NLRC5 Might Promote Endometrial Cancer Progression by Inducing PD-L1
Expression
Endometrial cancer (EC) is the most common cancer of uterine cancer, and defined as
the fifth leading cause of cancer death in the United States. In 2020, there were an estimated 79 420 new women are diagnosed with uterine
cancer and 16 880 deaths in the United States. Approximately 80% of EC patients developed symptomatic presentation and the
5-year survival rate of these cases was 75% to 90%. However, approximately 20% of EC patients are diagnosed with metastatic or
recurrent, and these kinds of cases led to considerably poorer outcomes with a
relative 5-year survival rate of 9% to 17%. Therefore, it is imperative to elucidate new molecular targets and
therapeutic approaches for EC. NOD-like receptor family caspase recruitment domain family domain-containing 5
(NLRC5) is a newly identified member of the NLR family. Human NLRC5 is located in the 16q13 locus and consists of 1866 amino acids
(aa) while mouse NLRC5 is at chromosome 8 and contains 1915 aa. NLRC5 possesses 3
structural domains including the N-terminal atypical caspase activation and
recruitment domain (CARD), which is completely distinct from the other NLRs, the
central NACHT domain, which contains the nucleotide-binding domain (NBD) and 27
leucine-rich repeats (LRRs) at the C-terminal. It has been demonstrated that NLRC5 is a negative regulator of inflammatory
response and type I interferon (IFN) production, and an activator and synergetic
component of the inflammasome. Moreover, NLRC5, known as an major histocompatibility complex (MHC) class I
transactivator, is a novel target for immune evasion in cancer. Downregulation of
NLRC5 in cancer is crucially correlated with decreased levels of MHC class I
molecules and impaired cytotoxic T cell activities. Recruitment of NLRC5 contributes
to the presentation of tumor antigens to CD8 + T cells, which further
increases the antitumor immunity. Intriguingly, in several cancer cells, accumulation of NLRC5 contributes to
cancer progression by promoting cancer cell proliferation, migration, and
invasion. , This interesting and even conflicting evidence might arise from the fact that the
exact function of NLRC5 could be cell type- and tumor microenvironment-dependent.
Our previous study in EC cells showed that NLRC5 may contribute to EC progression by
promoting cell migration and invasion. Nevertheless, the mechanism underlying NLRC5 in EC progression needs further
determined. The programmed cell death ligand 1 (PD-L1) receptor is one of the
important immune checkpoint proteins and is mainly expressed on mature cytotoxic T
lymphocytes in the tumor microenvironment. PD-L1 is also expressed on cancer cells and tumor intrinsic signaling pathways, and restricted by the tumor suppressor genes. Accumulating evidence shows that high expression of tumor cell-intrinsic
PD-L1 contributes to cancer initiation, metastasis, development, and recurrence in
multiple tumor types. , However, the mechanism related to NLRC5 downregulation in EC
patients remains unknown, and whether NLRC5 promotes EC progression by regulating
PD-L1. In our study, we detected the expressions of NLRC5 and PD-L1 in the endometrium
tissue microarray. Furthermore, we analyzed the expression correlation between NLRC5
and PD-L1. We also determined the correlation of expression levels of NLRC5 and
PD-L1 with different clinicopathologic features in EC patients. In addition, we
explore whether NLRC5 could regulate the tumor cell-intrinsic PD-L1 in EC cell
lines. Lastly, we explored the potential mechanism related to downregulated NLRC5 in
EC patients. Our evidence presented that NLRC5 was downregulated and PD-L1 was
upregulated in EC. Furthermore, there is a negative correlation between the
expressions of NLRC5 and PD-L1. Additionally, NLRC5 promotes the expression of PD-L1
in EC cells. The mutations of ANKRD20A2, C2orf42, ADGRB3, AVPR2, GOLGA6C, and IPPK
may lead to downregulation of NLRC5 in EC patients. 

Patients and Tissues Microarray The Anhui Medical University's Institutional Review Board approved the current
study (Approval No.: 20180023), and all patients provided written informed
consent. A tissue microarray composed of 60 EC endometrial tissue samples and 36
control endometrial tissues was analyzed. Clinical data collected for all EC
patients included histological subtype, lymph node metastasis, tumor grade,
tumor stage, and age. The 2009 Federation International of Gynecology and
Obstetrics (FIGO) criteria were used to validate tumor staging. The World Health
Organization (WHO) criteria for histological subtyping and tumor grading were
followed. Age and body mass index (BMI) were collected for all 36 participants
from who normal endometrial tissue samples were collected. Participants had not
undergone chemotherapy, radiotherapy, immunotherapy, or other treatments before
sample collection. Immunohistochemistry PD-L1 and NLRC5 expression levels were detected via immunohistochemistry as in
prior studies. Antigen retrieval was accomplished by microwaving tissue
microarray samples for 15 min in a citric saline solution after
deparaffinization with xylene and dehydration with ethanol. After 15 min of
treatment with 0.3% H 2 O 2 to quench endogenous peroxidase
activity, sections were blocked with 2% bovine serum albumin (BSA) and incubated
overnight at 4 °C with anti-NLRC5 antibodies (ab105411, Abcam, 1:100) or PD-L1
(ab213524, Abcam, 1:100). Sections were then washed, probed for 1 h at room
temperature with a biotinylated secondary antibody (G1210-2-A, Servicebio), and
protein detection was then visualized with 3′-diaminobenzidine
tetrahydrochloride. Following hematoxylin counterstaining, slides were
dehydrated, mounted, and 5 random fields of view per slide were imaged for
quantification (200x magnification) using a fluorescent microscope. Background
lighting was consistent for all samples. Image-Pro Plus 6.0 (Media Cybernetics,
Inc.) was used to examine staining intensity and found that dark brown staining
indicated a positive staining reaction. Cell Culture The American Type Culture Collection (ATCC) provided the HEC-1A human EC cell
line (Accession number: HTB-112), while the European Collection of Authenticated
Cell Cultures supplied the Ishikawa cells (Accession number: 99040201). At 37 °C
in a 5% CO 2 incubator, cells were cultured in Roswell Park Memorial
Institute (RPMI)-1640 (Invitrogen) containing 10% fetal bovine serum (FBS,
HyClone). Immunofluorescence Staining HEC-1A and Ishikawa cells were permeabilized for 20 min with 0.1% Triton X-100
(Thermo Fisher Scientific) after being fixed for 30 min with 4%
paraformaldehyde. Samples were then stained for 45 min at 37 °C with primary
anti-NLRC5 and anti-PD-L1 antibodies, rinsed (5 min/wash at room temperature)
with an immunohistochemical washing solution (Beyotime), and stained with
appropriate secondary antibodies for 45 min at 37 °C. Immunofluorescence
blocking solution (Sigma) was applied after 3 further washes as described above,
and slices were mounted and photographed using laser scanning confocal
microscopy (Nikon). Cell Transient Transfection The sequences of NLRC5 plasmid and siRNA-NLRC5 was performed as our previous study. HEC-1A and Ishikawa cells were transfected with these constructs using
Lipofectamine™ 2000 (Invitrogen) based on provided directions. Western Blotting Analysis To lyse cells, a protein extraction solution (Beyotime) was employed, followed by
a bicinchoninic acid (BCA) assay kit (SinoBio Biotech) to determine protein
quantities. Following centrifugation, lysates were transferred to polyvinylidene
fluoride (PVDF) membranes (Millipore Corp). After blocking with 5% nonfat milk
in tris buffered saline tween-20recipe (TBST) for 4 h at 37 °C, the blots were
treated overnight with PD-L1 (ab213524, Abcam, 1:1000), anti-NLRC5 (ab105411,
Abcam, 1:1000), and β-actin (GB12001, Servicebio, 1:5000) at 4 °C in primary
antibody dilution buffer (Beyotime), washed 3 times using TBST, and probed at
37 °C for 1 h with an horseradish peroxidase (HRP)-conjugated secondary antibody
(1:10 000). Protein bands were identified using an ECL kit (ECL-plus, Thermo
Scientific) after the blots were washed 3 times with TBST. Analyses were
repeated in triplicate a minimum of 3 times. Whole-exome Sequencing (WGS) From 3 EC patients, the genomic DNA was extracted from peripheral blood samples.
All gDNA samples met the purity requirements (OD260/280 > 1.8) and
concentration levels (50 ng/mL) necessary for sequencing. Upon fragmentation
with NEBNext dsDNA Fragmentase into 100 to 800 bp segments with a peak size of
∼250 bp, samples were treated to end repair, dA-Tailing, and adaptor ligation
with the Illumina NEBNext DNA Library Prep Reagent Set (acquired from New
England Biolabs). DNA fragments were separated using a 2% agarose gel
electrophoresis following adaptor ligation, and 300 to 400 bp fragments were
removed for further study. Ten cycles of PCR amplification with PE primers
(Illumina) and Phusion DNA polymerase (New England Biolabs) were performed on
the samples. The amplified sequences were then utilized to capture exome
sequences using an Illumina Truseq Exome Enrichment kit V6, which contained a
31.3 Mb CCDS (97.2% of the US NCBI CCDS Library) region covering approximately
20 794 genes within 62 Mb of coding exons (Illumina). After enrichment, 10
additional rounds of PCR amplification were performed as above, after which the
sizing of amplicons was checked and a BioAnalyzer 2100 was used to quantify
amplicon concentrations. The amplicons weresize-checked and quantitated using a
BioAnalyzer 2100, and then subjected to 2 ×150 bp paired-end massively parallel
sequencing using a Hiseq2500 SequencingSystem (Illumina). Statistical Analysis All data were analyzed using SPSS 23.0 (IL, USA), which is provided as
means ± SEM and analyzed using one-way ANOVA tests. To find significant
variations across groups, Duncan's multiple range tests were performed and to
investigate correlations between samples and Pearson’s correlation analysis was
utilized. P < .05 was the significance threshold. 

The Anhui Medical University's Institutional Review Board approved the current
study (Approval No.: 20180023), and all patients provided written informed
consent. A tissue microarray composed of 60 EC endometrial tissue samples and 36
control endometrial tissues was analyzed. Clinical data collected for all EC
patients included histological subtype, lymph node metastasis, tumor grade,
tumor stage, and age. The 2009 Federation International of Gynecology and
Obstetrics (FIGO) criteria were used to validate tumor staging. The World Health
Organization (WHO) criteria for histological subtyping and tumor grading were
followed. Age and body mass index (BMI) were collected for all 36 participants
from who normal endometrial tissue samples were collected. Participants had not
undergone chemotherapy, radiotherapy, immunotherapy, or other treatments before
sample collection. 

PD-L1 and NLRC5 expression levels were detected via immunohistochemistry as in
prior studies. Antigen retrieval was accomplished by microwaving tissue
microarray samples for 15 min in a citric saline solution after
deparaffinization with xylene and dehydration with ethanol. After 15 min of
treatment with 0.3% H 2 O 2 to quench endogenous peroxidase
activity, sections were blocked with 2% bovine serum albumin (BSA) and incubated
overnight at 4 °C with anti-NLRC5 antibodies (ab105411, Abcam, 1:100) or PD-L1
(ab213524, Abcam, 1:100). Sections were then washed, probed for 1 h at room
temperature with a biotinylated secondary antibody (G1210-2-A, Servicebio), and
protein detection was then visualized with 3′-diaminobenzidine
tetrahydrochloride. Following hematoxylin counterstaining, slides were
dehydrated, mounted, and 5 random fields of view per slide were imaged for
quantification (200x magnification) using a fluorescent microscope. Background
lighting was consistent for all samples. Image-Pro Plus 6.0 (Media Cybernetics,
Inc.) was used to examine staining intensity and found that dark brown staining
indicated a positive staining reaction. 

The American Type Culture Collection (ATCC) provided the HEC-1A human EC cell
line (Accession number: HTB-112), while the European Collection of Authenticated
Cell Cultures supplied the Ishikawa cells (Accession number: 99040201). At 37 °C
in a 5% CO 2 incubator, cells were cultured in Roswell Park Memorial
Institute (RPMI)-1640 (Invitrogen) containing 10% fetal bovine serum (FBS,
HyClone). 

HEC-1A and Ishikawa cells were permeabilized for 20 min with 0.1% Triton X-100
(Thermo Fisher Scientific) after being fixed for 30 min with 4%
paraformaldehyde. Samples were then stained for 45 min at 37 °C with primary
anti-NLRC5 and anti-PD-L1 antibodies, rinsed (5 min/wash at room temperature)
with an immunohistochemical washing solution (Beyotime), and stained with
appropriate secondary antibodies for 45 min at 37 °C. Immunofluorescence
blocking solution (Sigma) was applied after 3 further washes as described above,
and slices were mounted and photographed using laser scanning confocal
microscopy (Nikon). 

The sequences of NLRC5 plasmid and siRNA-NLRC5 was performed as our previous study. HEC-1A and Ishikawa cells were transfected with these constructs using
Lipofectamine™ 2000 (Invitrogen) based on provided directions. 

To lyse cells, a protein extraction solution (Beyotime) was employed, followed by
a bicinchoninic acid (BCA) assay kit (SinoBio Biotech) to determine protein
quantities. Following centrifugation, lysates were transferred to polyvinylidene
fluoride (PVDF) membranes (Millipore Corp). After blocking with 5% nonfat milk
in tris buffered saline tween-20recipe (TBST) for 4 h at 37 °C, the blots were
treated overnight with PD-L1 (ab213524, Abcam, 1:1000), anti-NLRC5 (ab105411,
Abcam, 1:1000), and β-actin (GB12001, Servicebio, 1:5000) at 4 °C in primary
antibody dilution buffer (Beyotime), washed 3 times using TBST, and probed at
37 °C for 1 h with an horseradish peroxidase (HRP)-conjugated secondary antibody
(1:10 000). Protein bands were identified using an ECL kit (ECL-plus, Thermo
Scientific) after the blots were washed 3 times with TBST. Analyses were
repeated in triplicate a minimum of 3 times. 

From 3 EC patients, the genomic DNA was extracted from peripheral blood samples.
All gDNA samples met the purity requirements (OD260/280 > 1.8) and
concentration levels (50 ng/mL) necessary for sequencing. Upon fragmentation
with NEBNext dsDNA Fragmentase into 100 to 800 bp segments with a peak size of
∼250 bp, samples were treated to end repair, dA-Tailing, and adaptor ligation
with the Illumina NEBNext DNA Library Prep Reagent Set (acquired from New
England Biolabs). DNA fragments were separated using a 2% agarose gel
electrophoresis following adaptor ligation, and 300 to 400 bp fragments were
removed for further study. Ten cycles of PCR amplification with PE primers
(Illumina) and Phusion DNA polymerase (New England Biolabs) were performed on
the samples. The amplified sequences were then utilized to capture exome
sequences using an Illumina Truseq Exome Enrichment kit V6, which contained a
31.3 Mb CCDS (97.2% of the US NCBI CCDS Library) region covering approximately
20 794 genes within 62 Mb of coding exons (Illumina). After enrichment, 10
additional rounds of PCR amplification were performed as above, after which the
sizing of amplicons was checked and a BioAnalyzer 2100 was used to quantify
amplicon concentrations. The amplicons weresize-checked and quantitated using a
BioAnalyzer 2100, and then subjected to 2 ×150 bp paired-end massively parallel
sequencing using a Hiseq2500 SequencingSystem (Illumina). 

All data were analyzed using SPSS 23.0 (IL, USA), which is provided as
means ± SEM and analyzed using one-way ANOVA tests. To find significant
variations across groups, Duncan's multiple range tests were performed and to
investigate correlations between samples and Pearson’s correlation analysis was
utilized. P < .05 was the significance threshold. 

General Data Endometrial tissues obtained from 60 patients with EC and 36 normal endometrial
tissues were used for the tissue microarray. Demographic characteristics are
summarized in . The 2 groups were matched for age (t = 1.169, P = .245) and BMI (t = 1.601, P = .113). Among
the EC patients, 49 (81.7%) were postmenopausal and 11 (18.6) were
premenopausal, and endometrioid histology was detected in 51 (85.0%) patients,
whereas serous histology was detected in 9 (15.0%) patients. According to the
FIGO criteria (2009), 20 (28.0%) patients had stage I EC, 28 (46.7%) had stage
II EC, 10 (16.7%) had stage III EC, and 2 (3.3%) had stage IV EC. Furthermore,
54 (90.0%) patients were in the G1-G2 stage, 50 (83.3%) patients showed
myometrial invasion, and 6 (10.0%) patients possessed lymph node metastasis. Comparisons of the Levels of NLRC5 and PD-L1 in the Normal Endometrium and
the Endometrium of EC Patients and Their Correlations With the Clinicopathologic
Features of EC Patients Immunohistochemistry analysis of tissue microarrays revealed that the NLRC5
expression in the endometrium of EC patients was significantly lower than that
in the normal endometrium (t = 5.053, P < .001), and PD-L1
was upregulated in the endometrium of EC patients when compared to that in
normal endometrium (t = 8.704, P < .001; , ). No
correlation was found between the level of NLRC5, PD-L1 and histology,
myometrial invasion, lymphatic node metastasis, FIGO stage (2009), and
histological grade in EC patients . Furthermore, there is no
correlation between NLRC5, PD-L1, and cumulative survival in EC patients . NLRC5 Promotes PD-L1 Expression in EC Cells Immunofluorescence staining analysis results showed that NLRC5 and PD-L1 are
co-located in the cytoplasm of HEC-1A and Ishikawa cells . There is a negative
expression correlation between NLRC5 and PD-L1 (r = −0.666, P < .001; ). Moreover, western blotting analysis results showed that
NLRC5 plasmid could promote NLRC5 protein expression effectively in HEC-1A cells
.
Overexpression of NLRC5 led to PD-L1 protein being upregulated in HEC-1A and
Ishikawa cells . siRNA-NLRC5 could inhibit NLRC5 protein expression effectively
in HEC-1A cells . Downregulation of NLRC5 inhibited PD-L1 protein expression in
HEC-1A and Ishikawa cells . These results indicated that NLRC5 played a positive
role in PD-L1 expression in EC cells. Mutations of ANKRD20A2, C2orf42, ADGRB3, AVPR2, GOLGA6C, and IPPK may Lead to
Downregulation of NLRC5 in EC Patients To assess the mutational statuses of the EC patients, WGS was performed for the 3
EC patients and their own peripheral blood, which was set as control group. The
top 10 mutated genes were shown in . However, we did not find
somatic mutations of NLRC5 in the samples. Furthermore, the mutation type was
shown in .
Additionally, using the muTarget database ( http://www.mutarget.com/ ),
we suggested that mutations of ANKRD20A2, C2orf42, ADGRB3, AVPR2, GOLGA6C, and
IPPK may lead to the downregulation of NLRC5 in EC patients . 

Endometrial tissues obtained from 60 patients with EC and 36 normal endometrial
tissues were used for the tissue microarray. Demographic characteristics are
summarized in . The 2 groups were matched for age (t = 1.169, P = .245) and BMI (t = 1.601, P = .113). Among
the EC patients, 49 (81.7%) were postmenopausal and 11 (18.6) were
premenopausal, and endometrioid histology was detected in 51 (85.0%) patients,
whereas serous histology was detected in 9 (15.0%) patients. According to the
FIGO criteria (2009), 20 (28.0%) patients had stage I EC, 28 (46.7%) had stage
II EC, 10 (16.7%) had stage III EC, and 2 (3.3%) had stage IV EC. Furthermore,
54 (90.0%) patients were in the G1-G2 stage, 50 (83.3%) patients showed
myometrial invasion, and 6 (10.0%) patients possessed lymph node metastasis. 

Immunohistochemistry analysis of tissue microarrays revealed that the NLRC5
expression in the endometrium of EC patients was significantly lower than that
in the normal endometrium (t = 5.053, P < .001), and PD-L1
was upregulated in the endometrium of EC patients when compared to that in
normal endometrium (t = 8.704, P < .001; , ). No
correlation was found between the level of NLRC5, PD-L1 and histology,
myometrial invasion, lymphatic node metastasis, FIGO stage (2009), and
histological grade in EC patients . Furthermore, there is no
correlation between NLRC5, PD-L1, and cumulative survival in EC patients . 

Immunofluorescence staining analysis results showed that NLRC5 and PD-L1 are
co-located in the cytoplasm of HEC-1A and Ishikawa cells . There is a negative
expression correlation between NLRC5 and PD-L1 (r = −0.666, P < .001; ). Moreover, western blotting analysis results showed that
NLRC5 plasmid could promote NLRC5 protein expression effectively in HEC-1A cells
.
Overexpression of NLRC5 led to PD-L1 protein being upregulated in HEC-1A and
Ishikawa cells . siRNA-NLRC5 could inhibit NLRC5 protein expression effectively
in HEC-1A cells . Downregulation of NLRC5 inhibited PD-L1 protein expression in
HEC-1A and Ishikawa cells . These results indicated that NLRC5 played a positive
role in PD-L1 expression in EC cells. 

To assess the mutational statuses of the EC patients, WGS was performed for the 3
EC patients and their own peripheral blood, which was set as control group. The
top 10 mutated genes were shown in . However, we did not find
somatic mutations of NLRC5 in the samples. Furthermore, the mutation type was
shown in .
Additionally, using the muTarget database ( http://www.mutarget.com/ ),
we suggested that mutations of ANKRD20A2, C2orf42, ADGRB3, AVPR2, GOLGA6C, and
IPPK may lead to the downregulation of NLRC5 in EC patients . 

EC cases are broadly classified into type I tumors, which express moderate-to-high
levels of estrogen receptor (ER) and account for ∼90% of all EC cases, and type II
tumors, which are estrogen-independent and generally more severe with respect to
their clinicopathological characteristics. Histological classification of EC tumors, however, is subject to relatively
poor reproducibility with overlapping IHC and morphological features among different
EC subtypes. The Proactive Molecular Risk Classifier for EC instead reclassified EC tumors
based on IHC findings into p53 WT, p53 abnormal, polymerase ε-mutated, and
mismatch-repair-deficient, with these subtypes being better able to predict patient
tumor responses to immunotherapeutic treatment. Clinical immunotherapy trials have explored the use of immune checkpoint
inhibitors for the treatment of EC, but most patients were found to exhibit unsatisfactory responses owing to the
incidence of immune escape. PD-L1 is a key immune checkpoint that can mediate immune evasion in a broad
range of cancers. Moreover, PD-L1 can serve as an oncogene to enhance initial tumor
development, growth, and metastatic progression through the activation of intrinsic
signaling pathways within these tumor cells. , The expression of PD-L1 on
the surface of tumor cells enables them to more effectively evade antitumor immune responses, interacting with PD-1 expressed on T cells surfaces to restrict the effector
functions of these cells, thus shielding tumors from immune-mediated rejection. PD-L1 inhibitor treatment has been linked to improved outcomes in many
cancers, including in EC, which indicated that PD-L1 represents a promising target
for immunotherapy in EC. , Overall, PD-L1 expression has been reported in 83% of primary
EC cases and 100% of metastatic EC cases. Here, significantly increased PD-L1 expression was detected in EC patients
when compared to normal endometrial tissues. As a result, PD-L1 overexpression may
contribute to EC formation, invasivity, metastasis, and/or immune evasion. The
specific mechanisms underlying PD-L1-mediated immune evasion, however, remain to be
fully clarified. NLRC5 serves as an MHC class I transactivator that modulates MHC class I-dependent
immunity. Yoshihama et al analyzed 7747 patients with 21 different types of solid
cancers and determined that NLRC4 represents a potential immune evasion-related
target, with the upregulation of this molecule being correlated with augmented MHC
class I expression, CD8 + T cell activation, and improved survival
outcomes in cancer patients. From a mechanistic perspective, somatic mutations, copy number deletion, and
promoter methylation can drive reductions in NLRC5 expression or activity, whereas
NLRC5 recruitment can restore the immunogenicity of tumors and drive enhanced
antitumor immune responses by rescuing MHC class I expression and thereby improving
tumor antigen presentation to CD8 + T cells. , No NLRC5 somatic mutations
were detected among EC samples in the present study, however. An analysis of the
muTarget database suggested that mutations in the ANKRD20A2, C2orf42, ADGRB3, AVPR2,
GOLGA6C, and IPPK genes may contribute to NLRC5 downregulation in patients with
EC. Both clinical HCC samples and HCC cell lines have been found to express NLRC5 at high
levels. In nude mice, NLRC5 knockdown was sufficient to dramatically reduce these
cells’ proliferation, motility, and invasivity, as well as their ability to form
tumors, promoting G0/G1 phase cell cycle arrest. NLRC5 overexpression, in contrast,
was sufficient to enhance HCC cell proliferative, migratory, and invasive activity
through the downstream activation of the Wnt/β-catenin signaling pathway. NLRC5 has recently been demonstrated to enhance the proliferation of HCC
tumor cells via activating the AKT/VEGF-A pathway, whereas NLRC5 knockdown was
linked to decreased in vivo HCC tumor development. Wang et al discovered that overexpression of the NLRC5 gene was connected to
more advanced staging and poorer prognostic outcomes in patients with clear cell
renal cell carcinoma (ccRCC). From a mechanistic perspective, NLRC5 was able to
promote ccRCC cell proliferative, migratory, and invasive activity through
Wnt/β-catenin pathway activation, while silencing NLRC5 led to the suppression of in vivo tumor growth through the inhibition of Wnt/β-catenin signaling. Recent work also suggests NLRC5 to be a miR-4319 target, with NLRC5
overexpression promoting positive miR-4319-related effects on esophageal squamous
cell carcinoma (ESCC) cell growth and cell cycle progression. These data thus further support a role for NLRC5 as a driver of
tumorigenesis, with NLRC5 inhibition thus representing a promising target for the
treatment of HCC, ccRCC, and other cancer types. NLRC5, however, may exhibit
tissue-specific regulatory roles. In a recent study, NLRC5 was found to be
downregulated in EC tissues relative to normal endometrial tissues, promoting AN3CA
cell invasion and migration through PI3K/AKT pathway activation. Even so, the role that NLRC5 plays in the context of EC development and
whether this role is regulated by PD-L1 has yet to be established. Prior studies showed that PD-L1-mediated tumor immune evasion was intimately
implicated in reduced activity of T cells, and PD-L1 inhibitor facilitated depletion
of tumor cells by rescuing CD8 + T cells. , Furthermore, it has
demonstrated that there was a negative correlation between MHC class I molecules and
PD-L1 expression on tumor tissues. Of special note is that a recent study indicated that NLRC5 variants could
interact with PD-L1 variants in colorectal cancer, which provides a novel biological
information to improve colorectal cancer risk management and immunotherapy. These studies provided a potential role of PD-L1 by NLRC5. In our study, we
found NLRC5 and PD-L1 was co-localization in the cytoplasm of HEC-1A and Ishikawa
cells. Furthermore, there is a negative correlation between NLRC5 and PD-L1.
Importantly, further investigations found that NLRC5 could promote the expression of
PD-L1 in EC cells. We suggest that the downregulation of NLRC5 in EC patients owning
to the mutations of ANKRD20A2, C2orf42, ADGRB3, AVPR2, GOLGA6C, and IPPK. Unlike the
NLRC5-mediated immune surveillance in the tumor microenvironment, NLRC5 may
contribute to the cell growth by promoting PD-L1 in the single EC cells. 

Our study presented a novel regulating role in EC cells that NLRC5 promotes PD-L1
expression, suggesting that inhibiting NLRC5 in combination with inhibiting PD-L1
may contribute to the therapy for EC. Nevertheless, the sample size in our study was
relatively small. Furthermore, our study is to focus on the role of NLRC5 in single
EC cells. On account of the critical role of NLRC5 in immune activation, which is
essential for tumor immunotherapy, simply to increase the expression of NLRC5 may
lead to tumorigenesis by promoting cell proliferation, migration, and invasion, and
if simply to reduce the expression of NLRC5 may inhibit NLRC5-mediated cancer immune
surveillance. Further experiments are needed to demonstrate the effect of promoting
or inhibiting NLRC5 expression as adjuvant therapy in the therapy for EC, including
immunotherapy. 

