AIRE is induced in oral squamous cell carcinoma and promotes cancer gene expression.

Autoimmune regulator (AIRE) is a transcriptional regulator that is primarily expressed in medullary epithelial cells, where it induces tissue-specific antigen expression. Under pathological conditions, AIRE expression is induced in epidermal cells and promotes skin tumor development. This study aimed to clarify the role of AIRE in the pathogenesis of oral squamous cell carcinoma (OSCC). AIRE expression was evaluated in six OSCC cell lines and in OSCC tissue specimens. Expression of STAT1, ICAM1, CXCL10, CXCL11, and MMP9 was elevated in 293A cells stably expressing AIRE, and conversely, was decreased in AIRE-knockout HSC3 OSCC cells when compared to the respective controls. Upregulation of STAT1, and ICAM in OSCC cells was confirmed in tissue specimens by immunohistochemistry. We provide evidence that AIRE exerts transcriptional control in cooperation with ETS1. Expression of STAT1, ICAM1, CXCL10, CXCL11, and MMP9 was increased in 293A cells upon Ets1 transfection, and coexpression of AIRE further increased the expression of these proteins. AIRE coprecipitated with ETS1 in a modified immunoprecipitation assay using formaldehyde crosslinking. Chromatin immunoprecipitation and quantitative PCR analysis revealed that promoter fragments of STAT1, ICAM1, CXCL10, and MMP9 were enriched in the AIRE precipitates. These results indicate that AIRE is induced in OSCC and supports cancer-related gene expression in cooperation with ETS1. This is a novel function of AIRE in extrathymic tissues under the pathological condition.

Introduction

The burden of oral cancer has significantly increased in many parts of the world, causing more than 145.000 death in 2012 worldwide [1]. Despite the advances in modern medicine, oral cancer can have devastating effects on critical life functions. About 90 percent of oral cancers are squamous cell carcinomas (OSCCs) that arise from keratinocytes of the oral mucosa.

Cancer development requires the acquisition of several properties, such as unlimited proliferation, vascularization, and invasion [2]. Aberrant growth control in cancer cells is the consequence of accumulated disorders in multiple cell-regulatory systems. Molecular analysis of OSCCs has uncovered genetic and epigenetic alterations in several distinct driver pathways, including mitogenic signaling and cell-cycle regulatory pathways, which lead to continual and unregulated proliferation of cancer cells. In addition, cancer cells recruit surrounding non-transformed cells, such as stromal cells and inflammatory cells, to create a tumor microenvironment that fosters tumor growth and invasion. Under the influence of interaction with the tumor microenvironment, cancer cells express a unique group of proteins that are absent or expressed at very low levels in normal cells. Differentially expressed proteins in OSCC compared to normal keratinocytes include various cytokines, chemokines (for example, CXCLs), extracellular matrix proteins, matrix remodeling enzymes (for example, matrix metalloproteases (MMPs)), cell adhesion molecules, and cytoskeletal proteins [3,4]. Among these factors that characterize cancer cells, we have been interested in keratin 17 (KRT17) because it is consistently and strongly induced in OSCC, and because of its unique functions. Keratins are a family of intermediate filaments that are indispensable for the structural stability of epithelial cells. KRT17 is regarded a “stress-response keratin” that is not expressed or at a very low level in normal squamous epithelium and is induced under pathological conditions, such as wound healing, inflammation, and cancer [5]. Interestingly, KRT17 has been recently reported to interact with AIRE [6]. AIRE was first identified as a causative gene for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome, which gives rise to multiple endocrine disorders, chronic mucocutaneous candidiasis, and various ectodermal defects.

AIRE consists of four domains, including a caspase recruitment domain/homogeneously staining region (CARD/HSR) domain that drives nuclear localization and oligomerization, an Sp100, AIRE, NucP41/75, DEAF1 (SAND) domain, and plant homeodomain 1 (PHD1) and PHD2 domains. The closest homolog of AIRE is Sp100, which shares the CARD, SAND, and PHD domains [7]. AIRE localizes in the nucleus in discrete, punctate structures that resemble promyelocytic leukemia nuclear bodies, which contain Sp100 family members. The nuclear localization and the presence of domains that are shared by transcription factors suggested a role for AIRE in the regulation of gene expression, and indeed, numerous in-vitro studies have demonstrated that AIRE functions as a transcriptional activator [7]. AIRE is expressed in medullary thymic epithelial cells (mTECs), in which it induces ectopic expression of peripheral tissue-specific antigens, thereby facilitating the elimination of self-reactive T cells. AIRE expression has been also reported in extrathymic tissues, including epidermal keratinocytes [8-11]. In the mouse epidermis, Aire is induced by inflammatory or tumorigenic stimuli, and stimulates the transcription of several chemokine genes, such as Cxcl10 and Cxcl11, and a gene encoding matrix-degrading enzyme, Mmp9, in keratinocytes [6]. These results suggest a novel role of AIRE in extrathymic epithelial tissues under pathological conditions; however, the significance of AIRE in other cancers remains to be elucidated.

The above findings inspired us to evaluate whether AIRE participates in the pathogenesis of OSCC. To this end, we first assessed the expression of AIRE in OSCC. Then, we explored the role of AIRE in the regulation of other genes associated with OSCC development.

Materials and methods

Chemically induced mouse model of tongue and esophageal cancer

Ten CD-1 mice were obtained from Sankyo Labo Service Co. (Tokyo, Japan) and were housed in groups of less than five in plastic cages in a temperature-controlled room with 12-h light/12-h dark cycles, and were provided free access to tap water and feed. To establish the cancer model, 4-nitroquinoline-1-oxide (4-NQO, MPN 147–03421, Lot AWQ5167, FUJIFILM Wako Chemical, Miyazaki, Japan) was added to the drinking water at a concentration of 100 μg/ml and administered to 6-week-old mice for 8 weeks. After the administration period, normal drinking water was given. The health of the animals was monitored daily. Behavioral changes were used as criteria to determine a humane endpoint. None of the mice reached the endpoint before the day of sacrifice. Mice were euthanized by cervical dislocation at 30 weeks of age, and tongues and esophagi were harvested. The animal studies were reviewed and approved by the institutional animal care and use committee of Tokyo Medical and Dental University (registration numbers: 0150213A and 0160316A).

Histology and immunohistochemistry

The tissues were fixed in 10% neutral buffered formalin for 24–48 h and embedded in paraffin. Four micrometer-thick tissue sections were cut and stained with hematoxylin and eosin. For immunohistochemistry, the tissue sections were incubated in antigen retrieval buffer (10 mM Tris (pH 9.0), 1 mM EDTA) at 120°C for 15 min. Endogenous peroxidase activity was quenched by 3% H2O2 for 10 min. The sections were incubated with primary antibody diluted 1:500 in TBST (10 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween 20) at 4°C overnight. After washing, the sections were incubated with a secondary antibody at room temperature for 1 h. 3,3′-Diaminobenzidine (DAB) was used for chromogenic detection. Primary antibodies used in this study were anti-AIRE (C-2, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for detection of human AIRE, anti-AIRE (M300, Santa Cruz Biotechnology) for detection of mouse AIRE, anti-GAPDH (D16H11, Cell Signaling Technology, Danvers, MA, USA), anti-STAT1 alpha (EPYR2154, Abcam, Cambridge, UK), anti-STAT1 phospho S727 (EPR3146, Abcam), anti-ICAM1 (EP1442Y, Abcam), anti-ETS1 (D8O8A, Cell Signaling Technology), anti-keratin 14 (LL002, Abcam), anti-Flag M2 (Sigma-Aldrich, St. Louis, MO, USA), and anti-HA (12CA5, Roche Diagnostics, Basel, Switzerland). Secondary antibodies used in this study were Envision FLEX+ (Dako, Glostrup, Denmark), HRP-donkey anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA), and HRP-rabbit anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 594 goat anti-rabbit IgG, and Alexa Fluor 594 goat anti-mouse IgG (Thermo Fisher Scientific).

Cell culture

Ca9-22 (derived from OSCC of the gingiva) and HSC-3 (derived from OSCC of the tongue, referred to as HSC3 hereafter) cell lines were obtained from the RIKEN Bioresource Center (Tsukuba, Japan). HSC5 (derived from SCC of the skin) and Ho-1-N-1 (derived from OSCC of the buccal mucosa, referred to as HO1N1 hereafter) were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). BHY (derived from OSCC of the gingiva) [12] was provided by Dr. Masato Okamoto (Tsurumi University). SAS (derived from OSCC of the tongue) [13] and HSC-4 (derived from OSCC of the tongue, referred to as HSC4 hereafter) [14] were provided by Dr. Masao Saito (Yamanashi University). 293A was purchased from Thermo Fisher Scientific Co. These cells were maintained in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (Sigma-Aldrich) with 10% fetal bovine serum at 37°C in the presence of 5% CO2. Primary human keratinocytes isolated from neonatal foreskins were obtained from Kurabo Co. (Osaka, Japan) and were maintained in HuMedia-KG2 (Kurabo) at 37°C in the presence of 5% CO2.

Plasmid constructs

A pET32a plasmid containing human AIRE cDNA was provided by Dr. Yoshitaka Yamaguchi (Keio University). The open reading frame of AIRE was recovered by PCR using this plasmid as a template and PrimeSTAR GXL DNA Polymerase (Takara, Shiga, Japan). The PCR product was digested with EcoR1 and inserted into pcDNA6-3xFlagN (in-house plasmid made using pcDNA6/V5-HisA (Thermo Fisher Scientific) as a backbone vector), or pAcGFP-C2 (Takara) to create N-terminally 3xFlag-tagged AIRE (Flag-AIRE), and GFP-tagged AIRE (GFP-AIRE), respectively. The plasmid construct targeting exon 2 of human AIRE (AIREKO) was created using pSpCas9(BB)-2A-GFP (a gift from Dr. Feng Zhang, #48138, Addgene, http://n2t.net/addgene:48138; RRID:Addgene_48138) as a backbone vector [15]. The guide sequence was GGAGCGCTATGGCCGGCTGC. pCMV-mFlagEts1 was a gift from Dr. Barbara Graves (# 86099, Addgene; http://n2t.net/addgene:86099; RRID:Addgene_86099) [16]. To remove the Flag tag, a fragment containing the coding region of mouse Ets1 was amplified by PCR, digested with BamH1 and Spe1, and inserted into pcDNA6/V5-HisA.

Generation of AIRE-overexpressing and AIRE-knockout cells

Transfection was carried out using Polyethylenimine Max (Polysciences, Warrington, PA, USA). To establish AIRE-overexpressing clones, 293A cells were transfected with Flag-AIRE and selected on blasticidin (5 μg/ml), and clones were isolated from colonies. To establish knockout clones, HSC3 cells were transfected with AIREKO, GFP-positive cells were sorted using BD FACSAria II (BD Biosciences, San Jose, CA, USA) 72 h after transfection to compensate for the low transfection efficiency of HSC3 cells, and expanded through limiting dilution. Clones harboring a nonsense mutation were determined by PCR-direct sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific).

Immunofluorescence staining of cultured cells

Cells cultured in a chamber slide were fixed with methanol for 10 min. The cells were permeabilized with 0.5% Triton X-100 for 15 min, washed in PBS, incubated with Image-iT FX Signal Enhancer (Thermo Fisher Scientific) at room temperature for 30 min, and then incubated with primary antibodies (1:500 dilution) for 3 h, followed by incubation with secondary antibodies (1:500 dilution) for 1 h. Nuclei were counterstained with propidium iodide or 4′,6-diamidino-2-phenylindole (DAPI).

Proliferation and migration assays

Cell proliferation was assessed by measuring metabolic activity using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Transwell migration assays were conducted using ThinCert cell culture inserts with 8 μm pore diameter (Greiner Bio-One, Wemmel, Belgium) according to the manufacturer’s instructions.

Northern blotting, RT-PCR, real-time RT-PCR, and cDNA microarray analysis

Total RNA was isolated from Ca9-22 and HSC3 cells using NucleoSpin (Macherey-Nagel, Düren, Germany). Northern blot analysis was conducted using DIG Easy Hyb and an RNA probe made with Dig RNA labeling Mixture (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. RNA was reverse-transcribed to cDNA using oligo(dT) primers and M-MuLV reverse transcriptase (Thermo Fisher Scientific). PCRs were run with PrimeSTAR GXL DNA Polymerase (Takara) using the following thermal cycling program: 98°C for 1 min, 30 cycles of 98°C for 10 s, 58°C for 15 s, and 68°C for 20 s, and 68°C for 2 min. GAPDH was used for normalization. Quantitative RT-PCR was conducted using Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific) and a LightCycler Nano system (Roche Life Science) using the following thermal cycling program: 95°C for 10 min, 40 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s. Primer sequences are available in S1 Table. For cDNA expression microarray analysis using SurePrint G3 Human GE 8x60K Ver.2.0 (Agilent Technologies, Santa Clara, CA, USA), total RNA was sent to a commercial service (Hokkaido System Science, Sapporo, Japan).

Western blot analysis

Cells were lysed in RIPA buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS and 0.5% sodium deoxycholate, containing protease inhibitor cocktail cOmplete (Sigma-Aldrich). Protein samples were subjected to 10% SDS-PAGE and transferred to Hybond-ECL (GE Healthcare, Pittsburgh, PA, USA). The blotted membranes were blocked in 2% non-fat milk for 30 min and then incubated with primary antibodies (1:2,000) at 4°C overnight, followed by incubation with secondary antibodies (1:20,000) at room temperature for 1 h, and visualized using ECL Select (GE Healthcare).

OSCC tissue specimens and immunohistochemistry

The study was reviewed and approved by the institutional ethics committee (registration number: D2012-078). As we used archival tissue specimens obtained for pathology diagnosis, the ethics committee approved waiver of the informed consent requirement in accordance with Ethical Guidelines for Clinical Studies by the Ministry of Health, Labor and Welfare of Japan. OSCC tissue specimens were randomly collected from sixty lateral tongue cancer cases from the archives of the Dental Hospital of Tokyo Medical and Dental University. Three tissue microarrays harboring 20 spots of 4 mm in diameter were constructed as previously described [17]. Fifty-one spots were fully evaluable after the loss of spots due to multiple sectioning. Histological evaluation was done as previously described [18]. Cancer-associated inflammation was scored based on the density of inflammatory cell infiltrates around tumor nests at the invasion front. Expression of AIRE, ICAM1, pSTAT1 and STAT1 in cancer was scored based on staining intensity in cancer versus normal epithelium, with “+/–” indicating that expression was undetected or detected but without noticeable upregulation in cancer, “+” indicating slight upregulation in cancer as revealed by a noticeable increase in staining intensity in cancer cells, and “++” indicating distinctive upregulation in cancer. All scorings were performed by two pathologists in a blinded manner, and the judgment was discussed until consent was obtained.

Immunoprecipitation and chromatin immunoprecipitation (ChIP)

Cells were crosslinked with 1% formaldehyde at room temperature for 10 min. The reaction was stopped by incubating the cells with 10% glycine for 15 min. The cells were rinsed twice with PBS and lysed in RIPA buffer. The cell lysate was sonicated for 5 min and centrifuged to pellet the debris. The cleared lysate was divided into two parts, one of which was used for immunoprecipitation and western blot analysis, and the other for immunoprecipitation and DNA quantitative PCR assay. For immunoprecipitation and western blotting, the lysate was incubated with anti-ETS1 antibody (1:100 dilution) at 4°C overnight. Dynabeads Protein G (Thermo Fisher Scientific) were added to the sample, which was incubated for an additional 1 h. The beads were washed with RIPA buffer three times using a magnet stand. Protein was eluted and crosslinks were reversed by heating the beads in SDS gel loading buffer (50 mM Tris-HCl [pH, 6.8], 2% SDS, 10% glycerol, 5% beta-mercaptoethanol, 0.01% bromophenol blue) at 95°C for 10 min. Eluted proteins were subjected to western blot analysis. For ChIP, the lysate was incubated with anti-AIRE antibody at 4°C overnight, then with Dynabeads Protein G for an additional 1 h. The sample was washed first with low-salt wash buffer (20 mM Tris-HCl [pH, 8.0], 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), then with high-salt wash buffer (20 mM Tris-HCl [pH, 8.0], 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), and then with LiCl wash buffer (10 mM Tris-HCl [pH, 8.0], 0.25 M LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate). DNA was eluted in elution buffer (100 mM NaHCO3, 1% SDS). Crosslinks were reversed by incubation at 65°C overnight, in the presence of RNaseA. Protein in the sample was digested with proteinase K at 60°C for 1 h, and DNA was purified by phenol/chloroform extraction. Primers for quantitative PCR analysis are available in S1 Table.

Statistical analysis

Quantitative data are presented as the mean ± standard error of at least three replicated experiments. Means were compared using Student’s t-test or the Mann-Whitney U test. For analysis of relative protein or gene expression data, the ratio t-test was used. P < 0.05 was considered statistically significant.

Results

AIRE is induced in chemically induced cancer of the upper digestive tract in mice

We first examined AIRE expression in the healthy mouse thymus by immunohistochemical staining. AIRE-expressing cells were detected specifically in some, but not all epithelioid cells in the medulla (Fig 1A). AIRE-positive cells were also positive for keratin 14 (KRT14), confirming that they were mTECs. AIRE localized in the nuclei in punctate regions, which were more clearly observed by fluorescence than by chromogenic detection (Fig 1A). This AIRE protein distribution was in agreement with previous reports [19,20], validating the immunohistochemical method for AIRE detection.

Fig 1

AIRE expression in esophageal tumors in mice.

A) AIRE expression in the thymic medullary epithelium. Hematoxylin and eosin staining (left panel), immunohistochemical staining using 3,3'-DAB (middle panel), and immunofluorescence double staining with KRT14 (right panel). Scale bar: 50 μm (left and middle panels), 10 μm (right panel). B) AIRE expression in neoplastic cells of the esophagus. Immunofluorescence staining (middle panel) and immunofluorescence double staining with KRT14 (right panel). PI: propidium iodide. Scale bar: 50 μm (left), 5 μm (middle and right panels). C) Expression of AIRE in neoplastic cells of the esophagus as revealed by immunohistochemical staining using DAB. Scale bar: 50 μm. A representative negative-control image using nonspecific rabbit IgG instead of anti-AIRE antibody is shown in S1 Fig.

We induced tumorigenesis in the upper digestive tract by administering the carcinogen 4-NQO to mice via the drinking water. Multiple tumors developed in the esophagi of all 4-NQO-treated mice (n = 5), whereas no tumor developed in control mice (n = 5). The tumors were histologically confirmed to be SCC or squamous intraepithelial neoplasia. We examined AIRE expression in these esophageal tumors. Although barely detectable in normal epithelium, AIRE expression was distinctively observed in neoplastic cells (Fig 1B and 1C). Nuclear dots as seen in the mTECs were evident in neoplastic cells based on immunofluorescence staining, but they were fewer than in the mTECs (Fig 1A and 1B). In chromogenic detection, cytoplasmic staining was observed in addition to nuclear staining (Fig 1C), the significance of which was uncertain.

AIRE is upregulated in human OSCC tissues and cell lines

We first examined AIRE expression in human SCC using 7 SCC cell lines (6 OSCCs and 1 skin SCC) and cultured primary keratinocytes as a normal reference. AIRE expression was examined by RT-PCR using an intron-spanning primer set targeting AIRE. AIRE mRNA was detected by RT-PCR in all the SCC cell lines examined as well as in normal keratinocytes. The level of expression was quantitated using real-time PCR analysis, which revealed that AIRE was upregulated in the oral cancer cell lines when compared with the normal keratinocytes (Fig 2A). Northern blot analysis failed to detect AIRE in any cells, suggesting a low amount of transcripts.

Fig 2

AIRE expression in OSCC.

A) Real-time RT-PCR analysis of various cancer cell lines. NE: normal epithelium from gingiva. GAPDH was used for normalization. Data are shown on a logarithmic scale as the mean ± SEM of technical triplicates. * P < 0.05, ** P < 0.01 by Student’s t-test. B) Upper panel: Western blot analysis of cancer cell lysates using anti-AIRE antibody. GAPDH was used as a loading control. Lower panel: Densitometric quantification of the western blot data. NE: normal epithelial primary cultured cells derived from neonatal foreskin. C) Nuclear localization of AIRE in cultured cells. Upper left and right panels: Ca9-22 cells were transfected with Flag-AIRE. The cells were fixed and subjected to immunofluorescence staining. Lower left panel: Ca9-22 cells were transfected with GFP-AIRE. The cells were examined under ultraviolet light. Lower right panel: endogenous AIRE in the nucleus of an HSC3 cell as revealed by immunofluorescence staining using laser scanning microscopy. PI: propidium iodide. Scale bar: 10 μm in upper left panel, 5 μm in lower right panel, scale bars were omitted in upper right and lower left panels. D) Representative images of immunohistochemical staining of AIRE in OSCC tissue specimens. Upper left, lower left, lower right panels: chromogenic detection using DAB. A representative negative-control image using nonspecific mouse IgG instead of anti-AIRE antibody is shown in S2 Fig. Upper right panel: fluorescence detection. Scale bar: 10 μm (upper left, upper right), 500 μm (lower left), 50 μm (lower right). E) Summary of immunohistochemical evaluation of AIRE in OSCC tissue specimens. Expression was compared between normal squamous epithelium and cancer in the same specimen and was scored as +/–: no upregulation in cancer; +: upregulation in cancer; ++: strong upregulation in cancer. Mann-Whitney U test. F) Comparison of AIRE expression with clinicopathological parameters (Mann-Whitney U test). “Weak” refers to cases with the score +/–or + in E), “strong” refers to the cases with score ++ in E).

Western blot analysis using an anti-AIRE antibody revealed that all the SCC cell lines examined expressed AIRE at substantially higher levels than did normal keratinocytes (Fig 2B). We further investigated the cellular localization of AIRE. For validation of the antibody in cultured-cell staining, Ca9-22 cells were transfected with Flag-AIRE and then stained using anti-AIRE or anti-FLAG antibody 48 h after transfection. Both antibodies produced identical nuclear-dot staining patterns (Fig 2C). The same nuclear localization pattern was observed in live cells expressing GFP-AIRE, and protein distribution was not altered by fixation. Although endogenous AIRE expression was weaker than exogenous expression and the size and number of nuclear dots were lower than those in transfected cells, nuclear dots in HSC3 cells were positive for AIRE as indicated by laser scanning microscopy (Fig 2C). Next, we examined AIRE expression in 51 OSCC tissue specimens. AIRE was detected by immunohistochemical staining in the nuclei and cytoplasm of cancer cells. Immunofluorescence staining also revealed a nuclear dot pattern of AIRE, with substantially less cytoplasmic signal (Fig 2D). Therefore, we regarded the cytoplasmic staining as nonspecific and scored AIRE expression in OSCC based on the nuclear staining intensity in comparison to normal epithelium. Twenty-seven cases showed a distinctive increase (“++”), 20 cases showed a weak increase (“+”), and four cases showed unremarkable change (“+/–”) in AIRE expression. These results indicated that AIRE expression is significantly increased in OSCC compared to normal epithelium (Fig 2E). Next, we examined whether AIRE expression was associated with clinicopathological parameters, including the differentiation grade, invasion pattern, lymph node metastasis, and level of inflammatory cell infiltration in the cancer stroma. OSCC with strong AIRE expression tended to exhibit severer inflammation (Fig 2F).

AIRE promotes the expression of STAT1, ICAM1, CXCL10, CXCL11, and MMP9

To investigate the effect of AIRE on other genes, we performed cDNA microarray analysis to screen for genes upregulated by AIRE. Genes whose expression was increased by more than 2-fold in AIRE-expressing compared to control 293A cells in at least three microarray spots are listed in Fig 3A. Among them, we focused on STAT1 and ICAM1 because these genes have been detected at considerable levels in our previous cDNA microarray analysis of HSC3 cells [21], and reportedly are overexpressed and play important roles in OSCC [22,23]. Western blot analysis confirmed the upregulation of STAT1 and ICAM1 in AIRE-expressing clones (Fig 3B). Phosphorylated STAT1 increased in proportion to STAT1 expression.

Fig 3

AIRE promotes the expression of ICAM1 and STAT1 in 293A cells.

A) Genes that were upregulated more than 2-fold in in 293/AIRE+ (C-3) when compared to non-transfected 293A cells in at least three spots on the cDNA microarray. B) Left panel: Western blot analysis of ICAM1 and STAT1 in 293/AIRE+ cells. Right panel: Densitometric quantification of the western blot data. ** P < 0.01, by ratio t test.

To further explore the role of AIRE in OSCC, we disrupted AIRE expression in HSC3 cells. First, we tried transient transfection of siRNA, but the knockdown efficiency at the protein level was less than 20%. Therefore, we used the CRISPR-Cas9 system for gene knockout, and we established three independent AIRE-knockout HSC3 cell clones (HSC3/AIRE-C1, C2, and C3, Fig 4A). There was no significant difference in cell proliferation between HSC3/AIRE cells and HSC3 control cells (Fig 4B). A Transwell migration assay revealed that the migration rate was significantly suppressed in HSC3/AIRE-C1 and C2 when compared to control cells (Fig 4C). HSC3/AIRE-C3 exhibited a lower rate of migration, although the difference was not statistically significant. Western blot analysis revealed that ICAM1, STAT1, and phosphorylated STAT1 (pSTAT1) expression were significantly decreased in HSC3/AIRE as compared to control cells (Fig 4A).

Fig 4

STAT1, and ICAM1 are upregulated in OSCC.

A) Left panel: Western blot analysis of ICAM1, pSTAT1, and STAT1 in HSC3/AIRE−clones (C1, C2, C3). GAPDH was used as a loading control. Right panel: Densitometric quantification of the western blot data. * P < 0.05; ** P < 0.01, by ratio t test. B) Cell proliferation of HSC3 and HSC3/AIRE−C1. Data are shown as the mean + SEM of biological triplicates. C2 and C3 also showed similar proliferation rates as HSC3. Student’s t-test. C) Transwell migration assay. Data are shown as the mean + SEM of biological triplicates. *P < 0.05, by Student’s t-test. D) Representative images of immunohistochemical staining. Scale bar: 50 μm. Representative negative-control images are shown in S2E Fig) Schematic summary of immunohistochemical expression or AIRE, ICAM1, and pSTAT1 in 51 cases of OSCC. Horizontal grids represent the cases. Filled squares denote upregulation compared to normal epithelium in the same specimen. Blank squares denote no upregulation, i.e., similar staining intensity as in normal epithelium.

To confirm these observations, we investigated the expression of ICAM1, and pSTAT1 in 51 OSCC tissue specimens by immunohistochemistry. In all cases examined, ICAM1, and pSTAT1 expression was increased in OSCC tissue compared to normal epithelium (Fig 4E). These findings supported the results of cell-culture experiments showing that AIRE promoted STAT1, and ICAM1 expression. AIRE stimulates the expression of the cancer-related proinflammatory genes MMP9, CXCL10, and CXCL11 [6]. In line herewith, we found significantly reduced expression of MMP9, CXCL10, and CXCL11 in HSC3/AIRE−cells (Fig 5A), and significant upregulation of MMP9, CXCL10, and CXCL11 in AIRE-expressing 293A cells (Fig 5B). These results suggested a common mechanism that regulates the expression of these cancer-related genes.

Fig 5

Relative expression of proinflammatory genes.

Gene expression in A) HSC3/AIRE–, and B) 293/AIRE+ in comparison to respective non-transformed cells as measured by real-time RT-PCR. GAPDH was used for normalization. Data are shown as the mean + SEM of technical triplicates and are representative of three independent experiments. *P < 0.05, ** P < 0.01, by Student’s t-test.

AIRE promotes cancer-related gene expression in cooperation with ETS1

Nuclear dots containing AIRE reportedly are different from nuclear dots containing Sp100, the closest homologue of AIRE. However, we found that AIRE and Sp100 colocalized in nuclear dots as revealed by double staining for AIRE and Sp100 in Flag-AIRE-transfected cells (S3 Fig). Sp100 physically interacts with ETS1 and mediates its transcriptional activity [24,25]. ETS1 is a member of the ETS family of transcription factors that physically and functionally interact with numerous molecules to control transcription [26]. Furthermore, various genes that we found to be positively correlated with AIRE in terms of expression (i.e., STAT1, ICAM1, MMP9, CXCL10, and CXCL11) have been previously reported as remarkably upregulated in cDNA microarray analysis of epidermal keratinocytes from Ets1 transgenic mice [27]. Therefore, we hypothesized that AIRE and ETS1 cooperatively contribute to expression regulation of these genes. The SCC lines expressed ETS1, along with STAT1 and ICAM1, at various levels (S4 Fig). We transiently transfected Flag-AIRE or/and Ets1 into 293A cells, which do not express ETS1 at a detectable level as indicated by western blot analysis, and we examined the expression of the aforementioned genes. Western blot analysis revealed increases in STAT1 and ICAM1 expression upon overexpression of AIRE or/and ETS1 (Fig 6A and 6B). Coexpression of AIRE and ETS1 resulted in enhanced expression of STAT1 and ICAM1. Quantitative RT-PCR revealed that CXCL10, CLCL11, and MMP9 expression was upregulated by ETS1 expression, and even more so upon cotransfection with Flag-AIRE (Fig 6C).

Fig 6

Physical and functional interaction of AIRE and ETS1.

A) Expression of STAT1, pSTAT1, and ICAM1 in 293A cells 48 h after transfection with Flag-AIRE or/and ETS1, or mock transfection. The blot shown is representative of three independent experiments. B) Densitometric analysis of data in A). Data are shown as the mean ± SEM of technical triplicates and are representative of three independent experiments. *P < 0.05, **P < 0.01, by ratio t-test. C) Relative gene expression of CXCL10, CXCL11, and MMP9 in comparison to mock transfection as revealed by real-time PCR. Data are shown as the mean ± SEM of triplicate wells and are representative of two independent experiments. * P < 0.05, ** P < 0.01, by ratio t-test. D) Immunoprecipitation and western blot analysis of ETS1 and AIRE. Formaldehyde crosslinking was performed prior to lysis. The lysates were sonicated to shear the DNA. Immunoprecipitation with the anti-ETS1 antibody was performed, and recovered protein was examined by western blot analysis. One tenth of the sample used for immunoprecipitation was loaded as a control. E) ChIP assay of 293A cells transiently transfected with Flag-AIRE. Enrichment of promoter fragments was measured by real-time PCR. Values were standardized to the input, and then to mock transfection. Relative values higher than 100 are indicated as 100< and a scale break was used in the Y-axis to allow intuitive interpretation of the relative expression. GAPDH was used as a reference. Data are representative of three independent experiments.

To examine the physical interaction between AIRE and ETS1, we cotransfected Flag-AIRE or/and Ets1 into 293A cells and used these cells for immunoprecipitation. Conventional immunoprecipitation using anti-ETS1 antibody did not show coprecipitation of AIRE with ETS1, which indicates that their interaction is weak or their functional cooperation is not mediated through direct physical interaction. ETS1 and AIRE may be recruited to nearby chromatin regions. To check this possibility, we performed a modified immunoprecipitation assay. Formaldehyde crosslinking was performed before cell lysis, and the DNA was sheared by sonication. Immunoprecipitation was conducted using the anti-ETS1 antibody, and recovered protein was examined by western blot analysis. AIRE coprecipitated with ETS1 (Fig 6D). We next performed conventional ChIP assays to assess whether AIRE was recruited to the promoters of the aforementioned genes. ChIP-quantitative PCR revealed that promoter fragments of STAT1, ICAM1, CXCL10, and MMP9 were enriched in the AIRE precipitate (Fig 6E). These results implicate that AIRE promotes cancer-related gene expression in cooperation with ETS1.

Discussion

Although accumulating evidence suggests that AIRE is expressed in extrathymic tissues, there are remarkable discrepancies across studies, depending on the techniques used. AIRE mRNA has been detected by RT-PCR in lymph nodes, tonsils, gut-associated lymphoid tissues, spleen, liver, testes, and ovaries [28]. Thymic AIRE mRNA expression is the highest and could also be detected by northern blotting [29,30]. As studies have concentrated mainly on lymphoid tissues, the skin and upper digestive tract mucosa have been left unexplored. Only a few studies have reported AIRE expression in epithelial tissues. In-situ hybridization and immunohistochemistry failed to detect AIRE expression in human skin [8], whereas AIRE expression has been detected by RT-PCR in HaCaT keratinocytes [11], and in A431 cells treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) [6]. TPA is an activator of the protein kinase C pathway, which induces inflammation, and acts as a strong carcinogen when topically applied to skin. Aire mRNA expression was barely detectable in normal mouse skin by in-situ hybridization, but was induced in the epidermis of TPA-treated mouse ear and genetically induced mouse skin tumor. Still, induced epidermal Aire expression was substantially lower than that in the thymus [6]. Our results showing that AIRE is expressed in OSCC are consistent with these previous observations, and suggest that AIRE is induced in the epithelium under pathological conditions.

We examined the expression of several tissue-specific antigens, including PTH, INS, GH1, which are induced in mTECs in an AIRE-dependent manner [7], in OSCC cell lines. None of these were detected in the OSCC cell lines examined or induced in AIRE-overexpressing cells. This is plausible considering the presumptive function of AIRE in reinstigating suspended transcription. Instead, we found evidence of upregulation of STAT1, and ICAM1 by AIRE. The coordinated overexpression of STAT1, and ICAM1 in OSCC is consistent with findings in previous studies [23,28,31-33]. Close correlations between STAT1, and ICAM1 expression in epithelial cells have been demonstrated in previous studies. Interferon gamma induces ICAM1 expression through activation of STAT1 [34-36]. Although the regulatory mechanism of STAT1 transcription is unclear, our finding that AIRE induces STAT1 expression suggests that AIRE may increase the expression of ICAM1 through STAT1 upregulation.

We found that AIRE stimulates MMP9, CXCL10, and CXCL11, which is consistent with findings by Hobbs and colleagues [6]. These authors demonstrated that AIRE and KRT17 are recruited to a specific region in the promoters of these proinflammatory genes carrying an NF-κB consensus motif [6]. Interestingly, the same spectrum of genes whose expression was dependent on AIRE in keratinocytes under stress in our study, including Stat1, Icam1, Mmp9, Cxcl10, and Cxcl11, was reportedly induced in keratinocytes of Ets1 transgenic mice [27]. Microarray analysis of Ets1 transgenic in comparison with wild-type mouse epidermis revealed the upregulation of Stat1 (2.4-fold), Icam1 (2.8-fold), Mmp9 (8.9-fold), Cxcl10 (8.2-fold), and Cxcl11 (3.3-fold) in the former [27]. Unfortunately, data on Aire expression were lacking in this previous microarray study, but the results in this and previous studies suggest that AIRE belongs to this group of genes that are induced in epithelial cells under stress condition.

AIRE regulates the transcription of numerous divergent genes whose expression is driven by various promoters and transcription factors. This suggests that AIRE is a broad transcriptional activator, rather than a specific DNA-binding transcription factor. The mechanism underlying this promiscuous transcriptional activation is not fully understood, but it has been proposed that AIRE is recruited to RNA polymerase II-rich regions where transcription of AIRE-responsive genes is initiated but interrupted, and AIRE releases the stalled RNA polymerase II complex to resume transcription [37-39]. Further, AIRE has been suggested to be preferentially recruited to so-called super-enhancers; genomic regions rich in enhancers and transcriptional regulators [40]. Through its global transcriptional activation, AIRE might assist cancer-associated gene expression and thus promote the cancer phenotype.

The regulatory mechanism of AIRE expression has not been clearly understood. The AIRE minimal promoter region contains binding domains for Sp1, AP-1, NF-Y [41], and Ets transcription factors [42]. Indeed, ETS1 and ETS2 enhanced TPA-induced AIRE promoter activity in HeLa cells [42]. Furthermore, AIRE expression is regulated by KRT17 in cooperation with the ribonucleoprotein hnRNPK [6]. As KRT17 was robustly induced in all the oral cancer tissues examined [31], the induction of AIRE in OSCC may be attributed to KRT17-mediated regulation. Further research is needed to elucidate the details of the mechanism of AIRE induction in cancer cells.

In conclusion, we demonstrated a novel role of AIRE in extra-thymic tissue under pathologic condition. AIRE is upregulated in OSCC and promotes the expression of cancer-related genes, at least in part by functional interaction with ETS1.

Sequences of the PCR primers used in this study.

(PDF)

Click here for additional data file.

Representative negative control image of immunohistochemical staining of mouse tissue.

Scale bar: 100 μm.

(PDF)

Click here for additional data file.

Representative negative control image of immunohistochemical staining of human OSCC.

Scale bar: 100 μm.

(PDF)

Click here for additional data file.

Sp100 and transfected AIRE in the nuclei of Ca9-22 cells as revealed by immunofluorescence staining and laser scanning microscopy.

There is a transfected cell in the center, in which AIRE colocalizes with endogenous Sp100. Note the Sp100 expression in the surrounding non-transfected cells. Scale bar: 5 μm.

(PDF)

Click here for additional data file.

Expression of STAT1, pSTAT1, ICAM1, and ETS1 in various OSCC cell lines.

Western blot analysis. GAPDH was used as a loading control.

(PDF)

Click here for additional data file.

Raw Images.

(PDF)

Click here for additional data file.

6 Nov 2019

PONE-D-19-24858

AIRE is induced in oral squamous cell carcinoma and promotes cancer gene expression.

PLOS ONE

Dear Dr Sakamoto,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Dec 21 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Roberto Mantovani

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

1. At this time, we request that you  please report additional details in your Methods section regarding animal care, as per our editorial guidelines.

(1) Please state the number of mice used in the study.

(2) Please state the source, product number and lot number of the 4-nitroquinoline-1-oxide (4-NQO) used in the study

Thank you for your attention to these requests.

2. Please provide additional information about each of the cell lines used in this work, including culture conditions (incubation temperature, CO2)  and any quality control testing procedures (authentication, characterisation, and mycoplasma testing). For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines.

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Experiments and in particular PCR and WB should be quantitative and statically significant. Statistical evaluation of the data should be provided. As detailed in the attached comments controls for immustaining should be provided.

In general Figures are of low quality and do not meet the scientific criteria to be accepted for publication in PlosONE.

Reviewer #2: In the manuscript “AIRE is induced in oral squamous cell carcinoma and promotes cancer gene

expression” the authors performed several experiments to demonstrated that AIRE, a thymic transcription factor, is expressed also in extra-thymic tissue (skin) and is overexpressed in OSCC. Their results indicate that AIRE induces in these tumors the expression of cancer-related genes and suggest it works through a functional interaction with ETS1.

Although the issue is interest and relevant, some results are very preliminary.

Comments

-In fluorescence AIRE is very poorly expressed in the nucleus in comparison with normal cells but in IHC the nuclei seem very positive. How do you explain it?

-The authors state that in most cases, KRT17, ICAM1, and pSTAT1 expression was increased in OSCC tissue compared to normal epithelium. Are these the same cases in which AIRE is overexpressed? If not how can you reconcile the results?

-AIRE induced expression of KRT17. Why the ko of KRT17 leads to downregulation of AIRE? Which is the molecular mechanism trough which KRT17 regulates AIRE? Is KRT17 recruited to the AIRE promoter? If this result is shown in the manuscript more experiments addressing the mechanism should be performed.

-Which is the molecular mechanism leading to the fact that ETS1 was induced in stable 293/AIRE+ clones and decreased in HSC3/AIRE– cells? Is AIRE recruited to the ETS1 promoter, too? Is AIRE regulating ETS1 expression, too? Again as for KRT17 if this result is shown in the manuscript more experiments addressing the mechanism should be performed.

-The results of figure 8 are only observational and do not implicate at all that AIRE is recruited to the chromosomal regions where ETS1 initiated transcription. re-ChIP experiments with or without the two proteins should be performed to demonstrate this.

-The genes are written sometimes in italicus sometimes no in a casual manner (ie: not according with gene or protein).

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Giulia Piaggio

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Sakamoto revision.docx

Click here for additional data file.

19 Dec 2019

Responses to the Reviewers’ comments

We thank the reviewers for the helpful suggestions. Changes made in the manuscript in response to the comments are highlighted in yellow.

Reviewer #1:

Q1: Experiments and in particular PCR and WB should be quantitative and statically significant. Statistical evaluation of the data should be provided. As detailed in the attached comments controls for immustaining should be provided.

A1: We thank the reviewer for this valuable comment. Following the reviewer's instructions, relevant figures were substantially revised. PCR and western blot results were quantitated, and statistical analysis, when relevant, was performed. Control images for immunostaining were provided in S1 Fig and S2 Fig.

Q2: Data from Hobbs RP et al. Nat. Genetics 2015 doi: 10.1038/ng.3355 demonstrated that AIRE induction and nuclear localization strongly depends upon functional interaction between Krt17 and hnRNPK. In this work, instead, the authors describe induction of Krt17 by AIRE thus suggesting a direct role of AIRE transcriptional factor in Krt17 induction. Thus, a critical point should be to understand the relevance of Krt17 in AIRE-induced transcriptional pathway or the potential existence of a regulatory feedback loop between them, a relevant point that has not been addressed by the authors.

The work is potentially interesting however I have several concerns especially about the way the data have been done and presented.

A2: We appreciate the reviewer’s constructive suggestion. We started this study inspired by the close relationship between KRT17 and AIRE. Several data using cultured cells indicated that a change in KRT17 expression was associated with a change in AIRE expression, although the mechanism of KRT17 upregulation in the AIRE-overexpressing cells was yet to be elucidated. On the other hand, Hobbs et al. have already shown that AIRE expression is induced by the cooperative action of KRT17 and hnRNPK. Thus, the induction of AIRE in oral cancer tissue would be explained by KRT17-mediated regulation, because KRT17 was robustly induced in all the oral cancer tissue examined. This possibility was added to the Discussion.

Our cell culture data implied at least four types of gene induction relationships, as follows: AIRE >> ETS1, ETS1 >> KRT17, KRT17 >> AIRE, AIRE >> KRT17.

As the reviewer pointed out, additional experiments are needed to clarify these relationships in terms of the underlying molecular mechanisms. Following the reviewers’ suggestions, we performed additional experiments, where we transiently transfected AIRE and ETS1 into 293A cells and confirmed that KRT17 mRNA expression was increased by both. Likely, there is a complex correlation between these genes, with bidirectional feedback and links to other signaling cascades. Each of the above questions is a big subject, and as the reviewer pointed out, the impact of KRT17 on AIRE function is also an important issue. That said, it would be too complicated to address all these questions in this paper, and this would obscure the main topic of the article. The main topic is that AIRE is overexpressed in oral squamous cell carcinoma and increases the expression of genes related to cancer behavior. Although the induction of AIRE in oral cancer cells may be the consequence of KRT17 upregulation, the function of KRT17 is not explicitly relevant to this story. Therefore, we decided to delete the KRT17 data from the paper. Data on ETS1 expression in AIRE-overexpressing clones were also deleted. We believe that the revised manuscript is more concise and readable. The biological complexity underlying the phenomena might be underrepresented, but these issues should be examined in detail in future studies.

Q3: The author should test if Krt17 knock-out or depletion plays any effects on AIRE-dependent gene regulation. This should easily clarify the role of Krt17 in this pathway.

A3: We thank the reviewer for this valuable suggestion. As mentioned above, KRT17 results were deleted from the present paper for it to be more concise. We agree that the role of Krt17 in AIRE-dependent gene regulation is an important topic. As the cells available in our repository either express both KRT17 and AIRE, or lack both, co-transfection of KRT17 and AIRE into 293A cells is a versatile experimental model to examine this topic. There seems to be a complex bidirectional regulatory feedback between the cancer genes, which may make it difficult to determine whether the effect is due to direct functional interaction or a secondary one via other signaling cascades. We would like to examine these questions in a future study.

Q4: Experiments in Fig. 2A and B should be quantitated and statically evaluation of the data should be provided.

A4: We performed real-time PCR in triplicate, and quantitative data were added in the revised manuscript.

Q5: In Fig. 3A, a panel showing mock transfected cells is missing. The experiment in Fig. 3C is not quantitative and should be repeated by quantitative PCR with appropriate internal controls.

A5: Real-time PCR was performed to evaluate KRT17 expression in AIRE-transfected 293A cells, which confirmed the upregulation. However, we chose not to include data related to the interaction between AIRE and KRT17 in this manuscript, and thus, Figure 3 was deleted.

Q6: In Fig. 3D the authors should explain why expression of Krt17 in clone C1 is so low given that AIRE is abundantly expressed compared to the other clones. Again, this experiment is not quantitative and statistics is not present.

A6: We thank the reviewer for this constructive comment. The level of expression of KRT17 and other genes did not seem to consistently parallel that of AIRE in the stable clones. We have frequently experienced such clone variation, which may be due to differences in integration sites.

Q7: In Fig. 4B, STAT1 levels appear to be comparable despite the different level of AIRE in C1, C2 and C3 clones.

A7: Such clone variation may be due to differences in integration sites. We assume that, as we did not identify the integration site or the epigenetic states of each clone, comparison between clones is not very informative. Therefore, we made comparisons between the non-transformed parent cells and the group of clones.

Q8: The WB presented in Fig. 4B is not quantitated.

A8: Densitometry results were added (Fig. 3A).

Q9: The WB in Fig. 7 should be quantitated, the AIRE panel in Fig. 7B

A9: This is overexpressed AIRE, and this is not an experiment to check the dose-dependency. Densitometry of AIRE and ETS1 is possible, but seems irrelevant in the context. The expression of other genes was quantitated and statistical analysis was performed (Fig. 6B, C).

Reviewer #2:

Q10: In fluorescence AIRE is very poorly expressed in the nucleus in comparison with normal cells but in IHC the nuclei seem very positive. How do you explain it?

A10: Immunohistochemical staining employs a signal enhancing technique, which can dramatically increase the staining intensity. However, intracellular localization tends to become blurred. As seen in Fig 1A, the dot pattern in TEC cells was clearly visible by immunofluorescence, whereas the nuclei were broadly stained by chromogenic substrate. In cancer cells, AIRE expression was weaker than the physiological expression in normal TEC cells. To obtain sufficient signal, we used a color development time of 5–10 min, while monitoring the color intensity. Longer incubation can increase non-specific staining, but no significant staining was observed in the control specimen in which the primary antibody was replaced with non-specific IgG.

Q11: The authors state that in most cases, KRT17, ICAM1, and pSTAT1 expression was increased in OSCC tissue compared to normal epithelium. Are these the same cases in which AIRE is overexpressed? If not how can you reconcile the results?

A11: We thank the reviewer for this constructive comment. KRT17, ICAM1, and pSTAT1 were increased in all the OSCC cases examined. As STAT1 staining was much weaker than that of pSTAT1, apparently because the STAT1 antibody is less optimal for immunohistochemical staining on formalin-fixed paraffin embedded tissue specimens, upregulation of STAT1 was not confirmed in all the cases. To avoid confusion, we decided to delete immunohistochemical results for STAT1. Overall, AIRE upregulation in OSCC was detected in 47/51 cases, and KRT17, ICAM1, pSTAT1 were upregulated in 51/51 cases. Thus, their co-upregulation is a common feature in OSCC.

Q12: AIRE induced expression of KRT17. Why the ko of KRT17 leads to downregulation of AIRE? Which is the molecular mechanism trough which KRT17 regulates AIRE? Is KRT17 recruited to the AIRE promoter? If this result is shown in the manuscript more experiments addressing the mechanism should be performed.

A12: We thank the reviewer for this constructive comment. AIRE downregulation in KRT17-KO cells is consistent with the data from Hobbs et al. that clearly demonstrated that AIRE is induced by Krt17 in association with hnRNPK. As mentioned above in response to a comment by reviewer #1, we considered that data related to KRT17 are better to be further analyzed in a future study. Therefore, data on KRT17 were removed from the manuscript.

Q13: Which is the molecular mechanism leading to the fact that ETS1 was induced in stable 293/AIRE+ clones and decreased in HSC3/AIRE– cells? Is AIRE recruited to the ETS1 promoter, too? Is AIRE regulating ETS1 expression, too? Again as for KRT17 if this result is shown in the manuscript more experiments addressing the mechanism should be performed.

A13: We thought that ETS1 upregulation in the AIRE-overexpressing clones is an interesting phenomenon that should be shown as additional information. Maybe there is a complex multidirectional feedback mechanism that allows the cells to adapt to a new state of balanced gene expression. However, this information might confuse the readers, and more experiments are definitely needed to understand the mechanisms underlying this finding. Therefore, we decided not to present this result in the current paper. The possibility that AIRE promotes ETS1 expression is to be examined in a future study.

Q14: The results of figure 8 are only observational and do not implicate at all that AIRE is recruited to the chromosomal regions where ETS1 initiated transcription. re-ChIP experiments with or without the two proteins should be performed to demonstrate this.

A14: Although the IP result suggested that ETS1 and AIRE exist as a complex, we agree that it is an overstatement that AIRE is recruited to the chromosomal regions where ETS1 initiates transcription. We attempted ChIP-re-ChIP experiments; however, unfortunately, they failed to provide direct evidence supporting the above hypothesis so far. We re-organized the current manuscript and decided to study the detailed mechanism in a future study. Accordingly, we deleted the data in the original Fig 8, and revised the manuscript to avoid any overstatements.

Q15: The genes are written sometimes in italicus sometimes no in a casual manner (ie: not according with gene or protein).

A15: We apologize for this mistake. The notation of genes and proteins has been carefully revised.

3 Jan 2020

AIRE is induced in oral squamous cell carcinoma and promotes cancer gene expression.

PONE-D-19-24858R1

Dear Dr. Sakamoto,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Roberto Mantovani

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Regrettably, the authors did not add all the informations requested by this reviewer. However, they decided to remove data from the previous version of the manuscript to make it more focused. Moreover, they add all the quantifications data requested. Therefore I think that the manuscript as it is in the revised version is acceptable for publication.

Reviewer #2: In the first version of the manuscript there were several preliminary data that would need further experiments. In this revised version, the authors deleted those preliminary data. The paper is now more focused, although simplified. In line with the policy of PONE, it can be accepted for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Viola Calabrò

Reviewer #2: Yes: Giulia Piaggio

9 Jan 2020

PONE-D-19-24858R1

AIRE is induced in oral squamous cell carcinoma and promotes cancer gene expression.

Dear Dr. Sakamoto:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Roberto Mantovani

Academic Editor

PLOS ONE