Pathogenesis of Nasopharyngeal Carcinoma

C H A P T E R

3 Pathogenesis of Nasopharyngeal Carcinoma: Histogenesis, EpsteineBarr Virus Infection, and Tumor Microenvironment C.M. Tsang1,2, K.W. Lo2, John M. Nicholls3, S.C.M. Huang1, S.W. Tsao1 1

School of Biomedical Sciences, University of Hong Kong, Hong Kong, China; 2Department of Anatomical and Cellular Pathology and State Key Laboratory of Translational Oncology, Chinese University of Hong Kong, Hong Kong, China; 3 Department of Pathology, University of Hong Kong, Hong Kong, China

O U T L I N E Cell Type Origin of Nasopharyngeal Carcinoma

The Dynamic Interaction of Tumor Microenvironment Cells With EpsteineBarr Virus-Infected Nasopharyngeal Carcinoma Cells Macrophages in Nasopharyngeal Carcinoma Cancer Associated Fibroblasts in Nasopharyngeal Carcinoma

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Histopathology and Classification of Nasopharyngeal Carcinoma 46 Altered Cell Signaling in Nasopharyngeal Carcinoma Aberrant Activation of NF-kB and STAT3 Inflammatory Pathways Altered PI3K-MAPK Signaling Pathway Aberrant NOTCH Pathways Activation of the Hedgehog Signaling Pathways Alterations in G1-S Cell Cycle Transition EpsteineBarr Virus Infection, BART-microRNAs, and Nasopharyngeal Carcinoma Pathogenesis The Contribution of Tumor Microenvironment to Nasopharyngeal Carcinoma Pathogenesis

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Conclusion

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Acknowledgments

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References

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Commentary on Chapter 3: The Pathogenesis of Nasopharyngeal Carcinoma: What We Know and What We Don’t Know

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Acknowledgment

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References

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CELL TYPE ORIGIN OF NASOPHARYNGEAL CARCINOMA Even though electron microscopic and immunohistochemical studies have demonstrated that nasopharyngeal carcinoma (NPC) is an epithelial tumor, the exact cell type giving rise to this tumor has not been clearly defined. Multiple epithelial cell types are present in the nasopharynx, located behind the nasal cavity. As a component of Waldeyer’s lymphoeipithelial ring, the stroma beneath the epithelium is richly infiltrated with lymphocytes; hence the original description of this tumor (which still exists in some textbooks today) was lymphoepithelioma.

Nasopharyngeal Carcinoma https://doi.org/10.1016/B978-0-12-814936-2.00003-1

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Copyright © 2019 Elsevier Inc. All rights reserved.

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3. PATHOGENESIS OF NASOPHARYNGEAL CARCINOMA

A detailed histopathological study performed many years ago showed that 60% of the epithelial lining of nasopharynx is made up of stratified squamous epithelium (largely unkeratinized) with the remaining epithelium made up of pseudostratified ciliated columnar epithelium.1 NPC rarely shows evidence of glandular differentiation, and in endemic regions squamous differentiation is uncommon.2 The highest incidence of NPC within the nasopharynx is found at the pharyngeal recess (Fossa of Rosenmu¨ller) that lies superiorly and posteriorly of the elevation of the Eustachian tube. This Fossa of Rosenmu¨ller is the lateral extension of nasopharynx, which is covered by mixed stratified epithelium and ciliated epithelium with patches of a discontinuous attenuated reticular epithelium.3 NPC may arise from a special transitional epithelium present in the nasopharyngeal regions referred to as reticular epithelium and, since this has certain morphological similarities with invasive NPC, it is possible that this may be the cell origin. Whether this special type of nasopharyngeal epithelium is particularly susceptible to EpsteineBarr virus (EBV) infection and subsequent transformation into NPC cells remains to be confirmed. However, unlike epithelial tumors that develop in other organs, there are few cases of “true” nasopharyngeal dysplasia, and if these do exist there are no large-scale series published that have demonstrated a progression from this dysplasia to invasive carcinoma. With the increasing use of circulating EBV-related tumor markers for early detection of NPC,4 it is possible that a clearer picture of these early lesions, and in particular the precursors to NPC, might be elucidated. Interestingly, allelic deletion of chromosomes 3p and/or 9p harboring the tumor suppressor genes RASSF1A and p16, respectively, were detected in the nasopharyngeal epithelium from high-risk southern Chinese but not in northern Chinese.5 Furthermore, these genetic alterations in nasopharyngeal epithelium could be detected before EBV infection, suggesting that EBV infection is a later event after genetic alterations have occurred in the premalignant nasopharyngeal epithelium.6 We have shown that inactivation of p16, which inhibits the CDK4/Cyclin D1 signaling cascade, is essential for cell cycle progression; or, that overexpression of Cyclin D1 in telomerase-immortalized nasopharyngeal epithelial cells supports stable and latent infection of EBV.7 All these observations suggest that genetic alterations present in premalignant nasopharyngeal epithelium in the high-risk NPC population predispose it to EBV infection and malignant transformation into NPC.

HISTOPATHOLOGY AND CLASSIFICATION OF NASOPHARYNGEAL CARCINOMA When NPC was first described at the end of the 19th century, it was classified as lymphoepithelioma because of its heavy infiltration of lymphocytes. The epithelial origin of NPC was confirmed by the presence of tonofilament and cell junctional structure, desmosomes, which are specific epithelial cell features. Figure 3.1 shows the typical histopathological features of NPC with infiltrating small lymphocytes. At present, NPC is classified by the World Health Organization (WHO) as keratinizing and nonkeratinizing squamous cell carcinoma based on morphology without the need for ancillary immunohistochemistry.8 Lymphoepitheliomas outside of nasopharyngeal regions have also been reported, and they are commonly associated with EBV infection and undifferentiated cancers.9

FIGURE 3.1 Nonkeratinizing undifferentiated carcinoma showing nests of pleomorphic polygonal cells with large vesicular nuclei. The stroma surrounding the tumor nests contains sheets of small lymphocytes.

HISTOPATHOLOGY AND CLASSIFICATION OF NASOPHARYNGEAL CARCINOMA

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FIGURE 3.2 Recurrent undifferentiated carcinoma which was removed by maxillary swing surgical excision. (A) This figure shows large nests of tumor cells infiltrating into the squamous lined stroma. (B) The tumor cells are positive for EBER in situ hybridization with no signal present in the stroma.

The nonkeratinizing NPC could be further classified into poorly differentiated and undifferentiated NPC, but this subclassification does not seem to have prognostic values for treatment responses.10 The nonkeratinizing NPC makes up 98% of NPC in the endemic areas and is closely associated with EBV infection. The close association of EBV infection in NPC is shown in Figure 3.2 and 3.3. Interestingly, EBV infection could also be detected in the basal nasopharyngeal epithelium associated with undifferentiated carcinoma (Figure 3.4). EBV infection can also be detected in the keratinizing NPC at a lower rate in endemic areas but not in nonendemic areas of NPC.8 The close association of EBV infection with lack of squamous or glandular differentiation strongly implicates that the special differentiation properties of NPC is involved in establishment of stable EBV infection. EBV infection in NPC is predominantly latency Type II with expression of a limited set of latent EBV genes including EBERs, LMP1, LMP2A, EBNA1, and abundant expression of BART-microRNAs. EBV infection of normal epithelium of the oropharynx and presumably nasopharynx is believed to be predominantly lytic, resulting in the release of EBV into saliva, and enabling transmission from individual to individual. A recent report has demonstrated the importance of epithelial differentiation in the induction of lytic infection of EBV.11 A switch from lytic to latent infection of EBV may represent an essential step in NPC pathogenesis. Alterations in cellular signaling as a result of acquired genetic changes in NPC cells, recruitment of tumor infiltrating leucocytes and other stromal cells into the special tumor microenvironment (TME) of NPC, together with the rich cytokine content of TME are all postulated to play important roles in the switch from lytic to latent EBV infection in NPC pathogenesis.

FIGURE 3.3 Nonkeratinizing differentiated carcinoma. (A) This tumour is more papillary in architecture than the nonkeratinizing undifferentiated carcinoma and the tumor nests are sharply demarcated from the lymphoid stroma. (B) In situ hybridization of EBV-encoded EBERs shows extensive positive signal in the surface epithelium.

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FIGURE 3.4 EBV infection in nonkeratinizing undifferentiated nasopharyngeal carcinoma. (A) Note the close extension of undifferentiated NPC cells to the surface epithelium. (B) In situ hybridization of EBV-encoded EBERs shows focal positivity of the basal epithelium. (C) Another case of undifferentiated carcinoma showing positive EBER signal in the basal epithelium.

ALTERED CELL SIGNALING IN NASOPHARYNGEAL CARCINOMA Aberrant Activation of NF-kB and STAT3 Inflammatory Pathways The unique lymphoepithelial-like histology of NPC implies the contribution of distinct signaling pathways deregulated in NPC tumorigenesis. The key inflammatory signaling pathways, NF-kB and JAK-STAT, are constitutively activated in this EBV-associated malignancy. Nuclear accumulation of abundant NF-kB and STAT3 signaling molecules were consistently demonstrated in EBV-positive NPC cell lines, patient-derived xenografts, and primary NPC.12e16 Studies have revealed that EBV latent genes dysregulate these cellular signals involved in promoting host cell survival, modulating tumor microenvironment, maintaining stemness properties, and regulating viral gene transcription and latency. The viral oncoprotein LMP1 functions as a constitutively active TNFR and interacts with multiple TRAF proteins (TRAF1, 2, 3, and 5), thereby activating both canonical and noncanonical NF-kB signaling pathways. The activated NF-kB signals promote inflammation in NPC and survival of EBVpositive NPC cells via modulating the expression of multiple chemokines, chemokine receptors, antiapoptotic genes, and transcription factors. The driver role of activated NF-kB signals in NPC tumorigenesis was strongly supported by the recent genomic sequencing studies that uncovered frequent alterations of upstream negative regulators of NF-kB pathways, including TRAF3, CYLD, NFKBIA, and NLRC5.17,18 Our study has detected somatic NF-kB pathway aberrations and LMP1-overexpression in more than 70% of tumors. Notably, the LMP1 expression

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and NF-kB pathway aberrations are mutually exclusive. Aside from directly binding and inactivating TRAF3 function, LMP1 induces phosphorylation of IKK complex and IkBa degradation to activate NF-kB signaling. Inactivation of the upstream negative regulators of NF-kB by somatic alterations could enhance NF-kB activation to promote survival of EBV-infected NPC cells. The occurrence of somatic mutations of NF-kB negative regulators may supplant the need for the cytotoxic LMP1 expression during NPC progression. Moreover, aberrant NF-kB activation involving p50/p50/BCL3 complex is commonly detected in NPC but not necessarily related to the expression of LMP1.12 NF-kB activation may contribute to the growth of NPC by multiple pathways. The EBV-encoded LMP1 is a potent activator of NF-kB signaling and has been implicated in mediating many tumorigenic properties of EBV-infected cells including cell survival and antiapoptosis. NF-kB signaling supports cell survival. Inhibition of NF-kB activation triggered apoptosis in EBV-transformed lyphoblastoid B cells.19 Besides, suppression of NF-kB by expressing an NF-kB inhibitor, IKBB, downregulated multiple proangiogenic factors including Rantes, Upar, IL6, and IL8 in epithelial cancer cells.20 This also implicates a role of proangiogenesis of NF-kB in NPC patients.20 A novel role of NF-kB is its involvement in mTOR signaling which plays an essential role in metabolic reprogramming for cell growth. The IKK complex is an important upstream regulator of canonical NF-kB signaling. The IKK-b in the IKK complex can directly inhibit the activity of Tsc1, an essential suppressor of mTOR signaling, by phosphorylation of the Ser 487 and Ser 511 of TSC1 to activate mTOR signaling.21 In the telomerase-immortalized nasopharyngeal epithelial cells, activation of NF-kB is coupled with mTOR signaling to support the growth of immortalized nasopharyngeal epithelial cells.22 The Cyclin D1 activation is downstream of NF-kB, which is upregulated in telomerase-immortalized NPE cells and can be suppressed by NF-kB inhibition.22 Importantly, upregulation of Cyclin D1 supports establishment of EBV infection in premalignant nasopharyngeal epithelial cells.7 We have recently shown that LMP1 activates NF-kB/mTOR signaling and is involved in enhancing glucose metabolism in EBV-infected nasopharyngeal epithelial cells by activating Glut-1.21 In addition to LMP1, the EBV-encoded RNAs, EBERs, also contribute to the activation of NF-kB signaling via interacting with TLR3.23 The abundant expression of EBERs in all NPC tumors suggests these viral noncoding RNAs as a strong intrinsic inducer for constitutive activation of NF-kB pathways. On the other hand, the binding of EBERs to TLR3 also triggers innate immune response and apoptosis in the EBV-infected cells via activation of IRF3 signaling pathway and type I interferon (IFN) expression. It is noted that both TRAF3 and CYLD play critical roles in this process. In EBV-positive cells in which TRAF3 and CYLD were inactivated either by LMP1 expression or somatic alterations, the EBERs-activated IRF3 signaling is suppressed. The inactivation of TRAF3 or CYLD allows constitutively active NF-kB pathways, while inhibiting innate immunity in EBV-infected NPC cells. Interestingly, the TRAF3 and CYLD gene alterations are also found in a subset of human papillomavirus (HPV)-positive head and neck squamous cell carcinoma with episomal HPV infection.24 These findings imply a critical role of aberrant NF-kB and TLR3 signal pathways in tumorigenesis of viral-associated cancers. Activation of STAT3 is observed in more than 70% of EBV-associated NPC and plays a causal role in driving NPC progression.15,25,26 As an oncogenic transcription factor, STAT3 activates multiple downstream targets for cell proliferation, survival, invasion, and stem cell maintenance. EBV infection and expression of LMP1 play an important role in activation of STAT3 signal in NPC cells.14,27,28 Somatic alterations in JAK-STAT3 pathway could also be detected in a small subset of NPC.17 Epigenetic suppression of DLEC1, a 3p22.2 tumor suppressor that diminishes the JAK2 and STAT3 binding, was recently reported to promote the activation of IL6/STAT3 signaling in the majority of NPC cases.29 Nevertheless, the constitutive STAT3 activation may be mainly driven by LMP1-induced PKCd signaling and EBNA1-modulated IL6R expression in EBV-infected epithelial cells.28,30 Notably, strong IL6 expression is commonly detected in the stromal lymphoid cells, but rarely in tumor cells.31 The STAT3 signaling in NPC cells is likely to be modulated by the NF-kB pathways and cytokine-induced inflammatory TME. In NPC, we have previously demonstrated that the major forms of NF-kB signaling include nuclear accumulation of p50/p50/BCL3 and p50/RelB, both of which could be induced by LMP1.13 The abundancy of this distinct p50/p50/BCL3 signal is believed to be a result of activation of both NF-kB and STAT3 signaling in EBVinfected NPC cells. In addition to promoting BCL3 expression, activated STAT3 may also contribute to the upregulation of p105/p50 in NPC cells via NOTCH3 signal.32 Furthermore, activated STAT3 may enhance p50-BCL3-DNA complex formation via upregulation of JAB1.16,33 The cross-talk between these two pathways may establish a distinct inflammatory signaling network in NPC.

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Altered PI3K-MAPK Signaling Pathway In addition to NF-kB/STAT3 signaling pathways, the PI3K/MAPK signaling cascade is also prominently altered in NPC. Somatic mutations of multiple key components including NRAS, PIK3CA, PTEN, and FGFR2 could be detected in recurrent NPC in recent genome sequencing studies of NPC.17,18,34 Notably, NPC patients with somatic oncogenic alterations in this pathway tended to have poorer survival than those without. Through whole exome sequencing analysis of paired primary and recurrent NPC, RAS mutations were shown to be a progression driver in two NPC patients.17 Notably, acquired clonal HRAS mutation was observed in a recurrent tumor after chemo-radiotherapy. Enrichment of these mutations in patients with recurrent NPC implied its potential role in cancer progression. The RAS mutations lead to constitutive activation of mitogen-activated protein kinase (MAPK) pathways, which may contribute to decreased treatment efficacy and resistance to chemotherapy and radiotherapy.35,36 Activation of PI3K-AKT-mTOR pathway is known to control multiple cellular processes and promote resistance to anticancer therapies in human cancers. Our studies have revealed that inactivating PTEN mutations, PIK3CA hotspot mutations and amplification were found in only 4%, 4%, and 20% of NPC, respectively.17,37 EBV-encoded LMP1 and LMP2A and epigenetic silencing of the INPP4B tumor suppressor may contribute to the activation of PI3K/AKT signaling pathway in a subset of NPC. In EBV-positive NPC cell line C666-1, restoration of INPP4B significantly suppressed the phosphorylation of AKT and mTOR, thereby inhibiting the growth of tumors in vivo.38 In contrast to the rare mutations of PIK3CA in NPC, whole-genome sequencing study revealed frequent PIK3CA mutations (80%) that distributed within all PIK3CA domains in EBV-associated gastric cancer (GC).39 Interestingly, somatic alterations involved in dysregulation of NF-kB pathways are uncommon in EBV-associated GC.17,39 The distinct mutational profiles of NPC imply the different roles of NF-kB and PI3K signaling pathways in the pathogenesis of the two EBVassociated epithelial cancers, NPC and GC.

Aberrant NOTCH Pathways Through comprehensive genomic analysis, the genetic abnormalities targeting NOTCH signaling pathways in NPC were unveiled by identifying mutations in the receptors (NOTCH1, NOTCH3; 4.7%) and negative regulator (FBXW7; 4%).17,34 The loss-of-function mutation of FBXW7 implies a potential oncogenic role of NOTCH signaling. However, the significance of the NOTCH1/NOTCH3 mutations in NPC is unclear. Nevertheless, upregulation of the NOTCH3 receptor and its ligands (JAG1 and DLL4) and constitutive activation of NOTCH3 signaling were found in almost all EBV-associated NPC.32 As an effector of STAT3 signal, the de novo expression of NOTCH3 receptor is believed to be a consequence of EBV-deregulated signaling cascades. The oncogenic roles of aberrant NOTCH3 signaling in regulation of cell proliferation, survival, and self-renewal capacity were reported in EBV-positive NPC cells.32 As a tumor-specific receptor, specific inhibitors targeting NOTCH3 signaling may have potential applications for NPC treatment.

Activation of the Hedgehog Signaling Pathways NPC frequently displays the deregulated hedgehog (HH) pathway, which is involved in maintenance of stem cell properties.40 HH signaling is activated through binding of its ligands, Sonic hedgehog (SHH), Indian hedgehog (IHH), and Desert hedgehog (DHH), to Patched (PTCH1), a transmembrane protein that represses the activity of smoothened (SMO). Binding of HH ligands to PTCH1 releases the repression of SMO and stimulates expression of various downstream effectors, which alter cell growth survival, differentiation, angiogenesis, and cell mobility.41 Expression of components of HH signaling was detected in NPC by microarray analysis, while somatic change of HH signaling components is rare.42 NPC tissue array revealed an increase in SHH expression in 60% of NPC (30 out of 50) relative to normal nasopharyngeal epithelium, while 95% of NPC (48 out of 50) expresses the HH receptor, PTCH1. Gene expression profiling of the EBV-positive NPC cell line, C666-1, revealed elevated HH activation with increased expression of SHH ligand, upregulation of positive regulators (GLI1, STK36, XIC2, and KIF3A) and decreased expression of negative regulators (SUFU, BTRC, and RAP23), as well as increased expression of genes downstream of HH signaling.40 All these may be involved in the cancer stem-like properties displayed by C6661.43 Moreover, EBV infection of epithelial cells is associated with activated HH signaling. Aberrant HH pathway activation in EBV-infected epithelial cells also revealed expression of stemness-associated genes including EBCD133, CD44, NANOG, BMI1, LRIG1, and others. Involvement of LMP1 and LMP2A in the induction of stemness properties in NPC has been reported in earlier studies.44,45 The ability of C666-1 to maintain EBV upon long-term propagation may be related to its stem cellelike properties. It remains to be determined whether EBV infection selectively expands cancer stem cell clones present in NPC. The role of HH signaling and other signaling pathways involved in driving the induction of stem cell properties in NPC remains to be defined.

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Alterations in G1-S Cell Cycle Transition According to earlier studies, Cyclin D1 (CCND1) amplification and p16 (CDKN2A) inactivation by homozygous deletion and promoter methylation were detected in w15% and w85% of primary tumors, respectively, implying gene alterations in G1-S cell cycle transition are common somatic events in NPC pathogenesis.46e48 Similar findings were also revealed in recent genomic sequencing studies.17 Aside from cell cycle dysregulation, p16 inactivation and Cyclin D1 overexpression also promote persistent EBV latent infection in nasopharyngeal epithelial cells and may be one of the early events in NPC development.7 TP53 mutations were reported to be rare in NPC in earlier studies, which may be related to inactivation of TP53 function by EBV gene products like EBNA1.49 Interestingly, recent comprehensive genomic studies have consistently detected TP53 mutations in 7%e10% of NPC.17,18,34 Our study has also shown that TP53 mutations were w2.3-fold enriched in recurrent/metastatic NPC compared to primary NPC,17 suggesting a selection during NPC progression for cancer clones harboring mutation of gene(s) involved in genomic instability.

EPSTEINeBARR VIRUS INFECTION, BART-MICRORNAS, AND NASOPHARYNGEAL CARCINOMA PATHOGENESIS The relative importance of lytic and latent EBV infection to NPC pathogenesis remains to be determined. In a CD34þ reconstituted humanized mouse model, injection of EBV into humanized mice resulted in the generation of CD20þ diffuse B-cell lymphoma.50 Interestingly, EBV defective in lytic reactivation (BZLF1 deleted) had a lower tumorigenicity in the humanized mice (2/14) compared to intact EBV capable of undergoing lytic infection (6/11). The underlying reason has not been clearly defined, but may involve both paracrine and immune suppressive factors associated with lytic EBV infection as a result of change of cytokine profile and immune modulation in TME. Multiple EBV latent genes are expressed in EBV-infected NPC cells including EBERs, LMP1, LMP2, EBNA1, and BART-microRNAs.51,52 The oncogenic properties of LMP1 have recently been extensively reviewed.53 The LMP1 is a potent activator of NF-kB signaling. Activation of NF-kB signaling mediates many of the tumorigenic properties of LMP1. The LMP2A could activate Akt signaling and transform HaCaT cells into tumorigenic cells.54 Both LMP1 and LMP2A have been reported to induce stem-like or stemness properties in infected epithelial cells.44,45 The EBNA1 could contribute to NPC pathogenesis through disruption of progressive multifocal leukoencephalopathy.55 It also competes with the tumor suppressor gene, p53, to bind to the USP7 protein and may impair the tumor suppression function of p53.56 The EBER induces IGF-1 in EBV-infected gastric cancer and is involved in innate immunity by interacting with RIG1 and TLR3 receptor to induce multiple downstream events.57 An important role of BART-microRNAs in NPC pathogenesis is emerging. The high levels of BART-microRNAs detected in NPC provide strong evidence of their involvement in NPC pathogenesis.58 BART-microRNAs enhance survival of EBV-infected NPC cells by targeting multiple cellular apoptotic proteins.59 The recent reports that BARTmicroRNAs potentiated tumorigenic growth of EBV-infected AGS cells (a gastric cancer cell line) strongly support their important roles in EBV-associated epithelial cancer.60,61 The expression of BART-microRNAs is positively selected in an EBV-infected AGS and NPC cells propagated in immune deficient animals.62 BART-microRNAs made up 26.2%e38.6% of total cellular microRNAs in four out of five NPC xenografts. The only NPC cell line that is capable of maintaining EBV episome in culture is C666-1. Interestingly, the expression of BART-microRNA is also high in C666-1 cells (29.3% of the total cellular microRNA),62 which is strongly implicating the selection of EBV-encoded BART-microRNAs expression to support growth of EBV-infected NPC cells. Interestingly, the expression of BART-microRNAs is not involved in the proliferation of B cells transformed by EBV and is not expressed in EBV-transformed B cells (type III latency). The commonly used EBV strain (B95-8) isolated from infectious mononucleosis for B cell transformation/immortalization is defective in the synthesis of BART-microRNAs due to a large genomic deletion in the BART region responsible for the synthesis of BART-microRNAs. BART-microRNAs have been reported to be involved in maintenance of viral latency and promote cell survival. An earlier study has reported that miR-BART20-5p targeted the BZLF1 (Zta) and BRLF1 (Rta) to stabilize viral latency in EBV-infected gastric carcinoma cells.64 BART-microRNAs are involved in regulation of ATM (ataxia telangiectasia mutated) expression, which is involved in repair of double-DNA strand breaks.62 The ATM is involved in lytic replication of EBV in epithelial cells.65 Four BART-microRNAs (miR-BART5-5p, miR-BARTT7-3p, miR-BART9-3p, miR-BART14-3p) cooperatively suppressed ATM activity and inhibited BZLF1 expression, hence supporting the involvement of BART-microRNAs to support latent EBV infection in NPC.62 The BART-microRNAs are also involved in optimizing the levels of viral genes, notably LMP1 in EBV-infected cells. The Cluster I miR-BARTs can

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negatively regulate LMP1 levels in EBV-infected cells to achieve an optimal level for cell survival.66 The BART-microRNAs also specifically target growth and apoptotic events. For example, the miR-BART3-5p can stimulate cell proliferation by targeting DICE1 30 -UTR, which has a tumor suppressor function and inhibits colony formation in tumor cells.67 Many studies have reported the important role of BART-microRNAs in suppressing cellular apoptosis, thus may enhance the survival of EBV-infected NPC cells. An earlier study has reported that the miR-BART5 targets a pro-apoptotic protein, the p53-upregulated modulator of apoptosis (PUMA).68 The combined action of Cluster I miR-BARTs alone or in combination with Cluster II miR-BARTs can downregulate BIM (Bcl-2 interacting mediation) of cell death.69 A more recent study has reported that BART-microRNAs target multiple apoptotic targets in cells including DICE1 and CASZ1a (by miR-BART3), OCT1 (by miR-BART6), CREBBP and SH2B3 (by miR-BART16), PAKS and TP53NP1 (by miR-BART22).59 Multiple studies also support a role of BART-microRNAs in promoting epithelial-mesenchymal transition (EMT) and enhancing invasive/metastatic properties of NPC cells. The miR-BART9 specifically targets E-cadherin to promote metastasis.70 The miR-BART7 also induces EMT by targeting E-cadherin and PTEN and induces relocation of Snail and b-catenin, through the PI3K/Akt/GSK signaling axis.71 The Snail and b-catenin are also targets of miRBART10-3p.72 Another study reported that mir-BART1 also induces tumor metastasis by activating the PTENdependent pathway.73 Moreover, a role of BART-microRNA expression in immune evasion has been implicated.58,74 The miR-BART2-5p can target the major histocompatibility complex class I-related chain B (MICB), which is a stress-induced ligand recognized by NK cells and CD8þT cells. Decreased expression of MICB in cell surfaces of EBV-infected NPC cells may suppress the cytolytic response following NKG2D receptor activation in NK cells.75 The miR-BART20-5p also targets the TBX2/T-bet, a transcription activation of gIFN and regulation of production of IL2 and Th2 cytokines.76 The miR-BART15-3p also inhibits the activation of NLRP3 inflammasome and the production of the proinflammatory cytokine IL-1.77 All these suggest a role of high-level expression of BART-microRNAs to support EBV infection, enhance survival, metastasis, and possibly immune evasion in NPC cells in patients. In regard to their potential functions in EBVassociated NPC pathogenesis, the BART-microRNAs are likely to represent potential therapeutic targets for NPC treatment.

THE CONTRIBUTION OF TUMOR MICROENVIRONMENT TO NASOPHARYNGEAL CARCINOMA PATHOGENESIS A characteristic feature of NPC is the presence of abundant tumor infiltrating leukocytes (TILs), predominantly T lymphocytes. A high percentage of the leukocytes are CD3þ T lymphocytes.78 At least 50% of the cellular components of primary NPC consist of infiltrated lymphocytes. NK cells make up about 5% of these infiltrating leucocytes.79,80 An analysis of leucocytes collected from NPC showed that 12% of these infiltrating leukocytes are T regulatory cells (CD4þ, CD25þ, Foxp3þ).81 A small percentage of B cells (CD19 þ CD20þ) are also present.82 Macrophages and dendritic cells are also present in the leucocyte infiltrates.83 The roles of these TIL on growth and invasive properties of NPC are complex and much remains to be elaborated. This heavy infiltration of TIL may not be involved to suppress growth of EBV-infected NPC cells. In fact, more and more evidence seems to support their role in promoting growth of NPC cells. The rich stroma of NPC has provided a unique TME, which may be essential to support the growth of NPC cells in patients and maintenance of stable EBV infection in NPC cells. NPC is known to be difficult to grow in culture or passage in immune-deficient animals. The absence of the unique NPC TME in cell culture conditions may be a major reason for the difficulty in establishment of EBV-positive NPC cell lines in culture. In fact, most reported NPC cell lines except one (C666-1) have lost their EBV episomes upon propagation in culture. Establishment of NPC patient-derived xenograft (PDX) to grow in vivo in immune-deficient animals also met with low success rate. A low rate of PDX establishment from primary NPC was reported by an earlier study by Pierre Busson (1 of 60 attempts).84 In contrast, metastatic NPC has a much higher rate of success (2 of 4 attempts). A rich TIL in NPC TME may be essential for the growth of primary NPC. Metastatic NPC generally has fewer TIL, which may suggest that metastatic NPC tumors are less dependent on the TME for growth. The metastatic NPC may have acquired additional genetic alterations, which render them less dependent on TME. Indeed, a more recent report also demonstrated a higher rate of establishment of PDX from metastatic NPC in immune-deficient mice.84,85 Defining the role of TME in NPC will contribute to our understanding of NPC pathogenesis and its progression and may lead to identification of new therapeutic targets for its treatment.

THE CONTRIBUTION OF TUMOR MICROENVIRONMENT TO NASOPHARYNGEAL CARCINOMA PATHOGENESIS

Survival

MIP-1 MIP-3

DC cell

PLA2G7 SDF-1 Treatment resistance

Chronic Inflammaon

MCP-1 IP10

Macrophages (type I & 2)

TGF-

Natural killer cell

MDSC

EBV-infected NPC cells

HGF

Angiogenesis

IL6 IL8

IFNSCF IL1-

IL10 IL12 CAF

IL18

IL1-

CD8+ T cell

B cell

Latent EBV infecon in NPC cells

T cell

Mast cell

Metabolism

Immune evasion

CD4+ cell (Treg, Th1, Th2, Th17, TH0,)

Exosomes Endothelial cells

53

Natural killer T cell

Matrix remodeling /invasion

FIGURE 3.5 Tumor microenvironment of nasopharyngeal carcinoma.

The Dynamic Interaction of Tumor Microenvironment Cells With EpsteineBarr Virus-Infected Nasopharyngeal Carcinoma Cells Much remains to be defined on the dynamic interaction of NPC cells with the infiltrated TME cells, which has been postulated to be involved in promoting growth and invasive properties of NPC cells.86 Figure 3.5 shows the presence of multiple stromal cell types and cytokines in the tumor microenvironment and their roles in the pathogenesis of NPC. Many proinflammatory cytokines, including MCP1(CCL2), MIP1-a (CCL3), MIP3-a (CCL20), IP10 (CXCL10), SDF-1 (CXCL12), HGF (hepatocyte growth factor), interferon g, interleukin 1a, interleukin 1b (lymphocyte activating factor), IL10, IL18, and others, are present in the TME of NPC. They are believed to be produced by NPC cells and/or by stromal cells in TME.3 The MCP-1 is produced by the CD68þ monocytes in NPC tumors, which is a chemo-attractant for monocytes, memory T cells, NK cells, and dendritic cells.87 The CD68þ monocytes also secrete MIP1-a to recruit CD8þ T cells. The MIP3-a is produced by the NPC cells and is a chemo-attractant for lymphocytes and dendritic cells signaling through the CCR6 receptor.88 The IP10 produced by NPC cells can induce chemotaxis of activated T cells.89 The SDF-1 is an agonist of the CXCR4 receptor.90,91 The HGF is an agonist of the Met oncogene, which is expressed in NPC. The HGF is produced by stromal cells in NPC and has broad effects on cell proliferation and invasive properties.92 The interferon g regulates the cellular immune response to antigen and is produced at abundant levels by the CD3þ T cells and CD94þ NK cells in NPC.89,93 The interleukin 1-a and b are proinflammatory cytokines produced by NPC cells.94,95 The IL10 is an important immunosuppressive cytokine that may promote immune evasions for NPC cells by suppressing the proliferation of antigen-specific Tcells and downregulating the class II MHC antigen on the antigen-presenting cells by inhibiting CTL (cytotoxic T lymphocyte) activity and IL-2 production from T-helper cells.96 The IL-18 is produced by NPC cells, which enhances production of g-interferon by activated T cells and promotes Th1 differentiation. The IL-18 as well as CXCL-10 can activate and induce IFN-g production by the infiltrating CXCR3þ T cells, which in turn stimulate CD68þ macrophages to produce IL-12 and IL-18, hence forming a positive feedback loop to recruit leucocyte infiltration into the TME.80 Some of these cytokine secretions are the results of EBV infection and are induced by EBV-encoded products expressed during latent and lytic infection. Interestingly, the EBV-encoded LMP1 is expressed at high levels in premalignant NP tissues suggestive of its important role at the early stage of NPC development.97 However, expression of LMP1 in later stages of NPC is heterogeneous. Additional genetic alterations in NPC during cancer progression may substitute some of the signaling events mediated by LMP1. Expression of LMP1 upregulates IL-8, MIP-1a, and

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MIP-1b to attract infiltration of various types of leucocytes into the TME.98 In addition to secretion of cytokines, a role of LMP1 in reprogramming cellular metabolism to modulate TME is emerging. We have recently reported that LMP1 expression in immortalized nasopharyngeal epithelial cells can upregulate GLUT-1 expression and its translocation to the lysosome to activate aerobic glycolysis results in the excessive production and secretion of lactate to the culture medium.99 Increase in lactate level in TME could modulate the immune environment of TME.100 The role of myeloid-derived suppression cells (MDSCs) in mediating immune suppression in human tumor has been implicated in human cancers.102 A recent study reported that LMP1-mediated aerobic glycolysis facilitates recruitment and expansion of MDSCs in NPC.101 The metabolic reprogramming by LMP1 contributes to increased expression of COX-2 and NLRP3 (nod-like receptor family protein 3), which are involved in the formation of inflammasomes leading to increased release of IL-1b, IL6, and GM-CSF into the TME to induce MDSC expansion. The EBV-encoded gene product, EBER, which is expressed at high levels in NPC, can inhibit interferon-stimulated gene activity by binding to PKR, a double-strand RNA-dependent protein kinase.103 Expression of lytic EBV genes is often detected in a small population of EBV-infected NPC cells. The involvement of EBV lytic proteins in immune evasion of EBV-infected B cells is well recognized.104 Their involvement in immune evasion in NPC cells has been implicated.105 The lytic EBV proteins may suppress the secretion of multiple antiviral cytokines. For example, the BZLF1 (Zta) and BRLF1 (Rta), which are the key immediate early lytic EBV genes involved in cellular switching of EBV latent infection to lytic infection, inhibit activation of interferon response genes and type 1 interferon production.106,107 The BZLF1 also inhibits JAK/STAT signaling through SOC3 to inhibit IFNg production by monocytes and induce a state of type I IFN-irresponsiveness.108 The Zta can bind to the IL-8 promoter and induce its expression.109 IL-8 is a potent chemoattractant for neutrophils. Zta can also upregulate secretion of an immune modulators such as GM-CSF (granulocyte-macrophage colony-stimulating factor) and prostaglandin E2.110 Furthermore, the EBV-infected NPC cells may release exosomes to evade immune detection. NPC-derived exosomes have also been shown to promote the activity of regulatory T cells, which further suppress the antitumor response of leucocyte infiltration.111 LMP1 and Galectin 9 were detected in these exosomes.112 LMP1 has broad anergic effects on immune cells including inhibition of T cell-proliferation, NK cell cytotoxicity, and IFN-g release,113 while Galectin-9 can suppress Th1 activity.114

Macrophages in Nasopharyngeal Carcinoma Macrophages are major players in tumor-related inflammation and producers of proinflammatory cytokines in NPC.87 Macrophages in the tumor tissues have been referred as tumor-associated macrophages (TAMs). The involvement of TAMs to support growth and invasive properties of cancer is well documented.115,116 Limited studies have so far assessed the prognostic value of macrophages in NPC. One study reported that an increased M2-polarized TAM in stroma correlates with poor prognosis in NPC.117 NPC patients with dense infiltration of CD163þ macrophages in the tumor stroma were associated with higher tumor (T) or nodal (N) stage. Another study showed that patients with low density of CD68þ macrophages had significantly longer progression-free survival (PFS) and overall survival (OS) than those with a high density of TAMs.118 Besides, high density of TAMs correlated with the levels of IL-6 in NPC tissues, and was associated with poor survival of NPC patients.118 There are a few studies investigating the interaction of macrophages and NPC cells. After coculturing with GMSFactivated macrophages, NPC cells showed decreased expression of E-cadherin, but increased expression of vimentin and EMT-transcription factors.119 It was also reported that primary monocytes and the monocyte cell line (THP-1) could be induced to upregulate the expression of proinflammatory cytokines and metastasisrelated genes after coculturing with the EBV-positive NPC cell line, C666-1, suggesting that the TAM may interact with EBV-infected NPC cells to modify their inflammatory and invasive properties to support cancer progression.120 Furthermore, the NPC cells were found to be enhanced in migratory capabilities through factors secreted by the macrophages. The phospholipase As Group 7 (PLAG7) was one of these macrophage-derived factors. EBVinfected NPC cells could produce VEGF and GM-CSF to attract monocytes to the tumor site and induce their differentiation into M2 macrophages, which support growth and promote metastasis of cancer cells.119 This study further showed the interactive roles between EBV-infected NPC cells and macrophages in promoting tumor invasive and metastatic properties using a humanized mouse model.119 Subcutaneous injection of EBVþve NPC cells (CNE-EBVþ) into the humanized mice led to increased macrophage infiltration in the xenografts, and increased GM-CSF and VEGF production compared with injection of EBVve NPC cells into the humanized mice. Interestingly, activation of EBV replication in the EBV-infected cells (by treatment with cigarette smoke extract) could increase metastasis in the lungs of the humanized mice, but not in those of nonhumanized mice. This suggests EBV reactivation may further modulate the invasive behavior

CONCLUSION

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of the tumor cells under the concerted effects with stromal macrophage. TAMs may also modulate the immune activity of CD8þ CTLs.121 One of the fundamental functions of CTL is to eradicate virally infected cells or tumor cells. Interestingly, TAMs in NPC were detected with the expression of an immunosuppressive protein, tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO), which can facilitate immune escape by impairing the cytotoxic action of CTL.121 In summary, there is a profound complexity of how TAMs promote tumorigenesis of NPC especially under the interaction of EBV infection. Further investigation on the detailed mechanisms of the protumor activity of TAMs, probably through the humanized mice model, is necessary for the advancement of TAM-directed anticancer therapy in NPC.

Cancer Associated Fibroblasts in Nasopharyngeal Carcinoma In addition to TIL, the NPC TME is also composed of other stromal cells such as cancer-associated fibroblast (CAF). CAF play important roles in supporting tumor growth.122e124 However, very little information is available on the properties of CAF isolated from NPC. We have collected a panel of NPC CAF and examined their secretion profiles by cytokine array. Immunofluorescence staining revealed positive staining for aSMA, which supports their biological properties as CAF. Our results revealed that high level of monocyte chemo-attractant protein 1 (MCP-1) was present in the conditioned medium harvested from NPC CAFs (Tsao et al., unpublished observations). The MCP-1 concentrations were confirmed by ELISA to range from 230 to 2776 pg/mL among the different CAFs. IL6 secretion has been detected in an NPC CAF line, which supports growth of C666-1 cells.125 TIMP-1 and TIMP-2 were also detected in conditioned medium collected from these CAFs. The high secretion of MCP-1 by CAFs was also observed in other cancer types.126 MCP-1, also called CCL2, is a small cytokine that is involved in recruitment of monocytes, T cells, and dendritic cells.127 MCP-1 has been reported to be involved in cancer progression. In breast cancer, MCP-1 could mediate the cross-talk between CAFs and cancer cells.128 MCP-1 has also been reported to upregulate the pro-survival signaling in head and neck squamous cell carcinoma.129 Interestingly, LMP1 could induce the expression of MCP-1 in an epithelial cell line.130 Meanwhile, exosomes containing a high abundance of LMP1 have been reported in NPC patients.131 Presumably, EBV-infected cells may be able to regulate MCP-1 expression in CAFs through secreted exosomes, and thus favor NPC progression. It has been reported that MCP-1 promotes metastasis in breast cancers through enhancing retention of TAMs.132 The expression level of tumor-derived MCP-1 has a positive correlation with TAM infiltration and tumor angiogenesis.133 Massive lymphoid infiltration is observed in NPC region. TAMs are speculated to be recruited by MCP-1. The cross-talk between NPC cells, TAMs, and CAFs requires further investigation as well. The role of CAF on the modulation of growth and invasive properties of NPC cells remains to be defined. The role of CAF to support latent infection of EBV would be another important aspect to be investigated.

CONCLUSION The unique anatomical site and the close association with EBV infection make NPC stand out as a distinct type of cancer in the head and neck region. An etiological role of EBV infection in NPC pathogenesis has long been postulated. The recent studies to define genetic alterations in NPC implicate the importance of NF-kB signaling in NPC. The common activation of NF-kB signaling suggests its involvement in NPC pathogenesis and possibly survival of EBV-infected NPC cells. At present, the detailed involvement of EBV infection in NPC pathogenesis and its influence on clinical behaviors of NPC remain to be defined. The expressions of various latent EBV genes or proteins, notably the BART-microRNAs, are believed to be involved in NPC pathogenesis. The high expression of BART-microRNAs in NPC suggests a selective growth advantage of EBV-infected NPC cells, notably in vivo. In addition to the genetic alterations present in NPC, the unique TME of NPC is likely to play an important role to support the growth of EBVinfected NPC cells in patients. Defining the functions of infiltrating lymphocytes and other stromal cell types present in the NPC TME and their interplay with NPC cells is a major challenge in the future, but it will advance our understanding of NPC pathogenesis leading to identification of novel therapeutic strategies for NPC treatment.

Acknowledgments The authors acknowledge the funding supports from Research Grant Council, Hong Kong: AoE NPC grant (AoE/M-06/08), Theme-based Research Scheme grant (T12-401/13-R) and Collaborative Research Fund (C7027-16G, C1013-15G) and General Research Fund (106130105, 17161116, 17120814, 779713, 779312, 17110315, 17111516, 14104415, 14138016, 14117316), and Health and Medical Research Fund (04151726, 13120872, 13142201, 05162386).

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References 1. Ali MY. Histology of the human nasopharyngeal mucosa. J Anat. 1965;99(Pt 3):657e672. 2. Nicholls JM, Agathanggelou A, Fung K, Zeng X, Niedobitek G. The association of squamous cell carcinomas of the nasopharynx with Epstein-Barr virus shows geographical variation reminiscent of Burkitt’s lymphoma. J Pathol. 1997;183(2):164e168. 3. Gourzones C, Klibi-Benlagha J, Friboulet L, Jlidi R, Busson P. Cellular interactions in nasopharyngeal carcinomas. In: Busson P, ed. Nasopharyngeal Carcinoma Advances in Experimental Medicine and Biology. New York: Springer; 2013:82e100. 4. Chan KCA, Woo JKS, King A, et al. Analysis of plasma Epstein-Barr virus DNA to screen for nasopharyngeal cancer. N Engl J Med. 2017; 377(6):513e522. 5. Lo KW, Chung GT, To KF. Acquired genetic and epigenetic alterations in nasopharyngeal carcinoma. In: Busson P, ed. Nasopharyngeal Carcinoma: Keys for Translational Medicine and Biology Landes Bioscience and Springer ScienceþBusiness Media. Landes Bioscience and Springer ScienceþBusiness Media; 2013. 6. Lo KW, To KF, Huang DP. Focus on nasopharyngeal carcinoma. Cancer Cell. 2004;5(5):423e428. 7. Tsang CM, Yip YL, Lo KW, et al. Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells. Proc Natl Acad Sci USA. 2012;109(50):E3473eE3482. 8. Chan J. Tumor of the nasopharynx. In: El-Naggar A, Chan J, Grandis J, Takata T, Slootweg P, eds. WHO Classification of Head and Neck Tumours. 4th ed. IARC Publication; 2017:63e76. 9. Tsao SW, Tsang CM, To KF, Lo KW. The role of Epstein-Barr virus in epithelial malignancies. J Pathol. 2015;235(2):323e333. 10. Nicholls J, Niedobitek G. Histopathological diagnosis of nasopharyngeal carcinoma: looking beyond the blue book. In: Busson P, ed. Nasopharyngeal Carcinoma Advances in Experimental Medicine and Biology. New York: Springer; 2013:10e22. 11. Nawandar DM, Wang A, Makielski K, et al. Differentiation-dependent KLF4 expression promotes lytic Epstein-Barr virus infection in epithelial cells. PLoS Pathogens. 2015;11(10):e1005195. 12. Thornburg NJ, Pathmanathan R, Raab-Traub N. Activation of nuclear factor-kappaB p50 homodimer/Bcl-3 complexes in nasopharyngeal carcinoma. Cancer Res. 2003;63(23):8293e8301. 13. Chung GT, Lou WP, Chow C, et al. Constitutive activation of distinct NF-kappaB signals in EBV-associated nasopharyngeal carcinoma. J Pathol. 2013;231(3):311e322. 14. Lo AK, Lo KW, Tsao SW, et al. Epstein-Barr virus infection alters cellular signal cascades in human nasopharyngeal epithelial cells. Neoplasia (New York, NY). 2006;8(3):173e180. 15. Ho Y, Tsao SW, Zeng M, Lui VW. STAT3 as a therapeutic target for Epstein-Barr virus (EBV): associated nasopharyngeal carcinoma. Cancer Lett. 2013;330(2):141e149. 16. Pan Y, Wang S, Su B, et al. Stat3 contributes to cancer progression by regulating Jab1/Csn5 expression. Oncogene. 2017;36(8):1069e1079. 17. Li YYCG, Lui VW, TO KF Ma BB, et al. Exome and genome sequencing of nasopharynx cancer identifies NF-kB pathway activating mutations. Nat Commun. 2016 (in print). 18. Zheng H, Dai W, Cheung AK, et al. Whole-exome sequencing identifies multiple loss-of-function mutations of NF-kappaB pathway regulators in nasopharyngeal carcinoma. Proc Natl Acad Sci USA. 2016;113(40):11283e11288. 19. Cahir-McFarland ED, Davidson DM, Schauer SL, Duong J, Kieff E. NF-kappa B inhibition causes spontaneous apoptosis in Epstein-Barr virus-transformed lymphoblastoid cells. Proc Natl Acad Sci USA. 2000;97(11):6055e6060. 20. Phoon YP, Cheung AK, Cheung FM, et al. IKBB tumor suppressive role in nasopharyngeal carcinoma via NF-kappaB-mediated signalling. Int J Cancer. 2016;138(1):160e170. 21. Lee DF, Kuo HP, Chen CT, et al. IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway. Cell. 2007; 130(3):440e455. 22. Zhu DD, Zhang J, Deng W, et al. Significance of NF-kappaB activation in immortalization of nasopharyngeal epithelial cells. Int J Cancer. 2016; 138(5):1175e1185. 23. Li Z, Duan Y, Cheng S, et al. EBV-encoded RNA via TLR3 induces inflammation in nasopharyngeal carcinoma. Oncotarget. 2015;6(27): 24291e24303. 24. Hajek M, Sewell A, Kaech S, Burtness B, Yarbrough WG, Issaeva N. TRAF3/CYLD mutations identify a distinct subset of human papillomavirus-associated head and neck squamous cell carcinoma. Cancer. 2017;123(10):1778e1790. 25. Hsiao JR, Jin YT, Tsai ST, Shiau AL, Wu CL, Su WC. Constitutive activation of STAT3 and STAT5 is present in the majority of nasopharyngeal carcinoma and correlates with better prognosis. Br J Cancer. 2003;89(2):344e349. 26. Lui VW, Wong EY, Ho Y, et al. STAT3 activation contributes directly to Epstein-Barr virus-mediated invasiveness of nasopharyngeal cancer cells in vitro. Int J Cancer. 2009;125(8):1884e1893. 27. Kung CP, Raab-Traub N. Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor through effects on Bcl-3 and STAT3. J Virol. 2008;82(11):5486e5493. 28. Kung CP, Meckes Jr DG, Raab-Traub N. Epstein-Barr virus LMP1 activates EGFR, STAT3, and ERK through effects on PKCdelta. J Virol. 2011; 85(9):4399e4408. 29. Li L, Xu J, Qiu G, et al. Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas. Theranostics. 2018;8(1):61e77. 30. Tempera I, De Leo A, Kossenkov AV, et al. Identification of MEF2B, EBF1, and IL6R as direct gene targets of Epstein-Barr virus (EBV) nuclear antigen 1 critical for EBV-infected B-lymphocyte survival. J Virol. 2016;90(1):345e355. 31. Zhang G, Tsang CM, Deng W, et al. Enhanced IL-6/IL-6R signaling promotes growth and malignant properties in EBV-infected premalignant and cancerous nasopharyngeal epithelial cells. PLoS One. 2013;8(5):e62284. 32. Man CH, Wei-Man Lun S, Wai-Ying Hui J, et al. Inhibition of NOTCH3 signalling significantly enhances sensitivity to cisplatin in EBVassociated nasopharyngeal carcinoma. J Pathol. 2012;226(3):471e481. 33. Dechend R, Hirano F, Lehmann K, et al. The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators. Oncogene. 1999;18(22):3316e3323. 34. Lin DC, Meng X, Hazawa M, et al. The genomic landscape of nasopharyngeal carcinoma. Nat Genet. 2014;46(8):866e871.

REFERENCES

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.

57

Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 2003;3(1):11e22. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer. 2011;11(11):761e774. Or YY, Hui AB, To KF, Lam CN, Lo KW. PIK3CA mutations in nasopharyngeal carcinoma. Int J Cancer. 2006;118(4):1065e1067. Yuen JW, Chung GT, Lun SW, Cheung CC, To KF, Lo KW. Epigenetic inactivation of inositol polyphosphate 4-phosphatase B (INPP4B), a regulator of PI3K/AKT signaling pathway in EBV-associated nasopharyngeal carcinoma. PLoS One. 2014;9(8):e105163. Xiao L, Hu Z, Dong X, et al. Targeting EpsteineBarr virus oncoprotein LMP1-mediated glycolysis sensitizes nasopharyngeal carcinoma to radiation therapy. Oncogene. 2014;33(37):4568. Port RJ, Pinheiro-Maia S, Hu C, et al. Epstein-Barr virus induction of the Hedgehog signalling pathway imposes a stem cell phenotype on human epithelial cells. J Pathol. 2013;231(3):367e377. Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cell renewal in carcinogenesis. Nature. 2004;432(7015):324e331. Hu C, Wei W, Chen X, et al. A global view of the oncogenic landscape in nasopharyngeal carcinoma: an integrated analysis at the genetic and expression levels. PLoS One. 2012;7(7):e41055. Lun SW, Cheung ST, Cheung PF, et al. CD44þ cancer stem-like cells in EBV-associated nasopharyngeal carcinoma. PLoS One. 2012;7(12): e52426. Kondo S, Wakisaka N, Muramatsu M, et al. Epstein-Barr virus latent membrane protein 1 induces cancer stem/progenitor-like cells in nasopharyngeal epithelial cell lines. J Virol. 2011;85(21):11255e11264. Kong QL, Hu LJ, Cao JY, et al. Epstein-Barr virus-encoded LMP2A induces an epithelial-mesenchymal transition and increases the number of side population stem-like cancer cells in nasopharyngeal carcinoma. PLoS Pathogens. 2010;6(6):e1000940. Hui AB, Or YY, Takano H, et al. Array-based comparative genomic hybridization analysis identified cyclin D1 as a target oncogene at 11q13.3 in nasopharyngeal carcinoma. Cancer Res. 2005;65(18):8125e8133. Lo KW, Cheung ST, Leung SF, et al. Hypermethylation of the p16 gene in nasopharyngeal carcinoma. Cancer Res. 1996;56(12):2721e2725. Lo KW, Huang DP, Lau KM. p16 gene alterations in nasopharyngeal carcinoma. Cancer Res. 1995;55(10):2039e2043. Saridakis V, Sheng Y, Sarkari F, et al. Structure of the p53 binding domain of HAUSP/USP7 bound to Epstein-Barr nuclear antigen 1 implications for EBV-mediated immortalization. Mol Cell. 2005;18(1):25e36. Ma SD, Hegde S, Young KH, et al. A new model of Epstein-Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas. J Virol. 2011;85(1):165e177. Elgui de Oliveira D, Muller-Coan BG, Pagano JS. Viral carcinogenesis beyond malignant transformation: EBV in the progression of human cancers. Trends Microbiol. 2016;24(8):649e664. Tsao SW, Tsang CM, Lo KW. Epstein-Barr virus infection and nasopharyngeal carcinoma. Philos Trans R Soc Lond Ser B Biol Sci. 2017;372(1732). Shair KHY, Reddy A, Cooper VS. New insights from elucidating the role of LMP1 in nasopharyngeal carcinoma. Cancers. 2018;10(4). Scholle F, Bendt KM, Raab-Traub N. Epstein-Barr virus LMP2A transforms epithelial cells, inhibits cell differentiation, and activates Akt. J Virol. 2000;74(22):10681e10689. Sivachandran N, Sarkari F, Frappier L. Epstein-Barr nuclear antigen 1 contributes to nasopharyngeal carcinoma through disruption of PML nuclear bodies. PLoS Pathogens. 2008;4(10):e1000170. Frappier L. EBNA1. Curr Top Microbiol Immunol. 2015;391:3e34. Iwakiri D, Takada K. Role of EBERs in the pathogenesis of EBV infection. Adv Cancer Res. 2010;107:119e136. Marquitz AR, Raab-Traub N. The role of miRNAs and EBV BARTs in NPC. Semin Cancer Biol. 2012;22(2):166e172. Kang D, Skalsky RL, Cullen BR. EBV BART microRNAs target multiple pro-apoptotic cellular genes to promote epithelial cell survival. PLoS Pathogens. 2015;11(6):e1004979. Qiu J, Smith P, Leahy L, Thorley-Lawson DA. The Epstein-Barr virus encoded BART miRNAs potentiate tumor growth in vivo. PLoS Pathogens. 2015;11(1):e1004561. Yang Y-C, Liem A, Lambert PF, Sugden B. Dissecting the regulation of EBV’s BART miRNAs in carcinomas. Virology. 2017;505:148e154. Lung RW, Hau PM, Yu KH, et al. EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma. J Pathol. 2018;244(4): 394e407. Sugden B. Epstein-Barr virus: the path from association to causality for a ubiquitous human pathogen. PLoS Biol. 2014;12(9):e1001939. Jung YJ, Choi H, Kim H, Lee SK. MicroRNA miR-BART20-5p stabilizes Epstein-Barr virus latency by directly targeting BZLF1 and BRLF1. J Virol. 2014;88(16):9027e9037. Hau PM, Deng W, Jia L, et al. Role of ATM in the formation of the replication compartment during lytic replication of Epstein-Barr virus in nasopharyngeal epithelial cells. J Virol. 2015;89(1):652e668. Lo AKF, To KF, Lo KW, et al. Modulation of LMP1 protein expression by EBV-encoded microRNAs. Proc Natl Acad Sci USA. 2007;104(41): 16164e16169. Lei T, Yuen KS, Xu R, et al. Targeting of DICE1 tumor suppressor by EpsteineBarr virus-encoded miR-BART3* microRNA in nasopharyngeal carcinoma. Int J Cancer. 2013;133(1):79e87. Choy EY-W, Siu K-L, Kok K-H, et al. An Epstein-Barr viruseencoded microRNA targets PUMA to promote host cell survival. J Exp Med. 2008; 205(11):2551e2560. Marquitz AR, Mathur A, Nam CS, Raab-Traub N. The EpsteineBarr Virus BART microRNAs target the pro-apoptotic protein Bim. Virology. 2011;412(2):392e400. Hsu C-Y, Yi Y-H, Chang K-P, Chang Y-S, Chen S-J, Chen H-C. The Epstein-Barr virus-encoded microRNA MiR-BART9 promotes tumor metastasis by targeting E-cadherin in nasopharyngeal carcinoma. PLoS Pathogens. 2014;10(2):e1003974. Cai LM, Lyu XM, Luo WR, et al. EBV-miR-BART7-3p promotes the EMT and metastasis of nasopharyngeal carcinoma cells by suppressing the tumor suppressor PTEN. Oncogene. 2015;34(17):2156e2166. Yan Q, Zeng Z, Gong Z, et al. EBV-miR-BART10-3p facilitates epithelial-mesenchymal transition and promotes metastasis of nasopharyngeal carcinoma by targeting BTRC. Oncotarget. 2015;6(39):41766. Cai L, Ye Y, Jiang Q, et al. Epstein-Barr virus-encoded microRNA BART1 induces tumour metastasis by regulating PTEN-dependent pathways in nasopharyngeal carcinoma. Nat Commun. 2015;6:7353. Raab-Traub N. Nasopharyngeal carcinoma: an evolving role for the Epstein-Barr virus. Curr Top Microbiol Immunol. 2015;390(Pt 1):339e363.

58

3. PATHOGENESIS OF NASOPHARYNGEAL CARCINOMA

75. Nachmani D, Stern-Ginossar N, Sarid R, Mandelboim O. Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells. Cell Host Microbe. 2009;5(4):376e385. 76. Lin TC, Liu TY, Hsu SM, Lin CW. Epstein-Barr virus-encoded miR-BART20-5p inhibits T-bet translation with secondary suppression of p53 in invasive nasal NK/T-cell lymphoma. Am J Pathol. 2013;182(5):1865e1875. 77. Haneklaus M, Gerlic M, Kurowska-Stolarska M, et al. Cutting edge: miR-223 and EBV miR-BART15 regulate the NLRP3 inflammasome and IL-1beta production. J Immunol (Baltimore, Md: 1950). 2012;189(8):3795e3799. 78. Herait P, Ganem G, Lipinski M, et al. Lymphocyte subsets in tumour of patients with undifferentiated nasopharyngeal carcinoma: presence of lymphocytes with the phenotype of activated T cells. Br J Cancer. 1987;55(2):135e139. 79. Ferradini L, Miescher S, Stoeck M, et al. Cytotoxic potential despite impaired activation pathways in T lymphocytes infiltrating nasopharyngeal carcinoma. Int J Cancer. 1991;47(3):362e370. 80. Hu H, Tang KF, Chua YN, et al. Expression of interleukin-18 by nasopharyngeal carcinoma cells: a factor that possibly initiates the massive leukocyte infiltration. Hum Pathol. 2004;35(6):722e728. 81. Lau KM, Cheng SH, Lo KW, et al. Increase in circulating Foxp3þCD4þCD25(high) regulatory T cells in nasopharyngeal carcinoma patients. Br J Cancer. 2007;96(4):617e622. 82. Agathanggelou A, Niedobitek G, Chen R, Nicholls J, Yin W, Young LS. Expression of immune regulatory molecules in Epstein-Barr virusassociated nasopharyngeal carcinomas with prominent lymphoid stroma. Evidence for a functional interaction between epithelial tumor cells and infiltrating lymphoid cells. Am J Pathol. 1995;147(4):1152e1160. 83. Giannini A, Bianchi S, Messerini L, et al. Prognostic significance of accessory cells and lymphocytes in nasopharyngeal carcinoma. Pathol Res Pract. 1991;187(4):496e502. 84. Busson P, Ganem G, Flores P, et al. Establishment and characterization of three transplantable EBV-containing nasopharyngeal carcinomas. Int J Cancer. 1988;42(4):599e606. 85. Hsu CL, Kuo YC, Huang Y, et al. Application of a patient-derived xenograft model in cytolytic viral activation therapy for nasopharyngeal carcinoma. Oncotarget. 2015;6(31):31323e31334. 86. Huang SCM, Tsao SW, Tsang CM. Interplay of viral infection, host cell factors and tumor microenvironment in the pathogenesis of nasopharyngeal carcinoma. Cancers. 2018;10(4). 87. Tang KF, Tan SY, Chan SH, et al. A distinct expression of CC chemokines by macrophages in nasopharyngeal carcinoma: implication for the intense tumor infiltration by T lymphocytes and macrophages. Hum Pathol. 2001;32(1):42e49. 88. Chang KP, Hao SP, Chang JH, et al. Macrophage inflammatory protein-3alpha is a novel serum marker for nasopharyngeal carcinoma detection and prediction of treatment outcomes. Clin Cancer Res. 2008;14(21):6979e6987. 89. Teichmann M, Meyer B, Beck A, Niedobitek G. Expression of the interferon-inducible chemokine IP-10 (CXCL10), a chemokine with proposed anti-neoplastic functions, in Hodgkin lymphoma and nasopharyngeal carcinoma. J Pathol. 2005;206(1):68e75. 90. Orimo A, Gupta PB, Sgroi DC, et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. 2005;121(3):335e348. 91. Wang N, Wu QL, Fang Y, et al. Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma: pattern of expression and correlation with clinical outcome. J Transl Med. 2005;3:26. 92. Qian CN, Guo X, Cao B, et al. Met protein expression level correlates with survival in patients with late-stage nasopharyngeal carcinoma. Cancer Res. 2002;62(2):589e596. 93. Niedobitek G, Agathanggelou A, Nicholls JM. Epstein-Barr virus infection and the pathogenesis of nasopharyngeal carcinoma: viral gene expression, tumour cell phenotype, and the role of the lymphoid stroma. Semin Cancer Biol. 1996;7(4):165e174. 94. Busson P, Braham K, Ganem G, et al. Epstein-Barr virus-containing epithelial cells from nasopharyngeal carcinoma produce interleukin 1 alpha. Proc Natl Acad Sci USA. 1987;84(17):6262e6266. 95. Huang YT, Sheen TS, Chen CL, et al. Profile of cytokine expression in nasopharyngeal carcinomas: a distinct expression of interleukin 1 in tumor and CD4þ T cells. Cancer Res. 1999;59(7):1599e1605. 96. Yao M, Ohshima K, Suzumiya J, Kume T, Shiroshita T, Kikuchi M. Interleukin-10 expression and cytotoxic-T-cell response in Epstein-Barrvirus-associated nasopharyngeal carcinoma. Int J Cancer. 1997;72(3):398e402. 97. Pathmanathan R, Prasad U, Sadler R, Flynn K, Raab-Traub N. Clonal proliferations of cells infected with EpsteineBarr virus in preinvasive lesions related to nasopharyngeal carcinoma. N Engl J Med. 1995;333(11):693e698. 98. Lai H-C, Hsiao J-R, Chen C-W, et al. Endogenous latent membrane protein 1 in EpsteineBarr virus-infected nasopharyngeal carcinoma cells attracts T lymphocytes through upregulation of multiple chemokines. Virology. 2010;405(2):464e473. 99. Zhang J, Jia L, Lin W, et al. Epstein-barr virus-encoded latent membrane protein 1 upregulates glucose transporter 1 transcription via the mTORC1/NF-kappaB signaling pathways. J Virol. 2017;91(6). 100. Colegio OR, Chu NQ, Szabo AL, et al. Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Nature. 2014; 513(7519):559e563. 101. Cai T-T, Ye S-B, Liu Y-N, et al. LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma. PLoS Pathogens. 2017;13(7):e1006503. 102. Kumar V, Patel S, Tcyganov E, Gabrilovich DI. The nature of myeloid-derived suppressor cells in the tumor microenvironment. Trends Immunol. 2016;37(3):208e220. 103. Nanbo A, Inoue K, Adachi-Takasawa K, Takada K. Epstein-Barr virus RNA confers resistance to interferon-alpha-induced apoptosis in Burkitt’s lymphoma. EMBO J. 2002;21(5):954e965. 104. Ressing ME, van Gent M, Gram AM, Hooykaas MJ, Piersma SJ, Wiertz EJ. Immune evasion by Epstein-Barr virus. Curr Top Microbiol Immunol. 2015;391:355e381. 105. Shen Y, Zhang S, Sun R, Wu T, Qian J. Understanding the interplay between host immunity and Epstein-Barr virus in NPC patients. Emerg Microb Infect. 2015;4(3):e20. 106. Bentz GL, Liu R, Hahn AM, Shackelford J, Pagano JS. Epstein-Barr virus BRLF1 inhibits transcription of IRF3 and IRF7 and suppresses induction of interferon-beta. Virology. 2010;402(1):121e128.

REFERENCES

59

107. Morrison TE, Mauser A, Wong A, Ting JP, Kenney SC. Inhibition of IFN-gamma signaling by an Epstein-Barr virus immediate-early protein. Immunity. 2001;15(5):787e799. 108. Michaud F, Coulombe F, Gaudreault E, Paquet-Bouchard C, Rola-Pleszczynski M, Gosselin J. Epstein-Barr virus interferes with the amplification of IFNalpha secretion by activating suppressor of cytokine signaling 3 in primary human monocytes. PLoS One. 2010;5(7):e11908. 109. Hsu M, Wu S-Y, Chang S-S, et al. Epstein-Barr virus lytic transactivator Zta enhances chemotactic activity through induction of interleukin-8 in nasopharyngeal carcinoma cells. J Virol. 2008;82(7):3679e3688. 110. Lee C-H, Yeh T-H, Lai H-C, et al. Epstein-Barr virus Zta-induced immunomodulators from nasopharyngeal carcinoma cells upregulate interleukin-10 production from monocytes. J Virol. 2011;85(14):7333e7342. 111. Mrizak D, Martin N, Barjon C, et al. Effect of nasopharyngeal carcinoma-derived exosomes on human regulatory T cells. JNCI: J Natl Cancer Inst. 2015;107(1). 112. Keryer-Bibens C, Pioche-Durieu C, Villemant C, et al. Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral latent membrane protein 1 and the immunomodulatory protein galectin 9. BMC Cancer. 2006;6(1):283. 113. Dukers DF, Meij P, Vervoort MB, et al. Direct immunosuppressive effects of EBV-encoded latent membrane protein 1. J Immunol. 2000;165(2): 663e670. 114. Lhuillier C, Barjon C, Niki T, et al. Impact of exogenous galectin-9 on human T cells: contribution of the T cell receptor complex to antigenindependent activation but not to apoptosis induction. J Biol Chem. 2015;290(27):16797e16811. 115. Caux C, Ramos RN, Prendergast GC, Bendriss-Vermare N, Menetrier-Caux C. A milestone review on how macrophages affect tumor growth. Cancer Res. 2016;76(22):6439e6442. 116. Mantovani A, Marchesi F, Malesci A, Laghi L, Allavena P. Tumour-associated macrophages as treatment targets in oncology. Nat Rev Clin Oncol. 2017;14(7):399e416. 117. Huang H, Liu X, Zhao F, et al. M2-polarized tumour-associated macrophages in stroma correlate with poor prognosis and Epstein-Barr viral infection in nasopharyngeal carcinoma. Acta Oto-laryngologica. 2017;137(8):888e894. 118. Liao Q, Zeng Z, Guo X, et al. LPLUNC1 suppresses IL-6-induced nasopharyngeal carcinoma cell proliferation via inhibiting the Stat3 activation. Oncogene. 2014;33(16):2098e2109. 119. Huang D, Song SJ, Wu ZZ, et al. Epstein-barr virus-induced VEGF and GM-CSF drive nasopharyngeal carcinoma metastasis via recruitment and activation of macrophages. Cancer Res. 2017;77(13):3591e3604. 120. Low HB, Png CW, Li C, Wang Y, Wong SB, Zhang Y. Monocyte-derived factors including PLA2G7 induced by macrophage-nasopharyngeal carcinoma cell interaction promote tumor cell invasiveness. Oncotarget. 2016;7(34):55473e55490. 121. Liu WL, Lin YH, Xiao H, et al. Epstein-Barr virus infection induces indoleamine 2,3-dioxygenase expression in human monocyte-derived macrophages through p38/mitogen-activated protein kinase and NF-kappaB pathways: impairment in T cell functions. J Virol. 2014; 88(12):6660e6671. 122. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006;6(5):392e401. 123. Bauer M, Su G, Casper C, He R, Rehrauer W, Friedl A. Heterogeneity of gene expression in stromal fibroblasts of human breast carcinomas and normal breast. Oncogene. 2010;29(12):1732e1740. 124. Navab R, Strumpf D, Bandarchi B, et al. Prognostic gene-expression signature of carcinoma-associated fibroblasts in non-small cell lung cancer. Proc Natl Acad Sci USA. 2011;108(17):7160e7165. 125. Tsang CM, Cheung YC, Lui VW, et al. Berberine suppresses tumorigenicity and growth of nasopharyngeal carcinoma cells by inhibiting STAT3 activation induced by tumor associated fibroblasts. BMC Cancer. 2013;13:619. 126. Subramaniam KS, Tham ST, Mohamed Z, Woo YL, Adenan NAM, Chung I. Cancer-associated fibroblasts promote proliferation of endometrial cancer cells. PLoS One. 2013;8(7):e68923. 127. Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 2009; 29(6):313e326. 128. Tsuyada A, Chow A, Wu J, et al. CCL2 mediates cross-talk between cancer cells and stromal fibroblasts that regulates breast cancer stem cells. Cancer Res. 2012;72(11):2768e2779. 129. Ji W-T, Chen H-R, Lin C-H, Lee J-W, Lee C-C. Monocyte chemotactic protein 1 (MCP-1) modulates pro-survival signaling to promote progression of head and neck squamous cell carcinoma. PLoS One. 2014;9(2):e88952. 130. Buettner M, Meyer B, Schreck S, Niedobitek G. Expression of RANTES and MCP-1 in epithelial cells is regulated via LMP1 and CD40. Int J Cancer. 2007;121(12):2703e2710. 131. Meckes DG, Shair KH, Marquitz AR, Kung C-P, Edwards RH, Raab-Traub N. Human tumor virus utilizes exosomes for intercellular communication. Proc Natl Acad Sci USA. 2010;107(47):20370e20375. 132. Kitamura T, Qian B-Z, Soong D, et al. CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages. J Exp Med. 2015;212(7):1043e1059. 133. O’Hayre M, Salanga CL, Handel TM, Allen SJ. Chemokines and cancer: migration, intracellular signalling and intercellular communication in the microenvironment. Biochem J. 2008;409(3):635e649.

Commentary on Chapter 3: The Pathogenesis of Nasopharyngeal Carcinoma: What We Know and What We Don’t Know Lawrence S. Young Warwick Medical School, University of Warwick, Coventry, United Kingdom

The last decade has seen an explosion in our knowledge about the pathogenesis of cancer, principally fuelled by the application of new technologies. As we have come to better understand the genetic and epigenetic landscape of tumors, we have also begun to appreciate the heterogeneity of cancer cells within a single tumor and the crucial role played by the microenvironment in tumor development and progression. A key issue remains the factors, both environmental and genetic, that drive the oncogenic process, and it is here that tumor virology provides important insights into pathogenesis and effective therapeutic interventions. As highlighted by Tsang et al.,1 the consistent presence of EpsteineBarr virus (EBV) in nasopharyngeal carcinoma (NPC) cells offers an important handle on the etiology of this tumor. Along with its unique geographic distribution, distinct site of origin within the upper pharyngeal recess, and profound lymphoid infiltrate, the intimate association of NPC with EBV infection provides an important opportunity to uncover the fundamental mechanisms driving oncogenesis in this tumor.

NASOPHARYNGEAL CARCINOMA PATHOGENESIS As discussed by Tsang et al.,1 the precise contribution of EBV infection to the development of NPC remains unclear. The presence of monoclonal EBV episomes in NPC tumor cells indicates that virus infection precedes the clonal expansion of the malignant cell population.2 However, the lack of epithelial EBV infection in normal nasopharyngeal biopsies from individuals at high risk of developing NPC suggests that epithelial infection may not be the initiating event in virus-associated carcinogenesis.3,4 EBV infection as detected by in situ hybridization to the EBER RNAs is found in high grade (severe dysplastic and carcinoma in situ) preinvasive lesions in the nasopharynx, but not in low grade disease or histologically normal nasopharyngeal epithelium.5,6 Both the high grade and carcinoma in situ lesions carry monoclonal EBV genomes.5 The difficulty in identifying these premalignant lesions, as pointed out by Tsang et al.,1 has precluded large-scale studies of nasopharyngeal dysplasias and so extrapolating these data to a definitive scheme of NPC pathogenesis is questionable. Multiple genetic changes have been found in NPC with frequent deletion of regions on chromosomes 3p, 9p, 11q, 13q, 14q, and 16q and promoter hypermethylation of specific genes on chromosomes 3p (RAS association domain family member 1 [RASSF1A], retinoic acid receptor b2 [RARb2]) and 9p (p16, p15, p14, DAP-kinase).7,8 Both 3p and 9p deletions have been identified in low grade dysplastic lesions and in normal nasopharyngeal epithelium from individuals at high risk of developing NPC in the absence of EBV infection suggesting that genetic events occur early in the pathogenesis of NPC and that these may predispose to establishment of latent EBV infection.6,9 This possibility is supported by in vitro data demonstrating that stable EBV infection of epithelial cells requires an altered, undifferentiated cellular environment10 and that cyclin D1 overexpression (a consequence of cyclin-dependent kinase inhibitor 2A [CDKN2A, which encodes p16INK4A] deletion on chromosome 9p and amplification of the cyclin D1 locus on chromosome 11q) facilitates persistent EBV infection of immortalized nasopharyngeal epithelial cells.11 Thus a scheme has been proposed whereby loss of heterozygosity occurs early in the pathogenesis of NPC possibly as a result of exposure to environmental cofactors such as dietary components (i.e., salted fish) creating low grade preinvasive lesions that after additional genetic and epigenetic events become susceptible to EBV infection. Once infected, EBV latent genes provide growth and survival benefits resulting in the development of NPC. Additional genetic and epigenetic changes occur after EBV infection and contribute to metastatic disease.4 While the precise timing of epigenetic events in the pathway of NPC development is unknown, some changes such as RASSF1A inactivation may occur early, while others (e.g., inactivation of RARB2, CDKN2A and death-associated protein kinase 1 [DAPK]) may be impacted by the ability of EBV to enhance the activity of the methylation machinery.12 Analysis of the genomic landscape in NPC has confirmed a role for chromatin modification in the carcinogenic process and identified additional somatic events including mutations in the ERBB-PI3K signaling pathway.13 A crucial role for the NF-kB pathway in NPC pathogenesis has been recently identified and this appears to be either a consequence of

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LMP1 expression or of genomic aberrations in negative regulators of the NF-kB signaling pathway.14,15 Studying the altered cell signaling landscape in NPC as discussed by Tsang et al.1 is providing important insights into the pathogenesis of NPC and the mutual contribution of genomic mutations versus EBV in targeting these pathways. But is this the whole story? Our understanding of the natural history of EBV infection remains somewhat muddled. While it is accepted that the seat of EBV persistence resides within the memory B cell population, the main site of virus replication remains obscure.4 Studies examining the tropism of EBV have uncovered an elegant mechanism whereby the composition of EBV surface glycoproteins mediating cell fusion differs depending on whether the virus is produced from a B cell or an epithelial cell, and this subsequently impacts the infectivity of EBV.16 Thus EBV produced from an epithelial cell is more infectious for a B cell and vice versa, suggesting an underlying teleological explanation for the dual tropism of EBV. Our previous contention from studies on oral hairy leukoplakia (OHL), an epithelial hyperplasia on the lateral tongue in which EBV lytic replication is observed, was that EBV infection is restricted to the differentiating superficial epithelial cells with no evidence of latent infection in the basal epithelial compartment.17 This observation is consistent with other studies in organotypic raft cultures demonstrating that EBV replication in differentiating epithelial cells is associated with the ability of differentiation-dependent cellular transcription factors to induce expression of the key immediate early EBV genes (BZLF1 and BRLF1).18 This study also found evidence of EBV infection in the undifferentiated basal cells of OHL and of normal tonsillar epithelium raising the possibility that, like human papillomavirus, low level latent EBV infection of basal epithelial cells could be a feature of persistent EBV infection that predisposes to NPC.

EPSTEINeBARR VIRUS STRAIN VARIATION A topic not considered by Tsang et al.1 is the possible role of EBV strain variation in the pathogenesis of NPC. The B cell-derived B95.8 strain of EBV was fully sequenced in 1984 and was, at that time, the largest DNA sequence (172 kilobases) ever determined. While variations in repeat regions of the EBV genome are observed among different EBV isolates, the genomes of viruses from different regions of the world or from patients with different virus-associated diseases appear to be very similar.3,4,19 Strain variation over the EBNA2-encoding (BamHI WYH) region of the EBV genome permits all virus isolates to be classified as either type 1 (EBV-1, B95.8-like) or type 2 (EBV-2, Jijoye-like).19 This genomic variation results in the production of two antigenically distinct forms of the EBNA2 protein with only 50% amino acid homology. Similar allelic polymorphisms (with 50%e80% sequence homology depending on the locus) related to the EBV type occur in a subset of latent genes, namely those encoding EBNA-LP, EBNA3A, EBNA3B, and EBNA3C.20 These differences have functional consequences as EBV-2 isolates are less efficient in in vitro B lymphocyte transformation assays compared with EBV-1 isolates.21 A combination of virus isolation and sero-epidemiological studies suggest that type 1 virus isolates are predominant (but not exclusively so) in many Western countries, whereas both types are widespread in equatorial Africa, New Guinea, and certain other regions.19,22 More recent studies have suggested that type 2 EBV uses T cells as an additional latency reservoir in healthy Kenyan children and that this infection may have been acquired via maternal saliva and breast milk.23 In addition to this broad distinction between EBV types 1 and 2, there is also minor heterogeneity within each virus type. Individual strains have been identified on the basis of changes, compared with B95.8, ranging from single base mutations to extensive deletions.19 While infection with multiple strains of EBV was originally thought to be confined to immunologically compromised patients, other studies have demonstrated that normal healthy seropositives can be infected with multiple EBV isolates and that their relative abundance and presence may vary over time.24 The possible contribution of EBV strain variation to virus-associated tumors remains unknown. A number of studies have failed to establish an epidemiological association between EBV strain variation and disease, concluding that the specific EBV gene polymorphisms detected in virus-associated tumors occur with similar frequencies in EBV isolates from healthy virus carriers from the same geographic region.4,22 However, these studies focused on specific regions of the EBV genome rather than comparing the entire viral DNA sequence. More recent work using nextgeneration sequencing (NGS) of EBV isolates from NPC biopsies have confirmed that, while there is a high level of overall similarity of the NPC-derived virus strains with the prototypical EBV genome, variation exists in viral genes that might result in functional differences.25e27 In this regard, an LMP1 variant containing a 10-amino acid deletion (residues 343 to 352), which was originally identified in Chinese NPC biopsies, has been found to have oncogenic and other functional properties distinct from those of the B95.8 LMP1 gene.28e30 More recent work has identified that EBNA1 derived from NPC has different properties than B95.8 EBNA1 that may impact the carcinogenic process.31 These studies, along with the observation that an EBV isolate cloned from NPC is more efficient at infecting epithelial cells and more lytic in B cells,32 supports the possibility that virus strain variation contributes to the risk of

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developing NPC. However, more biological studies using well-defined EBV variants are required along with more detailed NGS comparisons of tumor-derived EBV strains versus those derived from the blood of patients and from healthy normal donors.

TUMOR MICROENVIRONMENT As highlighted by Tsang et al.1 the prominent stroma in NPC has been poorly characterized. It may be that this stroma is required to support the growth of NPC explaining the difficulty in propagating NPC cells either as xenografts or as tumor cell lines in culture. This idea of the tumor microenvironment promoting tumor growth was suggested over 30 years ago by Thomas Tursz as the folie a deux, whereby the EBV-infected carcinoma cells elaborate soluble factors that attract and support the viability of stromal cells, and these infiltrating cells provide cytokines that enhance the growth and survival of the tumor cells.33 Studies of the tumor microenvironment in NPC and other tumors have since identified the complex interplay between tumor cells and stromal cells particularly as it relates to the immune landscape of cancer.34,35 The tumor immune microenvironment can influence cancer pathogenesis and the response to therapy with so-called hot tumors, providing an environment amenable to various immunotherapeutic interventions.36 As for NPC, the microenvironment appears to constitute an immune suppressive milieu as a consequence of restricted EBV gene expression, modulation of HLA expression in the carcinoma cells, the excretion of immunosuppressive cytokines, and the nature of the inflammatory cell infiltrate.34,37 The precise phenotype of NPC-infiltrating cells and their impact on tumor progression and response to therapy is beginning to be revealed.37 Specific studies examining the levels of tumor-infiltrating lymphocytes in NPC have identified correlations with patient survival,38 and more in-depth examination of the T cell genotype (T cell receptor rearrangements) suggests that differential immune responses and host immunity to NPC impacts prognosis.39 These studies bode well for the development of various immunotherapeutic interventions such as those employing immune checkpoint inhibitors.40

CONCLUSIONS AND FUTURE PERSPECTIVES EBV was discovered over 50 years ago and its DNA was fully sequenced in 1984. It remains the most common persistent virus infection in humans with over 95% of the population sustaining an asymptomatic life-long infection, testimony to the intimate interaction between EBV and the immune host. This relationship relies on the ability of EBV to persist in the memory B cell pool of normal healthy individuals and perturbation of this interaction results in virus-associated B cell tumors. The association of EBV with NPC is less clear and may be a consequence of the aberrant establishment of virus latency in epithelial cells that have already undergone premalignant genetic changes or the expansion of latently infected basal epithelial cells. The pattern of EBV gene expression in NPC is much more variable that previously believed, not only in terms of latent gene expression (e.g., LMP1), but also in the levels of lytic antigen expression,4,14,41 and this may have implications for disease progression and response to therapy. Whatever the precise role of EBV in the carcinogenic process, there is clearly the opportunity to exploit this association for the clinical benefit of NPC patients. The development of more efficient in vitro systems for studying EBV infection and replication in epithelial cells along with the use of recombinant forms of EBV is shedding light on the complex interplay between the virus and host. There is increasing evidence that EBV strain variation is important in the oncogenic process and further improvements in virus genome sequencing technologies, as well as the ability to clone wild-type EBV strains for biological characterization, will allow this key issue to be resolved. Improved in vitro and in vivo model systems will facilitate a better understanding of the host cellevirus interaction, particularly the role of the tumor microenvironment and the local cytokine milieu. Little is known about the processes responsible for the metastatic spread of NPC and how this is influenced by EBV infection. Studies on specimens of metastatic NPC and on circulating tumor cells42 are required to provide mechanistic insights that could impact the management of patients with recurrent or relapsed disease. All of these developments in our basic understanding of the interaction of EBV with epithelial cells and of the contribution of the local microenvironment to NPC will provide novel opportunities for therapeutic intervention. A deeper appreciation of the genomic, epigenomic, and transcriptomic landscape of NPC is providing more clues to the impact of EBV infection on the carcinogenic process. The application of these profiling technologies on

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individual NPC biopsies heralds the era of personalized medicine to guide the application of targeted therapy.43 Incorporating EBV profiling both at the genomic and transcriptional level into these studies as well as the ability to better characterize the tumor microenvironment will further enhance the development of novel individualized therapeutic strategies.

Acknowledgment The authors acknowledge the funding supports from Research Grant Council, Hong Kong: AoE NPC grant (AoE/M-06/08), Theme-based Research Scheme grant (T12-401/13-R) and Collaborative Research Fund (C7027-16G, C1013-15G, C4001-18G) and General Research Fund (106130105, 17161116, 17120814, 779713, 779312, 17110315, 17111516, 14104415, 14138016, 14117316), and Health and Medical Research Fund (04151726, 13120872, 13142201, 05162386, 05160386).

References 1. Tsang CM, Lo KW, Nicholls JM, Tsao SW. Pathogenesis of nasopharyngeal carcinoma: histiogenesis, EBV infection and tumour microenviroment. This book. 2. Raab-Traub N, Flynn K. The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell. 1986;47:883e889. 3. Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer. 2004;4:757e768. 4. Young LS, Yap LF, Murray PG. Epstein-Barr virus: more than 50 years old and still providing surprises. Nat Rev Cancer. 2016;16:789e802. 5. Pathmanathan R, Prasad U, Sadler R,K, Flynn K, Raab-Traub N. Clonal proliferations of cells infected with Epstein-Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. N Engl J Med. 1995;333:693e698. 6. Chan ASC, To KF, Lo KW, et al. High frequency of chromosome 3p deletion in histologically normal nasopharyngeal epithelia from Southern Chinese. Cancer Res. 2000;60:5365e5370. 7. Lo KW, Chung GTY, To KF. Deciphering the molecular genetic basis of NPC through molecular, cytogenetic, and epigenetic approaches. Semin Cancer Biol. 2012;22:79e86. 8. Lung HL, Cheung AKL, Ko JMY, Cheng Y, Stanbridge EJ, Lung ML. Deciphering the molecular genetic basis of NPC through functional approaches. Semin Cancer Biol. 2012;22:87e95. 9. Chan ASC, To KF, Lo KW, et al. Frequent chromosome 9p losses in histologically normal nasopharyngeal epithelia from Southern Chinese. Int J Cancer. 2002;102:300e303. 10. Knox PG, Li QX, Rickinson AB, Young LS. In vitro production of stable Epstein-Barr virus-positive epithelial cell clones which resemble the virus:cell interaction observed in nasopharyngeal carcinoma. Virology. 1996;215:40e50. 11. Tsang CM, Yip YL, Lo KW, et al. Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells. Proc Natl Acad Sci USA. 2012;109:3473e3482. 12. Li L, Zhang Y, Guo BB, et al. Oncogenic induction of cellular high CpG methylation by Epstein-Barr virus in malignant epithelial cells. Chin J Cancer. 2014;33:604e608. 13. Lin DC, Meng X, Hazawa M, et al. The genomic landscape of nasopharyngeal carcinoma. Nat Genet. 2014;46:866e871. 14. Li YY, Chung GTY, Lui VWY, et al. Exome and genome sequencing of nasopharynx cancer identifies NF-kB pathway activating mutations. Nat Commun. 2016;8:14121. 15. Zeng H, Dai W, Cheung AKL, et al. Whole-exome sequencing identifies multiple loss-of-function mutations of NF-kB pathway regulators in nasopharyngeal carcinoma. Proc Natl Acad Sci USA. 2016;113:11283e11288. 16. Hutt-Fletcher LM. The long and complicated relationship between Epstein-Barr virus and epithelial cells. J Virol. 2017;91. e01677e16. 17. Niedobitek G, Young LS, Lau R, et al. Epstein-Barr virus gene expression in oral hairy leukoplakia: virus replication in the absence of a detectable latent phase. J Gen Virol. 1991;72:3035e3046. 18. Nawander DM, Wang A, Makielski K, et al. Differentiation-dependent KLF4 expression promotes lytic Epstein-Barr virus infection in epithelial cells. PLoS Pathog. 2015;11(10):e1005195. 19. Tzellos S, Farrell PJ. Epstein-Barr virus sequence variation e biology and disease. Pathogens. 2012;1, 156e74. 20. Sample J, Young L, Martin B, Chatman T, Kieff E, Rickinson A. Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA- 3B, and EBNA-3C genes. J Virol. 1990;64:4084e4092. 21. Rickinson AB, Young LS, Rowe M. Influence of the Epstein-Barr vı´rus nuclear antigen EBNA2 on the growth phenotype of virus-transformed B cells. J Virol. 1987;61:1310e1317. 22. Khanim F, Yao QY, Niedobitek G, Sihota S, Rickinson AB, Young LS. Analysis of Epstein-Barr virus gene polymorphisms in normal donors and in virus-asssociated tumors from different geographic locations. Blood. 1996;88:3491e3501. 23. Coleman CB, Daud II, Ogolla SO, et al. Epstein-Barr virus type 2 infects T cells in healthy Kenyan Children. J Infect Dis. 2017;15:670e677. 24. Tierney RJ, Edwards RH, Sitki-Green D, et al. Multiple Epstein-Barr virus strains in patients with infectious mononucleosis: comparison of ex vivo samples with in vitro isolates by use of heteroduplex tracking assays. J Infect Dis. 2006;193:287e297. 25. Liu P, Fang X, Feng Z, et al. Direct sequencing and characterization of a clinical isolate of Epstein-Barr virus from nasopharyngeal carcinoma tissue by using next-generation sequencing technology. J Virol. 2011;85:11291e11299. 26. Kwok H, Wu CW, Palser AL, et al. Genomic diversity of Epstein-Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsy samples. J Virol. 2014;88:10662e10672. 27. Tu C, Zeng Z, Qi P, et al. Genome-wide analysis of eighteen Epstein-Barr viruses isolated from primary nasopharyngeal carcinoma biopsies. J Virol. 2017 (in press). 28. Edwards RH, Seillier-Moiseiwitsch F, Raab-Traub N. Signature amino acid changes in latent membrane protein 1 distinguish Epstein-Barr virus strains. Virology. 1999;261:79e95.

64

3. PATHOGENESIS OF NASOPHARYNGEAL CARCINOMA

29. Dawson CW, Eliopoulos AG, Blake SM, Barker R, Young LS. Identification of functional differences between prototype Epstein-Barr virusencoded LMP1 and a nasopharyngeal carcinoma-derived LMP1 in human epithelial cells. Virology. 2000;272:204e217, 2000. 30. Fielding CA, Sandvej K, Mehl AM, Brennan P, Jones M, Rowe M. Epstein-Barr virus LMP-1 natural sequence variants differ in their potential to activate cellular signaling pathways. J Virol. 2001;2001(75):9129e9141. 31. Dheekollu J, Malecka K, Wiedmer A, et al. Carcinoma-risk variant of EBNA1 deregulates Epstein-Barr virus episomal latency. Oncotarget. 2017; 8:7248e7264. 32. Tsai MH, Raykova A, Klinke O, et al. Spontaneous lytic replication and epitheliotropism define an Epstein-Barr virus strain found in carcinomas. Cell Rep. 2013;5:458e470. 33. Busson P, Braham K, Ganem G, et al. Epstein-Barr virus-containing epithelial cells from nasopharyngeal carcinoma produce interleukin 1 alpha. Proc Natl Acad Sci USA. 1987;84:6262e6266. 34. Tan GW, Visser L, Pan LP, van den Berg A, Diepstra A. The microenvironment in Epstein-Barr virus-associated malignancies. Pathogens. 2018;7: 40. https://doi.org/10.3390/pathogens7020040. 35. Thorsson V, Gibbs DL, Brown SD, et al. The immune landscape in cancer. Immunity. 2018;48:812e830. 36. Binnewies M, Roberts EW, Kersten K, et al. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med. 2018; 24:541e550. 37. Lu J, Chen X-M, Huang H-R, et al. Detailed analysis of inflammatory cell infiltration and the prognostic impact on nasopharyngeal carcinoma. Head Neck. 2018;40:1245e1253. 38. Wang Y-Q, Chen Y-P, Zhang Y, et al. Prognostic significance of tumor-infiltrating lymphocytes in nondisseminated nasopharyngeal carcinoma: a large-scale cohort study. Int J Cancer. 2018;142:2558e2566. 39. Chung Y-L, Wu M-L. Spatiotemporal homogeneity and distinctness of the T-cell receptor b-chain repertoires in Epstein-Barr virus-associated primary and metastatic nasopharyngeal carcinoma. Int J Cancer. 2018. https://doi.org/10.1002/ijc.31336 [Epub ahead of print]. 40. Ma BBY, Lim WT, Goh BC, et al. Antitumor activity of nivolumab in recurrent and metastatic nasopharyngeal carcinoma: an international, multicenter study of the mayo clinic phase 2 consortium (NCI-9742). J Clin Oncol. 2018;36:1412e1418. 41. Hu L, Lin Z, Wu Y, et al. Comprehensive profiling of EBV gene expression in nasopharyngeal carcinoma through paired-end transcriptome sequencing. Front Med. 2016;10:61e75. 42. Fu X, Shen C, Wang H, et al. Joint quantitative measurement of hTERT mRNA in both peripheral blood and circulating tumor cells of patients with nasopharyngeal carcinoma and its clinical significance. BMC Cancer. 2017;17:479. 43. Ali SM, Yao M, Yao J, et al. Comprehensive genomic profiling of different subtypes of nasopharyngeal carcinoma reveals similarities and differences to guide targeted therapy. Cancer. 2017. E-pub DOI 10.1002/cncr.30781.