Protein-protein interaction analysis reveals a novel cancer stem cell related target TMEM17 in colorectal cancer

Abstract

Background: Cancer stem cells (CSCs) are a small subpopulation of cells within tumors with stem cell property. Increased evidence suggest that CSCs could be responsible for chemoresistance and recurrence in colorectal cancer (CRC). However, a reliable therapeutic target on CSCs is still lacking.

Methods: Here we describe a two-step strategy to generate CSC targets with high selectivity for colon stem cell markers, specific proteins that are interacted with CSC markers were selected and subsequently validated in a survival analysis. TMEM17 protein was found and its biological functions in CRC cells were further examined. Finally, we utilized the Gene Set Enrichment Analysis (GSEA) to investigate the potential mechanisms of TMEM17 in CRC.

Results: By combining protein-protein interaction (PPI) database and high-throughput gene profiles, network analysis revealed a cluster of colon CSCs related genes. In the cluster, TMEM17 was identified as a novel CSCs related gene. The results of in-vitro functional study demonstrated that TMEM17 depletion can suppress the proliferation of CRC cells and sensitize CRC cells to chemotherapy drugs. Enrichment analysis revealed that the expression of TMEM17 is associated with the magnitude of activation of the Wnt/β-catenin pathway. Further validation in clinical samples demonstrated that the TMEM17 expression was much higher in tumor than normal tissue and was associated with poor survival in CRC patients.

Conclusion: Collectively, our finding unveils the critical role of TMEM17 in CRC and TMEM17 could be a potential effective therapeutic target for tumor recurrence and chemoresistance in the colorectal cancer (CRC).

Keywords: Cancer stem cell; Chemoresistance; Colorectal cancer; Protein–protein interaction; TMEM17.

Fig. 1

PPI network and survival analysis identified TMEM17 as a CSC related gene. a PPI network of the nine colon stem cell markers. Node size is -log2 transformed averaged P-values in 1000 randomization log-rank tests. Node color represents the frequency calculated by the number of times that the corresponding gene significantly associated with survival in the same analysis. Nodes with labels represent key genes related to CSC (frequency > 500). Edges represent physical PPIs between proteins obtained from BioGRID database. b The significant frequency of 11 CSC related genes in survival analysis (log-rank test, P < 0.05; frequency > 500). c TMEM17 expression is significantly higher in CRC samples than that in paired normal colon tissue (P = 0.002). d, e Kaplan–Meier survival analysis revealed that high TMEM17 expression was significantly correlated with tumor recurrence (d) and short overall survival (e)

Fig. 2

Depletion of TMEM17 suppressed CRC cells proliferation. a, b The transfection efficiency after depleting TMEM17 by siRNA in CRC cells were tested by RT-PCR (a) and immunoblotting (b). c Representative images of 48 h after CRC cells treated with si-TMEM17 in adherent culture. The bar = 200um. d Growth curves of CRC cells with depleting TMEM17 in a period of 5 days culture. *p < 0.05, **p < 0.01, ***p < 0.001, student’s T test (a), one-way ANOVA (d), as compared to the control group

Fig. 3

Targeting TMEM17 enhanced the sensitivity of chemotherapy drugs in CRC. a Growth curves of CRC cells with depleting TMEM17 and 5-Fu treatment in a period of 5 days culture. b Growth curves of CRC cells with depleting TMEM17 and oxaliplatin treatment in a period of 5 days culture. c Clonogenic assay of CRC cells with depleting TMEM17 and 5-Fu/oxaliplatin treatment in a period of 8 to 10 days culture. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA (a)

Fig. 4

Targeting TMEM17 suppressed CSC characteristic in CRC cells. a Genic depleting TMEM17 reduced cancer cell sphere formation, n = 3. The bar = 400um. b Tumorsphere formation assay of CRC cells with depleting TMEM17 and 5-Fu treatment, n = 3. c Tumorsphere formation assay of CRC cells with depleting TMEM17 and Oxaliplation treatment, n = 3. d Immunoblotting assay of the expression of TMEM17, EPCAM and LGR5 proteins from CRC cells with scramble or TMEM siRNA. (Left) Immunoblotting assay of the expression of TMEM17, EPCAM and LGR5 proteins from CRC cells with vector or TMEM17. (Right) e Immunoblotting assay of the expression of TMEM17, EPCAM and LGR5 proteins from CRC cells with adherent culture or tumorsphere culture. f Clonogenic assay of DLD1 and DLD1 oxaliplatin resistance cells. g Immunoblotting assay of DLD1 and DLD1 oxaliplatin resistance cells. Error bars represent ± SD. **P < 0.01, *P < 0.05, paired sample T test (a–c)

Fig. 5

Pathway enrichment analysis between differential expression groups of TMEM17. a Significantly dysregulated pathways were identified by GSEA in the CIT cohort for cancer hallmark pathways. Top ten pathways were selected for presentation by absolute enrichment score (positive score is green and negative score is red). b GSEA plot of the Hallmarks Wnt/ β-catenin signaling in the CIT cohort. c GSEA plot of Willert Wnt signalling in the CIT cohort. d Immunoblotting assay of the expression of TMEM17 and several Wnt signaling markers proteins from CRC cells with scramble or TMEM siRNA. (Left) Immunoblotting assay of the expression of TMEM17 and several Wnt signaling markers proteins from CRC cells with vector or TMEM17. (Right) e GSEA plot of Boquest Stem Cell signalling and Beier Glioma Stem Cell signalling in the CIT cohort

Fig. 6

TMEM17 is upregulated in CRC and is related to poor CRC survival. a Representative TMEM17 immunochemistry staining in CRC and normal colon tissue. The scale bar represents 100 μm. b Tissue microarray assay of TMEM17 expression in CRC and normal colon tissue. Error bars represent ± SD. ***P < 0.001, 2-tailed unpaired Student’s t test. c High TMEM17 expression was associated with poor overall survival in CRC patients