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. 2023 Jun;12(12):13586-13598.
doi: 10.1002/cam4.6056. Epub 2023 May 11.

Aberrant expression of NPPB through YAP1 and TAZ activation in mesothelioma with Hippo pathway gene alterations

Tatsuhiro Sato  1 , Ken Akao  1 , Ayuko Sato  2 , Tohru Tsujimura  2 , Satomi Mukai  1 , Yoshitaka Sekido  1   3
Affiliations

Aberrant expression of NPPB through YAP1 and TAZ activation in mesothelioma with Hippo pathway gene alterations

Tatsuhiro Sato et al. Cancer Med. 2023 Jun.

Abstract

Background: Mesothelioma is a neoplastic disease associated with asbestos exposure. It is highly malignant and has a poor prognosis; thus, early detection is desirable. Recent whole-genome analysis has revealed that mesothelioma is characterized by a high frequency of mutations in a set of genes involved in the Hippo pathway, such as NF2 and LATS2. However, a rapid, simple, and precise method for finding mesothelioma with these mutations has not yet been established.

Methods: Clustering of Hippo pathway gene alteration groups and the differential expression of each gene in mesothelioma patients were analyzed using The Cancer Genome Atlas database. Gene expression levels in various tumors and normal tissues were analyzed using public databases. Knockdown or transient expression of YAP1 or TAZ was performed to evaluate the regulation of gene expression by these genes. NT-proBNP was measured in the pleural effusions of 18 patients and was compared with NF2 expression in five cases where cell lines had been successfully established.

Results: NPPB mRNA expression was markedly higher in the group of mesothelioma patients with Hippo pathway gene mutations than in the group without them. NPPB expression was low in all normal tissues except heart, and was highest in mesothelioma. Mesothelioma patients in the high NPPB expression group had a significantly worse prognosis than those in the low NPPB expression group. NPPB expression was suppressed by knockdown of YAP1 or TAZ. NT-proBNP was abundant in the effusions of mesothelioma patients and was particularly high in those with impaired NF2 expression.

Conclusions: NPPB, whose levels can be measured in pleural effusions of mesothelioma patients, has the potential to act as a biomarker to detect NF2-Hippo pathway gene alterations and/or predict patient prognosis. Additionally, it may provide useful molecular insights for a better understanding of mesothelioma pathogenesis and for the development of novel therapies.

Keywords: NPPB; BNP; Hippo pathway; mesothelioma.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

FIGURE 1
FIGURE 1
Characteristics of the Hippo pathway gene alteration group in The Cancer Genome Atlas‐mesothelioma cohort. (A) Oncoprint of genetic alterations in 87 patients with mesothelioma; the group of patients with alterations in either NF2, LATS1, or LATS2 was classified as Altered group, and the other group as Unaltered group. In parentheses, the number of patients. Color differences indicate different types of genetic alterations. (B, C) Box plot showing gene expression levels of ANKRD1 (B), protein phosphorylation of YAP1 at serine 127 and total YAP1 protein expression levels (C) in Altered and Unaltered groups. (D, E) Kaplan–Meier curves of overall survival in Altered and Unaltered groups (D) or in ANKRD1 high‐expressing and low‐expressing groups (E).
FIGURE 2
FIGURE 2
High expression of NPPB in mesothelioma patients with Hippo gene alterations. (A) Volcano plot showing differentially expressed genes between Altered and Unaltered groups. Blue dots, genes with q‐value >0.05. (B, C) Box plot showing gene expression levels of NPPB (B) and NPR3 (C) in the Altered and Unaltered groups. (D) Box plot showing the expression levels of NPPB in 32 cancer types in TCGA database. Dots indicate outliers. (E) Violin plot showing the expression levels of NPPB in each of the human tissue types in the GTEx database. Outliers were eliminated.
FIGURE 3
FIGURE 3
Characteristics of groups divided according to NPPB expression levels in The Cancer Genome Atlas‐mesothelioma (TCGA‐MESO) cohort. (A) Oncoprint showing the genetic alterations of the patients in TCGA‐MESO cohort with high and low NPPB expression. Color differences indicate different types of genetic alterations. (B–D) Dot plots showing the correlation between NPPB and CLDN15 (B), VIM (C), or CLDN15/VIM (D) (C/V) gene expression values in TCGA‐MESO cohort data. Red lines represent regression lines. (E, F) Kaplan–Meier curves of overall survival (E) and progression‐free survival (F) in NPPB High and NPPB Low groups.
FIGURE 4
FIGURE 4
Reduced NPPB expression following YAP1 or TAZ knockdown. (A) NCI‐H2373 cells expressing shRNA targeting YAP1 (shYAP1 #1 and #2) or Scramble shRNA (shScr) as a control were generated using lentivirus and subjected to western blotting. (B, C) The expression ratio of ANKRD1 (B) or NPPB (C) in each cell was measured via qRT‐PCR. (D) The culture medium of NCI‐H2373 cells expressing shYAP1 or shScr was subjected to enzyme‐linked immunosorbent assay (ELISA) to assess the amount of secreted brain natriuretic peptide (BNP). (E–H) Using NCI‐H2373 cells expressing shTAZ (shTAZ #1, #2) or shScr, the same experiments as described in A–D were performed.
FIGURE 5
FIGURE 5
Induction of NPPB expression through YAP1 activation. (A) NCI‐H2373 cells or the cells expressing wild‐type YAP1 (YAP1 WT) or constitutively active YAP1 mutant (YAP1 S127A) fused with FLAG tag were generated using lentivirus and subjected to western blotting. (B, C) The expression ratio of ANKRD1 (B) or NPPB (C) in each cell was measured via qRT‐PCR. (D) The culture medium of NCI‐H2373 cells or the cells expressing YAP1 was subjected to enzyme‐linked immunosorbent assay (ELISA) to assess the amount of secreted brain natriuretic peptide (BNP).
FIGURE 6
FIGURE 6
Exploring the clinical value of NPPB expression levels. (A) NT‐proBNP levels were measured using pleural fluid collected from 18 patients. The NF2 expression ratios established from the pleural fluid in five cell lines are shown in color. (B, C) After purification of mRNA from cells treated with or without K‐975, the expression ratios of ANKRD1 (B) and NPPB (C) were measured via qRT‐PCR. (D, E) Dot plot showing the expression correlations of the indicated genes using The Cancer Genome Atlas‐mesothelioma cohort data. Red lines represent regression lines.
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