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Herpesvirus-Encoded G Protein-Coupled Receptor Signaling and its role in the Modulation of Glioblastoma Multiforme
Fan, T.S.
2019
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Fan, T. S. (2019). Herpesvirus-Encoded G Protein-Coupled Receptor Signaling and its role in the Modulation of
Glioblastoma Multiforme.
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Chapter II
Review
Viral G Protein-Coupled Receptors as
Modulators of Cancer Hallmarks
Tian Shu Fan1*, Jeffrey R. van Senten1*, Marco Siderius1, Martine J. Smit1
1Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal
Chemistry, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands
2
* T.S. Fan, and Jeffrey R. van Senten contributed equally to this work.
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Abstract
Herpesviruses encode transmembrane G protein-coupled receptors (GPCRs), which share structural homology to human chemokine receptors. These viral GPCRs include KSHV-encoded ORF74, EBV-encoded BILF1, and HCMV-encoded US28, UL33, and US27. Viral GPCRs hijack various signaling pathways and cellular networks, including pathways involved in the so-called cancer hallmarks. This
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eventually affects host cell proliferation, replication, and survival. In this review, we discuss how viral GPCRs hijack certain signaling routes, which play a role in the cancer hallmarks as recognized by Hanahan and Weinberg.
1. Introduction 1.1. Herpesviruses
Herpesviridae are ubiquitous double stranded DNA viruses, establishing life-long infections in their host that typically are asymptomatic. The latency of the infection allows these viruses to persist without being eliminated by the host’s immune system. Sporadically, upon decreased activity of the immune system, herpesviruses reactivate to ensure intra- and interhost virus spread. In immunosuppressed hosts or in infants with pre-mature immune system, inadequate immune surveillance can result in the development of severe pathologies upon reactivation of these opportunistic pathogens [1], [2].
Based on variation in their conserved glycoprotein H (gH) genes, herpesviruses are categorized in three subfamilies [3]. Human simplex virus (HSV) 1, HSV2 and varicella zoster virus form the α subfamily, human cytomegalovirus (HCMV) and Roseoloviruses HHV6 and HHV7 are β herpesviruses, and the γ subfamily consists of the Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV).
1.2. Herpesviruses and their GPCRs
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activated through ligand binding and couple predominantly to Gαi proteins. However,
viral homologs are fundamentally different and exhibit broad-spectrum G protein-coupling and constitutive activity [7].
Enhanced and/or constitutive (chemokine) GPCR signaling, via aberrant expression of GPCR ligands or increased expression of or mutations in cellular GPCRs or G proteins, has been linked to various pathological conditions and is frequently observed in cancer [8], [9]. This review focuses on modulation of carcinogenesis by the cancer-associated viruses EBV, HCMV and KSHV through their viral GPCRs. Even though the Roseoloviruses HHV6 and HHV7 both encode viral chemokine receptors, some of which are constitutively active, lack of correlation of receptor activity to cancer-related signaling prompted us to exclude these from the current work.
1.2.1. Kaposi’s sarcoma-associated herpesvirus
Involvement of viral GPCRs in oncogenic development is most clearly manifested in KSHV-driven malignancies; Kaposi’s sarcoma (KS), primary effusion lymphomas (PEL) and multicentric Castleman’s disease (MCD) [10], [11]. KSHV’s genome is around 140 kbp in size, which encodes over 80 open reading frames (ORFs), including one viral GPCR; ORF74 (also known as vGPCR) [12], [13]. This virally encoded GPCR is the driver of KSHV-associated malignancies and associated angio-proliferative phenotype [14]–[16]. ORF74 both displays ligand-induced as well as constitutive activity, coupling to Gαi, Gα13 or Gαq (Figure 1) [17]–[22]. The
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1.2.2. Human Cytomegalovirus
HCMV is the human herpesvirus with the largest genome (approximately 240 kbp). Although the size of its proteome is not yet fully understood, HCMV is estimated to encode over 200 proteins [33]. HCMV gene products (proteins and RNAs) are present in malignancies of diverse origin, e.g. breast cancer [34], colorectal cancer [35], glioma [36], lung cancer [37], mucoepidermoid carcinoma [38], neuroblastoma [39] and prostate cancer [40]. Unlike KSHV, HCMV is considered to be an oncomodulator, aggravating rather than driving tumorigenesis [41]. The HCMV genome contains four GPCR-encoding genes (UL33, UL78, US27 and US28), which were acquired via three gene capture events [42]. US27 and US28 originate from a single host gene and are products of gene duplication and subsequent divergence [43]. US28 is the only HCMV-encoded GPCR currently known to interact
Figure 1. Human herpesvirus-encoded GPCRs. GPCRs encoded by the human β- and γ-herpesviruses
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with chemokines (Figure 1) [44], [45]. UL33, US27 and US28 exhibit constitutive G protein-dependent signaling [46]–[48]. Where UL33 and US28 promiscuously couple to G proteins (Gαs, Gαi and Gαq, and Gαi, Gαq and Gα12/13, respectively) [46],
[48]–[52], US27 signaling is mediated via Gβγ heterodimers but the corresponding Gα class remains unknown [47]. Thus far, signaling by UL78 has not been reported. Nonetheless, similar to UL33, heteromerization of UL78 with both HCMV-encoded as well as host receptors influences cellular [53].
US28 protein is detected in tumor tissue samples derived from both glioblastoma and colorectal cancer patients [54]–[57]. Moreover, increased levels of US28 correlate with histological grade and reduced survival of colorectal cancer patients. Using transgenic mice and orthotopic xenograft models for glioblastoma or colon cancer, the capacity of US28 to accelerate tumor formation has been highlighted [57], [58]. Co-expression of an US28 agonist CCL2 augmented the development of neoplasms upon transgenic US28 expression in mouse intestinal endothelial cells, suggesting a contribution of ligand-mediated signaling [58]. Notably, tumor growth induced by US28 is not solely mediated via G protein signaling, as G protein-uncoupling of the receptor could not completely abolish US28-dependent growth [49]. Modulation of tumor formation by UL33, UL78 or US27 has not been reported.
1.2.3. Epstein-Barr Virus
The genome of EBV is around 170 kbp in size, encoding 84 genes. EBV is detected in B lymphocytes of Burkitt’s lymphoma patients (Epstein and barr 1964) and other tumors, including (non-)Hodgkin’s lymphoma, and epithelial cell tumors, gastric carcinoma [59]–[61] and nasopharyngeal carcinoma. Additionally, other EBV-related diseases have been described, e.g. infectious mononucleosis and post-transplant lymphoproliferative diseases.
Epithelial cells and circulating B-cells are sites of latent EBV infection in asymptomatic individuals [62]. It is hypothesized that EBV employs B-cell maturation pathways to achieve life-long persistence [63]. Consequently, infection of EBV in epithelial cells cannot be achieved in the absence of B lymphocytes [64]. EBV encodes a single viral GPCR, BILF1, which is expressed in various EBV-positive cell lines [65]. BILF1 remains an orphan receptor to this day and constitutively signals through Gαi proteins (Figure 1) [65], [66]. In NIH-3T3 cells, BILF1 expression induces
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also comprises of a G protein-independent component as indicated using non-G protein coupling mutants [67].
2. Herpesvirus-encoded GPCR biology and cancer hallmarks
In this review we link viral GPCR biology to the hallmarks of cancer, which describe ten key alterations in cell physiology enabling or supporting malignant growth [68]: sustaining proliferative signaling; evading growth suppressors; activating invasion and metastasis; enabling replicative immortality; inducing angiogenesis; resisting cell death; avoiding immune destruction; deregulating cellular energetics; genome instability and mutation; tumor-promoting inflammation
Although cancer hallmarks are listed as separate processes, signal transduction stimulating one hallmark may also affect other hallmarks due to integration into signaling networks. Identifying a single signal transduction pathway involved in a particular aspect of oncogenesis often is an oversimplification as signaling pathways are interconnected in networks. Examples of signaling hubs, modulated by viral GPCRs, affecting multiple hallmarks are depicted in Figure 2. Furthermore, it is important to note that the overall signaling output of a viral receptor strongly depends on the specific cellular background, resulting in different composition or priming of signaling networks. For example, viral GPCR-mediated signaling towards AKT may have different consequences based on the basal activity of the AKT kinase or the expression level/activity of the PTEN phosphatase counteracting AKT in a cell.
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Communication and Signaling (2016) 14:31).
2.1. Sustaining proliferative signaling
Sustained proliferative signaling is the most fundamental property of cancer cells. Different strategies are exploited to mediate growth independent from external growth factors. These include increasing the level of growth signals, tweaking the amount of growth factor receptors expressed on the cell surface, or affecting the downstream signaling pathways. In healthy tissue, homeostasis of growth-promoting signals is tightly regulated – ensuring a steady-state. The underlying processes controlling tissue integrity and architecture are regulated by endogenous (genetic) factors, as well as by paracrine signals. Many of these processes are disrupted in cancer cells [69].
PI3K/AKT signaling – The PI3K/AKT signaling pathway is involved in regulation
of cell growth, differentiation, and development. Consequently, it has often been found to be deregulated in cancer cells [70]. Signaling through this axis involves GSK3 and mTOR kinases, the activity of the latter is regulated by tuberous sclerosis complex (TSC).
ORF74 constitutively promotes proliferative signaling by activation of the TSC2/ mTOR pathway in an AKT-dependent manner. PI3K or mTOR inhibition reduces
Figure 2. Modulation of cancer hallmarks by GPCR-mediated cellular signaling pathways. Signal
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SVEC cell proliferation induced by ORF74 [71]. Since AKT activity is also increased in bystander endothelial cells in ORF74-induced tumors, Sodhi and co-workers postulated involvement of paracrine signaling through various cytokines (e.g. IL-6 and IL-10), angiogenic factors (e.g.VEGF1), growth factors (e.g. PDGF) and chemokines (e.g. CXCL8, CXCL9 and CXCL12) know to be excreted from ORF74 expressing cells [71]. Indeed, stimulation with conditioned medium from ORF74-expressing NIH-3T3 cells activates the AKT pathway in SVEC and primary endothelial cells, which could be blocked using the mTOR inhibitor Rapamycin [71], [72]. Moreover, S6 ribosomal protein phosphorylation, which is involved in cell cycle progression, was enhanced upon stimulation of endothelial cells with individual secreted factors at concentrations found in conditioned media from cells expressing ORF74 [71], [72].
MAPK/ERK signaling – Activation of the MAPK/ERK pathway is instrumental in
various cellular processes, including cell cycle regulation, survival, and cytokine secretion. Therefore, it is not surprising that deregulation of this pathway contributes to the progression of cancer [73].
ORF74 stimulates proliferation in COS-7 cells which is sustained via activation of the inositol phosphate (InsP)/PKC cascade involving Ras in a ligand-independent manner [74]. Modulation of MAP/ERK signaling by HCMV-encoded US28 has not been associated with enhanced proliferation, survival or cytokine secretion. However, CCL5-induced US28 activity resulted in Gα12-mediated ERK activation in
the context of smooth muscle cell migration [75], illustrating the context-dependency of viral GPCR-mediated signaling.
AP-1 signaling – Activation of transcription factor AP-1 has been linked to
proliferation in several types of cancer [76].
ORF74 activates AP-1 in PEL cells via two distinct pathways by coupling to Gαi,
signaling via the PI3K/AKT-Src axis, and by stimulating Gαq, activating ERK1/2
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viral GPCR input with particular cellular responses as output.
Hippo signaling – The Hippo pathway is involved in the control of tissue
homeostasis and organ size by regulating tissue-specific stem cells. This pathway plays a prominent role in tissue repair and regeneration, while dysregulation of the Hippo pathway is associated with cancer development. The activity of this pathway is mediated by specific sets of protein kinases, the MST1/2 kinases phosphorylate and activate Lats1/2 kinases, which in turn regulate the phosphorylation of YAP/ TAZ transcription factors. Various genes involved in cell survival and proliferation are regulated by YAP/TAZ.
ORF74 activates signaling to Lats1/2 via Gαq/11, Gα12/13, and RhoA, promoting
proliferation and migration in HEK293 and SVEC cells [79]. The importance of this pathway in ORF74-mediated oncogenesis was illustrated using YAP inhibitor Verteporfin, which impaired ORF74-mediated transformation and survival [80].
β-catenin signaling – β-catenin regulates genes involved in embryonic
development and tissue homeostasis regulated via Wnt. The canonical or Wnt/ β-catenin pathway drives β-catenin translocation to the nucleus in the presence of Wnt, acting as a transcription co-activator of TCF/LEF. Aberrant activation of β-catenin contributes to tumorigenesis by promoting cell cycle progression.
ORF74 and US28 are two viral GPCRs implicated in the activation of β-catenin. ORF74 enhances stability and nuclear translocation of TCF/LEF transactivator β-catenin in HUVEC cells [81]. Furthermore, it enhances TCF/LEF-driven transcription and mRNA levels of β-catenin target genes cyclin D1, MMP-9 and VEFGA. Effects of PI3K inhibition on TCF/LEF signaling suggest involvement of AKT-mediated inactivation of GSK-3β and stabilization of β-catenin in ORF74-driven Wnt signaling. HCMV-encoded US28 also modulates β-catenin signaling. This receptor stimulates inactivation of GSK-3β and concomitant activation of β-catenin and upregulation of Wnt target genes cMYC, survivin and cyclin D1 at mRNA and protein levels in mouse intestinal epithelial cells [58]. Moreover, HCMV infection elevates active β-catenin levels and TCF/LEF-mediated transcription in an US28-dependent manner in HFF and U373 glioblastoma cells, respectively [50]. Expression of US28 in HEK293T and NIH-3T3 cells enhances TCF/LEF-driven transcription via coupling to Gαq and Gα12/13 proteins and requires activation
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activity of β-catenin, signaling events involved appear to be distinct. Activation of β-catenin by ORF74 involves PI3K but is independent from COX-2 [81], whereas COX-2 inhibition but not PI3K inhibition reduced US28-mediated TCF/LEF-driven transcription [50].
CREB signaling – Cyclic AMP response element (CRE) is a transcription
regulatory sequence in the promoters of over hundred human genes. In tumor biology, activation of CRE binding protein (CREB), downstream of many growth related signaling pathways, is associated with tumor progression and CREB downregulation reduces cellular proliferation [82].
CREB activity is modulated by multiple viral GPCRs, i.e. BILF1, ORF74, UL33 and US28. Whether modulation is stimulatory or inhibitory depends on the predominant G protein-coupling of these receptors, as CREB can be differentially regulated via Gαproteins (Gαi, Gαs and Gαq). Furthermore, modulation of other CREB modulating
activities (e.g. PKA or PDE activity) may result in cell context-dependent effects. ORF74 promotes constitutive activation of CREB in COS-7, HEK293 and PEL cells [51], [77], [83]. In HEK293 cells, this activity can be potentiated upon CXCL1 stimulation or impaired by CXCL10 via modulation of either PLC, PKC and MAP kinases p38 and p44/42, but not Gαi [51]. Constitutive ORF74-mediated CREB
activation in PEL cells, however, is dependent on both Gαi and Gαq. PI3K and Src
are employed downstream of Gαi, whereas the circuitry leading from Gαq to CREB
runs via ERK1/2 [77]. On the contrary, in NIH-3T3 cells ORF74 inhibits cAMP production in a Gαi-dependent manner [67]. Although Gαi is generally expected to
inhibit adenylyl cyclase and thereby inhibit CREB, pertussis toxin (PTX)-sensitive activation of CREB is thought to be partly mediated by Gβγ as scavenging of Gβγ subunits reduced PTX sensitivity [77]. US28-expressing COS-7 and HEK293 cells show increased CRE-driven transcription, which can be negatively regulated by CX3CL1 stimulation [77], [83]. In HEK293 cells, the activation of CREB by US28 occurs mechanistically similar to ORF74, being sensitive to inhibition of PLC, PKC and MAP kinases p38 and p44/42 [51]. Moreover, ectopic US28 expression or HCMV (Towne) infection activate CREB in neuronal precursor cells, a model relevant for HCMV infection [84]. In addition to US28, HCMV encodes a second GPCR capable of triggering CREB activation [48]. UL33 stimulates CRE-driven transcription, comprising of both stimulatory Gαs and inhibitory Gαi signaling, when
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G proteins of the Rho family and p38. Furthermore, UL33 contributes to HCMV-mediated CREB activation upon infection of U373 glioblastoma cells. US27 failed to modulate CREB in COS-7 cells, hitherto the only report regarding US27 affecting CREB [85]. Constitutive coupling of BILF1 to Gαi negatively regulates cAMP
production in HEK293 and NIH-3T3 cells and inhibits CREB activity in HEK293 and COS-7 cells [65], [66], [86]. When evaluated in cell types relevant to EBV pathology, however, BILF1 stimulates CREB activity via activation of Gαi in Burkitt’s
lymphoma cell line HH514.c16 and lymphoblastoid B-cell line JY [65].
2.2. Evading growth suppressors
Cancer is usually characterized by cells which are able to uncontrollably divide. Therefore, tumor cells have to become insensitive to growth suppressors during cell division [68], [87]. Mutations in the tumorsuppressor p53 and molecular on/ off switch K-RAS, both major players in control of apoptosis and proliferation respectively, have most frequently been associated with the development of cancer [88]–[90]. These oncogenes are pivotal for the ability of cancer cells to ignore growth suppression signals.
To examine effects of HCMV-encoded GPCRs affecting insensitivity to growth suppressors, Tu et al. 2014 evaluated the effect of US27 on the expression of tumor suppressor p53 using a PCR array [91]. Expression of US27 correlated to downregulation of the transcripts of several p53 target genes. In HEK293 and HeLa cells US27 expression diminished protein levels were observed for cell cycle regulators p21 and SESN. Hence by this means this viral GPCR may have an impact on host cell physiology and influence growth.
2.3. Activating invasion and metastasis
Metastatic tumor formation is the main cause of high mortality rate of cancer patients. In order to metastasize, tumor cells gain migratory and invasive capabilities losing their cell-to-cell and cell-to-basement membrane contacts, and make use of matrix metalloproteinases (MMPs) to degrade the extracellular matrix (ECM). Metastasis is strongly depending on the expression of chemokine receptors on tumor cells and production of cognate chemokines at secondary sites [92].
Invasive phenotype – US28 expression in tissue derived from patients with
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invasiveness [54], [84]. Stimulation with US28 agonist CCL5 strengthened the invasive phenotype in glioblastoma cells, whereas US28 silencing in HCMV-infected glioblastoma cells prohibited CCL5-induced invasion [84]. The molecular mechanism underlying US28-promoted invasiveness remains to be elucidated.
Cell migration – Regulation of FAK by ORF74 is associated with increased
endothelial cell migration and inhibition of anoikis, contributing to the invasive phenotype. Anoikis describes programmed cell death which occurs as a response of cells losing contact to the ECM [93]. ORF74 expression in endothelial cells induces FAK-dependent phosphorylation of ERK1/2, which regulates membrane protrusions and focal adhesion turnover, and activates transcription factors AP-1 and NF-κB upon integrin engagement [94]. The latter transcription factors regulate the expression of a number of genes involved in migration e.g. cytoskeleton reorganization. Furthermore, increased activity of Src signaling mediating FAK phosphorylation is also observed upon expression of ORF74 in HEK293 cells [95], [96]. US28 induces cell migration in response to CCL2, CCL5 or CX3CL1 in US28-expressing or HCMV-infected smooth muscle cells, endothelial cells or fibroblasts [52], [75], [97], [98]. Cell migration mediated by US28 is ligand- and cell type-specific, which may be explained by the availability of G proteins. To transduce migratory signaling, US28 employs Src/FAK/Gbr2 via coupling to Gαq or Gα12/13
proteins, or RhoA/ROCK via Gα12/13 [52], [98]. CCL5 induces FAK activation via
Gα12 in US28-expressing fibroblasts, whereas CX3CL1 stimulates US28-mediated
pro-migratory FAK signaling through Gαq [98].
Degradation of extracellular environment – Degradation of the ECM or basement
membrane is an essential step for tumor cells to metastasize. Expression of ORF74 increases MMP-2 activity, an essential factor in endothelial and vascular remodeling, as well as ECM degradation [94], [99].These observations emphasize the complexity of invasion and metastasis. ORF74 contributes to this on several levels: metastatic cells have to ensure their survival during migration and clear the surrounding environment to be able to migrate.
Modulation of CXCR4 – Overexpression of chemokine receptor CXCR4 in cancer
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expression of CXCR4 upon ectopic expression and in HCMV-infected cells, through the constitutive activation of nuclear respiratory factor 1 (NRF-1) and binding of NRF-1 to the antioxidant response element (ARE) in the promoter of CXCR4 [47]. US27-induced ARE-driven transcription is mediated via activation of Gβγ proteins and PI3K, and was not observed for the G protein-uncoupled mutant US27R128A [47].
By elevating CXCR4 levels, US27 potentiates CXCL12-induced Ca2+ release and
CXCR4-mediated migration towards CXCL12 [100]. In contrast, BILF1, US28, UL33 and UL78 control CXCR4 in a negative manner., US28 lowers CXCR4 cell surface expression and impairs CXCL12-mediated signaling by affecting the recruitment of Gαi and β-arrestin2 to CXCR4 [101]. US28’s antagonizing effect on CXCR4
requires G protein coupling and is not the result of receptor heteromerization. BILF1, UL33 and UL78, on the other hand, directly interact with CXCR4 [53], [102]. BILF1 inhibits CXCL12 binding to CXCR4 and thereby CXCL12-induced signaling of CXCR4 [103]. Cross-regulation of CXCR4 is mediated through Gαi
scavenging by BILF1, as G protein uncoupling of BILF1 or co-expression of Gαi1
proteins restored CXCR4 signaling in response to CXCL12. Although UL33 and UL78 do not affect CXCR4 ligand binding, presence of these viral receptors lowers the efficacy and potency of CXCL12-induced inositol phosphate production and Ca2+ release mediated by CXCR4 [53]. Moreover, UL33, but not UL78, impaired
cell migration towards CXCL12.
2.4. Enabling replicative immortality
Telomeres cannot be copied completely during duplication of chromosomal DNA in healthy cells. As a consequence, shortening of telomeres limits proliferation of cells to a finite number of cell divisions, eventually resulting in replicative senescence and cell death after reaching the so-called Hayflick limit. Cancer cells circumvent this limitation acquiring the ability to maintain telomere length by induced telemorase activity, which is absent in normal cells [104].
Telomere stabilization – ORF74 expression in HUVEC endothelial cells is
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2.5. Inducing angiogenesis
When solid tumors become larger, growth and metastasis is only sustained when an adequate supply of oxygen and nutrients, and removal of metabolic waste products is ascertained. In order to achieve this, neovascularization mediated by a cellular process named angiogenesis is essential. Angiogenesis is dependent on migrating endothelial cells responding to activating (e.g. VEGF, FGF, TGF, TNF) or inhibiting cues (e.g. conformational changes in the basement membrane). In cancer, angiogenesis is a chronically activated, unbalanced process, characterized by the formation of many sprouting capillaries forming a chaotic network. These vessels differ from normal vessels in morphology and functionality, e.g. they form bidirectional blood flows [105]. One of the best characterized properties of ORF74 and US28 is their pro-angiogenic effect. Knockdown of ORF74 in endothelial bone marrow cells expressing the complete KSHV genome significantly reduced angiogenicity and allograft tumor formation, underlining the angioproliferative signaling by ORF74 [106].
VEGF signaling – Only a small fraction of ORF74-expressing cells is needed to
initiate VEGF secretion in bystander cells via paracrine routes [107], [108]. This is associated with the activation of mTOR and upregulation of HIF-1α/2α via various pathways, including PI3K/AKT, MEK/ERK, and p38 and IKKβ as discussed in section 2.1. Signaling via these mediators suggests that these cells are able to subvert the hypoxia response pathway by stimulating angiogenesis [71], [108]. The significance of VEGF signaling is emphasized by the observation of decreased ORF74-induced tumor growth upon VEGF inhibition in SVEC allografts in vivo [109]. ORF74 constitutive activity induces VEGF expression in NIH-3T3 cells via heme oxygenase-1 (HO-1) in a Gα12/13-dependent manner [110]. This is regulated
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cells [57]. Signaling circuitries leading to US28-mediated VEGF secretion involve Gαq, MAPKs p38 and p44/42, NF-κB, HIF-1, STAT3, IL-6 and COX-2, indicating
that multiple pathways activated by US28 converge at the level of VEGF production [49], [112]–[114]. BILF1 also constitutively promotes VEGF secretion in NIH-3T3 cells for which it relies on G protein-mediated signaling [67].
CXCL8 signaling – In addition to VEGF, both US28 and ORF74 stimulate another
angiogenic factor; CXCL8 (formerly known as IL-8), which is the endogenous ligand of CXCR2. These viral receptors elevate CXCL8 transcription in vitro and in xenograft tumors via the activation of NFAT [56]. In SVEC endothelial cells, ORF74 expression signals via the PI3K-Rac route, involving SWAP70, thereby stimulating CXCL8-driven endothelial permeability [115]. Furthermore, ORF74 expression in THP-1 cells stimulates CXCL8, CCL2, and growth factor FGF levels, stimulating cell migration and proliferation [116].
Angiopoietin signaling – KS lesions are characterized by leaky blood vessels,
associated with increased levels of angiopoietin 2 (ANGPT2) and angiopoietin like-4 (ANGPTL4) [109]. These vascular growth factors promote endothelial cell permeability, especially in combination with VEGF. ORF74 is one of the KSHV-encoded genes inducing the expression of ANGPT2 in lymphatic endothelial cells by activating MAPK signaling [117]. Additionally, the ORF74 secretome induces ANGPT2 expression in bystander cells [117]. Via stimulation of the Rho/ROCK pathway, ORF74 also elevates ANGPTL4 secretion in HMVEC cells [109]. Knockdown of ANGPTL4 in SVEC cells reduces ORF74-induced vascular permeability and allograft tumor growth. Double knockdown of ANGPTL4 and VEGF completely abolishes tumor growth induced by ORF74 in this model [109].
2.6. Resisting cell death
The ability of tumors to grow and expand is not only dependent on proliferation, but also on the rate of cell death. The most well described mechanism of programmed cell death is apoptosis [118].
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results in proliferation, whereas predominant Gα13 signaling triggers apoptosis.
Correspondingly, induction of apoptosis in COS-7 cells might be explained by the failure of US28 to signal to AKT in this cell type [121][77].
An additional survival mechanism was described in HEK293T and NIH3T3 cells, where ORF74 is able to crosstalk with receptor tyrosine kinase (RTK) insulin-like growth factor (IGF)-1 receptor [122]. As IGF-1R has previously been reported to contribute to KS survival by mediating anti-apoptotic signals in these cells, the ability of ORF74 to undergo crosstalk might be an contributing factor to cell death resistance as well [123].
NF-κB signaling – ORF74-mediated activation of NF-κB signaling in endothelial
cells leads to the rescue from apoptosis [124]. To activate NF-kB in Hela cells, ORF74 is mostly dependent on coupling to Gα13 and its signaling towards RhoA
and to a lesser extent Gαq-driven signaling [22]. In endothelial and COS-7 cells,
however, coupling to Gαi and Gαq also activates this transcription factor [18], [124].
Treatment of HUVEC cells with Wortmannin or AktK179M, inhibitors of PI3K and AKT respectively, impairs ORF74-mediated rescue from apoptosis in these cells [124]. Additionally, activation of NF-κB is also reported to regulate the transcription of several factors involved in pro-apoptotic signaling, including Bcl-2 and cyclin D1 [125], [126]. Transcription factors regulating genes of the Hippo pathway e.g. c-myc and Cdc64 are under control of NF-κB in endothelial cells as well [127]. Additionally, the Bcl2 gene is regulated by NF-κB, indicating that KSHV-encoded ORF74’s ability to regulate NF-κB is involved in survival signaling.
AP-1 signaling – As discussed earlier AP-1 is modulated to stimulate proliferation
via ORF74 activity. This transcriptional regulator combines functionality of Jun and Fos proteins [128], [129]. The latter proteins are also described to be modulated by HCMV-encoded US27, although no direct involvement of AP-1 is stated. HEK293 cells are more resistant to etoposide-induced apoptosis upon US27 expression, a response impaired upon G protein-uncoupling of US27 [91]. The anti-apoptotic properties of this receptor could involve pro-survival factors Bcl-x, Jun and Fos, whose expression is elevated by US27 [130].
2.7. Avoiding immune destruction
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result in the inhibition or enhancement of tumor growth. Immune regulation is a tightly regulated process, involving cell-mediated effects as well as the action of cytokines, pro- and anti-inflammatory chemokines, and interferons.
Many viruses, including the family of herpesviruses described here, are able to persist for life in their host requiring to stay under the radar of the immune system. To this end, viral chemokine receptors directly affect the immune cell response or indirectly mediate ligand sequestration and transport by scavenging of chemokines. By this means, they affect the chemokine-mediated response in their micro-environment. These processes will help tumor cells escape immune surveillance contributing to tumor development. Scavenging of chemokines by viral GPCRs, will also affect chemokine gradients and as a consequence, influence directed migration.
Chemokine scavenging – As a constitutively internalizing broad-spectrum
chemokine receptor, US28 is able to scavenge chemokines from the microenvironment of infected cells and thereby contribute to immune evasion by limiting recruitment of immune cells to sites of infection [44], [131]–[133]. Illustrating the chemokine sequestration capacity of US28 in relation to immune cell surveillance, HCMV depends on US28’s capacity to neutralize CCL2 and CCL5 for impairment of monocyte chemotaxis in response to infection of fibroblasts [44]. In contrast, US28 fails to affect monocyte adhesion to a monolayer of TNF-activated US28-expressing HUVEC cells, indicating that US28’s ability to scavenge chemokines is not sufficient to inhibit leukocyte arrest [134].
Little is known about the role of ORF74 in chemokine sequestration. One study in HEK293T cells reported the internalization of ORF74 upon binding of CXCL1 and CXCL8, but not CXCL10, in a β-arrestin-dependent manner [6].
COX-2 signaling – COX-2 is overexpressed in most types of cancer and activation
of the COX-2/PGE2/EP receptor axis plays a key role in multiple hallmarks of
cancer, like tumor-promoting inflammation and avoidance of the immune system, as reviewed in [135]. Of particular interest, enhanced PGE2 signaling contributes to
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COX-2 transcription is regulated via several signaling pathways activated by viral GPCRs; binding sites for CREB, NFAT and NF-κB are present in the COX-2 promoter (85). Although this may implicate that BILF1, ORF74, UL33 and US28 can regulate this cyclo-oxygenase, modulation of COX-2 is hitherto only reported for ORF74 and US28. ORF74 stimulates PGE2 production through increased transcription
and protein expression of COX-2 in HeLa and HUVEC cells [21], [56]. Moreover, COX-2 expression is elevated in ORF74-induced tumors in a transgenic mouse model expressing ORF74 in endothelial cells [56]. This receptor also induces COX-2 gene expression during lytic KSHV replication in the KS-derived SLK cell line. Upon ectopic expression or KSHV infection of endothelial cells, ORF74-mediated COX-2 protein expression is cyclosporin A-sensitive/calcineurin-dependent illustrating the contribution of NFAT signaling [56]. US28 promotes activation of the COX-2 promoter upon transfection of HEK293T, HUVEC and NIH-3T3 cells via NFAT- and NF-κB-dependent circuitries [56], [113]. Elevation of COX-2 mRNA upon HCMV infection of HFF cells is reduced upon infection using US28-deficient HCMV, indicating US28 also mediates COX-2 transcription in context of infection. Moreover, COX-2 is an important contributor to US28’s tumor-inducing properties, as COX-2 inhibitor celecoxib delays US28-driven tumor growth in xenograft nude mice and inhibits the VEGF secretion by US28-expressing NIH-3T3 cells [113].
Impairing antigen presentation – The human leukocyte antigen (HLA) system
is involved in immune response regulation by binding antigenic peptides and presenting them to immune cells. Herpesviruses are well known to impair the HLA system, contributing to immune evasion. During the lytic cycle of EBV, BILF1 contributes to downregulation of a wide range of HLA class I molecules via endocytosis and lysosomal degradation, modulating antigen presentation to CD8+
T cells [137], [138]. These properties might also be beneficial for cancer cells to evade the immune response, or at least decrease the number of immune cells at the EBV-infected tumor site.
2.8. Deregulating cellular energetics
Chapter II
as the Warburg effect, in which cells rely on glycolysis also in the presence of oxygen [139]. The evolutionary advantage of this reprogramming to cancer cells has long been unclear, but it might generate increased fitness scavenging nutrients into biomass [140]. It has been suggested that the metabolic changes in cancer cells are not passive responses, but rather regulated by oncogenes – metabolites themselves can be oncogenic and alterations in the metabolic profile might be an indirect response to proliferative and pro-survival signaling [141].
HIF-1/PKM2 signaling – Through activation of HIF-1, a key player in the
reprogramming of cancer metabolism [142], ORF74 and US28 are linked to metabolic reprogramming of host cells. ORF74 stimulates HIF-1 activity via PI3K, AKT and mTOR in NIH-3T3 fibroblast [71], [108]. In addition, this signaling circuitry is induced via paracrine mechanisms. Stimulation with supernatant derived from ORF74-expressing HMEC1 endothelial cells elevates HIF-1α levels in HUVEC cells. This is accompanied by the HIF-1-dependent expression of the metabolic protein pyruvate kinase M2 (PKM2) resulting in a strengthened pro-angiogenic response as exemplified by increased VEGF excretion [143] Allograft tumors formed by ORF74-expressing SVEC also show increased abundance of PKM2. Of note, treatment with mTOR inhibitor rapamycin or silencing of PKM2 expression reduces tumor growth in this model [143], [144]. US28, on the other hand, employs Gαq and CAMKII to activate AKT signaling [114]. Downstream of AKT, US28
Chapter II
β-catenin [149] and STAT3 [150].
2.9. Genome instability and mutations
Genomic stability is essential to mediate cell integrity and ensure a low mutation rate [151]. Genomic instability is responsible for the formation of precancerous lesions and drives tumor development as a result of a high rate of spontaneous, accumulating mutations.
MicroRNAs are short single-stranded RNAs that regulate gene expression on the transcript level. MicroRNA-34 (miR-34) is a highly conserved family of non-coding RNA acting downstream of p53. Deregulation of miR-43 has been associated with several types of cancer (deng 2018, li 2017). The only viral GPCR currently known to contribute to genome instability is ORF74. Expression of ORF74 in NIH-3T3 cells results in increased miR-34a activity and subsequently the downregulation of genome maintenance pathways, thereby promoting genomic instability. Knock-down of miR-34a in these cells restores the expression of genes involved in genome maintenance [152].
2.10. Tumor promoting inflammation
Tumors are infiltrated by cells of the innate and adaptive immune system [153]. The presence of these cells contributes to the onset and aggravation of cellular responses mediating cancer hallmarks. Immune cells supply bioactive molecules to the tumor microenvironment, including growth factors, survival factors, pro-angiogenic factors, and degrading enzymes that are needed for angiogenesis, invasion, and metastasis. Inflammatory cells can release reactive oxygen species, which increase the chances of mutations in nearby cancer cells as well. As has been discussed earlier, part of the signaling routes involved in mediating the inflammatory responses is tightly incorporated in the cellular signaling networks involved in oncogenic transformation and development.
NF-κB signaling – Transcription factors of the NF-κB family are regulators of divers
Chapter II
microenvironment that stimulates oncogenic development.
ORF74 couples to Gα13, Gαq [22], Gαi and Gαq depending on the cell type [18], [124].
Despite differences in G protein-coupling, the activation of NF-κB is linked to the secretion of cytokines, chemokines, and growth factors, affecting inflammation [20], [28], [155], [156]. Furthermore, IKKε is essential in ORF74-mediated transformation in vivo. IKKε links to a low-level persistent NF-κB activation and triggers a series of NF-κB-dependent paracrine mechanisms [157]. Furthermore, loss of IKKε completely abolished ORF74-induced expression of IL-6 and COX-2, among others.
Three HCMV-encoded GPCRs have been evaluated for NF-κB activation. Only US28, not UL33 nor US27, stimulates NF-κB-driven transcription in a Gαq-dependent
manner in COS-7 and HEK293T cells [85], [158]. Furthermore, US28 induces activation of NF-κB transcription factors in HCMV AD169-infected fibroblasts [113]. The impact of US28-induced NF-κB signaling is emphasized by the decrease of US28-mediated activation of STAT3 [112] and transcription of COX-2 and VEGF [113] upon NF-κB inhibition. Similar to US28 and ORF74, BILF1 activates NF-κB in COS-7 and HEK293 cells [65], [86]. This receptor, however, fails to stimulate the activation of NF-κB in Burkitt’s lymphoma cell line HH514.c16 and lymphoblastoid B-cell line JY, cell types relevant to EBV infection [65].
IL-6/STAT3 signaling – The cytokine IL-6 and its downstream target STAT3 are
important mediators of the immune response and inflammation, as they act on various signaling pathways involved in these processes.
Chapter II
transcription of oncostatin M [130]. ORF74 elevates IL-6 secretion from HUVEC, SLK and KSIMM endothelial cells in a yet uncharacterized manner [20], [159].
NFAT signaling - Transcription factors of the NFAT family are known regulators of
pro-inflammatory genes. Unlike NF-κB, however, the link between NFAT activation and inflammation-associated malignancies is less clear [160].
Activation of NFAT-driven transcription is shown for BILF1, ORF74 and US28. Stimulation of ORF74 (CXCL1 or CXCL10) and US28 (CX3CL1) modulate constitutive NFAT activation in HEK293 cells [51]. Moreover, NFAT activation is critical for ORF74- and US28-induced tumor formation; SVEC cells expressing ORF74 or US28 formed tumors in a xenograft mouse model, a cellular process sensitive to the calcineurin inhibitor cyclosporin A [56]. The mode of NFAT activation by the viral GPCRs is as yet not fully understood, different studies on activation of NFAT by ORF74 and US28 in HEK293(T) cells report contradicting results. ORF74 and US28 are proposed to activate NFAT through activation of Gαi, PLC, PKC,
the MAP kinases p38 and p44/42, and calcineurin, suggesting involvement of Gαi
and Gαq proteins [51]. In another study, calcium release from the ER is indicated
as receptor-mediated activation of NFAT via a non-canonical pathway as the activation is insensitive to both PLC inhibitor edelfosine and IP3 receptor inhibitor 2-APB [56]. Instead, ORF74 and US28 directly interact with sarcoplasmic reticulum calcium ATPase 2 (SERCA2), thereby inhibiting the ATPase activity of this negative regulator of NFAT. In HUVEC cells as well, ORF74 and US28 increase cytosolic calcium levels and promote calcineurin-dependent transcription with concomitant increase in protein levels of NFAT target genes, among which COX-2 [56]. Like in HEK293 cells, expression of ORF74 in PEL cell line BC3.14 stimulates NFAT by coupling to Gαi and Gαq, which is independent from PI3K [77]. In HUT 78 T
lymphocytes, on the other hand, NFAT activation by ORF74 is independent of calcineurin and mediated by blocking GSK-3-mediated nuclear export of NFAT via PI3K/AKT signaling [161]. Discrepancies also exist regarding the activation of NFAT by BILF1. Whereas one study reported NFAT activation upon BILF1 expression in HEK293 cells [86], others were unable to corroborate this finding in the same cellular background [56].
3. Conclusion
Chapter II
of cancer have indicated the interference of the signaling networks in affected cells to be of crucial importance in understanding the disease and determining therapeutic strategies. Characterization of the hallmarks of cancer has provided a set of cellular responses that explains behavior of tumor cells and predicts outcome of the disease. Understanding the underlying molecular mechanisms that mediate the development of cancerous phenotypes will help to define targets for therapeutic strategies. As affected networks provide possibilities to generate ‘back-up responses’ in case one component of the oncogenic network is therapeutically targeted, resistant phenotype are always a serious barrier of therapeutic success. To understand the integrated signaling network characterization of individual components is of crucial importance. Here we have described the role of exogenous, virally encoded GPCRs as mediators of network disturbances in the context of the cancer hallmarks. Understanding the mechanism of viral GPCR-mediated disruption of the cellular signaling network involved in cancer development, may lead to specific new therapeutic strategies targeting these receptor proteins along with their downstream effectors.
Chapter II
References
[1] and K. Y. Ann Arvin, Gabriella Campadelli-Fiume, Edward Mocarski, Patrick S. Moore, Bernard Roizman, Richard Whitley, Human Herpesviruses Biology,
Therapy, and Immunoprophylaxis. Cambridge University Press, 2007.
[2] H. F. Vischer, M. Siderius, R. Leurs, and M. J. Smit, “Herpesvirus-encoded
GPCRs: neglected players in inflammatory and proliferative diseases?,” Nat. Rev.
Drug Discov., vol. 13, no. 2, pp. 123–39, Feb. 2014.
[3] R. J. Stanton et al., “Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication,” J. Clin.
Invest., vol. 120, no. 9, pp. 3191–3208, 2010.
[4] P. Brunovskis and H. Kung, “Retrotransposition and Herpesvirus Evolution,” Virus
Genes, vol. 11, pp. 259–260, 1996.
[5] Ann-Sofie Mølleskov-Jensen, H. E. Farrell, N. Davis-Poynter, and M. T. Oliveira, “Chapter Twelve - Virus-Encoded 7 Transmembrane Receptors,” in Progress in
Molecular Biology and Translational Science, 2015, pp. 353–393.
[6] S. M. De Munnik, M. J. Smit, R. Leurs, and H. F. Vischer, “Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors,” Front.
Pharmacol., vol. 6, no. March, pp. 1–27, 2015.
[7] S. Montaner, I. Kufareva, R. Abagyan, and J. S. Gutkind, “Molecular Mechanisms Deployed by Virally Encoded G Protein–Coupled Receptors in Human Diseases,”
Annu. Rev. Pharmacol. Toxicol., vol. 53, no. 1, pp. 331–354, Jan. 2013.
[8] R. T. Dorsam and J. S. Gutkind, “G-protein-coupled receptors and cancer,” Nat.
Rev. Cancer, vol. 7, no. 2, pp. 79–94, 2007.
[9] G. D. Yancopoulos et al., “Effect of a Monoclonal Antibody to PCSK9 on LDL Cholesterol,” 2012.
[10] E. Cesarman, Y. Chang, P. S. Moore, J. W. Said, and D. M. Knowles, “Kaposi’s Sarcoma–Associated Herpesvirus-Like DNA Sequences in AIDS-Related Body-Cavity–Based Lymphomas,” N. Engl. J. Med., vol. 332, no. 18, pp. 1186–1191, 1995.
[11] J. Soulier, L. Grollet, and E. Oksenhendler, “Kaposi’s sarcoma-associated
herpesvirus-like DNA sequences in multicentric Castleman’s disease,” Blood, vol. 86, no. 4, pp. 1276–1280, 1995.
[12] L. N. Olp, A. Jeanniard, C. Marimo, J. T. West, and C. Wood, “Whole-Genome Sequencing of Kaposi ’ s Sarcoma-Associated Herpesvirus from Zambian Kaposi ’ s Sarcoma Biopsy Specimens Reveals Unique Viral Diversity,” vol. 89, no. 24, pp. 12299–12308, 2015.
[13] J. Russo et al., “Nucleotide sequence of the Kaposi sarcoma- associated herpesvirus ( HHV8 ),” Proc Natl Acad Sci U S A, vol. 93, no. December, pp. 14862–14867, 1996.
[14] M. Grisotto et al., “The human herpesvirus 8 chemokine receptor vGPCR triggers autonomous proliferation of endothelial cells,” J. Clin. Invest., vol. 21, pp. 1–7, 2010.
Chapter II
[16] T. Y. Yang et al., “Transgenic expression of the chemokine receptor encoded by human herpesvirus 8 induces an angioproliferative disease resembling Kaposi’s sarcoma.,” J. Exp. Med., vol. 191, no. 3, pp. 445–54, 2000.
[17] B. Abere and T. F. Schulz, “KSHV non-structural membrane proteins involved in the activation of intracellular signaling pathways and the pathogenesis of Kaposi’s sarcoma,” Curr. Opin. Virol., vol. 20, pp. 11–19, 2016.
[18] J. P. Couty, E. Geras-Raaka, B. B. Weksler, and M. C. Gershengorn, “Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor signals through multiple pathways in endothelial cells.,” J. Biol. Chem., vol. 276, no. 36, pp. 33805–11, 2001.
[19] S. Montaner, “Akt/TSC/mTOR activation by the KSHV G protein-coupled receptor: Emerging insights into the molecular oncogenesis and treatment of Kaposi’s sarcoma,” Cell Cycle, vol. 6, no. 4, pp. 438–443, 2007.
[20] S. Pati et al., “Activation of NF- κ B by the Human Herpesvirus 8 Chemokine Receptor ORF74 : Evidence for a Paracrine Model of Kaposi ’ s Sarcoma Pathogenesis Activation of NF-κB by the Human Herpesvirus 8 Chemokine Receptor ORF74 : Evidence for a Paracrine Model of Kap,” J. Virol., vol. 75, no. 18, pp. 8660–8673, 2001.
[21] B. D. Shelby et al., “Kaposi’s sarcoma associated herpesvirus G-protein coupled receptor activation of cyclooxygenase-2 in vascular endothelial cells,” Virol. J., vol. 4, pp. 1–9, 2007.
[22] L. W. Shepard et al., “Constitutive Activation of NF-κB and Secretion of
Interleukin-8 Induced by the G Protein-coupled Receptor of Kaposi’s Sarcoma-associated Herpesvirus Involve Gα13 and RhoA,” J. Biol. Chem., vol. 276, no. 49, pp. 45979–45987, 2001.
[23] C. Bais et al., “Kaposi’s sarcoma associated herpesvirus G protein-coupled receptor immortalizes human endothelial cells by activation of the VEGF receptor-2/ KDR,” Cancer Cell, vol. 3, no. 2, pp. 131–143, 2003.
[24] Y. Chang et al., “Identification of herpesvirus-like DNA sequences in
AIDS-associated Kaposi's sarcoma,” Science (80-. )., vol. 266, no. 5192, pp. 1865 LP – 1869, Dec. 1994.
[25] E. Cesarman et al., “Kaposi ’ s Sarcoma-Associated Herpesvirus Contains G Protein-Coupled Receptor and Cyclin D Homologs Which Are Expressed in Kaposi ’ s Sarcoma and Malignant Lymphoma,” J. Virol., vol. 70, no. 11, pp. 8218–8223, 1996.
[26] H.-G. Guo, M. Sadowska, W. Reid, E. Tschachler, G. Hayward, and M. Reitz, “Kaposi’s sarcoma-like tumors in a human herpesvirus 8 ORF74 transgenic mouse.,” J. Virol., vol. 77, no. 4, pp. 2631–9, 2003.
[27] D. Martin and J. S. Gutkind, Chapter 6 Kaposi’s Sarcoma Virally Encoded,
G-Protein-Coupled Receptor. A Paradigm for Paracrine Transformation, 1st ed.,
vol. 460, no. A. Elsevier Inc., 2009.
[28] S. Montaner et al., “The small GTPase Rac1 links the Kaposi sarcoma-associated herpesvirus vGPCR to cytokine secretion and paracrine neoplasia,” Blood, vol. 104, no. 9, pp. 2903–2911, 2004.
[29] S. Montaner et al., “The Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor as a therapeutic target for the treatment of Kaposi’s sarcoma,”
Chapter II
[30] P. J. Holst et al., “Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity,” vol. 108, no. 12, pp. 1789–1796, 2015.
[31] R. Chaisuparat, J. Hu, B. C. Jham, Z. A. Knight, K. M. Shokat, and S. Montaner, “Dual inhibition of PI3Kα and mTOR as an alternative treatment for Kaposi’s sarcoma,” Cancer Res., vol. 68, no. 20, pp. 8361–8368, 2008.
[32] H. Feng, Z. Sun, M. R. Farzan, and P. Feng, “Sulfotyrosines of the Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor promote
tumorigenesis through autocrine activation.,” J. Virol., vol. 84, no. 7, pp. 3351–61, 2010.
[33] N. Stern-Ginossar et al., “Decoding human cytomegalovirus.,” Science, vol. 338, no. 6110, pp. 1088–93, 2012.
[34] C. Taher, J. De Boniface, A. Mohammad, P. Religa, and J. Hartman, “High Prevalence of Human Cytomegalovirus Proteins and Nucleic Acids in Primary Breast Cancer and Metastatic Sentinel Lymph Nodes,” PLoS One, vol. 8, no. 2, 2013.
[35] L. Harkins et al., “Specific localisation of human cytomegalovirus nucleic acids and proteins in human colorectal cancer,” Lancet, vol. 360, no. 9345, pp. 1557–1563, Nov. 2002.
[36] C. S. Cobbs et al., “Human Cytomegalovirus Infection and Expression in Human Malignant Glioma.,” Cancer, vol. 62, pp. 3347–3350, 2002.
[37] L. Brouchet, S. Valmary, M. Dahan, A. Didier, P. Brousset, and B. Degano,
“Detection of oncogenic virus genomes and gene products in lung carcinoma,” Br.
J. Cancer, vol. 92, pp. 743–746, 2005.
[38] M. Melnick, P. P. Sedghizadeh, C. M. Allen, and T. Jaskoll, “Human
cytomegalovirus and mucoepidermoid carcinoma of salivary glands: Cell-specific localization of active viral and oncogenic signaling proteins is confirmatory of a causal relationship,” Exp. Mol. Pathol., vol. 92, no. 1, pp. 118–125, 2012. [39] N. Wolmer-Solberg et al., “Frequent detection of human cytomegalovirus in
neuroblastoma: A novel therapeutic target?,” Int. J. Cancer, vol. 133, no. 10, pp. 2351–2361, 2013.
[40] M. Samanta, L. Harkins, K. Klemm, W. J. Britt, and C. S. Cobbs, “High prevalence of human cytomegalovirus in prostatic intraepithelial neoplasia and prostatic carcinoma,” J. Urol., vol. 170, no. 3, pp. 998–1002, 2003.
[41] M. Michaelis and H. W. Doerr, “The Story of Human Cytomegalovirus and Cancer : Increasing Evidence and Open Questions,” Neoplasia, vol. 11, no. 1, pp. 1–9, 2009.
[42] M. S. Chee, S. C. Satchwell, E. Preddie, K. M. Weston, and B. G. Barrell, “Human cytomegalovirus encodes three G protein-coupled receptor homologues,” Nature, vol. 344, no. 6268, pp. 774–777, 1990.
[43] J. A. Scarborough, J. R. Paul, and J. V Spencer, “Evolution of the ability to modulate host chemokine networks via gene duplication in Human Cytomegalovirus (HCMV),” Infect. Genet. Evol., vol. 51, pp. 46–53, 2017.
Chapter II
[45] T. N. Kledal, M. M. Rosenkilde, and T. W. Schwartz, “Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28,” FEBS Lett., vol. 441, no. 2, pp. 209– 214, 1998.
[46] P. Casarosa et al., “Constitutive signaling of the human cytomegalovirus-encoded chemokine receptor US28,” J. Biol. Chem., vol. 276, no. 2, pp. 1133–1137, 2001. [47] J. M. Boeck, G. A. Stowell, C. M. O’Connor, and J. V. Spencer, “The human
cytomegalovirus US27 gene product constitutively activates antioxidant response element (ARE)-mediated transcription through Gβγ, phosphoinositide 3-kinase (PI3K), and nuclear respiratory factor 1 (NRF-1).,” J. Virol., no. September, 2018. [48] P. Casarosa et al., “Constitutive Signaling of the Human Cytomegalovirus-encoded
Receptor UL33 Differs from That of Its Rat Cytomegalovirus Homolog R33 by Promiscuous Activation of G Proteins of the G q , G i , and G s Classes *,” J. Biol.
Chem., vol. 278, no. 50, pp. 50010–50023, 2003.
[49] D. Maussang et al., “Human cytomegalovirus-encoded chemokine receptor US28 promotes tumorigenesis.,” Proc. Natl. Acad. Sci. U. S. A., vol. 103, no. 35, pp. 13068–73, Aug. 2006.
[50] E. V Langemeijer et al., “Constitutive β-catenin signaling by the viral chemokine receptor US28.,” PLoS One, vol. 7, no. 11, p. e48935, Jan. 2012.
[51] K. A. McLean, P. J. Holst, L. Martini, T. W. Schwartz, and M. M. Rosenkilde, “Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74,” Virology, vol. 325, no. 2, pp. 241–251, 2004.
[52] R. M. Melnychuk, D. N. Streblow, P. P. Smith, A. J. Hirsch, D. Pancheva, and J. a Nelson, “Human Cytomegalovirus-Encoded G Protein-Coupled Receptor US28 Mediates Smooth Muscle Cell Migration through G α Human Cytomegalovirus-Encoded G Protein-Coupled Receptor US28 Mediates Smooth Muscle Cell Migration through Gα12,” vol. 78, no. 15, pp. 8382–8391, 2004.
[53] K. Tadagaki et al., “Human cytomegalovirus-encoded UL33 and UL78
heteromerize with host CCR5 and CXCR4 impairing their HIV coreceptor activity,”
Blood, vol. 119, no. 21, pp. 1–3, 2012.
[54] Z. Cai et al., “Human cytomegalovirus-encoded US28 may act as a tumor promoter in colorectal cancer,” World J. Gastroenterol., vol. 22, no. 9, pp. 2789– 2798, 2016.
[55] L. Soroceanu et al., “Human cytomegalovirus US28 found in glioblastoma promotes an invasive and angiogenic phenotype,” Cancer Res., vol. 71, no. 21, pp. 6643–6653, 2011.
[56] J. Zhang et al., “Herpesviral G Protein-Coupled Receptors Activate NFAT to Induce Tumor Formation via Inhibiting the SERCA Calcium ATPase,” PLoS
Pathog., vol. 11, no. 3, pp. 1–26, 2015.
[57] R. Heukers et al., “The constitutive activity of the virally encoded chemokine receptor US28 accelerates glioblastoma growth,” Oncogene, 2018.
[58] G. Bongers et al., “The cytomegalovirus-encoded chemokine receptor US28 promotes intestinal neoplasia in transgenic mice,” J. Clin. Invest., vol. 120, no. 11, pp. 3969–3978, 2010.
Chapter II
5, pp. 337–350, 2010.
[60] G. R. Pearson, “Epstein-Barr virus and nasopharyngeal carcinoma,” J. Cell.
Biochem., vol. 53, no. S17F, pp. 150–154, 1993.
[61] L. C. F. Gequelin, I. N. Riediger, S. M. Nakatani, A. W. Biondo, and C. M. Bonfim, “Epstein-Barr virus,” Rev. Bras. Hematol. Hemoter., vol. 33, no. 5, pp. 383–388, 2011.
[62] E. M. Miyashita, B. Yang, G. J. Babcock, and D. A. Thorley-Lawson, “Identification of the Site of Epstein-Barr Virus Persistence In Vivo as a Resting B Cell,” J Virol., vol. 71, no. 7, pp. 4882–4891, 1997.
[63] D. A. Thorley-lawson and G. J. Babcock, “Epstein-Barr Virus : the Stealth Virus of Human B Cells,” Science (80-. )., vol. 65, no. 14, pp. 1433–1453, 1999.
[64] G. C. Faulkner, A. S. Krajewski, and D. H. Crawford, “The ins and outs of EBV infection.,” Trends Microbiol., vol. 8, no. 4, pp. 185–9, 2000.
[65] P. Beisser and D. Verzijl, “The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase,”
J. Virol., vol. 79, no. 1, pp. 441–449, 2005.
[66] S. J. Paulsen, M. A. Rosenkilde, J. Eugen-Olsen, and T. N. Kledal, “Epstein-Barr virus-encoded BILF1 is a constitutively active G protein-coupled receptor,” J.
Virol., vol. 79, no. 1, pp. 536–546, 2005.
[67] R. Lyngaa, K. Nørregaard, M. Kristensen, V. Kubale, M. M. Rosenkilde, and T. N. Kledal, “Cell transformation mediated by the Epstein-Barr virus G protein-coupled receptor BILF1 is dependent on constitutive signaling,” Oncogene, vol. 29, no. 31, pp. 4388–4398, 2010.
[68] D. Hanahan and R. a Weinberg, “Hallmarks of cancer: the next generation.,” Cell, vol. 144, no. 5, pp. 646–74, Mar. 2011.
[69] M. A. Feitelson et al., “Seminars in Cancer Biology Sustained proliferation in cancer : Mechanisms and novel therapeutic targets,” Semin. Cancer Biol., vol. 35, pp. S25–S54, 2015.
[70] J. A. Fresno Vara, E. Casado, J. de Castro, P. Cejas, C. Belda-Iniesta, and M. González-Barón, “PI3K/Akt signalling pathway and cancer.,” Cancer Treat. Rev., vol. 30, no. 2, pp. 193–204, Apr. 2004.
[71] A. Sodhi et al., “The Kaposi’s sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1α,” Cancer Res., vol. 60, no. 17, pp. 4873–4880, 2000.
[72] A. Sodhi et al., “The TSC2/mTOR pathway drives endothelial cell transformation induced by the Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor,” Cancer Cell, vol. 10, no. 2, pp. 133–143, 2006.
[73] L. Santarpia, S. L. Lippman, and A. El-Naggar, “Targeting the Mitogen-Activated Protein Kinase RAS-RAF Signaling Pathway in Cancer Therapy,” Expert opnin
Ther Targets, vol. 16, no. 1, pp. 103–119, 2012.
US28-Chapter II
induced Smooth Muscle Cell Migration is Mediated by Focal Adhesion Kinase and Src,” J. Biol. Chem., vol. 278, no. 50, pp. 50456–50465, 2003.
[76] S. Abd, E.-F. Ibrahim, A. Abudu, E. Jonhson, N. Aftab, and M. Fluck, “The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop,” vol. 9, no. 76, pp. 34259–34278, 2018. [77] M. L. Cannon and E. Cesarman, “The KSHV G protein-coupled receptor signals
via multiple pathways to induce transcription factor activation in primary effusion lymphoma cells,” Oncogene, vol. 23, no. 2, pp. 514–523, 2004.
[78] B. A. Krishna, M. S. Humby, W. E. Miller, and C. M. O. Connor, “Human cytomegalovirus G protein-coupled receptor US28 promotes latency by attenuating c-fos,” vol. 116, no. 5, pp. 1755–1764, 2019.
[79] Y. Liu, E. Carson-Walter, and K. A. Walter, “Targeting chemokine receptor CXCR7 inhibits glioma cell proliferation and mobility,” Anticancer Res., vol. 35, no. 1, pp. 53–64, 2015.
[80] Y. Liu-chittenden et al., “Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of TEAD-YAP,” Genes Dev., vol. 26, pp. 1300–1305, 2012.
[81] M. Angelova et al., “Kaposi’s sarcoma-associated herpesvirus G-protein coupled receptor activates the canonical Wnt/β-catenin signaling pathway,” Virol. J., vol. 11, no. 218, pp. 1–10, 2014.
[82] A. Steven and B. Seliger, “Control of CREB expression in tumors : from molecular mechanisms and signal transduction pathways to therapeutic target,” Oncotarget, vol. 7, no. 23, pp. 35454–35465, 2016.
[83] M. Waldhoer et al., “The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis,” J. Biol. Chem., vol. 278, no. 21, pp. 19473–19482, 2003.
[84] L. Soroceanu et al., “Human cytomegalovirus US28 found in glioblastoma promotes an invasive and angiogenic phenotype.,” Cancer Res., vol. 71, no. 21, pp. 6643–53, Nov. 2011.
[85] M. Waldhoer, T. N. Kledal, H. Farrell, and T. W. Schwartz, “Murine
Cytomegalovirus (CMV) M33 and Human CMV US28 Receptors Exhibit Similar Constitutive Signaling Activities,” J. Virol., vol. 76, no. 16, pp. 8161–8168, 2002. [86] K. Spiess et al., “Identification and Functional Comparison of
Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses,” J. Virol., vol. 89, no. 4, pp. 2253–2267, 2015.
[87] D. Hanahan and R. A. Weinberg, “The Hallmarks of Cancer,” Cell, vol. 100, pp. 57–70, 2000.
[88] M. Hajdúch, S. Jančík, J. Drábek, and D. Radzioch, “Clinical relevance of KRAS in human cancers,” J. Biomed. Biotechnol., vol. 2010, 2010.
[89] P. A. J. Muller and K. H. Vousden, “Mutant p53 in cancer: New functions and therapeutic opportunities,” Cancer Cell, vol. 25, no. 3, pp. 304–317, 2014. [90] L. Yang et al., “Mutations of p53 and KRAS activate NF-κB to promote
chemoresistance and tumorigenesis via dysregulation of cell cycle and
Chapter II
[91] C. C. Tu and J. V. Spencer, “The DRY box and C-terminal domain of the human cytomegalovirus US27 gene product play a role in promoting cell growth and survival,” PLoS One, vol. 9, no. 11, 2014.
[92] P. J. Sarvaiya, D. Guo, I. Ulasov, P. Gabikian, and M. S. Lesniak, “Chemokines in tumour progression and metastasis,” vol. 4, no. 12, 2013.
[93] Z. Cao, T. Livas, N. Kyprianou, M. Biochemistry, and C. Biology, “Anoikis and EMT: Lethal ‘Liaisons’ during Cancer Progression,” Crit. Rev. Oncol. Hematol., vol. 21, no. 3–4, pp. 155–168, 2016.
[94] M. He, T. Bakken, A. Kassimova, C. Boshoff, N. Philpott, and M. L. Cannon, “Focal adhesion kinase is required for KSHV vGPCR signaling,” Mol. Carcinog., vol. 51, no. 4, pp. 339–351, 2012.
[95] R. Li et al., “A mesenchymal-to-Epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts,” Cell Stem Cell, vol. 7, no. 1, pp. 51–63, 2010.
[96] P. Paoli, E. Giannoni, and P. Chiarugi, “Anoikis molecular pathways and its role in cancer progression,” Biochim. Biophys. Acta - Mol. Cell Res., vol. 1833, no. 12, pp. 3481–3498, 2013.
[97] D. N. Streblow et al., “The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration.,” Cell, vol. 99, no. 5, pp. 511– 520, 1999.
[98] J. Vomaske et al., “Differential Ligand Binding to a Human Cytomegalovirus Chemokine Receptor Determines Cell Type – Specific Motility,” PLoS Pathog., vol. 5, no. 2, 2009.
[99] B. Shan, C. A. Morris, Y. Zhuo, B. D. Shelby, D. R. Levy, and J. A. Lasky,
“Activation of proMMP-2 and Src by HHV8 vGPCR in human pulmonary arterial endothelial cells,” J. Mol. Cell. Cardiol., vol. 42, no. 3, pp. 517–525, 2007.
[100] K. L. Arnolds, A. P. Lares, and J. V. Spencer, “The US27 gene product of human cytomegalovirus enhances signaling of host chemokine receptor CXCR4,”
Virology, vol. 439, no. 2, pp. 122–131, 2013.
[101] T. Frank et al., “Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28,” Cell Commun. Signal., vol. 14, no. 1, pp. 1–16, 2016.
[102] P. Tschische, K. Tadagaki, M. Kamal, R. Jockers, and M. Waldhoer,
“Heteromerization of human cytomegalovirus encoded chemokine receptors,”
Biochem. Pharmacol., vol. 82, no. 6, pp. 610–619, Sep. 2011.
[103] S. Nijmeijer, R. Leurs, M. J. Smit, and H. F. Vischer, “The Epstein-Barr virus-encoded G protein-coupled receptor BILF1 hetero-oligomerizes with human CXCR4, scavenges Gαi proteins, and constitutively impairs CXCR4 functioning,”
J. Biol. Chem., vol. 285, no. 38, pp. 29632–29641, 2010.
[104] A. Bernal and L. Tusell, “Telomeres : Implications for Cancer Development,” Int. J.
Mol. Sci., vol. 2, no. 19, pp. 1–21, 2018.
[105] S. Ziyad and M. L. Iruela-Arispe, “Molecular Mechanisms of Tumor Angiogenesis,”
Genes and Cancer, vol. 2, no. 12, pp. 1085–1096, 2011.
Chapter II
[107] C. Bais et al., “G-protein-coupled receptor of Kaposi’s sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator,” Nature, vol. 391, 1998.
[108] B. C. Jham et al., “Amplification of the angiogenic signal through the activation of the TSC/mTOR/HIF axis by the KSHV vGPCR in Kaposi’s sarcoma,” PLoS One, vol. 6, no. 4, 2011.
[109] T. Ma et al., “Viral G protein-coupled receptor up-regulates Angiopoietin-like 4 promoting angiogenesis and vascular permeability in Kaposi’s sarcoma,” Proc.
Natl. Acad. Sci., vol. 107, no. 32, pp. 14363–14368, 2010.
[110] M. J. Marinissen, T. Tanos, M. Bolós, M. R. De Sagarra, O. A. Coso, and A.
Cuadrado, “Inhibition of heme oxygenase-1 interferes with the transforming activity of the Kaposi sarcoma herpesvirus-encoded G protein-coupled receptor,” J. Biol.
Chem., vol. 281, no. 16, pp. 11332–11346, 2006.
[111] M. J. Martín et al., “The Gα12/13family of heterotrimeric G proteins and the small GTPase RhoA link the Kaposi sarcoma-associated herpes virus G protein-coupled receptor to heme oxygenase-1 expression and tumorigenesis,” J. Biol. Chem., vol. 282, no. 47, pp. 34510–34524, 2007.
[112] E. Slinger et al., “HCMV-Encoded Chemokine Receptor US28 Mediates
Proliferative Signaling Through the IL-6 – STAT3 Axis,” Sci. Signal., vol. 3, no. 133, pp. 1–11, 2010.
[113] D. Maussang et al., “The human cytomegalovirus-encoded chemokine receptor US28 promotes angiogenesis and tumor formation via cyclooxygenase-2.,”
Cancer Res., vol. 69, no. 7, pp. 2861–9, Apr. 2009.
[114] R. H. de Wit, A. Mujić-Delić, J. R. van Senten, A. Fraile-Ramos, M. Siderius, and M. J. Smit, “Human cytomegalovirus encoded chemokine receptor US28 activates the HIF-1a-PKM2 axis in glioblastoma cells,” Oncotarget, vol. 7, no. 42, pp.
67966–67985, 2016.
[115] J. Dwyer et al., “The guanine exchange factor SWAP70 mediates vGPCR-induced endothelial plasticity,” Cell Commun. Signal., vol. 13, no. 1, pp. 1–7, 2015.
[116] M. Schwarz and P. M. Murphy, “Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor constitutively activates NF-kappa B and induces proinflammatory cytokine and chemokine production via a C-terminal signaling determinant,” J. Immunol., vol. 167, no. 1, pp. 505–13, 2001.
[117] R. J. Vart et al., “Kaposi’s sarcoma-associated herpesvirus-encoded interleukin-6 and G-protein-coupled receptor regulate angiopoietin-2 expression in lymphatic endothelial cells,” Cancer Res., vol. 67, no. 9, pp. 4042–4051, 2007.
[118] S. Elmore, “Apoptosis: A Review of Programmed Cell Death,” Toxicol Pathol, vol. 35, no. 4, pp. 495–516, 2007.
[119] O. Pleskoff et al., “The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis,” FEBS J., vol. 272, no. 16, pp. 4163–4177, 2005.
[120] S. Joshi et al., “Galpha(13) mediates human cytomegaolvirus-encoded chemokine receptor US28 induced cell death in melanoma,” Int. J. Cancer, vol. 93, no. 4, pp. 292–297, 2016.
