Clinical Colorectal Cancer Research Articles

Age, sex, and specific gene mutations affect the effects of immune checkpoint inhibitors in colorectal cancer

The effect of age and sex on the predictive value of colorectal cancer (CRC) patients treated with immune checkpoint inhibitors (ICIs) has been controversial, and the effect of specific gene mutations on the predictive value of CRC patients treated with ICIs remains to be explored. Our study analyzed the influence of the above factors on the overall survival (OS) of CRC patients receiving ICIs and explored the influencing mechanism of various predictive biomakers. We performed survival prognostic correlation analysis and bioinformatics analysis on the clinical CRC cohort receiving ICIs in from the Memorial Sloan Kettering Cancer Center (MSKCC) and the clinical and genetic data from The Cancer Genome Atlas (TCGA)-CRC dataset, including immunogenicity analysis, tumor immune microenvironment analysis, and gene set enrichment analysis and so on. We found that mutation count >11 mutation/Mb (tumor mutation burden, TMB-high) (HR = 0.22, 95 %CI: 0.09−0.53; P < 0.001), male (HR = 0.51, 95 %CI: 0.28−0.93; P = 0.029), RNF43-mutant (MT) (HR = 0.12, 95 %CI: 0.03−0.49; P = 0.003), CREBBP-MT (HR = 0.23, 95 %CI: 0.07−0.76; P = 0.016), NOTCH3-MT (HR = 0.17, 95 %CI: 0.04−0.74; P = 0018), PTCH1-MT (HR = 0.27, 95 %CI: 0.08−0.9; P = 0.033), CIC-MT (HR = 0.23, 95 %CI: 0.05−0.93; P = 0.040), DNMT1-MT (HR = 0.12, 95 %CI: 0.02−0.93; P = 0.043) and SPEN-MT (HR = 0.31, 95 %CI: 0.09−0.99; P < 0.049) are all related to longer OS, but age≤65 years (HR = 3.01, 95 %CI: 1.18–7.65; P = 0.021), APC-MT (HR = 2.51, 95 %CI: 1.12–5.63; P = 0.026) and TP53-MT (HR = 1.94, 95 %CI: 1.03–3.65; P = 0.041) are associated with shorter OS. The reason why positive predictive markers provide survival benefits to CRC may be related to higher immunogenicity such as TMB, highly expression of mRNA related to immune response, highly infiltrating immune-active cells such as CD8 + T cells, active immune-active pathways, and DNA damage repair pathways with an increased number of mutations.

Propranolol Suppresses the Growth of Colorectal Cancer Through Simultaneously Activating Autologous CD8+ T Cells and Inhibiting Tumor AKT/MAPK Pathway.

Propranolol suppresses tumor growth in a variety of preclinical solid tumor models, with a number of proposed cell signaling and immunological mechanisms. We want to confirm the potential mechanisms, including reduced phosphorylation of AKT/MAPK pathways, as well as enhanced CD8+ T-cell-mediated antitumor immune response. To clarify the mechanism of propranolol activity in colorectal cancer, the therapeutic activity of propranolol was then assessed in CT26WT tumors engrafted in BALB/C mice. Then the effect of propranolol treatment was also examined by randomizing patients undergoing surgical resection of a previously untreated colorectal cancer to propranolol or placebo group and treated for 1week prior to surgery. CT26WT tumor size was smaller after propranolol than vehicle control. Propranolol downregulated the expression of p-AKT/p-ERK/p-MEK in tumor tissue. The frequency of tumor CD8+ T cells was significantly elevated in propranolol-treated mice. The expression of GzmB/IFN-γ/T-bet in the CD8+ T-cell population was significantly increased in propranolol treated mice tumor tissue. In propranolol-treated surgical specimens, the expression of p-ERK was decreased and the frequency of CD8+ was significantly elevated. The expression of GzmB in the CD8+ T-cell population was significantly increased in propranolol-treated subjects. Together, these data show propranolol simultaneously activating autologous CD8+ T cells and decreasing the expression of p-AKT/p-ERK/p-MEK in mouse tumor models, while inhibiting the expression of p-ERK in clinical colorectal cancer. Effort is now needed to further dissect whether both pathways are required for antitumor effect, as the activity of this old drug is moved forward.

Fibronectin promotes tumor cells growth and drugs resistance through a CDC42-YAP-dependent signaling pathway in colorectal cancer.

Fibronectin (FN) is a high-molecular-weight glycoprotein of the extracellular matrix (ECM) that binds to membrane-spanning receptor proteins or other elements in ECM. The expression of FN could be involved in the cancer cells proliferation or migration, and the molecular mechanisms responsible for FN induced protumor signals begin to be elucidated. Here, we report that the elevated expression of FN was observed in those chemoresistant tumor tissues from patients with colorectal cancer. Consistently, FN culture significantly strengthened the proliferation of colorectal cancer cells, induced the colorectal tumor sustained growth and drug resistance in vitro and in vivo. In mechanism, FN could bind to integrin αvβ1, resulting the downstream cell division cycle 42/yes-associated protein 1 (CDC42/YAP-1) signaling pathway activation. The activation of CDC42/YAP-1 signal induces the upregulation of transcription factor SOX2, causing the sustained growth and drugs resistance in colorectal cancer. Blockade of integrin αvβ1 significantly suppressed the colorectal cancer growth and drugs resistance development in vitro and in vivo, which provides a new target for clinical colorectal cancer treatment.

Cholecystokinin type 2 receptor in colorectal cancer: diagnostic and therapeutic target.

Cholecystokinin type 2 receptor (CCK2R), which mediates the action of gastrin and cholecystokinin (CCK), has been demonstrated to promote the proliferation of colorectal cancer (CRC). A number of studies showed that CCK2R overexpressed in gastric cancer and pancreatic cancer but few in CRC. The correlation between CCK2R expression and clinicopathological characteristics is also not clear. This study investigated CCK2R expression in a wide range of cell lines and clinical CRC samples, and explored expression pattern and prognostic value of CCK2R in relation to clinicopathological parameters. The location and expression levels of CCK2R were measured by immunocytochemical (ICC), qRT-PCR and Western blot. The druggability and antineoplastic effects of CCK2R as a therapeutic target were investigated using an anti-CCK2R targeting recombinant toxin named rCCK8PE38 by CCK-8 assay. Compared with paracarcinoma tissues, tumor samples showed overexpression of CCK2R (p = 0.028) including both CRC tissue and plasma samples, with plasma detection showing a significant indication for CCK2R evaluation. Aberrant expression correlated significantly with histological type (p = 0.032) and p53 status (p < 0.01), and patients with CCK2R overexpression had significantly lower disease-free survival. Application of rCCK8PE38 demonstrated the specificity and druggability of CCK2R as a therapeutic target, providing a strategy for clinical case screening of drugs targeting CCK2R. This study highlighted the aberrant expression and clinical correlation of CCK2R and reveals its diagnostic, prognostic and treatment value in CRC. We hypothesize that CCK2R serve as a target for the diagnosis and treatment of this cancer.

Identification of Early Warning Signals at the Critical Transition Point of Colorectal Cancer Based on Dynamic Network Analysis.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Due to the lack of early diagnosis methods and warning signals of CRC and its strong heterogeneity, the determination of accurate treatments for CRC and the identification of specific early warning signals are still urgent problems for researchers. In this study, the expression profiles of cancer tissues and the expression profiles of tumor-adjacent tissues in 28 CRC patients were combined into a human protein–protein interaction (PPI) network to construct a specific network for each patient. A network propagation method was used to obtain a mutant giant cluster (GC) containing more than 90% of the mutation information of one patient. Next, mutation selection rules were applied to the GC to mine the mutation sequence of driver genes in each CRC patient. The mutation sequences from patients with the same type CRC were integrated to obtain the mutation sequences of driver genes of different types of CRC, which provide a reference for the diagnosis of clinical CRC disease progression. Finally, dynamic network analysis was used to mine dynamic network biomarkers (DNBs) in CRC patients. These DNBs were verified by clinical staging data to identify the critical transition point between the pre-disease state and the disease state in tumor progression. Twelve known drug targets were found in the DNBs, and 6 of them have been used as targets for anticancer drugs for clinical treatment. This study provides important information for the prognosis, diagnosis and treatment of CRC, especially for pre-emptive treatments. It is of great significance for reducing the incidence and mortality of CRC.

MICA ∗012:01 Allele Facilitates the Metastasis of KRAS-Mutant Colorectal Cancer.

Major histocompatibility complex (HLA) class I chain-related protein A (MICA) regulates immune surveillance through activation of NKG2D (natural killer group 2D) receptor. However, the genetic association, potential function, and predictive ability of MICA alleles with colorectal cancer (CRC) prognosis remain undefined. In this study, we characterized MICA alleles in tissue samples from 104 patients with CRC and 536 healthy controls and carried out genetic association studies by molecular and clinical CRC phenotypes. Preliminary sequence analysis revealed that MICA ∗009:01 or ∗049 alleles were significantly decreased in patients with CRC (p = 0.0049), and further stratification analysis indicated that MICA ∗012:01 allele was associated with patients with CRC and carrying KRAS codon 12 mutation (p = 0.027). The functional consequences of MICA alleles were examined via transfected CRC cell lines which showed that overexpression of MICA ∗012:01 enhanced the proliferation, invasion, and metastatic phenotype of CRC. Preliminary analysis of disease-free survival time in patients with and without MICA ∗012:01 suggest this allele may be predictive for poor prognosis of patients with KRAS codon 12 mutated CRC, as no somatic mutation of MICA gene was detected in CRC tumors compared to paracancerous tissues. Our study indicates that MICA ∗012:01 allele is associated with KRAS-mutated CRC, facilitates CRC invasion and metastasis, and possibly reduces the survival of patients with KRAS-mutated CRC.

Age, sex, and specific gene mutations affect the effects of immune checkpoint inhibitors in colorectal cancer.

e16103 Background: The effect of age and sex on the prognosis of colorectal cancer (CRC) patients treated with immune checkpoint inhibitors (ICIs) has been controversial, and the effect of specific gene mutations on the prognosis of patients treated with ICIs remains to be explored. Based on detailed clinical and genetic data, this study analyzed the influence of the above factors on the overall survival (OS) of CRC patients after ICI treatment and explored the influencing mechanism of various prognostic factors. Methods: We performed survival prognostic correlation analysis and bioinformatics analysis on the clinical CRC cohort receiving ICIs published by Samstein et al. and the clinical and genetic data from The Cancer Genome Atlas (TCGA)-CRC dataset, including immunogenicity and immunosignature analysis, DNA damage response and repair (DDR) pathway analysis, and gene set enrichment analysis (GSEA). Results: Both clinical characteristics and specific gene mutations may be prognostic factors for ICI treatment in CRC patients. Mutation count &gt; 11 (tumor mutation burden (TMB)-high), age &gt; 65, male, RNF43, CREBBP, NOTCH3, PTCH1, CIC, DNT1, and SPEN mutant (MT) were associated with longer OS, while APC-mMT and TP53-MT were significantly correlated with shorter OS. The reason why protective prognostic markers provide better survival benefits to CRC patients may be related to factors such as higher immunogenicity, highly infiltrated immunoactive cells and active immunoactive pathways. Conclusions: Our study is the first to elucidate comprehensive prognostic factors for the treatment of ICIs in CRC patients, from clinical characteristics such as age and sex to specific gene mutations, providing new ideas for immunotherapy in CRC.

Analysis of immune-related signatures of colorectal cancer identifying two different immune phenotypes: Evidence for immune checkpoint inhibitor therapy.

Immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment of numerous types of cancer, including colorectal cancer (CRC). Patients with CRC and deficient mismatch repair or high microsatellite instability could benefit from ICI treatment, although the response rate of most patients is low. Therefore, the immune subtyping of patients with CRC is required in order to determine the subtypes suitable for ICI treatment. The present study used a cohort of patients with CRC from The Cancer Genome Atlas (TCGA) to perform molecular subtyping, with results validated in three CRC cohorts from the Gene Expression Omnibus. Non-negative matrix factorization was used to achieve consensus molecular subtyping. The tumor immune dysfunction and exclusion algorithm was used to predict potential ICI therapy responses and gene set enrichment analysis was performed to define different pathways associated with the immune response. Two distinct subtypes of CRC were finally identified in TCGA cohorts, which were characterized as significantly different prognostic subtypes (low-risk and high-risk subtypes). Higher expression of programmed death-ligand 1, higher proportion of tumor-infiltrating lymphocytes and tumor mutation burden were significantly enriched in the low-risk subtype. Further pathway analysis revealed that the low-risk subtype was associated with immune response activation and signaling pathways involved in ‘antigen processing and presentation’. Three independent CRC cohorts were used to validate the above findings. In summary, two clinical CRC subtypes were identified, which are characterized by significantly different survival outcomes and immune infiltration patterns. The findings of the present study suggest that ICI treatment may be more effective in the low-risk CRC subtype.

USP38 regulates the stemness and chemoresistance of human colorectal cancer via regulation of HDAC3

Histone modification represents a crucial level of gene expression regulation and is actively involved in the carcinogenesis of human colorectal cancer. Histone acetyltransferases and deacetylases modulate the landscape of histone acetylation, which controls key genes of colorectal cancer pathology. However, the fine tune of histone deacetylases, especially the modification of histone deacetylases that facilitate colorectal cancer, remains elusive. Here, we identified that an ubiquitin-specific protease (USP), USP38, was downregulated in clinical colorectal cancer samples and colorectal cancer cell lines. Importantly, our results showed that USP38 was a specific deubiquitinase of histone deacetylase 3 (HDAC3), which cleaved the lysine 63 ubiquitin chain. Ubiquitination of HDAC3 resulted in a decreased level of histone acetylation and finally led to upregulation of cancer stem cell-related genes. In addition, our results demonstrated a tumor suppressor role of USP38 in colorectal cancer via inhibiting cancer stem cell populations. Most importantly, the ubiquitination level of HDAC3 was responsible for USP38 mediated regulation of cancer stem cell-related transcripts. Our data provided functional insights of USP38 and HDAC3 in colorectal cancer and revealed novel mechanisms of ubiquitination mediated epigenetic regulation.

Tenascin-C predicts poor outcomes for patients with colorectal cancer and drives cancer stemness via Hedgehog signaling pathway

BackgroundTenascin-C (TNC) is an extracellular matrix protein that is widely expressed in the stromal fibroblasts of various cancers. However, the roles of TNC in colorectal cancer (CRC) cells remain unclear.MethodsThe expression of TNC, cancer stem cell-like (CSC) and cell cycle markers, and Hedgehog (HH) signaling pathway genes were assessed in 100 paraffin embedded clinical CRC patient tissues using immunohistochemistry. The interaction between TNC and CSC marker or HH related genes in CRC cells were detected by immunofluorescence. Cell cycle distribution was measured by flow cytometry. Migration and invasion were evaluated by transwell assays. The expressions of TNC, CSC marker, and HH related proteins were analyzed by western blot.ResultsTNC expression was markedly upregulated in CRC tissues, and was associated with worse clinical outcomes. TNC overexpression was positively associated with CSC marker LSD1, cell cycle markers CDK4 and p16, and HH signaling pathway related genes SMO and GLI1 in clinical CRC tissue samples. TNC silencing downregulated the expression of the CSC marker LSD1, and the proliferation, migration, and invasion of CRC cells. Interestingly, the GLI1 inhibitor GANT61 strongly inhibited the expression of TNC in CRC cells.ConclusionsTNC may drive tumor progression and is involved in CSC properties via the HH signaling pathway. TNC has potential value in the evaluation of poor prognosis in CRC.

Abstract B06: Investigating the microbial etiology of Tanzanian esophageal squamous cell carcinoma

Abstract Background: Esophageal cancer is a leading cause of cancer-associated deaths worldwide. Notably, there is a disproportionately high incidence of esophageal squamous cell carcinoma (ESCC) throughout the eastern corridor of Africa. One possible explanation for the unique geographic distribution of ESCC may be the human microbiome, which has been shown to influence other gastrointestinal (GI) cancers, including gastric and colorectal cancer (CRC). Methods: To address this question, we conducted RNA sequencing (RNAseq) and whole-genome sequencing (WGS) of samples from 61 Tanzanian ESCC patients. We used the computational microbial identification and classification software PathSeq to conduct a microbial abundance analysis of these samples. Next, we implemented linear discriminant analysis with LEfSe to determine which, if any, microbial taxa are enriched in the ESCC tumors of Tanzanian patients relative to ESCC samples from North America patients available through The Cancer Genome Atlas (TCGA). Results: Analysis of these RNAseq and WGS data reveals an extremely high abundance of Fusobacterium, Prevotella, and Streptococcus in the tumors of a subset of these patients. Based on the WGS data in particular, 14, 45, and 27 of the 61 ESCC samples maintain at least 10% relative abundance of Fusobacterium, Prevotella, and Streptococcus, respectively. The RNAseq data are consistent and reveal that as much as 75% of all bacterially derived reads in these samples are from these genera. Furthermore, LEfSe analysis suggest that these bacteria are significantly enriched in Tanzanian ESCC samples compared to North American ESCC samples. Discussion: These data suggest that the microbiome may be involved in ESCC incidence in Tanzania. Fusobacterium, Prevotella, and Streptococcus are notable for their association with CRC and are correlated with distinct clinical and molecular CRC characteristics. Members of these bacterial genera have been observed to modulate the carcinogenesis of GI cancers in a variety of ways, including through the selective stimulation or inhibition of various classes of immune cells. Furthermore, species within these genera have been observed invasively within cancerous GI tissues. Current studies are under way to visualize these microorganisms in tumor tissue, to characterize their association with molecular subtypes of ESCC, and to understand the relationship between tumor-associated bacteria and bacteria in the oral microbiome of ESCC patients in Tanzania. Citation Format: Jason Nomburg, Susan Bullman, Eric Collisson, Beatrice Mushi, Msiba Seleka, Charlie Vaske, Yulia Newton, Amie Radenbuagh, Larry Akoko, James A. DeCaprio, Matthew Meyerson, Elia J. Mmbaga, Katherine Van Loon. Investigating the microbial etiology of Tanzanian esophageal squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B06.

Comparative lipidomics of 5-Fluorouracil\u2013sensitive and \u2013resistant colorectal cancer cells reveals altered sphingomyelin and ceramide controlled by acid sphingomyelinase (SMPD1)

5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat colorectal cancer. 5-FU is known to gradually lose its efficacy in treating colorectal cancer following the acquisition of resistance. We investigated the mechanism of 5-FU resistance using comprehensive lipidomic approaches. We performed lipidomic analysis on 5-FU–resistant (DLD-1/5-FU) and -sensitive (DLD-1) colorectal cancer cells using MALDI-MS and LC-MRM-MS. In particular, sphingomyelin (SM) species were significantly up-regulated in 5-FU–resistant cells in MALDI-TOF analysis. Further, we quantified sphingolipids including SM and Ceramide (Cer) using Multiple Reaction Monitoring (MRM), as they play a vital role in drug resistance. We found that 5-FU resistance in DLD-1/5-FU colorectal cancer cells was mainly associated with SM increase and Cer decrease, which are controlled by acid sphingomyelinase (SMPD1). In addition, reduction of SMPD1 expression was confirmed by LC-MRM-MS analysis and the effect of SMPD1 in drug resistance was assessed by treating DLD-1 cells with siRNA-SMPD1. Furthermore, clinical colorectal cancer data set analysis showed that down-regulation of SMPD1 was associated with resistance to chemotherapy regimens that include 5-FU. Thus, from our study, we propose that SM/Cer and SMPD1 are new potential target molecules for therapeutic strategies to overcome 5-FU resistance.

Reducing Patient Burden and Improving Data Quality With the New Cleveland Clinic Colorectal Cancer Quality of Life Questionnaire.

The Cleveland Clinic Colorectal Cancer Quality of Life Questionnaire was developed in response to the need for a new, fast, and comprehensive tool for evaluating quality of life in patients who have colorectal cancer. Available surveys such as the SF-12, SF-36, Functional Assessment of Cancer Therapy-Colorectal, and European Organization for Research and Treatment of Cancer are either too general to be informative or too lengthy to complete. The aim was to validate the Cleveland Clinic Foundation Colorectal Quality of Life Questionnaire. Data were obtained as part of a prospective randomized controlled trial. This was a worldwide multicenter study with 2 domestic and 5 international locations. This study randomly assigned 190 patients between the ages of 18 and 80 undergoing surgery for low rectal cancer. Of those randomly assigned, 142 with partially complete surveys were analyzed for selection bias and acceptability, and 95 with complete surveys were analyzed for survey validity. Patients received either a J-pouch, side-to-end anastomosis, or straight anastomosis. The study evaluated survey validity measures such as standardized Cronbach α for internal consistency and Spearman correlation coefficients for construct validity, convergent validity, and responsiveness. Univariate analyses were used to assess discriminative validity. Sufficient acceptability, construct, and convergent validity and responsiveness were achieved. All scores showed great internal consistency (Cronbach α >0.8). Superior discriminative ability was demonstrated by significant differences (p < 0.05) in 2 of 7 scores between neoadjuvant treatment groups, and in 6 of 7 scores between complication groups, none of which were detected by the SF-12 or Functional Assessment of Cancer Therapy-Colorectal surveys. Limitations included a small sample size, cultural differences, and failure to assess test-retest ability of the questionnaire. The Cleveland Clinic Colorectal Cancer Quality of Life Questionnaire is an efficient and reliable quality-of-life measure that better incorporates factors specific to colorectal cancer surgery. See Video Abstract at http://links.lww.com/DCR/B155. REDUCIENDO LA CARGA AL PACIENTE Y MEJORANDO LA CALIDAD DE DATOS CON EL NUEVO CUESTIONARIO DE CALIDAD DE VIDA EN CÁNCER COLORRECTAL DE CLEVELAND CLINIC (CCF-CAQL): El cuestionario de calidad de vida en cáncer colorrectal de Cleveland Clinic se desarrolló en respuesta a la necesidad de una herramienta nueva, rápida e integral para evaluar la calidad de vida en pacientes con cáncer colorrectal. Los cuestionarios disponibles como SF-12, SF-36, FACT-C y EORTC son demasiado generales para ser informativas o demasiado largas para completar.El objetivo fue validar el cuestionario de calidad de vida colorrectal de la Cleveland Clinic Foundation.Los datos se obtuvieron como parte de un ensayo prospectivo aleatorizado y controlado.Este fue un estudio multicéntrico mundial con dos sedes nacionales y cinco internacionales.Este estudio aleatorizó a 190 pacientes entre las edades de 18 y 80 sometidos a cirugía por cáncer rectal bajo. De aquellos aleatorizados, 142 con encuestas parcialmente completas se analizaron para determinar el sesgo de selección y la aceptabilidad, y 95 con encuestas completas se analizaron para determinar la validez de la encuesta.Los pacientes recibieron un reservorio en J, anastomosis latero-terminal o anastomosis termino-terminal.El estudio evaluó medidas de validez de la encuesta, como el Alfa de Cronbach estandarizado para la consistencia interna y los coeficientes de correlación de Spearman para la validez de construcción, la validez de convergencia y la capacidad de respuesta. Se utilizaron análisis univariados para evaluar la validez discriminativa.Se obtuvo suficiente aceptabilidad, construcción, validez de convergencia, y capacidad de respuesta. Todos los puntajes mostraron una gran consistencia interna (alfa de Cronbach > 0.8). Una capacidad discriminativa superior fue demostrada por diferencias significativas (p < 0.05) en dos de siete puntajes entre grupos de tratamiento neoadyuvante, y en seis de siete puntajes entre grupos de complicaciones, ninguno de los cuales fue detectado por SF-12 o FACT-C.Las limitaciones incluyeron un tamaño de muestra pequeño, diferencias culturales y la falta de evaluación de la confiabilidad test-retest del cuestionario.El Cuestionario de Calidad de Vida en Cáncer Colorrectal de Cleveland Clinic es una medida de calidad de vida eficiente y confiable que incorpora mejor factores específicos asociados a la cirugía de cáncer colorrectal. Consulte Video Resumen en http://links.lww.com/DCR/B155.

Exosomal MALAT1 sponges miR-26a/26b to promote the invasion and metastasis of colorectal cancer via FUT4 enhanced fucosylation and PI3K/Akt pathway

BackgroundExosomes are vesicles of endocytic origin released by various cell types and emerging as important mediators in tumor cells. Human metastases-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA known to promote cell proliferation, metastasis, and invasion in colorectal cancer (CRC).MethodsThe expression of MALAT1 was analyzed in CRC using qRT-PCR. FUT4 and fucosylation levels were detected in CRC clinical samples and CRC cell lines by immunofluorescent staining, western blot and lectin blot analysis. CRC derived exosomes were isolated and used to examine their tumor-promoting effects in vitro and in vivo.ResultsThe invasive and metastatic abilities of primary CRC cells were enhanced after exposure to exosomes derived from highly metastatic CRC cells, which increased the fucosyltransferase 4 (FUT4) levels and fucosylation not by directly transmitting FUT4 mRNA. Exosomal MALAT1 increased FUT4 expresssion via sponging miR-26a/26b. Furthermore, MALAT1/miR-26a/26b/FUT4 axis played an important role in exosome-mediated CRC progression. Exosomal MALAT1 also mediated FUT4-associated fucosylation and activated the PI3K/AKT/mTOR pathway.ConclusionsThese data indicated that exosomal MALAT1 promoted the malignant behavior of CRC cells by sponging miR-26a/26b via regulating FUT4 and activating PI3K/Akt/mTOR pathway.

SOX13 promotes colorectal cancer metastasis by transactivating SNAI2 and c-MET.

Metastasis is a major cause of high recurrence and poor survival of patients with colorectal cancer (CRC), although the mechanisms associated with this process remain poorly understood. In this study, we report a novel mechanism by which SOX13 promotes CRC metastasis by transactivating SNAI2 and c-MET. SOX13 overexpression was significantly correlated with more aggressive clinicopathological features of CRC and indicated poor prognosis in two independent cohorts of CRC patients (cohort I, n = 363; cohort II, n = 390). Overexpression of SOX13-promoted CRC migration, invasion, and metastasis, whereas SOX13 downregulation caused the opposite effects. Further mechanistic investigation identified SNAI2 and MET as important target genes of SOX13 using serial deletion and site-directed mutagenesis luciferase reporter and chromatin immunoprecipitation (ChIP) assays, as well as functional complementation analyses. In addition, SOX13 was shown to be a direct target of HGF/STAT3 signaling, and the c-MET inhibitor crizotinib blocked the HGF/STAT3/SOX13/c-MET axis, significantly inhibiting SOX13-mediated CRC migration, invasion and metastasis. Moreover, in clinical CRC tissues, SOX13 expression was positively correlated with the expression of SNAI2, c-MET, and HGF. CRC patients with positive coexpression of SOX13/SNAI2, SOX13/c-MET, or HGF/SOX13 exhibited a worse prognosis. In summary, SOX13 is a promising prognostic biomarker in patients with CRC, and blocking the HGF/STAT3/SOX13/c-MET axis with crizotinib could be a new therapeutic strategy to prevent SOX13-mediated CRC metastasis.

Identification of Core Gene Expression Signature and Key Pathways in Colorectal Cancer.

ObjectiveColorectal cancer (CRC) is considered the most prevalent malignant tumor that contributes to high cancer-related mortality. However, the signaling pathways involved in CRC and CRC-driven genes are largely unknown. We sought to discover a novel biomarker in CRC.Materials and MethodsAll clinical CRC samples (n = 20) were from Renmin Hospital of Wuhan University. We first selected MAD2L1 by integrated bioinformatics analysis of a GSE dataset. Next, the expression of MAD2L1 in tissues and cell lines was verified by quantitative real-time PCR. The effects of MAD2L1 on cell growth, proliferation, the cell cycle, and apoptosis were examined by in vitro assays.ResultsWe identified 683 shared DEGs (420 upregulated and 263 downregulated), and the top twenty genes (CDK1, CCNA2, TOP2A, PLK1, MAD2L1, AURKA, BUB1B, UBE2C, TPX2, RRM2, KIF11, NCAPG, MELK, NUSAP1, MCM4, RFC4, PTTG1, CHEK1, CEP55, DTL) were selected by integrated analysis. These hub genes were significantly overexpressed in CRC samples and were positively correlated. Our data revealed that the expression of MAD2L1 in CRC tissues is higher than that in normal tissues. MAD2L1 knockdown significantly suppressed CRC cell growth by impairing cell cycle progression and inducing cell apoptosis.ConclusionMAD2L1, as a novel oncogenic gene, plays a role in regulating cancer cell growth and apoptosis and could be used as a new biomarker for diagnosis and therapy in CRC.

Prognostic value of a hypoxia-related microRNA signature in patients with colorectal cancer.

Hypoxia has been particularly associated with poor prognosis in cancer patients. Recent studies have suggested that hypoxia-related miRNAs play a critical role in various cancers, including colorectal cancer (CRC). In the present study, we found 52 differentially expressed miRNAs in HT-29 cells under hypoxic conditions versus normoxic conditions by analyzing the profiles of miRNAs. Using Cox model, we developed a hypoxia-related miRNA signature consisting of four miRNAs, which could successfully discriminate high-risk patients in the Cancer Genome Atlas (TCGA) training cohort (n=381). The prognostic value of this signature was further confirmed in the TCGA testing cohort (n=190) and an independent validation cohort composed of formalin-fixed paraffin-embedded clinical CRC samples (n=220), respectively. Multivariable Cox regression and stratified survival analysis revealed this signature was an independent prognostic factor for CRC patients. Time-dependent receiver operating characteristic (ROC) analysis showed that the area under the curve (AUC) of this signature was significantly larger than that of any other clinical risk factors or single miRNA alone. A nomogram was constructed for clinical use, which incorporated both the miRNA signature and clinical risk factors and performed well in the calibration plots. Collectively, this novel hypoxia-related miRNA signature was an independent prognostic factor, and it possessed a stronger predictive power in identifying high-risk CRC patients than currently used clinicopathological features.

DNA sensors, crucial receptors to resist pathogens, are deregulated in colorectal cancer and associated with initiation and progression of the disease.

Background: DNA sensors are innate immune receptors that detect intracellular endogenous or exogenous DNA. They are critical to trigger immune response against DNA viral and intracellular bacterial infection, and are involved in inflammatory diseases and tumorigenesis. Recent accumulating evidences indicated that DNA sensors are also crucial for controlling the development of colorectal cancer (CRC). However, a systematic study on the expression profile of DNA sensors in CRC and their clinical significance are still lacking.Methods: We investigated the expression profile of DNA sensors in CRC and their clinical significance by taking advantage of clinical CRC samples, mouse AOM/DSS treatment model, and Oncomine ® bioinformatics platform.Results: Our study identified that the expression of DNA sensors, including AIM2, DAI, as well as inflammasome molecules ASC/IL-18, TLR9 and adaptor MyD88, and DDX60 decreased in human CRC, whereas the expression of DHX9, DHX36, and DDX41 significantly increased. Among them, the expression of AIM2/ASC/IL-18, MyD88, DAI, DHX36, and DDX60 were associated with cancer stages. In addition, we also performed correlation analysis between DNA sensors and their main signaling molecules to explore the possible mechanisms. The results showed that there were positive correlations between AIM2 and ASC/IL-18, DHX9 and MAVS, and TLR9 and MyD88 expression. In addition, the gene expression patterns of some DNA sensors were confirmed by Western-blot analysis.Conclusions: Our study revealed that the expression of multiple DNA sensors was deregulated in CRC and might be involved in tumor development. More importantly, the study identified that, among all these DNA sensors, AIM2, DAI, and DDX60 could be potentially critical for diagnosis, prognosis, and therapy of CRC and deserve further investigation.

CircCCT3 Modulates Vascular Endothelial Growth Factor A and Wnt Signaling to Enhance Colorectal Cancer Metastasis Through Sponging miR-613.

Colorectal cancer (CRC) has been suggested to be one of the leading cancer types all over the world. Till now, the molecular mechanism by which circCCT3 regulates CRC remains to be clarified. To detect mRNA and protein levels of various genes, Reverse Transcription-quantitative PCR and western blot were used in our study. Luciferase reporter assay was utilized to probe direct interaction between genes. We used transwell assay to assess the invasion ability of CRC cells. For apoptosis detection, immunofluorescence of CRC cells by Annexin V staining was performed. We carried out bioinformatic analysis to show higher expression of circCCT3 in human clinical CRC tumors. Low level of circCCT3 was closely associated with higher disease-free survival of CRC patients. Moreover, we found that circCCT3 was linked to advanced stage of CRC. miR-613 is the target of circCCT3 and responsible for circCCT3-modulated invasion and apoptosis of CRC cells. In addition, we identified WNT3 and vascular endothelial growth factor A (VEGFA) as downstream effectors of miR-613 in CRC cells. WNT3 and VEGFA overexpression resulted in partial rescue of miR-613-mediated phenotypes of CRC cells. In conclusion, we propose that circCCT3 contributes to CRC metastasis via miR-613/WNT3 or miR-613/VEGFA, promoting the development of therapeutical approaches for treating CRC.

MiR-223 Promotes Proliferation of Colon Cancer Cells by Down-Regulating Bcl-2-Like Protein 11 (BIM) Expression

Colorectal cancer is one of malignant tumors. microRNA plays an important role in various diseases. In this study, we evaluated miR-223's effect on the proliferation of colon cancer cells. Protein and RNA expression levels in patients with clinical colorectal cancer were determined by western blot and real-time quantitative PCR respectively. In addition, the mechanism of miR-223 action was explored by combining transfection methods in cell lines. Colon cancer tissues showed significantly elevated miR-223 expression compared with adjacent tissues. Meanwhile, FOXO3a and BIM protein levels were significantly lower in cancer tissues compared to adjacent tissues. In colon cancer cell lines, knockdown of miR-223 increased cell proliferation and decreased BIM expression. The luciferase reporter gene showed that miR-223 down-regulates BIM expression through targeting FOXO3a. In colon cancer cells, miR-223 can down-regulate BIM expression through FOXO3a, thereby promoting the proliferation of colon cancer cells, indicating that targeting miR-223-regulated FOXO3a pathway might lead to the development of a number of drugs, and it is feasible to have a purpose to regulate the behavior of malignant cells.