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[64]
while the rest are derived from the BART region . BART miRNAs are thought to play an important role in
EBVaGC and are highly abundant, accounting for up to 15-20% of the total miRNA pool in some EBVaGC
[65]
cell lines . Some studies have demonstrated that xenografts of EBV-infected GC cell lines, including AGS-
EBV and SNU719 cells, in immunocompromised mice show up to 10- or 100-fold overexpression of BART
miRNAs compared to their parental cell lines [66,67] . This suggests that BART miRNAs might be particularly
important for cancer progression in vivo. BART miRNAs are known to play a range of roles in GC,
targeting host cell apoptosis, cell cycle, and metastasis [68,69] . Their role in immune evasion is less well studied
in GC, although multiple immune-related functions have been attributed to BART miRNAs in other EBV-
[64]
associated cancers .

In contrast to BART miRNAs, which are expressed in all EBV latent stages, BHRF1 miRNAs are thought
to be expressed only in latency III and lytic replication. BHRF1 miRNAs are commonly reported as barely
[49]
detectable in GC tissues and cell lines [4,54,62] . However, Treece et al. analyzed miRNA expression in FFPE
tissues from 78 cases of gastric adenocarcinoma, including 20 EBVaGCs, and reported that BHRF1-2-5p
was significantly overexpressed in EBV-infected vs. EBV-negative cancers, although to a lower extent than
[52]
BART miRNAs. Marquitz et al. also detected low expression of BHRF1 miRNAs in sequencing from
EBV-infected GC cells (AGS-EBV). Both groups attributed the detection of BHRF1 miRNAs to possible
low levels of viral replication. Given that several studies have reported the expression of a subset of lytic
transcripts in EBVaGC, even in the absence of lytic replication, the level and importance of BHRF1 miRNA
expression remain to be determined. In an in vitro model of EBV-driven B-cell differentiation, BHRF1-2-5p
[70]
was found to downregulate PD-L1 . The same study identified potential binding sites for some
BART miRNAs in the PD-L1 3’-UTR, including BART19-3p, but overexpression of the miRNA did
not appear to influence PD-L1 expression. Four BART miRNAs, BART2-5p, BART7-3p, BART14-
3p, miR-BART22 were found to interact with PD-L1 mRNA in a high-throughput photoactivatable
ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) study in lymphoblastoid
cell lines, but no validation experiments have been performed [71,72] . A recent study reported that
miR-BART5-5p, which shares seed homology with the host miRNAs miR-18a-5p and miR-18b-5p,
leads to signal transducers and activators of transcription 3 (STAT3)-dependent transcriptional PD-
[73]
L1 upregulation by targeting the STAT3 inhibitor Protein Inhibitor Of Activated STAT 3 (PIAS3) .

INTRINSIC SIGNALING
PD-L1 expression can be dysregulated by oncogenic activation of signaling pathways like the JAK/STAT,
phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR),
Mitogen-activated protein kinase/ERK kinase (MEK)/ERK, and Jun/Activator protein 1 (AP-1) pathways.
These pathways act independently or synergistically to control PD-L1 expression, at the transcriptional,
post-transcriptional, and post-translational stage [74-76] . Their importance in PD-L1 regulation tends to vary
among different cancer types. The constitutive activation of an intrinsic signaling pathway in cancer is
usually the result of mutations or SVs in genes of key components or regulators of the pathway. In the case
of virus-associated cancers, viral proteins can also induce constitutive signaling in infected cells.

Signaling activation by host gene mutations
A frequently mutated gene in EBVaGC cancer is PIK3CA, which encodes a catalytic component of the
PI3K kinase [4,77] . The most common variants in PIK3CA are associated with increased PI3K signaling
[77]
activity . The PI3K pathway is thought to regulate PD-L1 expression in a tissue-specific manner. Loss of
Phosphatase and tensin homolog (PTEN) leads to activation of the PI3K pathway and induction of PD-
[79]
L1 expression in gliomas and colorectal cancer [78,79] . In gliomas, Parsa et al. showed that the PI3K/Akt/
mTOR pathway increases PD-L1 mRNA translation through polysomal recruitment. In the case of GC,
there is conflicting evidence for the importance of the PI3K pathway in PD-L1 regulation. Supporting the
importance of PI3K signaling in promoting PD-L1 expression, Kim et al. showed that the PI3K inhibitor
[80]