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Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

['Weikang Chen', 'Institute Of Human Virology', 'Zhongshan School Of Medicine', 'Sun Yat-Sen University', 'Guangzhou', 'Yao Ding', 'Dawei Liu', 'Department Of Pathology', 'The First Affiliated Hospital', 'Zhengzhou Lu']

Date: 2022-02 

Kaposi’s sarcoma (KS) is an angioproliferative and invasive tumor caused by Kaposi’s sarcoma-associated herpesvirus (KSHV). The cellular origin of KS tumor cells remains contentious. Recently, evidence has accrued indicating that KS may arise from KSHV-infected mesenchymal stem cells (MSCs) through mesenchymal-to-endothelial transition (MEndT), but the transformation process has been largely unknown. In this study, we investigated the KSHV-mediated MEndT process and found that KSHV infection rendered MSCs incomplete endothelial lineage differentiation and formed hybrid mesenchymal/endothelial (M/E) state cells characterized by simultaneous expression of mesenchymal markers Nestin/PDGFRA/α-SAM and endothelial markers CD31/PDPN/VEGFR2. The hybrid M/E cells have acquired tumorigenic phenotypes in vitro and the potential to form KS-like lesions after being transplanted in mice under renal capsules. These results suggest a homology of KSHV-infected MSCs with Kaposi’s sarcoma where proliferating KS spindle-shaped cells and the cells that line KS-specific aberrant vessels were also found to exhibit the hybrid M/E state. Furthermore, the genetic analysis identified KSHV-encoded FLICE inhibitory protein (vFLIP) as a crucial regulator controlling KSHV-induced MEndT and generating hybrid M/E state cells for tumorigenesis. Overall, KSHV-mediated MEndT that transforms MSCs to tumorigenic hybrid M/E state cells driven by vFLIP is an essential event in Kaposi’s sarcomagenesis.

Kaposi’s sarcoma manifests as multifocal lesions with spindle cell proliferation, intense angiogenesis, and erythrocyte extravasation. Although the origin and malignant nature of KS remain contentious, it is established that KSHV infection with concomitant viral oncogene expression in normal cell progenitors causes KS. The mechanism of KSHV oncogenesis could be revealed through a reproduction of KS by infection of normal cells. This study reports that the KSHV infection of mesenchymal stem cells initiates mesenchymal-to-endothelial transition (MEndT) that generates mesenchymal/endothelial (M/E) hybrid state cells. The hybrid M/E cells acquired tumorigenic phenotypes, including tumor initiation, angiogenesis, migration, and the potential to form KS-like lesions after transplanted in mice. This finding faithfully recapitulates Kaposi’s sarcoma where proliferating KS spindle cells and the cells that line KS-specific aberrant vessels are also found to exhibit the hybrid M/E phenotype. We also found that KSHV-encoded viral FLICE inhibitory protein (vFLIP) plays a crucial role in promoting MEndT and the generation of M/E state cells. These results provide a new layer of evidence for KSHV-infected MSCs being the cell source of KS spindle cells and reveal novel insight into KS pathogenesis and viral tumorigenesis.

Funding: This work is supported by National Natural Science Foundation of China grants 81530069, 81772177 to YY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Increasing evidence supports that KS derives from KSHV-infected MSC through MEndT. However, how KSHV infection drives mesenchymal stem cells for the MEndT process that leads to Kaposi’s sarcoma was largely unknown. In this study, we investigated the process of KSHV-mediated MEndT and the underlying mechanism. We found that KSHV infection initiates an endothelial lineage, but incomplete differentiation that generates premalignant cells with hybrid mesenchymal and endothelial phenotypes. Such hybrid M/E cells exhibit oncogenic properties and form KS-like lesions in kidney capsule transplantation. Finally, KSHV vFLIP was found to play critical roles in KSHV-induced MEndT and oncogenesis. These findings further support the hypothesis that KS tumor cells arise from KSHV-infected MSCs through MEndT.

KSHV can be found in all KS tumors and is present in all stages of KS (patch, plaque, and nodular). In the early (patch) stage, KSHV is found in spindle-like cells surrounding ectatic vessels, and in nodular KS, the virus is present in the vast majority of spindle cells surrounding slit-like vessels [ 22 ]. The majority of KSHV in KS lesions is in a latent phase where only a limited number of latent genes are expressed. In a small percentage of tumor cells, KSHV undergoes spontaneous lytic replication and expresses viral lytic genes. KSHV latency genes, including LANA, vCyclin, and vFLIP, are known to play roles in regulating cell proliferation and apoptosis evasion and endowing pro-angiogenic and inflammatory signals [ 6 , 23 ]. Some lytic viral proteins, such as vGPCR and vIL-6, exhibit tumorigenic activities and induce angiogenesis and inflammation [ 23 – 25 ]. vIL6 sufficiently induces BECs to differentiate to LECs via upregulating the expression of PROX1 [ 26 ], KSHV-encoded miRNAs induce LEC-to-BEC reprogramming via downregulating MAF [ 27 ], KSHV-initiated endothelial-to-mesenchymal transformation is mediated by vFLIP and vGPCR through MT1-MMP in 3D LECs culture system [ 28 ]. Thus, many viral genes are known to participate in KSHV-induced cell reprogramming and KS oncogenesis.

MSCs have been identified as a population of hierarchical postnatal stem cells with the potential to self-renew and differentiate into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, myoblasts, and neural cells [ 13 , 14 ]. MSCs can be induced to endothelial-like cells with angiogenic cytokines, including VEGF, bFGF, and angiopoietin [ 15 ]. The switch from mesenchymal to endothelial phenotype is referred to as Mesenchymal-to-Endothelial Transition (MEndT), which is a critical phase of embryonic organic development and also contributes to diseases. In an adult, MEndT contributes to neovascularization by inducing cardiac fibroblasts to generate endothelial cells after cardiac injury [ 16 ], and to cancer progression by enhancing angiogenesis [ 17 ]. Moreover, MEndT, like its reverse process EndMT, is not a binary switch but a dynamic transition, which generates many intermediate phenotypic states arrayed along the mesenchymal (M)-to-endothelial (E) spectrum including mesenchymal-like (M), endothelial-like (E), and hybrid M/E states. Studies suggested that tumor cells staying in different stages have different roles in tumor progression [ 18 – 21 ].

Kaposi’s sarcoma (KS) is the most common neoplasm in AIDS patients. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of this malignancy [ 1 ]. KSHV is also associated with other malignancies, including primary effusion lymphoma (PEL) [ 2 ], multicentric Castleman’s disease (MCD) [ 3 ]. Recent reports also suggest an involvement of KSHV in childhood osteosarcoma [ 4 ]. Kaposi’s sarcoma is a multicentric, oligoclonal neoplasm clinically presenting as red-purplish spots localize mainly in the oral cavity or skin [ 5 ]. The histological features of KS lesions are extremely complex. They consist of proliferating spindle tumor cells, immature and leaky vessels, and prominent inflammatory infiltrate. The cellular origin of KS spindle cells remains controversial. The current leading hypothesis is that KS spindle cells may derive from endothelial lineage, as they bear pan-endothelial markers (CD31, CD34, and CD36 and Factor VIII) and lymphatic endothelial markers (VEGFR3, LYVE-1 and PDPN). However, KS spindle cells also express other markers including smooth muscle cell (α-SAM), macrophage (CD68), dendritic cell (Factor XIII) and mesenchymal stem cell (Nestin and CD29) markers, suggesting that KS cells do not faithfully represent an endothelial cell lineage [ 6 ]. Besides, KS spindle cells display intriguing characteristics of progenitor or immature endothelial cells—the expression of endothelial progenitor cell markers and lack of Weibel-Palade bodies (WPB) regarded as a marker for mature vascular endothelium [ 7 , 8 ]. The remarkable heterogeneity of KS raised a hypothesis that KS spindle cells may originate from mesenchymal stem cells (MSCs) or precursors of vascular cells [ 9 , 10 ]. Recently, we found a series of evidence supporting the hypothesis. (i) An immuno-histochemistry analysis showed that AIDS-KS spindle cells express Neuroectodermal stem cell marker (Nestin) and oral MSC marker CD29, suggesting an oral/craniofacial MSC lineage of AIDS-associated KS. (ii) KSHV infection of oral MSCs effectively promotes multiple lineage differentiation, especially endothelial differentiation in vitro and in vivo. (iii) Gene expression profiling analysis showed that KSHV infection reprograms MSCs, resulting in mesenchymal-to-endothelial transition (MEndT) and rendering KSHV-infected MSC the closest distance to Kaposi’s sarcoma in gene expression profile. (iv) When implanted in mice, KSHV-infected MSCs were transformed into KS-like spindle-shaped cells with other KS-like phenotypes [ 11 ]. Moreover, KSHV-infected primary rat embryonic metanephric mesenchymal precursor cells (KMM) and mouse bone marrow-derived MSCs (KPα(+)S) growth in KS-like conditions efficiently form KS-like tumors in nude mice [ 10 , 12 ]. Taken together, increasing evidence supports the notion that Kaposi’s sarcoma may arise from KSHV-infected MSCs through MEndT. However, the underlying mechanism remains unclear.

Results

KSHV infection induces MSC differentiation into mesenchymal/endothelial hybrid state cells through MEndT in vitro The triple immunofluorescence assay of KS lesions revealed the hybrid M/E phenotype of KSHV-positive spindle-shaped tumor cells. This observation compelled us to hypothesize that KS spindle cells arose from mesenchymal stem cells and KSHV initiates an MEndT process converting cells from mesenchymal phenotype to hybrid M/E phenotype. To prove this hypothesis, we attempted to reproduce this process in cultured mesenchymal stem cells to investigate whether KSHV infection could induce reprogramming of MSCs, leading to endothelial-like or M/E hybrid cells and abnormal angiogenesis as observed in KS lesions. First, PDLSCs were grown in 2-D culture and infected with KSHV. The changes of the cells in mesenchymal and endothelial cell markers were examined at different time points using Western blot, RT-qPCR, and immunofluorescence assay (IFA). The result showed that some mesenchymal markers, such as COL1A1, α-SAM, and TAGLN, faded and endothelial markers PROX1 and PDPN increased starting at the fourth day post-infection (Fig 3A). In consistent with KS lesions, mesenchymal stem cell markers PDGFRA and Nestin remained unchanged after KSHV infection (Fig 3B). However, KSHV infection did not result in the substantial expression of endothelial markers CD31 and vWF, which are expressed in KS (Fig 3C), suggesting that the 2-D cell culture system may not faithfully represent the MEndT in tumors. PPT PowerPoint slide

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TIFF original image Download: Fig 3. KSHV infection initiates a differentiation process converting MSCs from mesenchymal phenotype to M/E hybrid state. (A) Cell lysates from mock- and KSHV-infected PDLSCs at indicated time points were immunoblotted for PDGFRA, COL1A1, α-SAM, TAGLN, PROX1, PDPN, VEGFA, and β-actin. (B) Relative mRNA levels of mesenchymal and endothelial related genes in mock- and KSHV-infected PDLSCs (K-PDLSCs) after 4 days infection. (C) Mock- and KSHV-infected PDLSCs (2-D) were immunostained for LANA, TAGLN, PDPN, vWF, and CD31 at 4 days post-infection. Scale bar, 50 μm. (D) A time course of K-PDLSC aggregating to form spheroid in non-adherent plates. Scale bar, 500 μm. (E) Expression of LANA, TAGLN, PDPN, vWF, and CD31 in mock- and KSHV-infected PDLSC spheroid (3-D) at 4 days post-infection. Scale bar, 50 μm. (F) The expression of endothelial markers in mock- and KSHV-infected PDLSCs under 2-D or 3D cell culture for 4d. (G) The mRNA expression level of TAGLN, α-SAM, Nestin, PDGFRA, PDPN, ICAM, PROX1, CD31, and VEGFA was analyzed by RT- qPCR in K-PDLSC spheroids in comparison with their parallel 2D culture. https://doi.org/10.1371/journal.ppat.1009600.g003 Three-dimensional (3-D) organotypic cultures allow the mimic function of living tissue and probably provide information encoded in tissue architecture. The 3-D culture was used in mesenchymal stem cells in that MSC spheroids display enhanced differentiation capability compared to 2-D culture [31,32]. We established a 3-D spheroid model by seeding mock- and KSHV-infected PDLSCs in a low attachment condition. As time went by, PDLSCs formed a decentralized network, and then numerous small aggregates progressively assembled into a single central spheroid (Fig 3D). Once aggregated, the spheroid did not change in size but was generally compacted. The expression spectrum of mesenchymal and endothelial cell markers in mock- and KSHV-infected PDLSC spheroids was examined by IFA. As shown in Fig 3E, the endothelial markers PDPN and CD31were induced, and mesenchymal marker TAGLN was decreased in KSHV-PDLSC spheroids compared with control spheroids. But PDGFRA expression remained unchanged between mock- and KSHV-infected PDKSC spheroids. The mesenchymal/endothelial marker profiles in 2-D culture and 3-D spheroids of KSHV-PDLSCs were compared using Western blot and RT-qPCR. Results showed that endothelial markers PROX1, PDPN, CD31, VEGFR2, and VEGFR3 increased, and the transcription of endothelial marker genes PDPN, ICAM, PROX1, CD31, and VEGFA were dramatically up-regulated in 3-D spheroids, confirming the occurrence of MEndT in KSHV-PDLSC spheroids (Fig 3F and 3G). Overall, the 3-D organotypic culture provides a suitable environment allowing the differentiation of KSHV-infected MSCs into hybrid M/E cells and demonstrating that KSHV infection of MSCs sufficiently induces the MEndT (or incomplete MEndT) process, generating hybrid M/E cells closely resembling the spindle cells in KS lesions.

KSHV-induced MEndT leads to incomplete endothelial differentiation Then, we asked if KSHV-induced hybrid M/E state cells have acquired functional characteristics of endothelial cells in addition to expressing endothelial markers. To this end, we evaluated KSHV-infected PDLSCs for their endothelial characteristics. Matrigel tubule formation assay was carried out for the acquisition of endothelial and angiogenesis properties and showed that KSHV-infected PDLSCs exhibited increased ability to form capillary-like structures in comparison to mock-infected PDLSCs (Fig 4A). The uptake of acetylated low-density lipoprotein (Ac-LDL) is a hallmark of endothelial cells and macrophages [33]. KSHV-infected PDLSCs were found to possess an Ac-LDL uptake capacity similar to HUVECs (Fig 4B). KSHV induced a notable outgrowth in KSHV-infected PDLSC spheroids whereas rare sprouting was observed in mock-infected PDLSCs (Fig 4C). Interestingly, KSHV-infected PDLSCs spontaneously formed many vessel-like structures in the 3D spheroids, but such structures were not seen in mock-infected PDLSC spheroids (Fig 4D). Immunofluorescence staining of these lumens showed the expression of pan-endothelial marker CD31 in KSHV-infected PDLSC spheroids but not in control spheroids (Fig 4E). PPT PowerPoint slide

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TIFF original image Download: Fig 4. The hybrid M/E state is enriched through KSHV-mediated MEndT and displays partial endothelial characteristics. (A) Tubule formation assays were performed with mock- and KSHV-infected PDLSCs (K-PDLSCs). Scale bar, 500 μm. (B) Mock- and KSHV-infected PDLSCs were incubated with DiI-acLDL for 4 hours. DiI-acLDL uptake (red), as well as KSHV-GFP infection (green), were analyzed with a fluorescence microscope. Scale bar, 100 μm. (C) Mock- and KSHV-infected PDLSC spheroids were embedded into Matrigel, and the sprouting length was analyzed by a Zeiss fluorescence microscope. Scale bar, 200 μm. (D) H&E staining of PDLSC and K-PDLSC spheroid sections. Scale bar, 50 μm. (E) The expression of CD31 in the spheroids of PDLSC and K-PDLSC was detected by IFA. Scale bar, 50 μm. (F) Mock- and KSHV-infected PDLSCs, along with HUVECs, were examined for Weibel–Palade bodies (WPBs) under a transmission electron microscope. Scale bar, 1 μm. Original magnification, ×2 (enlarged insets). https://doi.org/10.1371/journal.ppat.1009600.g004 Weibel–Palade bodies (WPBs) are mature endothelial cell-specialized organelles. To investigate the endothelial differentiation grade of KSHV-infected PDLSCs, we examined whether KSHV-infected PDLSCs displayed WPBs using a transmission electron microscope. Weibel–Palade bodies were not observed in both KSHV-infected PDLSCs and uninfected control PDLSCs (Fig 4F). Taken together, our findings indicate that KSHV infection can reprogram MSCs to acquire endothelial markers and functions through MEndT. However, KSHV-induced endothelial lineage differentiation is incomplete as cells retain specific mesenchymal markers and lack endothelial cell specialized organelles Weibel–Palade bodies (WPBs), exhibiting a striking resemblance to KS spindle cells [8].

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