Malignant Progression Of Colorectal Cancer Research Articles

A novel four‑gene biomarker for tobacco smoking -induced colorectal cancer progression.

Cigarette smoking greatly promotes the progression and poor prognosis of colorectal cancer (CRC) patients, with the molecular mechanism still not fully clear. In this study, CRC cells were exposed to tobacco specific nitrosamine 4‑(methylnitrosamino)‑1‑(3‑pyridyl) 1‑butanone (NNK), and the differentially expressed smoking-related genes were identified based on both NNK-induced CRC cells and a total of 763 CRC tissues from TCGA cohort. Cox regression analysis, ROC curve and Kaplan-Meier plot were used to establish the risk score model for CRC prognosis. Moreover, qRT-PCR, western blotting, colony formation, migration and invasion assays were performed to verify the core differentially expressed smoking-related gene and its molecular function in NNK-induced CRC progression. Results indicated NNK significantly enhanced CRC cell proliferation, migration and invasion. Moreover, a four-gene signature containing AKR1B10, CALB2, PLAC1, GNA15 was established as CRC prognosis marker. Among these four genes, AKR1B10 was further validated as the core gene, and its expression was significantly inhibited after NNK exposure in CRC cells. Results of gene enrichment analysis and western blotting suggested AKR1B10 might reduce the malignant progression of NNK-induced CRC cells through inhibiting Wnt signaling pathway by promoting E-Cadherin expression and inhibiting the expression of N-Cadherin, β-Catenin, Vimentin and Snail. In conclusion, a new four smoking-related genes can be jointly used as prognostic markers for CRC. AKR1B10 served as a tumor suppressor, can be used as a potential target to inhibit NNK-induced CRC malignant progression through regulating Wnt signaling pathway. This study demonstrates tobacco-derived NNK dependence would promote the malignant progression of colorectal cancer through regulating the expressions of AKR1B10/Wnt signaling pathway. And a novel four-gene signature is established for the prognosis prediction of smoking CRC patients. These findings have important translational implications given the continued use of tobacco and the difficulty in smoking cessation worldwide, which can be applied to alleviate the adverse effects induced by tobacco dependence on colorectal cancer patients.

Abstract 3790: HtrA3 promotes malignant progression of colorectal cancer via PI3K-AKT-FOXO1 signaling pathway

Abstract High-temperature requirement protease A3 (HtrA3) is known to contribute to the progression of various carcinomas through tissue-specific actions, yet its precise role in colorectal cancer (CRC) remains unclear. Our study revealed that HtrA3 is significantly upregulated in CRC at both mRNA and protein levels. TCGA database analysis showed that higher HtrA3 expression in colon adenocarcinoma is linked to poorer patient survival. Moreover, increased HtrA3 expression in HCT116 cells boosted their proliferation, migration, and invasion, while decreasing HtrA3 in SW480 cells had the reverse effect. In HCT116 cells with high HtrA3 levels, there was an increase in p-PI3K, p-AKT, and p-FOXO1, which reduced upon treatment with the PI3K inhibitor LY294002. Conversely, in SW480 cells with lowered HtrA3, these molecular levels decreased and rose again after treatment with the PI3K activator 740Y-P. HtrA3 also promoted cell proliferation, migration, and invasion in the presence of LY294002 and 740Y-P. In vivo experiments demonstrated that HtrA3 overexpression accelerated xenograft tumor growth. This study confirms that HtrA3’s overexpression in CRC cells promotes their proliferation, migration, and invasion by activating the PI3K-AKT-FOXO1 signaling pathway. Therefore, HtrA3 could be a significant factor in the advancement of CRC and may serve as a potential prognostic marker for guiding targeted CRC therapies. Citation Format: Jing Zheng, Xianhua Huang, Xian-e Peng, Yunli Wu. HtrA3 promotes malignant progression of colorectal cancer via PI3K-AKT-FOXO1 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3790.

DR6 Augments Colorectal Cancer Cell Growth, Invasion, and Stemness by Activating AKT/NF-κB Pathway.

This study aims to elucidate the role and mechanisms of Death Receptor 6 (DR6), a member of the tumor necrosis factor receptor superfamily, in the malignant progression of colorectal cancer (CRC). The association of DR6 expression levels and CRC patient survival was examined using the CRC cohort data from GEPIA database. The functional role of DR6 in CRC cells was investigated by performing loss-of-function and gain-of-function experiments based on CCK-8 proliferation assay, transwell migration and invasion assay, and sphere-forming assays. Xenograft model of CRC cells in nude mouse was established to evaluate the impact of DR6 knockdown on CRC tumorigenesis. Elevated expression of DR6 was correlated with an unfavorable prognosis in CRC patients. In vitro functional assays demonstrated that silencing DR6 considerably suppressed the proliferation, migration, invasion, and stemness of CRC cells, whereas its overexpression showed an opposite effect. DR6 knockdown also attenuated tumor formation of CRC cells in the nude mice. Mechanistically, silencing DR6 reduced the phosphorylation of AKT and NF-κB in CRC cells, and the treatment with an AKT activator (SC79) abrogated the inhibitory effects of DR6 knockdown on the malignant features of CRC cells. Our data suggest that DR6 contributes to the malignant progression of CRC by activating AKT/NF-κB pathway, indicating its clinical potential as a prognostic marker and therapeutic target for CRC.

ALKBH5-mediated upregulation of CPT1A promotes macrophage fatty acid metabolism and M2 macrophage polarization, facilitating malignant progression of colorectal cancer

m6A modification has been studied in tumors, but its role in host anti-tumor immune response and TAMs polarization remains unclear. The fatty acid oxidation (FAO) process of TAMs is also attracting attention.A co-culture model of colorectal cancer (CRC) cells and macrophages was used to simulate the tumor microenvironment. Expression changes of m6A demethylase genes FTO and ALKBH5 were screened. ALKBH5 was further investigated. Gain-of-function experiments were conducted to study ALKBH5's effects on macrophage M2 polarization, CRC cell viability, proliferation, migration, and more. Me-RIP and Actinomycin D assays were performed to study ALKBH5's influence on CPT1A, the FAO rate-limiting enzyme. AMP, ADP, and ATP content detection, OCR measurement, and ECAR measurement were used to explore ALKBH5's impact on macrophage FAO level. Rescue experiments validated ALKBH5's mechanistic role in macrophage M2 polarization and CRC malignant development.In co-culture, CRC cells enhance macrophage FAO and suppress m6A modification in M2 macrophages. ALKBH5 was selected as the gene for further investigation. ALKBH5 mediates CPT1A upregulation by removing m6A modification, promoting M2 macrophage polarization and facilitating CRC development. These findings indicate that ALKBH5 enhances fatty acid metabolism and M2 polarization of macrophages by upregulating CPT1A, thereby promoting CRC development.

Prognostic Model Associated with Necroptosis in Colorectal Cancer based on Transcriptomic Analysis and Experimental Validation.

Numerous studies have emphasised the importance of necroptosis in the malignant progression of colorectal cancer (CRC). However, whether necroptosis-related genes (NRGs) can be used to predict the prognosis of CRC remains to be revealed. Patients with CRC were divided into two clusters based on the expression of NRGs, and prognosis was compared between the two clusters. A prognostic model was established based on NRGs, and its predictive efficiency was validated using Kaplan-Meier (K-M) curves, receiver operating characteristic (ROC) curves and a nomogram. Immune infiltration, single-cell and drug sensitivity analyses were used to examine the effects of NRGs on the prognosis of CRC. The prognostic model served as a valid and independent predictor of CRC prognosis. Immune infiltration and single-cell analyses revealed that the unique immune microenvironment of CRC was regulated by NRGs. Drug sensitivity analysis showed that patients in the high- and low-risk groups were sensitive to different drugs. In addition, H2BC18 was found to play an important role in regulating the malignant progression of CRC. This study provides novel insights into precision immunotherapy based on NRGs in CRC. The NRG-based prognostic model may help to identify targeted drugs and develop more effective and individualised treatment strategies for patients with CRC.

SATB1 mediated tumor colonization and β-catenin nuclear localization are associated with colorectal cancer progression

ABSTRACT Colorectal cancer (CRC) is a malignancy with high incidence and poor prognosis. It is urgent to identify valuable biomarkers for early diagnosis and potent therapeutic targets. It has been reported that SATB1 is associated with the malignant progression in CRC. To explore the role of SATB1 in CRC progression and the underlying mechanism, we evaluated the expression of SATB1 in the paired CRC tissues with immunohistochemistry. The results showed that the expression of SATB1 in lymph node metastasis was higher than that in primary lesion, and that in distant organ metastasis was higher than that in primary lesion. The retrospective analysis showed that patients with high expression of SATB1 had a significantly worse prognosis than those with negative and moderate expression. In vitro experiments that employing SATB1 over-expressing and depleted CRC cell lines confirmed that SATB1 contributes to cell proliferation and colonization, while inhibiting cell motility. Furthermore, the tissue immunofluorescence assay, Co-IP and Western blot were conducted to reveal that SATB1 induced translocation of β-catenin and formed a protein complex with it in the nuclei. In conclusion, SATB1 mediated tumor colonization and β-catenin nuclear localization are associated with the malignant progression and poor prognosis of CRC.

TSC22D2 Regulates ACOT8 to Delay the Malignant Progression of Colorectal Cancer.

Colorectal cancer (CRC) is one of the cancers with high incidence and mortality rates worldwide. In China, there are approximately 400,000 new CRC cases each year, seriously endangering people's life and health. Transforming growth factor β-stimulated clone 22 domain family, member 2 (TSC22D2) is widely expression in cancers, but the role of TSC22D2 in CRC are still unknown. Real‑time quantitative PCR (qRT-PCR) and Western blot were applied to determine the TSC22D2 levels. CCK-8, colony formation and transwell assays were used to determine the proliferation and metastasis abilities of CRC cells in vitro. In vivo metastatic potential was assessed using a subcutaneously injected mouse model and. Western-blot and immunoprecipitation experiments were used to study the mechanism of TSC22D2‑mediated metastasis. We found TSC22D2 was deregulated in CRC tissues and cells and implied poor prognosis. Overexpression TSC22D2 significantly promoted CRC cells proliferation and tumorigenicity both in vitro and vivo, whereas knockdown TSC22D2 resulted in the opposite effects. Importantly using a co-immunoprecipitation (co-IP) assay combined with mass spectrometry analysis to identify TSC22D2-interacting acyl-coenzyme A thioesterases 8 (ACOT8), TSC22D2 maintained stability of ACOT8. Overexpression of TCC22D2 in CRC cells can promote the expression of ACOT8 and inhibit the proliferation and metastasis of CRC cells through EMT mechanism, highlighting the possibility of TSC22D2 as a potential target in CRC development. In summary, the present study revealed the inhibitory effect of TSC22D2 on the proliferation of colorectal cancer cells, suggesting that TSC22D2 may be an important tumor suppressor and a potential therapeutic target during colorectal carcinogenesis.

CYP1B1-AS1 Delays the Malignant Progression of Colorectal Cancer by Binding with NOP58.

Colorectal cancer (CRC) is a prevalent type of gastrointestinal cancer, and its poor prognosis is mainly attributed to the occurrence of invasion and metastasis. CYP1B1-AS1, as non-coding RNA, plays an important role in tumorigenesis and progression. However, the mechanism by which CYP1B1-AS1 acts in CRC is not yet understood. The objective of this study was to investigate how CYP1B1-AS1 contributes to the development of CRC, and provide a base for CRC diagnosis and treatment. RT-qPCR was used to detect the expression level of CYP1B1-AS1 in CRC and adjacent tissues. CCK-8, Edu, scratch healing, and transwell experiments were used to detect the changes of proliferation, migration, and invasion ability of CRC cells after overexpression or knockdown of CYP1B1-AS1 respectively. The RNA binding protein NOP58 combined with CYP1B1-AS1 was verified by RIP and RNA Pull-down experiments. Functional recovery experiments validated the interaction between CYP1B1-AS1 and NOP58 in CRC cells. The changes of EMT-related proteins were detected by Western blot, and the half-life of transcription factor SNAIL mRNA were detected by RT-qPCR after overexpression or knockdown of NOP58. CYP1B1-AS1 was found to be significantly downregulated in CRC compared to adjacent noncancerous tissues. Experiments conducted in vitro and in vivo confirmed that upregulation of CYP1B1-AS1 significantly inhibited the proliferation, migration, and invasion of CRC cells. In addition, CYP1B1-AS1 can directly bind to NOP58 and negatively regulate NOP58. The effect of overexpression CYP1B1-AS1 was reversed by NOP58 overexpression. NOP58 regulates the EMT process of CRC cells by affecting the stability of EMT-related transcription factor SNAIL mRNA, and then affects the progress of CRC. This research proves that CYP1B1-AS1 can inhibit the occurrence of EMT in CRC by binding with NOP58, thus delaying the progress of CRC. This finding indicates that CYP1B1-AS1 may be a novel biomarker to improve the diagnosis and treatment of CRC.

TP53INP2 modulates the malignant progression of colorectal cancer by reducing the inactive form of β-catenin

TP53INP2 (tumor protein p53-inducible nuclear protein 2), known as an autophagy protein, is essential for regulating transcription and starvation-induced autophagy, which plays a crucial role in the oncogenesis and progression of various cancers. The present study aims to investigate the expression pattern, function and prognostic value of TP53INP2 in colorectal cancer (CRC). Here, we report that low expression of TP53INP2 correlates with poor survival in CRC patients. TP53INP2 was significantly downregulated in CRC tissues compared with adjacent tissues. As the malignancy of CRC progresses, the expression level of TP53INP2 gradually decreased. Knockdown of TP53INP2 promoted CRC cell proliferation and tumor growth in mice. Mechanistically, TP53INP2 deficiency decreased phosphorylation of β-catenin on S33, S37, and T41, resulting in increased accumulation of β-catenin and enhanced nuclear translocation and transcriptional activity. Moreover, we further demonstrated that TP53INP2 sequestered TIM50, thereby inhibiting its activation of β-catenin. Taken together, our findings indicate that the downregulation of TP53INP2 promotes CRC progression by activating β-catenin and suggest that TP53INP2 may be a candidate therapeutic target for CRC.

EphA2 as a phase separation protein associated with ferroptosis and immune cell infiltration in colorectal cancer.

Colorectal cancer is one of the most common malignant tumors in the digestive system, and its high incidence and metastasis rate make it a terrible killer that threatens human health. In-depth exploration of the targets affecting the progression of colorectal cancer cells and the development of specific targeted drugs for them are of great significance for the prognosis of colorectal cancer patients. Erythropoietin-producing hepatocellular A2 (EphA2) is a member of the Eph subfamily with tyrosine kinase activity, plays a key role in the regulation of signaling pathways related to the malignant phenotype of various tumor cells, but its specific regulatory mechanism in colorectal cancer needs to be further clarified. Here, we found that EphA2 was abnormally highly expressed in colorectal cancer and that patients with colorectal cancer with high EphA2 expression had a worse prognosis. We also found that EphA2 can form liquid-liquid phase separation condensates on cell membrane, which can be disrupted by ALW-II-41-27, an inhibitor of EphA2. In addition, we found that EphA2 expression in colorectal cancer was positively correlated with the expression of ferroptosis-related genes and the infiltration of multiple immune cells. These findings suggest that EphA2 is a novel membrane protein with phase separation ability and is associated with ferroptosis and immune cell infiltration, which further suggests that malignant progression of colorectal cancer may be inhibited by suppressing the phase separation ability of EphA2.

Systematic analysis of integrated bioinformatics to identify upregulated THBS2 expression in colorectal cancer cells inhibiting tumour immunity through the HIF1A/Lactic Acid/GPR132 pathway

BackgroundTHBS2, a member of the extracellular matrix glycoprotein family, can effectively inhibit tumour growth and angiogenesis. This study aimed to investigate the biological role of THBS2 in various types of cancers and the mechanisms underlying the malignant progression of colorectal cancer (CRC).MethodsTHBS2 expression in pan-cancer tissues and cell lines was assessed using the HPA, TISCH and CCLE databases. The CIBERSORT, ESTIMATE, TIMER, xCell and ssGSEA (implemented using the IOBR R package) algorithms were used to calculate the proportion of tumour-infiltrating immune cells based on the expression profile of THBS2 in TCGA-COAD cohort. The clusterprofiler R package was used to implement GO and KEGG pathway enrichm SNVs were compared between the high- and low-THBS2-expression groups using the maftools R package. Additionally, immunotherapy responses were compared between the high- and low-THBS2-expression groups based on immunophenoscores (IPSs). CT26 cells were engineered to overexpress THBS2 (CT26-THBS2) to investigate its regulatory effects on HIF1 and cellular metabolism. The conditioned medium from CT26-THBS2 cells was collected to examine its effect on the M2 polarisation of RAW264.7 macrophages. Subsequently, in vitro experiments were performed to validate the inhibitory effects of M2-polarised macrophages on T-cell proliferation and cytotoxicity. A CT26-THBS2 tumour-bearing mouse model was constructed to validate the impact of high THBS2 expression in tumour cells on the tumour microenvironment in vivo.ResultsTHBS2 expression was upregulated in a majority of tumours, including COAD, and was positively associated with ESTIMATEScore, ImmuneScore and StromalScore. Furthermore, THBS2 expression was positively associated with angiogenesis and epithelial–mesenchymal transition and negatively associated with DNA repair, cell cycle and DNA replication in most tumours. THBS2 expression was considerably associated with progression-free interval (PFI) and positively associated with MSI in COAD. THBS2 methylation levels were remarkably lower in COAD tissues than in healthy tissues. The high expression of THBS2 in CT26 cells remarkably promoted the nuclear translocation of HIF1 and consequently enhanced lactate metabolism in cells. In vitro and in vivo experiments revealed that lactate released by tumour cells promoted M2 polarisation of macrophages, leading to inhibition of T-cell proliferation and cytotoxicity.ConclusionsTHBS2 expression is associated with PFI, immune cell infiltration, immune regulation, cell death, cell migration, epithelial–mesenchymal transition, angiogenesis and genomic variations in COAD. THBS2 may serve as a biomarker for immunotherapy in COAD. Upregulated THBS2 expression in CRC cells inhibits anti-tumour immunity through the HIF1A/lactic acid/GPR132 pathway.

Computational identification and clinical validation of a novel risk signature based on coagulation-related lncRNAs for predicting prognosis, immunotherapy response, and chemosensitivity in colorectal cancer patients.

Coagulation is critically involved in the tumor microenvironment, cancer progression, and prognosis assessment. Nevertheless, the roles of coagulation-related long noncoding RNAs (CRLs) in colorectal cancer (CRC) remain unclear. In this study, an integrated computational framework was constructed to develop a novel coagulation-related lncRNA signature (CRLncSig) to stratify the prognosis of CRC patients, predict response to immunotherapy and chemotherapy in CRC, and explore the potential molecular mechanism. CRC samples from The Cancer Genome Atlas (TCGA) were used as the training set, while the substantial bulk or single-cell RNA transcriptomics from Gene Expression Omnibus (GEO) datasets and real-time quantitative PCR (RT-qPCR) data from CRC cell lines and paired frozen tissues were used for validation. We performed unsupervised consensus clustering of CRLs to classify patients into distinct molecular subtypes. We then used stepwise regression to establish the CRLncSig risk model, which stratified patients into high- and low-risk groups. Subsequently, diversified bioinformatics algorithms were used to explore prognosis, biological pathway alteration, immune microenvironment, immunotherapy response, and drug sensitivity across patient subgroups. In addition, weighted gene coexpression network analysis was used to construct an lncRNA-miRNA-mRNA competitive endogenous network. Expression levels of CRLncSig, immune checkpoints, and immunosuppressors were determined using RT-qPCR. We identified two coagulation subclusters and constructed a risk score model using CRLncSig in CRC, where the patients in cluster 2 and the low-risk group had a better prognosis. The cluster and CRLncSig were confirmed as the independent risk factors, and a CRLncSig-based nomogram exhibited a robust prognostic performance. Notably, the cluster and CRLncSig were identified as the indicators of immune cell infiltration, immunoreactivity phenotype, and immunotherapy efficiency. In addition, we identified a new endogenous network of competing CRLs with microRNA/mRNA, which will provide a foundation for future mechanistic studies of CRLs in the malignant progression of CRC. Moreover, CRLncSig strongly correlated with drug susceptibility. We developed a reliable CRLncSig to predict the prognosis, immune landscape, immunotherapy response, and drug sensitivity in patients with CRC, which might facilitate optimizing risk stratification, guiding the applications of immunotherapy, and individualized treatments for CRC.

Peristalsis-Associated Mechanotransduction Drives Malignant Progression of Colorectal Cancer.

In the colorectal cancer (CRC) tumor microenvironment, cancerous and precancerous cells continuously experience mechanical forces associated with peristalsis. Given that mechanical forces like shear stress and strain can positively impact cancer progression, we explored the hypothesis that peristalsis may also contribute to malignant progression in CRC. We defined malignant progression as enrichment of cancer stem cells and the acquisition of invasive behaviors, both vital to CRC progression. We leveraged our peristalsis bioreactor to expose CRC cell lines (HCT116), patient-derived xenograft (PDX1,2) lines, or non-cancerous intestinal cells (HIEC-6) to forces associated with peristalsis in vitro. Cells were maintained in static control conditions or exposed to peristalsis for 24 h prior to assessment of cancer stem cell (CSC) emergence or the acquisition of invasive phenotypes. Exposure of HCT116 cells to peristalsis significantly increased the emergence of LGR5+ CSCs by 1.8-fold compared to static controls. Peristalsis enriched LGR5 positivity in several CRC cell lines, notably significant in KRAS mutant lines. In contrast, peristalsis failed to increase LGR5+ in non-cancerous intestinal cells, HIEC-6. LGR5+ emergence downstream of peristalsis was dependent on ROCK and Wnt activity, and not YAP1 activation. Additionally, HCT116 cells adopted invasive morphologies when exposed to peristalsis, with increased filopodia density and epithelial to mesenchymal gene expression, in a Wnt dependent manner. Peristalsis associated forces drive malignant progression of CRC via ROCK, YAP1, and Wnt-related mechanotransduction. The online version contains supplementary material available at 10.1007/s12195-023-00776-w.

Discovery of extracellular vesicle-delivered miR-185-5p in the plasma of patients as an indicator for advanced adenoma and colorectal cancer

BackgroundWe aimed to evaluate whether extracellular vesicles (EV)-derived microRNAs (miRNAs) can be used as biomarkers for advanced adenoma (AA) and colorectal cancer (CRC).MethodsWe detected the changes in the plasma EV-delivered miRNA profiles in healthy donor (HD), AA patient, and I-II stage CRC patient groups using miRNA deep sequencing assay. We performed the TaqMan miRNA assay using 173 plasma samples (two independent cohorts) from HDs, AA patients, and CRC patients to identify the candidate miRNA(s). The accuracy of candidate miRNA(s) in diagnosing AA and CRC was determined using the area under the receiver-operating characteristic curve (AUC) values. Logistic regression analysis was performed to evaluate the association of candidate miRNA(s) as an independent factor for the diagnosis of AA and CRC. The role of candidate miRNA(s) in the malignant progression of CRC was explored using functional assays.ResultsWe screened and identified four prospective EV-delivered miRNAs, including miR-185-5p, which were significantly upregulated or downregulated in AA vs. HD and CRC vs. AA groups. In two independent cohorts, miR-185-5p was the best potential biomarker with the AUCs of 0.737 (Cohort I) and 0.720 (Cohort II) for AA vs. HD diagnosis, 0.887 (Cohort I) and 0.803 (Cohort II) for CRC vs. HD diagnosis, and 0.700 (Cohort I) and 0.631 (Cohort II) for CRC vs. AA diagnosis. Finally, we demonstrated that the upregulated expression of miR-185-5p promoted the malignant progression of CRC.ConclusionEV-delivered miR-185-5p in the plasma of patients is a promising diagnostic biomarker for colorectal AA and CRC.Trial registration The study protocol was approved by the Ethics Committee of Changzheng Hospital, Naval Medical University, China (Ethics No. 2022SL005, Registration No. of China Clinical Trial Registration Center: ChiCTR220061592).

Genetic and nongenetic mechanisms for colorectal cancer evolution.

The stepwise accumulation of key driver mutations is responsible for the development and malignant progression of colorectal cancer in primary sites. Genetic mouse model studies have revealed combinations of driver gene mutations that induce phenotypic changes in tumors toward malignancy. However, cancer evolution is regulated by not only genetic alterations but also nongenetic mechanisms. For example, certain populations of metastatic cancer cells show a loss of malignant characteristics even after the accumulation of driver mutations, and such cells are eliminated in a negative selection manner. Furthermore, a polyclonal metastasis model has recently been proposed, in which cell clusters consisting of genetically heterogeneous cells break off from the primary site, disseminate to distant organs, and develop into heterogenous metastatic tumors. Such nongenetic mechanisms for malignant progression have been elucidated using genetically engineered mouse models as well as organoid transplantation experiments. In this review article, we discuss the role of genetic alterations in the malignant progression of primary intestinal tumors and nongenetic mechanisms for negative selection and polyclonal metastasis, which we learned from model studies.

Hydroxyphenylpyruvate Dioxygenase Is a Metabolic Immune Checkpoint for UTX-deficient Colorectal Cancer

Aberrant epigenetic events mediated by histone methyltransferases and demethylases contribute to malignant progression of colorectal cancer (CRC). However, the role of the histone demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in CRC remains poorly understood. UTX conditional knockout mice and UTX-silenced MC38 cells were used to investigate UTX function in tumorigenesis and development of CRC. We performed time of flight mass cytometry to clarify the functional role of UTX in remodeling immune microenvironment of CRC. To investigate metabolic interaction between myeloid-derived suppressor cells (MDSCs) and CRC, we analyzed metabolomics data to identify metabolites secreted by UTX-deficient cancer cells and taken up by MDSCs. We unraveled a tyrosine-mediated metabolic symbiosis between MDSC and UTX-deficient CRC. Loss of UTX in CRC resulted in methylation of phenylalanine hydroxylase, preventing its degradation and subsequently increasing tyrosine synthesis and secretion. Tyrosine taken up by MDSCs was metabolized to homogentisic acid by hydroxyphenylpyruvate dioxygenase. Homogentisic acid modified protein inhibitor of activated STAT3 via carbonylation of Cys 176, and relieved the inhibitory effect of protein inhibitor of activated STAT3 on signal transducer and activator of transcription 5 transcriptional activity. This in turn, promoted MDSC survival and accumulation, enabling CRC cells to acquire invasive and metastatic traits. Collectively, these findings highlight hydroxyphenylpyruvate dioxygenase as a metabolic checkpoint to restrict immunosuppressive MDSCs and to counteract malignant progression of UTX-deficient CRC.

In Vitro and In Vivo Models for Metastatic Intestinal Tumors Using Genotype-Defined Organoids.

It has been established that the accumulation of driver gene mutations causes malignant progression of colorectal cancer (CRC) through positive selection and clonal expansion, similar to Darwin's evolution. Following this multistep tumorigenesis concept, we previously showed the specific mutation patterns for each process of malignant progression, including submucosal invasion, epithelial mesenchymal transition (EMT), intravasation, and metastasis, using genetically engineered mouse and organoid models. However, we also found that certain populations of cancer-derived organoid cells lost malignant characteristics of metastatic ability, although driver mutations were not impaired, and such subpopulations were eliminated from the tumor tissues by negative selection. These organoid model studies have contributed to our understanding of the cancer evolution mechanism. We herein report the in vitro and in vivo experimental protocols to investigate the survival, growth, and metastatic ability of intestinal tumor-derived organoids. The model system will be useful for basic research as well as the development of clinical strategies.

SiRNA-based delivery nanoplatform attenuates the CRC progression via HIF1α-AS2

Colorectal cancer (CRC) is one of the most common malignancies and the leading causes of cancer-related deaths in the world. However, the molecular mechanisms of colorectal carcinogenesis and metastasis remain unknown. Long non-coding RNAs (lncRNAs) have been implicated to be in the initiation and malignant progression of CRC. In this study, we identified a novel oncogenic lncRNA HIF1α-AS2 in CRC patients by combination with lncRNA microarray and qRT-PCR. HIF1α-AS2 is upregulated progressively in adjacent non-tumor tissues to adenoma to CRC and contributes to CRC aggressive phenotypes. RNA-sequence assays and luciferase reporter assays were performed to reveal that upregulation of lncRNA HIF1α-AS2 may lead to the increased expression of HIF1α via competitively binding to miR-155–5p, miR-340–5p, miR-580–5p and miR-590–3p in CRC initiation, whereas HIF1α may work as a cellular sponge to directly interact with the RMRP promoter to promote RMRP transcription determined by chromatin immunoprecipitation and RNA-sequence assays. Data from RNA Binding Protein Immunoprecipitation and mass spectrometry revealed that RMRP-mediated an increased level of IGF2 expression promotes CRC progression and metastasis by competitively sponging IGF2R. Lastly, we developed mPEG-DSPE liposomes encapsulated with siRNA HIF1α-AS2, which exhibited desirable physicochemical properties along with narrow well-dispersion stability and significantly prevented the progression of CRC, indicating that mPEG-DSPE-liposome-encapsulated HIF1α-AS2 siRNA might be a potential biomarker and therapeutic target for CRC therapy.

Circ_0052184 Promotes Colorectal Cancer Progression via Targeting miR-604/HOXA9 Axis.

Background The mortality rate of colorectal cancer (CRC) ranks second. circRNAs are abnormal expression in some diseases, and their dysregulation is associated with cancer progression. Recent studies have shown that the malignant progression of colorectal cancer is inseparable from the abnormal expression of circRNAs. Methods First, the circ_0052184 expression in clinical tissue and cell samples was analyzed by qRT-PCR. Then, we constructed circ_0052184-silenced CRC cells and detected by qRT-PCR. Furthermore, the proliferation ability of cells was detected by colony formation assay. Cell migration ability was tested by wound healing assay and transwell assay. Cell invasion ability was detected by transwell assay. Results Expression of circ_0052184 was significantly increased in colorectal cancer cell lines and tissues. Silencing circ_0052184 affected the proliferation, migration, and invasion of colorectal cancer cells. miR-604 was targeted by circ_0052184. The downstream target of miR-604 was HOXA9, and silencing circ_0052184 inhibited HOXA9 expression. The existence of the circ_0052184/miR-604/HOXA9 regulatory network in colorectal cancer was validated. circ_0052184 promoted the occurrence and development of colorectal cancer by targeting the miR-604/HOXA9 axis. Conclusions Our study revealed that the molecular mechanism of circ_0052184 regulated the miR-604/HOXA9 axis, which might promote the malignant progression of colorectal cancer cells.

Circ_0056618 enhances PRRG4 expression by competitively binding to miR-411-5p to promote the malignant progression of colorectal cancer.

The purpose of this paper was to explore the role of circ_0056618 and associated mechanisms in colorectal cancer (CRC). The expression of circ_0056618, proline rich and Gla domain 4 (PRRG4) mRNA and miR-411-5p was measured by quantitative real-time PCR (qPCR).The protein levels of PRRG4 and epithelial-mesenchymal transition (EMT)-related markers were detected by western blot. Cell proliferation was assessed by cell counting kit-8, EdU, and colony formation assays. Cell migration and invasion were assessed by transwell assay. Cell apoptosis was detected by flow cytometry assay. The putative relationship between miR-411-5p and circ_0056618 or PRRG4 was verified by dual-luciferase reporter assay. The effects of circ_0056618 on tumor growth in vivo were determined by animal study. Circ_0056618 and PRRG4 was upregulated, while miR-411-5p was downregulated in CRC tumor tissues and cells. Circ_0056618 knockdown or PRRG4 knockdown inhibited CRC cell proliferation, migration/invasion, EMT, and survival. Circ_0056618 positively modulated PRRG4 expression by targeting miR-411-5p. MiR-411-5p absence or PRRG4 overexpression could rescue circ_0056618 knockdown-induced inhibition on proliferation, migration/invasion, and EMT in CRC cells. Animal assay showed circ_0056618 knockdown impeded tumor growth in vivo. Circ_0056618 promoted CRC growth and development by upregulating PRRG4 expression via competitively targeting miR-411-5p.