Evidence of Cooperation between Hippo Pathway and RAS Mutation in Thyroid Carcinomas

Abstract

Thyroid cancer incidences have been steadily increasing worldwide and are projected to become the fourth leading cancer diagnosis by 2030. Improved diagnosis and prognosis predictions for this type of cancer depend on understanding its genetic bases and disease biology. RAS mutations have been found in a wide range of thyroid tumors, from benign to aggressive thyroid carcinomas. Based on that and in vivo studies, it has been suggested that RAS cooperates with other driver mutations to induce tumorigenesis. This study aims to identify genetic alterations or pathways that cooperate with the RAS mutation in the pathogenesis of thyroid cancer. From a cohort of 120 thyroid carcinomas, 11 RAS-mutated samples were identified. The samples were subjected to RNA-Sequencing analyses. The mutation analysis in our eleven RAS-positive cases uncovered that four genes that belong to the Hippo pathway were mutated. The gene expression analysis revealed that this pathway was dysregulated in the RAS-positive samples. We additionally explored the mutational status and expression profiling of 60 RAS-positive papillary thyroid carcinomas (PTC) from The Cancer Genome Atlas (TCGA) cohort. Altogether, the mutational landscape and pathway enrichment analysis (gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genome (KEGG)) detected the Hippo pathway as dysregulated in RAS-positive thyroid carcinomas. Finally, we suggest a crosstalk between the Hippo and other signaling pathways, such as Wnt and BMP.

Keywords: Hippo pathway; RAS mutation; RNA-Seq; TCGA; thyroid cancer.

Figure 1

Workflow of the sample selection, screening and RNA-Sequencing pipeline analysis for the discovery cohort.

Figure 2

Mutational analysis and pathway enrichment in the RAS-positive cohort. (A) Forest plot shows representative genes that were recurrently mutated in the 11 RAS-positive groups or in the “others” group (samples negative for the driver mutation). The black points indicate genes exclusively mutated in the RAS cohort. The blue line shows genes mutated in both groups and the red line the ones mutated exclusively in the “others” group. Recurrent mutations were considered those mutations that occurred in at least two samples. *** p-value = 0.001, * p-value ≤ 0.05, NS = non-significant p-value (B) A KEGG pathway enrichment of the 126 exclusively mutated in the RAS cohort.

Figure 3

Amino acid change and localization relative to the respective protein domains in the four proteins (PAK1, RASSF4, HIPK2 and DVL1) whose corresponding genes belong to the Hippo pathway and are mutated in a RAS-positive cohort. Brackets from 1 to 5 indicate the number of samples positive for each mutation. In our RAS-positive samples, the alteration of each gene was present in two samples.

Figure 4

Mutational analysis and pathway enrichment (KEGG) in RAS-positive papillary thyroid carcinomas (PTC) from The Cancer Genome Atlas (TCGA) cohort. (A) A forest plot showing the eleven genes exclusively and recurrently mutated (present in at least two samples) in 60 RAS-positive samples. *** p-value ≤ 0.001, ** p-value ≤ 0.01, * p-value < 0.05, NS = non-significant p-value (B) KEGG pathway enrichment of the 11 exclusively mutated genes in rhe RAS-positive TCGA cohort.

Figure 5

Differential gene expression analysis in the RAS-positive group from the discovery cohort (11 samples) detected 1765 genes significantly up- and downregulated. The top 10% ranked genes were differentially expressed, which shows that the ones with the lowest adjusted p-values were submitted to GSEA and KEGG enrichment to gain insight into altered biological processes and pathways. (A) GSEA enrichment analysis of biological processes that contain upregulated genes are marked on the left panel and downregulated on the right panel. (B) Pathway enrichment analysis (KEGG) results showed pathways in which most of their genes are upregulated (left) or down-regulated (right) in the RAS-positive cohort.

Figure 6

Differential gene expression analysis in RAS-positive PTC samples from the TCGA cohort (60 samples). Enrichment analysis (GSEA and KEGG) was performed on the top 10% genes differentially expressed composing the ones with lowest adjusted p-values to gain insights into altered biological process and pathways. (A) GSEA enrichment analysis of the biological processes that contain upregulated genes are marked on the left panel and downregulated on the right panel. (B) Pathway enrichment analysis result showed pathways in which most of their genes are upregulated (left) or downregulated (right) in RAS-positive TCGA cohorts. (C) Common pathways associated with cancer progression differentially expressed in both RAS-positive discovery cohort and RAS-positive PTC from the TCGA cohort.

Figure 7

Hippo signaling pathway KEGG-based map, showing genes up- (red) and downregulated (green) detected by differential expression (adjusted p-value ≤ 0.05) performed in both the RAS-positive discovery cohort and RAS-positive TCGA cohort.

Figure 8

Hippo pathway simplified map, summarizing the findings of the mutation and differential gene expression analysis in our RAS-positive cohort supported by the RAS-positive TCGA data analysis. Inactivation of this pathway promotes YAP/TAZ translocation into the nucleus and targets gene expression, which leads to cellular survival and growth. Once Hippo is inactive and YAP/TAZ acting, a crosstalk may occur with the BMP and Wnt pathways.