Penetrating Exploration of Prognostic Correlations of the FKBP Gene Family with Lung Adenocarcinoma

The complexity of lung adenocarcinoma (LUAD), the development of which involves many interacting biological processes, makes it difficult to find therapeutic biomarkers for treatment. FK506-binding proteins (FKBPs) are composed of 12 members classified as conservative intracellular immunophilin family proteins, which are often connected to cyclophilin structures by tetratricopeptide repeat domains and have peptidyl prolyl isomerase activity that catalyzes proline from residues and turns the trans form into the cis form. Since FKBPs belong to chaperone molecules and promote protein folding, previous studies demonstrated that FKBP family members significantly contribute to the degradation of damaged, misfolded, abnormal, and foreign proteins. However, transcript expressions of this gene family in LUAD still need to be more fully investigated. In this research, we adopted high-throughput bioinformatics technology to analyze FKBP family genes in LUAD to provide credible information to clinicians and promote the development of novel cancer target drugs in the future. The current data revealed that the messenger (m)RNA levels of FKBP2, FKBP3, FKBP4, FKBP10, FKBP11, and FKBP14 were overexpressed in LUAD, and FKBP10 had connections to poor prognoses among LUAD patients in an overall survival (OS) analysis. Based on the above results, we selected FKBP10 to further conduct a comprehensive analysis of the downstream pathway and network. Through a DAVID analysis, we found that FKBP10 was involved in mitochondrial electron transport, NADH to ubiquinone transport, mitochondrial respiratory chain complex I assembly, etc. The MetaCore pathway analysis also indicated that FKBP10 was involved in "Ubiquinone metabolism", "Translation_(L)-selenoaminoacid incorporation in proteins during translation", and "Transcription_Negative regulation of HIF1A function". Collectively, this study revealed that FKBP family members are both significant prognostic biomarkers for lung cancer progression and promising clinical therapeutic targets, thus providing new targets for treating LUAD patients.


Introduction
In its advanced stages, lung cancer (LC) is the one of the most lethal types of cancer and is responsible for significant mortality worldwide. The 5-year overall survival (OS) of

Oncomine Gene Analysis for Expression Levels of FKBP Family Members in LUAD
The Oncomine platform (http://www.oncomine.org/, accessed on 22 November 2021) is an online cancer microarray bioinformatics database established to display transcriptional levels of target cancer and normal specimens from 715 datasets [42][43][44]. In this research, we analyzed individual mRNA expression levels of FKBP family members in various types of cancer via Oncomine with the setting of p < 0.01, fold change of 1.5, and gene rank in the top 10%.

Kaplan-Meier (KM) Plotter Survival Assessment of FKBP Gene Family Members
The KM plotter (http://kmplot.com/analysis/, accessed on 30 November 2021) is a visual bioinformatics database containing up to 54,000 genes in 21 cancer types including lung, ovarian, breast, and gastric cancers [47]. This public database can be used to conduct meta-analyses with TCGA, Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih. gov/geo/, accessed on 30 November 2021), and European Genome-phenome Archive (EGA) (https://ega-archive.org/, accessed on 30 November 2021) [48]. We adopted a pan-cancer platform to analyze the prognostic merits of transcriptional levels of individual FKBP family members in LUAD patients (n = 513) for OS and relapse-free survival (RFS) with the KM plotter choosing median values, a p log rank of <0.05, and hazard ratios (HRs) of >1.

cBioPortal Analysis of Genetic Alterations of FKBP Family Members in LUAD
cBioPortal (http://cbioportal.org/, accessed on 30 November 2021) is an online resource that integrates several cancer-related databases to analyze genetic alterations, DNA methylation, copy number changes, etc. [49][50][51] from more than 5000 cancer specimens in 20 cancer studies [44]. In this study, we explored genetic alterations of the FKBP gene family in LUAD with 503 complete samples from TCGA in cBioPortal.

Gene MANIA Was Used to Build Gene-Gene Interactions (GGIs) and Explore Their Functions
Gene MANIA (http://www.genemania.org/, accessed on 4 December 2021) is a versatile tool for predicting gene functions, analyzing gene lists, and recognizing the most interrelated genes such as Homo sapiens, based on more than 800 connections [52,53]. We examined GGI networks and functions of FKBP family numbers by Gene MANIA.

STRING Analysis of the FKBP Gene Family and Other Associations of Expressed Proteins
The purpose of the STRING informatics tool (https://string-db.org/, accessed on 4 December 2021) is to gather and integrate accessible sources of protein-protein interactions (PPIs) and conduct computational forecasts. The aim of this tool is to achieve a comprehensive analysis [54]. The newest version of STRING (11.5) for organisms, renewed on 12 August 2021, is nearly triple the size of the older version (11.0b) and covers 1,409,467,592,464 proteins and 20,052,394,042 interactions.   [55], and data visualization through online platform (http://www.bioinformatics.com.cn/srplot, accessed on 4 December 2021). These platform consists of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) and is composed of molecular functions (MFs), biological processes (BPs), and cellular components (CCs) [56]. The goal of KEGG is to assign biological functions to genes and genomes [57], while GO offers information about gene products, processes, and functions [58]. Together with the MetaCore platform, we mapped the intersection between these two sets of data in terms of related pathways and involved networks. A p value of <0.05 was considered significant, as previously described.
2.8. Tumor Immune Estimation Resource (TIMER) 1.0 Comprehensive Investigation of Components of Immune Cell Infiltration of FKBP Gene Family Members in LUAD TIMER 1.0 (http://timer.comp-genomics.org/, accessed on 4 December 2021) is a convenient server for the analysis and visualization of associations of target genes and related immune cells between tumor and normal samples from 10,897 samples in 32 cancer types [59,60]. In this research, we analyzed correlations of different FKBP family members in LUAD with the enrichment of immune cell infiltrates, including B cells, cluster of differentiation 8-positive (CD8 + ) T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells (DCs).

Statistical Analysis
We utilized TCGA Pan-Cancer Atlas, a dataset from cBioPortal, to obtain patient data and query the effects of the expressions of different FKBP family members on OS. For the survival analysis, a KM plotter was applied, with all default settings, and RFS was preferred, with automatic cutoff values and J best probe set. All possible cutoff values between the lower and upper quartiles were determined, and the best presenting threshold was subsequently used as the cutoff [51]. A log-rank p value of <0.05 was considered statistically significant.

Survival Analysis and Prognostic Values of FKBP Family Members in LUAD
To assess associations between distinct transcriptional levels of FKBP family members and survival rates of LUAD, we used the KM plotter database, and preferentially analyzed OS ( Figure 2). Outcomes revealed that five members of the FKBP gene family had correlations with poor prognoses among LUAD patients in the OS analysis including FKBP3  Table S2).

Survival Analysis and Prognostic Values of FKBP Family Members in LUAD
To assess associations between distinct transcriptional levels of FKBP family members and survival rates of LUAD, we used the KM plotter database, and preferentially analyzed OS (Figure 2). Outcomes revealed that five members of the FKBP gene family had correlations with poor prognoses among LUAD patients in the OS analysis including FKBP3 (p=0.0034; hazard ratio (HR)=1.55), FKBP4 (p=0.00051;

Analysis of GGIs and PPIs and Coexpression of Pathway Abundance of the FKBP Gene Family
Due to genetic diversity, GGIs have impacts on gene functions, relative pathways, and even the development of target drugs [72]. In this study, we analyzed GGI networks of FKBP family members with neighbor genes via GeneMANIA (Supplementary Figure S2A). Results showed that HECTD1, TTC6, and other correlated genes were intensely linked with FKBP family members in shared protein domains (55.30%); coexpression (31.22%); physical interactions (13.16%); predictions (0.23%); co-localization (0.08%); and genetic interactions (0.02%); and functions including drug binding, cis-trans isomerase activity, protein folding, peptidyl-proline medication, isomerase activity, negative regulation of calcium ion transport into the cytosol, and positive regulation of the sequestration of calcium ions. Additionally, because cellular life is based on a complicated network of functional influences among biomolecules [73], we also evaluated the PPIs of FKBP family members in Homo sapiens using the STRING database (Supplementary Figure S2B).
The current data revealed that mRNA levels of FKBP2, FKBP3, FKBP4, FKBP10, FKBP11, and FKBP14 were overexpressed in LUAD, and FKBP10 had connections to poor prognoses among LUAD patients in an OS analysis. Based on the above results, we selected FKBP10 to further conduct a comprehensive analysis of the downstream pathway and network. Next, to deeply analyze coexpressed genes with FKBP10, we downloaded an archive of genes coexpressed with FKBP10 in LUAD and chose data for the first 1000 small p values from cBioPortal before performing DAVID. We analyzed two different aspects. First, GO term enrichment (GOTERM) revealed several FKBP10-correlated pathways, including protein binding (p = 1.6 × 10 −16 ), isomerase activity (p = 8.6 × 10 −2 ), etc. (Supplementary Figure S3, Supplementary Table S3). GOTERM_BPs described biological events in which these coexpressed genes of FKBP10 were involved, including mitochondrial electron transport, NADH to ubiquinone, mitochondrial respiratory chain complex I assembly, etc.  Table S5). In another aspect of a KEGG analysis, outcomes indicated that there were 325 coexpressed genes (33.8%) in the non-alcoholic fatty liver disease (NAFLD) pathway (Supplementary Figure S6).

Levels of Immune Cell Infiltration of Different FKBP Family Members in LUAD Patients
The TME is extremely important for the existence of cancer and is composed of vessels, extracellular matrix (ECM), and various immune cells, which favor tumor invasion, proliferation, and metastasis. Therefore, the occurrence of cancer is closely related to immune cells [74,75]. To fully understand the associations be- Figure 5. MetaCore pathway analysis of the coexpression gene network of FKBP family members in lung adenocarcinoma (LUAD) patients. The MetaCore pathway analysis of "biological processes" revealed that "Ubiquinone metabolism"-related pathways were correlated with LUAD development.

Levels of Immune Cell Infiltration of Different FKBP Family Members in LUAD Patients
The TME is extremely important for the existence of cancer and is composed of vessels, extracellular matrix (ECM), and various immune cells, which favor tumor invasion, proliferation, and metastasis. Therefore, the occurrence of cancer is closely related to immune cells [74,75]. To fully understand the associations between FKBP family members and immune cell infiltration in LUAD, we evaluated the immunological microenvironment using the TIMER database (Supplementary Figure S7). Results indicated that

Discussion
In this study, we analyzed FKBP mRNA expressions, clinical phase IV, survival rates, genetic variants, and coexpressed genes. Moreover, we also determined correlations of infiltration levels of immune cells and FKBP gene expressions in LUAD. Although direct evidence still needs to be provided as to the biological functions of FKBP, such as cell models or patient tissue samples, to demonstrate the roles of FKBP in LUAD, based on our results, we can provide the concept that FKBP can potentially be a biomarker in LUAD.
By applying advances in high-throughput screening to cancer transcriptome profiling, alterations in the transcriptome patterns of FKBP gene families were found to be significantly associated with several types of malignancies [76][77][78][79][80]. FKBP gene expressions were found to be involved in tumor multi-stage progression along with other tumor-related factors. According to various database analyses, FKBP3, FKBP4, and FKBP10 were closely associated with LUAD. Previous studies determined that FKBP3 is a crucial oncogene in distinct cancers. The combination of FKBP3 and HDAC2 was related to oxaliplatin resistance in colorectal cancer via the PTEN/AKT pathway [18]. Downregulation of FKBP3 suppressed breast cancer [81], and FKBP3 expression was associated with poor survival in LUAD [82][83][84]. Furthermore, FKBP4 was reported to be related to breast cancer [20,85], colorectal cancer [22], prostate cancer [86], and lung cancer [21,87]. FKBP10 was connected with gastric cancer [88], stomach adenocarcinomas [89], papillary thyroid cancer [90], and lung cancer.
Our data were found to be consistent with those in previous research, as current findings indicated that mRNA levels of FKBP2, FKBP3, FKBP4, FKBP10, FKBP11, and FKBP14 were overexpressed in LUAD compared to healthy tissues. The OS analytical results revealed that five members of the FKBP gene family, viz., FKBP3, FKBP4, FKBP5, FKBP9, and FKBP10, had connections with poor prognoses among LUAD patients.
Based on the above results, we selected FKBP10 to further conduct a comprehensive analysis of the downstream pathway and network. Therefore, we explored the characteristics and functions of FKBP10 in more detail and discovered that FKBP10, also called FKBP65 (65-kDa), possesses four cytosolic PPI activities [88,91]. Moreover, downregulation of FKBP10 with collagen VI increased the formation of primary human lung fibroblasts (phLFs) [88]. Additionally, downregulation of FKBP10 suppressed tumorsphere formation by regulating protein translation [92]. Genes coexpressed with FKBP10 in TCGA LUAD were subsequently used to perform a pathway analysis. Through the DAVID analysis, we found FKBP10 to be involved in mitochondrial electron transport, NADH to ubiquinone and mitochondrial respiratory chain complex I assembly, etc. The Metacore pathway analysis also indicated that FKBP10 was involved in "Ubiquinone metabolism", "Translation_(L)-selenoaminoacids incorporation in proteins during translation", and "Tran-scription_Negative regulation of HIF1A function".
In recent years, cancer immunotherapy, a novel strategy that aims to activate and boost the immune system to directly recognize and eliminate tumor cells, has undergone tremendous developments, and is now regarded as a promising cancer treatment [93][94][95]. Increasing evidence indicates that the immunosuppressive environment mediated by tumorinfiltrating immune cells (TICs), such as regulatory T (Treg) cells and tumor-associated macrophages (TAMs), hinders the delivery of immunotherapies in LUAD. Our data suggested that TICs are strongly correlated with FKBP expressions. Therefore, this analysis of the tumor immune microenvironment could help develop clinical immunotherapies and provide accurate personalized treatment plans for patients.

Conclusions
Previous research had not fully explored the roles of FKBP family genes in LUAD. Consequently, this study represents the first work that specifically examined the roles of FKBP members in this disease, prior to providing a more-extensive and -incisive understanding of the potential therapeutic and prognostic value for LUAD patients.  Figure S7. Associations between FKBP family members and immune infiltration consisting of purity, B cells, cluster of differentiationpositive (CD8 + ) T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells from TCGA in lung adenocarcinoma (LUAD) by the TIMER database. * Means a partial correlation (r), and p < 0.05 indicates a significant difference; Supplementary Table S1: Significant changes in expressions of FKBP family members between different types of lung cancer and normal tissue; Supplementary   Data Availability Statement: The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare that they have no conflict of interest.