancer

Circular RNA (circRNA) is a non-coding RNA molecule that lacks polyadenylated tails and is highly stable, abundant, and conserved in human cells. CircRNAs can serve as a competing endogenous RNA (ceRNA) to sponge microRNAs (miRNA) and block their effects on target mRNA expression. CircRNAs also have possible relevance to cancer and therefore may be considered as ideal biomarkers for monitoring cancer progression. Of the about 300,000 predicted human circRNAs, only a few have validated biological functions related to cancer. To better understand the ceRNA role of circRNAs in colorectal cancer (CRC), we performed genome-wide circRNA-based RNA-sequencing (RNA-Seq) on nine CRC tumor samples and their paired histologically normal adjacent tissue samples. By profiling the mRNA expression in the same patients, we further explored the expression correlation between circRNAs and mRNAs generated from the same parental gene. Focusing on the concordant differential expression between circRNAs and mRNAs, we substantially reduced the regulatory noise. In total, we identified 694 circRNA-mRNA pairs that were consistently up or downregulated between tumor and normal tissues. These 694 circRNA-mRNA pairs are from 182 protein-coding genes associated with hormone responses and chemotaxis. Of these 182 genes, 43 are downstream targets of three highly conserved miRNAs (miR-410-3p, miR-135a, and miR-30a). Interestingly, these 43 genes are highly mutated in another cohort from eight independent CRC studies, which have significant effects on patient survival time. Focusing on miR-410-3p and its target oncogene MET, we experimentally validated the ceRNA regulatory motif of circMET. Notably, circMET is substantially upregulated in CRC cell lines and could promote cell proliferation and growth. By confirming the regulatory relationship between miR-410-3p and circMET, we propose a new mechanism for the observed sustained activation of MET in CRC. In conclusion, our work identifies a novel regulatory role of circMET and provides a potential diagnostic biomarker for CRC.

Changes in the Expression of SGO1 and SGO1-AS1 Genes in Colorectal Tumor Tissues, Compared to Healthy Tissues

Background and Aim: The protein encoded by the SGO1 gene is a member of the shugoshin family of proteins and protects the centromere during mitosis. lncRNAs are non-coding RNA with 200 nucleotides lengths, i.e., involved in regulating gene expression. The current study aimed to evaluate the expression of SGO1 and SGO1-AS1 in different stages of disease progression; we also compared their expression pattern in tumor tissues with healthy tissues in colorectal cancer patients. Methods &amp; Materials: In total, 40 tissue samples of patients with colorectal cancer were reported according to the examination and criteria with the approval of a pathologist. Besides, 40 normal tissues were sampled from a completely healthy part of the intestine of the same patients. After RNA extraction and cDNA synthesis, the Real-time RT-PCR technique was used to evaluate the expression of the desired genes in the study groups. ROC curve analysis was also used to determine the ability of each selected gene to diagnose the disease. Ethical Considerations: This study was approved by the Ethics Committee of Shahrekord Azad University (Code: IR.IAU.SHKREC.1398.020). Results The obtained data suggested that SGO1 significantly decreased in the colorectal cancer tumor samples (P&lt;0.001) and SGO1-AS1 LncRNA significantly increased expression, compared to adjacent healthy tissues. Additionally, in the age group of below 60 years, compared to the age group of over 60 years, SGO1 expression increased and SGO1-AS1 expression decreased. Based on the AUC obtained from the ROC diagram, it was found that the SGO1 gene with AUC=0.8041 and SGO1-AS1 with AUC=0.6364 could significantly distinguish a healthy population from patients with colorectal cancer. Conclusion: According to the collected results, SGO1 -AS1 and SGO1 were significantly reduced and increased in tumor tissue, respectively; however, only the SGO1 gene was introduced as a good marker for diagnosing colorectal cancer.

Alternative Splicing Events in Tumor Immune Infiltration in Colorectal Cancer

Despite extensive research, the exact mechanisms involved in colorectal cancer (CRC) etiology and pathogenesis remain unclear. This study aimed to examine the correlation between tumor-associated alternative splicing (AS) events and tumor immune infiltration (TII) in CRC. We analyzed transcriptome profiling and clinical CRC data from The Cancer Genome Atlas (TCGA) database and lists of AS-related and immune-related signatures from the SpliceSeq and Innate databases, respectively to develop and validate a risk model of differential AS events and subsequently a TII risk model. We then conducted a two-factor survival analysis to study the association between TII and AS risk and evaluated the associations between immune signatures and six types of immune cells based on the TIMER database. Subsequently, we studied the distribution of six types of TII cells in high- and low-risk groups for seven AS events and in total. We obtained the profiles of AS events/genes for 484 patients, which included 473 CRC tumor samples and 41 corresponding normal samples, and detected 22581 AS events in 8122 genes. Exon Skip (ES) (8446) and Mutually Exclusive Exons (ME) (74) exhibited the most and fewest AS events, respectively. We then classified the 433 patients with CRC into low-risk (n = 217) and high-risk (n = 216) groups based on the median risk score in different AS events. Compared with patients with low-risk scores (mortality = 11.8%), patients with high-risk scores were associated with poor overall survival (mortality = 27.6%). The risk score, cancer stage, and pathological stage (T, M, and N) were closely correlated with prognosis in patients with CRC (P < 0.001). We identified 6479 differentially expressed genes from the transcriptome profiles of CRC and intersected 468 differential immune-related signatures. High-AS-risk and high-TII-risk predicted a poor prognosis in CRC. Different AS types were associated with different TII risk characteristics. Alternate Acceptor site (AA) and Alternate Promoter (AP) events directly affected the concentration of CD4T cells, and the level of CD8T cells was closely correlated with Alternate Terminator (AT) and Exon Skip (ES) events. Thus, the concentration of CD4T and CD8T cells in the CRC immune microenvironment was not specifically modulated by AS. However, B cell, dendritic cell, macrophage, and neutrophilic cell levels were strongly correlated with AS events. These results indicate adverse associations between AS event risk levels and immune cell infiltration density. Taken together, our findings show a clear association between tumor-associated alternative splicing and immune cell infiltration events and patient outcome and could form a basis for the identification of novel markers and therapeutic targets for CRC and other cancers in the future.

Impact of preoperative antibiotics and other variables on integrated microbiome-host transcriptomic data generated from colorectal cancer resections.

BACKGROUNDIntegrative multi-omic approaches have been increasingly applied to discovery and functional studies of complex human diseases. Short-term preoperative antibiotics have been adopted to reduce site infections in colorectal cancer (CRC) resections. We hypothesize that the antibiotics will impact analysis of multi-omic datasets generated from resection samples to investigate biological CRC risk factors.AIMTo assess the impact of preoperative antibiotics and other variables on integrated microbiome and human transcriptomic data generated from archived CRC resection samples.METHODSGenomic DNA (gDNA) and RNA were extracted from prospectively collected 51 pairs of frozen sporadic CRC tumor and adjacent non-tumor mucosal samples from 50 CRC patients archived at a single medical center from 2010-2020. The 16S rRNA gene sequencing (V3V4 region, paired end, 300 bp) and confirmatory quantitative polymerase chain reaction (qPCR) assays were conducted on gDNA. RNA sequencing (IPE, 125 bp) was performed on parallel tumor and non-tumor RNA samples with RNA Integrity Numbers scores ≥ 6.RESULTSPERMANOVA detected significant effects of tumor vs nontumor histology (P = 0.002) and antibiotics (P = 0.001) on microbial β-diversity, but CRC tumor location (left vs right), diabetes mellitus vs not diabetic and Black/African Ancestry (AA) vs not Black/AA, did not reach significance. Linear mixed models detected significant tumor vs nontumor histology*antibiotics interaction terms for 14 genus level taxa. QPCR confirmed increased Fusobacterium abundance in tumor vs nontumor groups, and detected significantly reduced bacterial load in the (+)antibiotics group. Principal coordinate analysis of the transcriptomic data showed a clear separation between tumor and nontumor samples. Differentially expressed genes obtained from separate analyses of tumor and nontumor samples, are presented for the antibiotics, CRC location, diabetes and Black/AA race groups.CONCLUSIONRecent adoption of additional preoperative antibiotics as standard of care, has a measurable impact on -omics analysis of resected specimens. This study still confirmed increased Fusobacterium nucleatum in tumor.

Combination of GC-MS based metabolomics analysis with mouse xenograft models reveals a panel of dysregulated circulating metabolites and potential therapeutic targets for colorectal cancer.

BackgroundColorectal cancer (CRC) is a common gastrointestinal tumor with subtle, often undetectable early symptoms, which means that upon diagnosis, patients often present in the middle or late stages of disease. Therefore, the need for an effective biomarker for the early diagnosis and development of novel therapeutic targets is urgent to prolong patient survival time and reduce mortality.MethodsTwenty mice were randomly divided into patient-derived xenograft (PDX) model (transplantation of fresh CRC tumor samples) and control groups (10 mice in each group). All the animals were euthanized using isoflurane at the end of the experiment. Gas chromatography-mass spectrometry (GC-MS)-based metabolomic profiling was performed to investigate the differential metabolites in the serum, and publicly available gene expression data (GSE106582) were analyzed to determine dysregulated metabolic pathways. Joint pathway analysis was used to identify potential metabolic targets. Immunohistochemistry analysis was performed to confirm the presence of the identified targets at the protein level.ResultsA total of 96 differential circulating metabolites were identified, which were predominantly involved in amino acid metabolism. In particular, the serum levels of amino acids such as phenylalanine and aspartic acid were significantly downregulated in the PDX group, suggesting an increased consumption of amino acids in CRC. Moreover, both the mRNA and protein levels of the amino acid transporters, SLC7A5 and SLC1A5, were found to be upregulated in CRC.ConclusionsBy combining GC-MS-based metabolomics profiling with a PDX model of CRC our study successfully identified potential diagnostic circulating metabolites. Dysregulated amino acid metabolism was found to be a significant feature of CRC. The amino acid transporters, SLC7A5 and SLC1A5, were identified as potential metabolic therapeutic targets. This study furthers the understanding of the metabolic processes involved in CRC.

Comparison of immune activity profiles of microsatellite instable colon tumors to microsatellite stable tumors having high ColoType CMS1-score.

26 Background: Microsatellite instable (MSI) metastatic colorectal cancer (CRC) tumors have been found to be responsive to immune checkpoint inhibitors. Most MSI tumors are in consensus molecular subtype CMS1. Here, in a search for microsatellite stable (MSS) samples that may respond to checkpoint inhibitors, we used the ColoType CMS1-score to identify microsatellite stable (MSS) CRC tumors that have immune activity profiles similar to MSI tumors. Methods: This study was performed in Affymetrix cohort (n = 1,888) of primary CRC tumor samples assayed with hgu133plus2 Affymetrix microarrays. Degrees of infiltration of populations of immune cells were assessed with MCPcounter. Gene Set Enrichment Analysis (GSEA) using the Hallmark gene sets was used to compare biological features between subsets of samples. An 18-gene genomic score (PembroSig) of Ayers, et al, was used to predict response to Pembrolizumab. ColoType 40-gene signature identifies the CMS of a CRC tumor using values of continuous scores, one score for each of CMS1-4. CMS1+ denotes the samples with CMS1-score sufficiently high to predict it is in CMS1. A genomic score was used to predict MSI status for all samples in Affymetrix cohort; MSI predictions using said score had 97% agreement with clinically determined MSI status for 749 Affymetrix cohort samples. Results: The MSI score predicted 240 MSI samples in Affymetrix cohort, and 211 MSS samples in CMS1+ (CMS1+/MSS). The union of MSI and CMS1+/MSS compared to other samples exhibited elevated infiltration of cytotoxic lymphocytes (p &lt; 0.0001) and CD8 T-cells (p &lt; 0.0001), activity of interferon-gamma (p = 0.006) and interferon-alpha signaling (p = 0.012), inflammatory response (p = 0.012), and elevated PembroSig score (p &lt; 0.0001). CMS1+/MSS compared to MSI showed no significance difference in any of these measures. None of the immune regulatory genes PD-1, PD-L1, PD-L2, CTLA4, LAG3, and IDO1 were differentially expressed between CMS1+/MSS and MSI. Of note, the only Hallmark gene sets enriched in MSI compared to CMS1+/MSS were DNA repair (p = 0.002) and MYC targets V1 (p = 0.009) and V2 (p = 0.013). We further analyzed the biological differences between MSI samples in CMS1+ (CMS1+/MSI, n = 190) and MSI samples not in CMS1+ (CMS1-/MSI, n = 50). We found that CMS1+/MSI compared to CMS1-/MSI exhibited elevated cytotoxic lymphocyte infiltration (p &lt; 0.0001), interferon-gamma activity (p = 0.027), and PembroSig score (p &lt; 0.0001), highlighting the relationship between immune activity and CMS1-score. Conclusions: ColoType CMS1-score identified MSS samples in Affymetrix cohort (11%) with similar immune activity as MSI samples (13%) and equivalently high values of a genomic score predictive of Pembrolizumab response.

Overexpression of Nicotinamide N-methyltransferase mainly covers stroma of colorectal cancer and correlates with unfavorable survival by its product 1-MNA.

Background: Accumulating evidence indicates that Nicotinamide N-methyltransferase (NNMT) is abnormally expressed in tumor tissues of several cancers including colorectal cancer (CRC) and associated with cancer progression. However, the distribution characteristics and the clinical value of each part of NNMT expression in CRC are still not fully understood. The purpose of this study is to determine the distribution of NNMT expression and its association with survival in CRC.Methods: By using the cancer genome atlas (TCGA) and clinical proteomic tumor analysis consortium (CPTAC), we firstly analyzed the difference of gene and protein levels of NNMT between CRC and normal colorectal tissue. Then, NNMT protein expressions were detected in 18 intraepithelial neoplastic samples and 177 CRC tumor samples through immunohistochemistry in our study cohort. Furthermore, the relationship between NNMT expression and clinicopathological characteristics, overall survival (OS) and disease-free survival (DFS) of CRC patients were analyzed by Pearson χ2 test and log-rank test, respectively, in public datasets and our study cohort. Lastly, the function of NNMT and its product 1-methyl-nicotinamide (1-MNA) on migration and invasion in colorectal cancer cells was analyzed by wound healing assay and transwell assay.Results: We determined that higher NNMT expression in CRC tissues than normal tissues in both gene and protein level in TCGA and CPTAC datasets (all p < 0.05). In addition, the strong relationships of NNMT expression with stromal cells were found in the TCGA cohort. Fortunately, our cohort could validate that the expression of NNMT in tumor stroma cell was significantly higher than that in tumor cell (p < 0.0001), and both of them were significantly higher than that in adjacent normal tissue (ANT) (p < 0.0001 and p < 0.0001, respectively). Furthermore, the positive NNMT expression in tumor cell and stromal cell were associated with series of unfavorable clinical characteristics, including advanced TNM stage, lymph node metastasis, distant metastasis (all p < 0.05). Also, higher NNMT was associated with unfavorable survival both in our study and public datasets, including TCGA and two Gene Expression Omnibus (GEO) datasets (GSE33113 and GSE17538). Moreover, the functional experiments showed that stromal cells with high NNMT expression can secret 1-MAN to promote migration and invasion of CRC cells in vitro.Conclusions: In CRC, NNMT is overexpressed in tumor cells and stroma cells, and then mainly expressed in tumor stroma cells. Overexpression of NNMT in tumor cell and stroma cell both are associated with metastasis and unfavorable survival. Besides, stromal cells with high NNMT expression secrets 1-MAN to promote migration and invasion of CRC cells. Therefore, NNMT may be a potential prognostic indicator in CRC patients.

Clinicopathological Significance of Tumor Stem Cell Markers ALDH1 and CD133 in Colorectal Carcinoma

Background & Objective:Colorectal cancer (CRC) is the third most common cancer worldwide with a high mortality rate. The main causes of death in patients are recurrence and metastasis which are mainly attributed to the small subpopulation of cells within tumors called cancer stem cells (CSCs). This study aimed to evaluate the correlation between the expression of ALDH1 and CD133 as CSC associated markers and clinicopathological characteristics in CRC.Methods:In this cross-sectional study, a total of 483 CRC tumor samples were immunohistochemically stained for detection of CD133 and ALDH1 markers. Correlations of marker expression with clinicopathological factors were also evaluated.Results:There was a significant correlation between the luminal intensity of CD133 and neural invasion (P=0.05) and between the cytoplasmic intensity of CD133 and metastasis (P=0.05). In terms of H-score, a positive significant relation was observed between cytoplasmic expression of CD133 and lymph node (P=0.02), neural (P=0.04) and vascular invasion (P=0.02). The ALDH1 cytoplasmic expression showed a significant correlation with tumor size (P=0.001).Conclusion:Our findings showed that increased expression of CD133 and ALDH1 is associated with tumor progression and worse outcomes in CRC patients. These markers can be good candidates for localized targeting of CSCs using antibodies. Future researches need to be improved approaches for early detection of CRC, and treatment monitoring for CRC and other cancers.

Identification of Two Subgroups of FOLFOX Resistance Patterns and Prediction of FOLFOX Response in Colorectal Cancer Patients

To dissect gene expression subgroups of FOLFOX resistance colorectal cancer(CRC) and predict FOLFOX response, gene expression data of 83 stage IV CRC tumor samples (FOLFOX responder n = 42, non-responder n = 41) are used to develop a novel iterative supervised learning method IML. IML identified two mutually exclusive subgroups of CRC patients that rely on different DNA damage repair proteins and resist FOLFOX. IML was validated in two validation sets (HR = 2.6, p Value = 0.02; HR = 2.36, p value = 0.02). A subgroup of mesenchymal subtype patients benefit from FOLFOX. Different subgroups of FOLFOX nonresponders may need to be treated differently.