Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

Abstract When circulating tumor cells (CTCs) travel in circulation, they can be killed by detachment‐induced anoikis and fluidic shear stress (SS)‐mediated apoptosis. Circulatory treatment, which can make CTCs detached but also generate SS, can increase metastasis of cancer cells. To identify SS‐specific mechanosensors without detachment impacts, a microfluidic circulatory system is used to generate arteriosus SS and compare transcriptome profiles of circulating lung cancer cells with suspended cells. Half of the cancer cells can survive SS damage and show higher invasion ability. Mesotrypsin (PRSS3), protease‐activated receptor 2 (PAR2), and the subunit of activating protein 1, Fos‐related antigen 1 (FOSL1), are upregulated by SS, and their high expression is responsible for promoting invasion and metastasis. SS triggers PRSS3 to cleave the N‐terminal inhibitory domain of PAR2 within 2 h. As a G protein‐coupled receptor, PAR2 further activates the Gα i protein to turn on the Src‐ERK/p38/JNK‐FRA1/cJUN axis to promote the expression of epithelial–mesenchymal transition markers, and also PRSS3, which facilitates metastasis. Enriched PRSS3, PAR2, and FOSL1 in human tumor samples and their correlations with worse outcomes reveal their clinical significance. PAR2 may serve as an SS‐specific mechanosensor cleavable by PRSS3 in circulation, which provides new insights for targeting metastasis‐initiating CTCs.

added to a microfluidic circulatory system to undergo SS.    showing the levels of PRSS3 in A549 cells at 0 h and after 2 h of suspension, SS2 and SS15 treatment.(D) Representative IF staining images showing the subcellular localization of FRA1, p-FRA1, cJUN and p-cJUN before and after 10 h of suspension and SS treatment.Scale bar, 5 µm.(E) qPCR results showing the relative mRNA fold changes of PRSS3, PAR2 and FOSL1 in SS versus suspension after 0, 3, 5, 8, 10 and 12 h of treatment.The quantification results are the means ± SD from three independent experiments.The statistical analysis was performed compared with the value of 0 h and significant differences were determined by two-way ANOVA (E).**P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure S7. PRSS3 and AP-1 inhibitors significantly suppressed the resistance to SS, cell invasion and colony formation abilities of A-SSP6 cells. (A) Representative phase images
and quantified percentage of viable A-SSP6 cells before and after 10 h of circulation.Cells were pretreated with 10 µM PRSS3 inhibitor diminazene or AP-1 inhibitors SR11302 (10 µM) and T5224 (40 µM) for 24 h and then subjected to SS treatment and cocirculated with these inhibitors.Cells were treated with 0.1% DMSO in the control group.Scale bar, 100 µm.(B) Representative images and quantified relative colony area of the colonies formed by A-SSP6 cells under treatment with PRSS3 and AP-1 inhibitors.One thousand cells were seeded and allowed to grow for 7 days.The colony area in the control group was considered 1.0, and the relative colony area of each experimental group was calculated.Scale bar, 2 mm.(C) Representative images and quantification results of the invaded A-SSP6 cells pretreated with PRSS3 inhibitor and AP-1 inhibitors for 24 h.Twenty thousand cells were seeded in each insert and invaded for 18 h.Indicated inhibitors were also added into the inserts.Scale bar, 100 µm.The quantification results are the means ± SD from three independent experiments.Significant differences were determined by one-way ANOVA.***P < 0.001 and ****P < 0.0001.

Figure S2 .
Figure S2.SS induced cell death and enhanced migration, invasion and colony formation in other cancer cell lines.(A) Quantified cell viability of H1975, MDA-MB-231, MDA-MB-468 and HCT116 cells at 0 h and after 10 h of suspension and SS treatment.(B to D) Quantification results of the colony formation, Transwell migration and invasion abilities of H1975, MDA-MB-231, MDA-MB-468 and HCT116 cells under the indicated conditions.For the colony formation assay, 2 × 10 3 H1975 cells, 1 × 10 3 MDA-MB-231 cells, 5 × 10 3 MDA-MB-468 cells and 1 × 10 3 HCT116 cells were seeded in each well of 6-well plates and allowed to grow for 10 days.Ten thousand H1975 and HCT116 cells, and five thousand MDA-MB-231 and MDA-MB-468 cells were seeded in the Transwell migration assay.For the invasion assay, 2 × 10 4 H1975 and HCT116 cells, and 1 × 10 4 MDA-MB-231 and MDA-MB-468 cells were seeded.Cells were then allowed to migrate or invade for 18 h.The quantification results are

Figure S3 .
Figure S3.Volcano plots showing the differentially expressed genes in SUS vs. 0 h, SS vs. 0 h and SS vs. SUS.The thresholds were set as P < 0.05, a fold change ≥ 2 for upregulated genes and a fold change ≤ 0.5 for downregulated genes.

Figure S4 .
Figure S4.The levels of PRSS3, PAR2, FOSL1 and JUN were increased by SS in another NSCLC cell line H1975 and in more metastatic breast cancer cells.(A) Western blots showing the upregulation in protein levels of PRSS3, PAR2, FRA1, p-FRA1, cJUN and p-cJUN after SS compared with both 0 h and suspension conditions in H1975 cells.(B) Relative mRNA levels of PRSS3, PAR2, FOSL1 and JUN in 231-C3 cells compared with MCF7-C3 cells based on RNA-seq analysis.The quantification results are the means from three independent experiments.

Figure S5 .
Figure S5.Increases of PRSS3, PAR2, FOSL1 and JUN induced by SS were well validated.(A) Representative IF staining images of PAR2 in A549 cells under the indicated conditions.Scale bar, 5 µm.(B) Schematic showing that the fluorescent mCherry sequence was inserted into the N-terminus of PAR2 before the PRSS3 cleavage site.(C) Western blots

Figure S6 .
Figure S6.Knockdown of PRSS3, PAR2 and FOSL1 reduced the SS survival rate and colony formation of A-SSP6 cells.(A) Knockdown efficiencies of PRSS3, PAR2 and FOSL1 in A-SSP6 cells validated by qPCR (upper) and Western blotting (lower).(B) Representative phase images and quantified percentage of viable A-SSP6 cells transfected with shRNAs

Figure S8 .
Figure S8.Overexpression of PRSS3, PAR2 and FOSL1 in A549-C3 cells increased the SS survival rate and colony formation ability.(A) Efficiencies of overexpressing PRSS3, PAR2, FOSL1 and dual overexpressing FOSL1 and JUN as validated by qPCR (upper) and Western blotting (lower).(B and C) Representative phase images and quantified percentage of

Figure S10 .
Figure S10.The effects of knocking down FOSL1 on downstream molecules.(A) Western blots showing the change in FRA1 after treating A-SSP6 cells with the PI3K inhibitor dactolisib for 24 h.(B) Relative mRNA levels of EMT-promoting genes in A-SSP6-shFOSL1 cells compared with A-SSP6-shCtrl cells.The mRNA level of each gene in A-SSP6-shCtrl cells was normalized to 1.0.The quantification results are the means ± SD from three independent experiments.Significant differences were determined by Student's t test (B).*P < 0.05, ***P < 0.001 and ****P < 0.0001.ns, not significant.