CRC Tissues Research Articles

Exploring the Relationship between the Gut Mucosal Virome and Colorectal Cancer: Characteristics and Correlations.

The fecal virome has been reported to be associated with CRC. However, little is known about the mucosal virome signature in CRC. This study aimed to determine the viral community within CRC tissues and their contributions to colorectal carcinogenesis. Colonic mucosal biopsies were harvested from patients with CRC (biopsies of both neoplasia and adjacent normal tissue (CRC-A)) and healthy controls (HC). The shot-gun metagenomic sequencing of virus-like particles (VLPs) was performed on the biopsies. Viral community, functional pathways, and their correlations to clinical data were analyzed. Fluorescence in situ hybridizations (FISH) for the localization of viruses in the intestine was performed, as well as quantitative PCR for the detection of Torque teno virus load in human mucosal VLP DNA. A greater number and proportion of core species were found in CRC tissues than in CRC-A and HC tissues. The diversity of the mucosal virome in CRC tissues was significantly increased compared to that in HC and CRC-A tissues. The mucosal virome signature of CRC tissues were significantly different from those of HC and CRC-A tissues at the species level. The abundances of eukaryotic viruses from the Anelloviridae family and its sub-species Torque teno virus (TTV) were significantly higher in CRC patients than in HC. Furthermore, increased levels of TTV in the intestinal lamina propria were found in the CRC group. Multiple viral functions of TTV associated with carcinogenesis were enriched in CRC tissues. We revealed for the first time that the mucosal virobiota signature of CRC is characterized by a higher diversity and more eukaryotic viruses. The enrichment of TTV species in CRC tissues suggests that they may play an oncogenic role in CRC. Targeting eukaryotic viruses in the gut may provide novel strategies for the prevention and treatment of CRC.

Identification of Moesin (MSN) as a Potential Therapeutic Target for Colorectal Cancer via the β-Catenin-RUNX2 Axis.

CRC is the second leading cause of cancer-related death. The complex mechanisms of metastatic CRC limit available therapeutic choice. Thus, identifying new CRC therapeutic targets is essential. Moesin (MSN), a member of the ezrin-radixin-moesin family, connects the cell membrane to the actin-based cytoskeleton and regulates cell morphology. We investigated the role of MSN in the progression of CRC. GENT2 and oncomine were used to study MSN expression and CRC patient outcomes. MSN-specific shRNAs or MSN-overexpressed plasmid were used to establish MSN-KD and MSN overexpressed cell lines, respectively. SRB, migration, wound healing, and flow cytometry were used to test cell survival and migration. Propidium iodide and annexin V stain were used to analyze the cell cycle and apoptosis. MSN expression was found to be higher in CRC tissues than in normal tissues. Higher MSN expression is associated with poor overall survival, disease-free survival, and relapse-free survival rates in CRC patients. MSN silencing inhibits cell proliferation, adhesion, migration, and invasion in vitro, whereas MSN overexpression accelerates cell proliferation, adhesion, migration, and invasion. RNA sequencing was used to investigate differentially expressed genes, and RUNX2 was discovered as a possible downstream target for MSN. In CRC patients, RUNX2 expression was significantly correlated with MSN expression. We also found that MSN silencing decreased cytoplasmic and nuclear β-catenin levels. Additionally, pharmacological inhibition of β-catenin in MSN-overexpressed cells led to a reduction of RUNX2, and activating β-catenin signaling by inhibiting GSK3β rescued the RUNX2 downregulation in MSN-KD cells. This confirms that MSN regulates RUNX2 expression via activation of β-catenin signaling. Finally, our result further determined that RUNX2 silencing reduced the ability of MSN overexpression cells to proliferate and migrate. MSN accelerated CRC progression via the β-catenin-RUNX2 axis. As a result, MSN holds the potential to become a new target for CRC treatment.

Expression Profile of Microenvironmental Factors in the Interface Zone of Colorectal Cancer: Histological-Stromal Biomarkers and Cancer Cell-Cancer-Associated Fibroblast-Related Proteins Combined for the Assessment of Tumor Progression

Introduction: The characterization of tumor microenvironment (TME) related factors and their impact on tumor progression have attracted much interest. We investigated cancer cells and cancer-associated fibroblasts (CAFs) to evaluate biomarkers that are associated with neoplastic progression, observing them in different interface zones of colorectal cancer. Methods: On 357 CRC tissue microarrays, using immunohistochemistry, we examined the associations of podoplanin and α-SMA expressed in cancer cells and CAFs and evaluated them in different areas: tumor core, invasive front, tumor budding, tumor-stroma ratio (TSR) scoring, and desmoplastic stroma. Results: CAFs expressing α-SMA were found in more than 90% of the cases. Podoplanin+ was detected in cancer cells and CAFs, with positivities of 38.6% and 70%, respectively. Higher α-SMA+ CAFs and podoplanin+ cancer cells were observed predominantly at the TSR score area: 94.3% and 64.3% of cases, respectively. The status of podoplanin in CAFs+ was higher in the desmoplastic area (71.6%). Stroma-high tumors showed increased expression of α-SMA and podoplanin in comparison with stroma-low tumors. The status of podoplanin in cancer cells was observed in association with lymphatic invasion and distant metastasis. Conclusion: The substance of the CRC was composed predominantly of the surrounding stroma-α-SMA+ CAFs. Podoplanin expressed in the prognosticator zones was associated with unfavorable pathological features. The combination of histologic and protein-related biomarkers can result in a tool for the stratification of patients with CRC.

Gut Microbiota Conditioning Promotes Anti-Tumor Immunity and Responsiveness to Immunological Checkpoint Blockade in Colorectal Cancer

Abstract Background Colorectal cancer (CRC) infiltration by T lymphocytes is associated with prolonged survival. However, immune infiltration spontaneously occurs in <20% of tumors and no therapies able to promote it are currently available. CRC tissue is populated by gut bacteria translocated across the neoplastic epithelium. We previously showed that direct contact between tumor cells and bacteria induces overexpression of genes encoding immune cell-recruiting chemokines in tumor cells, and we identified a panel of bacterial species associated with enhanced chemokine expression, high T cell infiltration and favorable prognosis. Aims In this project we tested the ability of identified bacterial species to boost T-cell infiltration and promote tumor regression in orthotopic CRC experimental models. Methods Luciferase-expressing mismatch repair (MMR)-proficient or -deficient murine CRC cells from established cell lines were injected intra-cecum in syngeneic mice. Tumor growth was monitored by bioluminescence. Bacterial species of interest, defined as Operational Taxonomic Units (OTUs), were cultured under anaerobic conditions, and, following tumor development, were administered to tumor-bearing mice by oral gavage. In specific experiments, bacteria administration was combined with immunological checkpoint blockade (ICB), based on injection of anti-PD-1 antibodies. Immune cell infiltration was evaluated by flow cytometry, following tumor harvesting and enzymatic tissue digestion. Results In MMR-deficient tumors, known to be immunogenic and responsive to immunotherapy, administration of the bacterial species OTU8, was sufficient to reduce tumor growth. In MMR-proficient tumors, scarcely infiltrated by immune cells and poorly responsive to ICB, OTU8, did not per se affect tumor growth, but enhanced infiltration by cytotoxic CD8+ T cells, ultimately leading, in combination with anti-PD-1 antibodies, to tumor growth inhibition. Conclusions We demonstrated that administration of defined bacteria promotes endogenous immune responses against CRC. Gut microbiota conditioning might represent an innovative therapeutic strategy to enhance responsiveness to ICB and to broaden its indication beyond MMR-deficient tumors.

Circular RNA hsa_circ_0064559 affects tumor cell growth and progression of colorectal cancer

BackgroundColorectal cancer (CRC) is the second leading cause of cancer-related deaths globally. It is essential to identify new CRC-associated therapeutic targets and diagnostic biomarkers. Previous studies have demonstrated that a series of circular RNAs (circRNAs) play a crucial role in CRC pathogenesis. This study assessed the potential of hsa_circ_0064559 in tumor cell growth and progression of CRC.MethodsSix pairs of matched CRC and normal colorectal tissue samples were sequenced using the Affymetrix Clariom D array. Using RNA interference, the expression of thirteen circRNAs was knocked down in CRC cells. The proliferation of CRC cell lines (RKO and SW620 cells) was detected using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Apoptosis and cell cycle were determined by flow-cytometric analysis. An in vivo study uses nude mice to establish a CRC mouse model. The differentially expressed genes were analyzed using Affymetrix primeview human GeneChip array and verified by polymerase chain reaction.ResultsAffymetrix Clariom D array analysis revealed that thirteen circRNAs were upregulated in CRC. The proliferation of CRC cell lines was decreased, while the proportion of apoptotic and G1 phase cells was higher after hsa_circ_0064559 knockdown. In vivo xenograft nude mice model revealed that the volume and weight of the tumor were reduced by hsa_circ_0064559 knockdown. In Affymetrix primeview human GeneChip array, we found six upregulated genes (STAT1, ATF2, TNFRSF10B, TGFBR2, BAX, and SQSTM1) and two downregulated genes (SLC4A7 and CD274) related to apoptosis and proliferation of colorectal cancer cells after hsa_circ_0064559 knockdown.ConclusionsThe hsa_circ_0064559 knockdown could inhibit the proliferation, promote apoptosis in CRC cell lines in vitro, and inhibit the development of CRC tumors in vivo. The mechanism may be related to activating a wide range of signaling pathways. The hsa_circ_0064559 may be a potential biomarker for early diagnosis or prognosis of CRC and a novel drug target for CRC therapy.

MiR-509–5p promotes colorectal cancer cell ferroptosis by targeting SLC7A11

Background/AimColorectal cancer (CRC), is characterized by aberrant microRNA (miRNA) expression during their development and progression. Recently, miR-509–5p's role as a regulator of several malignancies has been highlighted. Its function in CRC, however, is exposed. This research aimed to determine the relative abundance of miR-509–5p and its biological function in colorectal cancer. MethodsThe expression of miR-509–5p in CRC cell lines and tissues, as well as neighboring normal tissues, was evaluated using real-time quantitative polymerase chain reaction (RT-PCR). 3-(4,5-dimethylthiazol-2-yl)− 2,5-diphenyl-2 H-tetrazolium bromide (MTT) was used to assess cell viability. The association between miR-509–5p and its predicted target in CRC cells was analyzed using bioinformatics tools. The levels of Solute carrier family seven number 11 (SLC7A11) were assessed using enzyme-linked immunosorbent assay (ELISA), while malondialdehyde (MDA) and iron content levels were determined colorimetrically. ResultsCompared to adjacent normal tissue and normal colorectal cell, there was a significant reduction in miR-509–5p expression in both CRC tissues and cells. miR-509–5p upregulation inhibited Caco-2 cell viability. SLC7A11 was predicted to be the cellular target of miR-509–5p. Interestingly, miR-509–5p’s overexpression suppressed both mRNA and protein levels of SLC7A11, whereas its downregulation boosted SLC7A11 gene expression. Finally, overexpressing miR-509–5p resulted in increased MDA and iron levels. ConclusionOur results demonstrate that miR-509–5p has CRC tumor suppressor functions through controlling the expression of SLC7A11 and promotion of ferroptosis providing a new therapeutic target for the treatment of CRC.

Identification of hub genes and pathways in lung metastatic colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most prevalent types of malignant tumours. Metastasis is the leading cause of cancer-related mortality, with lung metastases accounting for 32.9% of all metastatic CRCs. However, since the biological mechanism of lung metastatic CRC is poorly understood, limited therapeutic targets are available. In the present study, we aimed to identify the key genes and molecular processes involved in CRC lung metastasis.MethodsThe differentially expressed genes (DEGs) between primary and lung metastatic CRC patients were obtained from the Gene Expression Omnibus (GEO) database via the GEO2R tool. The enriched biological processes and pathways modulated by the DEGs were determined with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome Gene Sets analyses. The search tool Retrieval of Interacting Genes (STRING) and Cytoscape were used to construct a protein–protein interaction (PPI) network among DEGs.ResultsThe DEGs were enriched in surfactant metabolism, cell–cell communication and chemokine signaling pathways. The defined hub genes were included CLU, SFTPD, CCL18, SPP1, APOE, BGN and MMP3. Among them, CLU, SFTPD and CCL18 might be associated with the specific lung tropism metastasis in CRC. In addition, the expression and prognostic values of the hub genes in CRC patients were verified in database of The Cancer Genome Atlas (TCGA) and GEO. Moreover, the protein levels of the hub genes were detected in primary and lung metastatic CRC cells, serum or tissues. Furthermore, SFTPD was confirmed to facilitate cellular proliferation and lung metastasis in CRC.ConclusionThis bioinformatics study may provide a better understanding of the candidate therapeutic targets and molecular mechanisms for CRC lung metastasis.

Abstract 2202: Role of FadA secreting Fusobacterium nucleatum in initiation and progression of colorectal cancer

Abstract Colorectal cancer (CRC) is a global disease with poor prognosis. There is an alarming increase in incidence of CRC in younger patients along with well recognized racial disparities. Recent data implicate a role of microbiome in initiation and progression of CRC. Fusobacterium nucleatum (Fn), an oral opportunistic pathogen, has emerged as one of the common pathogens associated with CRC. Fn transformation from commensal to pathogen requires expression of amyloid-like FadA adhesin that plays a key role in its attachment to the colonic mucosal cells resulting in inflammatory, oncogenic, and metastatic changes. CRC initiates as polyps that go on to invade the mucosal layers and metastasize. This study analyzed the significance of Fn as a biomarker in CRC as well as to understand the etiology and metastasis of CRC. Deidentified and delinked formalin fixed paraffin embedded tissue sections from cancerous, precancerous and non-cancerous colorectal biopsies were analyzed for Fn presence and markers in epithelial to mesenchymal transition (EMT) in CRC through immunofluorescence. EMT was studied through biomarkers such as E-cadherin, which is an epithelial cell surface protein, vimentin as an intracellular filament protein normally expressed in mesenchymal cells, and N-cadherin, a cell adhesion molecule that is shown to express aberrantly in some solid tumor cells undergoing EMT. As expected, E-cadherin was abundantly expressed on the surface of epithelial cells while vimentin was found in the stromal cells in the non-cancerous tissue sections. Interestingly, the E-cadherin expression was lower in the cancerous tissue and some cancerous specimens also exhibited the vimentin positive foci within epithelial cells signifying partial EMT. N-cadherin expression was not observed in any of the specimens. The presence of Fn as analyzed through the FadA staining correlated with decline in the E-cadherin expression in some cancer specimens. To further analyze the significance of Fn, a meta-analytic study was conducted from 20 studies that indicated a significant presence of Fn not only in CRC tissues but also in fecal specimens from patients with CRC. A whole genome sequencing analysis corroborated with the abundance of Fn in CRC tissue specimens. These data suggest that analysis for Fn presence offers an attractive opportunity for intervention and targeted treatment for chemo resistant tumors. EMT analysis provides an additional corroboration in early detection of cancer progression which is pivotal in successful treatment of CRC. Citation Format: Angela Huang, Anjana Saxena, Mintoo Patel. Role of FadA secreting Fusobacterium nucleatum in initiation and progression of colorectal cancer [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 2202.

Abstract 1933: Transcriptomic profiling of Hispanic colorectal cancer disparities

Abstract Colorectal cancer (CRC) was reported as the third most diagnosed cancer worldwide with approximately 1.93 million new cases reported in 2020. Within the U.S., the incidence and death rates differ by ethnicity. Among Hispanics, CRC accounted for 12% and 8% of all estimated new cases of cancer and 11% and 9% of all cancer deaths in men and women, respectively, in 2018. Though CRC is preventable, and the overall survival rate is approximately 90% when detected at early stages, only about 39% of CRC patients are diagnosed in the early stages, mostly because screening rates in the US are low, especially among Hispanics. Access to health care for Hispanics is limited when compared to non-Hispanic Whites (NHWs); as a result, they tend to present with later-stage CRC, which likely accounts for their inferior survival after CRC. Essential to improving treatment, prognosis, and detection strategies is the identification and validation of new ethnicity-specific transcriptomic profiles. Here, we have conducted RNA sequencing from Hispanic and NHW CRC (n=10 each) and normal adjacent tissues (NATs; n=3 each), to identify the differentially expressed genes (DEGs) in these two populations. The Illumina sequencing reads were mapped against the reference human genome, and the read counts were normalized using the median of ratios method using DESeq2 software. The transcriptomic analysis revealed that as compared to the respective NATs, 7222 and 7873 genes were observed to be differentially expressed in NHW and Hispanic CRC tissues. Among these, 213 and 363 genes were unique to the NHW and Hispanic CRC cohorts, respectively. Within the Hispanic cohort, gender-based analyses revealed that 504 and 266 genes were differentially expressed in the female and male donors (n=5 each), respectively, as compared to the NHW counterparts. Further, 271 and 475 genes were found to be differentially expressed in early (stage I/II; n=5) and late (stage III/IV; n=3) stages of Hispanic CRC tissues, respectively, as compared to the NHW counterparts. Pathway enrichment analysis of the unique DEGs from the Hispanic and NHW cohorts was conducted using Ingenuity Pathway Analysis software. The genes involved in cysteine biosynthesis, ferroptosis, and GABA receptor signaling pathways were seen to be enriched in the NHW cohort, whereas the genes involved in glucocorticoid receptor (GR) signaling, bile, and androgen biosynthesis pathways were enriched in the Hispanic cohort. Previous studies have implicated, GR signaling to promote tumor heterogeneity and invasion in CRC via CDK1, or bile acids to regulate numerous genes including p53, CDK1, cyclooxygenase 2, IL8, and various miRNAs leading to CRC metastasis. As each pathway likely regulates specific cellular behaviors, the DEGs identified from this study will constitute potential ethnicity-specific biomarkers or attractive targets for the development of novel therapeutic interventions. Citation Format: Soumya Nair, Aditi Kulkarni, Sourav Roy. Transcriptomic profiling of Hispanic colorectal cancer disparities [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 1933.

Abstract 2113: The age-dependent intratissue accumulation of cholesterol, resulting in reduced squalene epoxidase, is associated with a high colorectal cancer risk population

Abstract Objective: we recently reported that cholesterol accelerates CRC aggravates via the dissociation of GSK3β/p53 by the degradation of squalene epoxidase (SQLE). However, despite its clinical reports, the underlying mechanisms between age, cholesterol, and CRC aggravation have never been evaluated in human cohorts. Design: tissue lysates and human specimens collected prospectively from CRC patients and controls were grouped according to CRC grade and divided into two groups based on age 50, the average risk for CRC. The intra-tissue content of total cholesterol and cholesteryl ester were measured using CRC lysates. We then used fluorescence-based multiplex immunohistochemistry with digital image analysis and machine learning to measure the quantitative expression of SQLE, GSK3β, p53wt, and p53mt. Results: through an epidemiological study, we estimated age-dependent increases in CRC incidence and the percentage of a group with high cholesterol around age 50. Considering this age-dependency, we demonstrated age and CRC-grade-dependent manner cholesterol accumulated inside CRC tissues, SQLE, p53wt, and p53mt degraded, and GSK3β inactivated. SQLE and GSK3β were clinically associated with increased risk and showed good diagnostic and prognostic performance in CRC patients older than 50. Furthermore, the combined value for four variables exhibited superior diagnostic performance in discriminating from CRC patients after age 50 to controls. Conclusion: this study provides new biological perceptions explaining the effects of age in the progression of CRC aggravation and potential biomarkers associated with a high CRC-risk population. Citation Format: Soo Young Jun, Ji-Yong Yoon, Jeong-Ju Lee, Nam-Soon Kim. The age-dependent intratissue accumulation of cholesterol, resulting in reduced squalene epoxidase, is associated with a high colorectal cancer risk population [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 2113.

Circ_0005615 Regulates the Progression of Colorectal Cancer Through the miR-873-5p/FOSL2 Signaling Pathway.

To determine the effects of circ_0005615 in CRC development and underneath mechanism. The expression levels of circ_0005615, microRNA-873-5p (miR-873-5p) and FOS-like antigen 2 (FOSL2) mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of exosome makers, proliferation-related makers and FOSL2 were detected by western blot or immunohistochemistry assay. Cell proliferation was evaluated by cell counting kit-8 (CCK-8) and cell colony formation assays. Cell migration and invasion were demonstrated by a transwell assay. Cell apoptosis was investigated by flow cytometry analysis. The binding relationship between miR-873-5p and circ_0005615 or FOSL2 was predicted by circular RNA interactome and targetscan online databases, respectively, and identified by dual-luciferase reporter assay. The impacts of circ_0005615 silencing on tumor formation were determined by in vivo tumor formation assay. Circ_0005615 expression was dramatically upregulated in serum exosomes of CRC patients compared with the control group. The CRC patients with a high circ_0005615 expression had a poor survival rate. Circ_0005615 and FOSL2 expressions were apparently increased, while miR-873-5p was decreased in CRC tissues or cells relative to control groups. Circ_0005615 knockdown inhibited cell proliferation, migration, and invasion, whereas promoted cell apoptosis in CRC; however, miR-873-5p inhibitor attenuated these impacts. Additionally, circ_0005615 acted as a sponge of miR-873-5p and miR-873-5p bound to FOSL2. FOSL2 overexpression restrained the effects of miR-873-5p mimic on CRC progression. Furthermore, circ_0005615 knockdown suppressed tumor growth in vivo. Circ_0005615 modulated CRC malignant progression by controlling FOSL2 expression through sponging miR-873-5p. This finding lays a foundation for the study on circRNA-mediated CRC therapy.

Characterization of the immune cell landscape in CRC: Clinical implications of tumour-infiltrating leukocytes in early- and late-stage CRC.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths globally. Tumour-infiltrating leukocytes play an important role in cancers, including CRC. We therefore sought to characterize the impact of tumour-infiltrating leukocytes on CRC prognosis. To determine whether the immune cell profile within CRC tissue could influence prognosis, we employed three computational methodologies (CIBERSORT, xCell and MCPcounter) to predict abundance of immune cell types, based on gene expression. This was done using two patient cohorts, TCGA and BC Cancer Personalized OncoGenomics (POG). We observed significant differences in immune cell composition between CRC and normal adjacent colon tissue, as well as differences in based on method of analysis. Evaluation of survival based on immune cell types revealed dendritic cells as a positive prognostic marker, consistently across methodologies. Mast cells were also found to be a positive prognostic marker, but in a stage-dependent manner. Unsupervised cluster analysis demonstrated that significant differences in immune cell composition has a more pronounced effect on prognosis in early-stage CRC, compared to late-stage CRC. This analysis revealed a distinct group of individuals with early-stage CRC which have an immune infiltration signature that indicates better survival probability. Taken together, characterization of the immune landscape in CRC has provided a powerful tool to assess prognosis. We anticipate that further characterization of the immune landscape will facilitate use of immunotherapies in CRC.

Patterns of Somatic Variants in Colorectal Adenoma and Carcinoma Tissue and Matched Plasma Samples from the Hungarian Oncogenome Program.

Analysis of circulating cell-free DNA (cfDNA) of colorectal adenoma (AD) and cancer (CRC) patients provides a minimally invasive approach that is able to explore genetic alterations. It is unknown whether there are specific genetic variants that could explain the high prevalence of CRC in Hungary. Whole-exome sequencing (WES) was performed on colon tissues (27 AD, 51 CRC) and matched cfDNAs (17 AD, 33 CRC); furthermore, targeted panel sequencing was performed on a subset of cfDNA samples. The most frequently mutated genes were APC, KRAS, and FBN3 in AD, while APC, TP53, TTN, and KRAS were the most frequently mutated in CRC tissue. Variants in KRAS codons 12 (AD: 8/27, CRC: 11/51 (0.216)) and 13 (CRC: 3/51 (0.06)) were the most frequent in our sample set, with G12V (5/27) dominance in ADs and G12D (5/51 (0.098)) in CRCs. In terms of the cfDNA WES results, tumor somatic variants were found in 6/33 of CRC cases. Panel sequencing revealed somatic variants in 8 out of the 12 enrolled patients, identifying 12/20 tumor somatic variants falling on its targeted regions, while WES recovered only 20% in the respective regions in cfDNA of the same patients. In liquid biopsy analyses, WES is less efficient compared to the targeted panel sequencing with a higher coverage depth that can hold a relevant clinical potential to be applied in everyday practice in the future.

Non-coding RNAs are correlated to TGF-β receptor type 2 in patients with colorectal cancer.

Multiple molecular expression alterations, in particular, in noncoding RNAs, play fundamental roles in the regulations of cellular processes and may relate to the occurrence and progression of CRC. In this study, we investigated the associations between TGFBR2, miR20a-5p and lncRNA LAMTOR5-AS1 in CRC patients. The colorectal cancer and adjacent normal tissue samples (n=34) were prepared from CRC patients. The associations between TGFBR2, miR20a-5p and lncRNA LAMTOR5-AS1 were predicted using bioinformatics tools. The expression levels of TGFBR2, miR20a-5p and lncRNA LAMTOR5-AS1 were measured using Quantitative Real-Time PCR technique. The TGFBR2 protein values were measured by western blotting method. The clinical importance of lncRNA LAMTOR5-AS1 was assessed using receiver operating characteristic (ROC) curve. The up-regulated levels of TGFBR2 (P = 0.02), TGFBR2 protein (P = 0.008) and lncRNA LAMTOR5-AS1 (P = 0.02) were significantly observed in CRC tissues as compared with the adjacent normal tissues. The miR20a-5p expression level (P = 0.009) was downregulated in CRC tissues. In addition, the miR20a-5p expression level was inversely correlated to the TGFBR2 gene (R² = 0.88, P <0.0001), protein (R² = 0.95, P <0.0001) and lncRNA LAMTOR5-AS1 gene (R² = 0.93, P <0.0001) expression levels. Based on the area under curve (AUC), the increase of lncRNA LAMTOR5-AS1 expression level with a sensitivity of 64.52 % and specificity of 65.52% was considered in CRC patients. We suggested that the miR20a-5p is inversely related to lncRNA LAMTOR5-AS so that it may be involved in the regulation of TGFBR2 expression level in CRC patients.

Comprehensive analysis of alfa defensin expression and prognosis in human colorectal cancer.

Colorectal cancer (CRC) is a serious threat to human health. Screening new biomarkers can provide basis for improving the prognosis and individualized treatment of CRC. Although some members of the defensin family were found increased in pancreatic cancer and CRC, their exact function and clinical significance remain unclear. In this study, the expression, correlation, mutation, and functional enrichment of several defensin family members in pancreatic cancer and CRC were analyzed using tumor public databases and verified in several patients. Results showed no significant correlation between the expression levels of DEFA1-4 and CRC. The expression levels of DEFA5 and DEFA6 significantly increased in CRC tissues compared with those in normal tissues. DEFA5 may be associated with better prognosis of CRC, while DEFA6 may be associated with poor prognosis. Immunohistochemistry (IHC) experiments showed that the expression of DEFA6 was significantly higher in adenoma than in normal mucosa and slightly higher in carcinoma than in normal mucosa. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that DEFAs were closely related to hsa05202: transcriptional misregulation in cancer and Hsa04015: Rap1 signaling pathway. DEFA5 may be a stable and good prognostic marker, and DEFA6 may be a poor prognostic marker in CRC of metastasis. Overall, DEFA5 and DEFA6 have a certain degree of sensitivity and specificity in predicting CRC.

Sodium Butyrate Induces CRC Cell Ferroptosis via the CD44/SLC7A11 Pathway and Exhibits a Synergistic Therapeutic Effect with Erastin.

Human normal intestinal epithelial cell line FHT and colorectal tumor cell line HCT-116 were treated with NaB alone or in combination with different programmed cell death inhibitors. Cell activity was then assessed with MTT assays and PI staining; ferroptosis with Fe2+, glutathione (GSH), and lipid peroxidation assays; signaling pathway screening with PCR arrays; and CD44, SCL7A11, and GPX4 expression with Western blotting. A CD44-overexpressing HCT-116 cell line was constructed to determine the effect of the overexpression of CD44 on NaB-induced ferroptosis. The synergistic effect of co-treatment with NaB and Erastin was assessed by isobolographic analysis. NaB induced apoptosis and ferroptosis in HCT-116 cells but only induced low-level apoptosis in FHC cells. Moreover, NaB significantly increased intracellular Fe2+ and promoted GSH depletion and lipid peroxidation in HCT-116 cells. Ferroptosis-related qPCR array analysis identified CD44/SLC7A11 as a potential effector molecular of NaB-induced ferroptosis. NaB significantly inhibited the expression of CD44 and SLC7A11 in mouse CRC tissues. A CD44 overexpressed HCT-116 cell line was used to verify that CD44/SLC7A11 was a key signaling pathway that NaB-induced GSH depletion, lipid peroxidation accumulation, and ferroptosis in HCT-116 cells. Examination of whether NaB can increase the effect of ferroptosis agents showed that NaB, in combination with Erastin, a ferroptosis inducer, further promoted HCT-116 cell death and increased changes of ferroptosis markers. Our results suggest that NaB induces ferroptosis in CRC cells through the CD44/SLC7A11 signaling pathway and has synergistic effects with Erastin. These results may provide new insights into CRC prevention and the combined use of NaB and ferroptosis-inducing agents.

The role of mechanical channel in the proliferation and migration of colon cancer

Piezo channel is the first family of mechanically gated cation channels found in mammals and has been shown correlated with the proliferation and metastasis of tumors in recent years. In a variety of cancer tissues, colon cancer tissues are particularly affected by mechanical stimulation. In this paper, we reported that the role of piezo1 in the proliferation and migration of colon cancer. Firstly, bioinformatics indicated that high levels of piezo1 expression existed in CRC tissues and were associated with poor prognosis. In vitro experiments, SW1116 cells were cultured in DMSO solutions, and CCK-8 assay and transwell assay were separately performed. It turned out that the inhibition of PIEZO1 by Dooku1 resulted in diminished cell proliferation and metastasis, while the activation of PIEZO1 by Yoda1 led to enhanced proliferation and migration of colon cells. Collectively, these findings demonstrated the facilitation role of piezo1 in colon cancer proliferation and migration, suggesting that piezo1 has the potential as a novel therapeutic target for drug design and treatment of colon cancer.

BST2 induced macrophage M2 polarization to promote the progression of colorectal cancer.

Background: Tumor-associated macrophages (TAMs) are one of the most prominent tumor-infiltrating immune cells in the tumor microenvironment (TME) of CRC and play a vital role in the progression of CRC. BST2 was predicted to be associated with the infiltration of TAMs. However, its potential function by which CRC cells and TAMs interact with each other still needs further investigation. Methods: The target genes in CRC were selected by bioinformatics screening. The level of bone marrow stromal cell antigen 2 (BST2) in CRC cells and tissues was determined by qRT‒PCR, Western blotting, and immunohistochemistry staining. In vitro and in vivo assays were applied to clarify the function of BST2. Results: In this study, according to bioinformatics analysis, a nomogram based on the risk score (constructed by BST2 and CAV1 (caveolin-1)) and clinical features was built and displayed satisfactory prognostic value. Upregulated BST2 was significantly related to Braf mutation, dMMR/MSI-H, CMS1 subtype, and immune response and was a potential biomarker for predicting immune checkpoint blockade therapy. Silencing BST2 in CRC obviously restrained CRC progression and M2 TAM polarization. The infiltration of TAMs was positively correlated with the high expression of BST2, and depletion of TAMs alleviated the protumoural effect of BST2 in CRC in vivo. In vitro experiments revealed that a reduction in BST2 in CRC inhibited CRC proliferation and migration and also M2 polarization. Conclusion: These findings indicated that BST2 played a vital role in CRC progression and might be a predictable marker for immunotherapy.

CD163 as a Potential Biomarker in Colorectal Cancer for Tumor Microenvironment and Cancer Prognosis: A Swedish Study from Tissue Microarrays to Big Data Analyses.

(1) Background: CD163, a specific macrophage receptor, affects the progression of malignant tumors. Unfortunately, the regulation and expression of CD163 are poorly understood. In this study, we determined the expressions of CD163 in TMA samples from CRC patients and combined them with patient data from several Swedish hospitals. (2) Methods: The expressions of CD163 in tissue samples from CRC patients were examined. After combining 472 CRC patients' gene expression and 438 CRC patients' clinical data with the TCGA database, 964 cases from the GEO database, and experimental expression data from 1247 Swedish CRC patients, we selected four genes (PCNA, LOX, BCL2, and CD163) and analyzed the tumor-infiltrating immune cells (TICs) and CRC prognosis. (3) Results: Based on histopathological TMA analysis, CD163 was strongly expressed in the stroma of both normal and cancer tissues, and the expressions in normal and cancer cells varied from negative to strong. The results from public databases show decreased expression of CD163 in cancer tissue compared to normal mucosa (|log FC| &gt; 1 and FDR &lt; 0.01), and it is a negative prognostic factor for CRC patients (p-value &lt; 0.05). Through tumor microenvironment (TME) analysis, we found a potential influence of CD163 on immune cell infiltration. Furthermore, the enrichment analysis indicated the possible interaction with other proteins and biological pathways. (4) Conclusions: CD163 is expressed differently in CRC tissue and is a negative prognostic factor. Its expression is associated with the TME and tumor purity of CRC. Considering all results, CD163 has the potential to be a predictive biomarker in the investigation of CRC.

CircPACRGL promoted cell proliferation, migration and invasion as well as inhibited cell apoptosis in colorectal cancer via regulation of the miR-330-3p/CNBP axis.

CircRNAs are a member of noncoding RNAs and have been verified to play an important regulatory role in cancers. In CRC, the regulatory mechanisms of various circRNAs have not been elucidated. The expression of circPACRGL and miR-330-3p was detected with qRT-PCR. The protein expression of CDK4, MMP-9, Bcl-2, Bax, cellular nucleic acid-binding protein (CNBP) and β-actin was measured with western blot. Cell proliferation was analyzed using MTT assay, colony formation assay, and EDU assay. Cell apoptosis was detected using flow cytometry. Cell migration and invasion were measured with wound healing and transwell invasion assay. Luciferase reporter assay and RIP assay was used to determine the relationship of among miR-330-3p, circPACRGL and CNBP in CRC cells. In this study, we found that circPACRGL and CNBP expressed high and miR-330-3p expressed low in CRC tissues and cells. Functional experiments showed that inhibition of circPACRGL reduced cell proliferation, migration and invasion in CRC. In addition, knockdown of circPACRGL contributed to cell apoptosis in CRC. Dual-luciferase report assay determined that circPACRGL was a miR-330-3p sponge molecular and CNBP was a target of miR-330-3p. Reversed experiments showed that the effects of sh-circPACRGL transfection on CRC cells were rescued by up-regulating CNBP expression. In this study, we for the first time found a novel regulatory network of circPACRGL in CRC. The results manifested that circPACRGL affected tumor growth by targeting miR-330-3p/CNBP axis in CRC, highlighting the potential of circPACRGL as a therapeutic target for colorectal cancer.