CRC Metastasis Research Articles

Identification of molecular anti-metastasis mechanisms of lycorine in colorectal cancer by RNA-seq analysis

BackgroundColorectal cancer (CRC) is one of the most common malignancies worldwide. Metastasis is the major cause of death in patients with CRC. Lycorine, the phenanthridine alkaloid most commonly found in spp of the Amaryllidaceae family, has shown promising anticancer activities with minor side effects. However, the effects and the detailed mechanism of lycorine against metastasis of CRC remains unclear. Study design/methodsThe purpose of this study was to investigate the effects of lycorine on CRC and characterize the molecular mechanisms observed in lycorine-treated CRC cells using RNA-sequencing. MTT assay, colony formation assay, acridine orange/ethidium bromide (AO/EB) staining and Annexin V-FITC/Propidium iodide (PI) staining were conducted to examine the effects of lycorine on cell proliferation and apoptosis in CRC cells. RNA sequencing, real-time PCR assays and western blot were performed. Migration and invasion abilities of lycorine-treated CRC cells were investigated by wound healing and transwell invasion assays. The mouse CRC lung metastasis model was established and was used to detect the effect of lycorine on CRC in vivo. ResultsOur results demonstrated that lycorine inhibited the proliferation and colony formation of CRC cells in a concentration-dependent manner. AO/EB staining and Annexin V-FITC/PI staining showed that lycorine induced apoptosis in a concentration-dependent manner. Lycorine also reduced lung metastasis of CRC in vivo. Moreover, transcriptomic analysis suggested that lycorine regulated the expression of 3556 genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was implicated according to the differentially expressed genes (DEGs), and multiple pathways including those of mitogen-activated protein kinase (MAPK), relaxin, Ras, phosphatidylinositol 3‑kinase (PI3K)-protein kinase B (Akt) and Wnt/β-catenin were selected by functional enrichment analyses. Furthermore, based on transcriptomic analysis, we found that the tumor necrosis factor (TNF) pathway and endoplasmic reticulum stress were responsible for lycorine-induced apoptosis. ConclusionsThese results obtained in this study demonstrated that lycorine has the potential to suppress CRC in vitro and in vivo through the lycorine-regulated multiple signaling pathways.

SK4 oncochannels regulate calcium entry and promote cell migration in KRAS-mutated colorectal cancer

BackgroundColorectal cancer (CRC) metastases are the main cause of CRC mortality. Intracellular Ca2+ regulates cell migration and invasion, key factors for metastases. Ca2+ also activates Ca2+-dependent potassium channels which in turn affect Ca2+ driving force. We have previously reported that the expression of the Ca2+ activated potassium channel KCNN4 (SK4) is higher in CRC primary tumors compared to normal tissues. Here, we aimed to investigate the role of SK4 in the physiology of CRC. ResultsSK4 protein expression is enhanced in CRC tissues compared to normal colon tissues, with a higher level of KCNN4 in CRC patients with KRAS mutations. At the cellular level, we found that SK4 regulates the membrane potential of HCT116 cells. We also found that its inhibition reduced store operated Ca2+ entry (SOCE) and constitutive Ca2+ entry (CCE), while reducing cell migration. We also found that the activity of SK4 is linked to resistance pathways such as KRAS mutation and the expression of NRF2 and HIF-1α. In addition, the pharmacological inhibition of SK4 reduced intracellular reactive oxygen species (ROS) production, NRF2 expression and HIF1α stabilization. ConclusionOur results suggest that SK4 contributes to colorectal cancer cell migration and invasion by modulating both Ca2+ entry and ROS regulation. Therefore, SK4 could be a potential target to reduce metastasis in KRAS-mutated CRC.

Tumor-derived exosomal microRNA-106b-5p activates EMT-cancer cell and M2-subtype TAM interaction to facilitate CRC metastasis

Epithelial-mesenchymal transition (EMT) is reported to involve in the crosstalk between tumor cells and tumor-associated macrophages (TAMs). Exosomes are considered as important mediators of orchestrating intercellular communication. However, the underlying mechanisms by which EMT-colorectal cancer (CRC) cells promote the M2 polarization of TAMs remain less understood. In this study, we found that EMT-CRC cells promoted the M2-like polarization of macrophages by directly transferring exosomes to macrophages, leading to a significant increase of the microRNA-106b-5p (miR-106b) level in macrophages. Mechanically, an increased level of miR-106b activated the phosphatidylinositol 3-kinase (PI3K)γ/AKT/mammalian target of rapamycin (mTOR) signaling cascade by directly suppressing programmed cell death 4 (PDCD4) in a post-transcription level, contributing to the M2 polarization of macrophages. Activated M2 macrophages, in a positive-feedback manner, promote EMT-mediated migration, invasion, and metastasis of CRC cells. Clinically, miR-106b was significantly elevated in CRC tissues and negatively correlated with the levels of PDCD4 in CRC specimens, and high expression of exosomal miR-106b in plasma was significantly associated with the malignant progression of CRC. Taken together, our results indicate that exosomal miR-106b derived from EMT-CRC cells has an important role in intercellular communication for inducing M2 macrophage polarization, illuminating a novel mechanism underlying CRC progression and offering potential targets for prevention of CRC metastasis.

Inhibitory effect of sodium butyrate on colorectal cancer cells and construction of the related molecular network

BackgroundSodium butyrate (NaB) is produced through the fermentation of dietary fiber that is not absorbed and digested by the small intestine.PurposeHere, we aimed to investigate the effects of NaB on the proliferation, invasion, and metastasis of CRC cells and their potential underlying molecular mechanism(s).MethodsThe cell counting kit-8 (CCK-8) assay and EdU assay were used to detect cell proliferation ability, flow cytometry was used to investigate the induction of apoptosis and cell cycle progression, and the scratch-wound healing and transwell assays were used to evaluate cell migration and invasion, respectively. The human CRC genome information for tissues and CRC cells treated with NaB obtained from the NCBI GEO database was reannotated and used for differential RNA analysis. Functional and pathway enrichment analyses were performed for differentially expressed lncRNAs and mRNAs. A protein-protein interaction (PPI) network for the hub genes was constructed using the Cytoscape software. Targeted miRNAs were predicted based on the lnCeDB database, and a ceRNA network was constructed using the Cytoscape software. The Kaplan-Meier method was used to analyze patient prognosis using the clinical information and exon-seq data for CRC obtained from the Broad Institute’s GDAC Firehose platform.ResultsNaB decreased the proliferation ability of CRC cells in a dose- and time-dependent manner. The number of apoptotic CRC cells increased with the increase in NaB concentrations, and NaB induced a G1 phase block in CRC cells. Moreover, NaB suppressed the migratory and invasive capabilities of CRC cells. There were 666 differentially expressed mRNAs and 30 differentially expressed lncRNAs involved in the CRC inhibition by NaB. The PPI network and ceRNA network were constructed based on the differentially expressed mRNAs and lncRNAs. Three differentially expressed mRNAs, including HMGA2, LOXL2, and ST7, were significantly correlated with the prognosis of CRC.ConclusionNaB induces the apoptosis and inhibition of CRC cell proliferation, invasion, and metastasis by modulating complex molecular networks. RNA prediction and molecular network construction need to be the focus of further research in this direction.

Cofilin-1, LIMK1 and SSH1 are differentially expressed in locally advanced colorectal cancer and according to consensus molecular subtypes

BackgroundColorectal cancer (CRC) is among the deadliest cancers, wherein early dissemination of tumor cells, and consequently, metastasis formation, are the main causes of mortality and poor prognosis. Cofilin-1 (CFL-1) and its modulators, LIMK1/SSH1, play key roles in mediating the invasiveness by driving actin cytoskeleton reorganization in various cancer types. However, their clinical significance and prognostic value in CRC has not been fully explored. Here, we evaluated the clinical contribution of these actin regulators according to TNM and consensus molecular subtypes (CMSs) classification.MethodsCFL-1, LIMK1 and SSH1 mRNA/protein levels were assessed by real-time PCR and immunohistochemical analyses using normal adjacent and tumor tissues obtained from a clinical cohort of CRC patients. The expression levels of these proteins were associated with clinicopathological features by using the chi square test. In addition, using RNA-Seq data of CRC patients from The Cancer Genome Atlas (TCGA) database, we determine how these actin regulators are expressed and distributed according to TNM and CMSs classification. Based on gene expression profiling, Kaplan–Meier survival analysis was used to evaluated overall survival.ResultsBioinformatic analysis revealed that LIMK1 expression was upregulated in all tumor stages. Patients with high levels of LIMK1 demonstrated significantly lower overall survival rates and exhibited greater lymph node metastatic potential in a clinical cohort. In contrast, CFL-1 and SSH1 have expression downregulated in all tumor stages. However, immunohistochemical analyses showed that patients with high protein levels of CFL-1 and SSH1 exhibited greater lymph node metastatic potential and greater depth of local invasion. In addition, using the CMSs classification to evaluate different biological phenotypes of CRC, we observed that LIMK1 and SSH1 genes are upregulated in immune (CMS1) and mesenchymal (CMS4) subtypes. However, patients with high levels of LIMK1 also demonstrated significantly lower overall survival rates in canonical (CMS2), and metabolic (CMS3) subtypes.ConclusionsWe demonstrated that CFL-1 and its modulators, LIMK1/SSH1, are differentially expressed and associated with lymph node metastasis in CRC. Finally, this expression profile may be useful to predict patients with aggressive signatures, particularly, the immune and mesenchymal subtypes of CRC.

Construction and prognostic analysis of miRNA-mRNA regulatory network in liver metastasis from colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most common cancers in the world, and liver metastasis is the leading cause of colorectal cancer-related deaths. However, the mechanism of liver metastasis in CRC has not been clearly elucidated.MethodsThree datasets from the Gene Expression Omnibus (GEO) database were analyzed to obtain differentially expressed genes (DEGs), which were subjected to functional enrichment analysis and protein-protein interaction analysis. Subsequently, mRNA-miRNA network was constructed, and the associated DEGs and DEMs were performed for prognostic analysis. Finally, we did infiltration analysis of growth arrest specific 1 (GAS1)-associated immune cells.ResultsWe obtained 325 DEGs and 9 differentially expressed miRNAs (DEMs) between primary CRC and liver metastases. Enrichment analysis and protein-protein interactions (PPI) further revealed the involvement of DEGs in the formation of the inflammatory microenvironment and epithelial-mesenchymal transition (EMT) during the liver metastases process in CRC. Survival analysis demonstrated that low-expressed GAS1 as well as low-expressed hsa-miR-33b-5p was a favorable prognostic indicator of overall survival. Further exploration of GAS1 revealed that its expression was interrelated with the infiltration of immune cells in tumor tissues.ConclusionsIn summary, DEGs, DEMs, and their interactions found in liver metastasis of CRC may provide a basis for further understanding of the mechanism of CRC metastasis.

LncRNA LINC00460 Functions as a Competing Endogenous RNA and Regulates Expression of BGN by Sponging miR-149-5p in Colorectal Cancer.

Background and Aim:There are an increasing number of studies indicating the important roles served by long non-coding RNAs (lncRNAs) in the development of different types of cancer. LINC00460 is a novel identified lncRNA that was found to be upregulated in colorectal cancer. However, the biological roles of LINC00460 in colorectal cancer have yet to be fully elucidated. This study was aimed to investigate the functions and molecular mechanisms of LINC00460 on colorectal cancer metastasis.Methods:Expression of LINC00460 and biglycan (BGN) in colorectal cancer tissues and cell lines were quantified by real time PCR or western blotting assay. Cell migration and invasion assays were performed to determine the effect of LINC00460 on tumor metastasis in vitro. The binding interaction between microRNA-149-5p and LINC00460 was revealed by luciferase reporter assay.Results:In the present study, lncRNA LINC00460 was shown to be upregulated in colorectal cancer tissues, and overexpression of LINC00460 significantly promoted metastasis of colorectal cancer in vitro. Furthermore, miR-149-5p interacted with LINC00460, and they negatively regulated expression of each other. Transfection of miR-149-5p mimics partially counteracted the tumor metastasis-promoting effects induced by LINC00460 overexpression. Finally, overexpression of LINC00460 upregulated the expression levels of biglycan, a target gene of miR-149-5p, which has also been identified as an oncogenic driver in colorectal cancer.Conclusion:Taken together, the present study demonstrated that LINC00460 promoted metastasis of CRC by sponging miR-149-5p and thereby affecting biglycan expression levels.

HDAC2 inhibits EMT-mediated cancer metastasis by downregulating the long noncoding RNA H19 in colorectal cancer

BackgroundEmerging evidence suggests that epithelial mesenchymal transition (EMT) and epigenetic mechanisms promote metastasis. Histone deacetylases (HDACs) and noncoding RNAs (ncRNAs) are important epigenetic regulators. Here, we elucidated a novel role of histone deacetylase 2 (HDAC2) in regulating EMT and CRC metastasis via ncRNA.MethodsThe expression of HDACs in CRC was analyzed using the public databases and matched primary and metastatic tissues, and CRC cells with different metastatic potentials (DLD1, HCT116, SW480 and SW620). Microarray analysis was used to identify differential genes in parental and HDAC2 knockout CRC cells. EMT and histone modifications were determined using western blot and immunofluorescence. Migration ability was assessed by transwell assay, and metastasis was assessed in vivo using a tail vain injection. Gene expression and regulation was assessed by RT-PCR, chromatin immunoprecipitation and reporter assays. Protein interaction was assessed by immunoprecipitation. Specific siRNAs targeting H19, SP1 and MMP14 were used to validate their role in HDAC2 loss induced EMT and metastasis.ResultsReduced HDAC2 expression was associated with poor prognosis in CRC patients and found in CRC metastasis. HDAC2 deletion or knockdown induced EMT and metastasis by upregulating the long noncoding RNA H19 (LncRNA H19). HDAC2 inhibited LncRNA H19 expression by histone H3K27 deacetylation in its promoter via binding with SP1. LncRNA H19 functioned as a miR-22-3P sponge to increase the expression of MMP14. HDAC2 loss strongly promoted CRC lung metastasis, which was suppressed LncRNA H19 knockdown.ConclusionOur study supports HDAC2 as a CRC metastasis suppressor through the inhibition of EMT and the expression of H19 and MMP14.

Metabolite secretions of Lactobacillus plantarum YYC-3 may inhibit colon cancer cell metastasis by suppressing the VEGF-MMP2/9 signaling pathway

BackgroundColorectal cancer (CRC) is a major clinical challenge, and the gut microbiome plays important roles in the occurrence and metastasis of CRC. Lactobacillus and their metabolites are thought to be able to suppress the growth of CRC cells. However, the antimetastatic mechanism of Lactobacillus or their metabolites toward CRC cells is not clear. Therefore, the aim of this study was to assess the inhibitory mechanism of cell-free supernatants (CFSs) of L. rhamnosus GG, L. casei M3, and L. plantarum YYC-3 on metastasis of CRC cells.ResultsYYC-3 CFS showed the highest inhibitory effect on CRC cell growth, invasion and migration, and inhibited MMP2, MMP9, and VEGFA gene and protein expression, and protein secretion. Furthermore, it suppressed the activities of MMPs by gelatin zymography. Moreover, the effective compounds in these CFSs were analyzed by Q Exactive Focus liquid chromatography–mass spectrometry.ConclusionsOur results showed that metabolite secretions of YYC-3 may inhibited cell metastasis by downregulating the VEGF/MMPs signaling pathway. These data suggest that treatment of CRC cells with metabolites from L. plantarum YYC-3 may reduce colon cancer metastasis.

Ciliated muconodular papillary tumor with a growing cavity shadow that mimicked colorectal metastasis to the lung: a case report

BackgroundCiliated muconodular papillary tumor (CMPT) is a rare papillary nodule tumor with benign and malignant characteristics that occurs in the peripheral lung.Case presentationA 70-year-old woman who underwent right hemicolectomy for colorectal cancer (CRC; pT3N0M0, p-stage II) 2 years prior, presented with a sub-centimeter growing cavity shadow on chest computed tomography (CT), which was suspected to be a CRC metastasis. Because positron emission tomography CT suggested there was no other site suspicious of recurrence, thoracoscopic resection with preoperative pleural dye marking was planned to remove the small lesion, which seemed to be hardly palpable on CT. Immediately after pleural dye marking adjacent to the lesion using cone beam CT in the hybrid operating room, thoracoscopic wedge resection was performed and the tumor was finally diagnosed as CMPT, characterized by the papillary growth of mucus-producing cells in the alveoli.ConclusionWe resected the non-palpable small lung lesions following preoperative marking using cone-beam CT in the hybrid operating room. This case highlights a rare cavitary CT image of a CMPT mimicking a metastatic lung tumor from colorectal cancer.

Identification of stool miR-135b-5p as a non-invasive diaognostic biomarker in later tumor stage of colorectal cancer

AimsColorectal cancer (CRC) is one of the most common cancers. However, the effective and non-invasive diagnostic biomarkers for the detection of CRC metastasis remain unsatisfied. Here, we aimed to evaluate the diagnostic performance of stool miR-135b-5p (a potential biomarker) for metastasis of CRC patients. Materials and methodsSerum and stool specimens from 77 patients with CRC and 29 normal controls were collected for miRNA purification. Real-time quantitative PCR was used for the relative quantification of miR-135b-5p expression. Receiver operating and characteristic (ROC) curve analysis was used to estimate the diagnostic performance of stool/serum miR-135b-5p for CRC metastasis. Dual-luciferase reporter assay was conducted to determine miR-135b-5p's target gene. Besides, a trans-well matrigel assay was performed to evaluate the invasion ability of HT-29 cell lines. Key findingsStool miR-135b-5p expression was dramatically up-regulated in CRC patients, and it effectively distinguished the CRC patients from normal controls with 74.1% of specificity and 96.5% of sensitivity. Moreover, stool miR-135b-5p exhibited a better diagnostic performance in distinguishing the different TNM stages of CRC patients than serum miR-135b-5p. In the molecular mechanism, our observations indicated that miR-135-5p directly targets the mRNA of ZNRF3, and then activates the Wnt pathway. Over-expression of ZNRF3 in HT-29 cells obviously reversed miR-135b-5p's effects on cell invasion and migration, indicating miR-135b-5p achieves its biological functions in a ZNRF3 dependent manner. SignificanceMiR-135b-5p may be a promising non-invasive biomarker for the diagnosis of CRC patients with TNM stage-III/IV and a potential candidate to develop an intervention strategy for colorectal cancer.

Abstract 2434: Epigenomic profiling of colorectal cancer to predict patients at high risk of metastasis

Abstract Colorectal cancer (CRC) is one of the biggest cause of death world-wide and the second-highest cause of death by cancer in New Zealand with a mortality rate of approximately four deaths/day and 90% of CRC-associated mortality is caused by metastasis. Development of metastasis and the modifications that occur during various stages in the CRC metastatic cascade remains obscure. Emerging evidence indicates that epigenetic alterations have a greater influence on the development of metastasis than genetic mutations, but the current understanding of epigenetic drivers that cause CRC metastasis is limited. In the current project, as a first step in identifying epigenetic drivers for metastasis, whole-genome methylation and transcriptome profiles from paired primary and metastatic samples from the same NZ patients will be compared. Subsequently, the epigenetic drivers will be validated, and it can potentially be used as CRC metastatic markers. Circulating tumour cells (CTCs) represent the conduit between primary and metastatic tumours. They could be used to identify driver epigenetic changes associated with epithelial-to-mesenchymal transition (EMT), migration and invasion which occur during the metastatic cascade. CTCs are obtained from the blood and would provide a clinically less invasive test rather than requiring tissue samples. The recent development of single-cell sequencing technologies has now made it feasible to identify epigenetic changes in individual CTCs. In the second part of this project, protocols for single-cell DNA methylation and expression analysis will be developed using human colon cancer cell lines as a model. These optimised protocols will then be applied to CTCs from patient blood samples to identify epigenetic changes that predispose patients to metastasis. Identification of epigenetic drivers will help to detect individuals at high risk for metastasis prior to metastasis being clinically apparent, allowing for more personalised treatment and contribute to improving survival. Citation Format: Aniruddha Chatterjee, John McCall, Parry Guilford, Sharon Pattison, Euan Rodger, Priyadarshana Ajithkumar. Epigenomic profiling of colorectal cancer to predict patients at high risk of metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2434.

Abstract 4962: Tumor heterogeneity driven by cancer stem cell expressing POU5F1

Abstract Background: The gene POU5F1, encoding the POU domain, class 5, is a transcription factor. and the expression is found in embryonic stem cells. In these pluripotent stem cells, POU5F1 gene controls self-renewal and differentiation, regulating the pluripotency and proliferation. Regarding the tumor heterogeneity and microenvironment, primary culture of cancer cells derived from the patient's tumor can provide important information of the “individual tumor,” representing the tumor heterogeneity. We focused on the gene expression and the relationship between colorectal cancer stem cells and the tumor heterogeneity, in the primary culture model. Methods: Using POU5F1 promoter-containing sequence, POU5F1-EGFP-positive cells were established by the construct with lentiviral vector, and we examined their sub-populations. The capacity to form distant metastasis in vivo was examined using the primary culture models. Results: We obtained clinical samples from surgically resected gastrointestinal cancers. They were mechanically and enzymatically digested and collected using customized filters. Obtained cells were cultured on a matrix-coated plate with chemically defined stem-cell culture medium. All these primary cultured cells grew and about 90% of them were successfully passaged. The morphology and gene expressions were similar to each parental clinical tumor. Using these primary tumors, POU5F1 expressing cells were examined. The POU5F1-EGFP-positive cells highly expressed stem cell-associated markers and they had self-renewal and differentiation abilities. POU5F1-high cells actively formed liver metastasis. Conclusion: POU5F1-positive cells have the self-renewal and differentiation abilities as cancer stem cells. The positive cells produce tumor heterogeneity. POU5F1 contributes to the ability forming liver metastasis in CRC. Keywords: colorectal cancer, tumor heterogeneity, metastasis, POU5F1. Citation Format: Norikatsu Miyoshi, Shiki Fujino, Masaru Sasaki, Takayuki Ogino, Hidekazu Takahashi, Mamoru Uemura, Chu Matsuda, Tsunekazu Mizushima, Hidetoshi Eguchi, Yuichiro Doki. Tumor heterogeneity driven by cancer stem cell expressing POU5F1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4962.

Abstract 2732: NLRP12 suppresses colorectal cancer progression and invasion via downregulation of the Wnt/b-catenin pathway

Abstract Metastatic CRC is associated with increased mortality, underscoring the importance of understanding the molecular mechanism of CRC invasion and metastasis. We recently demonstrated that mice deficient in NLRP12, an innate immune sensor, are susceptible to CRC with increased incidence of invasive adenocarcinoma. Here, we investigated the molecular mechanism of NLRP12-mediated suppression of cancer progression and invasion using mouse models and in vitro biochemical studies. Nlrp12−/− mice treated with azoxymethane plus dextran sodium sulfate developed higher tumor burden and invasive adenocarcinoma compared to WT mice. RNA-seq analysis showed significantly higher levels of protooncogenes, matrix metalloproteinases (Mmp 3, 7,8, 10, 12), and epithelial to mesenchymal transition (EMT) markers (Vim, Fn1, Foxc2, Zeb2, Ezh2) in Nlrp12−/− tumors. To understand the underlying mechanism of increased expression of cancer promoting genes in Nlrp12−/− tumors, we analyzed activation of various cell signaling pathways. To our surprise, there was no major difference in the activation NF-κB, ERK, JNK, and STAT3, which have previously been shown to be downregulated by NLRP12, between WT and Nlrp12−/- tumors. However, we observed strikingly higher levels of β-catenin and expression of Wnt target genes (Ctnnb1, Axin2, Yap1, Tcf3, Lgr5, etc.) in Nlrp12−/− tumors. Consistently, deletion of NLRP12 in APCmin/+ mice led to higher polyp development and activation of the Wnt/β-catenin pathway. Notably, hyperactivation of the Wnt/β-catenin pathway is a hallmark of CRC and about 80% of CRC patients carry mutations in the Wnt/β-catenin pathway. To further explore the link between NLRP12 and the Wnt/β-catenin pathway, we overexpressed NLRP12 in HEK293T cells and CRC cell line MC38. NLRP12-overexpressed cells showed reduced activation of β-catenin and expression of Wnt target genes following stimulation with Wnt3a. An opposite result was observed when NLRP12 was knocked out from these cell lines. Consistently, Nlrp12-KO MC38 cells exhibited increased proliferation, colony formation, and migration. These data for the first time demonstrate that NLRP12 is an intrinsic negative regulator of the Wnt/β-catenin pathway. Overall, these findings reveal a novel CRC suppression pathway involving NLRP12 and suggest that the NLRP12/β-catenin signaling axis could be therapeutically targeted for the treatment of CRC invasion and metastasis. Citation Format: Shahanshah Khan, Hasan Zaki. NLRP12 suppresses colorectal cancer progression and invasion via downregulation of the Wnt/b-catenin pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2732.

Abstract 4916: MUC13 enhances anchorage independent survival and cooperates with YAP1 and β-catenin towards colorectal cancer metastasis

Abstract Colorectal Cancer (CRC) is the 3rd most common and deadliest cancers in the United States. About 90% of all cancer related deaths are due to the development of metastatic sites in the body. Metastasis develops slowly over time, due to it being an inefficient process and having to overcome several natural defenses, anchorage independent growth aka Anoikis, being one of them. Understanding the mechanism overcoming Anoikis will help develop new therapeutic options to prevent the metastatic spread of cancer cells. Low adhesion cell culture model was developed and standardized to study the key pathways and proteins responsible for anchorage independent survival and metastasis progression in CRC. We identified mucin MUC13 in correspondence with YAP1 and β-catenin as key upregulated proteins in anchorage independent survival and metastasis progression. In an isogeneic CRC cell line model, overexpression of MUC13 in non-metastatic SW480 cells (originally expressing minimal MUC13) increased anchorage independent survival and enhanced tumorigenesis compared to SW480+Vector cells. Metastatic SW620 CRC cells (highly expressing MUC13) showed a decreased anchorage independent survival and tumorigenesis after knockdown using specific shRNA targeted against MUC13, compared to its vector. When co-cultured with fibroblast cells SW480+MUC13 cells showed an increase in spheroid size and percentage of live cells after 36hrs as compared to control cells. A decrease in spheroid size and live cells was observed with SW620+shMUC13 cells, compared to its control. We also observed an upregulation of key downstream β-catenin target genes c-Myc, Axin2 and an increase in YAP1 expression in MUC13 over-expressing cells. With MUC13 overexpression we observed and increase in the formation of YAP1/β-catenin survival complex within the nucleus at 36hrs compared to SW480+Vector. Enhanced interaction between MUC13/YAP1 and MUC13/β-catenin in the nucleus was also observed. In human CRC tissues, MUC13 and YAP1 expression was high in tumor compared to normal adjacent tumor. MUC13 and YAP1 expression in human CRC tissue was highest at Stage II. However, highest YAP1 expression was observed while MUC13 and/or β-catenin present in the nucleus. This supports the notion that MUC13 is a key factor in enhancing anchorage independence survival and CRC tumorigenesis through cooperation with YAP1 and β-catenin. This study for the first time demonstrates complex formation between MUC13, YAP1 and β-catenin, and define their role in CRC progression and metastasis. Citation Format: Kyle Doxtater, Radhika Sekhri, Utkarsh Mishra, Meena Jaggi, Manish Tripathi, Subhash Chauhan. MUC13 enhances anchorage independent survival and cooperates with YAP1 and β-catenin towards colorectal cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4916.

MPC1 Deficiency Promotes CRC Liver Metastasis via Facilitating Nuclear Translocation of β-Catenin.

Accumulating evidence has pointed out that metastasis is the leading cause of death in several malignant tumor, including CRC. During CRC, metastatic capacity is closely correlated with reprogrammed energy metabolism. Mitochondrial Pyruvate Carrier 1 (MPC1), as the carrier of transporting pyruvate into mitochondria, linked the glycolysis and TCA cycle, which would affect the energy production. However, the specific role of MPC1 on tumor metastasis in CRC remains unexplored. Here, by data mining of genes involved in pyruvate metabolism using the TCGA dataset, we found that MPC1 was significantly downregulated in CRC compared to nontumor tissues. Similar MPC1 expression pattern was also found in multiple GEO datasets. IHC staining in both human sample and AOM/DSS induced mouse CRC model revealed significant downregulation of MPC1. What is more, we found that MPC1 expression was gradually decreased in normal tissue, primary CRC, and metastasis CRC. Additionally, poor prognosis emerged in the MPC1 low expression patients, especially in patients with metastasis. Following, functional tests showed that MPC1 overexpression inhibited the motility of CRC cells in vitro and MPC1 silencing enhanced liver metastases in vivo. Furthermore, we uncovered that decreased MPC1 activated the Wnt/β-catenin pathway by promoting nuclear translocation of β-catenin to mediate the expression of MMP7, E-cadherin, Snail1, and myc. Collectively, our data suggest that MPC1 has the potential to be served as a promising biomarker for diagnosis and a therapeutic target in CRC.

LINC00941 promotes CRC metastasis through preventing SMAD4 protein degradation and activating the TGF-β/SMAD2/3 signaling pathway.

LINC00941 is a novel lncRNA that has been found to exhibit protumorigenic and prometastatic behaviors during tumorigenesis. However, its role in metastatic CRC remains unknown. We aimed to investigate the functions and mechanisms of LINC00941 in CRC metastasis. LINC00941 was shown to be upregulated in CRC, and upregulated LINC00941 was associated with poor prognosis. Functionally, LINC00941 promoted migratory and invasive capacities and accelerated lung metastasis in nude mice. Mechanistically, LINC00941 activated EMT in CRC cells, as indicated by the increased expression of key molecular markers of cell invasion and metastasis (Vimentin, Fibronectin, and Twist1) and simultaneous decreased expression of the main invasion suppressors E-cadherin and ZO-1. LINC00941 was found to activate EMT by directly binding the SMAD4 protein MH2 domain and competing with β-TrCP to prevent SMAD4 protein degradation, thus activating the TGF-β/SMAD2/3 signaling pathway. Our data reveal the essential role of LINC00941 in metastatic CRC via activation of the TGF-β/SMAD2/3 axis, which provides new insight into the mechanism of metastatic CRC and a novel potential therapeutic target for advanced CRC.

Exosomal circPACRGL promotes progression of colorectal cancer via the miR-142-3p/miR-506-3p- TGF-\u03b21 axis

BackgroundColorectal cancer (CRC) is the leading cause of cancer-related death worldwide. Exosome shave emerged as crucial regulators of intercellular communication and that abundant Circular RNAs (circRNAs) are enriched within exosomes. CircRNAs are novel members of noncoding RNAs regulating cancer proliferation and progression. However, the function and regulatory mechanism of cancer-derived exosomal circRNAs in CRC remains unclear.MethodsCRC cells-derived exosomes were characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blot. CCK-8, wound healing and transwell assays, and flow cytometry assays were conducted to assess whether exosomes would affect the proliferation, metastasis, and apoptosis of CRC cells, respectively. Moreover, we performed the RNA sequencing and RT-qPCR to identify circRNAs in exosome-stimulated CRC cells. Fluorescence in situ hybridization (FISH) assay was used to detect the cellular distribution of circPACRGL. Bioinformatic analyses (StarBase 2.0) were used to pool the miRNA targets of circPACRGL. Luciferase assays were performed to verify the direct interaction. Finally, flow cytometry was used to detect the differentiation of N1-N2 neutrophils.ResultsOur study identified a novel CRC-derived exosomal circRNA, circPACRGL. We found circPACRGL was significantly upregulated in CRC cells after tumor-derived exosomes addition. Moreover, circPACRGL serves as a sponge for miR-142-3p/miR-506-3p to facilitate the transforming growth factor-β1 (TGF-β1) expression. As a result, circPACRGL promoted CRC cell proliferation, migration and invasion, as well as differentiation of N1 to N2 neutrophils via miR-142-3p/miR-506-3p-TGF-β1 axis.ConclusionOur study, the first to reveal that cancer-derived exosomal circPACRGL plays an oncogenic role in CRC proliferation and metastasis, providing mechanistic insights into the roles of circRNAs in CRC progression and a valuable marker for CRC treatment.

Hypoxia-induced hsa_circ_0000826 is linked to liver metastasis of colorectal cancer.

Backgroundhsa_circ_0000826 has been previously linked to CRC through the competing endogenous RNA network; however, the upstream driver of hsa_circ_0000826 elevation remains unknown. In this study, we aim to elucidate the effect of hypoxia‐induced hsa_circ_0000826 on CRC tumorigenesis and metastasis.MethodsRNA scope assay was used to evaluate the expression of hsa_circ_0000826 in CRC cells under hypoxia condition. The effects of hsa_circ_0000826 on phenotypes of CRC cells were evaluated through cell migration and invasion assay. The nude, AOM‐DSS model mice and APCMin /+ mice were used to investigate the relationship between circ_0000826, hypoxia, and CRC in mice. A total of 100 CRC tissue samples, as well as the paired adjacent tissues, were collected, and qRT‐PCR assay was used to detect the expression of hsa_circ_0000826 in these samples.ResultsHypoxia‐induced hsa_circ_0000826 overexpression can increase the malignant phenotypes, tumor formation, and metastasis capability of CRC cells in vitro. mmu_circ_0000826 levels were significantly increased in the CRC tissues from AOM‐DSS and APC mice model under hypoxia conditions. Further, the hypoxia‐induced upregulation of mmu_circ_0000826 can also promote CRC tumorigenesis and liver metastasis in vivo. The expression of hsa_circ_0000826 in serum was significantly increased in CRC tissues in 100‐pair of CRC and according to the adjacent normal tissues by qRT‐PCR assays. Moreover, the expression levels of hsa_circ_0000826 in serum of patient with liver metastasis were significantly increased than those without metastasis.ConclusionOur results suggested that hsa_circ_0000826 was induced by the hypoxia in CRC, which can be a potential biomarker of CRC liver metastasis.

METTL14-mediated N6-methyladenosine modification of SOX4 mRNA inhibits tumor metastasis in colorectal cancer

BackgroundColorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide, and its main cause of death is distant metastasis. Methyltransferase-like 14(METTL14), a major RNA N6-adenosine methyltransferase, is involved in tumor progression via regulating RNA function. The goal of the study is to uncover the biological function and molecular mechanism of METTL14 in CRC.MethodsQuantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) assays were employed to detect METTL14 and SOX4 in CRC cell lines and tissues. The biological functions of METTL14 were demonstrated using in vitro and in vivo experiments. Chromatin immunoprecipitation (ChIP), Transcrptomic RNA sequencing (RNA-Seq), m6A-RNA immunoprecipitation sequencing (MeRIP-Seq), RNA immunoprecipitation and luciferase reporter assays were used to explore the mechanism of METTL14 action.ResultsMETTL14 expression was significantly downregulated in CRC and decreased METTL14 was associated with poor overall survival (OS). Both the univariate and multivariate Cox regression analysis indicated that METTL14 was an independent prognostic factor in CRC. Moreover, lysine-specific histone demethylase 5C(KDM5C)-mediated demethylation of histone H3 lysine 4 tri-methylation(H3K4me3) in the promoter of METTL14 inhibited METTL14 transcription. Functionally, we verified that METTL14 inhibited CRC cells migration, invasion and metastasis through in vitro and in vivo assays, respectively. Furthermore, we identified SRY-related high-mobility-group box 4(SOX4) as a target of METTL14-mediated m6A modification. Knockdown of METTL14 markedly abolished SOX4 mRNA m6A modification and elevated SOX4 mRNA expression. We also revealed that METTL14-mediated SOX4 mRNA degradation relied on the YTHDF2-dependent pathway. Lastly, we demonstrated that METTL14 might inhibit CRC malignant process partly through SOX4-mediated EMT process and PI3K/Akt signals.ConclusionsDecreased METTL14 facilitates tumor metastasis in CRC, suggesting that METTL14 might be a potential prognostic biomarker and effective therapeutic target for CRC.Graphical abstract