CD276 as a critical independent biomarker and immune checkpoint inhibitor target in epithelioid mesothelioma-TCGA study

Introduction

Background

Mesothelioma is a rare and refractory cancer (1,2). Its tumorigenesis is related to the exposure (3,4) of mesothelial cells (5) to asbestos following a latency period of 30–40 years (6,7). However, the mechanisms underlying mesothelioma tumorigenesis are not fully understood (3,5). Although some molecular targets and possible prognostic biomarkers for mesothelioma have been identified (8-10), they have been inconclusive. One reason for this inconclusiveness is that, despite the identification of tumor suppressor genes, mesothelioma-related oncogenes are not well understood (11-13). A combination of immune checkpoint inhibitors (ICIs), namely nivolumab and ipilimumab, which are programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors (14), and chemotherapy with cisplatin and pemetrexed (15) with/without bevacizumab, which is a vascular endothelial growth factor A (VEGFA) inhibitor (16), have been confirmed to be effective in a phase 3 study of mesothelioma. However, the overall survival (OS) of patients administered these treatments was 12–18 months, with many patients eventually developing resistance to treatment (14,15). Mesothelioma consists of epithelioid and nonepithelioid subtypes, including biphasic and sarcomatoid subtypes. The prognosis and treatment-associated OS vary according to the subtype (2,3,5-11,13,14).

Although ICIs such as nivolumab and ipilimumab have been demonstrated to be effective against nonepithelioid mesothelioma, their efficacy against epithelioid mesothelioma is unclear, and prognostic biomarkers are lacking (14). Identifying novel molecular targets and prognostic biomarkers for each subtype is essential for the personalized treatment of patients with mesothelioma. In a recent study, we demonstrated that ICIs targeting lymphocyte-activation gene 3 (LAG3) and PD-1 inhibitors could potentially contribute to better clinical outcomes, and LAG3 expression was identified as an independent prognostic biomarker for mesothelioma (17). With the development of novel ICIs, there has been improvement in cancer treatment outcomes (18). The B7 homolog 3 (B7-H3) protein encoded by CD276 was first reported in 2001; it is a member of the B7 family and a key immune checkpoint that is highly expressed on the membrane of normal and cancer cells (19). The structure of B7-H3 comprises an IgV domain, an IgC-like domain, a transmembrane region, and a diverse cytoplasmic tail (20). Although the receptor of B7-H3 has not been confirmed, B7-H3/CD276 is considered to play a key role in the escape of cancer cells from the immune system through the production of IL-10 and TGF-β1 (20). B7-H3/CD276 inhibits the proliferation, activity, and function of CD4/CD8+ T-cells, natural killer cells, macrophages, and dendritic cells and stimulates the function of regulatory T-cells (20,21). In addition, B7-H3/CD276 has been suggested to be associated with metabolic reprogramming via the glycolytic pathway (22), supporting the notion of the Warburg effect (23). It is also reportedly associated with the ferroptosis pathway that is correlated with cancer cell proliferation (24); angiogenesis via the NF-κB pathway (25); and the proliferation, migration, and invasion of cells via the JAK/STAT3, PIK3CA/AKT/mTOR and RAF/MEK pathways (26-28).

Rationale and knowledge gap

High expression of CD276 is correlated with a poor prognosis in various cancers (29,30), including mesothelioma. However, mesothelioma subtypes and multivariate Cox proportional hazards models were not considered in these analyses (31,32). Furthermore, CD276 inhibitors downregulated cancer cell proliferation in an in vivo study (33), and various clinical studies on CD276 inhibitors are currently ongoing (30).

Objective

While the exact significance of CD276 remains unknown in various cancer, particularly in each mesothelioma subtype, we hypothesize that CD276 could be a potentially novel biomarker and the therapeutic target for each mesothelioma subtype. Therefore, this study aimed to evaluate CD276 mRNA expression as a prognostic biomarker across various cancers and explore its potential as a therapeutic target for each mesothelioma subtype, considering the existing therapeutic strategies for mesothelioma using molecular targets of PD-1 (PDCD1), CTLA4, and VEGFA. Our study could provide a new perspective on the correlation between CD276 and various associated pathways in each mesothelioma subtype. We present this article in accordance with the REMARK reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1598/rc).

Methods

The Cancer Genome Atlas (TCGA) database

We extracted data from TCGA database, which is a cancer genomics project started by the National Cancer Institute in 2006, and evaluated the data retrospectively. CD276 mRNA expression between 17 types of cancer and normal tissues was analyzed using TCGA (https://cistrome.shinyapps.io/timer/), as previously described (17,34). To compare CD276 expression between cancer and the adjacent normal tissues, the mRNA expression was transformed to log2 transcripts per million (17,34). The CD276 mRNA expression and OS data of 36 human cancer types were analyzed using TCGA database (https://xena.ucsc.edu/), as previously described (17,34). To compare high and low CD276 expression in TCGA, the mean number of patients was used as a cutoff value. Number of patients, age, sex, disease stage, and histological evaluation from TCGA were used as clinical variables for assessing factors affecting the correlation between OS and high/low expression of CD276, PDCD1, CTLA4, and VEGFA mRNA, and the combinations of high/low expression of CD276 and PDCD1, CTLA4, VEGFA, and clinical variables (17). The correlation of CD276 mRNA expression with the expression of gene markers of tumor-infiltrating immune cells (TIICs) and the glycolytic, ferroptosis, NF-κB, JAK/STAT3, PIK3CA/AKT/mTOR, and RAF/MEK pathways in TCGA was also evaluated.

Statistical analysis

All statistical analyses were conducted using R 3.6.2 (The R Foundation for Statistical Computing, Vienna, Austria) and GraphPad PRISM 10.3 (GraphPad Software, La Jolla, CA, USA) (17,34). Comparison of mRNA expression between cancer and the adjacent normal tissues was performed using the Wilcoxon rank-sum test (17,34). The correlation of high/low CD276 mRNA expression with OS was evaluated via the Kaplan-Meier method using the log-rank test with the hazard ratio (HR) and 95% confidence interval (CI) (17,34). A multivariate Cox proportional hazards model adjusted for age, sex, and disease stage as basic data was used to evaluate factors affecting the correlation between OS and high/low CD276 mRNA expression (17,34).

The correlation between the mRNA expression of CD276 and expression of gene markers of TIICs and those of glycolytic, ferroptosis, NF-κB, JAK/STAT3, PIK3CA/AKT/mTOR, and RAF/MEK pathways was evaluated using the Spearman’s rank correlation coefficient using r (17). The r values estimated using the 95% CI indicated the following: 0.80–1.0, very strong correlation; 0.60–0.79, strong; 0.40–0.59, moderate; 0.20–0.39, weak; and 0.001–0.19, very weak (17). A multivariate linear regression model was used to confirm the factors affecting the correlation of CD276 mRNA expression using β value, which is the regression coefficient with a 95% CI. Results were considered statistically significant at P value <0.05 (17).

Ethical statement

We used data from an anonymized public open database, which was outside the scope of the Japanese ethical guidelines and was thus exempt from ethical scrutiny. Obtaining patient consent was waived by the Institutional Review Board of Tokyo Women’s Medical University for this retrospective study. This manuscript does not include data from any human or animal studies conducted by the authors. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki (as revised in 2013).

Results

Comparison of CD276 mRNA expression between cancer and adjacent normal tissues in human cancers

To determine CD276 mRNA expression in cancer, we compared CD276 mRNA expression between cancer tissues and adjacent normal tissues in various human cancers. We found that CD276 mRNA expression was higher in all cancers, including bladder cancer, breast cancer, cholangiocarcinoma, colon cancer, esophageal cancer, head and neck cancer, kidney chromophobe, kidney clear cell carcinoma, kidney papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, rectal cancer, thyroid cancer, and uterine endometrial carcinoma, than in normal tissues (P<0.001) (Figure 1).

Figure 1 Comparison of CD276 mRNA expression between cancer tissues and the adjacent normal tissues in various human cancers. Comparison of CD276 mRNA expression between cancer tissues and normal tissues in various human cancers was performed using TIMER (http://cistrome.org/TIMER/) and visualized using the Wilcoxon rank-sum test. CD276 mRNA expression in all cancer tissues, including bladder cancer, breast cancer, cholangiocarcinoma, colon cancer, esophageal cancer, head and neck cancer, kidney chromophobe, kidney clear cell carcinoma, kidney papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, rectal cancer, thyroid cancer, and uterine endometrial carcinoma, was higher than that in normal tissues (***, P<0.001). Red: cancer tissues; blue: normal tissues. TPM, transcripts per million.

Prognostic significance of CD276 mRNA expression in various cancers

As CD276 mRNA expression was higher in cancer tissues than in normal tissues, we compared the OS between patients with various cancers exhibiting high and those exhibiting low CD276 mRNA expression. We found that high CD276 mRNA expression was associated with a poor prognosis in various cancers (Table 1). In particular, upregulated CD276 mRNA expression was associated with a poor prognosis in adrenocortical carcinoma, bladder cancer, colon cancer, glioblastoma multiforme, head and neck cancer, kidney chromophobe, kidney clear cell carcinoma, kidney clear cell carcinoma, lower-grade glioma, liver cancer, lung adenocarcinoma, mesothelioma, ocular melanoma, and pancreatic adenocarcinoma (Table 1). These results indicate that CD276 mRNA is a novel prognostic biomarker for these cancers.

Prognostic significance of CD276 mRNA expression in mesothelioma subtypes

Owing to its association with a poor prognosis in various cancers, including mesothelioma, we investigated CD276 mRNA expression as a prognostic biomarker in each mesothelioma subtype: epithelioid and nonepithelioid, which comprises biphasic and sarcomatoid mesothelioma. In particular, we assessed the association between clinical variables and CD276 mRNA expression in 86 patients with mesothelioma. The characteristics of the patients (number of patients, age, sex, stage, histological evaluation, and high/low CD276 mRNA expression) are described in Table 2. The OS analysis using the Kaplan-Meier method demonstrated that upregulated CD276 mRNA expression was associated with a poor prognosis in epithelioid mesothelioma (HR =2.864, 95% CI: 1.550–5.291, P<0.001, Figure 2A), whereas it was not associated with a poor prognosis in biphasic and sarcomatoid mesothelioma (HR =1.549, 95% CI: 0.658–3.646, P=0.27, Figure 2B). As CD276 mRNA expression was associated with a poor prognosis in epithelioid mesothelioma, we performed further analysis of OS using a multivariate Cox proportional hazards model. We found that upregulated CD276 mRNA expression was associated with worse OS after adjusting the model for age, sex, and stage (HR =4.157, 95% CI: 2.014–8.921, Table 3), even when considering the currently available molecular-targeted therapeutic strategies. Therefore, our results suggest that CD276 mRNA expression is an independent prognostic biomarker and therapeutic target in epithelioid mesothelioma.

Figure 2 CD276 mRNA expression was associated with a poor prognosis in epithelioid mesothelioma. Overall survival analysis using the Kaplan-Meier method for CD276 expression in epithelioid (A) and biphasic and sarcomatoid (B) mesothelioma. The log-rank test demonstrated that upregulated CD276 mRNA expression was associated with a poor prognosis in epithelioid mesothelioma (HR =2.864, 95% CI: 1.550–5.291, P<0.001), whereas it was not associated with a poor prognosis in biphasic and sarcomatoid mesothelioma (HR =1.549, 95% CI: 0.658–3.646, P=0.27). HR, hazard ratio; CI, confidence interval.

Correlation between the mRNA expression of CD276 and expression of genes of each pathway in epithelioid mesothelioma

Our study demonstrated that CD276 mRNA expression is an independent prognostic biomarker and therapeutic target in epithelioid mesothelioma. Therefore, we explored the correlation between CD276 and the gene markers of TIICs and each pathway involved in epithelioid mesothelioma (Table 4). CD276 mRNA expression weakly correlated with the expression of 3 of the 37 markers of TIICs in epithelioid mesothelioma (Table 4). These results suggest that CD276 mRNA expression does not correlate with various TIICs in epithelioid mesothelioma. However, we identified a strong positive correlation between CD276 and PKM2 expression (r=0.603, 95% CI: 0.399–0.749, P<0.001). We also identified moderate positive correlations between the expression of CD276 and SLC2A1 (r=0.423, 95% CI: 0.175–0.620, P=0.001), HK1 (r=0.544, 95% CI: 0.323–0.709, P<0.001), GAPDH (r=0.440, 95% CI: 0.195–0.633, P=0.001), SLC3A2 (r=0.420, 95% CI: 0.171–0.618, P=0.001), SLC38A1 (r=0.489, 95% CI: 0.255–0.669, P<0.001), and VEGFC (r=0.473, 95% CI: 0.234–0.657, P<0.001) markers in epithelioid mesothelioma (Table 5, Table S1). These results illustrate that CD276 mRNA expression is associated with the glycolytic pathway. In addition, we confirmed the factors influencing the correlation between the expression of CD276 and that of SLC2A1, HK1, GAPDH, PKM2, PDP1, and LDHB in the glycolytic pathway using a multivariate linear regression model. After adjusting for six gene markers, we observed that the expression of CD276 mostly correlated with that of PKM2 (β=0.443, 95% CI: 0.087–0.799, P=0.01) (Table 6). PKM2 plays an important role in generating two molecules of adenosine triphosphate (ATP) in the glycolytic pathway, which is essential for metabolic reprogramming during cancer progression. Our results show that CD276 mRNA expression may regulate the glycolytic pathway in epithelioid mesothelioma.

Discussion

Key findings

In this study, upregulated CD276 mRNA expression correlated with a poor prognosis, and it was confirmed as an independent prognostic biomarker and therapeutic target in epithelioid mesothelioma. In addition, we demonstrated that CD276 mRNA expression may regulate the expression of genes associated with the glycolytic pathway, particularly PKM2.

Upregulated CD276 mRNA expression was associated with a poor prognosis in patients with epithelioid mesothelioma (Figure 2A,2B). CD276 mRNA expression was associated with a poor OS in epithelioid mesothelioma, as determined using the multivariate Cox proportional hazards model, even when available therapeutic strategies targeting PDCD1, CTLA4, and VEGFA were taken into consideration. Therefore, CD276 is an independent prognostic biomarker and a therapeutic target for epithelioid mesothelioma.

Strengths and limitations

Our study highlighted the significance of CD276 expression in epithelioid mesothelioma, which is a rare and refractory cancer. However, it had some limitations. First, as our study was retrospective and had a limited number of patients, prospective studies with larger populations will be necessary to validate the results. Second, our results were based on a clinical database; therefore, in vivo and in vitro studies will be necessary to clarify the significance and role of CD276 in epithelioid mesothelioma.

Comparison with similar research and explanations of findings

High expression of CD276 is correlated with a poor prognosis in mesothelioma, however, its subtypes and multivariate Cox proportional hazards models were not considered in these analyses (31,32). Further, age, sex, and stage were not associated with the prognosis of epithelioid mesothelioma, as determined using the multivariate Cox proportional hazards model. However, it should be noted that in multivariate analysis, a strong prognostic factor may mask the effects of other potential prognostic variables (35). As CD276 mRNA expression was identified to be a strong factor influencing OS, age, sex, and stage may not be prognostic markers.

CD276 mRNA expression does not correlate with various TIICs in epithelioid mesothelioma including PDCD1 and CTLA4 mRNA, which are the existing therapeutic target for mesothelioma, except for the expression of 3 of the 37 markers of TIICs. Further, the correlation of 3 markers of TIICs were weak correlation in epithelioid mesothelioma. In addition, CD276 mRNA expression does not correlate with VEGFA mRNA, which is another existing therapeutic target for mesothelioma. In fact, CD276 mRNA expression was independent prognostic biomarker using the multivariate Cox proportional hazards model, considering PDCD1, CTLA4, and VEGFA in epithelioid mesothelioma. Based on these results, CD276 mRNA may be the independent immune checkpoint different from immune checkpoint with existing ICIs and therapeutic target in epithelioid mesothelioma.

Notably, CD276 mRNA expression correlated well with gene markers of the glycolytic pathway (Table 5). This result indicates that CD276 regulates the glycolytic pathway in epithelioid mesotheliomas. The strongest correlation was identified between CD276 and PKM2, and a moderate correlation was identified between CD276 and SLC2A1 and HK1 (Table 5). PKM2 generates two ATP molecules via this pathway, which is essential for metabolic reprogramming for cancer progression (36). Thus, CD276 mRNA expression is strongly associated with PKM2 expression and ATP generation in the glycolytic pathway of epithelioid mesotheliomas. In addition, PKM2 and HK1, which encode enzymes that control two of the three irreversible reactions in the glycolytic pathway, play crucial roles (36) after glucose intake into the cytoplasm via GLUT1, encoded by SLC2A1 (37). However, irreversible reactions, including those controlled by PDP1, which weakly correlated with CD276 mRNA expression, lead to the tricarboxylic acid (TCA) cycle for ATP generation for metabolic reprogramming (38). A recent study has demonstrated that cancer cells rely on the TCA cycle and the glycolytic pathway for ATP generation (38). Therefore, our results suggest that CD276 mRNA expression may affect the TCA cycle and glycolytic pathway. Increased levels of PKM1, an enzyme that converts phosphoenolpyruvate to pyruvate, similar to PKM2, lead to upregulation of the glycolytic pathway and induction of the TCA cycle (39). However, the dataset used in this study did not include data on PKM1 mRNA expression, warranting studies on the correlation between CD276 and PKM1 in epithelioid mesotheliomas.

Implications and actions needed

Although evidence suggests that CD276 is associated with ferroptosis (24); angiogenesis (25); and the JAK2/STAT3, PIK3CA/AKT/mTOR, and RAF/MEK pathways (26-28) in various cancer, our results illustrated that CD276 expression was not well associated with these pathways in epithelioid mesothelioma. Based on the results of a previous in vivo study (33) and our study, CD276 is a target for ICIs in epithelioid mesothelioma and contributes to achieving better OS in patients with epithelioid mesothelioma.

Conclusions

Our study indicated that CD276 mRNA expression is associated with poor prognosis in various cancers, especially epithelioid mesothelioma. Moreover, CD276 mRNA expression was identified to be an independent prognostic biomarker in epithelioid mesothelioma. Our results showed that CD276 mRNA expression correlated with gene markers of the glycolytic pathway, especially PKM2. Thus, CD276 could be associated with the glycolytic pathway and contribute to ATP generation in epithelioid mesotheliomas. Based on these findings, CD276 is a target for ICIs in future therapeutic approaches; CD276 inhibitors could improve prognosis in patients with epithelioid mesothelioma. An ICIs against CD276 should be confirmed for obtaining a better prognosis in patients with epithelioid mesothelioma in the near future.

Acknowledgments

We thank Fumio Nakamura (Department of Biochemistry, Tokyo Women’s Medical University, Shinjuku, Tokyo, Japan) for his assistance and the patients who participated in this study.

Funding: This work was supported by JSPS KAKENHI (grant number 22K16203 to K.A.).

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1598/rc

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1598/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1598/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Ethical approval for the study was not required as we used data from an anonymized public open database and individual consent for this retrospective analysis was waived by the Institutional Review Board of Tokyo Women’s Medical University.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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