Dataset Information

Increased proton-sensing receptor GPR4 signalling promotes colorectal cancer progression by activating the hippo pathway.

ABSTRACT:

Background

Colorectal cancer (CRC) is one of the high incidences tumours and is ranked second in cancer-related mortality. Even though great progress has been made, there are no effective therapeutic strategies for late stage and metastatic CRC patients. Acidity is one characteristic of the tumour microenvironment. However, how cancer cells respond to this acidic environment surrounding them remains largely unknown, especially in colorectal cancer.

Methods

Proton sensor receptor expression was analysed in GEO and TCGA datasets. The expression of GPR4 in CRC specimens was confirmed by western blotting and immunohistochemistry (IHC). The role of GPR4 in CRC progression was analysed both in vitro and in vivo. Pharmacological intervention, immunofluorescence and gene set enrichment analyses were performed to reveal the underlying molecular mechanisms of GPR4.

Findings

We found that GPR4 was upregulated in CRC samples. In addition, its high expression correlated with late stage tumours and poor overall survival in patients. Furthermore, loss-of-function assays proved that GPR4 promoted CRC carcinogenesis and metastatic ability. Mechanistically, GPR4 was activated by extracellular protons in the tumour microenvironment and enhanced RhoA activation and F-actin rearrangement, leading to LATS activity inhibition, YAP1 nuclear translocation and oncogene transcription.

Interpretation

The expression of GPR4 is upregulated in colorectal cancer and is associated with shorter overall survival time in CRC patients. These findings reveal the novel roles of GPR4 in CRC progression and suggest GPR4 might be a new therapeutic target for CRC treatment.

SUBMITTER: Yu M

PROVIDER: S-EPMC6838423 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Publications

Increased proton-sensing receptor GPR4 signalling promotes colorectal cancer progression by activating the hippo pathway.

Yu Minhao M Cui Ran R Huang Yizhou Y Luo Yang Y Qin Shaolan S Zhong Ming M

EBioMedicine 20190914

<h4>Background</h4>Colorectal cancer (CRC) is one of the high incidences tumours and is ranked second in cancer-related mortality. Even though great progress has been made, there are no effective therapeutic strategies for late stage and metastatic CRC patients. Acidity is one characteristic of the tumour microenvironment. However, how cancer cells respond to this acidic environment surrounding them remains largely unknown, especially in colorectal cancer.<h4>Methods</h4>Proton sensor receptor e ...[more]

PMID: 31530502

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Project description:BackgroundColorectal carcinoma (CRC) is the third most common cancer and second most common cause of cancer-related deaths worldwide. Ribonucleic acid (RNA) N6-methyladnosine (m6A) and methyltransferase-like 3 (METTL3) play key roles in cancer progression. However, the roles of m6A and METTL3 in CRC progression require further clarification.MethodsAdenoma and CRC samples were examined to detect m6A and METTL3 levels, and tissue microarrays were performed to evaluate the association of m6A and METTL3 levels with the survival of patients with CRC. The biological functions of METTL3 were investigated through cell counting kit-8, wound healing, and transwell assays. M6A epitranscriptomic microarray, methylated RNA immunoprecipitation-qPCR, RNA stability, luciferase reporter, and RNA immunoprecipitation assays were performed to explore the mechanism of METTL3 in CRC progression.ResultsM6A and METTL3 levels were substantially elevated in CRC tissues, and patients with CRC with a high m6A or METTL3 levels exhibited shorter overall survival. METTL3 knockdown substantially inhibited the proliferation, migration, and invasion of CRC cells. An m6A epitranscriptomic microarray revealed that the cell polarity regulator Crumbs3 (CRB3) was the downstream target of METTL3. METTL3 knockdown substantially reduced the m6A level of CRB3, and inhibited the degradation of CRB3 mRNA to increase CRB3 expression. Luciferase reporter assays also showed that the transcriptional level of wild-type CRB3 significantly increased after METTL3 knockdown but not its level of variation. Knockdown of YT521-B homology domain-containing family protein 2 (YTHDF2) substantially increased CRB3 expression. RNA immunoprecipitation assays also verified the direct interaction between the YTHDF2 and CRB3 mRNA, and this direct interaction was impaired after METTL3 inhibition. In addition, CRB3 knockdown significantly promoted the proliferation, migration, and invasion of CRC cells. Mechanistically, METTL3 knockdown activated the Hippo pathway and reduced nuclear localization of Yes1-associated transcriptional regulator, and the effects were reversed by CRB3 knockdown.ConclusionsM6A and METTL3 levels were substantially elevated in CRC tissues relative to normal tissues. Patients with CRC with high m6A or METTL3 levels exhibited shorter overall survival, and METTL3 promoted CRC progression. Mechanistically, METTL3 regulated the progression of CRC by regulating the m6A-CRB3-Hippo pathway.

Project description:BackgroundFocal adhesion plays an essential role in tumour invasiveness and metastasis. Hippo component YAP has been widely reported to be involved in many aspects of tumour biology. However, its role in focal adhesion regulation in breast cancer remains unexplored.MethodsTissue microarray was used to evaluate YAP expression in clinical breast cancer specimens by immunohistochemical staining. Cell migration and invasion abilities were measured by Transwell assay. A cell adhesion assay was used to measure the ability of cell adhesion to gelatin. The focal adhesion was visualized through immunofluorescence. Phosphorylated FAK and other proteins were detected by Western blot analysis. Gene expression profiling was used to screen differently expressed genes, and gene ontology enrichment was performed using DAVID software. The gene mRNA levels were measured by quantitative real-time PCR. The activity of the THBS1-promoter was evaluated by dual luciferase assay. Chromatin immunoprecipitation (ChIP) was used to verify whether YAP could bind to the THBS1-promoter region. The prediction of potential protein-interaction was performed with the String program. The ChIP sequence data of TEAD was obtained from the ENCODE database and analysed via the ChIP-seek tool. The gene expression dataset (GSE30480) of purified tumour cells from primary breast tumour tissues and metastatic lymph nodes was used in the gene set enrichment analysis. Prognostic analysis of the TCGA dataset was performed by the SurvExpress program. Gene expression correlation of the TCGA dataset was analysed via R2: Genomics Analysis and Visualization Platform.ResultsOur study provides evidence that YAP acts as a promoter of focal adhesion and tumour invasiveness via regulating FAK phosphorylation in breast cancer. Further experiments reveal that YAP could induce FAK phosphorylation through a TEAD-dependent manner. Using gene expression profiling and bioinformatics analysis, we identify the FAK upstream gene, thrombospondin 1, as a direct transcriptional target of YAP-TEAD. Silencing THBS1 could reverse the YAP-induced FAK activation and focal adhesion.ConclusionOur results unveil a new signal axis, YAP/THBS1/FAK, in the modulation of cell adhesion and invasiveness, and provides new insights into the crosstalk between Hippo signalling and focal adhesion.

Dataset Information

Increased proton-sensing receptor GPR4 signalling promotes colorectal cancer progression by activating the hippo pathway.

Background

Methods

Findings

Interpretation

Publications

Increased proton-sensing receptor GPR4 signalling promotes colorectal cancer progression by activating the hippo pathway.

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