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Miliotis et al. J Cancer Metastasis Treat 2020;6:13 I http://dx.doi.org/10.20517/2394-4722.2020.12 Page 9 of 15
[81]
LY294002 reduced PD-L1 expression in three GC cell lines. In addition, Menyhárt et al. performed
hierarchical clustering to determine the mutations that could best stratify TCGA GC patients based on
PD-L1 expression. They showed that the mutation status of PIK3CA served as the best root node for the
stratification, while mutations in other immune-related genes like MEF2C, SLC11A1, and KIF15 could
help further refine it, suggesting potential interactions between these genes for the control of PD-L1
[82]
[81]
expression . However, Mimura et al. performed experiments with a panel of GC cell lines and
[77]
determined that the PI3K inhibitor wortmannin did not affect PD-L1 expression. Besides, Seo et al.
analyzed 112 EBV-positive GC samples and reported that PIK3CA mutations did not show any correlation
with PD-L1 expression or TIL abundance. Therefore, the importance of PIK3CA mutations and PI3K
signaling in the control of PD-L1 expression in GC and EBVaGC is still unclear, and may be dependent on
interactions with other mutations and signaling pathways.

Another gene that is commonly mutated in EBVaGC is AT-Rich Interaction Domain 1A (ARID1A),
which encodes an important component of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin
[80]
[4]
remodeling complex . Mutations in ARID1A are usually loss-of-function mutations. Kim et al. analyzed
273 GC samples and showed that ARID1A protein loss correlated with PD-L1 positivity (as defined by
IHC staining), independently of EBV or MSI status. They also showed that ARID1A knockdown in vitro
directly leads to PD-L1 overexpression, through the activation of the PI3K/Akt pathway. To account for
the variability in PD-L1 expression among ARID1A-mutated tumors, the authors looked for additional
mutations that could be acting synergistically with ARID1A mutations. They found KRAS mutations in the
three ARID1A-mutated MSI-H tumors with the highest PD-L1 expression, while two other samples that
harbored KRAS but not ARID1A mutations did not show elevated PD-L1 levels. These data further suggest
that PD-L1 expression is controlled by multiple oncogenic signaling pathways acting in coordination.

Signaling activation by viral latent proteins
The EBV latent membrane proteins (LMPs) are potent oncoproteins and are known to activate oncogenic
signaling cascades in EBV-associated malignancies. In epithelial cancers, LMP2A has been found to
regulate the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), PI3K/Akt, MEK/ERK,
and transforming growth factor beta (TGFβ) pathways [83-86] . In GC specifically, LMP2A has been reported
to lead to constitutive NFκB activation by inhibiting the expression of the nuclear factor of kappa light
polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), which negatively regulates NFκB activity [83,84] .
The mechanism of IκBα downregulation appears to be unrelated to promoter methylation and remains to
be determined. Independent studies have shown that NFκB promotes PD-L1 transcription in GC [87-89] , but
no direct association has been made between LMP2A, NFκB, and PD-L1. As mentioned above, LMP2A
is also thought to be important for promoting the DNA hypermethylated state observed in EBVaGC.
[90]
Hino et al. showed that LMP2A induces phosphorylation and activation of STAT3, which in turn leads
to overexpression of DNMT1, thus changing the DNA methylation landscape of the cell. They showed
that one of the targets of DNMT1 is PTEN, which encodes a negative regulator of the PI3K pathway. The
hypermethylation of the PTEN promoter reduces PTEN expression and leads to overactivation of the PI3K
pathway . Interestingly, PTEN is also known to be directly targeted by miR-BART1 and BART7-3p in
[90]
[93]
NPC [91,92] . Moon et al. reported that the small interfering RNA (siRNA)-mediated knockdown of LMP2A
did not affect PD-L1 expression in SNU719, an EBVaGC cell line. However, more studies with additional
methods of manipulating LMP2A expression and activity are required to elucidate its role in PD-L1
regulation.

INTERFERON GAMMA-INDUCIBLE PD-L1 EXPRESSION
+
Multiple studies have reported that GC patients with higher levels of CD8 TILs also have higher PD-L1
expression [94-96] . This suggests that PD-L1 overexpression in EBVaGC is at least partly a result of the
+
evolutionary pressure from the adaptive immune response acting against the cancer. When CD8 T cells are