Neuropilin-2 upregulation by stromal TGFβ1 induces lung disseminated tumor cells dormancy escape and promotes metastasis outgrowth

Abstract

Metastasis is the main cause of death from solid tumors. Therefore, identifying the mechanisms that govern metastatic growth poses a major biomedical challenge. Tumor microenvironment signals regulate the fate and survival of disseminated tumor cells (DTCs) in secondary organs. However, very little is known about the role of nervous system mediators in this process. We have previously reported that neuropilin-2 (NRP2) expression in breast cancer correlates with poor prognosis. Here, we show that NRP2 positively regulates the proliferation, invasion, and survival of breast and head and neck cancer cells in vitro. NRP2 deletion in tumor cells inhibits tumor growth in vivo and decreases the number and size of lung metastases by promoting lung DTCs quiescence. NRP2 deletion upregulates dormancy and cell cycle regulators expression and promotes DTCs reprograming into quiescence. Moreover, lung fibroblasts and macrophages induce NRP2 upregulation in DTCs through the secretion of TGFβ1. NRP2 facilitates lung DTC interaction with the extracellular matrix and promotes lung DTCs activation and metastasis. Therefore, we conclude that the TGFβ1-NRP2 axis is a new key dormancy-awakening inducer that promotes DTCs proliferation and lung metastasis development.

Keywords: Neuropilins; TGFβ; breast cancer; disseminated tumor cells; dormancy; head and neck cancer; metastasis.

Fig. 1

NRP2 deletion in proliferative cells alters cell cycle and decreases cell proliferation, by inducing p27 expression. A) Analysis of NRP2 mRNA relative expression in human healthy mammary epithelial (MCF10A) and BrCa cell lines. The bar plot shows relative quantification (RQ) values referred to MCF10A cells (n=3). The graph represents RQ mean values ± S.E.M.; **P < 0.01, ****P < 0.0001 comparing LDS (MDA-MB-231, MDA-MB-468, HCC1954, BT-549) vs HDS (BT-474, T-47D, MDA-MB-453, ZR-75-1) cell lines by t-Student’s test. B) Representative western blot analysis of NRP2 protein levels normalized with GAPDH. Protein quantifications are referred to MCF10A. n.d.=non detectable. (n=3). C) Analysis of NRP2 mRNA relative expression in HNSCC cell lines. The bar plot shows RQ mean values ± S.E.M. referred to T-HEp3 cells (n=3); ****P < 0.0001 comparing proliferative/LDS (T-HEp3, Lu-HEp3) vs dormant/HDS (BM-HEp3, D-HEp3) cell lines by t-Student’s test. D) Representative western blot analysis of NRP2 protein levels normalized with α-tubulin. Protein quantifications are referred to T-HEp3 cells (n=3). E) Representative western blot analysis of NRP2 and p27 protein levels normalized with α-tubulin in NRP2^KO cells (n=3). NRP2 and p27 quantifications are referred to the non-target control. F-G) MDA-MB-231 (F) and T-HEp3 (G) cells proliferation in control (Cas9, NTC) vs NRP2-deleted cells (NRP2^KO). Graphs represent the fold-change of proliferation mean ± S.E.M. referred to day 0 (n=2); *P < 0.05, **P < 0.01 comparing Cas9/NTC vs NRP2^KO by one-way ANOVA, Sidak’s test. H-I) Percentage (%) of MDA-MB-231 (H) and T-HEp3 cells (I) in each cell cycle’s phase in control (-) and NRP2-deleted (NRP2^KO) cells (+). The bar plots represent mean ± S.E.M. (n≥2); **P < 0.01, ****P < 0.0001 comparing Cas9/NTC (non-targeted cells) vs NRP2^KO by two-way ANOVA, Sidak’s test. J-K) Analysis of TFGBR3, SOX9, NR2F1, RARB, CDKN2A, CDKN2B and DEC2 mRNA relative expression in MDA-MB-231 (J) and T-HEp3 (K) NTC and NRP2-deleted (NRP2^KO) cells after 24h. The bar plot shows relative quantification (RQ) values referred to the NTC cells (n=3). The graph represents RQ mean values ± S.E.M.; *P < 0.05 by Mann Whitney test.

Fig. 2

NRP2 deletion inhibits T-HEp3 tumor growth. A, B) Left panels, representative images of colonies from anchorage-dependent growth assay in MDA-MB-231 (A) and T-HEp3 (B) cells. Right graphs, quantification of the total number of colonies. Graphs represent mean ± S.E.M. (n=1, triplicates); ns, non-significant, ****P < 0.0001, comparing non-target control (NTC) vs NRP2^KO by t-Student’s test. C) Diagram of the orthotopic mice in vivo experiment using T-HEp3 control (NTC) vs NRP2-depleted (NRP2^KO) cells. D) Graph representing T-HEp3 tumors volume (mm³) over time for each group. E) Left panel, representative T-HEp3 tumor images at the time of surgery. Middle and right panels, graphs showing PTs weight (g) (middle) and volume (cm³) (right) at the time of surgery. F-J) Left panels, representative immunofluorescence (IF) images of NRP2 (F), p27 (G), p21 (H), Ki67 (J) and cc3 (H) in T-HEp3 mice tumors. Scale bar: 50µm. Right panels, mean fluorescence intensity (mfi) quantification of NRP2, p27, p21 and cc3. In Ki67 graphs the positive area for Ki67 staining was quantified. Graphs represent mean ± S.E.M. (n=1, with 12 mice for NTC and 6 mice for NRP2^KO); *P < 0.05, **P < 0.01, ***P < 0.001 comparing NTC vs NRP2^KO by t-Student’s test.

Fig. 3

NRP2 induces cell motility and its expression is up-regulated in lung DTCs and lung metastases in vivo. A) Wound healing assay in MDA-MB-231 cells. Left panel, representative images from phase-contrast microscopy. Right panel, quantification of the wound area ratio after 0 and 24h migration in control (Cas9) and NRP2^KO MDA-MB-231 cells. Scale bar: 150µm. Graphs represent mean ± S.E.M. (n≥2); ****P < 0.0001, comparing Cas9 vs NRP2^KO by two-way ANOVA, Sidak’s test. B, C) Invasion assay in MDA-MB-231 (B) and T-HEp3 (C) cells. Left panels, representative images from phase-contrast microscopy. Scale bar: 200µm. Right panels, quantification of the invading cells area. Graphs represent mean ± S.E.M. (n=2); **P < 0.01 comparing Cas9/NTC vs NRP2^KO by t-Student’s test. D) Top and middle panels, representative IF images of vimentin (green) and NRP2 (red) staining of lung T-HEp3 DTCs in chick embryo (top; scale bar: 50µm) and mice (middle; scale bar: 50µm) lung sections. Bottom panel, representative IF images of NRP2 (green) and HER2 (red) staining in MMTV-Neu mice lung sections. Scale bar: 20µm. E) Upper panel, diagram of the tail vein in vivo injection using T-HEp3 cells. Lower panel, representative IF images of vimentin (pink) and NRP2 (green) in T-HEp3 lung DTCs from early (isolated 1 week post-inoculation) and late (isolated 3 weeks post-inoculation) mice tail vein injection in vivo models. Scale bar: 50µm. The graph represents the number of NRP2-positive lung DTCs mean ± S.E.M.; *P < 0.05 comparing early vs late by t-Student’s test. F) Upper panel, diagram of the orthotopic mice in vivo experiment using MDA-MB-453 mCherry+ cells. Lower panel, representative IF images of HER2 (red) and NRP2 (green) staining in lung MDA-MB-453 single DTCs and micrometastases (micromets). Scale bar: 50µm.

Fig. 4

Lung fibroblasts and macrophages-derived TGFβ1 drives NRP2 up-regulation. A) Representative western blot analysis of NRP2 protein levels normalized with GAPDH or β-tubulin (Housekeeping proteins (HKP)) after 24h treatment with SB431542 (5µM) and/or TGFβ1 (5ng/mL) (n=3). B) Representative western blot analysis of NRP2 protein levels normalized with GAPDH or β-tubulin after 24h treatment with lung CM and/or SB431542 (5µM) (n=2). C) Quantification of TGFβ1 levels by ELISA (pg/mL) in lung CM. The graph represents the TGFβ1 pg/mL mean values ± S.E.M (n=1, triplicates); ***P < 0.001 comparing control media vs lung CM by t-Student’s test. D) Representative western blot analysis of TGFβ1 protein levels released from the beads used for lung CM depletion used in E. Beads (-) refers to the control condition where control IgG-bound beads have been used for the assay whereas beads (+) refers to those beads linked to TGFβ1 antibody. E) Representative western blot analysis of NRP2 protein levels normalized with tubulin after 24h treatment with lung CM or TGFβ1-depleted lung CM (n=1). F) Quantification of TGFβ1 levels by ELISA (pg/mL) in tumor cells (T-HEp3, MDA-MB-231) and TME cells (macrophages, THP-1; fibroblasts, CCD19) conditioned media. The graph represents the TGFβ1 pg/mL mean values ± S.E.M (n=1, triplicates); ***P < 0.001, ****P < 0.0001 comparing tumor cells (T-HEp3 and 231) vs THP-1 or CCD19 by one-way ANOVA, Sidak’s test. G, H) Representative western blot analysis of NRP2 protein levels normalized with GAPDH or β-actin after 24h treatment with macrophages (THP-1) CM and SB431542 (5µM) (G) or fibroblasts (CCD19) CM and SB431542 (5µM) (H) (n=2). I) Left panel, representative IF images of αSMA in young and old mouse lungs (scale bar: 3µm). Right panels, αSMA mfi quantifications. J) TGFβ1 quantification by ELISA (pg/mL) in young and old mouse lung CM (n=1, triplicates). Graphs represent mean ± S.E.M.; *P < 0.05 comparing young vs old by one-way ANOVA, Sidak’s test. K) Representative western blot analysis of NRP2 protein levels normalized with GAPDH after 24h treatment with young or old mouse lung CM and SB431542 (5µM) (n=1). In all the western blots, NRP2 protein quantifications are referred to the non-treated control conditions.

Fig. 5

NRP2 deletion decreases lung metastases size and triggers quiescence in lung DTCs in vivo. A) Diagram of the orthotopic mice in vivo injection using T-HEp3 cells. B) Upper panel, representative IF images of vimentin (Vim.; green) and Ki67 (red) staining of T-HEp3 lung DTCs (scale bar: 100µm). Lower panel, quantification of lung micrometastases area (µm²) using ImageJ software. C) Quantification of the percentage (%) of Ki67-positive (proliferative) or Ki67-negative (dormant) single and doublet T-HEp3 cells per lung. D) Diagram of the tail vein mice in vivo injection using MDA-MB-231 or T-HEp3 cells. E) Upper panel, representative IF images of vimentin (Vim.; green) and Ki67 (red) staining of MDA-MB-231 lung DTCs 4 weeks (4w) after inoculation (scale bar: 50µm). Lower panel, quantification of lung macrometastases area (µm²) using ImageJ software. F) Quantification of the % of Ki67-positive or Ki67-negative single and doublet MDA-MB-231 cells per lung. G) Upper panel, representative IF images of vimentin (Vim.; green) and Ki67 (red) staining of T-HEp3 lung DTCs 2 weeks (2w) after inoculation (scale bar: 50µm). Lower panel, quantification of the % of Ki67-positive or Ki67-negative single and doublet T-HEp3 cells per lung. H) Upper panel, representative IF images of vimentin (Vim.; green) and Ki67 (red) staining of T-HEp3 lung DTCs 4 weeks (4w) after inoculation (scale bar: 50µm). Lower panel, quantification of lung macrometastases area (mm²) using ImageJ software. I) Quantification of the % of Ki67-positive or Ki67-negative single and doublet T-HEp3 cells per lung. Graphs represent mean ± S.E.M. (n≥5 lungs per group); ns, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 comparing NTC vs NRP2^KO by t-Student’s test.

Fig. 6

NRP2 deletion induces the expression of genes involved in cell cycle regulation and inhibits the expression of genes involved in remodeling of the extracellular matrix. A) Volcano plot showing the genes induced (red) and repressed (blue) in NRP2 KO cells. B)-Heat map that shows the genes differentially expressed between NRP2 KO and NTC cells. C) GO biological pathway enrichment analysis (GO:BP) using biological process databases for the genes differentially expressed between NTC and NRP2^KO cells. D-E) GSEA analysis in the GO biological process (D) and Kegg pathways (E) databases. Enrichment score (ES) plots for the indicated gene sets in the top most up-regulated genes in NRP2^KO cells. Vertical bars refer to individual genes in a gene set and their position reflects the contribution of each gene to the ES. F) Analysis of JAK2, AURORAK, TOP2A, BUB1 and WRN mRNA relative expression in T-HEp3 NTC and NRP2-deleted (NRP2^KO) cells after 24h. The bar plot shows relative quantification (RQ) values referred to the NTC cells (n=3). The graph represents RQ mean values ± S.E.M.; *P < 0.05 by Mann Whitney test. G) Analysis of overrepresentation of downregulated genes in NRP2^KO cells (p-adjusted value < 0.05, FC<0.5) in the databases "Reactome". The top 10 terms with the highest gene proportion are represented with a p-adjusted cut-off value of 0.05 and redundant terms removed. H) Venn diagram showing the genes whose expression correlates with high NRP2 expression in RNA sequencing data from head and neck cancer patient samples and downregulated genes in NRP2^KO cells. I) Heat map showing the expression of genes in common between those correlated with high NRP2 expression in patients and those inhibited in NRP2-deficient cells. J) Representative images of adhesion of T-HEp3 NTC and NRP2^KO cells to plastic, matrigel and collagen after 30 min. The graphs represent the mean value of the percentage of adhesion to plastic, matrigel and collagen ± S.D with respect to NTC cells (n=3, per sixfold for plastic /n=4, per sixfold for matrigel and collagen). K) Kaplan-Meier curve for distant metastases-free survival (DMFS) in a cohort of 2765 BrCa patients with high (red; n=1437) or low (black; n=1328) levels of NRP2. . L) Kaplan-Meier curve for DMFS in a cohort of 92 HNSCC patients with high (green; n=20) or low (blue; n=72) levels of NRP2. Analysis performed in collaboration with Dr. Camacho and Dr. Leon from Santa Creu i Sant Pau Hospital (Barcelona, Spain).