Epithelial-mesenchymal Transition In Colorectal Cancer Research Articles

N-acetylglucosaminyltransferase V drives colorectal cancer metastasis by facilitating ZO-1 ubiquitination and degradation

Increasing evidence supports the crucial role of Epithelial-Mesenchymal Transition (EMT) in cancer invasion and metastasis. N-acetylglucosaminyltransferase V (MGAT5), which is associated with multiantenna glycosylation, can contribute to tumorigenesis, yet its specific role in promoting colorectal cancer (CRC) metastasis remains unclear. Bioinformatics analysis of CRC datasets revealed that elevated MGAT5 expression was associated with EMT and a poor prognosis. In vitro experiments confirmed the pivotal role of MGAT5 as an EMT regulator in CRC cells. MGAT5 overexpression stimulated cell proliferation and migration, while MGAT5 knockdown had the opposite effect. Mechanistically, MGAT5 promoted EMT through multiantenna glycosylation of ZO-1, promoting its ubiquitination and reducing its expression. Clinically, MGAT5 upregulation in the CRC TMA correlated negatively with ZO-1 expression, which is indicative of malignancy and a poor prognosis. This study revealed that MGAT5 promotes EMT in CRC via interactions between multiple antenna glycosylation products and ZO-1 ubiquitination/degradation, indicating that MGAT5 could serve as a promising therapeutic target for CRC.Graphical

Reduced Proline-Rich Tyrosine Kinase 2 Promotes Tumor Metastasis by Activating Epithelial-Mesenchymal Transition in Colorectal Cancer.

Proline-rich tyrosine kinase 2 (PYK2) is involved in the occurrence, proliferation, migration, and invasion of various tumors. However, few studies have reported the role of PYK2 in colorectal cancer (CRC). To explore the effects of PYK2 on CRC metastasis and elucidate the detailed molecular mechanisms involved. The expression and prognosis value of PYK2 in CRC prognosis were analyzed using data from The Cancer Genome Atlas (TCGA). PYK2 was knocked down or overexpressed in human CRC cell line, HCT116. Cell proliferation, migration, invasion, and cycle changes were analyzed using CCK-8, Transwell, and flow cytometry assays. Western blotting and quantitative real-time PCR were performed to detect the mRNA and protein levels of cell proliferation and epithelial-mesenchymal transition (EMT) indicators. Fluorescence staining was performed to examine the cytoskeleton. Lower expression of PYK2 was observed in CRC tissues and associated with poor prognosis and metastasis in patients with CRC in TCGA database. PYK2 knockdown significantly induced the migration and invasion of CRC cells but did not affect cell proliferation or cycle. Immunofluorescence staining of phalloidin showed that the downregulation of PYK2 increased the cytoskeleton in CRC cells. Moreover, low expression of PYK2 induced the downregulation of E-cadherin and upregulation of snail and vimentin by activating Wnt/β-catenin signaling, thus promoting EMT in CRC cells. Low PYK2 expression was found in tumor tissues, especially metastases, and significantly correlated with patient prognosis. Moreover, decreased PYK2 induces EMT by activating Wnt/β-catenin signaling, which is the potential mechanism of CRC metastasis. Regulating the expression of PYK2 to suppress tumor cell metastasis may represent a promising therapeutic strategy for metastatic CRC.

PRNP is a pan-cancer prognostic and immunity-related to EMT in colorectal cancer.

Prion protein gene (PRNP) is widely expressed in a variety of tissues. Although the roles of PRNP in several cancers have been investigated, no pan-cancer analysis has revealed its relationship with tumorigenesis and immunity. Comprehensive analyses were conducted on The Cancer Genome Atlas (TCGA) Pan-Cancer dataset from the University of California Santa Cruz (UCSC) database to determine the expression of PRNP and its potential prognostic implications. Immune infiltration and enrichment analysis methods were used to ascertain correlations between PRNP expression levels, tumor immunity, and immunotherapy. Additionally, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) methods were employed to examine possible signaling pathways involving PRNP. In vitro experiments using CCK-8 assay, Wound healing assay, and Transwell assay to detect the effect of Cellular prion protein (PrPC) on proliferation, migration, and invasion in colorectal cancer (CRC) cells. The expression levels of epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, E-cadherin, Vimentin and Snail) were detected by western blot. Among most cancer types, PRNP is expressed at high levels, which is linked to the prognosis of patients. PRNP expression is strongly associated with immune infiltrating cells, immunosuppressive cell infiltration and immune checkpoint molecules. In addition to tumor mutation burden (TMB), substantial correlations are detected between PRNP expression and microsatellite instability (MSI) in several cancers. In vitro cell studies inferred that PrPC enhanced the proliferation, migration, invasion, and EMT of CRC cells. PRNP serves as an immune-related prognostic marker that holds promise for predicting outcomes related to CRC immunotherapy while simultaneously promoting cell proliferation, migration, and invasion activities. Furthermore, it potentially plays a role in governing EMT regulation within CRC.

E2F1 modulates RCCD1 expression to participate in the initiation and progression of EMT in colorectal cancer

ObjectiveMetastases in the advanced stages of colorectal cancer (CRC) present a major challenge to its treatment. Epithelial-Mesenchymal Transition (EMT) plays a crucial role in enhancing the metastasis and invasion ability of cancer cells. However, the progress of E2F transcription factor 1 (E2F1) and Regulator of chromatin condensation 1 (RCCD1) in CRC on EMT has not been studied. MethodsThe CRC differential expression data from The Cancer Genome Atlas database were analyzed by Gene Set Enrichment Analysis to verify the difference in expression of E2F1 and RCCD1 in cancerous and para-cancerous tissues.DNA-pull down and dual luciferase experiments confirmed that E2F1 regulates RCCD1. Western-blot and q-PCR experiments confirmed that E2F1 regulates RCCD1 and participates in the EMT-related progress of CRC.EDU, Wound healing and Transwell experiments verified the effects of regulation of E2F1 and RCCD1 on the proliferation, migration and invasion of CRC cells. ResultsE2F1 and RCCD1 are highly expressed in cancer tissues and cancer cells. E2F1 binds to the upstream promoter of RCCD1 to regulate RCCD1 and affect the expression of EMT-related targets in CRC cells. It also affects the proliferation, migration and invasion of CRC cells. ConclusionsE2F1 regulates the involvement of RCCD1 in CRC EMT and affects the proliferation, migration and invasion ability of CRC cells.

SIPA1 promotes epithelial-mesenchymal transition in colorectal cancer through STAT3 activation

Colorectal cancer (CRC) is the third leading cancer type worldwide and accounts for the second highest rate of cancer-related mortality. Liver metastasis significantly contributes to the mortality associated with CRC, but the fundamental mechanisms behind it remain unclear. Signal-induced proliferation-associated protein 1 (SIPA1), a GTPase activating protein, has been shown to promote metastasis in breast cancer. In this study, our objective was to explore the role of SIPA1 in regulating epithelial-mesenchymal transition (EMT) in CRC. The analysis of The Cancer Genome Atlas (TCGA) database revealed that the expression level of SIPA1 mRNA was notably upregulated and exhibited a positively correlated with EMT and STAT3 signaling pathways in CRC. Knockdown of SIPA1 impairs CRC cell proliferation and migration. Further studies on the reliance of SIPA1 on STAT3 signaling for EMT regulation have shown that SIPA1 stimulates the activation of STAT3, resulting in its nuclear translocation. The co-treatment of overexpressed SIPA1 with the STAT3 inhibitor STTITA has shown that SIPA1 regulates the expression of EMT-related markers through STAT3. Our study indicate that SIPA1 promotes CRC metastasis by activating the STAT3 signaling pathway, underscoring the potential of SIPA1 as a therapeutic target for metastatic CRC patients.

Integrative network analysis of transcriptomics data reveals potential prognostic biomarkers for colorectalcancer.

Cross-talk among biological pathways is essential for normal biological function and plays a significant role in cancer progression. Through integrated network analysis, this study explores the significance of pathway cross-talk in colorectal cancer (CRC) development at both the pathway and gene levels. In this study, we integrated the gene expression data with domain knowledge to construct state-dependent pathway cross-talk networks. The significance of the genes involved in pathway cross-talk was assessed by analyzing their association with cancer hallmarks, disease-gene relation, genetic alterations, and survival analysis. We also analyzed the gene regulatory network to identify the dysregulated genes and their role in CRC progression. Cross-talk was observed between immune-related pathways and pathways associated with cell communication and signaling. The PTPRC gene was identified as a mediator, facilitating interactions within the immune system and other signaling pathways. The rewired interactions of ITGA7 were identified as influential in the epithelial-mesenchymal transition in CRC. This study also highlighted the crucial link between cell communication and vascular smooth muscle contraction pathway in CRC progression. The survival analysis of identified gene clusters showed their significant prognostic value in distinguishing high-risk from low-risk CRC groups, and L1000CDS2 revealed seven potential drug molecules in CRC. Nine dysregulated genes (CTNNB1, EP300, JUN, MYC, NFKB1, RELA, SP1, STAT1, and TP53) emerge as transcription factors acting as common regulators across various pathways. This study highlights the crucial role of pathway cross-talk in CRC progression and identified the potential prognostic biomarkers and potential drug molecules.

Ubiquitin-specific peptidase 15 regulates the TFAP4/PCGF1 axis facilitating liver metastasis of colorectal cancer and cell stemness

The tumor recurrence and metastasis of colorectal cancer (CRC) are responsible for most of CRC-linked mortalities. It is an urgent need to deeply investigate the pathogenesis of CRC metastasis and look for novel targets for its treatment. The current study aimed to investigate the effects of ubiquitin-specific peptidase 15 (USP-15) on the CRC progression. In vivo, a mouse model of liver metastasis of CRC tumor was established to investigate the role of USP-15. In vitro, the migrated and invasive abilities of CRC cells were assessed by transwell assay. Cell stemness was evaluated by using sphere formation assay. The underlying mechanism was further explored by employing the co-immunoprecipitation, dual luciferase reporter assay, oligonucleotide pull-down assay, and chromatin immunoprecipitation assay. The results showed that USP-15 was upregulated in CRC patients with liver metastasis and high metastatic potential cell lines of CRC. Loss of USP-15 repressed the epithelial-to-mesenchymal transition (EMT), migration, invasion, and stemness properties of CRC cells in vitro. Downregulation of USP-15 reduced the liver metastasis of mice in vivo. USP-15 upregulation obtained the contrary effects. Subsequently, USP-15 deubiquitinated transcription factor AP-4 (TFAP4) and enhanced its protein stability. TFAP4 could transcriptionally activated polycomb group ring finger 1 (PCGF1). The pro-cancer effects of USP-15 were rescue by the knockdown of TFAP4 or PCGF1. In conclusions: USP-15 facilitated the liver metastasis by the enhancement of cell stemness and EMT in CRC, which was at least partly mediated by the deubiquitination of TFAP4 upon the upregulation of PCGF1.

Single-Cell and Spatial Transcriptome Profiling Identifies the Transcription Factor BHLHE40 as a Driver of EMT in Metastatic Colorectal Cancer.

Colorectal cancer is one of the most common malignant tumors in humans, with liver metastasis being the primary cause of mortality. The epithelial-mesenchymal transition (EMT) process endows cancer cells with enhanced metastatic potential. To elucidate the cellular mechanisms driving EMT in colorectal cancer, we analyzed single-cell RNA sequencing data from 11 nonmetastatic primary tumors (TnM) and 11 metastatic primary tumors (TM) from colorectal cancer patients. Compared with the TnM group, the TM samples showed elevated numbers of malignant epithelial cell and cancer-associated fibroblast (CAF) subsets that displayed enrichments of EMT, angiogenesis, and TGFβ signaling pathways. One specific TM-enriched subgroup of malignant epithelial cells underwent EMT to transdifferentiate into CXCL1+ CAFs that subsequently differentiated into SFRP2+ CAFs, which was validated by spatial transcriptomic and pseudotime trajectory analyses. Furthermore, cell-cell communication analysis identified BHLHE40 as a probable key transcription factor driving EMT that was associated with poor prognosis. Finally, in vitro and in vivo experiments functionally substantiated that BHLHE40 promoted the proliferation, invasion, migration, EMT, and liver metastasis of colorectal cancer cells. In summary, this study identified BHLHE40 as a key transcription factor regulating EMT that promotes liver metastasis in colorectal cancer. Significance: Integrated analysis of single-cell RNA sequencing and spatial transcriptomics in metastatic colorectal cancer provides insights into the mechanisms underlying EMT and cancer-associated fibroblast differentiation, which could help improve patient diagnosis and treatment.

METTL3 recruiting M2-type immunosuppressed macrophages by targeting m6A-SNAIL-CXCL2 axis to promote colorectal cancer pulmonary metastasis

BackgroundThe regulatory role of N6-methyladenosine (m6A) modification in the onset and progression of cancer has garnered increasing attention in recent years. However, the specific role of m6A modification in pulmonary metastasis of colorectal cancer remains unclear.MethodsThis study identified differential m6A gene expression between primary colorectal cancer and its pulmonary metastases using transcriptome sequencing and immunohistochemistry. We investigated the biological function of METTL3 gene both in vitro and in vivo using assays such as CCK-8, colony formation, wound healing, EDU, transwell, and apoptosis, along with a BALB/c nude mouse model. The regulatory mechanisms of METTL3 in colorectal cancer pulmonary metastasis were studied using methods like methylated RNA immunoprecipitation quantitative reverse transcription PCR, RNA stability analysis, luciferase reporter gene assay, Enzyme-Linked Immunosorbent Assay, and quantitative reverse transcription PCR.ResultsThe study revealed high expression of METTL3 and YTHDF1 in the tumors of patients with pulmonary metastasis of colorectal cancer. METTL3 promotes epithelial-mesenchymal transition in colorectal cancer by m6A modification of SNAIL mRNA, where SNAIL enhances the secretion of CXCL2 through the NF-κB pathway. Additionally, colorectal cancer cells expressing METTL3 recruit M2-type macrophages by secreting CXCL2.ConclusionMETTL3 facilitates pulmonary metastasis of colorectal cancer by targeting the m6A-Snail-CXCL2 axis to recruit M2-type immunosuppressive macrophages. This finding offers new research directions and potential therapeutic targets for colorectal cancer treatment.

PSAT1 mediated EMT of colorectal cancer cells by regulating Pl3K/AKT signaling pathway.

Background: The metastasis of colorectal cancer (CRC) is one of the significant barriers impeding its treated consequence and bring about high mortality, less surgical resection rate and poor prognosis of CRC patients. PSAT1 is an enzyme involved in serine biosynthesis. The studies showed that PSAT1 plays the part of a crucial character in the regulation of tumor metastasis. And Epithelial-Mesenchymal Transition (EMT) is a process of cell reprogramming in which epithelialcells obtain mesenchymal phenotypes. It is a crucial course in promoting cell metastasis and the progression of malignant tumors. The relationship between PSAT1 and EMT in colorectal cancer, as well as the underlying molecular mechanisms, remains enigmatic and warrants thorough exploration. These findings suggest that PSAT1 may serve as a promising therapeutic target for mitigating colorectal cancer metastasis and holds the potential to emerge as a valuable prognostic biomarker in forthcoming research endeavors. Materials and Methods: Utilizing TCGA dataset in conjunction with clinical CRC specimens, our initial focus was directed towards an in-depth examination of PSAT1 expression within CRC, specifically exploring its potential correlation with the adverse prognostic outcomes experienced by patients. Furthermore, we conducted a comprehensive investigation into the regulatory influence exerted by PSAT1 on CRC through the utilization of siRNA knockdown techniques. In the realm of in vitro experimentation, we meticulously evaluated the impact of PSAT1 on various facets of CRC progression, including cell migration, invasion, proliferation, and colony formation. In order to elucidate the intricate effects in question, we adopted a multifaceted methodology that encompassed a range of assays and analyses. These included wound healing assays, transwell assays, utilization of the Cell Counting Kit-8 (CCK-8) assay, and colony formation assays. By employing this diverse array of investigative techniques, we were able to achieve a comprehensive comprehension of the multifaceted role that PSAT1 plays in the pathogenesis of colorectal cancer. This multifarious analysis greatly contributed to our in-depth understanding of the complex mechanisms at play in colorectal cancer pathogenesis. Using WB and PCR experiments, we found that PSAT1 has a role in regulating EMT development in CRC.In terms of mechanism, we found that PSAT1 affected EMT by Regulating Pl3K/AKT Signaling Pathway. Results: Our investigation revealed a noteworthy down-regulation of PSAT1 expression in CRC specimens. Importantly, this down-regulation exhibited a significant positive correlation with the unfavorable prognosis of patients afflicted with CRC. Functionally, our study showcased that the siRNA-mediated knockdown of PSAT1 markedly enhanced various key aspects of CRC pathogenesis in an in vitro setting. Specifically, this included a substantial promotion of CRC cell migration, invasion, proliferation, and colony formation. Moreover, the silencing of PSAT1 also demonstrated a substantial promotion of the EMT process. Intriguingly, our research unveiled a hitherto unexplored mechanism underlying the regulatory role of PSAT1 in CRC and EMT. We have established, for the first time, that PSAT1 exerts its influence by modulating the activation of the PI3K/AKT Signaling Pathway. This mechanistic insight provides a valuable contribution to the understanding of the molecular underpinnings of CRC progression and EMT induction mediated by PSAT1. Conclusions: In unison, our research findings shed light on the previously uncharted and significant role of the PSAT1/PI3K/AKT axis in the initiation of the EMT process in CRC. Furthermore, our discoveries introduce a novel biomarker with potential implications for the clinical diagnosis and treatment of CRC.

Specific intracellular retention of circSKA3 promotes colorectal cancer metastasis by attenuating ubiquitination and degradation of SLUG

Our previous study demonstrated that tumor-suppressor circular RNAs (circRNAs) can be specifically secreted outside of colorectal cancer (CRC) cells within exosomes to maintain tumor cell fitness. However, whether tumor-driving circRNAs can be specifically retained in cells to facilitate tumor progression remains unknown. In this study, circRNA-seq showed that circSKA3 was significantly upregulated in CRC tissues but downregulated in serum samples from CRC patients. In addition, circSKA3 promoted CRC progression in vitro and in vivo and was retained in CRC cells via a specific cellmotif element. Interestingly, the cellmotif element was also the site of interaction of circSKA3 with SLUG, which inhibited SLUG ubiquitination degradation and promoted CRC epithelial–mesenchymal transition (EMT). Moreover, FUS was identified as a key circularization regulator of circSKA3 that bound to the key element. Finally, we designed and synthesized specific antisense oligonucleotides (ASOs) targeting circularization and cellmotif elements, which repressed circSKA3 expression, abolished the SLUG–circSKA3 interaction, and further inhibited CRC EMT and metastasis in vitro and in vivo.

Unraveling temporal and spatial biomarkers of epithelial-mesenchymal transition in colorectal cancer: insights into the crucial role of immunosuppressive cells

The occurrence and progression of tumors can be established through a complex interplay among tumor cells undergoing epithelial-mesenchymal transition (EMT), invasive factors and immune cells. In this study, we employed single-cell RNA sequencing (scRNA-seq) and spatially resolved transcriptomics (ST) to evaluate the pseudotime trajectory and spatial interactive relationship between EMT-invasive malignant tumors and immune cells in primary colorectal cancer (CRC) tissues at different stages (stage I/II and stage III with tumor deposit). Our research characterized the spatiotemporal relationship among different invasive tumor programs by constructing pseudotime endpoint-EMT-invasion tumor programs (EMTPs) located at the edge of ST, utilizing evolution trajectory analysis integrated with EMT-invasion genes. Strikingly, the invasive and expansive process of tumors undergoes remarkable spatial reprogramming of regulatory and immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), regulatory T cells (Treg), and exhausted T cells (Tex). These EMTP-adjacent cell are linked to EMT-related invasion genes, especially the C-X-C motif ligand 1 (CXCL1) and CXCL8 genes that are important for CRC prognosis. Interestingly, the EMTPs in stage I mainly produce an inflammatory margin invasive niche, while the EMTPs in stage III tissues likely produce a hypoxic pre-invasive niche. Our data demonstrate the crucial role of regulatory and immunosuppressive cells in tumor formation and progression of CRC. This study provides a framework to delineate the spatiotemporal invasive niche in CRC samples.Graphical

Role of Epithelial to Mesenchymal Transition in Colorectal Cancer.

The epithelial-mesenchymal transition (EMT) is a cellular reprogramming process that occurs during embryonic development and adult tissue homeostasis. This process involves epithelial cells acquiring a mesenchymal phenotype. Through EMT, cancer cells acquire properties associated with a more aggressive phenotype. EMT and its opposite, mesenchymal-epithelial transition (MET), have been described in more tumors over the past ten years, including colorectal cancer (CRC). When EMT is activated, the expression of the epithelial marker E-cadherin is decreased and the expression of the mesenchymal marker vimentin is raised. As a result, cells temporarily take on a mesenchymal phenotype, becoming motile and promoting the spread of tumor cells. Epithelial-mesenchymal plasticity (EMP) has become a hot issue in CRC because strong inducers of EMT (such as transforming growth factor β, TGF-β) can initiate EMT and regulate metastasis, microenvironment, and immune system resistance in CRC. In this review, we take into account the significance of EMT-MET in CRC and the impact of the epithelial cells' plasticity on the prognosis of CRC. The analysis of connection between EMT and colorectal cancer stem cells (CCSCs) will help to further clarify the current meager understandings of EMT. Recent advances affecting important EMT transcription factors and EMT and CCSCs are highlighted. We come to the conclusion that the regulatory network for EMT in CRC is complicated, with a great deal of crosstalk and alternate paths. More thorough research is required to more effectively connect the clinical management of CRC with biomarkers and targeted treatments associated with EMT.

Artificial down-regulation of RPL34 restricts the proliferation and metastasis of colorectal cancer by suppressing the JAK2/STAT3 signaling pathway.

The highly conserved ribosomal protein L34 (RPL34) has been reported to play an essential role in the progression of diverse malignancies. RPL34 is aberrantly expressed in multiple cancers, although its significant in colorectal cancer (CRC) is currently unclear. Here, we demonstrated that RPL34 expression was higher in CRC tissues than in normal tissues. Upon RPL34 overexpression, the ability of proliferation, migration, invasion and metastasis of CRC cells were significantly enhanced in vitro and in vivo. Furthermore, high expression of RPL34 accelerated cell cycle progression, activated the JAK2/STAT3 signaling pathway, and induced the epithelial-to-mesenchymal transition (EMT) program. Conversely, RPL34 silencing inhibited these CRC malignant progression. Utilizing immunoprecipitation assays, we identified the RPL34 interactor, the cullin-associated NEDD8-dissociated protein 1 (CAND1), a negative regulator of Cullin-RING ligases. CAND1 overexpression reduced the ubiquitin level of RPL34 and stabilized RPL34 protein. CAND1 silencing in CRC cells resulted in a decrease in the ability of proliferation, migration and invasion. CAND1 overexpression promoted CRC malignant phenotypes and induced EMT, and RPL34 knockdown rescued CAND1-induced CRC progression. In summary, our study indicates that RPL34 acts as a mediator, is stabilized by CAND1, promotes proliferation and metastasis may in part through the activation of the JAK2/STAT3 signaling pathway and induction of EMT in CRC.

The B56γ3-containing protein phosphatase 2A attenuates p70S6K-mediated negative feedback loop to enhance AKT-facilitated epithelial-mesenchymal transition in colorectal cancer

BackgroundProtein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC).MethodsPolyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein–protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients.ResultsWe showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC.ConclusionsOur finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC.CUkGCcvqWD-y5KjYaB3kRRVideo

Roles of miR-4442 in Colorectal Cancer: Predicting Early Recurrence and Regulating Epithelial-Mesenchymal Transition.

Early recurrence in patients with colorectal cancer (CRC) is associated with a poor prognosis. We aimed to identify circulating microRNAs that are biomarkers of early CRC recurrence and elucidate their functions. We identified miR-4442 as a candidate biomarker by microRNA array analysis comparing preoperative and postoperative plasma levels in patients with CRC, with and without recurrence. The association between preoperative plasma miR-4442 levels, clinicopathological features, and recurrence-free survival was analyzed in 108 patients with CRC after curative surgery. Furthermore, cell-function analyses were performed, and the involvement of miR-4442 in regulating epithelial-mesenchymal transition (EMT) was examined. Preoperatively plasma miR-4442 levels were associated with CRC recurrence and exhibited an incremental increase with earlier recurrence dates. Moreover, miR-4442 demonstrated high sensitivity and specificity as a potential biomarker for early CRC recurrence. The expression of miR-4442 in cancer tissues of patients with metastatic liver cancer from CRC was higher than in normal liver, CRC, and normal colorectal tissues. The overexpression of miR-4442 promoted the proliferative, migratory, and invasive activities of CRC cells, decreased levels of RBMS1 and E-cadherin, and increased levels of N-cadherin and Snail1. Plasma miR-4442 is a clinically useful biomarker for predicting the early recurrence of CRC. Furthermore, miR-4442 regulates EMT in CRC by directly targeting the messenger RNA of RBMS1.

High glucose promotes the progression of colorectal cancer by activating the BMP4 signaling and inhibited by glucagon-like peptide-1 receptor agonist

BackgroundThe detailed molecular mechanism between type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) is still uncertain. Bone morphogenetic protein 4 (BMP4) dysregulation is implicated in T2DM and CRC, respectively. This study aims to investigate whether BMP4 can mediate the interaction of CRC with T2DM.MethodsWe firstly explored the expression of BMP4 in The Cancer Genome Altas (TCGA) databases and CRC patients with or without DM from the Shanghai Tenth People’s Hospital. The diabetic model of CRC cell lines in vitro and the mice model in vivo were developed to explore the BMP4 expression during CRC with or without diabetes. Further inhibition of BMP4 to observe its effects on CRC. Also, glucagon-like peptide-1 receptor agonist (GLP-1RA) was used to verify the underlying mechanism of hypoglycemic drugs on CRC via BMP4.ResultsBMP4 expression was upregulated in CRC patients, and significantly higher in CRC patients with diabetes (P < 0.05). High glucose-induced insulin resistance (IR)-CRC cells and diabetic mice with metastasis model of CRC had increased BMP4 expression, activated BMP4-Smad1/5/8 pathway, and improved proliferative and metastatic ability mediated by epithelial-mesenchymal transition (EMT). And, treated CRC cells with exogenously BMP inhibitor-Noggin or transfected with lentivirus (sh-BMP4) could block the upregulated metastatic ability of CRC cells induced by IR. Meanwhile, GLP-1R was downregulated by high glucose-induced IR while unregulated by BMP4 inhibitor noggin, and treated GLP-1RA could suppress the proliferation of CRC cells induced by IR through downregulated BMP4.ConclusionsBMP4 increased by high glucose promoted the EMT of CRC. The mechanism of the BMP4/Smad pathway was related to the susceptible metastasis of high glucose-induced IR-CRC. The commonly used hypoglycemic drug, GLP-1RA, inhibited the growth and promoted the apoptosis of CRC through the downregulation of BMP4. The result of our study suggested that BMP4 might serve as a therapeutic target in CRC patients with diabetes.

HUNK inhibits epithelial-mesenchymal transition of CRC via direct phosphorylation of GEF-H1 and activating RhoA/LIMK-1/CFL-1

Epithelial-mesenchymal transition (EMT) is associated with the invasive and metastatic phenotypes in colorectal cancer (CRC). However, the mechanisms underlying EMT in CRC are not completely understood. In this study, we find that HUNK inhibits EMT and metastasis of CRC cells via its substrate GEF-H1 in a kinase-dependent manner. Mechanistically, HUNK directly phosphorylates GEF-H1 at serine 645 (S645) site, which activates RhoA and consequently leads to a cascade of phosphorylation of LIMK-1/CFL-1, thereby stabilizing F-actin and inhibiting EMT. Clinically, the levels of both HUNK expression and phosphorylation S645 of GEH-H1 are not only downregulated in CRC tissues with metastasis compared with that without metastasis, but also positively correlated among these tissues. Our findings highlight the importance of HUNK kinase direct phosphorylation of GEF-H1 in regulation of EMT and metastasis of CRC.

Abstract 3599: Molecular characterization of mouse colorectal cancer cell lines with high potential of peritoneal metastasis

Abstract Background: Peritoneal metastasis occurs in approximately 7% of colorectal cancer (CRC) patients and is associated with worse prognosis compared to non-peritoneal metastasis. In CRC patients with peritoneal metastasis, administration of systemic chemotherapy only slightly improves overall survival. Therefore, a better understanding of the biology of peritoneal metastasis and the development of new molecular therapeutics for CRC patients with peritoneal metastasis are urgently needed. Materials and Methods: CT26 mouse CRC cell lines with low metastatic potentials (CT26-N5) and high metastatic potentials to peritoneum (CT26-P6) were established after several rounds of in vivo selection by orthotopic transplantation of CT26 cells into the syngeneic BALB/c mice. Transcriptomic and proteomic analyses were conducted on CT26-N5 and CT26-P6 cells. Results: Orthotopic transplantation of CT26-P6 cells exhibited significantly increased peritoneal metastasis compared to CT26-N5 cells, while growth of primary tumors was not different between these CT26 sublines. In addition, CT26-P6 cells had higher cell migration and invasion potential in vitro than CT26-N5 cells. Integrated analyses of transcriptome and proteome resulted in identification of molecular signatures associated with peritoneal metastasis. An actin-binding protein Advillin, encoded by the Avil gene, was markedly increased at both mRNA and protein levels in CT26-P6 cells compared to CT26-N5 cells. Knockdown of Avil using siRNA or shRNA in CT26-P6 cells significantly reduced cell migration and invasion in vitro and occurrence of peritoneal metastasis in vivo. Gene Set Enrichment Analysis on transcriptome datasets from CT26-P6 cells and CT26-N5 cells identified interferon gamma response as the most significantly enriched pathway in CT26-cells. In addition, Immunoprecipitation coupled with LC-MS/MS identified potential interaction between Avil and a molecule X that regulates epithelial-mesenchymal transition in CRC. Consistent with the findings in mouse CT26 cells, AVIL knockdown in CRC patient-derived cells (PDCs) suppressed cell migration and invasion in vitro. While AVIL expression was induced by interferon gamma in CRC PDCs, JAK inhibitors, Momelotinib and Ruxolitinib, significantly reduced interferon gamma-induced AVIL expression, suggesting regulation of AVIL by interferon gamma. Conclusion: Molecular characterization of mouse colorectal cancer cell lines with different metastatic potential identified AVIL as a potential therapeutic target in CRC patients with peritoneal metastasis. Regulation of AVIL expression by interferon gamma may suggest functional relevance of tumor immune microenvironment in the development of peritoneal metastasis in CRC. Citation Format: Haruki Mori, Hisanori Isomura, Shuang Zhou, Taisuke Kajino, Yuichi Abe, Takashi Kinoshita, Seiji Natsume, Yusuke Sato, Akira Ouchi, Toru Miyake, Waki Hosoda, Koji Komori, Yasuhiro Shimizu, Masaji Tani, Ayumu Taguchi. Molecular characterization of mouse colorectal cancer cell lines with high potential of peritoneal metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3599.

Src-FAK Signaling Mediates Interleukin 6-Induced HCT116 Colorectal Cancer Epithelial–Mesenchymal Transition

Colorectal cancer is one of the most prevalent and lethal malignancies, affecting approximately 900,000 individuals each year worldwide. Patients with colorectal cancer are found with elevated serum interleukin-6 (IL-6), which is associated with advanced tumor grades and is related to their poor survival outcomes. Although IL-6 is recognized as a potent inducer of colorectal cancer progression, the detail mechanisms underlying IL-6-induced colorectal cancer epithelial-mesenchymal transition (EMT), one of the major process of tumor metastasis, remain unclear. In the present study, we investigated the regulatory role of IL-6 signaling in colorectal cancer EMT using HCT116 human colorectal cancer cells. We noted that the expression of epithelial marker E-cadherin was reduced in HCT116 cells exposed to IL-6, along with the increase in a set of mesenchymal cell markers including vimentin and α-smooth muscle actin (α-SMA), as well as EMT transcription regulators-twist, snail and slug. The changes of EMT phenotype were related to the activation of Src, FAK, ERK1/2, p38 mitogen-activated protein kinase (p38MAPK), as well as transcription factors STAT3, κB and C/EBPβ. IL-6 treatment has promoted the recruitment of STAT3, κB and C/EBPβ toward the Twist promoter region. Furthermore, the Src-FAK signaling blockade resulted in the decline of IL-6 induced activation of ERK1/2, p38MAPK, κB, C/EBPβ and STAT3, as well as the decreasing mesenchymal state of HCT116 cells. These results suggested that IL-6 activates the Src-FAK-ERK/p38MAPK signaling cascade to cause the EMT of colorectal cancer cells. Pharmacological approaches targeting Src-FAK signaling may provide potential therapeutic strategies for rescuing colorectal cancer progression.