PD-L1, an Important Immune Checkpoint Regulator, Is Suppressed by miR-34a in Head and Neck Squamous Cell Carcinoma

Background: Key molecules regulating the immune checkpoint have shed light on the efforts to control several cancers. Recently, immune checkpoint inhibitors for cancer therapy such as antibodies against programmed cell death 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), and cytotoxic T-lymphocyte associated protein 4 (CTLA4) have been developed. In head and neck squamous cell carcinomas (HNSCCs), such immune checkpoint inhibitors have come into clinical use and are expected to improve patients’ prognoses. Recently, miR-34a has been shown to be a downstream micro RNA of TP53 that regulates PD-L1 in several types of cancer. To reveal the correlations between miR-34a and PD-L1 in HNSCCs in terms of clinical signicance, we analyzed 19 HNSCC cell lines. Methods: We measured the expression levels of miR-34a and PD-L1 in 10 HNSCC cell lines as well as in 9 of their derived acquired cisplatin (CDDP) resistant cell lines by qRT-PCR and Western blotting. Results were further analyzed by their TP53 statuses. We also investigated the changes in PD-L1 expression levels after miR-34a precursor- or inhibitor-mediated forced expression or suppression in HNSCC cell lines. Results: We observed inverse correlations between both mRNA and protein expression levels of miR-34a and PD-L1. No signicant differences in miR-34a levels were observed with regard to TP53 status. Forced expression of miR-34a decreased PD-L1 expression, and suppression of miR-34a increased PD-L1 expression. Conclusion: Our present results suggest that miR-34a negatively regulates PD-L1 expression, possibly in a TP53 independent manner in HNSCC.

PD-L1, an Important Immune Checkpoint Regulator, Is Suppressed by miR-34a in Head

Background
Head and neck squamous cell carcinoma (HNSCC) is the sixth most commonly diagnosed cancer and the eighth most common cause of cancer death worldwide [1]. Although molecular target therapy has been utilized lately, surgical resection and/or a combination of cisplatin-based chemo-radiotherapy or sole radiotherapy are the major therapeutic strategies for patients with HNSCC. However, poor survival rates are still reported in advanced stage patients. Recently, functions of immune checkpoint proteins such as programmed cell death 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1) and cytotoxic Tlymphocyte associated protein 4 (CTLA-4) in the cancer cells have been elucidated, and inhibitory antibodies against those immune checkpoint molecules have been developed [2,3]. In HNSCC, anti-PD-1 antibody nivolumab and pembrolizumab have been shown to be clinically effective [4,5].
Micro RNAs (miRNAs) are a class of small noncoding RNA molecules, typically 18-25 nucleotides long, known to be among the factors regulating gene functions. miRNAs are found to play important roles in proliferation, differentiation, and migration [6,7], and some of them work critically for suppression of carcinogenesis. miR-34a is known to be one of the downstream molecules of TP53 and functions as a tumor suppressor [8][9][10][11]. It could be a prognostic marker in sinonasal squamous cell carcinoma treated with cisplatin (CDDP)-based chemoradiotherapy [12]. A number of genes targeted by miR-34a have been identi ed [13][14][15], and PD-L1 was included in one such target gene in non-small cell lung cancer and acute myeloid leukemia [16,17]. In this study, we investigated the relationship between miR-34a and PD-L1 in HNSCC.
Culture conditions of these 19 cell lines were described previously [18,19].

Rna Preparation
Preparation of miRNAs and total RNAs were done using mirVana miRNA Isolation kit (Applied Biosystems, Foster City, CA) and the RNeasy Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturers' instructions, and the quality of the extracted RNAs was monitored by Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Quantitative Reverse Transcription-polymerase Chain Reaction (qrt-pcr) qRT-PCR experiments were performed according to methods described previously [12]. In brief, quanti cation of miR-34a was assessed by TaqMan microRNA assay kit and an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Results were normalized by ∆Ct methods, and the RNU44 level was monitored as the internal control. Using ROX Mix (Thermo Fisher Scienti c, Tokyo, Japan), 2 µg of total RNA from each sample was used for cDNA synthesis and qRT-PCR analyses for detection of PD-L1 expression levels. Signals were detected by an ABI PRISM 7000 Sequence Detection System, and expression levels were normalized by ∆∆Ct methods using beta-2-microglobulin (B2M) as the internal control [20]. The PD-L1 primer pairs and the probe used for qRT-PCR are as follows: forward primer, 5'-CCC AGT TCT GCG CAG CTT C-3'; reverse primer, 5'-AGC AAA TAT CCT CAT CTT TCT G-3'; and probe, 5'-CGC GCT TCT GTC CGC CTG CAG-3'. All of these experiments were performed in triplicate.

Western Blotting
Western blotting analyses were performed as described previously [21]. In brief, total proteins from the collected cell were extracted, both before treatment and 16 hours after treatment, with 1 µg/ml of doxorubicin, then electrophoresed on a SuperSep Ace 5-20% polyacrylamide gradient gel (Wako, Osaka, Japan), transferred to a PVDF membrane (ATTO, Tokyo, Japan), and blocked by skim milk. Then the membrane was incubated overnight at 4 o C with one of the following primary antibodies: rabbit antihuman PD-L1 monoclonal antibody [28 − 8] (Abcam, Tokyo, Japan), mouse anti-human p53 monoclonal antibody DO-7 (DAKO, Glostrup, Denmark), or mouse anti-ActB monoclonal antibody (Sigma, St. Louis, MO). Horseradish peroxidase (HRP) conjugated secondary antibodies (Amersham Biosciences, Little Chalfont, UK) were used against corresponding primary antibodies (rabbit or mouse). Signals were visualized using Enhanced ChemiLuminescence (ECL) Detection Reagent (Amersham) and digitally processed using ImageQuant LAS 4000 (GE Healthcare, Tokyo, Japan). The band intensities were digitalized by the ImageQuant software (GE Healthcare), and relative intensities were calculated by normalization using β-actin (ACTB) as the internal control.
Transfection of miR-34a precursor or inhibitor to the cell lines Cell lines were seeded into six-well plates at a density of 2 × 10 5 cells per well and grown overnight. Then either the miR-34a precursor (Ambion Pre-miR miRNA Precursor) or the miR-34a inhibitor (Ambion Anti-miR miRNA Inhibitor) was transfected at the nal concentration of 12.5 nM using 7.5 µL RNAiMAX transfection reagent. After 72 hours, cell pellets were collected for further investigation.

Statistical analysis
Statistical analyses were done using Microsoft excel for Mac (ver. 16.32 (19120802)) and R for Mac (R 3.5.2 GUI 1.70 El Capitan build (7612)). The Spearman's rank correlation, Kruskal-Wallis test and Welch's t test analyses were carried out. Signi cant difference was considered as P < 0.05.

Expression Analyses Of Pd-l1 Mrna And Protein
We Analyses of the relationships between miR-34a and PD-L1 expressions The relationships between miR-34a and PD-L1 mRNA and protein were analyzed (Figs. 2c and 2d). No discrepancies between mRNA and protein expression levels were evident in PD-L1. HSC2, HSC3, HSC3CR, SAS, and SASCR showed high PD-L1 mRNA expression relative to miR-34a. In contrast, RPMI2650, HSQ89, IMC4 and IMC4CR showed higher expressions of miR-34a relative to PD-L1. It is notable that (1) we observed signi cant inverse associations between expression levels of miR-34a and PD-L1 (mRNA: r = -0.4684 P = 0.04476, protein: r = -0.7439 P = 0.0003931), and that (2) we did not observe any coherence of miR-34a expression levels between parental and resistant cells.
Forced expression of miR-34a Next, we induced forced expression using miR-34a precursor into the cell lines with lower expressions of miR-34a. We selected three cell lines, HSC2, HSC3 and HSC3CR, and analyzed their expressional changes of PD-L1 after introduction of miR-34a precursor. We observed signi cantly decreased PD-L1 mRNA expression levels in response to the increase of miR-34a in HSC3 and HSC3CR (Figs. 3a and 3b). These results are in good accordance with those in Fig. 2.

Inhibition of miR-34a
In an opposite manner, we also performed suppression experiments using miR-34a inhibitor. We selected four cell lines with the higher expression of miR-34a, RPMI2650, HSQ89, IMC4 and IMC4CR; results are shown in Figs. 3c and 3d. We observed signi cantly increased PD-L1 mRNA expression levels in response to the decrease in miR-34a levels in RPMI2650 and IMC4CR. Again, these results are in good accordance with those in Fig. 2.

Elucidation of miR-34a in association with TP53
Because the regulation of miR-34a in association with TP53 has been reported [22], the statuses of TP53 in 10 HNSCC cell lines and a representative CDDP resistant RPMI2650CR were sequenced for exons 5-8 to detect mutations. Results are shown in Fig. 4a. Then we analyzed protein expression levels of TP53 by Western blotting with and without doxorubicin treatments; results are shown in Fig. 4a. Three of ve cell lines without TP53 mutation (RPMI2650, IMC4, and IMC3) increased their protein expression levels after doxorubicin treatment, but no signi cant differences of miR-34a levels were detected irrespective of TP53 status (Fig. 4b).

Discussion
In this study, we demonstrated an inverse correlation between expression levels of miR-34a and PD-L1 in HNSCC cells. Forced expression of miR-34a decreased PD-L1 expression, and introduction of an inhibitor of miR-34a increased PD-L1. These results suggest that miR-34a is one of the important molecules in regulation of PD-L1 in HNSCCs, possibly in an TP53 independent manner.
PD-L1 is reported as a ligand that speci cally binds to PD-1 [23]. Its important role is to suppress the immune system by binding to PD-1. A number of cancer cells express PD-L1, which results in escape from immune cells, and such cancer cells can survive. Some reports have demonstrated a signi cant association between PD-L1 expression and poor prognosis in various malignant tumors [24][25][26][27][28]. Chen et al. [29] showed that PD-L1 expression was regulated by oncogenic pro-survival signaling pathways, such as MAPK or PI3K/Akt pathways. Several investigators have also reported correlations between miR-34a and PD-L1 expressions. Wang Xi et al. [16] suggested that miR-34a can regulate PD-L1 expression by targeting PD-L1 mRNA in acute myeloid leukemia, and Cortez et al. [17] found that TP53 regulates PD-L1 via miR-34a, which directly binds to the 3' untranslated region of PD-L1 in non-small cell lung cancer.
miR-34a is known as a tumor suppressing miRNA and its role as the tumor suppressor in HNSCC has been reported [30,31]. In sinonasal squamous cell carcinomas, miR-34a expression can be an independent prognostic biomarker in patients undergoing cis-diamminedichloroplatinum treatment [12].
One of the key ndings in the present study is that the expression levels of PD-L1 and miR-34a inversely associated in HNSCC; this result suggests that the PD-L1 opposes miR-34a. This is in good agreement with previous reports that miR-34a may downregulate PD-L1 expression in squamous cell carcinoma [16,17]; this should lead to a better prognosis.
Various clinical trials of the immune checkpoint inhibitors have been performed recently, and the expression levels of the immune checkpoint proteins such as PD-L1 or tumor mutation burden are sometimes discussed as indications of such immune checkpoint inhibitors in HNSCCs [32]. We analyzed miR-34a, which has been reported as one of the downstream genes of TP53 [22], but the expression levels of miR-34a were found to be independent of TP53 statuses in the present study; expression levels of miR-34a varied among cells with wild-type TP53, and no signi cant associations were observed. It is reported that methylation-induced silencing of miR-34a is associated with chemoresistance in prostate cancer [33] and that miR-34a promoter methylation and TP53 polymorphisms may be associated with colorectal cancer pathogenesis [34]. However, the numbers of available cells were limited and further investigations are needed.
In this study, we demonstrated that miR-34a is one of the important molecules in regulation of immune checkpoint proteins, but further genetic and epigenetic studies, including upstream and downstream molecules of the miR-34a family, will contribute to better clinical management of patients with HNSCC.

Conclusion
In HNSCC cells, miR-34a negatively controls the PD-L1 function, possibly independently from TP53 status. These results may contribute to open a pathway for novel and e cient ways to treat patients with HNSCC.

Consent for publication
Not applicable.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors have no competing interests to declare.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupplementaryFig4a.tif SupplementaryFig2b.tif