Identification of therapeutic targets and prognostic biomarkers among STAT family in glioblastoma

Background: Glioblastoma (GBM) is the most common and aggressive primary brain malignancies with high incidence and mortality. The aberrant activation of STAT signaling was confirmed to result in tumor pathogenesis and progress by regulating cell cycle, cell survival, and immune response. Methods: The clinical significance of and regulation network of STAT family in GBM were explored with several web applications or database. Results: The level of STAT1/3/5A/5B/6 were increased in GBM while STAT4 level was decreased. GBM patients with high expression of STAT1/2/3/5A/6 and low expression of STAT4/5B had a worse overall survival. Among the STAT family, STAT 4 and STAT6 were the top two frequently mutated genes. Correlation suggested a low to moderate correlation among STAT family. STAT family were also involved in the activation or inhibition of the famous cancer related pathways. Immune infiltrates analysis suggested that STAT5A level showed significantly correlated with the abundance of immune cells and the level of immune gene biomarkers. GO functions and KEGG pathways analysis revealed that STAT5A was involved in immune response-regulating signaling pathway, neutrophil and lymphocyte mediated immunity, single-stranded DNA binding, cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, NF-kappa B signaling pathway, and TNF signaling pathway. Moreover, we also identified several Kinase and transcription factor targets of STAT5A in GBM. Conclusions: Our results revealed the therapeutic targets, prognostic biomarkers and regulation network of STAT family in GBM, laying the foundation for further studies about STAT family in therapy and prognosis of GBM.


Background
Glioblastoma (GBM) is the most common and aggressive primary brain malignancies, accounting for about 45.6% of brain malignancies [1]. In 2020, an estimated of 23,890 patients would be initially diagnosed with breath malignancies in America alone [2]. No significant progress was made in the standard of care in the past of two decades [3].
Moreover, the prognosis of GBM patients was poor, with a median overall survival less than 20 months and 5-year-survival of 5% [4,5]. The exploration and identification of novel biomarkers for the prognosis and therapy, and a combination of current treatments, are the most promising strategies for the management of GBM.
Signal transducer and activator of transcription (STAT) signaling pathways are crucial for the function, proliferation, and apoptosis of immune cells, such as CD8 + cells and dendritic cells [6]. The aberrant activation of STAT signaling was confirmed to result in tumor pathogenesis and progress by regulating cell cycle, cell survival, and immune response [7,8]. STAT family consist of STAT1-4, STAT5A/5B, and STAT6. Previous studies revealed that STAT1 acted as an anti-oncogene in GBM under hypoxia and inhibited angiogenesis [9]. STAT3 interacted with FOXM1 could confer radio-resistance in GBM [10].
These evidences demonstrated the vital function of that STAT in the occurrence and progression of GBM. However, the potential application value and mechanism of STAT family in therapy and prognosis of GBM still not be clarified.
In this study, we performed a systematic exploration about the level and prognostic value of STAT family in GBM. Besides, single nucleotide variation (SNV), correlation, cancerrelated pathway, drug sensitivity, Immune infiltrates, and function analysis of STAT family in GBM were also conducted. Our study may reveal the potential application value and mechanism of STAT family in therapy and prognosis of GBM.

Oncomine
The Oncomine (www.oncomine.org/) is a web application for translational bioinformatics services [11]. It contains 700 + independent datasets, which could help researcher interrogate gene expression profiles, and biological interactions. Using Oncomine web application with the P < 0.05 and fold-change = 2, STAT family mRNA level in GBM were detected.  [14]. Various customizable functions, including immune cell infiltrates and immune gene biomarker correlation analysis of STAT family in GBM, were explored by TIMER. Table 3 showed the immune gene biomarkers and their corresponding immune cells, which had been referred in previous studies [15][16][17].  CGGA (the Chinese Glioma Genome Atlas, www.cgga.org.cn) is a database for exploring brain tumors with 2,000 samples from Chinese cohorts [18]. We used "mRNAseq_693" of CGGA to explore the prognostic value of STAT family, and their correlation with immune gene biomarkers in GBM.

Expression and prognosis analysis of STAT family in GBM.
Oncomine and GEPIA was allowed to performed expression analysis of STAT family in GBM.
As listed in Fig. 1 [24]. We observed that the expression of STAT6 were significantly upregulated GBM, and the fold change was 2.103 (p = 0.004).
Results from GEPIA indicated an increased level of STAT1, STAT3, and STAT5A, and a decreased level of STAT4 in GBM tissues compared with brain tissues ( Fig. 2A). Table 1 The mRNA levels of STAT family in glioblastoma (ONCOMINE).  (Fig. 2B). Table 2 The prognostic value of STAT family in glioblastoma (PrognoScan As shown in Fig. 3A, missense mutation was the most common variant classification and C > T was the most common SNV class. Among the STAT family, STAT 4 and STAT6 were the top two frequently mutated genes (Fig. 3A). Correlation analysis of STAT family demonstrated a low to moderate correlation among STAT family (Fig. 3B). Figure 3C showed the result of STAT family in the famous cancer related pathways. The STAT family played significant functions in GBM mainly by activating apoptosis pathway, EMT pathway, hormone ER pathway, RAS/MAPK pathway, and RTK pathway, and inhibiting cell cycle pathway, DNA damage response pathway, hormone AR pathway, and P13K/AKT pathway (Fig. 3C). The results of drug sensitivity analysis were shown in Fig. 4. GBM patients with low expression of STAT5B show resistance to 59 drugs or small molecules.

STATs
Furthermore, GBM patients with low expression of STAT5A show resistance to 42 drugs or small molecules (Fig. 4).

Immune infiltrates analysis of STAT5A in GBM.
Above results suggested that STAT5A expression was increased in GBM and acted as potential prognostic biomarker. Moreover, STAT5A was involved in the activation and inhibition of famous cancer related pathways, and drug resistance. Thus, STAT5A may play a significant role the pathogenesis and progress, and we select it for further analysis. We next analyzed the correlation between STAT5A and immune infiltrates in GBM because of the important role of STAT family in immune response [25,26], As presented in Fig. 5 The results of immune gene biomarkers correlation analysis revealed that STAT5A expression showed strong correlatio gene biomarkers expression in GBM, which were presented in Table 3. For gene biomarkers of CD8 + T cell, data of TIMER revealed that 3.4. Enrichment analysis of STAT5A in THCA. Figure 6A showed the genes were significantly correlated with STAT5A in GBM. Among these genes, the top 50 most significant genes positively (Fig. 6B) and negatively (Fig. 6C) associated with STAT5A were also extracted. Moreover, Supplementary Fig. 1  The kinase and transcription factor target networks of STAT5A in GBM were also explored.
The results were listed in Table 4. The data suggested kinase LCK, SYK, LYN, HCK, and TTK as the most five significant kinase network targets. With the construction of a PPI network, we found that kinase LCK target network were involved in the regulation of immune response, antigen receptor-mediated signaling pathway, T cell activation and receptor signaling pathway (Fig. 7A). As for transcription factor target, the most five significant targets were V$ELF1_Q6, V$IRF_Q6, V$PEA3_Q6, V$PU1_Q6, and V$NERF_Q2. With the construction of a PPI network, we found that kinase LCK target network were involved in the regulation of immune response, regulation of lymphocyte and leukocyte activation, and B cell activation (Fig. 7B). Table 4 The kinase and transcription factor targets of STAT5A in glioblastoma (LinkedOmics

Discussion
The STAT family could mediate various biological processes, including cellular immunity, invasion, differentiation, and apoptosis [27]. Activated by diverse cytokines, STAT signaling played a central role in immunity, cell death, and cancer pathogenesis and progression [8]. Extensive studies have revealed that STAT family exert important functions in cancers, such as hepatocellular carcinoma. However, the study about the all the STAT family in GBM has never been performed. So, our study was carried out. Expression analysis revealed that the level of STAT1/3/5A/5B/6 were increased in GBM while STAT4 level was decreased. GBM patients with high expression of STAT1/2/3/5A/6 and low expression of STAT4/5B had a worse overall survival. These results were consistent with previous studies. Balaram et al also found that STAT1 is upregulated in GBM and correlated with a worse prognosis [28]. Another study suggested STAT3 as a potential biomarker for the prognosis of GBM [29]. Therefore, we suggested that STAT family may act as potential prognostic biomarkers in GBM.
Cancer-related pathways analysis revealed that The STAT family played significant functions in GBM mainly by activating apoptosis pathway, EMT pathway, hormone ER pathway, RAS/MAPK pathway, and RTK pathway, and inhibiting cell cycle pathway, DNA damage response pathway, hormone AR pathway, and P13K/AKT pathway. Adetola et al also revealed that STAT3 inhibition induced apoptosis in cancer cells [30]. In hepatocellular carcinoma, STAT1 was also supposed as tumor-suppressor, and induced G0/G1 cell cycle arrest and apoptosis of tumor cell [31]. Therefore, STAT family may regulate the pathogenesis and progress of GBM via these pathways.
In our study, we also observed that STAT5A level showed significantly correlated with the abundance of immune cells and the level of immune gene biomarkers. Actually, these immune cells and immune gene biomarkers had been reported to be therapy target and play a significant role in various types of cancer. Immune cells, including dendritic cells and CD8 + cytotoxic T lymphocytes, could function as promising targets for the therapy of bone metastasis [32]. In breast cancer, CD8 + T cells and regulatory T cells were suggested as the reliable prognostic biomarker [33]. CTLA4 and PD-1 had been suggested as immune checkpoint for cancer immunotherapy [34]. Therefore, we suggested STAT5A as potential therapeutic target for cancer immunotherapy.
We next performed GO functions and KEGG pathways analysis, which revealed that Single-stranded DNA-binding were also involved in cell-cycle checkpoint activation following DNA damage in cancer [37]. Therefore, STAT5A may affect the pathogenesis and progress of GBM via these signaling pathways.
There is no gainsaying that some limitations were found in our study. First, the sample size in our study was relatively small, and it would be better if a larger cohort study were performed. Moreover, our study was short of clinicopathological features analysis.

Conclusion
In conclusion, our study revealed the expression and prognostic value of STAT family.
Immune infiltrates analysis suggested that STAT5A level showed significantly correlated Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.   The drug sensitivity analysis of STAT family in GBM. The negative correlation means that low expression of gene is resistant to the drug, vise verse.

Figure 5
Immune cell infiltration analysis of STAT5A in GBM.

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download. Supplementary Fig 1.tif