Instigation of Notch Signaling in the Pathogenesis of Kaposi's Sarcoma-associated Herpesvirus and Other Human Tumor Viruses

Fang Cheng; Pirita Pekkonen; Päivi M Ojala


Future Microbiol. 2012;7(10):1191-1205. 

In This Article

Biological Significance of Notch Signaling in KSHV-associated Pathologies

Notch Engagement in KSHV-associated Tumors & in Experimental KS Models

Human KS tumors overexpress multiple Notch pathway components, including the ligand Jagged1, the Notch receptors 1, 2 and 4 and the targets HES-1 and HEY-1.[49] In line with this, KSHV-infected primary lymphatic endothelial cells (K-LECs) have been shown to express several Notch components at the mRNA level, namely JAG1, DLL4, NOTCH4, HEY1 and HES1.[50] In addition, studies by Curry et al. indicate that the growth of primary KS tumor cells and experimental lesions in mice can be inhibited by blocking Notch signaling using γ-secretase inhibitors (GSIs),[49,51] which block the processing of Notch and thus production of NICD1. These data suggest that activated Notch signaling might be important for the survival of the KS tumor cells (Figure 2). It is important, however, to note that GSIs have other targets besides the four Notch receptors.[52] Therefore, further experiments with more specific targeting of the Notch pathway (e.g., by RNAi) complemented with restoration of expression of NICD are necessary to validate the need of Notch for the growth of KSHV-infected cells.

Figure 2.

Notch involvement in Kaposi's sarcoma-associated herpesvirus pathogenesis.
Notch pathway activation controls multiple steps of the KSHV pathogenesis. (A) KSHV-induced Notch promotes and is needed for survival and proliferation of the KS tumor cells, PEL cells and KSHV-infected cells both in culture and experimental mouse models. (B) vGPCR- and vFLIP-mediated Notch activation results in mesenchymal reprogramming and endothelial-to-mesenchymal transition of the KSHV-infected LECs. (C) Notch activity can counteract VEGF-C-dependent (lymph)angiogenesis in KSHV-infected endothelial cells. (D) The KSHV early lytic gene RTA controls the viral lytic replication cycle and virus production through interaction with the Notch effector RBP-Jκ.
KS: Kaposi's sarcoma; KSHV: Kaposi's sarcoma-associated herpesvirus; LEC: Lymphatic endothelial cell; PEL: Primary effusion lymphoma.

Notch has also been implicated in the pathogenesis of the other KSHV-associated malignancies, such as PEL. The activated intracellular domain of Notch1 (NICD1) was shown to aberrantly accumulate in latently KSHV-infected PEL cells and result in increased proliferation (Figure 2).[53] Accumulation of NICD1 was dependent on LANA, which was shown to interact with SEL10, which is an E3 ligase and mediates the ubiquitin-dependent degradation of NICD. LANA interrupts this ubiquitin-dependent degradation pathway by competing with NICD for the association with SEL10.[54] Growth of the KSHV-infected PEL cell lines BCBL-1, BC-3 and JSC-1 in vitro was dramatically arrested at the G1 phase by treatment with GSIs.[53] Furthermore, GSI treatment resulted in necrosis as well as apoptosis in tumors generated by the xenotransplanted PEL cell lines. This implies that targeted downregulation of abnormal Notch signaling could have therapeutic potential for KSHV-related PELs.[55]

KSHV has evolved specific mechanisms to induce and upregulate the Notch ligands in lymphatic endothelial cells (LECs). Jagged1 expression is mostly upregulated in late-stage KS lesions, where latent infection predominates.[49] In endothelial cells, the expression of Jagged1 is known to be regulated by the NF-κB pathway.[56] Fittingly, expression of the latent KSHV gene product vFLIP, a well-established inducer of NF-κB signaling, leads to NF-κB-dependent Jagged1 expression in LECs. Furthermore, another Notch ligand, DLL4, is upregulated by the viral lytic protein vGPCR via the activation of ERK. Both vFLIP and vGPCR activate Notch signaling through the Notch4 and Notch1 receptors (Figure 1). Induction of these Notch ligands in KSHV-infected LECs leads to Notch activation and cell cycle quiescence in the neighboring uninfected cells and promotes the survival of the infected cells.[50] Liu et al. have also demonstrated ERK- and NF-κB-dependent upregulation of DLL4 and Jagged1 by vFLIP in human umbilical vein endothelial cells and by vGPCR in 293T cells, respectively.[57] Notably, and in contrast to the findings by Emuss et al.,[50] in this report, expression of vFLIP was shown to upregulate DLL4, whereas vGPCR induced Jagged1, suggesting that the engagement of different Notch pathway components could be cell type-specific.

Notch in Mesenchymal Reprogramming by KSHV

LECs are derived from venous blood endothelial cells (BECs) during embryonic development. The LECs retain a plastic, reprogrammable phenotype, and the identity of LECs can be reverted to BECs by subtle alterations in the expression of a few master regulators of the LEC fate (Notch signaling, Coup-TFII and homeobox transcription factor Prox1, among others).[58,59] BEC/LEC lineage switches have been observed in various human malignancies, but the LEC plasticity is thought to be limited to different endothelial cell types. Recent findings from our group and others suggest LEC plasticity beyond the endothelial fate by demonstrating that KSHV-induced Notch signaling can modify the LEC transcriptome and thereby the cell identity.[57,60,61] The reprogramming of primary LECs occurs via KSHV-induced endothelial-to-mesenchymal transition (EndMT) through Notch-dependent signaling elicited by vFLIP and vGPCR (Figure 2).[60] EndMT resembles EMT, a process implicated in tumor invasion and metastasis.[62] In our system, the virus-induced EndMT requires a 3D cross-linked matrix where cell-to-cell interactions in vivo are mimicked by assembling the KSHV-infected primary LECs into 3D spheroids embedded in clotted fibrin scaffolds. When LECs are grown on tissue culture dishes (2D culture), KSHV infection induces spindle formation in the cells,[63] which leads to activation of Notch signaling[50] and downregulation of LEC markers,[64,65] as well as reprogramming towards BECs.[66] In our experimental model, EndMT is not detected in the primary 2D K-LECs,[60] thus exemplifying the context-dependent consequences of Notch signaling. This is in contrast to the reports by Liu et al.[57] and Gasperini et al.[61] demonstrating the capability of KSHV-activated Notch to induce mural cell characteristics and EndMT already in 2D. This apparent discrepancy could be due to the differences in experimental systems, as Liu et al. used oncogene (HPV E6/E7)-immortalized LECs, whereas in the study by Gasperini et al., the authors used dermal microvascular endothelial cells that represent a mixture of BECs and LECs. These findings, however, further underscore the context-dependent role of the Notch pathway in KSHV pathogenesis.

Our work with the 3D K-LECs also reveals a previously unrecognized signaling axis by demonstrating Notch to be the upstream regulator of MT1-MMP. EndMT and the upregulation of membrane type 1 matrix metalloproteinase leads to increased invasiveness of the K-LECs, higher viral load and possibly malignant progression.[60] As the KS tumor cells have been known to express markers of the mesenchyme for a long time, in addition to the well-characterized endothelial component, it is possible that KSHV-induced EndMT is the source of the heterogeneity in the KS tumors and plays an important role in the KS pathogenesis. Endothelial cells undergoing EndMT have recently been identified as a source of cancer-associated fibroblasts (CAFs) or stem cells in experimental tumor model systems.[67,68] CAFs produce growth factors, chemokines and extracellular matrix and behave as key determinants of the tumor microenvironment and malignant progression of cancer. In light of these recent findings, investigations of Notch-related mechanisms in KS progression in the context of CAFs and even cancer stem cells are highly relevant to pursue.

Notch–VEGF Interplay & KSHV Pathogenesis

The Notch pathway is intimately involved in both physiological and pathological tumor angiogenesis.[48,69,70] During physiological angiogenesis, VEGF-A induces DLL4 in the sprouting endothelial tip cells, leading to Notch pathway activation in the adjacent stalk cells. This leads to downregulation of the receptor for VEGF-A (VEGFR-2) in the stalk cells and inhibition of excess branching and angiogenesis.[71,72] A recent report, however, fundamentally changed the understanding of the cross-talk between Notch and VEGFR-2 in angiogenesis. By using inducible loss-of-function mouse models for Dll4 and VEGFR-2 in combination with inhibitors in vivo, this work demonstrates that a substantial level of endothelial Notch signaling is maintained without VEGFR-2 function. While deletion of the Dll4 gene prominently increased sprouting and vascular density, this increase in angiogenesis was not abolished by also targeting Vegfr2 (Kdr and Flk1). By contrast, VEGFR-3, the main receptor for VEGF-C, was strongly modulated by Notch, which allowed VEGFR-3 to induce angiogenesis only in cells with low levels of Notch activation.[73] Further proof of the intimate interplay between Notch and VEGFs is provided by studies demonstrating that Notch signaling antagonizes VEGF function and inhibits angiogenic sprouting of human umbilical vein endothelial cells (BECs)[74] and lymphangiogenic sprouting of LECs.[75] The Notch pathway may also play a role in the highly vascular phenotype of KS tumors[49,57] and in KSHV-induced lymphangiogenesis.[76] Interestingly, and in support of the antagonizing role of VEGFs on Notch, stimulation of K-LECs by VEGF-A/C induces a dramatic outgrowth of capillary sprouts in 3D, and a simultaneous inhibition of EndMT (Figure 2).[60] This suggests that the mesenchymal sprouting of K-LECs represents a distinct biological process that suppresses (lymph)angiogenic sprouting in the absence of VEGF-A/C stimulation. These results further support the central role of Notch signaling in cell fate determination, in this case between the lymphangiogenic and mesenchymal cell fates. This may also partially explain the heterogeneity of the cell types within the KS tumors in vivo, as the fate of the infected cells may be controlled through the balance of extrinsic factors and intrinsic properties of the infected cells.

RBP-Jκ as an Important Determinant of the KSHV Replication Program

RTA is necessary for the switch from latency to active lytic replication in the KSHV life cycle. RTA interacts with RBP-Jκ, the major downstream effector of the Notch signaling pathway.[77–79] This binding is essential for viral reactivation as demonstrated by infection of RBP-Jκ-knockout murine fibroblasts. Although KSHV was able to establish latency in the RBP-Jκ-knockout murine fibroblasts, RTA was not capable of transactivating lytic promoters, leading to a completely abolished lytic reactivation. This could, however, be restored by ectopic expression of RBP-Jκ.[80]

Interaction of RTA with RBP-Jκ results in the replacement of RBP-Jκ's intrinsic repressive action and activation of its target promoters in the viral genome. The activation is mediated by the C-terminal domain of RTA that binds to the central repression domain of RBP-Jκ,[77] an interaction site also used by the NICD fragment. In addition, RTA also binds the N-terminal domain of RBP-Jκ. It is tempting to speculate that upon interaction with RBP-Jκ, RTA could replace the repressor complex at the target site and thereby convert RBP-Jκ from repression to activation (see Figure 1 for a depiction of this hypothesis). However, there is no experimental data supporting this hypothesis to date. RBP-Jκ cannot activate viral transcription in the absence of RTA as demonstrated by the use of a constitutively active fusion protein RBP-Jκ/VP16, which did not transactivate viral promoters independently, but needed interaction with RTA for increased binding affinity to its recognition sites on the viral promoters. This interaction requires RTA to bind the DNA. However, the transactivation function of RTA is not needed since cotransfection of RBP-Jκ and an RTA mutant lacking the transactivation domain robustly restored transcription of the lytic genes. Full transactivation capacity therefore requires intact DNA binding sites for the RBP-Jκ–RTA complexes at the viral promoters and the formation of the trimeric complex RBP-Jκ–RTA–viral DNA.[81,82] A further demonstration of the importance of RBP-Jκ in coordinating the KSHV lytic gene expression program came from a recent extensive search of predicted RBP-Jκ binding motifs in the KSHV genome. At least 260 matches were found in the KSHV genome, including previously unidentified ones that demonstrated RBP-Jκ response to RTA. This approach further corroborated the role of the Notch effector in coordinating the KSHV lytic gene expression and will help to define a signature motif for functional RBP-Jκ sites within the viral genome.[83] Although expression of a constitutively active form of the endogenous upstream regulator of RBP-Jκ, NICD1, can also cooperate with RTA,[81] only RTA, and not NICD1 alone, was shown to be capable of transactivating productive cycle promoters in reporter assays in uninfected cells.[81,82] Interestingly, two reports have demonstrated robust induction of expression of a number of viral genes in response to NICD introduction.[84,85] In one of these, however, inducible expression of NICD was not capable of evoking the full repertoire of lytic viral gene expression and thereby lytic replication.[84,85] One possible explanation for the inability of activated Notch to fully reactivate KSHV is that RBP-Jκ is not broadly and constitutively bound to KSHV DNA during latency in the absence of RTA expression.[81,82] In fact, RTA seems to engage Notch signaling in a ligand-independent manner, suggesting that it may be interacting with the noncanonical rather than the canonical Notch pathway.

Another viral lytic gene product, vIRF4, was recently identified to be a novel interaction partner of RBP-Jκ. vIRF4 binds the hydrophobic pocket of RBP-Jκ through a short peptide motif that closely resembles a motif found in NICD. It is therefore possible that vIRF4 may compete with Notch for RBP-Jκ binding and thereby antagonize Notch signaling (Figure 1).[86] The biological consequences of this interaction are currently unknown.

KSHV exploits the Notch signaling pathway not only to promote lytic gene expression, but also to control the balance between establishment of latency and viral reactivation. LANA is the major protein expressed during KSHV latency[87,88] and is needed to tether the KSHV episome/genome to the host cell chromosomes, for episomal DNA replication and to ensure segregation of the new episomes into daughter cells during mitosis.[89,90] Lan et al. have described a feedback mechanism[91] where RTA contributes to the establishment of KSHV latency by activating LANA expression at the early stages of infection, utilizing RBP-Jκ to transactivate the LANA promoter.[92] In addition, LANA engages Notch signaling to actively establish and maintain this latency. This was reported to involve antagonizing NICD function via interaction with RBP-Jκ,[85] which occurs through a recently identified conserved RBP-Jκ binding domain in the LANA protein (Figure 1).[93] This interaction competes with RTA binding to RBP-Jκ, and impedes the ability of RTA to autoactivate its own promoter. Taken together, the Notch signaling pathway appears to be a critical component of the LANA–RTA feedback loop in the control of KSHV reactivation.

Besides directly affecting KSHV viral lytic gene expression, Notch signaling has been implicated in modulating the host cellular microenvironment to be more favorable for virus replication and viral reproduction. Our recent work with the 3D K-LECs reveals that the Notch-induced EndMT is accompanied by an increase in viral loads, as well as in the expression of a panel of lytic (e.g., vGPCR and vIL-6) and latent genes (e.g., LANA and vFLIP) over the parental 2D K-LEC cultures (Figure 2).[60] This suggests that the cell–cell interactions and increased Notch signaling in the 3D culture provided a favorable milieu for virus replication and maintenance of infection. This was supported by a reduction in expression of lytic genes following inhibition of Notch by a GSI, DAPT. KSHV RTA can also regulate RBP-Jκ-mediated cellular gene expression of the surface glycoproteins CD21 and CD23a, which was shown to facilitate coinfection of the cells by another gammaherpesvirus EBV, and to provide a favorable milieu for viral reproduction.[94,95]

In summary, KSHV exploits the Notch pathway components to control the viral replication cycle, proliferation, survival and differentiation of infected cells, as well as to modulate the host cell microenvironment (see Figure 2). Given that Notch has a key role in many fundamental processes in mammalian development, it is perhaps not surprising that KSHV has evolved to employ this conserved signaling pathway to advance the critical steps of its pathogenesis.