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Kaposi sarcoma herpesvirus promotes endothelial-to-mesenchymal transition through Notch-dependent signaling - PubMed

  • ️Sun Jan 01 2012

Kaposi sarcoma herpesvirus promotes endothelial-to-mesenchymal transition through Notch-dependent signaling

Paola Gasperini et al. Cancer Res. 2012.

Abstract

Endothelial-to-mesenchymal transition (EndMT) is now widely considered a pivotal contributor to cancer progression. In this study, we show that the Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a sufficient cause of EndMT, potentially helping to explain the aggressiveness of KS that occurs commonly in AIDS patients. Upon KSHV infection, primary dermal microvascular endothelial cells lost expression of endothelial markers and acquired expression of mesenchymal markers, displaying new invasive and migratory properties along with increased survival. KSHV activated Notch-induced transcription factors Slug and ZEB1, and canonical Notch signaling was required for KSHV-induced EndMT. In contrast, KSHV did not utilize the TGFβ signaling pathway, which has also been linked to EndMT. Within KS lesions, KSHV-infected spindle cells displayed features compatible with KSHV-induced EndMT including a complex phenotype of endothelial and mesenchymal properties, Notch activity, and nuclear ZEB1 expression. Our results show that KSHV engages the EndMT program to increase the invasiveness and survival of infected endothelial cells, traits that likely contribute to viral persistence and malignant progression. One important implication of our findings is that therapeutic approaches to disrupt the Notch pathway may offer novel approaches for KS treatment.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1

Repression of endothelial markers and upregulation of mesenchymal markers in endothelial cells infected with KSHV. Relative mRNA levels of CD31, VE-cadherin, CD34, CD146, NG-2, PDGFRB, SMA, and acta2 in (A) DMVECs and (B) EAHY926 cell line (EAHY) control (none), infected with KSHV (KSHV), or cultured with TGFβ1 (TGFβ1); mRNA levels were measured by quantitative PCR. RNAs were extracted 12 days after KSHV infection or treatment with 5 ng/mL of TGFβ1. RNA from MSCs is tested as a control. mRNA levels normalized for GAPDH are expressed as fold change relative to control cells. Experiments are representative of 8 (DMVECs) and 6 (EAHY) conducted. C, cell lysates from control and KSHV-infected DMVECs (12 days after infection) were immunoblotted for CD31, VE cadherin, Tie-2, Vimentin, PDGFRB, SMA proteins, and β-actin (loading control). The experiment is representative of 3 conducted. D, fluorescence microscopy images of uninfected DMVECs (left) and KSHV-infected (12 days postinfection) DMVECs (right) stained with phalloidin and DAPI or (E) immunostained for vimentin and DAPI; original magnification 25×. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2
Figure 2

KSHV promotes functional change in endothelial cells. A, wound closure by KSHV-infected EAHY cells compared with uninfected EAHY cells. Images from scratch assay; original magnification 10×. B, wound closure (% wound area) as a function of time by uninfected (control) and KSHV-infected (KSHV) EAHY cells. Each data point is the average (±SD) wound closure from 3 fields per well in 3 independent experiments (±SD); C, mean relative wound closure (± SD) by control and KSHV-infected EAHY at time 0- and 18-hour incubation (3 experiments). D, growth curves of uninfected (control) and KSHV-infected (KSHV) EAHY cells over 48-hour culture (0.1% FBS) from IncuCyte imaging [expressed as % mean (±SD) confluency (3 fields per well; 3 wells; representative experiment]. E, bright-field images of uninfected and KSHV-infected EAHY (50 hours after seeding at equal density); original magnification 10×. Chemotaxis of (F) EAHY cells toward FBS (10%) and (G) DMVECs toward PDGF (50 ng/mL). Cells were seeded (IBIDI chambers) and cell movement was imaged overnight every 10 minutes. Lines reflect tracks of cells that moved away or toward the chemoattractant; grey symbol (+) represent the average movement of the cell population. Representative results from 3 experiments. Cells were used on day 10 postinfection.

Figure 3
Figure 3

Analysis of CD31, CD146, and PDGFR expression in KS tissues. A, fluorescence microscopy images of normal skin tissue (left) and KS (stage T1) tissue (right) immunostained for CD31 (red) and LANA (green); nuclei are visualized with DAPI (blue). CD31bright endothelial cells lining vessels are visualized in the normal skin and in KS tissue. The spindle-like cells visualized in the KS tissue (LANA-positive) are typically CD31low or CD31−. Original magnification 32×; the larger boxed panel represents a magnification of the smaller boxed panel. Images from normal skin tissue (B) and KS (stage T1) tissue (C) immunostained for CD146 (pink), CD31 (red), and LANA (green); nuclei are visualized with DAPI (blue). In normal skin, CD31+CD146+ cells are LANA− and mark endothelial cells lining blood vessels. In KS tissue, the LANA+CD146+CD31low/neg cells are widely distributed in the tumor tissue; original magnification 40×; the merged images are magnified in the panel furthest to the right. Normal skin (D) and KS (stage T1) tissue (E) immunostained for PDGFRβ (red), CD146 (pink), and LANA (green); nuclei are stained with DAPI (blue). In normal skin, CD146+ PDGFRβ+ cells mark vessels and do not express LANA. In KS tissue, CD146+PDGFRβ+LANA+ cells are widely distributed within the tumor tissue. The right panel reflects merged images; the inset reflects a magnification within the panel; original magnification 40×.

Figure 4
Figure 4

Expression of SMA and CD31 in KS tissue and normal skin. A, fluorescence microscopy images of normal skin (top panels) and KS (stage T0) tissue (bottom) immunostained for SMA (yellow), CD31 (red), and LANA (green); nuclei are stained with DAPI (blue). In normal skin, SMA+CD31+ cells mark the vessel wall. In KS tissue, SMA+ cells are widely distributed. B, microscopy images of KS (stage T0) tissue displaying a cluster of CD31bright cells expressing nuclear LANA+. C, microscopy images showing coexpression of SMA (yellow) and low-level CD31 (red) in a LANA+ cell. Original magnification of all images, 60×.

Figure 5
Figure 5

Effects of KSHV infection on TGFβ1 secretion and signaling. A, levels of TGFβ1 were measured by ELISA in supernatants from 36-hour cultures of uninfected or KSHV-infected cells (DMVECs and EAHY). Results are expressed as means (± SD) of 2 experiments tested in duplicate. B, TGFβ receptor 2 (TGFBR2) in cell lysates of DMVECs (top) and EAHY (bottom) cells at the indicated time points (d3 = day3) after KSHV infection or culture without infection detected by immunoblotting. The membranes were reprobed for actin. The experiment is representative of 5 (DMVECs) or 3 (EAHY) conducted. C, phosphorylated (P) SMAD2/3, total SMAD2/3, and actin detected by immunoblotting in cell lysates of DMVECs and EAHY cultured for 9 days with or without infection with KSHV. Experiment representative of 3 conducted. The bar graph reflects the mean relative ratios (± SD) of P-SMAD2/3/total SMAD2/3 band intensities detected by immunoblotting in 3 experiments.

Figure 6
Figure 6

KSHV infection promotes activation of the Notch pathway. A, expression levels of Hey1 and Hey2 mRNAs in (DMVECs) and EAHY cells uninfected or infected with KSHV. RNAs were extracted 12 days after KSHV infection or culture without infection. The results from quantitative PCR are expressed as fold change relative to the uninfected cells. The experiment is representative of 3 conducted. B, fluorescence microscopy images of KS (stage T0) tissue immunostained for Hey2 (red) and LANA (green); nuclei are stained with DAPI (blue); original magnification 32×. The inset reflects a magnification showing the coexpression of LANA and Hey2 immunostaining. C, effects of the γ-secretase inhibitor DAPT (5 μmol/L throughout the culture period) on the levels of Hey1, Hey2, NG-2, PDGFRB, SMA, and acta2 mRNAs in the EAHY926 cells uninfected or KSHV-infected (12-day culture after achieving more than 90% cell infection as detected by GFP). The experiment is representative of 4 conducted.

Figure 7
Figure 7

KSHV infection promotes the expression of SNAIL, SLUG, TWIST, ZEB1, and ZEB2 transcription factors. RNA was extracted from DMVECs (A) and EAHY cells (B) cultured (12 days) in medium only or with TGFβ (5 ng/mL) or after KSHV infection (~90% cells infected). RNA from MSCs was a control. Results reflect fold increase in mRNA (quantitative PCR) relative to control cells after normalization for GAPDH. The results in A and B are representative of 3 to 5 experiments conducted. C, effects of the γ-secretase inhibitor DAPT (10 μg/mL) on KSHV-induced expression of SLUG and ZEB1 in EAHY cells (experimental conditions described in the legend to Fig. 6C). D, images of KS (stage T1) tissue immunostained for ZEB1 (pink) and LANA (green); nuclei are stained with DAPI (blue); original magnification (32×). E, magnification of the inset linked by the arrow showing an area of the tissue in which LANA− cells express mostly cytoplasmic ZEB1 staining. F, magnification of the inset linked by the arrow showing an area of KS tissue with LANA+ cells, in which both LANA and ZEB1 are mostly coexpressed in the nuclei; individual stains in E and F are merged in the right panel. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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