Loss of stretch-activated channels, PIEZOs, accelerates non-small cell lung cancer progression and cell migration

Abstract

PIEZO channels are stretch-activated channels involved in wound sealing and cell proliferation in many cell types. A recent study focussing on lung cancer (LC), using next-generation sequencing analysis, has indicated that PIEZO functions were implicated in LC development. However, the expression and role of PIEZO channels in non-small cell LC (NSCLC) progression require elucidation. In the current study, we investigated the gene expression and alteration frequency in human NSCLC tissue, accessed the prognostic roles of PIEZO channels in NSCLC patients, and further studied the effect of PIEZOs in NSCLC cell proliferation and tumor growth in vivo The mRNA expression of PIEZO1 and 2 was clearly decreased in NSCLC tumor tissue compared with that in matched adjacent non-tumor tissue. In human NSCLC tissues, PIEZO1 gene expression exhibits a highly deep deletion rate, and PIEZO2 mainly exhibits mutation in gene expression. High mRNA expression of PIEZO channels was found to correlate with better overall survival (OS) for NSCLC patients, especially for patients with lung adenocarcinoma (LUAD), but not for patients with lung squamous cell carcinoma (LUSC). The prognostic role of PIEZO channels was more sensitive in female patients than male patients, and more sensitive in patients at earlier stages than patients at latter stages. Knockdown of PIEZO1 or PIEZO2 in NSCLC cells significantly promoted cell migration in vitro and tumor growth in vivo These results indicate the critical prognostic values of the PIEZO channels in NSCLC. This information will be beneficial to understand the pathological mechanism of NSCLC and to generate effective therapeutic approaches for NSCLC patients.

Keywords: NSCLC; PIEZO; Prognostic roles; cell migration.

Figure 1. Expression of PIEZO1 and 2 in normal and tumor tissues from human NSCLC patients

mRNA and protein expression of PIEZO1 and 2 in lung tumors was compared with the expression in adjacent normal tissues. (A,B) Expression levels of PIEZO1 in human lc tissues compared with normal tissues in GSE10072 (A) and GSE19804 (B). (C,D) Expression levels of PIEZO2 in human LC tissues compared with normal tissues in GSE10072 (C) and GSE19804 (D). (E,F) mRNA expression of PIEZO1 (E) and PIEZO2 (F) in cancer tissue and adjacent normal tissues from human NSCLC patients were collected from Jilin Hospital. The mRNA expression was analyzed by RT-qPCR, and normalized to the expression of GAPDH. (G,H) Protein expression of PIEZO1 (G) and PIEZO2 (H) in cancer tissue and adjacent normal tissues. Upper panel indicates the representative images of Western blot from cancer tissue (C1–C3) and adjacent normal tissues (N1–N3), and the lower panel shows the quantitation of protein expression (n=5). The normalized expression of PIEZO1 and PIEZO2 is displayed as mean ± S.D. *P<0.05.

Figure 2. Alteration frequency of PIEZO1 and 2 in NSCLC

The alteration of PIEZO1 and 2 were visualized using the cBioPortal for Cancer Genomics database. Mutation, deletion, and amplification are shown in different colors. (A) Alteration frequency of PIEZO1 in NSCLC. (B) Alteration frequency of PIEZO2 in NSCLC.

Figure 3. Prognostic value of the expression of PIEZOs in NSCLC

(A,B) Prognostic value of PIEZO1 expression in NSCLC patients (A, n=1432) and LUAD patients (B, n=488). (C,D) Prognostic value of PIEZO2 expression in NSCLC patients (C, n=671) and LUAD patients (D, n=461).

Figure 4. Prognostic value of the mRNA expression of PIEZOs in female and male patients

(A,B) Prognostic value of PIEZO1 mRNA expression in female patients (A, n=575) and male patients (B, n=746). (C,D) Prognostic value of PIEZO2 mRNA expression in female patients (C, n=251) and male patients (D, n=309).

Figure 5. Knockdown of PIEZO gene expression promotes cell migration in A549 cells

A549 cells were transfected with Veh shRNA and shRNA target PIEZO1 or PIEZO2, and the stable cell line was selected and maintained in culture medium with puromycin as described in the ‘Materials and methods’ section. The mRNA expression of PIEZO1 and 2 in these cells was analyzed using RT-qPCR, and the protein expression of GAPDH, PIEZO1, and 2 in these cells was analyzed using Western blot. Cell migration in A549 cells with and without knockdown of PIEZO genes was analyzed by scratch assay as described in the ‘Materials and methods’ section. (A,E) mRNA expression of PIEZO1 (A) and PIEZO2 (E) in A549 cells. (B,F) Protein expression PIEZO1 (B) and PIEZO2 (F) in A549 cells; upper panel indicates the representative images of Western blot, and the lower panel shows the quantitation of protein expression. (C,D) Cell migration in A549 cells with stable knockdown of PIEZO1. Representative images (C) and quantitation of migration (D) in A549 cells with stable knockdown of PIEZO1. (G,H) Cell migration in A549 cells with stable knockdown of PIEZO2. Representative images (G) and quantitation of migration (H) in A549 cells with stable knockdown of PIEZO2. Data are displayed as mean ± S.D. of gene expression in different cells. *, P<0.05; **, P<0.01, cells with stable knockdown of PIEZO1 or 2 compared with cells with stable transfection of Veh shRNA.;

Figure 6. Knockdown of the expression of PIEZOs promotes tumor growth in vivo

Nude mice were subcutaneously inoculated with A549 cells stably transfected with sh-PIEZO1 (n=8), sh-PIEZO2 (n=8), or Veh shRNA (n=8). The subcutaneous tumor size was recorded every week. Data are presented as mean ± S.D. of the measurement of each group. The subcutaneous tumors were isolated and measured. After inoculation, the tumor volumes were measured every week. *P<0.05. (A) Tumor growth in nude mice inoculated with A549 cells with stable knockdown of PIEZO1. Right panel shows the xenografts from mice with subcutaneous inoculation of A549 cells with or without knockdown of PIEZO1. sh-PIEZO1 (n=5) and Veh shRNA (n=3). (B) qPCR quantitation of mRNA expression of human PIEZO1 from the xenografts with or without stable knockdown of PIEZO1. (C) Tumor growth in nude mice inoculated with A549 cells with stable knockdown of PIEZO2. Right panel shows the xenografts from mice with subcutaneous inoculation of A549 cells with or without knockdown of PIEZO2. sh-PIEZO1 (n=3) and Veh shRNA (n=3). (D) qPCR quantitation of mRNA expression of human PIEZO2 from the xenografts with or with stable knockdown of PIEZO2. *, P<0.05, cells with stable knockdown of PIEZO1 or 2 compared with cells with stable transfection of Veh shRNA.