Therapeutic Target For Colorectal Cancer Research Articles

Influence of Clinical Factors on miR-3613-3p Expression in Colorectal Cancer.

Colorectal cancer (CRC) is the second most common cause of cancer-related death globally. Because of a tendency to be an asymptomatic primary tumor and therefore resulting in late detection, most CRC patients are diagnosed in the advanced stage. Several miRNAs have the potential to become novel noninvasive biomarkers measured as diagnostic and prognostic indicators of CRC to guide surgical therapies and promote the understanding of the carcinogenesis of CRC. Since the change of miR-3613-3p was associated with several types of cancer other than colorectal cancer, there is a lack of functional evidence and the results are inconsistent. We conducted a pilot microarray study in which we noted a decreased expression of miR-3613-3p in colorectal cancer cells, then we confirmed the expression of miR-3613-3p by qPCR on a group of 83 patients, including 65 patients with colorectal cancer, 5 with a benign tumor and 13 from the control group. We noted that in both malignant and benign tumors, miR-3613-3p is downgraded relative to the surrounding tissue. As a result of the study, we also observed colorectal tumor tissue and surrounding tissue in patients with colorectal cancer who received radiotherapy before surgery, which showed a significantly higher expression of miR-3613-3p compared to patients who did not receive radiotherapy. In addition, we noted that the tissue surrounding the tumor in patients with distant metastases showed a significantly higher expression of miR-3613-3p compared to patients without distant metastases. The increased expression of miR-3613-3p in patients after radiotherapy suggests the possibility of using this miR as a therapeutic target for CRC, but this requires confirmation in further studies.

KIF18A improves migration and invasion of colorectal cancer (CRC) cells through inhibiting PTEN signaling.

Kinesin family member 18A (KIF18A) is involved in the development of a variety of human malignancies. However, we have never known the influences of KIF18A on colorectal cancer (CRC). The study is designed to investigate the effect and molecular mechanism of KIF18A on the progression of colorectal cancer. We have not only analyzed the database using GEO, but have examined the effect of KIF18A on the development of CRC by subcutaneous tumorigenesis in nude mice. HE staining was used to observe tumor size. Besides, we make use of Western blotting to monitor the expression of related proteins. In addition, the scratch wound assay and Transwell assay were conducted to detect the effect of KIF18A on the migration and invasion of CRC cells. The results of GEO database analysis suggested that KIF18A had a positive correlation with the growth of CRC. The results of subcutaneous tumorigenesis and HE staining in nude mice explained that KIF18A promoted the progression of CRC. Both scratch wound assay and Transwell indicated that the migration and invasion of CRC could be promoted by KIF18A. The results of Western blot illustrated that KIF18A could forward the migration and invasion of CRC cells, and inhibit PTEN, which promoted the activation of PI3K/Akt signaling pathway, thus bringing about the expression of MMP2 and MMP9. In conclusion, KIF18A can further the activation of PI3K/Akt signaling pathway by means of inhibiting PTEN transcription. Therefore, it is inferred that that KIF18A is a therapeutic target for CRC.

A novel EGFR inhibitor, HNPMI, regulates apoptosis and oncogenesis by modulating BCL-2/BAX and p53 in colon cancer.

Colorectal cancer (CRC) is the second most lethal disease, with high mortality due to its heterogeneity and chemo-resistance. Here, we have focused on the epidermal growth factor receptor (EGFR) as an effective therapeutic target in CRC and studied the effects of polyphenols known to modulate several key signalling mechanisms including EGFR signalling, associated with anti-proliferative and anti-metastatic properties. Using ligand- and structure-based cheminformatics, we developed three potent, selective alkylaminophenols, 2-[(3,4-dihydroquinolin-1(2H)-yl)(p-tolyl)methyl]phenol (THTMP), 2-[(1,2,3,4-tetrahydroquinolin-1-yl)(4-methoxyphenyl)methyl]phenol (THMPP) and N-[2-hydroxy-5-nitrophenyl(4'-methylphenyl)methyl]indoline (HNPMI). These alkylaminophenols were assessed for EGFR interaction, EGFR-pathway modulation, cytotoxic and apoptosis induction, caspase activation and transcriptional and translational regulation. The lead compound HNPMI was evaluated in mice bearing xenografts of CRC cells. Of the three alkylaminophenols tested, HNPMI exhibited the lowest IC50 in CRC cells and potential cytotoxic effects on other tumour cells. Modulation of EGFR pathway down-regulated protein levels of osteopontin, survivin and cathepsin S, leading to apoptosis. Cell cycle analysis revealed that HNPMI induced G0/G1 phase arrest in CRC cells. HNPMI altered the mRNA for and protein levels of several apoptosis-related proteins including caspase 3, BCL-2 and p53. HNPMI down-regulated the proteins crucial to oncogenesis in CRC cells. Assays in mice bearing CRC xenografts showed that HNPMI reduced the relative tumour volume. HNPMI is a promising EGFR inhibitor for clinical translation. HNPMI regulated apoptosis and oncogenesis by modulating BCL-2/BAX and p53 in CRC cell lines, showing potential as a therapeutic agent in the treatment of CRC.

Knockdown of long non-coding RNA LINC01123 plays a molecular sponge on miR-625-5p to inhibit the process of colorectal cancer cells via LASP1.

Colorectal cancer (CRC) at an advanced stage of cancer has a lower 5-year survival rate. Research on the molecular biological mechanisms of CRC is helpful for disease prevention and treatment. Long non-coding RNAs (lncRNAs) were shown to be suitable as therapeutic targets for CRC. Previously, our research team found that LINC01123 promoted proliferation and metastasis in CRC by regulating miR-625-5p and the LIM and SH3 protein 1 (LASP1). Therefore, this study speculated that the molecular sponge effect of LINC01123 on miR-625-5p affected the process of CRC via regulating LASP1. The LINC01123-silenced CRC cell models (using the LOVO and SW480 cells) and xenograft tumor models were established to verify the above conjecture. As a result, it was found that silencing LINC01123 inhibited viability, proliferation, metastasis, and invasion but promoted apoptosis in LOVO and SW480 cells. Additionally, the knockdown of LINC01123 inhibited the LASP1, N-cadherin, PCNA, and Bcl-2 protein levels and raised the E-cadherin, Bax, and Caspase-3 protein levels in vitro. Furthermore, it showed that LINC01123, as a molecular sponge, targeted the miR-625-5p/LASP1 axis. The results of the xenograft tumor assay further verified the above effects of LINCO1123-silenced on tumor growth in vivo. And the miR-625-5p mimics treatment promoted the aforementioned effects of silencing LINC01123 on CRC cells while overexpressing LASP1 has an antagonistic effect to silencing LINC01123. In conclusion, this study suggests that silencing LINC01123 inhibits the process of CRC via sponging to the miR-625-5p/LASP1 axis. This finding hopes to provide research fundamentals on the biological mechanism study of CRC.

MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation

BackgroundThe Makorin ring finger protein 1 (MKRN1) gene, also called RNF61, is located on the long arm of chromosome 7 and is a member of the RING finger protein family. The E3 ubiquitin ligase MKRN1 is closely linked to tumour development, but the exact mechanism needs to be elucidated. In this study, we aimed to investigate the specific mechanism and role of MKRN1 in colorectal cancer (CRC) development.MethodsMKRN1 expression in CRC was analysed using the Cancer Cell Line Encyclopaedia and the Cancer Genome Atlas (TCGA) databases. Rectal tumour tissues were frozen to explore the MKRN1 expression in CRC and its clinical significance. The impact of MKRN1 on CRC cell proliferation and migration was observed using CCK8, colony formation, wound healing, and transwell assays. A combination of MKRN1 quantitative proteomics, ubiquitination modification omics analysis, and a string of in vitro and in vivo experiments revealed the potential mechanisms by which MKRN1 regulates CRC metastasis.ResultsMKRN1 expression was significantly elevated in CRC tissues compared to paracancerous tissues and was positively linked with prognosis (P < 0.01). MKRN1 downregulation inhibits CRC cell proliferation, migration, and invasion. Conversely, MKRN1 overexpression promotes the proliferation, migration, and invasion of CRC cells. Mechanistically, MKRN1 induces epithelial-mesenchymal transition (EMT) in CRC cells via ubiquitination and degradation of Smad nuclear-interacting protein 1 (SNIP1). Furthermore, SNIP1 inhibits transforming growth factor-β (TGF-β) signalling, and MKRN1 promotes TGF-β signalling by degrading SNIP1 to induce EMT in CRC cells. Finally, using conditional knockout mice, intestinal lesions and metastatic liver microlesions were greatly reduced in the intestinal knockout MKRN1 group compared to that in the control group.ConclusionsHigh MKRN1 levels promote TGF-β signalling through ubiquitination and degradation of SNIP1, thereby facilitating CRC metastasis, and supporting MKRN1 as a CRC pro-cancer factor. The MKRN1/SNIP1/TGF-β axis may be a potential therapeutic target in CRC.

Role of ELK1 in regulating colorectal cancer progression: miR-31-5p/CDIP1 axis in CRC pathogenesis.

Colorectal cancer (CRC) is a malignant tumor that affects the digestive system. With the increased of modernization of society, the incidence of colorectal cancer has increased throughout the world. As a transcription factor, ELK1 has been widely studied in colorectal cancer. However, there are still many unknown factors regarding its specific mechanism of action.This study explored the role of ELK1 and its downstream pathway in CRC pathogenesis. Based on clinical samples, this study examined miR-31-5p expression in CRC cells and its impact on malignant behaviors (migration, invasion, apoptosis) and autophagy. The promoter sequence of miR-31-5p was obtained from the UCSC database, and ELK1 was identified as its transcription factor. In ELK1-knockdown CRC cells, miR-31-5p was overexpressed, and its response in malignant behaviors and autophagy was analyzed. The target gene CDIP1 was predicted and verified using a dual-luciferase assay. The influence of CDIP1 on malignant behavior in CRC cells was assessed, and CDIP1 siRNA was used as a rescue treatment for miR-31-5p inhibition. The role of ELK1/miR-31-5p in tumor growth was validated in vivo. miR-31-5p expression was upregulated in the colorectal cancer tissues and cells. The knockdown of miR-31-5p markedly inhibited cancer cells' malignant behaviors and mediated autophagy. ELK1 was confirmed to bind with the miR-31-5p promoter and enhance miR-31-5p transcription. miR-31-5p was found to bind with the CDIP1 3'UTR and inhibit CDIP1 expression. CDIP1 siRNA partially rescued the effects of miR-31-5p knockdown on cell metastatic ability, autophagy, and apoptosis. Based on the in vivo experiments, results showed that the ELK1/miR-31-5p axis positively regulated tumor growth in nude mice. Our findings indicate that ELK1 regulates the progression of colorectal cancer via an miR-31-5p/CDIP1 axis, and the ELK1/miR-31-5p/CDIP1 axis could be a therapeutic target for colorectal cancer.

Titin as a potential novel therapeutic target in colorectal cancer.

Colorectal cancer (CRC) is identified as a primary cause of death around the world. The current chemotherapies are not cost-effective. Therefore, finding novel potential therapeutic target is urgent. Titin (TTN) is a muscle protein that is critical in hypertrophic cardiomyopathy. However, its role in CRC is not well understood. The study focused on exploring the possible role of TTN in CRC carcinogenesis. TTN mRNA and protein expression levels presented an obvious downregulation in CRC tissue samples, relative to normal control (p < 0.05). TTN expression significantly correlated with the clinical stage (normal vs. Stage 1, p < 0.05; normal vs. Stage 4, p < 0.05), node metastasis (normal vs. N1, p < 0.05; N1 vs. N2, p < 0.05), histological type (normal vs. adenocarcinoma, p < 0.05), race (Caucasian vs. Asian, p < 0.05; African-American vs. Asian, p < 0.05) and TP53 mutation (normal vs. TP53 mutation, p < 0.05), considering The Cancer Genome Atlas database. However, for patients who had higher TTN expression, the overall survival was remarkably shorter than patients who had low TTN expression. Furthermore, TTN was lowly expressed in four CRC cell lines. TTN overexpression facilitated CRC cells in terms of the proliferation, metastasis and invasion. Based on gene set enrichment analysis, the ERB pathway might be responsible for TTN-related CRC. Besides, TTN was involved in the response to azacitidine. Overall, TTN might serve as a potential novel therapeutic target for treating and overcoming chemotherapy resistance in CRC.

Novel heavily fucosylated glycans as a promising therapeutic target in colorectal cancer

BackgroundColorectal cancer (CRC) is highly prevalent and lethal globally, and its prognosis remains unsatisfactory. Drug resistance is regarded as the main cause of treatment failure leading to tumor recurrence and metastasis. The overexpression of fucosylated epitopes, which are usually modifications of glycoproteins, was reported to occur in various epithelial cancers. However, the effects of treatments that target these antigens in colorectal cancer remain unclear.MethodsThis study investigated the expression of heavily fucosylated glycans (HFGs) in 30 clinical samples from patients with CRC and other normal human tissues. The complement-dependent cytotoxicity was explored in vitro through treatment with anti-HFG monoclonal antibody (mAb) alone or in combination with chemotherapeutic agents. In vivo inhibitory effects were also examined using a xenograft mouse model.ResultsImmunohistochemistry staining and western blotting revealed that HFG expression was higher in human colorectal cancer tissues than in normal tissues. In DLD-1 and SW1116 cells, which overexpress fucosylated epitopes, anti-HFG mAb produced observable cytotoxic effects, especially when it was combined with chemotherapeutic agents. The xenograft model also demonstrated that anti-HFG mAb had potent and dose-dependent inhibitory effects on colorectal tumor growth.ConclusionsAs a novel cancer antigen, HFGs are a promising treatment target, and the implementation of anti-HFG mAb treatment for CRC warrants further investigation.

Lnc-LRRTM4 promotes proliferation, metastasis and EMT of colorectal cancer through activating LRRTM4 transcription

Numerous mechanisms have shown that long noncoding RNAs (lncRNAs) promote the development of colorectal cancer (CRC), but the role of lnc-LRRTM4 in the progression of CRC remains unclear. In this article, we found that lnc-LRRTM4 was highly expressed in CRC tissues and cell lines and that lnc-LRRTM4 could promote the proliferation and metastasis of CRC cells. These consequences were achieved by lnc-LRRTM4 directly binding to the promoter of LRRTM4 to induce its transcription. Moreover, lnc-LRRTM4 enhanced the growth of CRC cells in vivo by promoting cell cycle progression and reducing apoptosis. Taken together, our results revealed that lnc-LRRTM4 promotes the proliferation and metastasis of CRC cells, suggesting that it may be a potential diagnostic and therapeutic target for CRC.

Long noncoding RNA LINC01594 inhibits the CELF6-mediated splicing of oncogenic CD44 variants to promote colorectal cancer metastasis

Long noncoding RNAs (lncRNAs) play critical roles in tumorigenesis and tumor metastasis. However, the underlying mechanisms of lncRNAs in colorectal cancer (CRC) need further exploration. By using data from The Cancer Genome Atlas (TCGA) and GEO databases, we identified a novel CRC-related lncRNA, LINC01594, that is significantly upregulated in CRC and associated with poor prognosis. In vitro and in vivo, gain- and loss-of-function experiments demonstrated that LINC01594 promotes metastasis in CRC. LINC01594 functions as a DNMT1 scaffold, increasing the level of CELF6 promoter methylation. LINC01594 also competitively binds the transcription factor p53, decreasing CELF6 expression. This inhibited the exon skipping of CD44 V4–V7 induced by CELF6. In summary, this study highlights a novel CRC biomarker and therapeutic target, LINC01594, and the findings suggest that the LINC01594-CELF6-CD44 axis might serve as a biomarker and therapeutic target in CRC.

Transglutaminase 2 is associated with adverse colorectal cancer survival and represents a therapeutic target

Molecular markers for predicting prognosis of colorectal cancer (CRC) patients are urgently needed for effective disease management. We reported previously that the multifunctional enzyme Transglutaminase 2 (TGM2) is essential for CRC cell survival by inactivation of the tumor suppressor p53. Based on these data, we determined the clinical relevance of TGM2 expression and explored its potential as prognostic marker and therapeutic target in CRC. We profiled TGM2 protein expression in tumor samples of 279 clinically characterized CRC patients using immunohistochemical staining. TGM2 expression was upregulated in matched tumor samples in comparison to normal tissue. A strong TGM2 expression was associated with advanced tumor stages and predicted worse prognosis regarding progression-free and overall-survival, even at early stages. Inhibition of TGM2 in CRC cell lines by the inhibitors LDN27219 and Tyrphostin resulted in a strong reduction of cancer cell proliferation and tumorsphere formation in vitro by induction of p53-mediated apoptosis. Primary patient-derived tumorsphere formation was significantly reduced by inhibition of TGM2. Treatment of mice with TGM2 inhibitors exhibited a significant deceleration of tumor progression. Our data indicate that high TGM2 expression in CRC is associated with worse prognosis and may serve as a therapeutic target in CRC patients with strong TGM2 expression.

CCDC85C suppresses colorectal cancer cells proliferation and metastasis through activating GSK-3β and promoting β-catenin degradation

Coiled-coil domain-containing 85C (CCDC85C) is a member of the DIPA family and contains a pair of conserved coiled-coil motifs, which was found to be related to a therapeutic target for colorectal cancer, however, its biological effects require further elucidation. This study aimed to determine the effect of CCDC85C on Colorectal Cancer (CRC) progression and to explore the related mechanism. pLV-PURO plasmid was used to construct CCDC85C-overexpressing cells while CRISPR-CasRx was used to construct CCDC85C knockdown cells. Effects of CCDC85C on cell proliferation, cycle and migration were examined using cell counting kit-8 assay, flow cytometry, wound healing assay and transwell assay. Immunofluorescence staining, immunoprecipitation, Western blot, co-immunoprecipitation and qPCR were performed to explore the mechanism. The overexpression of CCDC85C inhibited the proliferation and migration of HCT-116 and RKO cells in vitro and in vivo, but its knockdown promoted the proliferation of HCT-116 and RKO cells in vitro. Moreover, co-immunoprecipitation experiment confirmed that CCDC85C binding with GSK-3β in RKO cells. Excess CCDC85C promoted phosphorylation and ubiquitination of β-catenin. Our results suggested that CCDC85C binds to GSK-3β to promote its activity and facilitates ubiquitination of β-catenin. β-catenin degradation is responsible for the inhibitory effect of CCDC85C on CRC cell proliferation and migration.

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.

RP11-296E3.2 acts as an important molecular chaperone for YBX1 and promotes colorectal cancer proliferation and metastasis by activating STAT3

BackgroundRP11-296E3.2 is a novel long noncoding RNA (lncRNA) associated with colorectal cancer (CRC) metastasis, that was reported in our previous clinical studies. However, the mechanisms of RP11-296E3.2 in colorectal tumorigenesis remain elusive.MethodsRNA sequencing (RNA-seq), Fluorescence in situ hybridization (FISH), Transwell assays and others, were performed to evaluate the function of RP11-296E3.2 for proliferation and metastasis in vitro. In situ and metastatic tumor models were performed to evaluate the function of RP11-296E3.2 for proliferation and metastasis in vivo. RNA-pulldown, RNA-interacting protein immunoprecipitation (RIP), tissue microarray (TMA) assay, a luciferase reporter assay, chromatin immunoprecipitation (ChIP) and others were performed to explore the mechanisms by which RP11-296E3.2 regulates CRC tumorigenesis.ResultsRP11-296E3.2 was confirmed to be associated with CRC cell proliferation and metastasis in vitro and in vivo. Mechanistically, RP11-296E3.2 directly bound to recombinant Y-Box Binding Protein 1 (YBX1) and enhanced signal transducer and activator of transcription 3 (STAT3) transcription and phosphorylation. YBX1 promoted the CRC cell proliferation and migration, while knockdown of RP11-296E3.2 attenuated the effects of YBX1 on CRC cell proliferation, and metastasis and the expression of several related downstream genes. We are the first to discover and confirm the existence of the YBX1/STAT3 pathway, a pathway dependent on RP11-296E3.2.ConclusionTogether, these novel findings show that the RP11-296E3.2/YBX1 pathway promotes colorectal tumorigenesis and progression by activating STAT3 transcription and phosphorylation, and suggest that RP11-296E3.2 is a potential diagnostic biomarker and therapeutic target in CRC.

Knockdown of UBQLN1 Functions as a Strategy to Inhibit CRC Progression through the ERK-c-Myc Pathway

Purpose: Colorectal cancer (CRC) is characterized by the absence of obvious symptoms in the early stage. Due to the high rate of late diagnosis of CRC patients, the mortality rate of CRC is higher than that of other malignant tumors. Accumulating evidence has demonstrated that UBQLN1 plays an important role in many biological processes. However, the role of UBQLN1 in CRC progression is still elusive. Methods and results: we found that UBQLN1 was significantly highly expressed in CRC tissues compared with normal tissues. Enhanced/reduced UBQLN1 promoted/inhibited CRC cell proliferation, colony formation, epithelial–mesenchymal transition (EMT) in vitro, and knockdown of UBQLN1 inhibited CRC cells’ tumorigenesis and metastasis in nude mice in vivo. Moreover, the knockdown of UBQLN1 reduced the expression of c-Myc by downregulating the ERK-MAPK pathway. Furthermore, the elevation of c-Myc in UBQLN1-deficient cells rescued proliferation caused by UBQLN1 silencing. Conclusions: Knockdown of UBQLN1 inhibits the progression of CRC through the ERK-c-Myc pathway, which provides new insights into the mechanism of CRC progression. UBQLN1 may be a potential prognostic biomarker and therapeutic target of CRC.

Association of intermediates of bioenergetic pathways and plasma metabolomics with colorectal cancer (CRC): Diagnosis, stage, and survival.

e15575 Background: Metabolomics is an evolving technique that can offer a non-invasive method for comprehensive identification of metabolic biomarkers. We evaluated the feasibility of measuring plasma metabolites in a longitudinal, observational cohort of patients (pts) with stage I-IV CRC for association with disease characteristics and overall survival (OS). Methods: In this retrospective analysis of prospectively collected blood samples from a single-institute cohort of pts with stage I-IV CRC, up to 150 metabolites of central carbon metabolism were analyzed by mass spectrometry and high-performance liquid chromatography. All blood samples were collected at pre-chemotherapy baseline. Analytes were compared by relative area under the curve (AUC) and differences evaluated by ANOVA. Blood samples were collected from two non-CRC pts to serve as healthy controls. Comparison of plasma metabolite levels were stratified by diagnosis of CRC vs. healthy control, non-metastatic CRC (stage I-III) vs. metastatic CRC (stage IV), and short OS vs. long OS (defined by the median threshold OS of 24.8 months in the cohort). Results: Plasma metabolomics was performed on a total of 32 pts with CRC. The median age of the cohort was 65 years (range 20-90) with the majority being female (53%) and having stage IV disease (78%). Of 25 pts with stage IV CRC, 88% had pre-chemotherapy blood samples collected in the first-line setting and 44% were KRAS mutant compared to 37% KRAS WT and 19% unknown KRAS status. Compared to healthy controls, CRC pts had higher levels of glutamate, nucleotides UMP and CMP, and nucleosides hypoxanthine and inosine (all p &lt; 0.05). When stratified by stage, stage I-III CRC pts had higher levels of the non-essential amino acid (AA) serine and essential AAs threonine and methionine, while stage IV had higher levels of the aldopentose ribose (all p &lt; 0.05) Those with short OS had increased levels in intermediates of glycolysis (lactate, UDP-Glc), glutamine metabolism (GlcNAc), MTA cycle (SAM, 5-Methioadenosine), and pentose phosphate pathway (R5P) as well as increased levels of proline (non-essential AA), phenylalanine (essential AA), thymine (nucleobase), and UDP (nucleotide) compared to pts with long OS (all p &lt; 0.05). Notably, both stage IV and short OS pts had trends in increased intermediates of glutamine metabolism (glutamate, a-KG, UDP-GlcNAc) that did not reach significance. Conclusions: We show that pts with CRC compared to healthy controls and those with stage IV and short OS appear to be enriched in intermediates of multiple bioenergetic pathways including AA synthesis, nucleotide synthesis, glycolysis, methyl donor biology, and glutamine signaling. These findings are hypothesis-generating and can inform more focused evaluation of potential biomarkers and therapeutic targets in CRC.

Forkhead box protein D2 suppresses colorectal cancer by reprogramming enhancer interactions.

Somatic stem cells contribute to normal tissue homeostasis, and their epigenomic features play an important role in regulating tissue identities or developing disease states. Enhancers are one of the key players controlling chromatin context-specific gene expression in a spatial and temporal manner while maintaining tissue homeostasis, and their dysregulation leads to tumorigenesis. Here, epigenomic and transcriptomic analyses reveal that forkhead box protein D2 (FOXD2) is a hub for the gene regulatory network exclusive to large intestinal stem cells, and its overexpression plays a significant role in colon cancer regression. FOXD2 is positioned at the closed chromatin and facilitates mixed-lineage leukemia protein-4 (MLL4/KMT2D) binding to deposit H3K4 monomethylation. De novo FOXD2-mediated chromatin interactions rewire the regulation of p53-responsive genes and induction of apoptosis. Taken together, our findings illustrate the novel mechanistic details of FOXD2 in suppressing colorectal cancer growth and suggest its function as a chromatin-tuning factor and a potential therapeutic target for colorectal cancer.

MEX3A promotes angiogenesis in colorectal cancer via glycolysis

ABSTRACT As a gastrointestinal malignancy, colorectal cancer (CRC) is a main cause of cancer-related deaths worldwide. Mex-3 RNA-binding family member A (MEX3A) is upregulated in multiple types of tumors and plays a critical role in tumor proliferation and metastasis. However, the function of MEX3A in CRC angiogenesis has not been fully understood. Hence, the aim of this study was to explore the role of MEX3A in CRC angiogenesis and investigate its underlying mechanisms. MEX3A expression in CRC was first investigated by bioinformatics means and then measured by qRT-PCR and Western blot. CCK-8 assay was employed to test cell viability. Angiogenesis assay was used to assess angiogenesis. The protein levels of VEGF, FGF and SDF-1 were evaluated using Western blot. The expression levels of MYC, HK2 and PGK1 were investigated by qRT-PCR. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were determined by Seahorse XP 96. The levels of pyruvate, lactate, citric acid and malate were measured by corresponding kits. Bioinformatics analysis demonstrated high MEX3A expression in CRC tissues and MEX3A enrichment in glycolysis and angiogenesis pathways. Cell assays showed high MEX3A expression in CRC cells and its promoting effects in CRC cell proliferation and glycolysis as well as angiogenesis. Rescue experiment confirmed that glycolysis inhibitor 2-DG could offset the promoting effects of MEX3A on the proliferation, angiogenesis and glycolysis of CRC cells. In conclusion, MEX3A could facilitate CRC angiogenesis by activating the glycolytic pathway, suggesting that MEX3A may be a novel therapeutic target for CRC.

Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer

BackgroundMethylation of the p16 promoter resulting in epigenetic gene silencing—known as p16 epimutation—is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis.MethodsWe established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious.ResultsMice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone.ConclusionsOur study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

The E3 ubiquitin ligase SCF (FBXW10)-mediated LATS2 degradation regulates angiogenesis and liver metastasis in colorectal cancer.

F-box and WD repeat domain containing 10 (FBXW10) is a member of the FBXW subgroup that contains the WD40 domain. FBXW10 has been rarely reported in colorectal cancer (CRC) and its mechanism is unclear. To investigate the role of FBXW10 in CRC, we conducted in vitro and in vivo experiments. Through the database and our clinical samples, we found that FBXW10 expression was up-regulated in CRC, and it was positively correlated with CD31 expression. CRC patients with high FBXW10 expression levels had a poor prognosis. Overexpression of FBXW10 up-regulated cell proliferation, migration and vascular formation, while knockdown of FBXW10 had the opposite effects. Studies on the mechanism of FBXW10 in CRC showed that FBXW10 could ubiquitinate large tumor suppressor kinase 2 (LATS2) and promote its degradation with the Fbox region of FBXW10 played an essential role in this process. In vivo studies demonstrated that knockout of FBXW10 inhibited tumor proliferation and reduced liver metastasis. In conclusion, our study proved that FBXW10 was significantly overexpressed in CRC and was involved in the pathogenesis of CRC by affecting angiogenesis and liver metastasis. Mechanistically, FBXW10 degraded LATS2 through ubiquitination. Therefore, FBXW10-LATS2 can be used as a therapeutic target for CRC in subsequent studies.