Metastasis-associated colon cancer 1 drives pancreatic cancer via p53/Notch signaling

Figure 1 Expression of metastasis-associated colon cancer 1 among multiple cancer. A: Y-axis showing the metastasis-associated colon cancer 1 (MACC1) the expression levels of mRNA in The Cancer Genome Atlas (TCGA). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001; B: As depicted from the figure boxplots showing minimum, and maximum, and median, and quartiles, each point showing one sample only more details are similar to Figure 1A, but group samples are paired. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001; C: Depicts the expression level of MACC1 in normal and tumor tissues and these details were collected from two different datasets known as Genotype-Tissue Expression and TCGA. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001; D: In various organs the distribution and expression of MACC1; E: It further depicts the validation of different protein levels externally, and this was performed using a CPTAC database as per requirements; F: Here, the Y-axis indicates the levels of genetic expression: Analysis based on logistic regression for TCGA and TCGA-Genotype-Tissue Expression, where red is 1 or greater number, blue shows values between 0 and 1, a white circle denotes no significance, and no collection of relevant data set was depicted by an empty value); G: Diagnostic value of MACC1 for distinguishing different tissues (tumor and normal); H: Wilcoxon rank-sum test showing the differences in MACC1 expression in microsatellite instability. AUC: Area under the curve; GTEx: Genotype-Tissue Expression; MACC1: Metastasis-associated colon cancer 1; TCGA: The Cancer Genome Atlas.

Figure 2 Altered genetic expression of metastasis-associated colon cancer 1. A: Metastasis-associated colon cancer 1 (MACC1) frequency of mutations in different types of tumors; B: The details of the mutation information; C: Number of cases and sites of genetic alterations of MACC1 in pancreatic cancer collected from cBioPortal; D: Histogram shows the frequency of somatic copy number alteration for MACC1 in each cancer type; E: The inter-connections between the MACC1 mRNA expression level and altered genetic expression/regulation; F: The Spearman’s correlation between somatic copy number alteration and the expression of MACC1; G: Spearman’s correlation of MACC1 between promoter methylation and transcriptional expression and (red represent positive and blue represent negative correlations); H: Heatmap showing hypermethylated (red) and hypomethylated (blue) MACC1 (Wilcoxon rank-sum test); I: Visualization of spearman correlation coefficients by radar map for MACC1 and 10 genomics features. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. MACC: Metastasis-associated colon cancer.

Figure 3 Metastasis-associated colon cancer 1 expression correlates with infiltration of immunological factors. A: The heatmap showed a prominent correlation of expression level of metastasis-associated colon cancer 1 (MACC1) with chemokine, immune-stimulatory, major histocompatibility complex genes, chemokine receptor, and immune-inhibitor; B: The Tracking Tumor Immunophenotype scores differences calculation between low expression MACC1 group and high expression MACC1 group. The correlation between MACC1 and tumor microenvironment feature scores was visualized by radar map, and the overall negative correlation trend was observed; C: Evaluation of the correlation of MACC1 expression with cancer immune infiltration seven software were used; D: Cell sources of MACC1 in pan-cancer at the single-cell level.

Figure 4 The function mediated by metastasis-associated colon cancer 1 and its regulatory mechanism. A: Metastasis-associated colon cancer 1 (MACC1) was negative in a large number of cells, indicating that cell growth was inhibited after MACC1 knockout; B: Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed for differential-expression genes (UP); C: Enrichment differences of MACC1 in 50 HALLMARK and 85 metabolism gene sets; D: MACC1 correlation with 14 tumor-related pathway scores and 14 cancer markers, where main correlation is positive; E: The Cancer Proteome Atlas data suggested that CLAUDIN7 had a higher expression intensity in the group with high expression of MACC1 mRNA.

Figure 5 Clinical relevance of metastasis-associated colon cancer 1. A: Metastasis-associated colon cancer 1 (MACC1) expression correlation with overall survival, disease-specific survival, disease-free interval and progression-free interval on the basis of univariate Cox regression and Kaplan-Meier models (red shows that MACC1 is a risk factor, and green arrow showed MACC1 is a protective factor); B-E: Cox survival analysis results of four survival periods are shown in forest maps; F and G: Log rank test results of kidney renal papillary cell carcinoma and pancreatic adenocarcinoma are shown in Kaplan-Meier curves inner packages.

Figure 6 Analysis of resistance to drug based on the expression of metastasis-associated colon cancer 1. A: Analysis for the sensitivity of drug through different databases; B: Cancer Therapeutics Response Portal; C: Genomics of drug sensitivity in cancer; D: Predicting potential compounds to target metastasis-associated colon cancer 1 (MACC1). Visualized the top 5 selected compounds on the basis of analysis through connectivity map, that could potentially target MACC1; E and F: Those with high expression of MACC1 survived better; G: The expression of MACC1 was higher in the complete or partial response group; H: The proportion of complete remission or partial remission group with elevated expression of MACC1; I: The compounds with significance between high expression of MACC1 and low expression of MACC1 in Cancer Therapeutics Response Portal database.

Figure 7 Upregulated expression of metastasis-associated colon cancer 1 promoted tumor proliferation, migration, invasion and resist cell apoptosis in pancreatic cancer cells. A: Left: The mRNA level of metastasis-associated colon cancer 1 in pancreatic cancer tissues and paracancerous tissue from our local cohort. Right: Correlation expression level between expression of metastasis-associated colon cancer 1 and clinical American Joint Committee on Cancer stages or pathological grading from our local cohort; B: Proliferation of PANC-1 and ASPC after transfection was evaluated using cholecystokinin-8 assay. ^aP < 0.05, ^bP < 0.01; C: Some enriched proliferation-related pathway from gene set enrichment analyses were listed; D: The representative images of the colony formation, transwell migration and invasion in transfected PANC-1 and ASPC cells were presented; E: Apoptosis analysis of transfected pancreatic cancer cells were performed by flow cytometry; F: Some enriched related pathway from gene set enrichment analyses were listed. FDR: False discovery rate; KEGG: Kyoto Encyclopedia of Genes and Genomes; MACC1: Metastasis-associated colon cancer 1; NES: Normalized enrichment score.

Figure 8 Metastasis-associated colon cancer 1 activated Notch and p53 signaling pathway. A and B: Hallmarks of gene set enrichment analyses revealed that the expression level of Metastasis-associated colon cancer 1 significantly correlates with Notch and p53 signaling pathway; C: The luciferase assay showed the effect of Metastasis-associated colon cancer 1 on luciferase activity of Notch1 and p53 promoter in ASPC-1; D: Up: Western blot result about the expression of Notch pathway-related protein, including Notch1, Hey1 and Hes1 in downregulated ASPC-1, and western blot result about the expression of p53 pathway-related protein downregulated in ASPC-1. Down: The gray density about the expression of Notch pathway-related protein, including Notch1, Hey1 and Hes1 in downregulated ASPC-1 (n = 3), and the gray density about the expression of p53 pathway-related protein (n = 3). ^aP < 0.05, ^bP < 0.01. FDR: False discovery rate; KEGG: Kyoto Encyclopedia of Genes and Genomes; MACC1: Metastasis-associated colon cancer 1; NES: Normalized enrichment score; NS: Not significant.