Primary Colorectal Cancer Samples Research Articles

Exploring somatic mutations in POLE and POLD1: Their role in colorectal cancer pathogenesis and potential therapeutic strategies

Colorectal cancer (CRC) is one of the major causes of morbidity and mortality worldwide, resulting from the accumulation of genetic and epigenetic alterations in several oncogenes and tumor suppressor genes. Recent studies have identified germline and somatic mutations in the exonuclease domain regions of both epsilon polymerase (POLE) and delta polymerase (POLD1) genes in CRCs. We sought to examine the mutation of these genes in a series of sporadic CRCs. To do this, we extracted DNA from 100 primary CRC samples and 40 corresponding normal tissue samples, which had been previously characterized for clinicopathological and molecular features. We employed a combination of quick-multiplex consensus (QMC)-polymerase chain reaction (PCR) and co-amplification at lower denaturation temperature (COLD)-PCR, followed by high-resolution melting (HRM) analysis and Sanger sequencing, to investigate the exonuclease domain regions of POLE and POLD1 genes for somatic mutations that may potentially alter the proofreading activities of these genes. In silico predictions of the functional significance of the identified genetic alterations were performed using the protein variation effect analyzer, sorting intolerant from tolerant, and PON-P2 algorithms. We identified a total of eight new and non-recurrent somatic variants in the endonuclease domains of POLE and two in POLD1. Nine out of ten variants caused amino acid substitutions, whereas one resulted in a stop codon. Although no significant associations or correlations were found between the POLE/POLD1 mutations and the clinicopathological or molecular features of the CRC cases, most of the POLE/POLD1-mutated cases were microsatellite stable (90%) and aneuploid (80%). Furthermore, in silico analyses showed that nine of the ten variants would likely cause some adverse effect on protein function. Ten somatic variants with predicted proofreading activity-altering effects have been identified in the endonuclease domains of POLE and POLD1 in CRC using a combination of QMC-PCR, COLD-PCR, HRM analyses, and Sanger sequencing.

Identification and validation of metastasis-related gene ZG16 in the prognosis and progression in colorectal cancer.

Metastasis remains the leading cause of mortality among colorectal cancer (CRC) patients. Identification of new metastasis-related genes are critical to improve colorectal cancer prognosis. Data on mRNA expression in metastatic and primary CRC was obtained from the Gene Expression Omnibus (GEO) database, including GSE81986, GSE41568, GSE71222, GSE21510, and GSE14333. Additionally, data concerning mRNA expression in colon cancer (COAD) and adjacent normal tissues were acquired from The Cancer Genome Atlas (TCGA) database. Hub genes were identified by weighted gene co-expression network analysis (WGCNA) and differential gene expression analysis. Moreover, we assessed the impact of hub gene expression on both overall survival (OS) and disease-free survival (DFS) in patients and identified ZG16 as a potential target. We generated CRC cell lines transfected with lentivirus OE-ZG16 to investigate proliferation, invasion, and migration in vitro. To further elucidate the involvement of ZG16, we utilized gene set enrichment analysis (GSEA) to identify enriched pathways, which were subsequently validated via Western blot analysis. Five datasets containing primary and metastatic CRC samples from GEO database and CRC samples from TCGA database were included in this study and 29 hub genes were identified by WGCNA and differentially expressed gene (DEG) analysis. Low expression of the hub genes (CLCA1 and ZG16) was associated with poor DFS and OS. We confirmed the low expression of ZG16 in CRC using external database and IHC analysis at both transcriptional and protein levels. In addition, the expression of ZG16 was notably elevated in NCM460 cells in comparison to CRC cell lines. The overexpression of ZG16 in CRC cells has been shown to inhibit the proliferation, invasion, and migration of CRC cells. Furthermore, the overexpression of ZG16 has been found to suppress the activation of the epithelial-mesenchymal transition (EMT) and Wnt/β-catenin signaling pathways in CRC. ZG16 may serve as a promising therapeutic target for metastatic CRC treatment.

RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis.

The N6-methyladenosine (m6A) RNA-binding protein YTHDF1 is frequently overexpressed in colorectal cancer and drives chemotherapeutic resistance. To systematically identify druggable targets in colorectal cancer with high expression of YTHDF1, this study used a CRISPR/Cas9 screening strategy that revealed RUVBL1 and RUVBL2 as putative targets. RUVBL1/2 were overexpressed in primary colorectal cancer samples and represented independent predictors of poor patient prognosis. Functionally, loss of RUVBL1/2 preferentially impaired the growth of YTHDF1-high colorectal cancer cells, patient-derived primary colorectal cancer organoids, and subcutaneous xenografts. Mechanistically, YTHFD1 and RUVBL1/2 formed a positive feedforward circuit to accelerate oncogenic translation. YTHDF1 bound to m6A-modified RUVBL1/2 mRNA to promote translation initiation and protein expression. Coimmunoprecipitation and mass spectrometry identified that RUVBL1/2 reciprocally interacted with YTHDF1 at 40S translation initiation complexes. Consequently, RUVBL1/2 depletion stalled YTHDF1-driven oncogenic translation and nascent protein biosynthesis, leading to proliferative arrest and apoptosis. Ribosome sequencing revealed that RUVBL1/2 loss impaired the activation of MAPK, RAS, and PI3K-AKT signaling induced by YTHDF1. Finally, the blockade of RUVBL1/2 by the pharmacological inhibitor CB6644 or vesicle-like nanoparticle-encapsulated siRNAs preferentially arrested the growth of YTHDF1-expressing colorectal cancer in vitro and in vivo. Our findings show that RUVBL1/2 are potential prognostic markers and druggable targets that regulate protein translation in YTHDF1-high colorectal cancer. Significance: RUVBL1/2 inhibition is a therapeutic strategy to abrogate YTHDF1-driven oncogenic translation and overcome m6A dysregulation in colorectal cancer.

New treatment alternatives for primary and metastatic colorectal cancer by an integrated transcriptome and network analyses

Metastatic colorectal cancer (CRC) is still in need of effective treatments. This study applies a holistic approach to propose new targets for treatment of primary and liver metastatic CRC and investigates their therapeutic potential in-vitro. An integrative analysis of primary and metastatic CRC samples was implemented for alternative target and treatment proposals. Integrated microarray samples were grouped based on a co-expression network analysis. Significant gene modules correlated with primary CRC and metastatic phenotypes were identified. Network clustering and pathway enrichments were applied to gene modules to prioritize potential targets, which were shortlisted by independent validation. Finally, drug-target interaction search led to three agents for primary and liver metastatic CRC phenotypes. Hesperadin and BAY-1217389 suppress colony formation over a 14-day period, with Hesperadin showing additional efficacy in reducing cell viability within 48 h. As both candidates target the G2/M phase proteins NEK2 or TTK, we confirmed their anti-proliferative properties by Ki-67 staining. Hesperadinin particular arrested the cell cycle at the G2/M phase. IL-29A treatment reduced migration and invasion capacities of TGF-β induced metastatic cell lines. In addition, this anti-metastatic treatment attenuated TGF-β dependent mesenchymal transition. Network analysis suggests IL-29A induces the JAK/STAT pathway in a preventive manner.

Mismatch repair protein deficiency and its implications on distant metastasis in colorectal cancer: A comprehensive analysis.

While previous studies have indicated variability in distant metastatic potential among different mismatch repair (MMR) states in colorectal cancer (CRC), their findings remain inconclusive, especially considering potential differences across various ethnic backgrounds. Furthermore, the gene regulatory networks and the underlying mechanisms responsible for these variances in metastatic potential across MMR states have yet to be elucidated. We collected 2058 consecutive primary CRC samples from the South West of China and assessed the expression of MMR proteins (MLH1, MSH2, MSH6, and PMS2) using immunohistochemistry. To explore the inconsistencies between different MMR statuses and recurrence, we performed a meta-analysis. To delve deeper, we employed Weighted Gene Co-expression Network Analysis (WGCNA), ClueGo, and iRegulon, pinpointing gene expression networks and key regulatory molecules linked to metastasis and recurrence in CRC. Lastly, both univariate and multivariate Cox regression analyses were applied to determine the impact of core regulatory molecules on metastasis. Of the samples, 8.2% displayed deficient MMR (dMMR), with losses of MLH1 and PSM2 observed in 40.8% and 63.9%, respectively. A unique 24.3% isolated loss of PMS2 without concurrent metastasis was identified, a result that diverges from established literature. Additionally, our meta-analysis further solidifies the reduced recurrence likelihood in dMMR CRC samples compared to proficient MMR (pMMR). Two gene expression networks tied to distant metastasis and recurrence were identified, with a majority of metastasis-related genes located on chromosomes 8 and 18. An IRF1 positive feedback loop was discerned in the metastasis-related network, and IRF1 was identified as a predictive marker for both recurrence-free and distant metastasis-free survival across multiple datasets. Geographical and ethnic factors might influence peculiarities in MMR protein loss. Our findings also highlight new gene expression networks and crucial regulatory molecules in CRC metastasis, enhancing our comprehension of the mechanisms driving distant metastasis.

The epigenetic downregulation of LncGHRLOS mediated by RNA m6A methylase ZCCHC4 promotes colorectal cancer tumorigenesis

Backgroundm6A modification is currently recognized as a major driver of RNA function that maintains cancer cell homeostasis. Long non-coding (Lnc) RNAs control cell proliferation and play an important role in the occurrence and progression of colorectal cancer (CRC). ZCCHC4 is a newly discovered m6A methyltransferase whose role and mechanism in tumors have not yet been elucidated.MethodsThe EpiQuik m6A RNA methylation kit was used to detect the level of total RNA m6A in six types of digestive tract tumors. The Kaplan-Meier method and receiver operating characteristic curve were used to evaluate the prognostic and diagnostic value of the newly discovered m6A methyltransferase, ZCCHC4, in CRC. The effects on CRC growth in vitro and in vivo were studied using gain- and loss-of-function experiments. The epigenetic mechanisms underlying ZCCHC4 upregulation in CRC were studied using RIP, MeRIP-seq, RNA pull-down, and animal experiments.ResultsWe reported that the ZCCHC4-LncRNAGHRLOS-KDM5D axis regulates the growth of CRC in vitro and in vivo. We found that ZCCHC4 was upregulated in primary CRC samples and could predict adverse clinical outcomes in patients with CRC. Mechanistically, ZCCHC4 downregulated LncRNAGHRLOS to promote CRC tumorigenesis. As a downstream molecule of LncRNAGHRLOS, KDM5D directly controls CRC cell proliferation, migration, and invasion.ConclusionThis study suggests that the ZCCHC4 axis contributes to the tumorigenesis and progression of CRC and that ZCCHC4 may be a potential biomarker for this malignancy.

DNA Methylome and Transcriptome Maps of Primary Colorectal Cancer and Matched Liver Metastasis

Sequencing-based genome-wide DNA methylation, gene expression studies and associated data on paired colorectal cancer (CRC) primary and liver metastasis are very limited. We have profiled the DNA methylome and transcriptome of matched primary CRC and liver metastasis samples from the same patients. Genome-scale methylation and expression levels were examined using Reduced Representation Bisulfite Sequencing (RRBS) and RNA-Seq, respectively. To investigate DNA methylation and expression patterns, we generated a total of 1.01 × 109 RRBS reads and 4.38 × 108 RNA-Seq reads from the matched cancer tissues. Here, we describe in detail the sample features, experimental design, methods and bioinformatic pipeline for these epigenetic data. We demonstrate the quality of both the samples and sequence data obtained from the paired samples. The sequencing data obtained from this study will serve as a valuable resource for studying underlying mechanisms of distant metastasis and the utility of epigenetic profiles in cancer metastasis.

Dissecting the heterogeneities of the tumor microenvironment between metastatic and nonmetastatic primary colorectal cancer patients by single-cell RNA sequencing

One of the main factors hampering the long-term prognosis of colorectal cancer (CRC) patients is distant metastasis. However, the driving factors of CRC metastasis have not been clarified at the single-cell level, which limits the in-depth study of accurate prediction and prevention of CRC metastasis to improve the prognosis. Heterogeneities in the tumor microenvironment (TME) between metastatic and nonmetastatic CRC were investigated by single-cell RNA (scRNA) sequencing data. In detail, 50,462 single cells from 20 primary CRC samples, including 40,910 cells from nonmetastatic CRC (M0 group) and 9552 cells from metastatic CRC (M1 group), were systematically analyzed in this study. Based on the single-cell atlas, we revealed that cancer cells and fibroblasts accounted for relatively high proportions in metastatic CRC compared with nonmetastatic CRC. Moreover, two specific cancer cell subtypes (FGGY+SLC6A6+ and IGFBP3+KLK7+ cancer cells) and three specific fibroblast subtypes (ADAMTS6+CAPG+, PIM1+SGK1+ and CA9+UPP1+ fibroblasts) in metastatic CRC were identified. The functional and differentiation characteristics of these specific cell subclusters were elucidated by enrichment and trajectory analyses. These results provide fundamental knowledge for future in-depth research to screen effective methods and drugs to predict and prevent CRC metastasis to improve prognosis.

Abstract 4647: Potential of TIMP3 regulating the versican status in the colorectal cancer tumor microenvironment

Abstract Background: In colorectal cancer (CRC), versican (VCAN) accumulation has been found to negatively correlate with CD8+ tumor infiltrating lymphocyte (TIL) abundance and its proteolysis into versikine (Vkine) positively correlates with TIL abundance. TIMP3 has demonstrated the ability to inhibit several ADAMTS proteases able to cleave VCAN to Vkine. Here we investigate the correlation of TIMP3 accumulation in CRCs with the VCAN status and CD8+ T cell infiltration. Methods: Matched primary and metastatic CRC samples from 156 patients who had resected oligometastatic CRC were stained for VCAN, Vkine, TIMP3, and CD8+ TILs. VCAN, Vkine, and TIMP3 were scored using an intensity binning system ranging from 0-3+ and T cells in the epithelium were reported per high power field (HPF). Tumors were designated as high (2 or 3+) or low (0 or 1) for both VCAN and Vkine and positive (+; 1-3) or negative (0) for TIMP3. Tumors designated VCAN low and Vkine high are considered VCAN proteolytic predominant (VPP) and all other combinations are considered VCAN proteolytic weak (VPW). Results: Across the whole dataset, 52% of cancers were VCAN high and 24% were VPP. TIMP3 was detected in 33% of cancers. The frequency of TIMP3 was similar between primary and metastatic tumors (39% and 29%, respectively). Of the VCAN high cancers, 42% were TIMP3+ whereas 61% of the TIMP3+ samples are VCAN high. Of the Vkine high cancers, 44% were TIMP3+ and 36% of VPP cancers were TIMP3+. The prevalence of the VPP phenotype was similar between the TIMP3+ and negative cancers. Additionally, among the TIMP3+ samples 68% were Vkine high versus 46% in TIMP3 negative cancers (p=0.001). VCAN proteolysis correlates with higher CD8+ TIL abundance, thus we assessed the number of CD8+ TILs in TIMP3+ and negative tumors. The TIMP3+ cancers had a median of 2 CD8+ TILs/HPF and the TIMP3 negative cancers has a median of 3 CD8+ TILs/HPF. For VPP cancers, those that were also TIMP3+ had a median of 3 CD8+ TILs/HPF compared to TIMP3 negative cancers which had a median of 6 CD8+ TILs/HPF (p=0.2). Conclusions: TIMP3 correlates with higher levels of VCAN accumulation but does not correlate with lower levels of proteolysis. Future studies are needed to better clarify the impact of TIMP3 and other mediators in regulating VCAN accumulation and proteolysis in the tumor microenvironment. Citation Format: Anna Field, Sean Kraus, Kristina Matkowskyj, Dustin Deming, Cheri A. Pasch, Wei Zhang. Potential of TIMP3 regulating the versican status in the colorectal cancer tumor microenvironment. [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 4647.

Cyclin‐dependent kinase 12‐mediated super‐enhancers as promising targets in cancer therapy

Cyclin‐dependent kinase 12‐mediated super‐enhancers as promising targets in cancer therapy

A Colorectal Cancer 3D Bioprinting Workflow as a Platform for Disease Modeling and Chemotherapeutic Screening.

Colorectal cancer (CRC) is the third most common malignancy and has recently moved up to the second leading cause of death among carcinomas. Prognosis, especially for advanced diseases or certain molecular subtypes of CRC, remains poor, which highlights the urgent need for better therapeutic strategies. However, currently, as little as 0.1% of all drugs make it from bench to bedside because of the inherently high false-positive and false-negative rates of current preclinical and clinical drug testing data. Therefore, the success of developing novel treatment agents lies in the introduction of improved preclinical disease models which resemble in vivo carcinomas closer, possess higher predictive properties, and offer opportunities for individualized therapies. Aiming to address these needs, we have established an affordable, flexible, and highly reproducible 3D bioprinted CRC model. The histological assessment of Caco-2 cells in 3D bioprints revealed the formation of glandular-like structures which show greater pathomorphological resemblance to tumors than monolayer cultures do. RNA expression profiles in 3D bioprinted cells were marked by upregulation of genes involved in cell adhesion, hypoxia, EGFR/KRAS signaling, and downregulation of cell cycle programs. Testing this 3D experimental platform with three of the most commonly used chemotherapeutics in CRC (5-fluoruracil, oxaliplatin, and irinotecan) revealed overall increased resistance compared to 2D cell cultures. Last, we demonstrate that our workflow can be successfully extended to primary CRC samples. Thereby, we describe a novel accessible platform for disease modeling and drug testing, which may present an innovative opportunity for personalized therapeutic screening.

Expression of neurofibromin 1 in colorectal cancer and cetuximab resistance.

Neurofibromin 1 (NF1) is a tumor suppressor that has been previously reported to regulate RAS-MAPK signaling. The present study investigated the possible relationship between NF1 expression and anti-EGFR antibody (cetuximab) sensitivity in colorectal cancer cell lines. In addition, primary or metastatic colorectal cancer samples from patients treated with cetuximab were assessed for the association of cetuximab sensitivity. The quantities of the NF1 transcript, NF1-related pathway enrichment and NF1 mutation profile were measured and investigated using RNA sequencing and targeted DNA sequencing. Based on growth inhibition and colony formation assay results, cell lines were designated to be cetuximab-sensitive (NCI-H508 and Caco2) or cetuximab-resistant (KM12C and SM480). Western blotting revealed NF1 was highly expressed in cetuximab-sensitive cell lines whilst there was little expression in their cetuximab-resistant counterparts. Knocking down NF1 expression using small interfering RNA in the cetuximab-sensitive cell lines enhanced the phosphorylation of MEK and ERK according to western blotting. NF1 knockdown also reduced apoptosis, as observed by the decreased number of apoptotic bodies by DAPI nuclear staining and reduced cleavage of caspase and poly-(ADP ribose) polymerase. NF1 overexpression by transfection with GTPase-activating protein-related domain subunit rendered the cetuximab-resistant cell lines, KM12C and SW480, more susceptible to cetuximab-induced apoptosis. RNA sequencing of 111 RAS and BRAFV600 wild-type tumor samples collected from cetuximab-treated patients with metastatic colorectal cancer revealed that the pre-treatment NF1 expression levels were not associated with the cetuximab response. However, tumor samples obtained after cetuximab treatment displayed slightly lower NF1 transcript levels compared with those in the pre-treatment samples, suggesting that exposure to the anti-EGFR antibody may be associated with reduced NF1 expression levels. Next-generation sequencing revealed that the frequency of inactivating mutations in NF1 were rare (1.8%) in patients with colorectal cancer and were not associated with the protein expression levels of NF1 except for in a small number of cases (0.5%), where the biallelic inactivation of NF1 was observed. To conclude, the present study showed that modification of NF1 expression can affect sensitivity to cetuximab in colorectal cancer cell lines, though a limitation exists in terms of its potential application as a biomarker for RAS and BRAF V600 wild-type tumors.

Genome-Wide Identification of m6A-Associated Single-Nucleotide Polymorphisms in Colorectal Cancer.

BackgroundN6-methyladenosine (m6A)-associated single-nucleotide polymorphisms (SNPs) play important roles in cancers, with previous research suggesting potential associations between m6A-SNPs and cancer. However, the relationship between the genetic determinants of m6A modification and colorectal cancer (CRC) remains unclear.MethodsAn integrative method combining raw data and summary statistics of genome-wide association studies with expression quantitative trait loci (eQTL) and differential expression data was applied to screen potential candidate CRC-associated m6A-SNPs.ResultsA total of 402 m6A-SNPs were identified as being associated with CRC (p < 0.001), with 98 showing eQTL signals. In particular, three genes were found to harbor CRC-associated m6A-SNPs: rs178184 in NOVA1, rs35782901 in HTR4, and rs60571683 in SLCO1B3. These genes were differentially expressed in at least one publicly available dataset (p < 0.05), with NOVA1 (p = 3.41×10−11) and HTR4 (p = 5.56×10−7) being significantly downregulated in CRC (dataset: GSE89076), and SLCO1B3 was significantly overexpressed (datasets: GSE32323 [p = 3.27×10−5], GSE21510 [p = 1.09×10−6], and GSE89076 [p = 7.63×10−6]).ConclusionThis study identified three m6A-SNPs (rs178184, rs35782901, and rs60571683) that may be associated with CRC. However, the lack of analysis of primary CRC samples in order to further elucidate the underlying pathogenesis is a major limitation of this study. Future investigations are needed to validate these CRC-associated m6A-SNPs and explore the m6A-mediated pathogenic mechanism in CRC.

Combined Targeting of AKT and mTOR Synergistically Inhibits Formation of Primary Colorectal Carcinoma Tumouroids In Vitro: A 3D Tumour Model for Pre-therapeutic Drug Screening.

Pre-therapeutic analysis of three-dimensional spheroid cultures of primary tumour samples is a promising approach of assessing susceptibility to potential treatment. The phosphatidylinositol-3-kinase/AKT serine/threonine kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway is frequently activated in colorectal cancer (CRC). In previous work, we showed combined inhibition of AKT and mTOR to be highly synergistic in cell lines from patients with hepatocellular carcinoma and cholangiocarcinoma in vitro as well as in vivo in murine xenograft tumour models. Patient-derived xenograft colorectal carcinoma cell lines HROC80 T1 M1, HROC147 T0 M1, HROC147Met, HROC277 T0 M1 and HROC277Met2 were treated with AKT inhibitor MK2206, mTOR inhibitor RAD001 or the combination of both drugs. The sensitivity of these cell lines to inhibition was evaluated by calculation of combinatory indices after bromodeoxyuridine assays and analysis of the respective pathways by western blotting. Furthermore, the dual inhibition of AKT and mTOR was confirmed in vivo in a xenograft mouse model. Additionally, primary CRC samples of four patients were embedded in a three-dimensional matrix and the sensitivity of these samples was analyzed by measurement of the spheroid area. In this study, we demonstrate that combined treatment with MK2206 and RAD001 resulted in strong synergistic effects on growth of several primary CRC cell lines and reduced the growth of a patient-derived CRC xenograft in a xenotransplantation mouse model in vivo. Interestingly, the response to treatment varied between cell lines derived from the primary lesion and a liver metastasis of the same patient. In addition, combined treatment with AKT and mTOR inhibitors resulted in a synergistic inhibition of tumouroid growth in all four of the primary patient samples, analyzed in a three-dimensional spheroid model in vitro. Our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of cell lines and primary tumour cells from patients with CRC and may be a promising approach for the treatment of CRC.

Functional relationship between CFTR and RAC3 expression for maintaining cancer cell stemness in human colorectal cancer.

CFTR mutations not only cause cystic fibrosis, but also increase the risk of colorectal cancer. A putative role of CFTR in colorectal cancer patients without cystic fibrosis has so far, however, not been investigated. RAC3 is a nuclear receptor coactivator that has been found to be overexpressed in several human tumors, and to be required for maintaining cancer stemness. Here, we investigated the functional relationship between CFTR and RAC3 for maintaining cancer stemness in human colorectal cancer. Cancer stemness was investigated by analysing the expression of stem cell markers, clonogenic growth and selective retention of fluorochrome, using stable transfection of shCFTR or shRAC3 in HCT116 colorectal cancer cells. In addition, we performed pathway enrichment and network analyses in both primary human colorectal cancer samples (TCGA, Xena platform) and Caco-2 colorectal cancer cells including (1) CD133+ or CD133- side populations and (2) CFTRwt or CFTRmut cells (ConsensusPathDB, STRING, Cytoscape, GeneMANIA). We found that the CD133+ side population expresses higher levels of RAC3 and CFTR than the CD133- side population. RAC3 overexpression increased CFTR expression, whereas CFTR downregulation inhibited the cancer stem phenotype. CFTR mRNA levels were found to be increased in colorectal cancer samples from patients without cystic fibrosis compared to those with CFTR mutations, and this correlated with an increased expression of RAC3. The expression pattern of a gene set involved in inflammatory response and nuclear receptor modulation in CD133+ Caco-2 cells was found to be shared with that in CFTRwt Caco-2 cells. These genes may contribute to colorectal cancer development. CFTR may play a non-tumor suppressor role in colorectal cancer development and maintenance involving enhancement of the expression of a set of genes related to cancer stemness and development in patients without CFTR mutations.

IgG Fc Binding Protein (FCGBP) is Down-Regulated in Metastatic Lesions and Predicts Survival in Metastatic Colorectal Cancer Patients

BackgroundThe liver is the most frequent site for metastatic spread in colorectal cancer (CRC) patients, and these patients have much poorer prognosis than those without metastasis. Previous studies have shown that IgG Fc binding protein (FCGBP) plays important roles in tumorigenesis, progression, and prognosis, but its role in CRC metastasis remains unclear.PurposeIn this study, we are aimed to explore the significance of FCGBP in liver metastatic CRC (LMCRC) patients.MethodsWe analyzed the expression of FCGBP RNA between CRC primary samples and liver metastatic samples in the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA). Next, we assessed the expression of FCGBP protein in 135 paired primary CRC (PC) samples and LMCRC samples. Finally, we explored the relationship between the expression features and clinicopathological characteristics.ResultsThe expression data of FCGBP were obtained from the GEO and TCGA databases. FCGBP RNA expression was evaluated between primary lesions (PC) and liver metastatic lesions (LM). FCGBP RNA was down-regulated in PC and LM, and especially lower in LM (p<0.001). Next, the expression of FCGBP protein was evaluated by an immunohistochemistry array in 135 paired primary tumor tissues and metastatic tissues. We found that FCGBP protein was down-regulated in primary lesions and metastatic lesions, especially in metastatic lesions. According to the immunohistochemistry score (SI), each cohort was divided into FCGBP-positive (SI=4–12) and FCGBP-negative (SI=0–3) groups. In both groups, the levels of CEA (PC group, 3.880 vs 77.049, p<0.001; LM group, 3.890 vs 14.239, p=0.008) and CA19-9 (PC group, 8.610 vs 111.700, p<0.001; LM group, 7.660 vs 19.380, p=0.037) were lower than those in the FCGBP-negative group. FCGBP positivity in the LM cohort was an independent risk factor in both overall survival (HR 1.573, 95% Cl [1.017–2.433], p=0.042) and disease-free survival (HR 1.869, 95% Cl [1.256–2.781], p=0.002).ConclusionThis study found a relationship between FCGBP expression and clinical information of LMCRC patients, and found that FCGBP expression decreased with disease development. The expression of FCGBP in liver metastasis is associated with both the overall and progression-free survival. Our results show that FCGBP could be a promising prognostic factor for LMCRC.

Glutamate receptor ionotropic, kainate 1 serves as a novel tumor suppressor of colorectal carcinoma and predicts clinical prognosis.

Colorectal cancer (CRC) is one of the most malignant cancers worldwide. However, the mechanisms of initiation and development of CRC are still largely unclear. The present study aimed to investigate the biological function and prognosis of glutamate receptor ionotropic, kainate 1 (GRIK1) in CRC. GRIK1 expression levels were analyzed in tissue microarrays containing 80 primary CRC samples using immunohistochemistry (IHC). The association between GRIK1 expression levels, clinicopathological factors and the prognosis was also investigated using Spearman's correlation analysis and Kaplan-Meier analysis, respectively. After genetic knockdown or overexpression of GRIK1, invasion/migration assays, proliferation assay, soft agar/colony formation assays, western blotting, reverse transcription-quantitative PCR and tumor xenograft models were used to investigate the function of GRIK1 both in vitro in two CRC cell lines, HCT116 and SW620, and in vivo. The results revealed that the expression levels of GRIK1 were significantly downregulated in CRC samples. Furthermore, IHC analysis indicated that the downregulated expression levels of GRIK1 were significantly associated with lymph node status and tumor size. In addition, patients with CRC with low GRIK1 expression levels demonstrated a consistently poor overall survival. The overexpression of GRIK1 inhibited the proliferation, colony formation, migration, invasion and epithelial-mesenchymal transition of HCT116 cells in vitro. In contrast, the genetic knockdown of GRIK1 promoted the proliferative, colony forming, migratory and invasive abilities of SW620 cells in vitro. Moreover, the overexpression of GRIK1 inhibited tumor growth, and liver and lung metastasis of CRC in vivo. In conclusion, the findings of the present study suggested that GRIK1 may serve as a tumor suppressor in CRC, and upregulated expression levels of GRIK1 may predict an improved prognosis for patients with CRC.

DSTYK Promotes Metastasis and Chemoresistance via EMT in Colorectal Cancer.

ObjectiveTumor metastasis and resistance to chemotherapy are two critical factors that contribute to the high death rate of colorectal cancer (CRC) patients. Metastasis is facilitated by the epithelial-mesenchymal transition (EMT) of tumor cells, which has emerged not only as a fundamental process during metastasis, but is also a key process leading to chemoresistance of cancer cells. However, the underlying mechanisms of EMT in CRC cell remain unknown. Here, we aim to assess the role of dual serine/threonine and tyrosine protein kinase (DSTYK) in CRC metastasis and chemoresistance.MethodsTo study the role of DSTYK in TGF-β-induced EMT, we employed techniques including Crispr/Cas9 knockout (KO) to generate DSTYK KO cell lines, RT-PCR to detect the mRNA expression, immunofluorescence analyses, and western blots to detect protein levels of DSTYK in the following 4 cell lines: control LS411N-TβRII and LS411N-TβRII/DSTYK KO, control LS513 and LS513/DSTYK KO cells, treated with/without TGF-β. The effects of DSTYK on apoptosis were investigated by MTT assays, flow cytometry assays, and TUNEL assays. The expression of DSTYK in CRC patients and its correlation with EMT markers were determined by bioinformatics analysis. For in vivo analysis, both xenograft and orthotopic tumor mouse models were employed to investigate the function of DSTYK in chemoresistance and metastasis of tumors.ResultsIn this study, we demonstrate that the novel kinase DSTYK promotes both TGF-β-induced EMT and the subsequent chemoresistance in CRC cells. DSTYK KO significantly attenuates TGF-β–induced EMT and chemoresistance in CRC cells. According to the Gene Expression Omnibus (GEO) database, the expression of DSTYK is not only positively correlated to the expression of TGF-β, but proportional to the death rate of CRC patients as well. Evidently, the expression of DSTYK in the metastatic colorectal cancer samples from patients was significantly higher than that of primary colorectal cancer samples. Further, we demonstrate in mouse models that chemotherapeutic drug treatment suppresses the growth of DSTYK KO tumors more effectively than control tumors.ConclusionOur findings identify DSTYK as a novel protein kinase in regulating TGF-β–mediated EMT and chemoresistance in CRC cells, which defines DSTYK as a potential therapeutic target for CRC therapy.

Identification of biomarkers for the diagnosis and treatment of primary colorectal cancer based on microarray technology.

BackgroundPrimary colorectal cancer (PCRC) is one of the most common malignant tumors in clinic, and is characterized by high heterogeneity occurring between tumors and intracellularly. Therefore, this study aimed to explore potential gene targets for the diagnosis and treatment of PCRC via bioinformatic technology.MethodsGene Expression Omnibus (GEO) was used to download the data used in this study. Differently expressed genes (DEGs) were identified with GEO2R, and the gene set enrichment analysis (GSEA) was implemented for enrichment analysis. Then, the researchers constructed a protein-protein interaction (PPI) network, a significant module, and a hub genes network.ResultsThe GSE81558 dataset was downloaded, and a total of 97 DEGs were found. There were 23 up-regulated DEGs and 74 down-regulated DEGs in the PCRC samples, compared with the control group. The PPI network included a total of 42 nodes and 63 edges. One module network consisted of 11 nodes and 25 edges. Another module network consisted of 4 nodes and 6 edges. The hub genes network was created by cytoHubba using GCG, GUCA2B, CLCA4, ZG16, TMIGD1, GUCA2A, CHGA, PYY, SST, and MS4A12.ConclusionsTen hub genes were found from the genomic samples of patients with PCRC and normal controls by bioinformatics analysis. The hub genes might provide novel ideas and evidence for the diagnosis and targeted therapy of PCRC.

Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer.

Somatic copy number alterations play a critical role in oncogenesis. Loss of chromosomal regions containing tumor suppressors can lead to collateral deletion of passenger genes. This can be exploited therapeutically if synthetic lethal partners of such passenger genes are known and represent druggable targets. Here, we report that VPS4B gene, encoding an ATPase involved in ESCRT‐dependent membrane remodeling, is such a passenger gene frequently deleted in many cancer types, notably in colorectal cancer (CRC). We observed downregulation of VPS4B mRNA and protein levels from CRC patient samples. We identified VPS4A paralog as a synthetic lethal interactor for VPS4B in vitro and in mouse xenografts. Depleting both proteins profoundly altered the cellular transcriptome and induced cell death accompanied by the release of immunomodulatory molecules that mediate inflammatory and anti‐tumor responses. Our results identify a pair of novel druggable targets for personalized oncology and provide a rationale to develop VPS4 inhibitors for precision therapy of VPS4B‐deficient cancers.