Piezo1 promotes melanoma cell viability
We analysed the copy number of Piezo1 in several tumour cells, and Piezo1was widely expressed in multiple tumour cell lines (Fig. S1A). Bioinformatics analysis was also used to explore the expression level of Piezo1 in melanoma. We found that the Piezo1 mRNA expression level was upregulated in metastatic melanoma and primary melanoma compared with normal skin tissues in TCGA (Fig. S1B). Human Protein Atlas website datasets showed that the upregulation of Piezo1 was correlated with the shorter overall survival of melanoma patients (Fig. S1C). To investigate the role of Piezo1 in melanoma cells, the stable A375 cell line was established successfully with Piezo1 shRNA, Piezo1 knockdown of B16 was performed with (mouse)-Piezo1 siRNA, and the efficiency of knockdown was determined by qRT-PCR and western blot analysis (Fig. 1a, b). Furthermore, we selected two cell lines with significant knockdown effects for subsequent experiments. When control cells were stimulated with Yoda1 (an agonist of Piezo1, 10 µM), intracellular [Ca2+]i recordings showed a transient increase in the free cytosolic Ca2+ concentration. However, in the Piezo1 knockdown group, the intracellular calcium signal weakened significantly, and enhancement of the signal was very slow (Fig. 1c). The results of this experiment suggest that the Piezo1 ion channel protein is functional in melanoma cells.
To determine whether Piezo1 affects melanoma cell proliferation, a CCK-8 assay was applied to continuously monitor changes in cell viability at 0, 24, 48, 72, and 96 h. As shown in Fig. 1d, knockdown of Piezo1 suppressed the viability of melanoma cells. DNA replication activity is usually considered to represent proliferation capacity and was determined by cell staining with Ki67 antibody. There was reduced Ki67 staining after Piezo1 knockdown (Fig. 1e). Taken together, these results indicated that Piezo1 loss may suppress melanoma cell viability.
Knockdown of Piezo1 inhibits the migration, invasion and transendothelial migration of melanoma cells
Previous studies have clarified that intracellular calcium signalling is also associated with reorganization of the cytoskeleton such as F-actin, stress fiber and the formation of lamellipodia, which play a vital role in regulating tumour invasion and metastasis [3, 12]. Our result confirmed that activation of the mechanically sensitive Piezo1 ion channel promotes inward calcium flow. Therefore, the potential role of Piezo1 in the migration and invasion capacity of melanoma cells was explored. The results suggested that efficient knockdown of Piezo1 significantly impaired the wound healing rate and invasiveness ability compared with the control group (Fig. 2a-c). Aaaitionally, a transendothelial migration assay was used to verify the role of Piezo1 in transendothelial migration of tumour cells. The results showed that the migration ability of Piezo1 knockdown cells crossing the HUVACs was significantly reduced (Fig. 2d).
Piezo1 inhibition blocks invasion, metastasis and cell cycle related genes in vitro
The data indicated that Piezo1 inhibition suppressed melanoma cell viability. In addition, expression of the cell cycle-related CDK2 and cyclinD1 genes decreased after Piezo1 knockdown. However, P21 and PTEN, classical tumour suppressors were increased (Fig. 3a). Numerous studies have suggested that epithelial-to-mesenchymal transition (EMT) contributes to early-stage dissemination of cancer cells and is pivotal for invasion and metastasis of melanoma [13, 14]. In addition, matrix metalloproteinases (MMP2, MMP9) play vital roles in tissue remolding and cancer metastasis [15]. The results showed that mesenchymal-related gene (N-cadherin and E-cadherin), invasion-and metastasis-related gene (MMP2 and MMP9) expression in melanoma cells were significantly decreased after Piezo1 loss (Fig. 3b). Taken together, our observations indicated that Piezo1 is critical in malignant tumour events, including invasion and metastasis.
Piezo1 inhibition inactivates the PI3k/AKT pathway
Evidence indicates that, PI3K/AKT signalling is frequently activated in most malignant cancers, and regulates tumour cell proliferation, adhesion, survival, migration and invasion [2, 16, 17]. In addition, activation of PI3K/AKT induces tumour cell migration and invasion through degradation of the MMPs-mediated matrix [18]. We analysed several possible candidates that respond to mechanical biosignalling pathways by the online bioinformatics GEPIA websites and found that Piezo1 is associated with AKT pathway molecules (Fig. 2a). Therefore, we treated melanoma cells with Yoda1 (a Piezo1 activator) at various concentrations (Supplementary Fig. 2) and selected 10 µM as the most effective concentration. The results indicated that phosphorylation of AKT was decreased in melanoma cells after knockdown of Piezo1 (Fig. 3c). Furthermore, we treated melanoma cells with Yoda1 to investigate which pathways were associated with the activation of Piezo1. AKT pathway was activated after the administration of Yoda1 (Fig. 3d-e). The above results indicate that the phosphorylation of AKT requires the activation of Piezo1. Taken together, these data revealed that the level of activation of the mechanical ion channel Piezo1 increased the activation of the PI3K/AKT pathway.
Piezo1 promotes the melanoma cell proliferation, metastasis and invasion via the PI3K/AKT signaling pathway
To further clarify whether the function of Piezo1 in regulating the malignant progression of melanoma was dependent on the PI3K/AKT pathway, we treated wild-type A375 cells with the PI3K inhibitor LY294002, the Piezo1 activator Yoda1 and both. Blocking PI3K not only inhibited migration, invasion and transendothelial migration in control cells but also significantly reduced the migration, invasion and transendothelial migration induced by Piezo1 activation with Yoda1 (Fig. 4a-d). Moreover, as shown in Fig. 4e-f, p-AKT, MMP2, and N- cadherin protein levels were increased by the Piezo1 activator Yoda1, while LY294002 treatment reversed the expression of p-AKT but also MMP2, and N-cadherin. These results collectively indicated that the oncogenic property of Piezo1 was mediated by activation of the PI3K/AKT signalling pathway.
Knockdown of Piezo1 attenuated melanoma metastasis and transendothelial migration in vivo
Based on the results obtained from in vitro experiments, we verified the role of Piezo1 in vascular extravasation of tumour cells in vivo using the lung metastasis mouse model. By injecting B16 cells (siPiezo1and siNC fluorescently labelled) into mice and collecting lung tissue for frozen sectioning after 6 hours, we surprisingly found that the tumor cells that the number of tumour cells that penetrated the blood vessels was 50% lower in the Piezo1-silenced group than in the control group. (Fig. 5a).
The tumour metastasis-promoting role of Piezo1 in melanoma was verified in vivo. We established a lung metastasis mouse model using the tail-vein injection model of B16 cell-lines. Two weeks later, the fluorescence signals of tumour cells in mice were tracked using a bioluminescence imaging system, and the results showed that there were less tumour signals in Piezo1-silenced tumour cells. Since melanoma appears as black spots on lung tissue, the mice were sacrificed, and lung tissue was collected. The results showed that the lungs of the shPiezo1 mice had only a few scattered black spots, while the control mice had evenly spaced spots (Fig. 5b, c). These data suggest that the Piezo1 channel may promote the metastasis of melanoma in vivo. Therefore, Piezo1 plays an important role in the transvascular migration of tumour cells and provides a basis for distant metastasis of the tumour.