ASF1B May Regulate the Tumor Microenvironment and Epithelial-mesenchymal Transition in Malignant Mesothelioma to Induce the Differentiation of Sarcomatoid Phenotype as a Prognosis Target

Tianqi Chen First Teaching Hospital of Tianjin University of Traditional Chinese Medicine https://orcid.org/00000002-8762-9682 Yingjie Jia First Teaching Hospital of Tianjin University of Traditional Chinese Medicine Xiaojiang Li First Teaching Hospital of Tianjin University of Traditional Chinese Medicine Fanming Kong First Teaching Hospital of Tianjin University of Traditional Chinese Medicine Yafei Yin (  m18222453423@163.com ) Chengdu Second People's Hospital Shanqi Guo First Teaching Hospital of Tianjin University of Traditional Chinese Medicine


Introduction
The mesothelium is a cell monolayer that is spread over the surface of serosal organs and cavities. The primary function of mesothelium is to protect tissues and organs from damage or infection. The tumor of the mesothelium is called a malignant mesothelioma (MM). It is a rare malignant tumor with a median survival of 6 to 12 months. 1 . The incidence of MM has increased constantly and is estimated to increase continuously around the world. The exposure to asbestos is the main cause of this devastating disease.
With the recent developmental works such as reconstruction and restoration, exposure to asbestos is evitable in some countries where mesothelioma will remain a health burden until 2030. 2 According to the present clinical and laboratory studies, Mesothelioma is an aggressive cancer that occurs in the mesothelium and is strongly associated with asbestos exposure. Fibrous mineral erionite, carbon nanotubes and gene mutations, radiation and Simian Virus 40 are considered to be the most hazardous factors that contribute to the development of mesothelioma. 3 Histologically, mesothelioma can be classi ed into three subgroups which include epithelioid, sarcomatoid and biphasic types. The type of epithelioid or sarcomatoid is morphologically characterized by the presence of polygonal or spindle-like cells. The underlying mechanism for the development of histologically different mesothelioma types is yet to be elucidated. The current MM diagnosis includes two positive and two negative histochemical markers. However, there is little data available on markers that could help to distinguish sarcomatoids types or further stratify prognoses amongst histologic variants which makes diagnosis even more di cult. In addition to the de nition of multiple morphological patterns of these subtypes, the primary lung cancer and pleural metastases are similar in clinic presentation and histological appearance which make MM diagnosis even more challenging. 4 Epithelial-mesenchymal transition (EMT), which is related to tumor proliferation and disease progression, represents a reversion in embryological development. 5 Several studies have proved that EMT plays a signi cant role in the morphological characteristics of malignant mesothelioma and eventually lead to the development of sarcomatoid patterns. [5][6] . In order to enhance the diagnostic accuracy, it is important to elaborate the function of oncogenic genomic changes in MM as predictive biomarkers and future therapeutic targets.  15 . In TCGA, there were no paired normal tissues of MM and as such the differentially expressed genes could not be determined.
UALCAN is an interactive web-portal to perform the in-depth analyses of TCGA gene expression data (http://ualcan.path.uab.edu/analysis.html) 16 . The boxplots were drawn by UALCAN with the correlation between the expression or methylation of ASF1B and different ages of the patients, nodal metastasis status and tumor histology. The cBio Cancer Genomics Portal (cbioportal, http://www.cbioportal.org/) 17 was also used to investigate the relationship of mRNA expression with the level of ASF1B methylation.

Gene ontology enrichment and similar gene mutation
The UniProt-GOA database (http://www.ebi.ac.uk/GOA/) 18 was used to perform the GO analysis on ASF1B. Subsequently, the LinkedOmics (http://www.linkedomics.org/login.php) as used to nd the correlated genes and to identify the downregulated and upregulated genes and present them in the form of a heat map. 19 . Furthermore, PPI network was constructed to nd the interaction of those hub genes in STRING (https://string-db.org/). Moreover, the related microRNAs have also been detected in LinkedOmics and the survival of MM patients affected by those miRNAs were investigated by the OncomiR (http://www.oncomir.org/oncomir/survival_custom.html) 20 .

Analysis of the relative abundance of tumor in ltrating immune cells (TIICs)
The Cibersortx (https://cibersortx.stanford.edu/index.php) was used to identify the different immune cells

GSEA pathway analysis
Gene set enrichment analysis (GSEA) is commonly used to reveal biological pathways by interpreting gene expression data. In this study, GSEA was used to analyze the differential signaling pathways of the activation of ASF1B low and high expression groups in MM patients. LinkedOmics (http://www.linkedomics.org/) is a comprehensive multiomics analysis platform for the TCGA database.
The LinkedOmics was used to nd the ASF1B correlated genes, including downregulated and upregulated genes, and to verify the pathway enrichment of GSEA. The minimum number of genes (size) was set to 3 and simulation to 500, and p < 0.05 is taken as a measure of signi cant enrichment.

Statistical analysis
The correlation between ASF1B expression and the clinicopathological parameters was assessed by Pearson's Chi-square test. The independent indicators related to OS were identi ed using Cox proportional hazards model, and the hazard ratios (HR) with 95% con dence intervals (CI) were also calculated. The correlation between the ASF1B mRNA expression and the ASF1B DNA methylation level (or the miRNAs expression) was evaluated by linear regression analysis. SPSS 22.0 and R version 3.6.2 were used for statistical analyses. A two-tailed p value less than 0.05 was considered to be statistically signi cant. Area under the curve (AUC) was 0.9326 (p<0.0001) and the cut-off value was 7.635 (Speci city:87.27%, Sensitivity:95.12%). The speci city and AUC were calculated for the discrimination of mesothelioma from the paired normal controls in GSE51024.

The relationship between ASF1B expression and some clinicopathological features
In TCGA-MM, there were no compared tissue of mesothelioma so we could not determine the differential expression of ASF1B between normal and tumor tissues. However, the survival analysis was performed, and it was found that in low-expression group, patients tend to have longer OS than high-expression group. (p<0.05, Fig 2).
Through COX univariate analysis, it was found that the reduced expression of ASF1B was signi cantly related to longer overall survival. (P<0.001) This means that the low ASF1B expression level is a signi cant protective factor for the survival. (Table 2). Other clinical information like stage, gender, laterality and histology showed no signi cant difference.
Previous studies have shown that pathological type is an independent predictor of survival of malignant mesothelioma. The degree of differentiation was also an independent predictor of survival of malignant mesothelioma. 23 Although there is no signi cant difference (p>0.05), our univariate Cox regression analysis indicated that sarcomatoid mesothelioma might be a hazard factor as compared to the epithelioid type (HR=2.00, 95%CI=[0.79, 5.11]).
A correlation analysis was performed on 84 patients with clinical case characteristics (age of the parents, tumor histology, status of nodal metastasis, clinical stage) using the UALCAN (Fig. 3).
The results showed that the increase in ASF1B expression was signi cantly correlated with histology (Yingjie Jia Figure3D, 3E, 3F). The results were veri ed by the data in GEO (Yingjie Jia Figure3E)(p<0.05), Oncomine (Yingjie Jia Figure3F)(p<0.05). The results showed that signi cant upregulation of ASF1B in biphasic mesothelioma and sarcomatoid mesothelioma, which are considered to be more malignant. And this could be one explanation of how ASF1B induces the progression of mesothelioma.

GO enrichment, KEGG pathways and comparable mutant genes
The GO enrichment includes three parts-molecular function, biological process, cellular component, and the ASF1B mainly function in histone binding in nucleus and chromatin (Table 3).
MSigDB enrichment analysis was performed for this study, to screen for signaling pathways with major variations caused by the expression of ASF1B (p < 0.05). Subsequently, the KEGG pathway analysis by the LinkedOmics platform (Yingjie Jia Figure. 5) was compared with the GSEA(Yingjie Jia Figure 6), and those pathways mainly functioned in the proliferation of cells. The main pathways modulated by ASF1B were mainly related to cell cycle and include chromosome segregation, organelle ssion, spindle organization, DNA replication and mitotic cell cycle phase transition. At the same time, ASF1B also could downregulate the process of cargo loading into vesicle, protein activation cascade, response to interleukin-6 and platelet-derived growth factor receptor signaling pathway.
In GSEA between the high and low AFS1B expression datasets to screen out the differentially activated signaling pathways, ASF1B modulated the gene replication, nucleotide excision repair, and base excision repair to name a few. STRING shows that these closely related genes were all function in the process of TATA box binding protein associated factor (TAF), Centromere kinetochore component CENP-T histone fold and core histone H2A/H2B/H3/H4(Yingjie Jia Figure 7C).
We investigated the relative expression of miRNAs and found that hsa-miR-503, hsa-miR-130b, hsa-miR-301b, hsa-miR-196b were positively correlated to the expression of ASF1B and they all could signi cantly shorten the survival of MM patients. In contrary, hsa-miR-29c, hsa-miR-195, hsa-miR-100, hsa-miR-30d were negatively correlated to the expression of ASF1B and could prolong the OS time. (Yingjie Jia Figure   8)

Association between ASF1B expression and composition of TIICs
In the database pf TCGA, macrophage M1, macrophage M2, CD8 T cell, T cell follicular helper and regulatory T cell showed a signi cant increase in ASF1B high-expression group. In contrary, the NK cells resting, monocytes, eosinophils, neutrophils, dendritic cells activated, CD4 T memory resting showed signi cant decrease under high expression of ASF1B.
In the database of GEO, the regulatory T cells (Tregs), macrophage M1, macrophage M0, dendritic cells resting showed a signi cant increase in ASF1B high-expression group. In contrary, the B cells naïve, plasma cells, monocytes, dendritic cells activated showed a decrease under the effect of high ASF1B expression (Yingjie Jia Figure 9 and Yingjie Jia Figure 10). The detail proportion of immune cells data can be obtained in the supplement table 2(GEO) and supplement table 3(TCGA).
The results of the present study revealed that under the regulatory in uence of ASF1B, the microenvironment changed a lot. These results were veri ed by GEO and TCGA. Two databases showed that under the effect of ASF1B, the changes of DC cells, Tregs and the subgroup of macrophages indicated the immune suppression in the tumour microenvironment. More than that, many other immune cells need further investigated.

Discussion
The present study revealed that the incidence of malignant pleural mesothelioma is comparatively higher in male (82.10%), generally present on the right side (59.5%) and in people with asbestos exposure history (83.3%). The prevalence of MM has sharply increased over the last 50 years, with a ratio of 4.6:1 from male to female. These ndings in agreement with the results of previous studies.
To nd out the subtypes of mesothelial, epithelial, or sarcomatous distinction in malignant cells, pathological diagnosis was done with the help of immunohistochemical analysis [37]. The reported diagnostic yield from CT guided biopsy ranged from 60% to 85% with multiple attempts and the highest yields were acquired by open or thoracoscopic pleural biopsy. [38] Since MM has a partial broblastic phenotype within EMT, this can partly explain why it is a highly invasive and chemoresistant cancer, and can be utilized to distinguish epithelioid from sarcomatoid MM 25  Additionally, studies have proved that Tregs induce TGF-β-mediated EMT and subsequent metastasis of several types of tumor cells such as melanoma and hepatocellular carcinoma and also accelerates the radiation-induced pulmonary brosis. 28 Till date there is not a single study that reports the relationship between dendritic cell and EMT in mesothelioma. Nonetheless, it has been proved that dendritic cell take part in the EMT in some speci c cancer cells such as breast cancer cells. 25,29 Furthermore, DC phenotype and its production of anticancer cytokine IL-12 could trigger changes in the microenvironment of colon cancer.
ASF1 is the evolutionarily most conserved histone H3/H4 chaperone protein from yeast to humans. It effects the heterochromatin silencing and is involved in multiple functions related to chromatin. Its chaperones contribute to chromatin functions by linking histone H3/H4 to DNA. Additionally, it acts as an essential cofactor in histone acetylation on certain histone H3/H4 residues. 30 ASF1B is a member of the H3/H4 family of histone chaperone proteins, and functions similarly to ASF1. Tousled-like kinase family of cell cycle-regulated kinases are its downstream molecules. ASF1B also play a key role in modifying the chromatin nucleosome structure by maintaining continuous supply of the histones at nucleosome assembly sites. In the present study, it was found that ASF1B may change the tumor microenvironment and changes the percentage of the macrophage and some other immune cells such as dendritic cell, monocyte and T cells regulatory (Tregs).
In a previous study, sarcomatoid MM was more likely found to have an M2-like protumor phenotype than epithelioid MM. Our results indicate that high-ASF1B group with higher inhibitory immune cell in ltration (Tregs and macrophage, etc) may result in the phenotype like biphasic mesothelioma or sarcomatoid mesothelioma with EMT, which is related to poor overall survival outcomes and extent of malignancy.
Moreover, we investigated the relative expression of miRNAs and found that hsa-miR-503, hsa-miR-130b, hsa-miR-301b, hsa-miR-196b were positively correlated to the expression of ASF1B and they all could signi cantly shorten the survival of MM patients. In contrary, hsa-miR-29c, hsa-miR-195, hsa-miR-100, hsa-miR-30d were negatively correlated to the expression of ASF1B and could prolong the OS time.
Previous reports have indicated that miR-503-5p is related to the proliferation and invasion of various cancer cells. It was recently found to regulate EMT and also affected the metastasis and prognosis of hepatocellular carcinoma. 31 Until now, the relationship between hsa-miR-29c, hsamiR-30 and malignant mesothelioma has been extensively studies. In epithelial mesothelioma, miR-29c* levels have been documented to be elevated relative to sarcomatoid MM. In addition, the miR-29 family itself has been associated with EMT and the expression of B7-H3 proteins, suppressing mesothelioma immune escape. 3233 . Besides, hsa-miR-30c has been found to be correlated with survival in sarcomatoid tumors. 34 However, other miRNAs like hsa-miR-195, hsa-miR-100 have not been fully investigated in the mesothelioma. In the present study, it was found that those miRNAs which were closely related to the ASF1B could affect the survival of MM patients, which needs to be explored further.
The microRNAs take a fundamental part in the regulation of gene expression. Histone modi cations, meanwhile, could also modify microRNAs. While numerous studies have been devoted to exploring the miRNA-regulated EMT and epigenetic regulations, their synchronized effect has not been thoroughly examined. To decide if there is a combinatorial action of ASF1B with histone modi cation and miRNA regulations to mediate the microenvironment and the EMT process, further research endeavors are urgently required.

Conclusion
Collectively, ASF1B could be a predictive factor for mesothelioma patients with the sensitivity of 95.12% and speci city of 87.27%. The expression level of ASF1B is negatively related to the survival of MM patients, which could be considered as a hazardous factor in MM patients. Moreover, ASF1B could mediate the tumor immune in ltrating microenvironment, which may lead to EMT and eventually causing increase in the incidence of biphasic or sarcomatoid mesothelioma, rather than the epithelioid phenotype.

Figure 2
The overall survival and disease free survival in TCGA