Diagnostic Pathology: Open Access
Open Access

About 85% of all lung cancers are Non-Small-Cell Lung Cancers (NSCLCs), which also happen to be the primary cause of cancer-related mortality globally [1,2]. Lung Adenocarcinoma (LUAD) is the most common histological subtype and accounts for most lung cancer-related fatalities [3,4]. The overall prognosis for LUAD patients remains unsatisfactory despite significant breakthroughs in treatment, such as immunotherapy, molecular targeted therapy and radiation [5]. Immunotherapies, particularly Immune Checkpoint Blockades (ICB) that target Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) or Programmed cell Death protein 1 (PD-1), its Ligand (PD-L1), or both, dramatically increase Overall Survival (OS) in patients with a variety of cancer types, including LUAD [6,7]. However, a sizable fraction of LUAD patients receiving ICB treatment were unable to produce an objective response, highlighting the need for the identification of predictive biomarkers for selection of patients who may gain from this treatment [8]. Although ICB has shown effectiveness as an oncologic treatment for LUAD [9], distinct genomic and immunological landscapes may influence the response to ICB [10]. Thus, comprehending the correlations between particular gene expression and the immune micro environmental characteristics of tumors should aid in the creation of reliable biomarkers for immunotherapy, improve the clinical response, and increase the population that benefits [11,12]. The smallest but most functionally complex member of the Inhibitor of Apoptosis Protein (IAP) family is BIRC5, commonly referred to as survivin [13]. By physically blocking direct connections with molecules that promote apoptosis and indirectly reducing caspase-9 activation, BIRC5 plays a critical role in protecting cells from both intrinsic and extrinsic apoptotic pathways [14]. By regulating the mitotic apparatus, ensuring appropriate chromosomal segregation and protecting microtubule integrity, BIRC5 functionally supports the growth of malignant cells by promoting safe and effective cell division [15]. Hypoxia stimulates the expression of BIRC5, which is closely associated with angiogenesis and cell division. The majority of human malignancies have high levels of BIRC5 expression, which is closely linked to tumor growth, recurrence, resistance to chemotherapy and a poor prognosis. Nevertheless, BIRC5 expression in LUAD and its prognostic and predictive significance for the results of ICB treatment have not yet been thoroughly examined. Thus, we used information from The Cancer Genome Atlas (TCGA) to do this study in order to describe the expression of BIRC5, its correlation with clinicopathological characteristics in LUAD, as well as its predictive and prognostic values, as well as the protein expression from Human Pathology Atlas (HPA) online database. To clarify the relationship between tumor micro-environmental features and BIRC5 expression, we analyze the bulk RNA and single-cell sequencing data to investigate the connections between BIRC5 expression level and enriched biological pathways and the immune cells’ infiltrations.

Patients and databases

The TCGA database provided gene expression data for 535 LUAD and 59 normal lung tissues. An additional 57 LUAD and matched normal lung tissues were grouped for prognostic value and clinicopathological feature analysis. To investigate BIRC5 expression across the two databases, transcriptomic data in Transcripts per Million (TPM) formats from TCGA and GTEx was consistently processed. Furthermore, using the Oncomine database, BIRC5 expression data from adjacent normal tissues as well as pan-cancer tissues were obtained and analyzed. LUAD and normal lung tissues' BIRC5 protein levels were assessed using the Human Protein Atlas (HPA) database.

BIRC5-related functional enrichment analysis

Utilizing the STRING website, we examined BIRC5-binding proteins and set certain conditions, such as a minimum required interaction score of low confidence, active interaction sources from publications and a network type of complete network, to find the binding proteins. We found 20 experimentally verified proteins that bind to BIRC5, after limiting the maximum number of interactors shown to 20 and adjusting the significance of network edges to evidence. Furthermore, we chose the top 100 potential genes by using GEPIA2 to assess genes that showed BIRC5-like expression patterns in LUAD. Next, using the DAVID website, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment investigations on the BIRC5, 20 BIRC5-binding proteins and 100 putative genes.

Single-cell sequencing data analysis

We investigated the functional state of cancer cells in relation to BIRC5 expression, with the use of CancerSEA database, a dedicated platform for single cell sequencing data analysis. A heatmap was designed based on the findings of the correlation study that was run to investigate the relationship between BIRC5 expression and several tumor functional features. To assess the connection between BIRC5 and tumor cell functionality even more, we created t-distributed Stochastic Neighbor Embedding (t-SNE) diagrams for every single cell from the Cancer SEA website.

Differential gene expression analysis

To identify Differentially Expressed Genes (DEGs) between high and low SIRPg expression groups, the "edgeR" R program was used. To identify the most significant DEGs, a cutoff gene expression fold change of >1.5 or versus low BIRC5 expression group.

Pathway enrichment analysis

To carry out the pathway enrichment study, we used three different approaches: GO, KEGG and GSEA. The Molecular Signature Database provided the curated gene sets of known signaling pathways. The GO word analysis was performed using the R package "cluster Profiler," and the relevant pathways between the high and low SIRPg expression groups were examined using the GSEA software version 4.1.0.

Infiltration of immune cells

We used the single-sample Gene Set Enrichment Analysis (ssGSEA) approach to measure the amount of immune cell infiltration in tumor tissues. The LUAD expression profile data was exposed to immunological datasets that included 24 types of immunocytes and the R package "GSVA" was used to measure the infiltration levels [16]. Additionally, the relationship between immune cells and the two groups as well as the correlation between BIRC5 and other immune cells were assessed using the Spearman correlation and Wilcoxon rank sum test.

Public datasets of anti-PD-1/PD-L1 treated study

The response data that were accessible, along with the clinical and bulk RNA-seq data of 54 NSCLC patients who received PD-1/PD-L1 inhibition monotherapy, were obtained from other publications [1,17,18]. The Complete Response (CR), Partial Response (PR), Stable Disease (SD), or Progressing Disease (PD) were the clinical responses that were evaluated using the Response Evaluation Criteria in Solid Tumors (RECIST). Patients who underwent PD-1/PD-L1 blockade with CR, PR, or SD were classified as responders. Patients who got PD-1/PD-L1 blockade in conjunction with PD were classified as non-responders. The definitions of Overall Survival (OS) and Progression-Free Survival (PFS) were in line with the relevant published research. The survival was assessed using the Kaplan-Meier curves with two-sided log-rank tests and a Cox proportional hazards model with computed Hazard Ratios (HRs) and 95% Confidence Intervals (CIs).

Statistical analysis

BIRC5 expression was examined using the Wilcoxon rank-sum test in both the LUAD and normal lung groups. Based on the median expression of BIRC5, two groups of LUAD patients were created. Using the Wilcoxon rank sum or Kruskal-Wallis tests, the clinicopathological characteristics of the high and low BIRC5 expression groups were compared. A Pearson correlation analysis was performed to assess the relationship between the abundance of immune cells, the expression of immunological markers and BIRC5 expression. Depending on the available cohort, survival outcomes were measured using Overall Survival (OS), Disease-Free Survival (DFS), or Progression-Free Survival (PFS). The prognostic and predictive significance of BIRC5 expression was evaluated through the Kaplan-Meier method, the log-rank test and Cox proportional hazards regression. To determine the predictive importance of genes with differential expression, a Receiver Operating Characteristic (ROC) curve was generated using the "plotROC" tool. The predictive value nomogram of LUAD patients was plotted using the R function "rms." In all two-sided statistical significance tests, p values or FDR q values

Characterization of BIRC5 expression in LUAD

First, we utilized the transcriptome datasets from the TCGA program to examine the different expression levels of BIRC5 between the normal lung and LUAD tissues. Table 1 provided the specifics of 535 LUAD patients along with the expression of BIRC5. The expression level of BIRC5 was significantly greater in LUAD tissues compared to normal lung samples (p<0.001; Figure 1A). In the paired tissue samples, LUAD showed a significantly higher expression level of BIRC5 than normal lung tissues (p<0.001; Figure 1B). We conducted a systematic analysis using transcriptomic datasets from the TCGA program to investigate BIRC5 expression across various cancer types. Our findings revealed that BIRC5 was overexpressed in most cancers, such as glioblastoma multiforme, bladder urothelial carcinoma, bone cancer and cholangiocarcinoma (see Figure 1C). We assessed the expression levels of BIRC5 in LUAD and normal lung tissues using the HPA database. Compared to matched normal lung tissues, we found that LUAD had increased protein levels of BIRC5 (Figure 1D). Additionally, BIRC5 expression was considerably higher in tumors with pathological stages II–IV than in those with pathological stages I (p<0.001) (Table 1 and Figures 1A-1D).

Figure 1: The expression level of BIRC5. (A) Comparison of BIRC5 expression level between LUAD and normal lung tissues; (B) BIRC5 expression level in 57 matched LUAD and normal lung tissues; (C) Pan-cancer analysis of BIRC5 expression in various cancers and adjacent normal tissues; (D) Human Protein Atlas (HPA) database were applied to characterize the BIRC5 protein expression. Note: **p<0.01; ***p<0.001.

Table 1: Baseline characteristics comparison between high and low BIRC5 expression groups.

Additionally, we found that elevated expression of BIRC5 was linked with increased T stage (P<0.001), N and M stages (p<0.001), smoking history (p<0.05) and young age (p<0.05) (Figures 2A-2F).

Figure 2: Correlation between BIRC5 expression and clinicopathological features. (A) Clinical stage; (B) T stage; (C) N stage; (D) M stage; (E) Smoking history; (F) Age. Note: *p<0.05; **p<0.01; ***p<0.001.

We investigated the relationship between BIRC5 expression and patient survival rates in order to ascertain the prognostic significance of BIRC5 in LUAD. Significantly lower OS (HR=1.79, p<0.001; Figure 3A), DFS (HR =1.98, p<0.001; Figure 3B) and PFS (HR=1.58, p=0.001; Figure 3C) were all associated with increased expression of BIRC5. Furthermore, an Area Under the Curve (AUC) value of 0.96 was found using the ROC analysis of LUAD to assess the predictive importance of BIRC5 (Figures 3A-3D).

Figure 3: : Survival analysis of clinical outcomes between patients with high and low BIRC5 expression. (A) Kaplan-Meier plot of overall survival; (B) Kaplan-Meier plot of disease-specific survival; (C) Kaplan-Meier plot of progression-free survival; (D) ROC analysis of LUAD to determine the predictive significance of BIRC5.

Analysis of BIRC5 co-expression network and enriched pathway

Using the STRING website, we determined and experimentally verified the binding proteins of BIRC5 in order to explore the co-expression network and pathways where BIRC5 is enriched in LUAD. Twenty proteins were detected, according to the results, binding to BIRC5:MED1, MED6, MED18, MED30, MED31, LAMTOR5, H3F3A, H3F3B, AURKB, AURKC, HFE, INCENP, CDCA8, HIST3H3, CASP3, CASP7, CASP9, DIABLO, XIAP, and BIRC2 (Figure 4A). Next, we chose the 100 genes that were most closely associated with BIRC5 out of the GEPIA2 database. On the 448 genes, GO and KEGG enrichment analysis were performed. These genes were substantially linked to chromosome segregation, mitotic sister chromatid segregation, nuclear division, ATPase activity, microtubule motor activity, tubulin binding, microtubule binding and other processes, according to GO enrichment analysis (Figures 4B-4D). Furthermore, we observed that BIRC5 was connected to the development of tumors through the p53 signaling pathway, cellular senescence, cell cycle, pyrimidine metabolism, DNA replication and progesterone-mediated oocyte maturation (Figure 4E). When combined, these findings supported the traditional function of BIRC5 as described in earlier studies Figures 4A-4E.

Figure 4: Co-expression network of BIRC5 and enrichment pathway analysis. (A) The binding proteins of BIRC5 by STRING website; (B-D) Gene Ontology (GO) enrichment analysis of biological process, cellular component, molecular function; (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis based on BIRC5 binding proteins and interactive genes.

Expression level of BIRC5 in single cells and its effect on functional status

An essential technique for examining the intricate network of tumor, immunological, endothelial and stromal cells in malignancies is single-cell transcriptome sequencing. Using the CancerSEA website, we examined the relationship between BIRC5 expression and tumor function in LUAD at the single-cell level. We found that a number of critical physiological processes, including the cell cycle, DNA damage, repair, hypoxia, inflammation, and proliferation, were strongly linked with the expression of BIRC5 (Figure 5A). Moreover, our study demonstrated a correlation between BIRC5 expression and the following in LUAD: Invasion, proliferation, DNA damage response, cell cycle progression, invasion, Epithelial-Mesenchymal Transition (EMT) and inflammation (Figure 5B). Using t-SNE diagrams, we were also able to see the BIRC5 expression profiles in individual LUAD cells (Figure 5C). These results imply that BIRC5 may play a critical role in the development of LUAD by regulating important biological processes as proliferation, DNA damage/repair, hypoxia, the cell cycle and inflammation (Figures 5A-5C).

Figure 5: Expression pattern of BIRC5 in single cells and its correlation with tumor functional status. (A) Heatmap showed the correlation between BIRC5 expression and different tumor functional status based on CancerSEA database; (B) Correlation between BIRC5 expression and seven significantly different functional states based on CancerSEA database; (C) t-SNE diagram of BIRC5 expression profiles in single cells.

Univariate and multivariate analyses

We next performed the univariate and multivariate analyses by modifying the common clinicopathological characteristics after observing the negative correlation between high BIRC5 expression with the clinical outcomes of LUAD patients. The findings demonstrated that in univariate analyses, tumor status, residual tumor, T, N and M stages, as well as pathological stage, were substantially linked with OS (Supplemental Table 1). Poor OS was also significantly correlated with high BIRC5 expression (HR=1.75, 95% CI 1.30-2.36, p<0.001). The results of multivariate analyses showed an independent relationship between shorter OS and increased BIRC5 expression (HR=1.44, 95% CI 1.01-2.28, p=0.043).

In order to examine the effect of BIRC5 expression on OS based on age, sex, and risk variables for anatomic neoplasm subdivision, we conducted a subgroup analysis. We found that increased BIRC5 expression was substantially linked with inferior survival outcomes in these subgroups stratified by age, gender and anatomic neoplasm classification (Supplemental Figure 1). The results presented above show a substantial correlation between BIRC5 expression levels and LUAD patients' clinical outcomes. In order to quantify the odds of each individual survival, we consequently created a predictive nomogram based on these characteristics (Supplemental Figure 2A). The 1-3 years and 5 years survival estimates closely matched the ideal line, according to the predictive model's calibration curve (Supplemental Figure 2B). These findings demonstrate that BIRC5 expression-based model could provide accurate estimation of overall prognosis.

Enriched pathways in tumor with high BIRC5 expression

Using bulk RNA sequencing data from 535 LUAD patients, we ran GSEA to determine the distinct pathways enriched between high and low BIRC5 expression groups. The findings showed that mRNA splicing, DNA repair, and translation were among the enriched pathways linked to malignancies with elevated BIRC5 expression (Supplemental Figure 3). A further possible mechanistic connection between BIRC5 expression and the cell cycle, DNA damage/repair, hypoxia, inflammation and cell proliferation was discovered using single-cell sequencing data analysis.

Immune infiltration analysis

In order to visualize the immunological infiltration landscape, we deconvoluted the bulk RNA sequencing data of 535 LUAD patients in order to identify the immune profiles of tumors with different degrees of BIRC5 expression. The results of the deconvolution analysis showed that the infiltration levels of various immunosupportive cells, such as B cells, T helper 17 cells, CD8+ T cells, macrophages, dendritic cells and NK cells in LUAD, were negatively correlated with BIRC5 expression. Conversely, the infiltration levels of regulatory T cells (Tregs) were positively correlated with BIRC5 expression (Figure 6). These findings imply that tumors expressing a high level of BIRC5 would have a cool tumor immune milieu.

Figure 6: The correlation between immune cell infiltrating and BIRC5 expression level in LUAD1.