Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion

Abstract

In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2^KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.

Keywords: CAF; PKN2; Rho GTPases; cancer-associated fibroblasts; matrisome; pancreatic cancer; protein kinase N2; tumour microenvironment.

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Figure 1

PKN2 loss reduces PSC growth and myofibroblast differentiation (A) Growth of immortalized WT and PKN2^KO PSCs relative to WT PSCs on day 4 as assessed by MTT assay (n = 3; unpaired t test). (B) Density of WT and PKN2^KO PSCs grown to confluency (8 days post-seeding) relative to maximum density of WT cells (n = 3; unpaired t test). (C) Western blot of cyclin D1, proliferating cell nuclear antigen (PCNA), and housekeeping HSC70 in WT and PKN2^KO PSCs (n = 5). (D) Percentage of WT and PKN2^KO PSCs in G1, S, and G2 of the cell cycle (n = 3; two-way ANOVA with Sidak's test). ns, not significant. (E and F) Representative images and quantification of gel contraction from embedded WT and PKN2^KO PSCs treated with 5 ng/mL TGF-β1 or vehicle for 72 h using the formula (1 − ratio of gel size/well size) × 100. Scale bar represents 5 mm; (n = 2). (G and H) Representative images and quantification of absolute number of α-SMA fibers in WT and PKN2^KO PSCs treated with vehicle or 5 ng/mL TGF-β1 for 72 h. Scale bar represents 25 μm. Quantification is relative to vehicle-treated WT PSCs using MATLAB algorithm (n = 3). (I and J) Representative images (I) and quantification (J) of Oil Red O staining (arrows) of immortalized PSCs plated on glass coverslips and treated with vehicle or ATRA daily for 4 days (n = 3; scale bar represents 25 μm). (F, H, and J) Statistics are two-way ANOVA with Tukey's multiple comparisons test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 2

Deletion of PKN2 promotes a CAF-like ECM signature in PSCs (A and B) Differentially expressed (DE) ECM and adhesion gene transcripts (QIAseq) in PKN2^KO PSCs relative to WT PSCs treated with vehicle (A) or 5 ng/mL TGF-β1 (B) for 72 h. Log2 fold change and p values determined by DESeq2 (n = 3; p < 0.05). (C) DE gene transcripts in PKN2^KO PSCs relative to WT PSCs treated with vehicle or 5 ng/mL TGF-β1 for 72 h; transcripts in bold were at least halved or doubled in expression between WT and KO (n = 3; p < 0.05). (D) Comparison of transcriptomic expression data between WT and PKN2^KO PSCs and CAF expression data from Öhlund et al. (2017), using the panel of DE ECM genes with significance greater than p < 0.05 (C). Concurrence of changes between the two datasets is indicated in the righthand side bar (concur). (E) qPCR analysis of mRNA expression of Il6, Lif, Cxcl1, Plin2, and Pparγ in WT and PKN2^KO PSCs expressed as fold change to WT for each gene (n = 4; ^∗p < 0.05; ratio paired t-rest).

Figure 3

PKN2 modulates TEAD-driven transcription and nuclear localization of the mechanosensor YAP (A) Schematic showing potential downstream targets of PKN2 involved in myofibroblast differentiation. (B–D) Normalized expression of SRF (B), TEAD (C), or SMAD (D) responsive Firefly luciferase reporter in WT and KO PSCs starved in 0.5–1% serum or treated with 5 ng/mL TGF-β1 or 10% serum. Values are normalized to a Renilla luciferase control per sample and presented relative to WT serum-starved PSCs (n = 5; two-way ANOVA with Tukey's correction). (E) qPCR analysis of expression of indicated genes in PKN2 WT and KO PSCs expressed as a fold change to WT control (n = 4). (F) Immunofluorescent images of YAP1 localization (green) in WT and PKN2^KO PSCs plated at low and high density on glass coverslips for 48 h (minimum of 100 cells/condition; n = 3; scale bar represents 50 μm). (G) Percentage of WT and PKN2^KO PSCs with YAP-positive nuclei plated at both high and low density (n = 3; unpaired t test). (H) Quantification by Python CellProfiler algorithm of YAP nuclear intensity for indicated number of cell neighbors (n = 3; two-way ANOVA with Sidak's test). (I and J) Representative western blot and quantification of p-YAP S112 and total YAP expression in WT and PKN2^KO PSCs plated at low and high density (n = 3; two-way ANOVA with Tukey's multiple comparisons test). (K and L) Western blot and quantification of p-SMAD2/3 induction with 5 ng/mL TGF-β1 for indicated time points; quantification expressed relative to untreated WT PSCs (n = 3; unpaired t test). (M) Western blot analysis of p-p70 S6K, total p70 S6K, p-ERK1/2, and total ERK in WT and PKN2^KO PSCs starved in 1% serum and treated with vehicle or 5 ng/mL TGF-β1 for 4 h (n = 2). For statistics: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; and ^∗∗∗∗p < 0.0001.

Figure 4

PKN2 loss reduces PSC-led cancer cell invasion but promotes cancer cell outgrowth. (A) Bright-field (top; scale bar represents 200 μm) and live-cell confocal z stack projections (bottom; scale bar represents 100 μm) of spheroids (n > 16) containing H2B-RFP TB32048 PDAC cells (red) and H2B-GFP WT or PKN2^KO PSCs (green) embedded in Matrigel matrix for 3 days after siRNA treatment. (B and C) Area of fibroblast-led invasion (B) or cancer cell outgrowth (C) per spheroid, normalized to total spheroid area and expressed as fold change relative to WT control (n > 16 spheroids/condition; one-way ANOVA with Tukey's multiple comparisons test). (D) Bright-field (top panel) and confocal (bottom panels) images of spheroids containing TB32048 cancer cells with WT or PKN2^KO PSCs transduced with either empty vector (EV), YAP WT (YAP), or YAP S6A (S6A) vectors. Dotted white lines indicate core area of spheroid. (E and F) Quantification of area of PSC-led (E) or epithelial (F) invasion, normalized to total spheroid area per spheroid, relative to EV (n = 3; two-way ANOVA with Tukey's multiple comparisons test). (G) Dual luciferase analysis of TEAD reporter on WT PSCs transduced with EV, YAP, or S6A YAP. Data expressed as Firefly or Renilla luminescence for each well relative to EV (n > 3; two-way ANOVA with Tukey's multiple comparisons test; ∗p < 0.05, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001).

Figure 5

Deletion of stromal PKN2 in vivo promotes pancreatic tumor invasion (A) Schematic of experimental model for orthotopic pancreatic tumor development in inducible conditional PKN2^KO mice; Rosa26 CreERT2 was induced with tamoxifen in PKN2 WT, HET, or KO mice; n = 8–11/group; d, days. (B and C) Quantification of primary tumor volume (B), with representative pictures of tumors alongside spleens (C). (D–F) Quantification of the number (D) and volume (E) of secondary tumors found associated with the peritoneum and the number of mice with (gray) or without (white) these foci (F). (G) Quantification of the number of diaphragm nodules found per mouse. ∗p < 0.05; one-way ANOVA with Sidak's multiple comparison's test. (H) Quantification of the number of animals with (gray) or without (white) sites of invasion observed in cross-sections of the tumor (^∗p < 0.05; chi-squared test for distribution of invasive sites across genotypes). (I) Representative H&E staining of abutted region of tumor with healthy pancreas in the WT (left) and invasive tumor region of a tumor in a PKN2^KO mouse (right; scale bar represents 50 μm). (J–M) Sirius Red (scale bar represents 500 μm), α-SMA (scale bar represents 200 μm), and endomucin (scale bar represents 200 μm) staining (J) of primary tumors with respective quantification of positive stain per pixel area (K and L) or vessel count (M). (M) ∗p<0.05; one-way ANOVA with Sidak's multiple comparison's test.

Figure 6

Enhanced tumor invasion in PKN2^KO mice is associated with a pro-metastatic matrisome score (A) Unsupervised clustering of PKN2 WT and KO tumors based on their expression of the 22 MI genes defined by Pearce et al. (B) MI score of WT and PKN2^KO tumors (n = 5–6 tumors/group; ^∗p < 0.05; unpaired t test). (C) Pseudocolor overlay of MI ECM proteins VCAN, FN1, COMP, and CTSB at the edge or invasive front of tumors in a representative PKN2 WT (top) or PKN2^KO tumor (bottom, left panels). Cathepsin B staining (right panels) of tissue sections used in the overlay is shown; calibration bar in overlay indicates the number of overlapping ECM proteins at each pixel (T, tumor; NP and arrows indicate healthy pancreatic acini; area within white dotted lines indicates edge or invasive area). (D) PKN2^KO matrisome signature genes based on high-confidence PSC and orthotopic DE gene set. (E) Kaplan-Meier analysis of TCGA-PAAD patients with high (red) or low (blue) expression of PKN2^KO matrisome score. (F) Hazard ratio (HR) scores with 95% confidence interval (CI) determined by multivariate Cox proportional hazards model across TCGA tumor datasets. HR > 1; high PKN2^KO matrisome score associated with poor prognosis. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. (G) Hallmark GSEA analysis of RNA-seq data from TCGA-PAAD stratified PKN2^KO matrisome score compared with WT versus PKN2^KO orthotopic tumors.