Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Oct 25;7(43):69489-69506.
doi: 10.18632/oncotarget.11060.

Novel regulatory role of neuropilin-1 in endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma

Pratiek N Matkar  1   2 , Krishna Kumar Singh  3   4   2 , Dmitriy Rudenko  1 , Yu Jin Kim  1 , Michael A Kuliszewski  1 , Gerald J Prud'homme  5 , David W Hedley  6 , Howard Leong-Poi  1   2
Affiliations

Novel regulatory role of neuropilin-1 in endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma

Pratiek N Matkar et al. Oncotarget. .

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumor desmoplasia, which is in part responsible for its aggressiveness. Endothelial cells have been shown to display cellular plasticity in the form of endothelial-to-mesenchymal transition (EndMT) that serves as an important source of fibroblasts in pathological disorders, including cancer. Angiogenic co-receptor, neuropilin-1 (NRP- 1) actively binds TGFβ1, the primary mediator of EndMT and is involved in oncogenic processes like epithelial-to-mesenchymal transition (EMT). NRP-1 and TGFβ1 signaling have been shown to be aberrantly up-regulated in PDAC. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis in human PDAC xenografts. Loss of NRP-1 in HUVECs limited TGFβ1-induced EndMT as demonstrated by gain of endothelial and loss of mesenchymal markers, while maintaining endothelial cell architecture. Knockdown of NRP-1 down-regulated TGFβ canonical signaling (pSMAD2) and associated pro-fibrotic genes. Overexpression of NRP-1 exacerbated TGFβ1-induced EndMT and up-regulated TGFβ signaling and expression of pro-fibrotic genes. In vivo, loss of NRP-1 attenuated tumor perfusion and size, accompanied by reduction in EndMT and fibrosis. This study defines a previously unrecognized role of NRP-1 in regulating TGFβ1-induced EndMT and fibrosis, and advocates NRP-1 as a therapeutic target to reduce tumor fibrosis and PDAC progression.

Keywords: endothelial cell; endothelial-to-mesenchymal transition; fibrosis; neuropilin-1; pancreatic cancer.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. NRP-1 levels positively correlate with EndMT and fibrosis markers in human PDAC xenografts
Five human PDAC xenografts were obtained for this study. (A) Tumor tissues were stained with H&E staining to recognize tissue type and the morphology; for NRP-1 levels (indicated by brown color) by immunohistochemistry and for extent of fibrosis by Masson's trichrome staining (indicated by blue color). Representative images reveal varying levels of tissue differentiation (Scale bar = 100 μm), NRP-1 expression (Scale bar = 50 μm) and extent of fibrosis in the xenografts (Scale bar = 100 μm). (B) Quantification of NRP-1 levels and collagen content using the ImageJ software (expressed in AU, arbitrary units). NRP-1 levels positively correlate with EndMT markers and pro-fibrotic genes at the transcript (C) and protein level (D) as determined by qPCR and western blotting respectively. Correlation was determined by Spearman's Rho test (positive correlation: 0.8 < R ≤ 1, R = correlation coefficient). For C, data has been normalized to tissue sample having lowest expression of the respective genes that are plotted. (E) Representative immunohistochemistry images showing correlation between NRP-1 level and EndMT markers in tumor tissues having the lowest and highest NRP-1 expression as determined previously (Scale bar = 100 μm). (F) Summary of NRP-1-EndMT correlation.
Figure 2
Figure 2. Loss of NRP-1 inhibits TGFβ1- induced EndMT in HUVECs. HUVECs were cultured and transfected with 5 nM of either siNRP-1 or scramble control
Total RNA and protein was extracted from the transfected HUVECs at 24, 48 and 72 h post-transfection. (A) qPCR data demonstrate successful silencing of NRP-1 (~85% reduction) in siNRP-1 transfected HUVECs after 24 h. All qPCR data are presented as fold change to the scrambled control. (B) NRP-1 silencing was confirmed at protein level by immunoblotting at 24, 48 and 72 h after transfection. GAPDH was used as a loading control. (C) Representative images from capillary-like tube formation assay demonstrating reduced number of tubes in siNRP-1 compared to scramble control at 6 h. (D) Tube formation ability of HUVECs on MatrigelTM was quantified as number of nodes and number of tubes, showing that siNRP-1 caused a significant anti-angiogenic effect as compared to scramble control. *p < 0.05 and ***p < 0.001 vs. scramble control. n = 5 in triplicate. (E) Confluent monolayer of HUVECs exhibits typical cobblestone morphology in scramble control under light microscope (20X, scale = 50 μm). TGFβ1 stimulation (10 ng/ml) caused marked morphological changes with the cells becoming enlarged and spindle shaped in scramble control transfected cells. These morphological changes were inhibited to some extent in siNRP-1 transfected TGFβ1-stimulated HUVECs. (F) Fluorescent microscopy images (scale = 10 μm) stained with alpha-actinin (α-actinin; green color), demonstrating cytoskeletal protein re-organization in scramble control transfected TGFβ1-stimulated HUVECs. Nuclei were stained with DAPI (blue color). HUVECs were transfected with scramble control and siNRP-1, and total RNA and protein was extracted at 24 h and 48 h, respectively. qPCR analysis demonstrates significant changes in the EndMT markers at transcript level (G) and protein level (H) by immunoblotting. These data were further confirmed by immunofluorescence for NRP-1 and EndMT markers (I) (scale = 20 μm; scale for magnified image = 10 μm) 48 h post-transfection. Nuclei were stained by DAPI (blue). (J) qPCR data analysis demonstrates significant down-regulation of TGFβ1, TGFBR1 and TGFBR2 and TGFβ1-responsive genes; Slug, Collagen I and CTGF at transcript level and (K) TGFβ1, TGFBR1, TGFBR2, pSMAD2, SMAD2 and (H) pro-fibrotic genes CTGF, Collagen I and Slug at protein level by immunoblotting upon NRP-1 silencing. *p < 0.05, **p < 0.01, ***p < 0.0001 vs. scramble control. n = 3–4 in triplicate.
Figure 3
Figure 3. Lentivirus-mediated NRP-1 overexpression exacerbates TGFβ1-induced EndMT in HUVECs
HUVECs were cultured and transduced with either lentiNRP-1 or blank/empty control lentivirus (lentiControl). Total RNA and protein was extracted from the transduced HUVECs at 24, 48 and 72 h post-transduction. (A) qPCR data demonstrate successful overexpression of NRP-1 in lentiNRP-1 transduced HUVECs after 48 h. All qPCR data are presented as fold change to the scrambled control. (B) NRP-1 overexpression was confirmed at protein level by immunoblotting. (C) Representative images from capillary-like tube formation assay demonstrating increased number of tubes in lentiNRP-1 compared to lentiControl at 6 h time point. (D) Tube formation ability of HUVECs on MatrigelTM was quantified as number of nodes and number of tubes and showed that lentiNRP-1 caused a significant pro-angiogenic effect as compared to lentiControl. *p < 0.05 and **p < 0.01 vs. lentiControl. n = 5. (E) Confluent monolayer of HUVECs exhibits typical cobblestone morphology in scramble control under light microscope (scale = 50 μm). TGFβ1 stimulation caused marked morphological changes with the cells becoming enlarged and spindle shaped in cells transduced with lentiControl. Similar morphological changes were observed in lentiNRP-1 transduced HUVECs upon TGFβ1 stimulation. (F) Fluorescent microscopic images (scale = 10 μm) stained with alpha-actinin (α-actinin; green color), demonstrating cytoskeletal protein re-organization in lentiNRP-1 transduced HUVECs. Nuclei were stained with DAPI (blue color). HUVECs were transduced with lentiControl and lentiNRP-1, and total RNA and protein was extracted at 48 h. (G) qPCR analysis demonstrates significant changes in the EndMT markers at transcript level and protein level by immunoblotting (H). (I) These data were further confirmed by immunofluorescence for NRP-1 and EndMT markers (Scramble: scale = 20 μm, scale for magnified image = 10 μm; LentiNRP-1: scale = 10 μm, scale for magnified image = 10 μm) 48 h post-transduction in the photomicrographs. Nuclei were stained by DAPI (blue). (J) qPCR data analysis demonstrates significant down-regulation of TGFβ1, TGFBR1, TGFBR2 and (G) TGFβ1-responsive genes Slug, Collagen 1 and CTGF at transcript level and (K) TGFβ1, TGFBR1, TGFBR2, pSMAD2, SMAD2 and (H) pro-fibrotic genes Slug, Collagen I and CTGF at protein level by immunoblotting upon NRP-1 overexpression. Nuclei were stained by DAPI (blue) *p < 0.05, **p < 0.01 and ***p < 0.001 vs. lentiControl. n = 3–4 in triplicate.
Figure 4
Figure 4. Loss of NRP-1 inhibits EndMT and fibrosis in vivo and results in reduced tumor growth
Total RNA and protein was extracted from the tumor tissues. (A) qPCR data and (B) immunoblotting demonstrate successful silencing of NRP-1 in shNRP-1 minicircle delivered animals. (A) qPCR analysis demonstrates significant changes in the EndMT markers at transcript level and protein level (B) by immunoblotting. (C) These data were further confirmed by immunohistochemistry for NRP-1 and EndMT markers (scale = 100 μm; indicated by brown color). The tumor tissues were also stained with H&E to recognize tissue type and the morphology, and extent of fibrosis by Masson's trichrome staining (indicated by blue color). (C) Representative images reveal ductal morphology, reduced NRP-1 levels and reduced extent of fibrosis in in shNRP-1 minicircle delivered animals. (D) Tumor volumes (ml) as measured by volumetric assessment, with smallest tumor volumes seen in the shNRP-1 minicircle group as compared to scramble group. (E) Representative contrast-enhanced ultrasound perfusion images of orthotopic pancreatic tumors on day 28 (before gene delivery) and day 56 (28 days after gene delivery). Images are color coded, with bright yellow/orange signifying the highest acoustic signal and greatest perfusion. Tumor area (F) and perfusion (G, H) is greater and more complete throughout the tumor in scramble minicircle group, while reduced perfusion seen in tumors treated with shNRP-1 minicircle. (G,H) At day 28 there were no significant differences in microvascular blood volume (MBV) and microvascular blood flow (MBF) between groups. Normalized tumor blood volume (G) and blood flow (H) at day 56. At day 56, tumor blood volume and flow was significantly reduced in shNRP-1 minicircle group compared with scramble control group. *p < 0.05, **p < 0.01 vs. scramble. n = 4 for each group. Tumor area and volume data are expressed as mean ± SEM.
Figure 5
Figure 5. A putative schematic diagram depicting the complex interaction of NRP-1 and TGFβ-signaling pathway in EndMT and tumor fibrosis
Endothelial cell-specific loss of NRP-1 down-regulates and deactivates TGFβ signaling and its receptors leading to reduced pSMAD2, Slug, CTGF and Collagen 1. The reduction in the levels of transcription factor Slug further up-regulates the expression of endothelial markers like VE-cadherin and CD31, limiting EndMT. Contrastingly, overexpression of NRP-1 exacerbates the TGFβ-induced EndMT and associated tumor fibrosis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65:5–29. - PubMed
    1. Jemal A, Center MM, DeSantis C, Ward EM. Global patterns of cancer incidence and mortality rates and trends. Cancer Epidemiol Biomarkers Prev. 2010;19:1893–1907. - PubMed
    1. Weir HK, Thompson TD, Soman A, Moller B, Leadbetter S. The past, present, and future of cancer incidence in the United States: 1975 through 2020. Cancer. 2015;121:1827–1837. - PMC - PubMed
    1. Yadav D, Lowenfels AB. The epidemiology of pancreatitis and pancreatic cancer. Gastroenterology. 2013;144:1252–1261. - PMC - PubMed
    1. Malvezzi M, Bertuccio P, Levi F, La Vecchia C, Negri E. European cancer mortality predictions for the year 2014. Ann Oncol. 2014;25:1650–1656. - PubMed

MeSH terms