Progression Of Human Colorectal Cancer Research Articles

Dehydroepiandrosterone suppresses human colorectal cancer progression through ER stress-mediated autophagy and apoptosis in a p53-independent manner.

Colorectal cancer (CRC) is one of the primary contributors to cancer-related fatalities, with up to 80% of advanced CRC cases exhibiting mutations in the p53 gene. Unfortunately, the development of new compounds targeting mutant p53 is quite limited. The anticancer effects of Dehydroepiandrosterone (DHEA) on various cancers have been reported. However, the suppressive effect of DHEA on CRC cells harboring wild-type or mutant p53 gene remains controversial. This study emphasized revealing the suppressive mechanism and the effect of DHEA on CRC cell tumorigenesis in the presence of wild-type or mutant p53 gene. We demonstrate that DHEA causes CRC cell death and cell cycle arrest in a dose and time-dependent manner. Notably, DHEA exhibits similar inhibitory effects on CRC cells regardless of the p53 gene status. Further study reveals that DHEA induces endoplasmic reticulum (ER) stress and triggers PERK/eIF2/ATF4/CHOP UPR signaling pathway to activate autophagy followed by apoptosis, which was confirmed by suppression of 4-phenylbutyric acid (an ER stress inhibitor) or knockdown either ATF4 or CHOP. DHEA-induced apoptosis was attenuated by silencing ATG5 gene in either p53+/+ or p53-/- CRC cells, indicating autophagy regulation of apoptosis. Furthermore, DHEA treatment accompanied by bafilomycin A1 (a blocker of autophagosome degradation) leads to the accumulation of ATF4, CHOP, DR5, and p21 levels in CRC cells, implying that the degradative autophagy machinery regulates these four molecules. Consistently, DHEA demonstrates its inhibitory effect by suppressing CRC tumor formation in vivo. Altogether, we provide compelling evidence that DHEA is a potential therapeutic candidate for CRC patient treatment regardless of the p53 status through ER stress-PERK-autophagy-apoptosis axis.

Interleukin-6-Positive Immune Cells as a Possible New Immunologic Marker Associated With the Colorectal Cancer Prognosis.

Chronic inflammation creates tumor microenvironment (TME) that facilitates colorectal cancer (CRC) cell proliferation, migration, metastasis, and tumor progression. Interleukin-6 (IL-6) is a proinflammatory cytokine with a pleiotropic effect on CRC development. We aimed to evaluate IL-6 expression in tumor cells and in immune cells in TME, to assess the serum level and IL6 -174 G/C genotype distribution and to correlate the results with selected morphologic and clinical parameters that may add useful information in understanding the mechanisms of human CRC progression. A total of 153 patients with CRC were recruited in the current study. We assessed the IL-6 serum concentration through the ELISA method, the expression of IL-6 in tumor and in immune cells by immunohistochemical and double immunofluorescence staining, the MSI status by immunоhistochemistry for 4 mismatch repair (MMR) proteins, and the genotype distributions for IL6 -174G/C (rs1800795) single-nucleotide polymorphism through PCR-RFLP method. Our results showed that serum IL-6 level were increased in CRC patients as compared with healthy controls (P<0.0001), and in patients with cancers with advanced histologic type (type IV). However, the higher concentration (above the median of 55.71pg/mL) was with borderline association with longer survival of the patients after surgical therapy (P=0.055, Log rank test). We also found that IL-6+ immune cells prevailed in the invasive front (IF) of tumors compared with the tumor stroma (TS) (P<0.0001). More IL-6+ cells were recruited in the tumors with less advanced histologic type (I+II), with stronger inflammatory infiltrate in the IF, in early pTNM stages (I+II), without lymph node and distant metastases and the higher levels of IL-6+ cells, especially in the IF, were associated with longer survival (P=0.012). The results of our study suggest that although the serum levels of IL-6 are higher in CRC, the increased IL-6+ cells in tumor microenvironment, both in the invasive front and in tumor stroma, as well as the higher serum levels are associated with good prognostic variables and longer survival of the patients mainly in the early stages of CRC.

TRIM6 Reduces Ferroptosis and Chemosensitivity by Targeting SLC1A5 in Lung Cancer.

Ferroptosis, a newly identified form of cell death, plays critical roles in the development and chemoresistance of lung cancer. Tripartite motif 6 (TRIM6) acts as an E3-ubiquitin ligase and can promote the progression of human colorectal cancer. The present study is aimed at investigating its role and potential mechanisms in lung cancer. Lentiviral vectors were used to overexpress or knock down TRIM6 in human lung cancer cells. Cell survival, colony formation, lipid peroxidation, intracellular iron levels, and other ferroptotic markers were examined. The role of TRIM6 on ferroptosis and chemosensitivity was further tested in mouse tumor xenograft models. TRIM6 was highly expressed in human lung cancer tissues and cells, and its expression in the lung cancer cells was further increased by ferroptotic stimulation. TRIM6 overexpression inhibited, while TRIM6 silence promoted erastin- and RSL3-induced glutaminolysis and ferroptosis in the lung cancer cells. Mechanistically, TRIM6 directly interacted with solute carrier family 1 member 5 to promote its ubiquitination and degradation, thereby inhibiting glutamine import, glutaminolysis, lipid peroxidation, and ferroptotic cell death. Moreover, we observed that TRIM6 overexpression reduced the chemotherapeutic effects of cisplatin and paclitaxel. In contrast, TRIM6 silence sensitized human lung cancer cells to cisplatin and paclitaxel in vivo and in vitro. Our findings for the first time define TRIM6 as a negative regulator of ferroptosis in the lung cancer cells, and TRIM6 overexpression enhances the resistance of human lung cancer cells to chemotherapeutic drugs. Overall, targeting TRIM6 may help to establish novel strategies to treat lung cancer.

GOLM1 facilitates human colorectal cancer progression and metastasis via activating the AKT/GSK3β/EMT axis.

GOLM1 (Golgi membrane protein 1), a key tumor progression- and metastasis-related marker, is highly expressed in a variety of epithelium-derived human cancers. However, its expression and functions in human colorectal cancer (CRC) have been rarely explored. The present study verified the high expression of GOLM1 within CRC tissues and cell lines. GOLM1 was positively correlated with vascular invasion, TNM stage, and lymph node metastasis among CRC cases. In vitro experiments showed that GOLM1 downregulation inhibited the growth, migration, and invasion of Caco-2 and HCT116 cells, while the overexpression of GOLM1 facilitated the growth, migration, and invasion of SW480 cells. In vivo experiments revealed that the knockdown of GOLM1 reduced the growth of nude mouse xenografts and lung metastasis of HCT116 cells. Furthermore, GOLM1 was found to be a motivator for the epithelial-mesenchymal transition (EMT) phenotype and the AKT/GSK3β pathway in CRC cells. Finally, MK2206, an AKT inhibitor, could markedly reverse GOLM1-elicited proliferation, migration, invasion, and EMT phenotype by inhibiting the AKT/GSK3β pathway. Collectively, our data indicate that GOLM1 facilitates human CRC progression and metastasis via activating the AKT/GSK3β/EMT axis. Most importantly, our study makes substantial support for the clinical translation of GOLM1 in CRC target therapy.

Identification of HAGHL as a novel metabolic oncogene regulating human colorectal cancer progression.

Cancer development remains the most challenging obstacle in colorectal cancer (CRC) treatment. The current study aims to identify and demonstrate novel oncogenes for CRC. The CRC data of the Cancer Genome Atlas database and the Gene Expression Omnibus database were subjected to bioinformatics analysis to identify the novel potential diagnostic and prognostic biomarkers for CRC. Immunohistochemical assay, western blot, and quantitative PCR (qPCR) were used to analyze hydroxyacylglutathione hydrolase-like (HAGHL) gene expression in CRC tissues and cultured CRC cells. D-Lactate colorimetric assay was applied to determine concentration of D-lactate in supernatants from CRC tissues and cell culture medium. Cell counting kit-8 (CCK-8) assay, flow cytometry, tumor xenografts experiment, and TUNEL staining analysis were performed to evaluate the function of HAGHL in CRC. We comprehensively analyzed the CRC data of the Cancer Genome Atlas database and the Gene Expression Omnibus database, and identified several novel potential diagnostic and prognostic biomarkers for CRC, including HAGHL, DNTTIP1, DHX34, and AP1S3. The expression of HAGHL, the strongest oncogenic activity gene, is positively related to D-lactate levels in CRC tissues and negatively associated with patient prognosis. HAGHL downregulation suppressed the production of D-lactate and induced apoptosis, resulting in inhibition of cell proliferation in vitro. In vivo experiment showed that knockdown of HAGHL induced cell apoptosis and inhibited tumor growth. These findings suggest that HAGHL acts as a novel metabolic oncogene and demonstrate the underlying mechanism by which HAGHL regulates CRC progression, highlighting its utility as a diagnostic and prognostic factor and as a potential therapeutic target for the treatment of CRC.

DLGAP1-AS2 promotes human colorectal cancer progression through trans-activation of Myc.

Substantial evidence suggests that non-coding RNA plays a vital role in human cancer, especially long non-coding RNA (lncRNA) with a length greater than 200nt. Herein, we found a lncRNA facilitating human colorectal cancer (CRC) progression. DLGAP1-AS2 was significantly increased in CRC tissues and cell lines. Knockdown of DLGAP1-AS2 inhibited CRC cell proliferation, migration, invasion in vitro, and tumor growth in vivo. The subcellular localization of DLGAP1-AS2 was translocated from the cytoplasm of normal cells to the nucleus of CRC cells due to reduced levels of N6-methyladenosine (m6A) modification. Further, through the screening of a series of signal pathways, we found that Myc pathway was involved in the effect of DLGAP1-AS2. Silencing of DLGAP1-AS2 markedly reduced Myc mRNA and protein levels. Blockade of Myc effectively abolished the enhanced aggressive behaviors of CRC cells caused by DLGAP1-AS2 overexpression. Mechanistically, DLGAP1-AS2 directly bound CTCF, a well-known transcriptional repressor of Myc, resulting in reduced binding of CTCF on Myc promoter and activating Myc transcription. The second hairpin structure of DLGAP1-AS2 was critical for the interaction between DLGAP1-AS2 and CTCF in the nucleus. Taken together, our study reveals the oncogenic regulatory axis of DLGAP1-AS2/CTCF/Myc in CRC, implying a promising targeted therapy for clinical application.

Significance of NOTCH1 Expression in the Progression of Human Lung and Colorectal Cancers.

Lung and colorectal cancers are the most common types of cancer characterized by a poor prognosis and a high mortality rate. Mutations in the genes encoding components of the main intra- and extracellular signaling pathways, in particular the NOTCH1 gene (Notch1, a member of the Notch family of receptors), play one of the key roles in progression of these malignancies. Notch signaling is involved in maintaining homeostasis of the intestinal epithelium and structural and functional lung elements. Therefore, it is not surprising that the constitutive activity and hyperactivity of Notch signaling due to somatic mutations in genes coding for the products directly involved into its activation, could lead to the progression of these cancer types. The aim of our study was to investigate how the NOTCH1 downregulation via RNA interference (RNAi) affects the phenotype, characteristics, and Notch-dependent signaling of human A549 lung and HCT116 colorectal carcinoma cells. Several small harpin RNAs (shRNAs) were selected using the bioinformatic analysis and tested for their ability to suppress the NOTCH1 expression. The most efficient one was used to produce the A549 and HCT116 cells with NOTCH1 knockdown. The obtained cell lines demonstrated decreased proliferation rates, reduced colony-forming capacity under adhesive conditions, and decreased migration activity in a Boyden chamber. The NOTCH1 knockdown also significantly decreased expression of some Notch signaling target genes potentially involved in the acquisition and maintenance of more invasive and malignant cell phenotype. In vivo experiments in immunodeficient athymic female Balb/c nu/nu mice confirmed the results obtained in vitro: the NOTCH1 inhibition decreased the growth rates of the subcutaneous xenografts formed by A549 and HCT116 tumor cells. Therefore, downregulation of the gene encoding the Notch1 receptor potentially reduces malignant characteristics of human lung and colorectal carcinoma cells.

Machine learning phenomics (MLP) combining deep learning with time-lapse-microscopy for monitoring colorectal adenocarcinoma cells gene expression and drug-response

High-throughput phenotyping is becoming increasingly available thanks to analytical and bioinformatics approaches that enable the use of very high-dimensional data and to the availability of dynamic models that link phenomena across levels: from genes to cells, from cells to organs, and through the whole organism. The combination of phenomics, deep learning, and machine learning represents a strong potential for the phenotypical investigation, leading the way to a more embracing approach, called machine learning phenomics (MLP). In particular, in this work we present a novel MLP platform for phenomics investigation of cancer-cells response to therapy, exploiting and combining the potential of time-lapse microscopy for cell behavior data acquisition and robust deep learning software architectures for the latent phenotypes extraction. A two-step proof of concepts is designed. First, we demonstrate a strict correlation among gene expression and cell phenotype with the aim to identify new biomarkers and targets for tailored therapy in human colorectal cancer onset and progression. Experiments were conducted on human colorectal adenocarcinoma cells (DLD-1) and their profile was compared with an isogenic line in which the expression of LOX-1 transcript was knocked down. In addition, we also evaluate the phenotypic impact of the administration of different doses of an antineoplastic drug over DLD-1 cells. Under the omics paradigm, proteomics results are used to confirm the findings of the experiments.

Comparative Study of the Role of Interepithelial Mucosal Mast Cells in the Context of Intestinal Adenoma-Carcinoma Progression.

Simple SummaryThe role of mast cells in the tumor microenvironment (TME) remains controversial but has become increasingly evident and explored as a possible therapeutic target. In this study, we investigated the underexplored mast cell heterogeneity of intestinal cancer by applying standardized mast cell subtyping during adenoma-carcinoma progression. We immunohistochemically evaluated and scored the occurrence of interepithelial mucosal mast cells (ieMMCs) in tumors of frequently employed genetically engineered mouse models and in human colonic adenomas and carcinomas. We found a decrease of ieMMCs from colonic low-grade adenomas to carcinomas. Moreover, mouse models based on altered Wnt signaling showed higher ieMMC scores than models based on altered MAPK signaling. Our descriptive study indicates that ieMMCs play a special role in the molecular TME related to adenoma-carcinoma progression. Furthermore, it emphasizes the need for adequate immunohistochemical methodology and experimental setup when investigating the functional role of mast cell populations of the TME.Mast cells (MCs) are crucial players in the relationship between the tumor microenvironment (TME) and cancer cells and have been shown to influence angiogenesis and progression of human colorectal cancer (CRC). However, the role of MCs in the TME is controversially discussed as either pro- or anti-tumorigenic. Genetically engineered mouse models (GEMMs) are the most frequently used in vivo models for human CRC research. In the murine intestine there are at least three different MC subtypes: interepithelial mucosal mast cells (ieMMCs), lamina proprial mucosal mast cells (lpMMCs) and connective tissue mast cells (CTMCs). Interepithelial mucosal mast cells (ieMMCs) in (pre-)neoplastic intestinal formalin-fixed paraffin-embedded (FFPE) specimens of mouse models (total lesions n = 274) and human patients (n = 104) were immunohistochemically identified and semiquantitatively scored. Scores were analyzed along the adenoma-carcinoma sequence in humans and 12 GEMMs of small and large intestinal cancer. The presence of ieMMCs was a common finding in intestinal adenomas and carcinomas in mice and humans. The number of ieMMCs decreased in the course of colonic adenoma-carcinoma sequence in both species (p < 0.001). However, this dynamic cellular state was not observed for small intestinal murine tumors. Furthermore, ieMMC scores were higher in GEMMs with altered Wnt signaling (active β-catenin) than in GEMMs with altered MAPK signaling and wildtypes (WT). In conclusion, we hypothesize that, besides stromal MCs (lpMMCs/CTMCs), particularly the ieMMC subset is important for onset and progression of intestinal neoplasia and may interact with the adjacent neoplastic epithelial cells in dependence on the molecular environment. Moreover, our study indicates the need for adequate GEMMs for the investigation of the intestinal immunologic TME.

SHMT2 Drives the Progression of Colorectal Cancer by Regulating UHRF1 Expression.

Introduction Serine hydroxymethyltransferase 2 (SHMT2) has a critical role in serine-glycine metabolism to drive cancer cell proliferation. Yet, the function of SHMT2 in tumorigenesis, especially in human colorectal cancer (CRC) progression, remains largely unclear. Materials and Methods CRC and paired normal samples were collected in the Department of Colorectal Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, and assessed by real-time polymerase chain reaction (qPCR) analysis, western blot (WB), and immunohistochemistry (IHC). Moreover, SHMT2 expression in human CRC cells was identified by qPCR and WB. The CRC cell proliferation, migration, and invasion after SHMT2 knockdown were explored through in vitro and in vivo assays. mRNA-seq assays were used to investigate the underlying mechanisms behind the SHMT2 function. Results It was found that SHMT2 mRNA and protein were overexpressed in CRC tissue compared to the levels in normal mucosa. Positive expression of SHMT2 was significantly correlated with TNM stage and lymph node metastasis, and elevated expression of SHMT2 resulted as an independent prognostic factor in patients with CRC. SHMT2 knockdown impaired the proliferation of CRC in vitro and in vivo and induced cell cycle arrest by regulating UHRF1 expression. Conclusion Taken together, our findings reveal that UHRF1 is a novel target gene of SHMT2, which can be used as a potential therapeutic strategy for CRC therapy.

B-Myb accelerates colorectal cancer progression through reciprocal feed-forward transactivation of E2F2

B-Myb is an important transcription factor that plays a critical role in gene expression regulation and tumorigenesis. However, its functional implication in colorectal cancer remains elusive. In this study, we found that B-Myb was significantly upregulated at both mRNA and protein levels in colorectal cancer samples compared to non-tumor counterparts. B-Myb overexpression accelerated cell proliferation, cell cycle progression and cell motility in colorectal cancer cells, and promoted tumor growth in orthotopic nude mouse models in vivo. In contrast, B-Myb depletion inhibited these malignant phenotypes. Mechanistic investigations revealed that E2F2 was a novel transcriptional target of B-Myb and is essential to B-Myb-induced malignant phenotypes. Notably, B-Myb and E2F2 exhibited positive expression correlation, and interacted with each other in colorectal cancer cells. In addition to their autoregulatory mechanisms, B-Myb and E2F2 can also directly transactivate each other, thus constituting consolidated reciprocal feed-forward transactivation loops. Moreover, both B-Myb and E2F2 are required for the activation of ERK and AKT signaling pathways in colorectal cancer cells. Taken together, our data clarified a critical role for B-Myb in colorectal cancer and unraveled an exquisite mutual collaboration and reciprocal cross regulation between B-Myb and E2F2 that contribute to the malignant progression of human colorectal cancer.

A Metabolic Model of Intestinal Secretions: The Link between Human Microbiota and Colorectal Cancer Progression.

The human gut microbiota plays a dual key role in maintaining human health or inducing disorders, for example, obesity, type 2 diabetes, and cancers such as colorectal cancer (CRC). High-throughput data analysis, such as metagenomics and metabolomics, have shown the diverse effects of alterations in dynamic bacterial populations on the initiation and progression of colorectal cancer. However, it is well established that microbiome and human cells constantly influence each other, so it is not appropriate to study them independently. Genome-scale metabolic modeling is a well-established mathematical framework that describes the dynamic behavior of these two axes at the system level. In this study, we created community microbiome models of three conditions during colorectal cancer progression, including carcinoma, adenoma and health status, and showed how changes in the microbial population influence intestinal secretions. Conclusively, our findings showed that alterations in the gut microbiome might provoke mutations and transform adenomas into carcinomas. These alterations include the secretion of mutagenic metabolites such as H2S, NO compounds, spermidine and TMA (trimethylamine), as well as the reduction of butyrate. Furthermore, we found that the colorectal cancer microbiome can promote inflammation, cancer progression (e.g., angiogenesis) and cancer prevention (e.g., apoptosis) by increasing and decreasing certain metabolites such as histamine, glutamine and pyruvate. Thus, modulating the gut microbiome could be a promising strategy for the prevention and treatment of CRC.

The Promising Effect of Peucedanum chenur Chloroformic Extract on Prevention of Human Colorectal Cancer Progression by Modulating miR-135b, miR-21, and APC Genes.

The therapeutic use of herbal medicines for the diseases, including cancer, is increasing due to their lower side effects. The present research evaluated the effect of Peucedanum chenur chloroformic extract (PCCE) on cell proliferation against HCT-116 human colorectal cancer cell line. The cytotoxic effect of PCCE was evaluated by MTT assay. The activity of the Wnt/B-catenin pathway was assayed through measuring the expression of miR-135b, miR-21, and APC genes by real-time PCR. The flow cytometry and scratch tests were used to study the cell cycle and cell migration, respectively. Also, the antioxidant activity of PCCE was measured by DPPH and iron-chelating tests. The results showed the downregulation of miR-135b and miR-21 and overexpression of the APC gene. Furthermore, PCCE decreased the free radicals, cell migration, and cell proliferation. The antioxidant activity of PCCE was confirmed by standard tests. Altogether, our findings suggest that purified compounds of PCCE could be developed as a potent chemo-preventive drug for the treatment of CRC.

Role of tumor-associated macrophages at the invasive front in human colorectal cancer progression.

Macrophages are an essential component of antitumor activity; however, the role of tumor‐associated macrophages (TAMs) in colorectal cancer (CRC) remains controversial. Here, we elucidated the role of TAMs in CRC progression, especially at the early stage. We assessed the TAM number, phenotype, and distribution in 53 patients with colorectal neoplasia, including intramucosal neoplasia, submucosal invasive colorectal cancer (SM‐CRC), and advanced cancer, using double immunofluorescence for CD68 and CD163. Next, we focused on the invasive front in SM‐CRC and association between TAMs and clinicopathological features including lymph node metastasis, which were evaluated in 87 SM‐CRC clinical specimens. The number of M2 macrophages increased with tumor progression and dynamic changes were observed with respect to the number and phenotype of TAMs at the invasive front, especially at the stage of submucosal invasion. A high M2 macrophage count at the invasive front was correlated with lymphovascular invasion, low histological differentiation, and lymph node metastasis; a low M1 macrophage count at the invasive front was correlated with lymph node metastasis. Furthermore, receiver operating characteristic curve analysis revealed that the M2/M1 ratio was a better predictor of the risk of lymph node metastasis than the pan‐, M1, or M2 macrophage counts at the invasive front. These results suggested that TAMs at the invasive front might play a role in CRC progression, especially at the early stages. Therefore, evaluating the TAM phenotype, number, and distribution may be a potential predictor of metastasis, including lymph node metastasis, and TAMs may be a potential CRC therapeutic target.

Colonic epithelial cathelicidin (LL-37) expression intensity is associated with progression of colorectal cancer and presence of CD8+ T cell infiltrate.

Colorectal cancer (CRC) remains a leading cause of cancer mortality. Here, we define the colonic epithelial expression of cathelicidin (LL‐37) in CRC. Cathelicidin exerts pleotropic effects including anti‐microbial and immunoregulatory functions. Genetic knockout of cathelicidin led to increased size and number of colorectal tumours in the azoxymethane‐induced murine model of CRC. We aimed to translate this to human disease. The expression of LL‐37 in a large (n = 650) fully characterised cohort of treatment‐naïve primary human colorectal tumours and 50 matched normal mucosa samples with associated clinical and pathological data (patient age, gender, tumour site, tumour stage [UICC], presence or absence of extra‐mural vascular invasion, tumour differentiation, mismatch repair protein status, and survival to 18 years) was assessed by immunohistochemistry. The biological consequences of LL‐37 expression on the epithelial barrier and immune cell phenotype were assessed using targeted quantitative PCR gene expression of epithelial permeability (CLDN2, CLDN4, OCLN, CDH1, and TJP1) and cytokine (IL‐1β, IL‐18, IL‐33, IL‐10, IL‐22, and IL‐27) genes in a human colon organoid model, and CD3+, CD4+, and CD8+ lymphocyte phenotyping by immunohistochemistry, respectively. Our data reveal that loss of cathelicidin is associated with human CRC progression, with a switch in expression intensity an early feature of CRC. LL‐37 expression intensity is associated with CD8+ T cell infiltrate, influenced by tumour characteristics including mismatch repair protein status. There was no effect on epithelial barrier gene expression. These data offer novel insights into the contribution of LL‐37 to the pathogenesis of CRC and as a therapeutic molecule.

CSK-homologous kinase (CHK/MATK) is a potential colorectal cancer tumour suppressor gene epigenetically silenced by promoter methylation.

Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFK genes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATK but not CSK was significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATK expression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATK promoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATK was also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATK mRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATK as a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATK hypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.

Secreted phosphoprotein-1 accelerates the progression of human colorectal cancer through activating β-catenin signaling.

Colorectal cancer (CRC) is a common malignant tumor of the digestive tract and one of the leading causes of cancer-associated mortality. Secreted phosphoprotein-1 (SPP-1) is overexpressed in CRC and promotes cancer progression, but the underlying mechanisms underlying SPP-1 function remain unclear. The present study aimed to explore the effects of Wnt/β-catenin signaling in SPP-1-induced CRC progression. The expression patterns of SPP-1 in CRC tissues were examined using reverse transcription-quantitative (RT-q)PCR, western blotting and immunohistochemistry. SPP-1 expression in cells was assessed using RT-qPCR and western blotting. Cell-Counting Kit-8, flow cytometry and tumor-burdened mice experiments were used to determine cell proliferation, apoptosis and in vivo tumor formation abilities. The results showed that SPP-1 expression was markedly elevated in CRC tissues and cells compared with that in normal colorectal tissues and cells. High expression of SPP-1 was associated with advanced clinical process and low overall survival rate in patients with CRC. Besides, SPP-1 could interact with β-catenin and positively regulated β-catenin protein expression, and enhanced its nuclear accumulation. Moreover, SPP-1-upregulation significantly enhanced cell proliferation and in vivo tumor formation ability, and reduced apoptosis, whereas these effects were all abolished when β-catenin was silenced. Overall, the present study revealed that SPP-1 promoted the progression of CRC in a β-catenin-dependent manner.

The DDX39B/FUT3/TGF\u03b2R-I axis promotes tumor metastasis and EMT in colorectal cancer

DDX39B is a member of the DEAD box (DDX) RNA helicase family required for nearly all cellular RNA metabolic processes. The exact role and potential molecular mechanism of DDX39B in the progression of human colorectal cancer (CRC) remain to be investigated. In the present study, we demonstrate that DDX39B expression is higher in CRC tissues than in adjacent normal tissues. Gain- and loss-of-function assays revealed that DDX39B facilitates CRC metastasis in vivo and in vitro. Mechanistically, RNA-sequencing (RNA-seq) and RNA-binding protein immunoprecipitation-sequencing (RIP-seq) showed that DDX39B binds directly to the FUT3 pre-mRNA and upregulates FUT3 expression. Splicing experiments in vitro using a Minigene assay confirmed that DDX39B promotes FUT3 pre-mRNA splicing. A nuclear and cytoplasmic RNA separation assay indicates that DDX39B enhances the mRNA export of FUT3. Upregulation of FUT3 accelerates the fucosylation of TGFβR-I, which activates the TGFβ signaling pathway and eventually drives the epithelial–mesenchymal transition (EMT) program and contributes to CRC progression. These findings not only provide new insight into the role of DDX39B in mRNA splicing and export as well as in tumorigenesis, but also shed light on the effects of aberrant fucosylation on CRC progression.

Cytoplasmic SHMT2 drives the progression and metastasis of colorectal cancer by inhibiting β-catenin degradation.

Introduction: Serine hydroxymethyltransferase 2 (SHMT2) plays a critical role in serine-glycine metabolism to drive cancer cell proliferation. However, the nonmetabolic function of SHMT2 in tumorigenesis, especially in human colorectal cancer (CRC) progression, remains largely unclear.Methods: SHMT2 expression in human CRC cells was identified by western blot and immunofluorescence assay. The CRC cell proliferation, migration, and invasion after SHMT2 knockdown or overexpression were explored through in vitro and in vivo assays. Immunofluorescence, mRNA-seq, co-immunoprecipitation, chromatin immunoprecipitation-qPCR and immunohistochemistry assays were used to investigate the underlying mechanisms behind the SHMT2 nonmetabolic function.Results: We demonstrated that SHMT2 was distributed in the cytoplasm and nucleus of human CRC cells. SHMT2 knockdown resulted in the significant inhibition of CRC cell proliferation, which was not restored by serine, glycine, or formate supplementation. The invasion and migration of CRC cells were suppressed after SHMT2 knockdown. Mechanistically, SHMT2 interacted with β-catenin in the cytoplasm. This interaction inhibited the ubiquitylation-mediated degradation of β-catenin and subsequently modulated the expression of its target genes, leading to the promotion of CRC cell proliferation and metastasis. Notably, the lysine 64 residue on SHMT2 (SHMT2K64) mediated its interaction with β-catenin. Moreover, transcription factor TCF4 interacted with β-catenin, which in turn increased SHMT2 expression, forming an SHMT2/β-catenin positive feedback loop. In vivo xenograft experiments confirmed that SHMT2 promoted the growth and metastasis of CRC cells. Finally, the level of SHMT2 was found to be significantly increased in human CRC tissues. The SHMT2 level was correlated with an increased level of β-catenin, associated with CRC progression and predicted poor patient survival.Conclusion: Taken together, our findings reveal a novel nonmetabolic function of SHMT2 in which it stabilizes β-catenin to prevent its ubiquitylation-mediated degradation and provide a potential therapeutic strategy for CRC therapy.

A mathematical model of the circadian clock and drug pharmacology to optimize irinotecan administration timing in colorectal cancer

Scheduling anticancer drug administration over 24 h may critically impact treatment success in a patient-specific manner. Here, we address personalization of treatment timing using a novel mathematical model of irinotecan cellular pharmacokinetics and –dynamics linked to a representation of the core clock and predict treatment toxicity in a colorectal cancer (CRC) cellular model. The mathematical model is fitted to three different scenarios: mouse liver, where the drug metabolism mainly occurs, and two human colorectal cancer cell lines representing an in vitro experimental system for human colorectal cancer progression. Our model successfully recapitulates quantitative circadian datasets of mRNA and protein expression together with timing-dependent irinotecan cytotoxicity data. The model also discriminates time-dependent toxicity between the different cells, suggesting that treatment can be optimized according to their cellular clock. Our results show that the time-dependent degradation of the protein mediating irinotecan activation, as well as an oscillation in the death rate may play an important role in the circadian variations of drug toxicity. In the future, this model can be used to support personalized treatment scheduling by predicting optimal drug timing based on the patient’s gene expression profile.