KSHV is the causative agent of KS, a malignancy characterized by the presence of virus-infected spindle cells, mixed in with inflammatory cells and aberrant slit-like capillaries. The nature of KS spindle cells and exactly how KSHV promotes KS development is still controversial. Here we show that endothelial cells infected with KSHV lose expression of mature endothelial phenotypic markers and acquire phenotypic and functional markers of mesenchymal cells. We find that this transdifferentiation program is associated with activation of a group of transcription factors linked to EMT and is critically driven by KSHV-induced Notch signaling. Consistent with these observations, KSHV-infected spindle cells within KS biopsies display a complex and variable phenotype that includes endothelial and mesenchymal cell markers, express the EMT-inducing transcription factor ZEB1, and have evidence of Notch activation. These novel findings clarify the cell source of KS spindle cells and explain the reasons for previous controversy, provide the first example of virus-induced EndMT, and contribute new insights into KS pathogenesis and the mechanisms and potential roles of EndMT.
Activation of Notch signaling was previously reported in KSHV-infected cells (41
) and was attributed to a direct stimulatory effect by KSHV-RTA (32
), or to a Jag1/Dll4-mediated effect by vGPCR and vFLIP gene products (33
), but its role as a mediator of KSHV infection and disease has remained unclear. Notch signaling can regulate the expression of KSHV genes (42
) and promote quiescence in uninfected cells neighboring the KSHV-infected cells, thus providing a growth advantage to the KS cells (33
). In this view, KS cells provide the Notch ligands Jag1 and Dll4, which induce Notch signaling in uninfected cells. Other studies using the KS cell lines SLK or KS IMM, which are not infected with KSHV, or using a KSHV-infected, but E6-E7 immortalized lymphatic endothelial cell line suggested that Notch signaling provides a survival advantage to the cells (41
). The current results show that Notch signaling plays a critical role in promoting KSHV-induced EndMT and that this transformation confers on endothelial cells increased motility and resistance to death, a set of traits that likely underline the characteristic ability of KS cells to infiltrate locally. Increased VEGF (5
) likely contributes to KSHV-induced EndMT but does not explain the increased cell motility to the mesenchymal-selective promigratory factor PDGF (24
). Interestingly, VEGF contributes to EndMT during heart development (44
EndMT is deployed during morphogenesis of the heart enabling endocardial cells (with an endothelial phenotype) to generate the heart cushion cells from which the mesenchymal portion of cardiac septa and valves is formed (13
). Here, the EndMT process results in the generation of terminally differentiated cells. Similarly, the EndMT program has been linked to organ fibrosis and to ectopic tissue ossification from endothelial cells (15
). In cancer, EndMT was implicated as a source of cancer-associated fibroblasts, which may promote tumor progression by modifying the tumor micro-environment (18
). By contrast, EndMT induced by KSHV reprograms endothelial cells to acquire either a mixed endothelial–mesenchymal phenotype or predominantly mesenchymal phenotype, without progression to mature fibroblasts, fat, cartilage, or bone cells. This maturation block is determined by KSHV itself, and it is likely attributable to KSHV-LANA inactivation of glycogen synthase kinase 3, which regulates key participants in cell differentiation (26
). Consistent with this phenotype, KS lesions do not normally include fibrous, cartilage, bone, or fat tissue, and the virus-infected KS cells display a complex endothelial–mesenchymal phenotype that is not encountered in normal tissues. Thus, KSHV-induced EndMT is more similar to EMT occurring in epithelial malignancies in displaying different stages of mesenchymal cell differentiation within a tumor (23
). Occasionally, aggressive KS has been associated with “woody” cutaneous edema and fibroma-like nodules (45
). The pathogenesis of these lesions has remained unclear. Our results suggest the possibility that EndMT may lead KS cells to differentiate into fibrous tissue, perhaps in conjunction with loss of viral infection.
TGFβ2 is an inducer of EntMT in development and in mature endothelial cells (18
), but we find no evidence that TGFβ signaling is deployed in KSHV-induced EndMT. This is consistent with the observation that the TGFβ signaling is blocked in KSHV-infected cells by epigenetic mechanisms (47
). Other factors from the KS microenvironment (perhaps the inflammatory component) may contribute to KSHV-induced EndMT, as KS spindle cells are not fully autonomous and require growth factors from the microenvironment for growth (5
). Activation of an EMT program in tumor cells often relies on the interplay between cancer cells and the neighboring stroma (23
), and many factors contribute to EMT, including fibroblast growth factor, hepatocyte growth factor, IL-6, and insulin-like growth factors (22
Signaling downstream of TGFβ converge in the stimulation of the EndMT-inducing transcription factors Slug and Snail, which coordinate change in lineage marker expression and function (19
). These and other transcription factors, including ZEB1, ZEB2, and Twist are effectors of EMT (22
). Notch signaling can induce expression of Snail, Slug, and ZEB1 (28
). ZEB1 can reciprocally activate Notch signaling, and Snail and Twist cooperate in the regulation of expression and stability of ZEB1 during EMT (50
). We find increased expression of Snail, Slug, Twist, ZEB1, and ZEB2 in KSHV-induced EndMT, and link expression of Snail, ZEB1, and ZEB2 to Notch signaling regulation. ZEB1 is abundant in KS lesions, including the KSHV-infected spindle cells where the nuclear localization is consistent with activity.
In spite of often displaying an indolent clinical course, KS can be aggressive and difficult to manage in patients with AIDS. We find that KS cells have phenotypic traits consistent with EndMT, and that KSHV can induce Notch-dependent EndMT in vitro. Mesenchymal transformation likely plays an important role in KS pathogenesis as it does in many cancer types, suggesting that inhibition of Notch signaling should be considered for the treatment of KS.