Tumor Tissues Of Colorectal Cancer Patients Research Articles

Research on the Effect and Mechanism of Selenium on Colorectal Cancer Through TRIM32.

The intake of selenium (Se) in the human body is negatively correlated with the risk of colorectal cancer (CRC), but its mechanism in the occurrence and development of CRC is not clear. This study aimed to evaluate the therapeutic effect of Se on CRC, and explore the anti-tumor effect of Se supplementation on CRC and its molecular mechanism. In this study, we utilized colony formation assay, cell scratch test, Transwell migration, and flow cytometry to assess cell proliferation, migration, and apoptosis. Our findings demonstrate that Se effectively suppresses the growth and proliferation of CRC cell lines HCT116 and SW480 and promoting cellular apoptosis. In vivo experiments demonstrated a significant inhibitory effect of Se on tumor growth. CRC-related datasets were extracted from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases for differential expression analysis of TRIM32 and survival analysis. We found that TRIM32 was highly expressed in tumor tissues of CRC patients and correlated with a poor prognosis. Furthermore, through RNA sequencing analysis, we identified TRIM32 as a gene that was significantly decreased after Se treatment in HCT116 cells. This finding was subsequently validated by Western blot results. Moreover, TRIM32 knockdown combined with Se treatment significantly inhibited cell growth proliferation and migration and further induced apoptosis of colorectal cancer cells. In conclusion, our findings provided evidence that Se inhibited the growth of colorectal cancer cells by down-regulating TRIM32.

Abstract 6683: The abundance of Fusobacteria nucleatum in tumor tissue is a prognostic factor in patients of right-sided colorectal cancer

Abstract Background: A significant association between the gut microbiome and colorectal carcinogenesis, as well as cancer progression. It has been reported that Fusobacteria nucleatum (F. nucleatum) is one of the most famous bacteria associated with colorectal cancer (CRC). It has been reported that the abundance of F. nucleatum is associated with colorectal cancer-specific survival (CSS), however it remains unclear for association with F. nulceatum and relapse-free survival (RFS). If F. nucleatum has a significant involvement in tumor progression of colorectal cancer, the abundance of F. nucleatum should be associated with not only CSS but also RFS in colorectal cancer. In this study, we measured the bacterial mass of F. nucleatum in tumor tissue of CRC patients in Stage II-III to explore the differences in prognosis. Methods: We included patients with CRC in stage II-III who underwent surgical treatment at Nippon Medical School Hospital between January 2017 and December 2020 except patients who received preoperative chemoradiotherapy and had multiple cancers. We measured the absolute of F. nucleatum copy number of the DNA exacted tumor tissues using droplet digital PCR. Results: 253 patients with CRC were included. 128 patients had pathological stage II and 125 patients had pathological stage III. Among 253 patients, 104 patients had right-sided CRC, 89 patients had left-sided, and 60 patients had rectal cancer. F. nucleatum was detected in 166 patients (65.6%), and the median of F. nucleatum copy number was 1.08 (range; 0.00 -2791.3). F. nucleatum copy number was more abundant in right-sided CRC than in left-sided and rectal cancer (p= 0.02, p=0.01). There was no significant difference between stage II CRC and stage III. We categorized patients into two groups based on the median cut point of F. nucleatum copy number: the F. nucleatum-high group and the F. nucleatum-low group for comparison with clinicopathological features and prognosis. Patients with high abundance of F. nucleatum exhibited T4 of tumor depth (p=0.03), right-sided CRC (p=0.01), longer tumor circumference (p <0.001), longer tumor length (p <0.001), and no lymph node metastasis (p=0.024). There was no significant difference in relapse-free survival between the F. nucleatum-high group and F. nucleatum-low group among whole patients (p=0.53). Analyzing divided tumor location, in right-sided CRC high abundance of F. nucleatum was significantly associated with shorter relapse-free survival than low abundance (p=0.02), while in left-sided and rectal cancer there was no significant difference (p=0.61). Conclusion: F. nucleatum was more abundant in right-sided CRC and associated with relapse-free survival only in right-sided CRC. F. nucleatum may make a greater impact on right-sided CRC than on left-sided and rectal cancer. Citation Format: Toshimitsu Miyasaka, Takeshi Yamada, Sho Kuriyama, Akihisa Matsuda, Kay Uehara, Seiichi Shinji, Yasuyuki Yokoyama, Goro Takahashi, Takuma Iwai, Kohki Takeda, Shintaro Kanaka, Hiroshi Yoshida. The abundance of Fusobacteria nucleatum in tumor tissue is a prognostic factor in patients of right-sided 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 6683.

The multispecies microbial cluster of Fusobacterium, Parvimonas, Bacteroides and Faecalibacterium as a precision biomarker for colorectal cancer diagnosis.

The incidence of colorectal cancer (CRC) has increased worldwide, and early diagnosis is crucial to reduce mortality rates. Therefore, new noninvasive biomarkers for CRC are required. Recent studies have revealed an imbalance in the oral and gut microbiomes of patients with CRC, as well as impaired gut vascular barrier function. In the present study, the microbiomes of saliva, crevicular fluid, feces, and non-neoplastic and tumor intestinal tissue samples of 93 CRC patients and 30 healthy individuals without digestive disorders (non-CRC) were analyzed by 16S rRNA metabarcoding procedures. The data revealed that Parvimonas, Fusobacterium, and Bacteroides fragilis were significantly over-represented in stool samples of CRC patients, whereas Faecalibacterium and Blautia were significantly over-abundant in the non-CRC group. Moreover, the tumor samples were enriched in well-known periodontal anaerobes, including Fusobacterium, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Co-occurrence patterns of these oral microorganisms were observed in the subgingival pocket and in the tumor tissues of CRC patients, where they also correlated with other gut microbes, such as Hungatella. This study provides new evidence that oral pathobionts, normally located in subgingival pockets, can migrate to the colon and probably aggregate with aerobic bacteria, forming synergistic consortia. Furthermore, we suggest that the group composed of Fusobacterium, Parvimonas, Bacteroides, and Faecalibacterium could be used to design an excellent noninvasive fecal test for the early diagnosis of CRC. The combination of these four genera would significantly improve the reliability of a discriminatory test with respect to others that use a single species as a unique CRC biomarker.

Deletion of IL-27p28 induces CD8 T cell immunity against colorectal tumorigenesis

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, characterized by molecular and clinical heterogeneity. Interleukin (IL)-27, a heterodimeric cytokine composed of p28 and EBI3 subunits, has been reported to exert potent antitumor activity in several cancer models. However, the precise role of IL-27 in the pathogenesis of CRC remains unclear. Here, we show that during the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC development, IL-27p28 levels are dramatically increased in peripheral blood and tumor tissues, and the cytokine is mainly produced by tumor-infiltrating myeloid cells. IL-27p28 deficient mice display tumor resistances in both inflammation-associated CRC model and syngeneic MC38 colon cancer model. Administration with IL-27p28 neutralizing antibody also reduces the tumor formation in AOM/DSS-treated mice. Mechanically, CD8+ T cells in IL-27p28-/- mice exhibit enhanced tumor infiltration and cytotoxicity, which can be largely attributed to activation of the Akt/mTOR signaling pathway. Furthermore, selective depletion of CD8+ T cells in IL-27p28-/- mice markedly accelerate tumor growth and almost abrogate the protective effects of IL-27p28 deficiency. Most interestingly, the expression of IL-27p28 is also upregulated in tumor tissues of CRC patients and those with high expression of IL-27p28 tend to have a poorer overall survival. Our results suggest that loss of IL-27p28 suppresses colorectal tumorigenesis by augmenting CD8+ T cell-mediated anti-tumor immunity. Targeting IL-27p28 could be developed as a novel strategy for the treatment of colorectal cancers.

Amino Acid Profiles in the Biological Fluids and Tumor Tissue of CRC Patients.

Amino acids are the building blocks of proteins and essential players in pathways such as the citric acid and urea cycle, purine and pyrimidine biosynthesis, and redox cell signaling. Therefore, it is unsurprising that these molecules have a significant role in cancer metabolism and its metabolic plasticity. As one of the most prevalent malign diseases, colorectal cancer needs biomarkers for its early detection, prognostic, and prediction of response to therapy. However, the available biomarkers for this disease must be more powerful and present several drawbacks, such as high costs and complex laboratory procedures. Metabolomics has gathered substantial attention in the past two decades as a screening platform to study new metabolites, partly due to the development of techniques, such as mass spectrometry or liquid chromatography, which have become standard practice in diagnostic procedures for other diseases. Extensive metabolomic studies have been performed in colorectal cancer (CRC) patients in the past years, and several exciting results concerning amino acid metabolism have been found. This review aims to gather and present findings concerning alterations in the amino acid plasma pool of colorectal cancer patients.

LncRNA VLDLR-AS1 Gene Expression in Colorectal Cancer in Patients from East Azerbaijan Province, Iran

Background: Colorectal cancer (CRC) is the third most common cancer that frequently spreads to other parts of the body, with a low chance of recovery and a high mortality rate. Long non-coding RNAs are considered significant prognostic and diagnostic indicators because they are dysregulated in various cancers and have distinctive expression patterns and high tissue- and cell-specificity. VLDLR-AS1 lncRNA deregulation has been associated with several malignancies. Objectives: This study aimed to evaluate the expression levels of VLDLR-AS1 in CRC. It was the first time this assessment was conducted. Methods: We studied 188 samples, including 94 tumor samples and 94 paired adjacent non-tumor tissues. Total RNA was extracted, and its quantity and purity were assessed. TaKaRa PrimeScript 1st Strand cDNA Synthesis Kit (Kusatsu, Japan) was used for the reaction. The StepOnePlus Real-Time PCR System (Applied Biosystems) was set up to assess the relative expression of VLDLR-AS1. Results: According to the qRT-PCR data, the VLDLR-AS1 expression levels in CRC tissues were significantly lower than in tumor margins (P < 0.0001). In addition, receiver operating characteristic curve analysis demonstrated that VLDLR-AS1 expression can discriminate between tumor and non-tumor samples with the sensitivity and specificity of 72.34% and 51.06%, respectively (P = 0.03, AUC = 0.6274). However, no significant association was found between the expression levels of VLDLR-AS1 and clinicopathological features in CRC patients. Conclusions: The results of this study indicated that VLDLR-AS1 lncRNA is significantly downregulated in the tumor tissues of CRC patients compared to healthy tumor margin tissues. This evidence shows the potential of this gene as a promising biomarker for the early detection of CRC development.

Circular RNA ZNF800 (hsa_circ_0082096) regulates cancer stem cell properties and tumor growth in colorectal cancer

BackgroundCancer stem cells form a rare cell population in tumors that contributes to metastasis, recurrence and chemoresistance in cancer patients. Circular RNAs (circRNAs) are post-transcriptional regulators of gene expression that sponge targeted microRNA (miRNAs) to affect a multitude of downstream cellular processes. We previously showed in an expression profiling study that circZNF800 (hsa_circ_0082096) was up-regulated in cancer stem cell-enriched spheroids derived from colorectal cancer (CRC) cell lines.MethodsSpheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo.ResultsCircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth.ConclusionsCircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.

Bifidobacterium adolescentis induces Decorin+ macrophages via TLR2 to suppress colorectal carcinogenesis

BackgroundThe interplay between gut microbiota and tumor microenvironment (TME) in the pathogenesis of colorectal cancer (CRC) is largely unknown. Here, we elucidated the functional role of B. adolescentis and its possible mechanism on the manipulation of Decorin+ macrophages in colorectal cancer.MethodsThe relative abundance of B. adolescentis in tumor or para-tumor tissue of CRC patients was analyzed. The role of B. adolescentis was explored in the CRC animal models. The single cell-RNA sequencing (scRNA-seq) was used to investigate the myeloid cells subsets in TME. The expression level of TLR2/YAP axis and its downstream Decorin in macrophages were tested by Western blot and qRT-PCR. Knockdown of Decorin in Raw264.7 was performed to investigate the effect of Decorin+ macrophages on subcutaneous tumor formation. Multi-immunofluorescence assay examined the number of Decorin+ macrophages on the CRC tissue.ResultsWe found that the abundance of B. adolescentis was significantly reduced in tumor tissue of CRC patients. Supplementation with B. adolescentis suppressed AOM/DSS-induced tumorigenesis in mice. ScRNA-seq and animal experiment revealed that B. adolescentis increased Decorin+ macrophages. Mechanically, Decorin was activated by TLR2/YAP axis in macrophages. The abundance of B. adolescentis was correlated with the number of Decorin+ macrophages and the expression level of TLR2 in tumor tissue of CRC patients.ConclusionsThese results highlight that B. adolescentis induced Decorin+ macrophages and provide a novel therapeutic target for probiotic-based modulation of immune microenvironment in CRC.

Prognostic value of the levels of CTLA-4 and its ligand B7.2 in patients with colorectal cancer

Aim. To develop a computer program to determine the probability of colorectal cancer based on the assessment of the levels of CTLA-4 and its ligand B7.2.Materials and methods. The study included 44 patients with colorectal cancer (CRC) and 25 patients with benign tumors of the colon. The control group consisted of 25 individuals who had been operated for colon injury. We determined the levels of CTLA-4 and B7.2 in the blood serum and in the supernatants of tumor tissue and lymph node homogenates using flow cytofluorometry.Results. We found that the level of CTLA-4 in the blood serum increased by 2.77 times in CRC patients compared to the control group (p < 0.001). The concentration of CTLA-4 in the tumor tissue in patients with CRC was 2.34 times higher than in the control group (p = 0.007). The concentration of the B7.2 ligand in the blood serum of patients with CRC exceeded this parameter in the control group by 2.51 times (p = 0.002). The concentration of B7.2 in the tumor tissue of CRC patients was 1.68 times higher (p = 0.004) than in the control group. The analysis of the obtained data determined the parameters that have prognostic value in the structure of the diagnostic model. Using these parameters, we developed a computer program to determine the probability of CRC in the patient.Conclusion. The data obtained demonstrate an increase in the levels of CTLA-4 and its ligand B7.2 in the serum and tumor tissue of patients with CRC.

ATG4B and pS383/392-ATG4B serve as potential biomarkers and therapeutic targets of colorectal cancer

BackgroundAutophagy related protease 4B (ATG4B) is a protease required for autophagy processing, which is strongly implicated in cancer progression. Phosphorylation of ATG4B is crucial for activation of its protease activity. However, little is known about the relationship of ATG4B and its phosphorylated form at Ser 383 and 392 sites (pS383/392-ATG4B), with clinical outcomes, particularly in colorectal cancer (CRC).MethodsThe ATG4B gene expression in CRC patients was obtained from The Cancer Genome Atlas (TCGA) database to analyze its clinical relevance. Tissue microarrays composed of 118 CRC patient specimens were used to determine the associations of ATG4B and pS383/392-ATG4B protein levels with prognosis. The biological functions of ATG4B in CRC cells were inspected with cell proliferation, mobility and spheroid culture assays.ResultsATG4B gene expression was elevated in tumor tissues of CRC patients compared to that in adjacent normal tissues and high level of ATG4B expression was associated with poor survival. Similarly, protein levels of ATG4B and pS383/392-ATG4B were highly correlated with worse overall survival and disease-free survival. Stratification analysis results showed that high level of ATG4B had significantly higher risk of mortality in males and elderly patients compared to those female patients and patients 60 years or younger. In contrast, multivariate Cox’s regression analysis indicated that high level of pS383/392-ATG4B was significantly linked to unfavorable overall survival and disease-free survival of males and elderly patients, whereas, it had no correlation with female patients and patients 60 years or younger. Moreover, high level of ATG4B was positively associated with increased mortality risk in patients with advanced AJCC stages (III and IV) and lymph node invasion (N1 and N2) for both overall survival and disease-free survival. Nevertheless, high level of pS383/392-ATG4B was positively correlated with increased mortality risk in patients with early AJCC stages (I and II) and without lymph node invasion (N0). In addition, silencing ATG4B attenuated migration, invasion, and further enhanced the cytotoxic effects of chemotherapeutic drugs in two and three-dimensional cultures of CRC cells.ConclusionsOur results suggest that ATG4B and pS383/392-ATG4B might be suitable biomarkers and therapeutic targets for CRC.

Abstract 2120: Exploring the genomic landscape of colorectal cancer for enabling clinical genomics informed and real-world data based clinical development

Abstract Background: Activation of the MAPK-signaling by RAS- (KRAS or NRAS) alterations is an important driver event in colorectal cancer (CRC) tumorigenesis that determines treatment selection (1).Real-world data (RWD) mining is an important tool to inform patient selection and stratification (2,3). Here, RWD genomics data mining was conducted using the Foundation Medicine database to explore RAS and BRAF-alterations in CRC patients and to investigate the general alterations’ landscape - including rare alterations. The prevalence of microsatellite instability (MSI) and tumor mutational burden (TMB) were also explored. Methods: The prevalence of gene alterations and their co-occurrence with RAS/BRAF-alterations in tumor tissue of CRC patients (N=42800) were investigated using the FoundationInsightsTM web platform, which includes harmonized results from the FoundationOneCDx, FoundationOneHeme, and PD-L1 assays from 2012 to March 2022. Data was collected from FMI solid and heme tests from 2012 to March 2022. Alterations that render a particular gene ‘altered’ included single nucleotide variants (SNVs) or insertion-deletion mutations (Indels), collectively referred to as short variants (SVs), rearrangements (RE) and copy number events (CN). TMB (cut-off 10 mut/MB) and MSI status were also characterized. Results: Altogether, 36.9% of CRC samples were RAS/BRAF-WT and 63.1% had a known or likely functional RAS/BRAF-alteration, respectively. Of all KRAS and NRAS alterations (SV, CN and RE regardless of annotation as functional or presence in a hotspot), SVs in exons 2, 3 and 4 constituted 96.1% and 94.2% of all alterations, respectively. Relative to all samples the potentially targetable KRAS p.G12C, p.G12D and p.G12V mutations were present in 3.6%, 15.1% and 10.2% of samples, respectively. The p.G12E variant was present in less than ten samples. The five most frequently altered potentially targetable genes were PIK3CA (19.6%), PTEN (8.7%), ERBB2 (4.7%), EGFR (2.5%) and FGFR1 (2.0%). NTRK-alterations (NTRK1/2/3) were present in 0.9%, ROS1-alterations in 0.4%, RET-alterations in 0.6% and ALK-alterations in 0.5% of samples, respectively. The prevalence of immuno-oncology biomarkers was also explored; overall, 9.6% of samples were TMB-high, 6.1% MSI-high and 0.6% POLE-altered. MSI-high or TMB-high samples were significantly more abundant in the RAS/BRAF-altered subset. In the RAS/BRAF-WT subset 4.6% were MSI-high and 7.9% TMB-high, whereas in the RAS/BRAF-altered subset 7.1% were MSI-high and 10.6% TMB-high. Conclusion: RWD based clinical genomic profiling confirms that the vast majority of RAS-alterations in CRC are SVs in exons 2, 3 and 4. Both TMB-high and MSI-high samples are significantly more abundant in the RAS/BRAF-altered cohort. RWD confirms that PI3K-, ERBB- and FGFR-signaling pathways are frequently altered in CRC. RWD based clinical genomic profiling has the potential to support comprehensive clinical study design as well as patient selection/stratification. Citation Format: Markus Schulze, XiaoZhe (Janet) Wang, Jawad Hamad, Julia F. Hopkins, Julia F. Hopkins, Jürgen Scheuenpflug, Zheng Feng. Exploring the genomic landscape of colorectal cancer for enabling clinical genomics informed and real-world data based clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2120.

NF-κB Activator 1 downregulation in macrophages activates STAT3 to promote adenoma-adenocarcinoma transition and immunosuppression in colorectal cancer

BackgroundAdenoma-adenocarcinoma transition is a key feature of colorectal cancer (CRC) occurrence and is closely regulated by tumor-associated macrophages (TAMs) and CD8+ T cells. Here, we investigated the effect of the NF-κB activator 1 (Act1) downregulation of macrophages in the adenoma-adenocarcinoma transition.MethodsThis study used spontaneous adenoma-developing ApcMin/+, macrophage-specific Act1-knockdown (anti-Act1), and ApcMin/+; anti-Act1 (AA) mice. Histological analysis was performed on CRC tissues of patients and mice. CRC patients’ data retrieved from the TCGA dataset were analyzed. Primary cell isolation, co-culture system, RNA-seq, and fluorescence-activated cell sorting (FACS) were used.ResultsBy TCGA and TISIDB analysis, the downregulation of Act1 expression in tumor tissues of CRC patients negatively correlated with accumulated CD68+ macrophages in the tumor. Relative expression of EMT markers in the tumor enriched ACT1lowCD68+ macrophages of CRC patients. AA mice showed adenoma-adenocarcinoma transition, TAMs recruitment, and CD8+ T cell infiltration in the tumor. Macrophages depletion in AA mice reversed adenocarcinoma, reduced tumor amounts, and suppressed CD8+ T cell infiltration. Besides, macrophage depletion or anti-CD8a effectively inhibited metastatic nodules in the lung metastasis mouse model of anti-Act1 mice. CRC cells induced activation of IL-6/STAT3 and IFN-γ/NF-κB signaling and the expressions of CXCL9/10, IL-6, and PD-L1 in anti-Act1 macrophages. Anti-Act1 macrophages facilitated epithelial-mesenchymal-transition and CRC cells’ migration via CXCL9/10-CXCR3-axis. Furthermore, anti-Act1 macrophages promoted exhaustive PD1+ Tim3+ CD8+ T cell formation. Anti-PD-L1 treatment repressed adenoma-adenocarcinoma transition in AA mice. Silencing STAT3 in anti-Act1 macrophages reduced CXCL9/10 and PD-L1 expression and correspondingly inhibited epithelial-mesenchymal-transition and CRC cells’ migration.ConclusionsAct1 downregulation in macrophages activates STAT3 that promotes adenoma-adenocarcinoma transition via CXCL9/10-CXCR3-axis in CRC cells and PD-1/PD-L1-axis in CD8+ T cells.

Bovine meat and milk factor protein expression in tumor-free mucosa of colorectal cancer patients coincides with macrophages and might interfere with patient survival.

Bovine milk and meat factors (BMMFs) are plasmid-like DNA molecules isolated from bovine milk and serum, as well as the peritumor of colorectal cancer (CRC) patients. BMMFs have been proposed as zoonotic infectious agents and drivers of indirect carcinogenesis of CRC, inducing chronic tissue inflammation, radical formation and increased levels of DNA damage. Data on expression of BMMFs in large clinical cohorts to test an association with co-markers and clinical parameters were not previously available and were therefore assessed in this study. Tissue sections with paired tumor-adjacent mucosa and tumor tissues of CRC patients [individual cohorts and tissue microarrays (TMAs) (n= 246)], low-/high-grade dysplasia (LGD/HGD) and mucosa of healthy donors were used for immunohistochemical quantification of the expression of BMMF replication protein (Rep) and CD68/CD163 (macrophages) by co-immunofluorescence microscopy and immunohistochemical scoring (TMA). Rep was expressed in the tumor-adjacent mucosa of 99% of CRC patients (TMA), was histologically associated with CD68+/CD163+ macrophages and was increased in CRC patients when compared to healthy controls. Tumor tissues showed only low stromal Rep expression. Rep was expressed in LGD and less in HGD but was strongly expressed in LGD/HGD-adjacent tissues. Albeit not reaching statistical significance, incidence curves for CRC-specific death were increased for higher Rep expression (TMA), with high tumor-adjacent Rep expression being linked to the highest incidence of death. BMMF Rep expression might represent a marker and early risk factor for CRC. The correlation between Rep and CD68 expression supports a previous hypothesis that BMMF-specific inflammatory regulations, including macrophages, are involved in the pathogenesis of CRC.

MicroRNA-455-3p accelerate malignant progression of tumor by targeting H2AFZ in colorectal cancer

ABSTRACT Colorectal cancer (CRC) becomes the second leading cause of cancer-related deaths in 2020. Emerging studies have indicated that microRNAs (miRNAs) play a key role in tumorigenesis and progression. The dysfunctions of miR-455-3p are observed in many cancers. However, its biological function in CRC remains to be confirmed. By sequencing serum sample, miR-455-3p was found to be up-regulated in CRC patients. RT-qPCR demonstrated that the miR-455-3p expression was both higher in the serum and tumor tissues of CRC patients. Furthermore, it indicated that miR-455-3p had the ability in promoting cell proliferation, suppressing cell apoptosis, and stimulating cell migration. In vivo experiments also showed that miR-455-3p promoted tumor growth. Additionally, H2AFZ was proved as the direct gene target of miR-455-3p by dual-luciferase assay. Taken together, miR-455-3p functioned as a tumor promoter in CRC development by regulating H2AFZ directly. Thus, it has enormous potential as a biomarker in the diagnosis of CRC.

Fecal gene detection based on next generation sequencing for colorectal cancer diagnosis.

BACKGROUNDColorectal cancer (CRC) is one of the most common malignancies worldwide. Given its insidious onset, the condition often already progresses to advanced stage when symptoms occur. Thus, early diagnosis is of great significance for timely clinical intervention, efficacy enhancement, and prognostic improvement. Featuring high throughput, fastness, and rich information, next generation sequencing (NGS) can greatly shorten the detection time, which is a widely used detection technique at present.AIMTo screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC, and to establish a technological platform for CRC screening, diagnosis, and efficacy monitoring through fecal DNA detection. METHODSNGS was used to sequence the stool DNA of patients with CRC, which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease, as well as the tumor tissues of CRC patients. Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened, and their significances in diagnosing CRC and predicting patients' prognosis were comprehensively evaluated. RESULTSHigh mutation frequencies of TP53, APC, and KRAS were detected in the stools and tumor tissues of CRC patients prior to surgery. Contrastively, no pathogenic mutations of the above three genes were noted in the postoperative stools, the normal controls, or the benign intestinal disease group. This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients. The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent. Compared to the postoperative stools and the stools in the two control groups, the pathogenic mutation frequencies of TP53 and KRAS were significantly higher for the preoperative stools (χ2 = 7.328, P < 0.05; χ2 = 4.219, P < 0.05), suggesting that fecal TP53 and KRAS genes can be used for CRC screening, diagnosis, and prognostic prediction. No significant difference in the pathogenic mutation frequency of the APC gene was found from the postoperative stools or the two control groups (χ2 = 0.878, P > 0.05), so further analysis with larger sample size is required. Among CRC patients, the pathogenic mutation sites of TP53 occurred in 16 of 27 preoperative stools, with a true positive rate of 59.26%, while the pathogenic mutation sites of KRAS occurred in 10 stools, with a true positive rate of 37.04%. The sensitivity and negative predictive values of the combined genetic testing of TP53 and KRAS were 66.67% (18/27) and 68.97%, respectively, both of which were higher than those of TP53 or KRAS mutation detection alone, suggesting that the combined genetic testing can improve the CRC detection rate. The mutation sites TP53 exon 4 A84G and EGFR exon 20 I821T (mutation start and stop positions were both 7579436 for the former, while 55249164 for the latter) were found in the preoperative stools and tumor tissues. These "undetected" mutation sites may be new types of mutations occurring during the CRC carcinogenesis and progression, which needs to be confirmed through further research. Some mutations of "unknown clinical significance" were found in such genes as TP53, PTEN, KRAS, BRAF, AKT1, and PIK3CA, whose clinical values is worthy of further exploration.CONCLUSIONNGS-based fecal genetic testing can be used as a complementary technique for the CRC diagnosis. Fecal TP53 and KRAS can be used as specific genes for the screening, diagnosis, prognostic prediction, and recurrence monitoring of CRC. Moreover, the combined testing of TP53 and KRAS genes can improve the CRC detection rate.

Fusobacterium nucleatum Affects Cell Apoptosis by Regulating Intestinal Flora and Metabolites to Promote the Development of Colorectal Cancer.

Background/AimsIntestinal flora, especially Fusobacterium nucleatum (Fn), can affect the development of colorectal cancer (CRC). In this study, we examined the composition of intestinal flora and their metabolites in the tissues, serum and feces of CRC patients.Materials and MethodsCRC tissues, adjacent normal colonic tissues, fecal and serum samples were collected from CRC patients who received surgical treatment between January 2018 and January 2020. Fecal and serum samples were collected from healthy individuals for comparison. In addition, fecal samples were collected from BALB/c female mice. SW480, a human CRC cell line, was utilized for in vitro studies. The experiments involved 1H-NMR-based metabolomics analysis, targeted and untargeted mass spectrometry analysis, and intestinal flora 16S rDNA V4 region sequencing.ResultsThe abundance of Bacteroides and propionic acid concentration were decreased and that of Lactobacillus and lactic acid concentration were increased in CRC tissues. In addition, the abundances of Ruminococcus, Prevotella, and Sutterell were decreased in CRC patients. The levels of leucine and isoleucine were decreased in the serum and tumor tissues of CRC patients. Aspartate, glutamate and glutathione levels were elevated in the tissues of CRC patients only. The serum glutamine, tyrosine, valine, alanine, and histidine levels were decreased significantly. Lactic acid inhibited and propionic acid promoted apoptosis among SW480 CRC cells.ConclusionFn affected the apoptosis of CRC cells and promoted the progression of CRC by affecting the distribution of intestinal flora, which altered the concentrations of metabolites such as lactic acid, propionic acid. Intestinal flora could regulate amino acid metabolism.

Fusobacterium nucleatum promotes colorectal cancer cells adhesion to endothelial cells and facilitates extravasation and metastasis by inducing ALPK1/NF-κB/ICAM1 axis

ABSTRACT Metastasis is the leading cause of death for colorectal cancer (CRC) patients, and the spreading tumor cells adhesion to endothelial cells is a critical step for extravasation and further distant metastasis. Previous studies have documented the important roles of gut microbiota-host interactions in the CRC malignancy, and Fusobacterium nucleatum (F. nucleatum) was reported to increase proliferation and invasive activities of CRC cells. However, the potential functions and underlying mechanisms of F. nucleatum in the interactions between CRC cells and endothelial cells and subsequent extravasation remain unclear. Here, we uncovered that F. nucleatum enhanced the adhesion of CRC cells to endothelial cells, promoted extravasation and metastasis by inducing ICAM1 expression. Mechanistically, we identified that F. nucleatum induced a new pattern recognition receptor ALPK1 to activate NF-κB pathway, resulting in the upregulation of ICAM1. Interestingly, the abundance of F. nucleatum in tumor tissues of CRC patients was positively associated with the expression levels of ALPK1 and ICAM1. Moreover, high expression of ALPK1 or ICAM1 was significantly associated with a shorter overall survival time of CRC patients. This study provides a new insight into the role of gut microbiota in engaging into the distant metastasis of CRC cells.

Evolution of Mutational Landscape and Tumor Immune-Microenvironment in Liver Oligo-Metastatic Colorectal Cancer.

Simple SummaryAbout 10% of colorectal cancer patients presents with oligo-metastatic disease. The aim of our study was to assess genetic and immunologic dynamics underlying the oligo-metastatic status, evaluating genotype-phenotype correlations in a clean and homogeneous clinical model of liver-limited metastatic colorectal cancer. We show that loss of KRAS and SMAD4 mutations characterizes the oligo-metastatic disease while a progressive mutational evolution (gain in KRAS, PI3KCA, BRAF and SMAD4) is observed in poly-metastatic evolving disease. Furthermore, high granzyme-B+ T-cells infiltration is found in oligo-metastatic lesions. This study can support innovative strategies to monitor clinical evolution and to induce regressive genetic trajectories in cancer.Genetic dynamics underlying cancer progression are largely unknown and several genes involved in highly prevalent illnesses (e.g., hypertension, obesity, and diabetes) strongly concur to cancer phenotype heterogeneity. To study genotype-phenotype relationships contributing to the mutational evolution of colorectal cancer (CRC) with a focus on liver metastases, we performed genome profiling on tumor tissues of CRC patients with liver metastatic disease and no co-morbidities. We studied 523 cancer-related genes and tumor-immune microenvironment characteristics in primary and matched metastatic tissues. We observed a loss of KRAS and SMAD4 alterations and a high granzyme-B+ T-cell infiltration when the disease did not progress. Conversely, gain in KRAS, PIK3CA and SMAD4 alterations and scarce granzyme-B+ T-cells infiltration were observed when the tumor evolved towards a poly-metastatic spread. These findings provide novel insights into the identification of tumor oligo-metastatic status, indicating that some genes are on a boundary line between these two clinical settings (oligo- vs. poly-metastatic CRC). We speculate that the identification of these genes and modification of their evolution could be a new approach for anti-cancer therapeutic strategies.

Comprehensive ceRNA network analysis and experimental studies identify an IGF2-AS/miR-150/IGF2 regulatory axis in colorectal cancer

Recently, a growing body of studies has demonstrated that long non-coding RNA (lncRNA) can act as microRNA (miRNA) sponges to regulate protein-coding gene expression and play essential roles in tumor initiation and progression. In the present study, we constructed a competitive endogenous RNA (ceRNA) network and identified potential regulatory axes in colorectal cancer (CRC) through both bioinformation and experimental validation. Firstly, we obtained differentially expressed (DE) lncRNAs, miRNAs, and mRNAs by analyzing the RNA expression profiles of CRC retrieved from The Cancer Genome Atlas (TCGA) database and CRC patients' data from affiliated Hospital of Jiangnan University, respectively. Then, we established a ceRNA regulatory network of CRC that includes 23 lncRNAs, 7 miRNAs and 244 mRNAs. To further identify these lncRNA-miRNA-mRNA regulatory axes which might play vital roles in CRC tumorigenesis and prognosis, we performed additional analyses using comprehensive bioinformatic methods. Several ceRNA regulatory axes, which consist of 2 lncRNAs, 2 miRNAs and 5 mRNAs, were obtained from the network. Finally, the interactions and correlations among these ceRNA networks were validated by experiments on CRC cell lines and clinical tumor tissues, and a potential IGF2-AS/miR-150/IGF2 axis that perfectly conform to the ceRNA theory was determined. According to the qRT-PCR results, miR-150 overexpression remarkably decreased IGF2-AS and IGF2 expression. Meanwhile, IGF2-AS expression was positively correlated with IGF2 expression in tumor tissue of CRC patients. Besides, dual luciferase reporter assays indicated that miR-150 could bound to IGF2-AS and the 3′UTR of and IGF2. In general, the constructed novel IGF2-AS/miR-150/IGF2 network might provide potential mechanisms of CRC development, and could act as a promising target for CRC treatment.

Increasing the expression of programmed death ligand 2 (PD-L2) but not 4-1BB ligand in colorectal cancer cells.

Immune checkpoint (ICP) molecules modulate the immune response by either inducing or preventing T cell activation. Over-expression of some ICPs on malignant cells has been shown to regulate anti-tumor immune responses. We aimed to investigate the expression levels of two immune checkpoint molecules which have not been studied extensively in patients with colorectal cancer (CRC). Programmed Death Ligand 2 (co-inhibitory) and 4-1BB ligand (co-stimulatory) were assessed in tumor tissues of CRC patients compared to the adjacent normal tissues. Following tissue excision during surgical operation from 21 CRC patients, RNA extraction, cDNA synthesis and semi-quantitative real-time PCR were done for measuring the expressions of PD-L2 and 4-1BBL genes. In protein level, indirect immunohistochemistery (IHC) was performed on tissue sections. We revealed that PD-L2 was expressed in about 81% CRCs and insignificantly correlated with the tumor differentiation grade. Although a 3.25-fold change in the gene expression of PD-L2 was found in tumor tissues compared to the adjacent normal tissues (P = 0.005), but decreased level of 4-1BBL in counterpart tissues was not significant. Our results were confirmed by IHC for PDL-2 (P = 0.02) and 4-1BBL, however it was not statistically significant for the latter one. Although not significant, we could find an association between the elevated expression of PD-L2 and the tumor differentiation grade. Increased expression of negative regulator of the anti-tumor immune responses like PD-L2, as a prominent way of tumor escape, can be considered for cancer immunotherapy approaches in CRC patients using blocking monoclonal antibodies.