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Shrestha et al. Hepatoma Res 2019;5:32 I http://dx.doi.org/10.20517/2394-5079.2019.24 Page 11 of 17

regulation of immune checkpoint molecules as reported in several instances earlier in this review. EMT is
[94]
also known to promote immune resistance to NK cell-mediated lysis . An EMT inducer, TGF-β, promotes
immunosuppression by several mechanisms including impaired maturation, differentiation or activation
of innate and adaptive immune cells, inhibition of cytotoxic T-cell functions and dysregulating cytokine
[94]
production . The association of EMT and immunosuppression in tumor cells has also been reported
in HCC. A study reported that hypoxia-induced EMT promotes overexpression of CCL20 resulting in
+
[96]
+
reduced CD4 and CD8 T-cell proliferation along with increased immunosuppressive Treg cells . A study
[86]
has reported that Snail-induced EMT is associated with immunosuppression in cancer patients .
In addition, a study has shown that there is an association between EMT score of tumor cells and
[97]
expression of immune checkpoint molecules such as PD-1, PD-L1, PD-L2, B7-H3 and others . Several
lung cancer studies have reported the association between EMT and immune checkpoint molecules. One
of the earlier studies in lung adenocarcinoma reported that EMT was strongly associated with upregulation
of multiple targetable immune checkpoint molecules such as PD-1, PD-L1, PD-2, CTLA-4, BTLA, B7-H3
[98]
and TIM-3 . Similarly, another study in lung adenocarcinoma demonstrated that EMT phenotype was
related to PD-L1 overexpression . Notably, a significant correlation between mesenchymal phenotype
[99]
with expression of immune checkpoint molecules such as PD-1, PD-L1, CTLA-4, OX40L and PD-L2
[97]
was confirmed in lung cancer . MUC1-C has been reported to simultaneously induce EMT and the
[93]
expression of PD-L1 in non-small cell lung cancer . In lung cancer cell lines, induction of EMT through
downregulation of miR-200s and ZEB1 overexpression resulted in increased PD-L1 expression . A very
[100]
[101]
interesting study by David et al. utilized M7824, a bifunctional fusion protein, inhibiting PD-L1 and TGF-β
to demonstrate that TGF-β-induced immunosuppression in non-small cell lung cancer was mediated by
[102]
PD-L1 upregulation. Chae et al. reported reduced infiltration of immune cells with antitumor functions
and increased infiltration of immune cells with immunosuppressive functions in mesenchymal non-small
cell lung cancer. This study further reported increased expression of immune checkpoint molecules CTLA-4
[102]
and TIM-3 in mesenchymal lung adenocarcinoma and lung squamous cell carcinoma .
Furthermore, PD-L1 expression was closely related with EMT as higher PD-L1 expression was observed
[103]
in oral squamous cell carcinoma cells co-cultured with mesenchymal phenotypes . In breast cancer,
[104]
Noman et al. revealed that ZEB-1/miR200 or Snai1 simultaneously induced EMT and upregulated the
[105]
expression of PD-L1. Chen et al. demonstrated that EMT positive human esophageal cancer tissues
had higher PD-L1 expression compared to an EMT negative subgroup. Similar studies have shown
an association between EMT and immune checkpoint expression in several cancers including thymic
carcinoma , melanoma , adeno cystic carcinoma , extrahepatic cholangiocarcinoma and renal cell
[107]
[106]
[108]
[109]
[110]
carcinoma .
Many studies have reported several pathways involved in the regulation of PD-L1 by EMT. PD-L1
expression in non-small cell lung carcinoma was regulated by DNA methylation in a TGF-β1 dependent
[111]
manner and by NF-κB/IKKε signalling pathway in a TNF- α dependent manner . Another study in lung
cancer demonstrated that p-Smad2 dependent TGF-β signalling is involved in PD-L1 overexpression .
[101]
Epidermal growth factor also induced EMT and PD-L1 expression in breast cancer and salivary adenoid
cystic carcinoma cells [108,112] .

In HCC, a significant association of EMT phenotype with PD-L1 expression was reported in 422 HCC
[25]
patients . The study confirmed that high risk HCC patients had significantly higher expression of
mesenchymal marker Vimentin and lower expression of the epithelial marker E-cadherin along with
[25]
elevated expression of PD-L1 . Moreover, the combined coordinate expression of PD-L1 with E-cadherin
[25]
and Vimentin was associated with poor overall survival and recurrence-free survival . This study
suggested that patients with an EMT phenotype may benefit from PD-1/PD-L1 blockade therapy. In vitro