PRSS3/Mesotrypsin and kallikrein-related peptidase 5 are associated with poor prognosis and contribute to tumor cell invasion and growth in lung adenocarcinoma

Abstract

Serine proteases have been implicated as key drivers and facilitators of lung cancer malignancy, and while these proteins represent straightforward targets for therapeutic inhibitors, identification of optimal points for intervention has been complicated by the complex networks in which these enzymes function. Here we implicate a signaling pathway consisting of PRSS3/mesotrypsin and kallikrein-related peptidase 5 (KLK5) in lung adenocarcinoma malignancy. We show that elevated PRSS3/mesotrypsin expression is prognostic for poor outcome for patients with lung adenocarcinoma, and that genetic or pharmacologic targeting of PRSS3/mesotrypsin reduces lung adenocarcinoma cell invasiveness and proliferation. We further show that genetic targeting of KLK5, a known target of PRSS3/mesotrypsin, phenocopies the effect of PRSS3/mesotrypsin knockdown, and also that elevated expression of KLK5 is similarly prognostic for outcome in lung adenocarcinoma. Finally, we use transcriptional profiling experiments to show that PRSS3/mesotrypsin and KLK5 control a common malignancy-promoting pathway. These experiments implicate a potential PRSS3/mesotrypsin-KLK5 signaling module in lung adenocarcinoma and reveal the potential therapeutic benefit of selectively targeting these pathways.

Figure 1

PRSS3 is prognostic of poor survival and cancer progression in lung adenocarcinoma (LAC) but not squamous cell carcinoma (SCC). Univariate Kaplan-Meier survival analyses of NSCLC patients are plotted, stratified by PRSS3 mRNA expression. (a,b) High PRSS3 expression was significantly associated with (a) poor OS and (b) poor PFS in analyses including all NSCLC patients. (c,d) Analyses restricted to patients with LAC also showed highly significant association of PRSS3 expression with (c) poor OS and (d) poor PFS, and revealed greater hazard ratios. (e,f) Analyses of the subset of patients with SCC did not show significant association of PRSS3 expression with clinical outcomes. Analyses were conducted using the KM Plotter online tool (http://kmplot.com/analysis/) and included data pooled from 13 cohorts; additional details are described in the Materials and Methods.

Figure 2

PRSS3 silencing inhibits invasion and growth of PC9 lung adenocarcinoma cells. (a) Baseline expression of PRSS3, determined by qRT/PCR normalized to GAPDH, is plotted for a panel of lung cancer cell lines. Expression levels are normalized to the average for LAC-derived cell lines. LAC, lung adenocarcinoma; SSC, squamous cell carcinoma; LCLC, large cell lung carcinoma. (b) PRSS3 expression is effectively suppressed by two targeted lentiviral shRNA constructs, PRSS3-KD1 and PRSS3-KD2, relative to cells transduced with the non-target control virus (NT), as assessed by qRT/PCR normalized to GAPDH expression. (c) Knockdown of PRSS3 significantly inhibits PC9 cellular invasion in Matrigel transwell assays. Representative images of the invasion assay are shown above each condition. (d) Knockdown of PRSS3 significantly inhibits PC9 cell growth as assessed by MTT assays. Error bars show SEM. ^**P ≤ 0.01; ^***P ≤ 0.001.

Figure 3

Mesotrypsin inhibition suppresses invasion and growth of PC9 lung adenocarcinoma cells. (a) Treatment of PC9 cells with mesotrypsin inhibitor diminazene over a range of concentrations (shown below the x-axis of the graph) significantly inhibited PC9 cellular invasion in Matrigel transwell assays. Cells in which PRSS3 expression was silenced by shRNA construct PRSS3-KD1 were evaluated for comparison. Representative images of the invasion assay are shown above each condition. (b) Treatment of PC9 cells with mesotrypsin inhibitor diminazene over a range of concentrations (shown below the x-axis of the plot) significantly inhibited PC9 cell growth as assessed by MTT assays. Cells in which PRSS3 expression was silenced by shRNA construct PRSS3-KD1 were evaluated for comparison. Error bars show SEM. ^*P ≤ 0.05; ^**P ≤ 0.01; ^***P ≤ 0.001.

Figure 4

KLK5 silencing inhibits invasion of PC9 lung adenocarcinoma cells. (a) KLK5 expression is effectively suppressed by two targeted lentiviral shRNA constructs, KLK5-KD1 and KLK5-KD2, relative to cells transduced with the non-target control virus (NT), as assessed by qRT/PCR normalized to GAPDH expression. (b) KLK7 expression is effectively suppressed by two targeted lentiviral shRNA constructs, KLK7-KD1 and KLK7-KD2, relative to cells transduced with the non-target control virus (NT), as assessed by qRT/PCR normalized to GAPDH expression. (c) Knockdown of KLK5 significantly inhibits PC9 cellular invasion in Matrigel transwell assays. (d) By contrast, knockdown of KLK7 has no significant impact on PC9 cellular invasion in Matrigel transwell assays. Representative images of the invasion assays are shown above each condition. Error bars show SEM. **P ≤ 0.01; ***P ≤ 0.001.

Figure 5

KLK5 is prognostic of poor survival and cancer progression in lung adenocarcinoma (LAC). Univariate Kaplan-Meier survival analyses of LAC patients are plotted, stratified by KLK5 mRNA expression, showing association of high KLK5 expression with (a) poor OS and (b) poor PFS. Analyses were conducted using the KM Plotter online tool (http://kmplot.com/analysis/) and included data pooled from 10 cohorts; additional details are described in the Materials and Methods.

Figure 6

PRSS3 and KLK5 drive expression of highly overlapping gene sets in lung adenocarcinoma cells. (a) Heat map of 2416 genes differentially expressed between NT (control), PRSS3 KD, and KLK5 KD (P < 0.05, FC > 1.5). (b) Overlap of significantly differentially expressed genes (P < 0.05, FC > 1.5; N = 184) between PRSS3 KD vs NT (N = 1345) and KLK5 KD vs NT (N = 1255). (c) Knockdown of PRSS3 or KLK5 significantly decreases expression of WNT6. (d) Knockdown of PRSS3 or KLK5 significantly decreases expression of SNAI3. (e) Knockdown of PRSS3 or KLK5 significantly increases expression of CD83.