Colorectal Cancer Tumor Tissues Research Articles

MiR-4780 Derived from N2-Like Neutrophil Exosome Aggravates Epithelial-Mesenchymal Transition and Angiogenesis in Colorectal Cancer.

Despite significant advances in diagnostic methods and treatment strategies, the prognosis for patients with advanced colon cancer remains poor, and mortality rates are often high due to metastasis. Increasing evidence showed that it is of significant importance to investigate how the tumor microenvironment participates in the development of colorectal cancer (CRC). In this manuscript, neutrophils were sequentially stimulated with all-trans retinoic acid and transforming growth factor-β in turn to induce the neutrophil polarization. Differentially expressed miRNA in neutrophil exosomes have been sequenced by microarray profile, and the effect of N2-like neutrophil-derived exosomal miR-4780 on epithelial-mesenchymal transition (EMT) and angiogenesis was investigated. In our results, we found that neutrophils were enriched in CRC tumor tissue and that CD11b expression correlated with tumor site and serous membrane invasion. At the same time, we demonstrated that internalization of N2 exosomes exacerbated the viability, migration, and invasion of CRC cell lines and inhibited apoptosis. To further investigate the molecular mechanism, we analyzed the miRNA expression profile in the N2-like neutrophils, which led to the selection of hsa-miR-4780 for the subsequent experiment. The overexpression of miR-4780 from N2-like neutrophil-derived exosomes exacerbated EMT and angiogenesis. Moreover, miR-4780 can regulate its target gene SOX11 to effect EMT and angiogenesis in CRC cell lines. CRC with liver metastasis model also validated that aberrant expression of miR-4780 in N2-like neutrophil exosomes exacerbated tumor metastasis and development of tumor via EMT and angiogenesis. In conclusion, our current findings reveal an important mechanism by which mR-4780 from N2-like neutrophil exosomes exacerbates tumor metastasis and progression via EMT and angiogenesis.

The Pathogenicity of Fusobacterium nucleatum Modulated by Dietary Fibers-A Possible Missing Link between the Dietary Composition and the Risk of Colorectal Cancer.

The dietary composition has been approved to be strongly associated with the risk of colorectal cancer (CRC), one of the most serious malignancies worldwide, through regulating the gut microbiota structure, thereby influencing the homeostasis of colonic epithelial cells by producing carcinogens, i.e., ammonia or antitumor metabolites, like butyrate. Though butyrate-producing Fusobacterium nucleatum has been considered a potential tumor driver associated with chemotherapy resistance and poor prognosis in CRC, it was more frequently identified in the gut microbiota of healthy individuals rather than CRC tumor tissues. First, within the concentration range tested, the fermentation broth of F. nucleatum exhibited no significant effects on Caco-2 and NCM460 cells viability except for a notable up-regulation of the expression of TLR4 (30.70%, p < 0.0001) and Myc (47.67%, p = 0.021) and genes encoding proinflammatory cytokines including IL1B (197.57%, p < 0.0001), IL6 (1704.51%, p < 0.0001), and IL8 (897.05%, p < 0.0001) in Caco-2 cells exclusively. Although no marked effects of polydextrose or fibersol-2 on the growth of F. nucleatum, Caco-2 and NCM460 cells were observed, once culture media supplemented with polydextrose or fibersol-2, the corresponding fermentation broths of F. nucleatum significantly inhibited the growth of Caco-2 cells up to 48.90% (p = 0.0003, 72 h, 10%) and 52.96% (p = 0.0002, 72 h, 10%), respectively in a dose-dependent manner. These two kinds of fibers considerably promoted butyrate production of F. nucleatum up to 205.67% (p < 0.0001, 6% polydextrose at 24 h) and 153.46% (p = 0.0002, 6% fibersol-2 at 12 h), which explained why and how the fermentation broths of F. nucleatum cultured with fibers suppressing the growth of Caco-2 cells. Above findings indicated that dietary fiber determined F. nucleatum to be a carcinogenic or antitumor bacterium, and F. nucleatum played an important role in the association between the dietary composition, primarily the content of dietary fibers, and the risk of CRC.

Biglycan regulated colorectal cancer progress by modulating enteric neuron-derived IL-10 and abundance of Bacteroides thetaiotaomicron

Biglycan (BGN) is a proteoglycan with branch chains and highly expressed in enteric neurons in the tumor tissue of colorectal cancer (CRC), which is negatively associated with survival rates in patients with CRC. However, how the proteoglycan promotes the progress of CRC through interacting with bacteria and regulating the immune response of enteric neurons remains largely unknown. In the present study, we found that biglycan deficiency changed tumor distribution in a colitis-associated colon cancer model. Furthermore, we revealed that BGN deficiency inhibits tumor growth in an allograft tumor model and the migration of cancer cell by upregulating interleukin-10 expression in enteric neurons. Significantly, we demonstrated that biglycan deficiency enriched the abundance of Bacteroides thetaiotaomicron through competing with it for chondroitin sulfate to inhibit CRC progress. Our work provided new insights into the interaction between host proteoglycan and gut microbiota as well as the role of enteric neurons in the tumor microenvironment.

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.

Comparative Analysis of Gene Expression Profile in Tumor and Healthy Tissue in Patients with Colorectal Cancer

INTRODUCTION: Colorectal cancer (CRC) is a sure leader among malignant neoplasms (12.3%), without taking into account gender. Five-year survival rate in stage I CRC is 91%, in stage IV 14%. The currently existing treatment methods are helpless to significantly reduce mortality the approaches should be personalized and include the use of molecular genetic methods.&#x0D; AIM: To perform a comparative evaluation of expression profile of samples of tumor and healthy colon tissue in CRC.&#x0D; MATERIALS AND METHODS: The material for the study was 19 samples of tumor tissue taken from the pathologically altered colonic mucosa of 19 patients with CRC, and 7 samples of healthy tissue taken 10 cm12 cm distally or proximally from the visual boundary of the tumor. Biopsy materials were homogenized using a mechanical method. The quality and quantity of ribonucleic acid in the eluted solution were evaluated using IMPLEN nanospectrophotometer (Germany). Gene expression was evaluated using microchip kit SurePrint G3 HumanGeneExpv3 ArrayKit (Agilent, USА). Microchips were scanned on InnoScan 1100 AL apparatus (США) with subsequent image processing in Mapix Software program (USA).&#x0D; RESULTS: The analysis of expression profile demonstrated 505 differentially expressed genes, 337 of which showed reduced expression and 168 enhanced expression in the tumor material. The highest expression was demonstrated by genes bound with miRNA: hsa-miR-29b-3p and hsa-miR-1-5p, and also genes Н19, FOXQ1, INHBA, MMP1, CDH3, CXCL2, MDFI, THBS2. On the contrary, genes TMIGD1, GUCA2B, ZG16, AQP8, SLC4A4, CDKN2B-AS1, CA4, СА1 demonstrated a low expression in the tumor material. Expression of genes responsible for functioning of signal pathways: IL-17, NF-kappa B, TNF, was increased in tumor samples. Genes responsible for signal pathways Fatty acid degradation, Drug metabolism cytochrome P450, Metabolic pathways, Fatty acid metabolism and Steroid hormone biosynthesis, showed reduced expression.&#x0D; CONCLUSION: Significant differences were found in the expression profile of tumor and healthy tissue in patients with CRC. A comparative analysis of gene enrichment and the data of the international databases permitted to identify a number of terms, genes, clusters that can be used in future in search for predictors of prognosis and of response to treatment.

The B56γ3-containing protein phosphatase 2A attenuates p70S6K-mediated negative feedback loop to enhance AKT-facilitated epithelial-mesenchymal transition in colorectal cancer

BackgroundProtein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC).MethodsPolyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein–protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients.ResultsWe showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC.ConclusionsOur finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC.CUkGCcvqWD-y5KjYaB3kRRVideo

Profound Diversity of the N-Glycome from Microdissected Regions of Colorectal Cancer, Stroma, and Normal Colon Mucosa

• Striking diversity of N -glycosylation was found between cancerous, stromal and healthy mucosa cells. • Cancer showed increased α2-6 sialylation and monoantennary N -glycans and decreased bisection compared to healthy mucosa. • N -glycans carrying (sialyl-)Lewis A/X were exclusively found in cancer. • Stroma showed higher fucosylation and (α2-3) sialylation and lower oligomannosidics, monoantennaries, Lewis A/X and sulfation compared to cancer. Aberrant glycosylation is considered to be a hallmark of colorectal cancer (CRC), as demonstrated by various studies. While the N -glycosylation of cell lines and serum has been widely examined, the analysis of cancer-associated N -glycans from tissues has been hampered by the heterogeneity of tumors and the complexity of N -glycan structures. To overcome these obstacles, we present a study using laser capture microdissection that makes it possible to largely deconvolute distinct N- glycomic signatures originating from different regions of heterogeneous tissues including cancerous, stromal, and healthy mucosa cells. N -glycan alditols were analyzed by means of porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry, enabling the differentiation and structural characterization of isomeric species. In total, 116 N- glycans were identified that showed profound differences in expression among cancer, stroma, and normal mucosa. In comparison with healthy mucosa, the cancer cells showed an increase in α2-6 sialylation and monoantennary N -glycans, as well as a decrease in bisected N -glycans. Moreover, specific sialylated and (sialyl-)Lewis A/X antigen-carrying N -glycans were exclusively expressed in cancers. In comparison with cancer, the stroma showed lower levels of oligomannosidic and monoantennary N -glycans, Lewis A/X epitopes, and sulfation, as well as increased expression of (core-)fucosylation and α2-3 sialylation. Our study reveals the distinct N -glycomic profiles of different cell types in CRC tumor and control tissues, proving the necessity of their separate analysis for the discovery of cancer-associated glycans.

SLC9A5 promotes tumor growth and cell motility via ACOX1-mediated peroxisomal fatty acid oxidation

Growing evidence suggests a strong association between decreased lipid catabolism and the development of cancer. Solute carrier family 9 member A5 (SLC9A5) plays a regulatory role in colorectal function. However, the specific involvement of SLC9A5 in colorectal cancer (CRC) remains unclear, as well as its potential connection to lipid catabolism. We found that SLC9A5 exhibited significantly higher expression in CRC tumor tissues compared to adjacent paratumor tissues, as confirmed through analysis of the TCGA database and validation on a CRC tissue chip using IHC. Furthermore, in vitro experiments showed that knockdown of SLC9A5 resulted in suppressed cell proliferation, migration, and invasion. Then we performed bioinformatics analysis and found that SLC9A5 was significantly enriched in peroxisomal fatty acid oxidation (FAO) pathway and negatively correlated with its first rate-limiting enzyme acyl-CoA oxidases (ACOX). Interestingly, the expression of ACOX1, as well as FAO process indicated by changes in very long chain fatty acid levels, were enhanced upon SLC9A5 knockdown in CRC cells. Moreover, the attenuated tumor growth, migration, invasion, and increased FAO observed after SLC9A5 knockdown could be reversed by simultaneous knockdown of both SLC9A5 and ACOX1. In summary, these findings reveal the oncogenic role of SLC9A5 in CRC, particularly in relation to ACOX1-mediated peroxidation, and might serve as a promising therapeutic target for inhibiting the progression of colorectal cancer.

Differences in tissue-associated bacteria between metastatic and non-metastatic colorectal cancer.

Accumulated evidence indicates that the intestinal microbiota plays crucial roles in the initiation and progression of colorectal cancer (CRC). However, the effects of the tissue-associated microbiota on CRC metastasis are poorly defined. The aim of this study was to explore the differences in bacteria between metastatic and non-metastatic CRC tissues and identify potential bacterial species that associate with CRC metastasis. 16S rDNA amplicon high-throughput sequencing was used to test the intestinal tissue-associated microbiota in patients with metastatic CRC (n = 48) and non-metastatic CRC (n = 44). The microbial diversity and differential species were analysed by standard microbiological methods, and then the differential bacteria were confirmed by qPCR. Receiver operating characteristic (ROC) curves were plotted to evaluate the ability of the differential bacteria in predicting the metastasis of CRC. In addition, the microbial compositions of tumor-adjacent tissues from the metastatic and non-metastatic CRC groups were analysed. The α- or β-diversity of microbial community between the metastatic and non-metastatic CRC groups did not exhibit significant differences. However, some bacterial abundances between two groups showed significant differences. At the phylum level, Bacteroidota and Desulfobacterota were significantly higher in the metastatic group than in the non-metastatic group, while Proteobacteria was significantly decreased in the metastatic group. At the genus level, Bacteroides (mainly composed of Bacteroides fragilis and Bacteroides uniformis) was significantly higher in the metastatic group than in the non-metastatic group, while Streptococcus and Escherichia-Shigella were significantly decreased. The ROC curves of the selected bacteria showed area under the curve (AUC) values ranging from 0.598 to 0.69; when CEA and the selected bacteria were combined, the AUC values increased from 0.678 to 0.705. In addition, the bacterial composition of tumor-adjacent tissues from the metastatic and non-metastatic CRC groups were also different, and the differential bacteria were consistent with those between metastatic and non-metastatic CRC tumor tissues. The bacterial composition of tumor and tumor adjacent tissue from the metastatic CRC group was different from that of the non-metastatic CRC group; in particular, Bacteroides was increased, and Streptococcus was decreased. These findings are helpful to further reveal the mechanism of CRC metastasis and provide new ideas for the clinical diagnosis and treatment of CRC metastasis.

Predictive value of inflammatory regulators TGFb1 and CXCL8 in tumor tissue in colorectal cancer

Background. Colorectal cancer is ranked third in terms of incidence and second in terms of mortality around the world. Molecular markers of chemoresistance allow to determine the prognosis of the disease and sensitivity of the tumor to drugs.Aim. To assess the predictive value of expression of regulators of tumor-associated inflammation TGFb1 and CXCL8 in the tumor tissue in colorectal cancer.Materials and methods. Patients were divided into 3 groups: group I included patients without relapse of the disease, group II encompassed patients with relapse of the disease (within 6–16 months after the end of chemotherapy), group III included patients with disease progression. Expression of TGFb1 and CXCL8 in the tumor tissue before treatment in patients with stage II–III colorectal cancer (n = 77) was determined using quantitative realtime polymerase chain reaction (PCR) on the Bio-Rad CFX-96 Touch Real-Time PCR Detection System (USA). Statistical data processing was performed using Statistica 13.0 software (StatSoft, USA).Results. We found that in samples of poorly differentiated colorectal cancer, the level of TGFb and CXCL8 mRNA was significantly higher than in moderately and well differentiated tumors. We did not reveal any relationship of the level of TGFb1 and CXCL8 transcripts in tumor samples of patients with stage II–III colorectal cancer with age and the presence of mutations in the EGFR (Epidermal Growth Factor Receptor) signaling pathway (RAS, BRAF). We found a strong positive correlation between the levels of TGFb1 and CXCL8 transcripts for the entire sample of patients with colorectal cancer. We have found that the expression of TGFb1 and CXCL8 genes was significantly higher in the tumor tissue of patients with disease progression.Conclusion. Overexpression of TGFb1 and CXCL8, which are involved in the mechanism of tumor-associated inflammation, can be considered as a negative prognostic factor for the progression-free interval when using the FOLFOX / XELOX regimen for the treatment of colorectal cancer.

STAT2-induced linc02231 promotes tumorigenesis and angiogenesis through modulation of hnRNPA1/ANGPTL4 in colorectal cancer.

Long non-coding RNAs (lncRNAs) play a critical role in regulating various human diseases including cancer. In colorectal cancer (CRC), there are still some undervalued lncRNAs with potential functions and mechanisms that need to be clarified. The purpose of this project is to study the role of linc02231 in the progression of CRC. The proliferation of CRC cells was evaluated using the CCK8 assay, colony formation assay, and EdU assay. Cell migration was examined through wound healing and Transwell analyses. The impact of linc02231 on angiogenesis was determined through a tube formation assay. Western blotting was used to detect the expression of specific proteins. A mouse xenograft model is established to observe the effect of linc02231 on the in vivo growth of CRC cells. Target genes of linc02231 are screened using high-throughput sequencing. The transcriptional activity of STAT2 on linc02231 and the binding activity between linc02231/miR-939-5p/hnRNPA1 were analyzed by luciferase assay. Based on public databases and comprehensive bioinformatics analysis, we found that lncRNA linc02231 was upregulated in CRC tumour tissues, which is consistent with our clinical results. Linc02231 promoted the proliferation and migration of CRC cells in vitro and their tumorigenicity in vivo. Furthermore, linc02231 promotes the angiogenic ability of human umbilical vein endothelial cells (HUVECs). Mechanistically, the transcription factor STAT2 binds to the promoter region of linc02231 and activates its transcription. Linc02231 also competes with miR-939-5p for binding to the pro-oncogenic target gene hnRNPA1, preventing its degradation. hnRNPA1 prevents the maturation of ANGPTL4 messenger RNA, leading to impaired tumor angiogenesis and increased metastasis of CRC. The expression of linc02231, which is induced by STAT2, has been found to enhance the proliferation, metastasis, and angiogenesis of CRC by binding to miR-939-5p and increasing the expression of hnNRPA1 while suppressing ANGPTL4. These findings suggest that linc02231 could serve as a potential biomarker and therapeutic target for CRC.

A senescence-based prognostic gene signature for colorectal cancer and identification of the role of SPP1-positive macrophages in tumor senescence.

Senescence is significantly associated with cancer prognosis. This study aimed to construct a senescence-related prognostic model for colorectal cancer (CRC) and to investigate the influence of senescence on the tumor microenvironment. Transcriptome and clinical data of CRC cases were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Senescence-related prognostic genes detected by univariate Cox regression were included in Least Absolute Shrinkage and Selection Operator (LASSO) analysis to construct a model. The efficacy of the model was validated using the receiver operating characteristic (ROC) curve and survival analysis. Differentially expressed genes (DEGs) were identified and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed. CIBERSORT and Immuno-Oncology Biological Research (IOBR) were used to investigate the features of the tumor microenvironment. Single-cell RNA-seq data were used to investigate the expression levels of model genes in various cell types. Immunofluorescence staining for p21, SPP1, and CD68 was performed with human colon tissues. A seven-gene (PTGER2, FGF2, IGFBP3, ANGPTL4, DKK1, WNT16 and SPP1) model was finally constructed. Patients were classified as high- or low-risk using the median score as the threshold. The area under the ROC curve (AUC) for the 1-, 2-, and 3-year disease-specific survival (DSS) were 0.731, 0.651, and 0.643, respectively. Survival analysis showed a better 5-year DSS in low-risk patients in the construction and validation cohorts. GO and KEGG analyses revealed that DEGs were enriched in extracellular matrix (ECM)-receptor interactions, focal adhesion, and protein digestion and absorption. CIBERSORT and IOBR analyses revealed an abundance of macrophages and an immunosuppressive environment in the high-risk subgroup. Low-risk patients had higher response rates to immunotherapy than high-risk patients. ScRNA-seq data revealed high expression of SPP1 in a subset of macrophages with strong senescence-associated secretory phenotype (SASP) features. Using CRC tumor tissues, we discovered that SPP1+ macrophages were surrounded by a large number of senescent tumor cells in high-grade tumors. Our study presents a novel model based on senescence-related genes that can identify CRC patients with a poor prognosis and an immunosuppressive tumor microenvironment. SPP1+ macrophages may correlate with cell senescence leading to poor prognosis.

Abstract 2241: Transcriptomic analysis of tetraspanins in colorectal cancer

Abstract Introduction: Transmembrane proteins and their overexpression have been highly involved in malignancies. In particular, Tetraspanins (TSPANs)—a family of four-span transmembrane proteins—have been proposed as “master organizers” of multi-molecular membrane complexes by associating with specific partners and thus contributed to the progression of many cancer types. To date, transcriptomic analysis of all human TSPANs in colorectal cancer (CRC) has remained unclear. Here, we focused on utilizing next-generation RNA sequencing (RNA-Seq) to explore the expression level of TSPANs in primary tumor tissues of CRC, which is a complex disease and the second cause of cancer mortality worldwide. Experimental Procedures: Resected primary tumor tissues of CRC were collected from 100 patients along with the written informed consents. Later, total RNA was extracted from all frozen patient tissues. RNA-Seq of those RNA samples was next done before the transcriptomic analysis of TSPANs in CRC was performed. Immunofluorescence (IF) and Immunohistochemistry (IHC) stains were also employed to confirm the target protein expression in formalin-fixed paraffin-embedded tissue (FFPE) slides from CRC patients. Results: As a result of transcriptomic analysis of TSPANs in CRC tissues, among 33 human TSPANs, the high expression levels (transcript per million above the average of all TSPANs) of TSPAN1, TSPAN8, TSPAN24, TSPAN29 and TSPAN30 were observed in the patient-derived CRC tissues. Notably, the expression level of TSPAN8 was comparable with that of EPCAM, which is a well-known tumor marker. Moreover, the high expression of TSPAN8 protein could be clearly observed in both IF and IHC-stained FFPE slides from CRC patients. Our result highlights the possible relevance of these overexpressed TSPANs in CRC development. Conclusions: This study is the first RNA-Seq-based transcriptomic analysis of all human TSPANs in CRC. Our finding suggests the significant overexpression of particular TSPANs, especially TSPAN8, in CRC. Further analyses will be beneficial for translational research in CRC. The overexpression of TSPANs may suggest a framework to further explore diagnostic, prognostic, and predictive surface biomarkers, therapeutic targets, and other applications for CRC. Citation Format: Thanawat Suwatthanarak, Pariyada Tanjak, Amphun Chaiboonchoe, Onchira Acharayothin, Kullanist Thanormjit, Tharathorn Suwatthanarak, Watsaphon Tangkullayanone, Manop Pithukpakorn, Vitoon Chinswangwatanakul. Transcriptomic analysis of tetraspanins in 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 2241.

Do Elevated YKL-40 Levels Drive the Immunosuppressive Tumor Microenvironment in Colorectal Cancer? Assessment of the Association of the Expression of YKL-40, MMP-8, IL17A, and PD-L1 with Coexisting Type 2 Diabetes, Obesity, and Active Smoking

The influence of chitinase-3-like protein 1 (YKL-40 or CHI3L1) expression on the immunological properties of the tumor microenvironment, which may affect the effectiveness of immunotherapy, is currently not sufficiently understood in colorectal cancer (CRC). The aim of this study was to investigate the relationship between YKL-40 expression and the immunological properties of the tumor microenvironment in CRC. We performed in silico analysis, including analysis of immune cell infiltration scores and the immune landscape depending on YKL-40 expression, gene set enrichment analysis (GSEA), and analysis of three Gene Expression Omnibus (GEO) datasets. In 48 CRC tissue homogenates and the surgical margin, we analyzed the expression of YKL-40, MMP8, IL17A, and PD-L1. Moreover, we analyzed the expression of YKL-40 in tissue homogenates retrieved from patients with coexisting diabetes, obesity, and smoking. The expression of YKL-40 was significantly higher in CRC tumor tissue compared to healthy tissue and correlated with MMP-8, IL17A, and PD-L1 expression. In silico analysis revealed an association of YKL-40 with disease recurrence, and GSEA revealed a potential link between elevated YKL-40 expression and immunosuppressive properties of the tumor microenvironment in CRC.

Overexpression and Role of HHLA2, a Novel Immune Checkpoint, in Colorectal Cancer.

The study aimed to investigate correlations between HHLA2 levels and parameters, including microsatellite instability (MSI) status, CD8+ cells, and histopathological features: budding, tumor-infiltrating lymphocytes (TILs), TNM scale, grading, cytokines, chemokines, and cell signaling moleculesin colorectal cancer (CRC). Furthermore, the immune infiltration landscape and HHLA2-related pathways in colorectal cancer using available online datasets were analyzed. The study included 167 patients diagnosed with CRC. Expression of HHLA2 was detected by immunohistochemistry method (IHC) and enzyme-linked immunosorbent assay (ELISA). The IHC was used to evaluate the MSI and CD8+ status. The budding and TILs were measured using a light microscope. The concentrations of cytokines, chemokines, and cell signaling molecules were measured to analyze the data by the Bio-Plex Pro Human cytokine screening panel, 48 cytokine assay, and principal component analysis (PCA). Geneset enrichment analysis (GSEA) was conducted to identify HHLA2-related pathways. The biological function of HHLA2 was predicted by Gene Ontology (GO). Analysis of the immune infiltration landscape of HHLA2 in colorectal cancer was made by the web-based tool Camoip. High HHLA2 expression was detected in CRC tumor tissues compared to the adjacent noncancerous tissues. The percentage of HHLA2-positive tumors was 97%. GSEA and GO showed that HHLA2 upregulation correlated with cancer-related pathways and several biological functions. Tumor-infiltrating lymphocytes score correlated positively with IHC HHLA2 expression level percentage. There was a negative correlation between HHLA2, anti-tumor cytokines and pro-tumor growth factors. This study provides a valuable insight into the role of HHLA2 in CRC. We reveal the role of HHLA2 expression as well as a stimulatory and inhibitory immune checkpoint in colorectal cancer. Further research may verify the therapeutic values of the HHLA2-KIR3DL3/TMIGD2 pathway in colorectal cancer.

MiR-7-5p/KLF4 signaling inhibits stemness and radioresistance in colorectal cancer

Resistance to radiotherapy remains a major unmet clinical obstacle in the treatment of locally advanced rectal cancer. Cancer stem cells (CSCs) are considered to mediate tumor development and radioresistance. However, the role of CSCs in regulating resistance to radiotherapy in colorectal cancer (CRC) remains largely unknown. We established two radioresistant CRC cell lines, HCT116-R and RKO-R, using fractionated irradiation. Analysis using miRNA sequencing and quantitative real-time PCR confirmed lower levels of miR-7-5p in both of the radioresistant cells compared to their parental cells. Subsequently, we validated that miR-7-5p expression was decreased in cancerous tissues from radiotherapy-resistant rectal cancer patients. The Cancer Genome Atlas (TCGA) database analyses revealed that low miR-7-5p expression was significantly correlated with poor prognosis in CRC patients. Overexpression of miR-7-5p led to a rescue of radioresistance and an increase in radiation-induced apoptosis, and attenuated the stem cell-like properties in HCT116-R and RKO-R cells. Conversely, knocking down miR-7-5p in parental HCT116 and RKO cells suppressed the sensitivity to radiation treatment and enhance cancer cell stemness. Stemness-associated transcription factor KLF4 was demonstrated as a target of miR-7-5p. Rescue experiments revealed that miR-7-5p/KLF4 axis could induce radiosensitivity by regulating CSCs in colorectal cancer cells. Furthermore, we used CRC tumor tissues which exhibited resistance to neoadjuvant radiotherapy to establish a patient-derived xenograft (PDX) mouse model. Tail vein injection of magnetic nanoparticles carrying miR-7-5p mimics into the PDX mice significantly inhibited tumor growth with or without irradiation treatment in vivo. Our current studies not only demonstrate an anti-cancer function of miR-7-5p in regulating CSC properties and radiosensitivity in colorectal cancer, but also provide a novel potential strategy for delaying or reverse radiation resistance in preoperative radiotherapy of CRC patients.

PKCδ promotes the invasion and migration of colorectal cancer through c-myc/NDRG1 pathway.

Colorectal cancer (CRC) is the third cause of expected cancer deaths both in men and women in the U.S. and the third most commonly diagnosed cancer in China Targeted therapy has been proven to improve overall survival for unresectable metastatic CRC. But the location of the primary tumor or the presence of various core driver gene mutations that confer resistance may limit the utility of targeted therapy. Therefore, it is of great significance to further elucidate novel mechanisms of invasion and metastasis of CRC and find potential novel therapeutic targets. Protein Kinase C Delta (PKCδ) plays an important role in various diseases, including tumors. In CRC, the function of PKCδ on proliferation and differentiation is mostly studied but various research results were reported. Therefore, the role of PKCδ in CRC needs to be further studied, especially in tumor invasion and metastasis in CRC which few studies have looked into. The expression of PRKCD was analyzed by the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases and Immunohistochemical (IHC). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) enrichment analysis were used to explore the biological functions and pathways related to PRKCD. Lentivirus transfection was used to construct CRC cell lines with overexpression and knock-down of PKCδ or N-myc Downstream Regulated Gene 1 (NDRG1). Cell invasion and migration assay, wound healing assay were used to detect the function of PKCδ and NDRG1 in the invasion and migration of cells. Flow cytometry analysis was used to detect the influence of PKCδ on the CRC cell cycles .Immunofluorescence histochemistry ,Immunoprecipitation Assay and qPCR were used to detect the relationship of PKCδ and NDRG1. Xenograft model was used to verify the role of PKCδ in vivo. PKCδ is overexpressed in CRC and could promote Epithelial-Mesenchymal Transition (EMT) and the invasion and migration of CRC in vitro. We confirmed that PKCδ and the tumor suppressor factor NDRG1 had a co-localization relationship in CRC. PKCδ inhibited NDRG1 transcription and protein expression. Overexpressing NDRG1 could inhibit the function of PKCδ in promoting tumor invasion and migration. PKCδ could regulate c-Myc, one transcription factor of NDRG1, to down-regulate NDRG1. In vivo, overexpressing PKCδ could promote xenograft growth and volume. Thus, our results showed that PKCδ reduced the expression of NDRG1 through c-Myc, promoting the invasion and migration of CRC through promoting EMT. The increased expression of PKCδ in CRC tumor tissue could promote the invasion and migration of tumor cells, and one of the mechanisms may be regulating c-Myc to inhibit the expression of NDRG1 and promote EMT.

Long Non-Coding RNA BCAR4 Promotes Oxaliplatin Resistance in Colorectal Cancer by Modulating miR-484-3p/RAB5C Expression

Background: Oxaliplatin-based chemotherapy resistance is a major cause of recurrence in patients with colorectal cancer (CRC). Increasing evidence indicates that lncRNA BCAR4 is involved in the occurrence and development of various cancers. However, the effect of BCAR4 on CRC chemotherapy resistance remains unclear. Methods: Real-time quantitative PCR and Western blotting were used to detect the expression levels of gene and protein, respectively. The role of BCAR4 in drug resistance was evaluated by cell viability and apoptosis experiments. Luciferase reporter assay and Western blot analysis confirmed the relationship between BCAR4, miR-483-3p, and RAB5C. Results: Luciferase reporter assay and Western blotting analysis confirmed the relationship among BCAR4, miR-483-3p, and RAB5C. The results showed that the expression levels of BCAR4 and RAB5C were increased in CRC tumor tissue. The expression levels of BCAR4 were increased in patients with chemotherapy resistance. Functional analysis showed that knockdown of BCAR4 reduced the expression levels of proteins related to stemness, decreased the activity of cells, and promoted apoptosis of CRC cells, while overexpression of RAB5C reversed these effects. Moreover, the results showed that BCAR4 promoted oxaliplatin resistance by inhibiting cell apoptosis. Mechanistically, BCAR4 sponged miR-483-3p and promoted the expression of RAB5C. Knockdown of BCAR4 reduced tumor size and enhanced cell sensitivity to oxaliplatin in vivo. Conclusion: The results suggested that BCAR4/miR-483-3p/RAB5C axis has the potential to be explored as a novel therapeutic target for CRC treatment.

ANP32A Knockdown Attenuates the Malignant Biological Behavior of Colorectal Cancer Cells by Suppressing Epithelial-mesenchymal Transition and ERK Activation.

Acidic leucine rich nuclear phosphoprotein-32A (ANP32A) protein has a variety of functions, such as regulating cell differentiation, influencing cell apoptosis and cell cycle progression. Our previous study demonstrated that high expression of ANP32A was found in the tumor tissues of colorectal cancer (CRC) patients and was positively associated with tumor grading. However, the function and underlying mechanisms of ANP32A in CRC metastasis have not been fully explored. In this study, we found that ANP32A knockdown significantly attenuated the migration and invasion, and epithelial-mesenchymal transition (EMT) in cells. Further mechanistic studies revealed that ANP32A knockdown inhibited the expression of β-catenin and phosphorylated-ERK. The immunofluorescent staining experiment has revealed that ANP32A was expressed in the cell membrane, cytosol and nucleus, and its expression was positively associated with β-catenin expression levels. Moreover, the ability of cell migration and invasion was inhibited, the expression of E-cadherin was enhanced following ANP32A knockdown, and these affects were abolished by an ERK activator PMA, enhanced by an ERK inhibitor PD98059. Moreover, our animal experiment also demonstrated that silenced ANP32A inhibited CRC cell growth, multi-organ metastasis, ERK activation and EMT progression in vivo. Collectively, these findings demonstrated that ANP32A promotes CRC progression and that may be a promising target for the anti-metastasis treatment of CRC.

Identification of Biomarkers Associated with the Prognoses of Colorectal Cancer Patients

Introduction: Colorectal cancer (CRC) is a common cancer. As metastasis and recurrence are main causes of CRC death, it is of great significance to find prognostic biomarkers. Methods: Data related to CRC were collected from GEO database. The patients were grouped based on clinical information, and the differentially expressed genes (DEGs) were obtained by differential analysis. GO and KEGG pathway enrichment analyses were conducted based on DEGs. Cox combined with LASSO regression analysis was applied to screen out the key genes that used to build the prognostic model. Survival curve and receiver operating characteristic curve were employed to evaluate the validity and reliability of the model. Cox regression analysis was applied to determine the independence of risk score. GSEA and GSVA analyses were performed on patients with different risks according to the risk model scores, and the prognostic nomogram was plotted combined with clinical data. Also, qRT-PCR was applied to examine the expression status of the screened signatures in clinical cases. Results: We obtained 302 DEGs by dividing CRC patients into early-stage and advanced-stage groups. The results of enrichment analyses demonstrated that the DEGs were mainly concentrated in tissues of extracellular matrix, epithelial cell proliferation, and cell adhesion-related pathways. Regression identified 9 hub genes notably correlated with prognosis, including CLK1, SLC2A3, LIPG, EPHB2, ATOH1, PLCB4, GZMB, CKMT2, and CXCL11. The validation of the risk model proved that the risk model was accurate and could independently determine the prognosis of patients. Finally, differences were found in pathway activity of extracellular matrix secretion, plaque secretion, Notch signaling pathway, and tight junctions in high-risk and low-risk patients. In addition to LIPG and CKMT2, other feature genes were notably overexpressed in CRC tumor tissues. Conclusion: The results proved that the expression levels of the 9 biomarkers could be used to predict the prognosis of CRC patients.