Identification of CD24 as a potential diagnostic and therapeutic target for malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy of the pleura that is currently incurable due to the lack of an effective early diagnostic method and specific medication. The CDKN2A (p16) and NF2 genes are both frequently mutated in MPM. To understand how these mutations contribute to MPM tumor growth, we generated NF2/p16 double-knockout (DKO) cell clones using human MeT-5A and HOMC-B1 mesothelial cell lines. Cell growth and migration activities were significantly increased in DKO compared with parental cells. cDNA microarray analysis revealed differences in global gene expression profiles between DKO and parental cells. Quantitative PCR and western blot analyses showed upregulation of CD24 concomitant with increased phosphorylation of AKT, p70S6K, and c-Jun in DKO clones. This upregulation was abrogated by exogenous expression of NF2 and p16. CD24 knockdown in DKO cells significantly decreased TGF-β1 expression and increased expression of E-cadherin, an epithelial–mesenchymal transition marker. CD24 was highly expressed in human mesothelioma tissues (28/45 cases, 62%) and associated with the loss of NF2 and p16. Public data analysis revealed a significantly shorter survival time in MPM patients with high CD24 gene expression levels. These results strongly indicate the potential use of CD24 as a prognostic marker as well as a novel diagnostic and therapeutic target for MPM.


Introduction
Malignant pleural mesothelioma (MPM) is an aggressive malignancy of the pleura that is associated with asbestos exposure after 30-40 years of latency 1,2 . Patients with MPM are usually diagnosed at an advanced stage of the disease and their prognosis remains poor. The median survival after diagnosis is 6-12 months and the standard treatment agents, pemetrexed and cisplatin, are relatively ineffective at increasing survival time 2,3 . Despite the restricted and banned use of asbestos, MPM is increasingly being diagnosed in young individuals and women 4,5 . Other risk factors, including exposure to erionite fibers, therapeutic ionizing radiation to the chest, and germline BRCA1-associated protein 1 (BAP1)inactivating mutations, have been causally linked to MPM 1 . Therefore, new therapies based on improving patient survival are still required 6,7 .
Molecular biological studies in MPM have revealed frequent genetic alterations of tumor suppressor genes, including neurofibromatosis 2 (NF2), cyclin-dependent kinase inhibitor 2A (p16), and BAP1 [8][9][10][11][12][13][14][15] . Furthermore, multiplex molecular analyses with whole-exome sequencing, highdensity array comparative genomic hybridization, and immunohistochemistry (IHC) have disclosed that somatic BAP1 mutations and deletions were present in >60% of MPM patients 16,17 . We recently reported that fibroblast growth factor receptor 2 is highly expressed in NF2knockout mesothelial cell lines and is a candidate molecule for the development of therapeutic and diagnostic strategies targeting MPM 18 . A previous study using NF2 and p16 double-knockout (DKO) mice indicated that these genes, when mutated, contribute to mesothelioma development 19 . However, it remains unclear how these complex mutations contribute to tumor formation in MPM. We established NF2/p16-DKO cell clones in human immortalized mesothelial cell lines and identified several genes regulated by NF2 and p16.
CD24 is a glycosylphosphatidylinositol-linked sialoprotein that was shown to be present in the cell membranes of B lymphocyte precursors, neutrophils, neuronal cells, and some epithelial cells, and is highly expressed in several types of cancers [20][21][22][23][24][25][26][27][28][29][30] . It was also shown to be an independent prognostic marker of reduced patient survival in ovarian cancer and non-small cell lung cancer 21,22 . Furthermore, CD24 is reportedly related to epithelial-mesenchymal transition (EMT) in ovarian and pancreatic cancers 31,32 . The present study found that CD24 is highly expressed in NF2/16-DKO cell clones and human MPM tissues, highlighting it as a potential diagnostic and therapeutic target for MPM.

Gene expression change induced by disruption of NF2/p16
To identify genes involved in enhanced cell growth and migration, we performed comprehensive cDNA microarray analysis in the DKO, NF2-KO, and p16-KO clones, as well as the parental cells. To compare gene expression profiles, normalized values of raw microarray data were calculated and clustered according to differential gene expression. Expression of 29 genes was upregulated >20.0-fold (Supplementary Table S3) and 76 genes were downregulated <0.05-fold (Supplementary Table S4). Clustering of the 105 genes showed a distinct gene expression pattern among the DKO, NF2-KO, and p16/p14-KO clones, and the parental cells (Fig. 3a). To further confirm the effect of NF2/p16 on changes to gene expression, we generated clones exogenously expressing NF2 in the NF2-KO clone (exogenous NF2/NF2-KO), exogenous p16 in the p16-KO clone (exogenous p16/p16-KO), exogenous NF2 and p16 in the DKO clone (exogenous NF2 and p16/DKO). We then performed quantitative real-time PCR (qRT-PCR) analysis for four cell surface receptor genes (PTN, CD24, BMP7, and CADM1), because their protein products are easily detectable by molecular diagnosis and are also related to cell survival, proliferation, or tumor growth. The qRT-PCR results revealed increased expression of PTN, CD24, BMP7, and CADM1 genes in the DKO clones compared with the parental cells and NF2-KO and p16-KO clones (Fig. 3b). Furthermore, increased expression of PTN, CD24, and BMP7 was abrogated in the DKO clones exogenously expressing NF2 and p16 (exogenous NF2 and p16/DKO) (Fig. 3b), strongly suggesting that the affected gene products were downstream of NF2 and p16 signaling.
Effect of NF2/p16 DKO on CD24 expression and cell cyclerelated molecules CD24 has been implicated in the pathogenesis of a wide range of human cancers and emerged as a novel anticancer application in a panel of solid cancers [33][34][35][36] . Therefore, we focused on CD24 as its expression was increased in DKO cells (Fig. 4a). Exogenous expression of NF2 and p16 resulted in reduced CD24 expression in NF2 and p16/ DKO clones (Fig. 4a), indicating that CD24 expression is negatively regulated by NF2/p16 signaling in mesothelial cells.
Next, the phosphorylation levels of AKT, p70S6K, and c-Jun were investigated and quantified to determine cell growth signaling. As shown in Fig. 4a, b, we observed increased phosphorylation of cellular growth and proliferation-related signaling molecules, including AKT, p70S6K, and c-Jun, in the DKO clones. PTN, BMP7, and CADM1 protein products, as well as CD24, were increased in the DKO clones (Fig. 4a). To confirm the effect of NF2 and p16 on the expression of these signaling proteins, we utilized DKO clones exogenously expressing NF2 and p16 (exogenous NF2 and p16/DKO) (Fig. 4a). In the exogenous NF2 and p16/DKO clones, increased expression of CD24, PTN, BMP7, CADM1, p-pAKT, p-p70S6K, and p-c-Jun was downregulated, strongly suggesting that these proteins function downstream of NF2 and p16 signaling. Different expression patterns between CADM1 mRNA and its protein were observed in HOMC-B1 cells. Different expression patterns of PTN and BMP7 proteins from their mRNAs in MeT-5A and HOMC-B1 cells were also observed. The difference would be, in part, due to a post-transcriptional modification.
CD24 knockdown downregulates TGF-β1 and cell proliferation in the absence of NF2/p16 gene expression To investigate the involvement of CD24 in cell proliferation, we knocked down CD24 in the DKO clones and parental cells. MTT assay showed that cell growth was significantly decreased in the CD24 shRNA-DKO cells compared with the DKO clones (Fig. 5a). Furthermore, colony formation also decreased in the CD24 shRNA-DKO cells ( Supplementary Fig. S1). These results strongly suggest that CD24 may play an important role in cell growth and clonogenicity of mesothelioma cells with loss of p16 and NF2 expression.
SMAD2 in DKO cells compared to parental cells and subsequent decrease after treatment with the inhibitor were also observed (Fig. 5c). Furthermore, DKO clones with higher CD24 expression showed a spindle cell morphology, which is a morphological feature of EMT (Fig. 5d). Knockdown of CD24 also led to a change in cell morphology from spindle cell to circle shape, as well as an increased number of cell-cell contacts. To obtain further experimental evidence for the EMT phenotype, immunofluorescence analyses were performed to examine the expression of desmoplakin, one of the desmosomes 37 . A loss of desmoplakin expression in DKO cells and a mesothelioma cell line compared to mesothelial cells was found ( Supplementary Fig. S2). Taken together, these results prompted us to speculate that CD24 induces the EMT phenotype in mesothelial cells with loss of NF2 and p16.

IHC analysis of CD24 expression in patients with MPM
To investigate the role of CD24 in human MPMs, we performed immunohistochemical analysis to examine protein expression levels of CD24 in 45 MPMs and three normal mesothelium tissue samples (Table 1 and Fig. 6a). Microscopic analysis detected 8 strong (3+), 20 moderate (2+), and 6 weak (1+) CD24-positive signals among 45 MPM tissue samples (Fig. 6b), whereas CD24 expression was not detectable in three normal mesothelium tissues (Table 1 and Fig. 6a, left panels). Also, the CD24 expression pattern in three subtypes (epithelioid, sarcomatous, and biphasic) 1 is summarized in Table S5. A high frequency of CD24 expression was observed in the epithelioid type. To further validate CD24 expression in MPM patients, we analyzed a public cohort of 86 patients from The Cancer Genome Atlas Mesothelioma. Our analysis revealed that overall survival in MPM patients with higher CD24 expression levels was shorter than that in those with low CD24 expression levels (Fig. 6c). According to the log 2 value of CD24 probe, patients were divided into two groups: high (log 2 value > 6.938, n = 42) and low (log 2 value < 6.938, n = 44) CD24 expression. Thus, high CD24 expression was significantly associated with overall survival (P < 0.0001).
Furthermore, NF2 and p16 expression, together with CD24 expression in human MPM tissue arrays, was immunohistochemically examined. CD24-positive expression showed 20 (100%) of 20 cases with both NF2-and p16negative expression, whereas CD24-positive expression showed 8 (42%) of 19 cases with both NF2-and p16-positive expression (Supplementary Table S6 and Supplementary Fig.  S3). These results suggest that CD24-positive expression might be associated with the loss of NF2 and p16 expression in MPM tissues.

Discussion
NF2, p16, and BAP1 are the three major tumor suppressor genes that frequently undergo genomic alterations in MPMs. In the present study, we generated both NF2 and p16-knockout isogenic clones using the human normal mesothelial cell lines, MeT-5A and HOMC-B1, and showed that loss of NF2 and p16 expression enhanced cell growth, clonogenicity, and migration activities with global changes in gene expression. Our findings strongly suggest that CD24 expression occurs downstream of NF2 and p16 in mesothelial cells and is correlated with overall survival of MPM patients.
CD24 is frequently overexpressed in several types of solid tumors and is correlated with poor prognosis 22,25,28,38,39 . Thus, CD24 could be a potential target for a monoclonal antibody-mediated therapy. Antibodymediated therapy toward cell surface antigens, such as CD20, has been used as treatment for non-Hodgkin's lymphoma 40,41 . Therefore, the present study focused on CD24, as its expression is increased in DKO clones. Knockdown of CD24 reduced cell growth and clonogenicity. CD24 was also reported to be related to EMT, which is an important step leading to invasion, migration, and resistance to apoptosis of various cancer cells 28,31,32 . EMT phenotypes are characterized by decreased expression of E-cadherin as an epithelial marker, and increased expression of TGF-β1 as an inducer of EMT and Snail, Twist, Slug, vimentin, and N-cadherin as mesenchymal markers, and are responsible for poor prognosis in patients with various cancers 42 . In the present study, knockdown of CD24 decreased the expression of TGF-β1, Snail, and N-cadherin, and increased the expression of E-cadherin in DKO clones, strongly suggesting that CD24 may contribute to the EMT phenotype in the mesothelial cells. To further explore the relationship between CD24 and TGF-β1, we examined the effect of TGF-β1 inhibitor in the DKO cells. Knockdown of CD24 decreased the (see figure on previous page) Fig. 2 Cellular phenotype of parent, NF2-KO, p16-KO, and NF2/p16-DKO-Met-5A and HOMC-B1 cells. a MTT analysis of the growth rate in parental cells, NF2-KO cell clones, p16-KO cell clones, and NF2/p16-DKO cell clones. Data are mean ± SEM (n = 3). *P < 0.05, statistically significant difference between parental and DKO cells. b Representative soft agar colony formation assays are shown. Right bar graphs represent the number of stained colonies. Scale bar = 100 μm. Data represent mean ± SEM (n = 3). *P < 0.05, statistically significant difference. c Representative migration assays using a Boyden chamber are shown. The right bar graph represents the number of stained colonies. Scale bar = 200 μm. Data represent mean ± SEM (n = 3). Asterisks (*) indicate statistically significant differences (*P < 0.05). b Quantitative real-time PCR (qRT-PCR) analysis. Four genes that were upregulated in DKO cell, as detected using cDNA microarray analysis, were selected for qRT-PCR analysis using the SYBR green method. Relative gene expression levels are shown after normalization to GAPDH mRNA expression. Mean values were compared with the normal control value to calculate the relative amounts of transcripts. Data represent mean ± SEM (n = 3). Asterisks (*) indicate statistically significant differences (*P < 0.05).
expression of TGF-β1; however, TGF-β1 inhibitor did not alter the expression of CD24, indicating that CD24 functions upstream of TGF-β1. In addition, DKO clones with higher expression of CD24 showed a spindle cell morphology, which is a morphological feature of EMT.
We also observed that knockdown of CD24 changed the morphology of spindle cells to circles. Thus, our results suggest that CD24 contributes to EMT phenotypes in mesothelial cells. Our results indicate that CD24 expression is closely associated with changes in both gene  Supplementary Table S1. The cell lysates obtained were used for western blotting analysis. GAPDH was used as an internal control. b Quantitation of p-AKT, p70S6K, and c-Jun in MeT-5A and HOMC-B1 cells. p-AKT, p70S6K, and c-Jun expression levels were quantified by measuring the ratio between phosphorylated and total levels. Data are mean ± SEM (n = 3). *P < 0.05, statistically significant difference. expression and cell morphology characteristic of EMT phenotypes. Desmosome is a cell structure specialized for cell-to-cell adhesion 37 . The loss of expression of many desmosome proteins has been reported in different cancers and may contribute to EMT 43,44 . Thus, the expression of desmoplakin, one of the desmosomes, was analyzed, and loss of desmoplakin expression was found in DKO cells and mesothelioma cell lines, providing further experimental evidence for the EMT phenotype 45 ; therefore, we propose that CD24 expression induces the EMT phenotypes involved in the pathogenesis of MPM.
IHC analysis showed that CD24 was commonly expressed in 62% of human MPM tissues. Furthermore, analysis of public data revealed that MPM patients with higher expression of the CD24 gene showed a significantly shorter survival time. In addition, loss of NF2 and p16 resulted in increased expression of CD24, and subsequent rescue of the two genes decreased its expression in the MeT-5A and HOMC-B1 cell lines. More importantly, CD24 expression was associated with the loss of NF2 and p16 expression in human MPM tissues. These results indicate that CD24 expression is intimately regulated by NF2 and p16 expression. Furthermore, knockdown of CD24 in the DKO clones led to retardation of cell growth and colony formation, strongly suggesting that CD24 may play a pivotal role in the proliferation and clonogenicity of DKO cells.
In the present study, global gene expression changes in response to loss of NF2 and p16 in the MeT-5A and HOMC-B1 human mesothelial cell lines were demonstrated. CRISPR/Cas9-mediated loss of NF2 and p16 enhanced cell proliferation and CD24 expression, and disruption of CD24 reduced the proliferation of the cells. Although the molecular mechanisms underlying the upregulation of CD24 via loss of NF2 and p16 remain unclear, CD24 was commonly expressed in MPM tissues and was associated with poor prognosis of MPM patients. Therefore, CD24 may be used as a prognostic marker as well as a novel diagnostic and therapeutic target for MPM. Further studies are warranted to develop a new therapeutic approach for the treatment of MPM.

Construction of RNAi vectors and expression vectors
RNA interference vectors were constructed by inserting shRNA oligonucleotides into the pLentiLox3.7 plasmid (Addgene) under the control of the U6 promoter. One shRNA oligonucleotide was designed for the target sequence of the hairpin loop of CD24 (sh, 5′-TGCATTG ACCACGACTAA-3′). A control shRNA vector was also constructed using a scrambled (scr) CD24 sequence (scr, 5′-GGATAAACTAAGGGATAGGAA-3′). DKO and parent cells (1 × 10 6 cells) were nucleofected with 1 μg of each vector using a 4D-Nucleofector instrument (Lonza Japan). After 48 h of incubation, cell lysates were prepared and used for western blot analysis.
(see figure on previous page) Fig. 5 CD24 knockdown reduces cell proliferation and induces gene expression and morphological changes of EMT phenotype in DKO cells. a Effect of CD24 shRNA on cell proliferation. DKO cells (Met-5A and HOMC-B1) and parent cells were transfected with CD24 shRNA and control shRNA vectors. b Effect of CD24 shRNA on protein expression. c Effect of TGF-β1 inhibitor (vactosertib) on CD24 expression. Cells were treated with 2.5 μM vactosertib for 24 h and cell lysates were used for western blot analysis. GAPDH or total protein was used as an internal control. d Effect of CD24 shRNA on morphological changes in DKO cells and parent cells. DKO cells and parent cells were transfected with CD24 shRNA and control shRNA vectors. After 48 h of incubation, photomicrographs were taken depicting cell morphological in control shRNA-parent, CD24 shRNA-parent cells, control shRNA-DKO cells, and CD24 shRNA-DKO cells. Scale bar = 100 μm. All data are mean ± SEM (n = 3). *P < 0.05, statistically significant difference. The NF2/pcDNA3.1 vector was transfected into NF2-KO clones, the p16/pcDNA3.1 vector was transfected into p16-KO clones, and the NF2/pcDNA3.1 and p16/ pcDNA3.1 vectors were transfected into DKO clones using the 4D-Nucleofector System. After transfection, cells were incubated for 48 h, washed with phosphatebuffered saline (PBS), and lysed in loading buffer. The lysates were used for Western blot analysis.

Quantitative real-time PCR
qRT-PCR analysis for PTN, CD24, BMP7, and CADM1 was performed using SYBR Green I as previously described 48 . Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. The sequences of the primers for PTN, CD24, BMP7, and CADM1 used in this study are summarized in Supplementary Table S1.
cDNA microarray analysis cDNA microarray analysis was performed according to the manufacturer's instructions (Agilent Technologies). In brief, cDNA synthesis and cRNA labeling with cyanine 3 (Cy3) dye were performed using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies). Cy3-labeled cRNA was purified, fragmented, and hybridized on a Human Gene Expression 4 × 44K v2 Microarray Chip containing 27,958 Entrez Gene RNAs using a Gene Expression Hybridization kit (Agilent Technologies). Raw and normalized microarray data were submitted to the Gene Expression Omnibus database at the National Center for Biotechnology Information (accession number GSE116000; https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE116000). Gene set enrichment analysis was performed according to the instructions.

Cell growth assay
Cell growth rate was determined using MTT assay. Briefly, cells (1 × 10 3 per well) were seeded into 96-well plates and cultured for the indicated times. Subsequently, 10 μL of MTT solution (5 mg/mL; Sigma-Aldrich) was added to each well and cells were further incubated for 4 h. Next, cell lysis buffer was added to the wells to dissolve the colored formazan crystals produced by MTT. The relative optical density (OD) at 595 nm was calculated by dividing the OD on day 0 at each time point (days 0, 1, 3, 5, and 7). The absorbance was measured at 595 nm using a SpectraMAX M5 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

Soft agar colony formation assay
Soft agar colony formation assay was performed as described previously 49 . Then, cells (200 per well) were seeded in 6-well plates. After 14 days, the cells were stained with MTT and imaged. The number of colonies was counted using Colony Counter software (Keyence, Tokyo, Japan). Data are presented as mean ± SEM (n = 3).

Migration assay
Cells (2.5 × 10 5 per well) were seeded in Boyden chambers in 24-well plates (8 μm for 24-well plates; Millipore, Tokyo, Japan) and the culture medium was added into the lower chambers. After 24 h, cells were stained with crystal violet and imaged. The number of colonies was counted manually under a microscope.

Western blot analysis
Western blot analysis was performed as described previously 49 . The antibodies used are listed in Supplementary  Table S2. Immune complexes were detected using Immuno Star LD (Wako Pure Chemical Industries, Ltd, Osaka, Japan) in conjunction with a LAS-4000 image analyzer (GE Healthcare, Tokyo, Japan).

Immunofluorescence
MeT-5A, HOMC-B1, DKO-MeT-5A, DKO-HOMC-B1, and Y-MESO-9 cells were cultured on glass coverslips and Fig. 6 Immunohistochemical (IHC) analysis of CD24 expression. a Representative IHC results showing CD24 expression in two MPM tissues (right panels, cases 6 and 33) and two normal mesothelium tissues (left panels, cases 46 and 47). b Summary of IHC results in MPM tissues. Immunoreactivity was independently evaluated by two investigators (S.K. and H.M.). Staining intensity was scored as strong (3+), moderate (2+), weak (1+), or negative (0). The number of cases and their staining intensities are shown in the right panel. c Kaplan-Meier analysis was conducted to assess the value of CD24 in overall survival of MPM patients in TCGA mesothelioma obtained from the UCSC Xena database. Fluorescence values above the median were considered high CD24 expression, whereas fluorescence values below the median were considered low CD24 expression. Scale bar = 100 μm. fixed using 4% paraformaldehyde solution for 20 min at room temperature. Cells were permeabilized with PBS containing 0.1% Triton X-100 and blocked using PBS containing 7% serum for 30 min. Then, cells were incubated with γ-catenin (desmoplakin; BD Transduction Laboratories, San Jose, CA, USA; 1:100 dilution) for 2 h at room temperature followed by fluorescence staining with anti-mouse IgG Alexa Fluor® 594 (Abcam, Cambridge, UK; 1:200 dilution) and Hoechst (Dojindo, Kumamoto, Japan; 1:200 dilution) for 1 h at room temperature. Images were acquired using BZ-II (Keyence) with a fluorescence microscope (BZ-X9000; Keyence).

Statistical analysis
The statistical significance between groups was determined using one-way analysis of variance and Dunnett's comparison. Statistical analyses were performed using SPSS 23.0 program (SPSS, Inc., Chicago, Illinois, USA). Results are expressed as mean ± SEM.