Non–small cell lung cancer (NSCLC) has one of the highest mortality rates among malignant tumors globally, which accounts for approximately 80% of all lung cancers (Siegel et al., 2020). The two predominant histological phenotypes of NSCLC are lung adenocarcinoma (LUAD, ~50% of cases) and lung squamous cell carcinoma (LUSC, ~40% of cases) (Davidson et al., 2013; Langer et al., 2015). Unfortunately, currently, available biomarkers mainly reflect sex and age variations (Tsao et al., 2012) but cannot accurately identify the stage or prognosis of a tumor. Consequently, NSCLC remains difficult to detect at an early stage, and most patients are commonly diagnosed when the cancer has already progressed to an advanced stage (40% of NSCLC cases are diagnosed at stage IV) and thus not eligible for curative treatments. Therefore, the prognosis of NSCLC remains poor with a 5-year survival rate of only 2%–13% (Liu et al., 2020). Currently, the primary treatment of NSCLC is surgery, radiotherapy, and chemotherapy (Upadhya et al., 2021). Because of tumor heterogeneity, the current biomarkers used to predict NSCLC prognosis have some limitations; thus, it is necessary to explore new biomarkers as diagnostic and prognostic indicators to effectively improve survival and individualized treatment.

Inhibitors of apoptosis proteins (IAPs) are among the most extensively studied molecular and therapeutic targets in treating cancers, and their dysregulated expression has been reported in NSCLC (De-Xuan et al., 2017; Mazur et al., 2018). IAPs play essential roles in preventing apoptosis or programmed cell death. To date, eight IAPs have been identified in mammalian cells (BIRC1–8; see Table 1). The common feature of IAP family members is the presence of one or more baculoviral IAP repeats (Kumar et al., 2020). In addition to inhibiting apoptosis, IAPs play various biological roles, including regulation of innate immunity and inflammation, cell proliferation, cell migration, and apoptosis (Ji et al., 2018; Khan et al., 2021). Accordingly, IAPs act as pivotal regulators in oncogenesis by directly or indirectly affecting apoptosis through intrinsic and extrinsic cell death signaling pathways (Ji et al., 2018; Khan et al., 2021). Therefore, dysregulation of IAPs may lead cells toward cancerization (Yang and Wang, 2016; Yang et al., 2020; Zhang et al., 2021).

Downregulating BIRC2 expression indirectly induces NSCLC cell apoptosis by preventing the formation of the caspase-8–activating platform (Yang and Wang, 2016; Jian et al., 2019). Moreover, the positive rates of BIRC4 mRNA expression in pathological tissues of patients with NSCLC were reported to be significantly higher than those in the para-cancerous tissues (De-Xuan et al., 2017). BIRC5 is strongly expressed in different types of tumors but is not expressed or is only expressed at low levels in most normal differentiated tissues (Xiao and Li, 2015; Mazur et al., 2018). These findings suggest a role of IAPs in NSCLC. However, the underlying mechanism and functions of IAPs in different subtypes of NSCLC or at different stages of cancer progression have yet to be fully elucidated.

RNA and DNA research, an essential component of biological and biomedical studies, has been revolutionized with the development of microarray technology, providing vast molecular data for comparative analysis. However, to the best of our knowledge, bioinformatics analysis of IAPs has yet to be applied for NSCLC. In this study, we comprehensively analyzed the expression of IAPs in patients with NSCLC using public datasets to determine their expression patterns, potential functions, and distinct prognostic value. This study can provide new insight into understanding the molecular mechanisms of IAPs in NSCLC toward development of drugs to inhibit aberrantly expressed IAPs that can help to induce apoptosis in cancerous cells. Moreover, exploring biomarker to diagnose lung cancer and distinguish stages is very necessary.

Although the role of IAPs in tumor development and progression has been partially confirmed in NSCLC, further bioinformatics analysis has yet to be performed (Rashed et al., 2019; Frazzi, 2021). This is the first study to comprehensively explore the transcriptional profiles, prognostic values, interactions, and functional enrichment of IAPs in different subtypes of NSCLC and across different tumor stages. Our findings can provide guidance for the development of IAPs as markers in the prevention, treatment, and prognosis for patients with NSCLC.

There has been no evidence of a role of BIRC1 in NSCLC until now, and little has been reported of any association of BIRC1 in cancer. A PTV-loaded nanocarrier was developed to trigger the apoptosis of glioblastoma multiforme cells by reducing the mRNA levels of NFKB, IL6, BIRC1, and BIRC5 (Psc et al., 2021). Mig-6 exerts a tumor-suppressor function in murine endometrial cancer through downregulation of BIRC1 expression (Kim et al., 2019). In our study, the GEPIA dataset revealed that the expression of BIRC1 was lower in LUSC than that in normal tissues. Moreover, BIRC1 mRNA expression was significantly different at least between two stages of LUAD and was associated with OS or PFS in patients with NSCLC. These data suggest that downregulation of BIRC1 possibly plays a tumor-suppressor function in NSCLC development.

A previous study showed that BIRC2 expression regulates the apoptosis and survival of NSCLC cells: downregulating BIRC2 expression indirectly induces NSCLC cell apoptosis by preventing formation of the caspase-8–activating platform (Yang and Wang, 2016; Jian et al., 2019). The overexpression of BIRC2, regulated by Pellino-1, contributes to the oncogenesis of A549 and H1299 cells, which are both LUAD cell lines, and promotes cancer cell survival (Jeon et al., 2016; Xv et al., 2021). Consistently, we found that downregulated BIRC2 expression was associated with the prolonged survival time of patients with LUAD.

In our study, only BIRC3 expression was positively correlated with the OS of patients with LUAD. There is substantial evidence pointing to the pro-survival and anti-apoptotic roles of BIRC3 in cancer cells; however, not all data are consistent (Frazzi, 2021). An in vitro study showed that RNA-binding motif 10 overexpression inhibited the malignant behaviors of A549 and H1299 cells by inducing the expression of AKT2, BIRC3, and JUN (Guan et al., 2017). However, Dubois et al. (2019) reported that overexpression of BIRC3 regulated by RASSF1A depletion decreased the rate of cancer cell apoptosis. Similarly, upregulation of BIRC3 expression via Pellino-1 overexpression in A549 and H1299 cells promoted lung oncogenesis and survival, and BIRC3 also demonstrated a strong positive correlation with Pellino-1 in human LUAD tissues (Yang and Wang, 2016). Therefore, the mechanism of BIRC3 in cancer needs further study.

Surprisingly, we did not identify a specific role of BIRC4 in the patients with LUAD or LUSC on the basis of the databases analyzed in this study. However, several in vitro studies have suggested an anti-NSCLC role of BIRC4. Hydrogen gas was suggested to promote the apoptosis of A549 cells by reducing the expression of BIRC4 (Zhang et al., 2020a). Combined with other drugs in treating NSCLC in vitro, TRAIL induced cell apoptosis by inhibiting BIRC4 expression and increasing cytotoxicity (Deok et al., 2018; Kim et al., 2019). Moreover, the positive rate of BIRC4 mRNA expression in the pathological tissues of NSCLC patients was significantly higher than that in paracancerous tissues (De-Xuan et al., 2017). Although the expression of BIRC4 varies in vitro and in vivo, further in vitro experiments can represent an important starting point to better understand its regulation mechanisms and functions in vivo.

Unlike other IAPs, BIRC5 is strongly expressed in most tumors but is not expressed or is expressed at only low levels in most normal differentiated tissues (Xiao and Li, 2015; Mazur et al., 2018). Consistently, we found that BIRC5 expression levels were significantly higher in tumor tissues than in normal tissues. Previous studies have suggested BIRC5 as a predictive biomarker in NSCLC, especially for LUAD (Zhang et al., 2020b; Haakensen et al., 2020). In addition, in the present study, BIRC5 emerged as the most significant IAP that could be developed as a marker for preventing and treating NSCLC patients. Low expression of BIRC5 mRNA was also previously positively correlated with NSCLC patient survival (Cao et al., 2019; Nitschkowski et al., 2019; Rashed et al., 2019; Zhang et al., 2020b).

BIRC6 has been suggested as a progression marker in NSCLC (Dong et al., 2013; Gharabaghi and Asadi, 2016), which was also associated with the PFS of the patients with NSCLC in our study. However, previous studies did not distinguish among different subtypes of NSCLC. Here, we show that BIRC6 expression actually shows an opposite association with prognosis in patients with LUAD and LUSC: Increased BIRC6 expression was significantly associated with the PFS of patients with LUAD, whereas decreased BIRC6 was significantly associated with the PFS of patients with LUSC. This suggests that BIRC6 is a potential biomarker for differentiating different types of NSCLC. No specific roles of BIRC7 and BIRC8 in NSCLC were identified in this study or in the literature to date.

From GO and KEGG enrichment analysis, we found that all eight members of the IAP family are enriched in ubiquitin-protein transferase activity, and most of them (six of eight) are enriched in ubiquitin-mediated proteolysis. The ubiquitin–proteasome system has become a key system of pathogenesis in several cancers (Senft et al., 2018). Thevebioside (an active ingredient from Traditional Chinese Medicine) was reported to inhibit the tumor growth of NSCLC through inhibiting SRC-3–mediated IGF-1R–PI3K-AKT signaling via ubiquitination to induce cellular apoptosis (Yao et al., 2020). In addition, deregulation of APC/C (a representative E3 ligase) together with its co-activators cell division cycle 20 (CDC20) or CDC20-like protein 1 (CDH1) has been associated with cancers (Jeon et al., 2016). Overexpression of Pellino-1 (an E3 ubiquitin ligase) is dependent on the expression of BIRC3 in human lung cancer cells, resulting in increased cell survival and colony forming ability (Jeon et al., 2016). SKP2 promotes programmed cell death protein 4 degradation through phosphorylation and ubiquitination, resulting in increased proliferation and radiation tolerance of breast cancer cells (Li et al., 2019). In addition, IAPs were also found to play a role in the NOD-like receptor signaling pathway and NF-κB signaling pathway (Mann and Oakley, 2005; Liu et al., 2019; Kumar et al., 2021). Thus, we speculate that the dysregulation of IAPs has more effective role in the inflammatory response.

In this study, we systematically analyzed the expression and prognostic value of IAPs in different subtypes of NSCLC, which can help to provide a more thorough understanding of the molecular biological properties of this cancer. Our results indicate that BIRC1 and BIRC5 are potential diagnostic markers for both LUAD and LUAC. BIRC1, BIRC2, and BIRC5 are potential prognostic markers for LUAD, whereas BIRC2 and BIRC6 are prognostic markers for patients with NSCLC. From GO and KEGG enrichment analysis, we found that most IAP members are associated with ubiquitin and apoptosis. These highlight new targets for the early detection, treatment, and management of NSCLC.