Malignant Progression Of Colorectal Cancer Research Articles

Mutant p53 in colon cancer.

The accumulation of genetic alterations in driver genes is responsible for the development and malignant progression of colorectal cancer. Comprehensive genome analyses have revealed the driver genes, including APC, KRAS, TGFBR2, and TP53, whose mutations are frequently found in human colorectal cancers. Among them, the p53 mutation is found in ~60% of colorectal cancers, and a majority of mutations are missense-type at ‘hot spots’, suggesting an oncogenic role of mutant p53 by ‘gain-of-function’ mechanisms. Mouse model studies have shown that one of these missense-type mutations, p53 R270H (corresponding to human R273H), causes submucosal invasion of intestinal tumors, while the loss of wild-type p53 has a limited effect on the invasion process. Furthermore, the same mutant p53 promotes metastasis when combined with Kras activation and TGF-β suppression. Importantly, either missense-type p53 mutation or loss of wild-type p53 induces NF-κB activation by a variety of mechanisms, such as increasing promoter accessibility by chromatin remodeling, which may contribute to progression to epithelial–mesenchymal transition. These results indicate that missense-type p53 mutations together with loss of wild-type p53 accelerate the late stage of colorectal cancer progression through the activation of both oncogenic and inflammatory pathways. Accordingly, the suppression of the mutant p53 function via the inhibition of nuclear accumulation is expected to be an effective strategy against malignant progression of colorectal cancer.

Crosstalk between TF/FVIIa and EGFR signaling in colorectal cancer cells

ABSTRACTTF/FVIIa (Tissue Factor/Active Coagulation factor VII) and EGFR (Epidermal Growth Factor Receptor) signaling both promote malignant progression of colorectal cancer. However, the crosstalk of these two signaling pathways in human colorectal cancer cells remains unclear. Here we detected the changes of mRNA profile in human colorectal cancer cell SW620 exposed to FVIIa. Microarray showed that mRNA levels of EGFR ligands were significantly upregulated. Western blot analysis confirmed the upregulation of EGFR ligands and the phosphorylation of EGFR at tyrosine-845 in colorectal cancer cells exposed to FVIIa. However, knockdown of TF by RNAi could block the upregulation of EGFR ligands induced by FVIIa stimulation. On the other hand, the expression of components of TF/FVIIa signaling was significantly upregulated in LoVo cells stimulated by EGF. However, the crosstalk between the two signaling pathways could not be detected in HT-29 colon cancer cells bearing wild-type KRAS. Taken together, our study suggest that the crosstalk between TF/FVIIa and EGFR signaling pathways in colon cancer cells depends on KRAS mutation.

High expression of TFEB is associated with aggressive clinical features in colorectal cancer.

ObjectivesThe transcription factor EB (TFEB), a member of the micropthalmia family, has been found to be associated with autophagy and upregulated in some kinds of tumors. However, very few studies focused on TFEB in colorectal cancer (CRC). TFEB expression status and its relevance to clinical features in CRC would be analyzed in this study.Materials and methodsReal-time PCR, Western blot, and immunohistological staining were used to evaluate TFEB expression in CRC tissues and adjacent normal tissues, and the role of TFEB in CRC cell lines was investigated in vitro and in vivo.ResultsTFEB was expressed at lower level in CRC tissues than normal in both mRNA and protein level. However, there were significantly positive correlations between TFEB expression in cancer tissues and malignant progression of CRC. Cancers with TFEB overexpression always had deeper infiltration and higher lymphatic metastasis rate. Furthermore, patients with high TFEB levels always had poor survival, and higher TFEB expression could be a predictor of survival in multivariate analysis. Meanwhile, knockdown TFEB by shRNA or knockout TFEB by sgRNA in CRC cell lines could significantly inhibit cell proliferation and migration in amino acid-free medium. In addition, we found a positive relationship between TFEB and Beclin1 expression, and silencing TFEB inhibited Beclin1 expression in CRC cells.ConclusionTFEB expression correlated with aggressive clinical features in CRC, and higher TFEB expression could be a prognostic factor and potential treatment target of CRC.

A Novel lncRNA, LINC00460, Affects Cell Proliferation and Apoptosis by Regulating KLF2 and CUL4A Expression in Colorectal Cancer

Emerging evidence has proven that long noncoding RNAs (lncRNAs) play important roles in human colorectal cancer (CRC) biology, although few lncRNAs have been characterized in CRC. Therefore, the functional significance of lncRNAs in the malignant progression of CRC still needs to be further explored. In this study, through analyzing TCGA RNA sequencing data and other publicly available microarray data, we found a novel lncRNA, LINC00460, whose expression was significantly upregulated in CRC tissues compared to adjacent normal tissues. Consistently, real-time qPCR results also verified that LINC00460 was overexpressed in CRC tissues and cells. Furthermore, high LINC00460 expression levels in CRC specimens were correlated with larger tumor size, advanced tumor stage, lymph node metastasis and shorter overall survival. In vitro and in vivo assays of LINC00460 alterations revealed a complex integrated phenotype affecting cell growth and apoptosis. Mechanistically, LINC00460 repressed Krüppel-like factor 2 (KLF2) transcription by binding to enhancer of zeste homolog 2 (EZH2). LINC00460 also functioned as a molecular sponge for miR-149-5p, antagonizing its ability to repress cullin 4A (CUL4A) protein translation. Taken together, our findings support a model in which the LINC00460/EZH2/KLF2 and LINC00460/miR-149-5p/CUL4A crosstalk serve as critical effectors in CRC tumorigenesis and progression, suggesting new therapeutic directions in CRC.

Targeting IL-17 for cancer-associated inflammation and immunity

Abstract Interleukin-17A (IL-17A) is a proinflammatory cytokine whose expression has been linked to rapid malignant progression of colorectal cancer (CRC) and resistance to therapy. Ablation of IL-17A or its type A receptor (IL-17RA) in mouse models of colonic tumorigenesis confirmed their tumor-promoting function. IL-17RA signals directly within transformed colonic epithelial cells (enterocytes) to promote early tumor development. IL-17RA engagement activates ERK, p38 MAPK and NF-κB signaling and promotes the proliferation of tumorigenic enterocytes who just lost expression of the APC tumor suppressor. Although IL-17RA signaling also controls production of IL-6, this mechanism makes only a partial contribution to colonic tumorigenesis. Combined treatment with chemotherapy, which induces IL-17A expression, and an IL-17A neutralizing antibody enhanced the therapeutic responsiveness of established colon tumors. Consistent with the notion that IL-17 promotes tumor-associated inflammation and inhibits anti-tumor immunity, ablation of IL-17RA resulted in reduced infiltration of CD11b+ cells and elevated numbers and activity of cytotoxic T lymphocytes in colorectal tumors. Using anti-IL-17 as an adjuvant therapy may improve the efficacy and safety of current cancer immunotherapeutic schemes.

MTA3 regulates malignant progression of colorectal cancer through Wnt signaling pathway.

MTA3 overexpression has been implicated in carcinogenesis. The aim of the present study was to explore the clinical significance and biological roles of MTA3 in human colorectal cancer and colorectal cancer cells. A total of 80 cases of colorectal cancer tissues were examined by immunohistochemistry for MTA3 protein expression. We analyzed the relationship between MTA3 and clinical factors and the results showed that MTA3 was overexpressed in 51.25% (41/80) cancer cases. There was significant associations between MTA3 overexpression and advanced TNM stage (p = 0.0086) and Ki67 index (p = 0.001). We overexpressed MTA3 in LoVo cells and depleted its expression in HCT15 cells. The results showed that MTA3 promoted cancer cell proliferation, invasion, migration, and cell cycle progression, and inhibited 5-fluorouracil-induced apoptosis in LoVo cell line. MTA3 depletion in HCT15 cell line showed the opposite effects. In addition, we found that MTA3 positively regulated cell cycle proteins including cyclin D1 and cyclin E. It also upregulated Bcl2 and downregulated Bax expression. Furthermore, we found that MTA3 could activate Wnt signaling pathway by upregulating Wnt target proteins. Our results demonstrated that MTA3 overexpression contributes to colorectal cancer carcinogenesis, progression, and chemoresistance. MTA3 could serve as a potential therapeutic target in colorectal cancer.

Epigenetically regulated miR-145 suppresses colon cancer invasion and metastasis by targeting LASP1.

MiR-145 is a tumor-suppressive microRNA that participates in the malignant progression of colorectal cancer (CRC). Although miR-145 has been reported to inhibit proliferation and to induce apoptosis of CRC cells, the reports about its role in invasion and metastasis are controversial. The regulation of miR-145 its own expression also requires further elucidation. In this study, we firstly found that miR-145 is markedly downregulated in the metastatic tumors of CRC patients. Then through gain- and loss-of function studies, we demonstrated that miR-145 suppresses the invasion and metastasis of CRC cells. We also provided experimental evidences which include direct binding assays and verifications on tissue specimens to confirm that LIM and SH3 protein 1 (LASP1) is a direct target of miR-145. Furthermore, we identified the core promoter regions of miR-145 and observed the cooperation between histone methylation and transcription factors through binding to these core promoter regions to regulate the expression of miR-145 in CRC cells. Our study provides an insight into the regulatory network in CRC cells, thus offering new targets for treating CRC patients.

Abstract 4337: Novel potent and selective orally available CDK8/19 kinase inhibitors

Abstract CDK8 and its paralog CDK19 are cyclin-dependent kinases and together with CDK7 and CDK9 belongs to the group of C-terminal domain (CTD) kinases that phosphorylate the CTD of RNA polymerase II, thus regulating transcription. CDK8 together with its partner Cyclin C, MED12 and MED13 are components of multi-protein Mediator complex which couples action of transcription factors with the molecular machinery that carries out transcription, e.g. CDK8 couple basal transcriptional machinery to sequence-specific transcription factors such as Notch, p53, β-catenin, and also repress the transcription of other genes. As Mediator independent roles, CDK8 has been shown to act as part of a separate complex behaving as a histone kinase. Moreover, CDK8 has been identified as a major kinase in the response to IFN signaling mediated STAT1-S727 phosphorylation. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer being a marker of poor prognosis. Moreover, in gastric cancer CDK8 expression and the delocalization of β-catenin expression showed a significant positive correlation with carcinogenesis and tumor progression, especially lymph node metastasis. Recently, gene amplification of CDK8, CDK19, CCNC and MED13 in breast cancers has been related with poor response to adjuvant therapy. These results suggest that CDK8 inhibitors may become a unique class of anticancer drugs that could increase the efficacy of cancer therapy by blocking chemotherapy-induced production of tumor-promoting secreted factors. We have carried out a medium throughput screening campaign which led to the discovery of several low nanomolar hits belonging to 3 different chemical series. Later on, we have performed a Hit Generation phase, designing novel inhibitors within a patentable chemical space. Here, we have taken into account information from the chemical structures of the screening hits, X-ray structure of CDK8 protein co-crystalized with sorafenib, docking studies, chemical feasibility and intellectual property. We have identified a novel chemical series of CDK8/19 inhibitors. After a HtL exploration we have reached lead compounds with potency in the picomolar range and high selectivity versus a panel of 468 protein kinases. The leads display cellular activity by blocking β−catenin reporter activity and STAT1 phoshorylation in the low nanomolar range. Lead compounds have been screened in a panel of tumoral cell lines, showing a defined profile in terms of sensitivity with GI50s which range from nanomolar to low micromolar values. These inhibitors induce cell death in a dose response manner. The novel chemical series show a drug-like profile in terms of solubility, permeability, CYP450 inhibition and hERG. The identified lead compounds are orally bioavailable, with good clearance, volume of distribution and exposure levels. A selected lead compound has been used in in vivo PK-PD studies showing positive results. Citation Format: Carmen Blanco Aparicio, Oliver Renner, Elena Gomez-Casero, Maria Isabel Albarran, Antonio Cebria, Borja Barrera, Enara Aguirre, Maria del Carmen Rodriguez de Miguel, Manuel Urbano, Ana Isabel Hernandez, Cristina Gomez de la Oliva, Virginia Rivero, Rosario Concepcion Riesco, Sonia Martinez Gonzalez, Joaquin Pastor. Novel potent and selective orally available CDK8/19 kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4337.

Abstract A86: LRP16, interacting with PKR, prolongs NF-kappaB activation and drives colorectal cancer tumorigenesis

Abstract NF-kappaB frequently activated in colorectal cancer (CRC) is linked to rapid malignant progression of CRC and is also crucial for the maintenance of homeostasis and therapy resistance. However, the underlying mechanisms how NF-kappaB activation and the crosstalk of NF-kappaB with other transcription factors are controlled in CRC remain incompletely understood. Here, we show that LRP16 physically interacts with double-stranded RNA-dependent protein kinase (PKR) functions as a signal transducer in signaling pathways activated by different stimuli by a variety of effectors, including NF-kappaB. Overexpression of LRP16 confers PKR autophosphorylation leading to the phosphorylation and activation of the NF-kappaB complex. Modulation of NF-kappaB mediated gene expression in response to diverse signals is coordinated by PKR through the phosphorylation and activation of IKK complex and this process does not require PKR kinase activity. We identified PKR, an essential regulatory subunit of the IKK complex. Silencing PKR expression by RNA interference blocked LRP16 induced expression of NF-kappaB target genes. These cells were also not protected from apoptosis in response to chemotherapy. In vivo, overexpression of LRP16 promotes CRC tumorigenesis, in turn, inhibition of LRP16 or PKR blunts CRC tumorigenesis. These results demonstrated that LRP16 likely functions by recruiting PKR to the IKK complex and thus serves a regulatory function for NF-kappaB activation, suggesting that a novel role for LRP16 as mediator of chemotherapy resistance modulated in part through the protective effects of NF-kappaB activation and may be a novel strategy in the future to overcome chemotherapy resistance in CRC. Citation Format: Xiaolei Li, Zhiqiang Wu, Weidong Han. LRP16, interacting with PKR, prolongs NF-kappaB activation and drives colorectal cancer tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A86.

Abstract 1663: Selective CDK8 inhibitor SEL120-34A alters expression of interferon-related DNA damage resistance signature genes in colorectal cancer

Abstract CDK8 (cyclin-dependent kinase 8) is a kinase component of a multi - protein Mediator complex, involved in transcription control. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer (CRC). Herewith we present molecular insights into mechanism of action of SEL120-34A - a selective small molecule inhibitor of CDK8 kinase. Biochemical and binding studies indicated that SEL120-34A selectively binds and inhibits enzymatic activity of CDK8 in the low nM range. Recently CDK8 has been described as a regulator of STAT1 activity in NK cells where by phosphorylating STAT1 serine 727 (Ser727) influences a possible immunoescape mechanism in various cancers. Consistently, SEL120-34A and other recently reported selective CDK8 inhibitors could repress phosphorylation of STAT1 at a Ser727 at low nM concentrations in cancer cells without any significant changes on tyrosine sites directly regulated by JAK kinases. SEL120-34A inhibited expression of several STAT1 dependent genes in CRC cell lines, stimulated by various cytokines and growth factors. These results were further corroborated with specific CDK8 siRNA knockdown experiments and chromatin immunoprecipitation studies showing CDK8 occupancy on promoters of SEL120-34A regulated genes. In order to better characterize in vivo mechanism of action, mice bearing HCT116 and Colo205 xenograft tumors were treated with SEL120-34A and gene expression changes were measured with microarrays in excised tumors. In animals treated with the CDK8 inhibitor a dose dependent repression of STAT1 Ser727 was observed. The functional analyses of significantly (adj. p. value < 0.05) altered genes with Gene Ontology revealed that those with reduced expression belong to interferon I pathway and type I interferon-mediated signaling pathway terms. This subset of STAT regulated genes was further characterized as an interferon-related DNA damage resistance signature (IRDS) - a prosurvival pathway which correlated strongly with resistance to radiation and chemotherapy in various tumors. Consistently, SEL120-34A has shown very potent cytotoxic synergy with standard of care drugs in CRC, particularly in cells stimulated with interferons. Taken together, for the first time we have shown that selective CDK8 inhibitors are potent regulators of STAT related - IRDS signaling pathway in vitro and in vivo. In addition to previously reported stand-alone efficacy of CDK8 inhibitors in vivo, we provide also a combination treatment rationale for CRC. Citation Format: Tomasz Rzymski, Michał Mikula, Małgorzata Szajewska-Skuta, Eliza Zyłkiewicz, Łukasz Sapała, Izabela Dolata, Agata Kitlińska, Krzysztof Goryca, Aleksandra Grochowska, Aleksandra Cabaj, Agnieszka Dreas, Katarzyna Kucwaj, Artur Białas, Adam Radzimierski, Aniela Gołas, Renata Windak, Jerzy Ostrowski, Krzysztof Brzózka. Selective CDK8 inhibitor SEL120-34A alters expression of interferon-related DNA damage resistance signature genes in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1663. doi:10.1158/1538-7445.AM2015-1663

Prognostic relevance of LGALS3BP in human colorectal carcinoma.

BackgroundA previous report has shown that LGALS3BP (also known as 90K or Mac-2 BP) has antitumor activity in colorectal cancer (CRC) via suppression of Wnt signalling with a novel mechanism of ISGylation-dependent ubiquitination of β-catenin. The role of LGALS3BP in CRC prognosis was investigated.MethodsThe role of LGALS3BP on CRC progression and clinical prognosis was analyzed by combining cell cultures, in vitro assays, and immunohistochemistry.ResultsSilencing of LGALS3BP in HCT-116 human colon cancer cells resulted in enhanced β-catenin expression that was reversed by addition of human recombinant LGALS3BP. Moreover, intra-tumor delivery of LGALS3BP reduced tumor growth of xenografts originating from LGALS3BP-silenced HCT-116 cells. Finally, in a series of 196 CRC patients, LGALS3BP expression in tumor tissue associated with clinical outcome. Patients with high LGALS3BP expression had lower risk of relapse and a longer overall survival time than those with low LGALS3BP expression. Multivariate analyses confirmed LGALS3BP expression status as the only independent prognostic factor of survival.ConclusionsThese results provide evidence that low expression of LGALS3BP participates in malignant progression of CRC and implicates poor prognosis, highlighting its augmentation as a potential therapeutic approach.

Expression of HAX-1 in colorectal cancer and its role in cancer cell growth.

Colorectal cancer (CRC) is one of the most common types of cancer worldwide. Hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) has been found to be involved in several types of cancer. However, the role of HAX-1 in CRC remains to be elucidated. The aim of the present study was to investigate whether the expression of HAX-1 is associated with the progression of CRC, and to determine the effects of HAX-1 on the apoptosis and proliferation of CRC cells. Tumor tissues and adjacent noncancerous tissues were collected from 60 patients with CRC, following the provision of informed consent. The expression levels of HAX-1 and the association with clinical and pathological characteristics were then analyzed. The expression levels of HAX-1 were significantly higher in the cancerous tissues from the patients with CRC, particularly in tissues of an advanced stage of cancer. In addition, HAX-1 expression was associated with malignant progression and poor prognosis. Furthermore, SW480 CRC cells, overexpressing HAX-1, exhibited increased resistance to camptothecin in vitro, and promoted proliferation in vitro and in vivo. By contrast, HAX-1 knockdown significantly decreased the proliferation. In addition, the expression levels of ki-67 and phosphorylatedakt were inhibited following HAX-1 knockdown. In conclusion, the expression levels of HAX-1 were increased in cancerous tissue from patients with CRC, and were associated with progression of the disease. These results suggested that HAX-1 may contribute to chemotherapy resistance and malignant progression in CRC.

N-cadherin, a novel prognostic biomarker, drives malignant progression of colorectal cancer.

Recent studies have indicated that the epithelial-mesenchymal transition (EMT) is a key molecular mechanism involved in the development of colorectal cancer (CRC). N-cadherin is a mesenchymal marker of the EMT and has been closely linked to several human malignancies. However, its role in CRC has remained elusive. In the present study, qRT-PCR and western blot analysis indicated that N-cadherin expression was higher in tumor tissues than in that in their adjacent normal tissues. Immunohistochemical evaluation of N-cadherin and E-cadherin (an epithelial marker of the EMT), indicated that N-cadherin expression was significantly associated with tumor differentiation, tumor size as well as tumor, nodes and metastasis stage. Correlation analysis suggested the expression of N-cadherin was negatively correlated with that of E-cadherin in CRC tissues. Kaplan-Meier analysis indicated that patients with high N-cadherin expression had a significantly lower overall survival and disease-free survival rate than those with low N-cadherin expression, while the opposite was found for E-cadherin. Of note, the present study found that high N-cadherin expression was an independent prognostic factor for CRC. In vitro assays showed that N-cadherin was widely expressed in CRC cell lines and silencing of N-cadherin suppressed the proliferation and migration of the CRC cell line HT-29 by upregulating E-cadherin, suggesting a potential role of N-cadherin in inducing EMT. In conclusion, the present study suggested that N-cadherin has the potential of serving as a novel prognostic predictor and a promising therapeutic target for CRC.

Interleukin-17 Receptor A Signaling in Transformed Enterocytes Promotes Early Colorectal Tumorigenesis

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine linked to rapid malignant progression of colorectal cancer (CRC) and therapy resistance. IL-17A exerts its pro-tumorigenic activity through its type A receptor (IL-17RA). However, IL-17RA is expressed in many cell types, including hematopoietic, fibroblastoid, and epithelial cells, in the tumor microenvironment, and how IL-17RA engagement promotes colonic tumorigenesis is unknown. Here we show that IL-17RA signals directly within transformed colonic epithelial cells (enterocytes) to promote early tumor development. IL-17RA engagement activates ERK, p38 MAPK, and NF-κB signaling and promotes the proliferation of tumorigenic enterocytes that just lost expression of the APC tumor suppressor. Although IL-17RA signaling also controls the production of IL-6, this mechanism makes only a partial contribution to colonic tumorigenesis. Combined treatment with chemotherapy, which induces IL-17A expression, and an IL-17A neutralizing antibody enhanced the therapeutic responsiveness of established colon tumors. These findings establish IL-17A and IL-17RA as therapeutic targets in colorectal cancer.

Abstract SY40-01: Tumor elicited inflammation and malignant progression in colorectal cancer

Abstract Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the US and other Western countries. Whereas 2% of CRC arises in the context of pre-existing inflammatory bowel disease (IBD), especially ulcerative colitis (UC), and is known as colitis associated cancer (CAC), most CRC, including familial and sporadic cases, is found in individuals that do not show any signs of IBD. Yet, expression profiling has revealed the same inflammatory gene signature, which depends on activation of NF-κB and STAT3, in both CAC and CRC, findings that generate a question regarding the origin of the CRC-elicited inflammatory response. In early experiments we have shown that activation of NF-κB, which leads to production of the pro-inflammatory cytokine IL-6, a potent activator of STAT3, plays a critical role in the development of CAC. More recently we investigated the origin and role of inflammation in the development and progression of sporadic CRC, most of which is driven by loss of the tumor suppressor APC and activation of the β catenin signaling pathway. We focused our studies on IL-23, a heterodimeric cytokine, composed of a unique p19 subunit and a p40 subunit which it shares with IL-12. As observed by others, we also found that IL-23 expression is strongly elevated in CRC relative to adjacent non-tumor tissue and have extended these findings to the CPC-APC mouse model of colorectal tumorigenesis. In CPC-APC mice, the major source of IL-23 expression in colorectal adenomas are tumor associated macrophages (TAM). Importantly, ablation of IL-23 p19 either in all cells or only in bone marrow (BM)-derived cells attenuates the development and slows down the progression of CRC in CPC-APC mice and similar results were observed upon ablation of IL-23 receptor (IL-23R). As IL-23R is not expressed on adenoma epithelial cells, IL-23 must exert its pro-tumorigenic effect via an indirect mechanism. Indeed, IL-23 signaling promotes the polarization of IL-17 producing T cells (Th17) and the production of IL-6, both of which contribute to the development and progression of sporadic colorectal tumors in mice. Importantly, molecular epidemiological studies carried out by Galon, Fridman and their co-workers have revealed that an “IL-23-Th17” signature that is upregulated in about 10% of human CRC patients, and that the presence of this signature in stage I/II CRC is associated with very poor prognosis and a marked decrease in disease free survival. We investigated the mechanism responsible for the specific induction of IL-23 in TAM and found it to depend on Toll like receptor (TLR) - MyD88 signaling, which appears to be activated in response to components of the colonic microflora that permeate the adenomas. We also detected eubacterial 16S RNA in both mouse and human colonic adenomas, as well as increased penetrance of bacterial endotoxin (LPS) into adenomas relative to surrounding non-cancerous tissue. The development of colorectal adenomas in both mice and men is associated with loss of protective mucins and junctional adhesion proteins and this is likely to be the primary mechanism that accounts for the selective entry of microbial products into the tumor and induction of the tumor promoting “IL-23-Th17” signature. Future studies should focus on the therapeutic value of anti-IL-23 or anti-IL-17 interventions and the genetic or environmental causes of the large variation in the magnitude of IL-17 production amongst human CRC patients.

Angiopoietin-like protein 2 negatively regulated by microRNA-25 contributes to the malignant progression of colorectal cancer.

Angiopoietin-like protein2(ANGPTL2) is associated with tumor progression while dysregulation of its expression has been observed in various types of cancer. However, the expression and role of ANGPTL2 remain exclusive in colorectal cancer(CRC). In the present study, we determined the expression levels ofANGPTL2 in CRC tissues and cells. The roles of ANGPTL2 and miR-25 in the migration and invasion of CRC SW620 and HCT-116 cells were also investigated using transwell assays or scratch wound assays. The results showed that ANGPTL2 increased with metastatic progression. Increased ANGPTL2 and decreased microRNA-25(miR-25) expression were found to coexist in CRC. The functional studies revealed that knockdown of ANGPTL2 reduced colony formation, and the invasive and migratory abilities of human CRC SW620 and HCT-116 cells. Similarly, overexpression of miR-25 resulted in reduced colony formation, invasion and migration in both cell lines. The overexpression of miR-25 led to a decreased ANGPTL2 mRNA and protein expression, whereas the downregulation of miR-25 resulted in increased ANGPTL2 mRNA and protein expression, in SW620 and HCT-116 cells. miR-25 directly targeted ANGPTL2 by binding to its 3'‑UTR, as determined by the dual luciferase reporter assay. To the best of our know-ledge, the results of this study suggest for the first time that the abnormal upregulation of ANGPTL2 in CRC is associated with miR-25 downregulation. Additionally, miR-25‑mediated ANGPTL2 promoted the malignant progression of CRC. The present study provides evidence supporting ANGPTL2 and miR-25 as diagnostic ortherapeutic targets for CRC.

Expression of HAX-1 in human colorectal cancer and its clinical significance

Colorectal cancer (CRC) remains one of the most common cancers worldwide. HS1-associated protein X-1 (HAX-1) has been highlighted as an important marker in many types of cancers. However, little is known about the role of HAX-1 in CRC. The aim of this study was to analyze the correlation of HAX-1 expression with the clinicopathological features of CRC. The protein and mRNA levels of HAX-1 were examined by immunohistochemistry (IHC) and real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) in CRC tissues and adjacent noncancerous tissues. Survival curves were made with follow-up data. The relations of the prognosis with clinical and pathological characteristics were analyzed. Using IHC and RT-qPCR, we showed that HAX-1 expression was significantly higher in CRC tissues than in adjacent noncancerous tissues (P < 0.05). High HAX-1 expression was significantly associated with lymph node metastasis (P = 0.034) and tumor (T) node (N) metastasis (M) stage (P = 0.028) of patients with CRC. The Kaplan-Meier survival curves indicated that overall survival was significantly worse in CRC patients with HAX-1 overexpression. Multivariate analysis showed that high HAX-1 expression was an independent predictor of overall survival. In conclusion, our data for the first time provide a basis for the concept that overexpression of HAX-1 may contribute to the malignant progression of CRC and predict poor prognosis for patients with this disease. HAX-1 might be an important marker for tumor progression and prognosis, as well as a potential therapeutic target of CRC.

Hypoxia-inducible factor-1α modulates the down-regulation of the homeodomain protein CDX2 in colorectal cancer

Hypoxia-inducible factor-1alpha (HIF-1alpha) is the main active subunit of HIF-1, which promotes tumor cell survival and critical steps involved in tumor progression and aggressiveness. To clarify the possible involvement of the HIF-1alpha subunit and homeodomain protein CDX2 in the development and progression of colorectal cancer (CRC), we examined in vivo the immunohistochemical expression of HIF-1alpha and its topological correlation with CDX2 expression in colorectal adenocarcinoma. We then examined the in vitro effect of hypoxia mimicked by CoCl2 on mRNA and protein expression of HIF-1alpha and CDX2 using two human colon cancer cell lines, SW480 and LS174T. In addition, hypoxia-induced changes in the mRNA expression of Snail were also analyzed. Of the 62 cases of CRC examined, 43 (69.4%) and 39 (62.9%) were positive for CDX2 and HIF-1alpha, respectively, such that their expression was correlated with differentiation grade, tumor stage and lymph node metastasis (chi2 test, p<0.01). Furthermore, HIF-1alpha expression observed in 10 of the 16 (62.5%) poorly differentiated CRCs showed a topological correlation with loss of CDX2 expression. Real-time PCR and western blotting demonstrated that CDX2 expression was decreased by CoCl2 (100 microM) in both SW480 and LS174T cells. mRNA and protein expression of HIF-1alpha and mRNA expression of Snail was increased by hypoxia in both colon cancer cell lines. In conclusion, the present observations support that hypoxia-inducible factor-1alpha induces down-regulation of CDX2 in colon carcinoma cells and that Snail may be involved in this regulation process. These findings suggest that hypoxia plays an important role in the malignant progression of CRC.

Colorectal carcinogenesis: Review of human and experimental animal studies

This review gives a comprehensive overview of cancer development and links it to the current understanding of tumorigenesis and malignant progression in colorectal cancer. The focus is on human and murine colorectal carcinogenesis and the histogenesis of this malignant disorder. A summary of a model of colitis-associated colon tumorigenesis (an AOM/DSS model) will also be presented. The earliest phases of colorectal oncogenesis occur in the normal mucosa, with a disorder of cell replication. The large majority of colorectal malignancies develop from an adenomatous polyp (adenoma). These can be defined as well-demarcated masses of epithelial dysplasia, with uncontrolled crypt cell proliferation. When neoplastic cells pass through the muscularis mucosa and infiltrate the submucosa, they are malignant. Carcinomas usually originate from pre-existing adenomas, but this does not imply that all polyps undergo malignant changes and does not exclude de novo oncogenesis. Besides adenomas, there are other types of pre-neoplasia, which include hyperplastic polyps, serrated adenomas, flat adenomas and dysplasia that occurs in the inflamed colon in associated with inflammatory bowel disease. Colorectal neoplasms cover a wide range of pre-malignant and malignant lesions, many of which can easily be removed during endoscopy if they are small. Colorectal neoplasms and/or pre-neoplasms can be prevented by interfering with the various steps of oncogenesis, which begins with uncontrolled epithelial cell replication, continues with the formation of adenomas and eventually evolves into malignancy. The knowledge described herein will help to reduce and prevent this malignancy, which is one of the most frequent neoplasms in some Western and developed countries.

Heterogeneous expression of Wnt/β‐catenin target genes within colorectal cancer

Invasion of common colorectal adenocarcinomas is coupled with a transient loss of epithelial differentiation of tumor cells. Previously, we have shown that dedifferentiated tumor cells at the invasive front (IF) accumulate the transcriptional activator beta-catenin in the nucleus, in contrast to cells of the tumor center. To characterize the cells of these two morphogenic tumor areas, gene expression profiling was performed. Our study demonstrates that intratumorous heterogeneity in colorectal cancer correlates with differential expression of 510 genes between the central tumor region (TC) and the IF. Many genes differentially expressed at the IF are involved in cellular invasion processes like cell motility, cell adhesion and extracellular matrix interaction. This in vivo analysis shows overexpression of known Wnt/beta-catenin target genes either in the entire tumor tissue (compared to normal mucosa) or specifically at the IF. Thus, even though all tumor cells overexpress beta-catenin, the existence of at least 2 groups of Wnt/beta-catenin target genes selectively activated in different tumor regions is suggested. The concomitant high expression of inflammation- and tissue repair-related genes at the IF supports the hypothesis that an inflammation-activated microenvironment may trigger selective Wnt/beta-catenin target gene expression and contribute to the malignant progression of colorectal cancer.