Tumor Microenvironment-Based Drug Discovery: A Novel Insight into Bladder Cancer Immunotherapy

Bladder cancer (BCa) is one of the most common urological malignant tumors, with a worldwide incidence of 573,278 cases in 2020, ranking eleventh among all tumors. Despite the availability of approved immune checkpoint inhibitors for BCa, the response rates to these drugs remain limited, ranging from 20% to 40%. This underscores the critical need for discovering new and effective immunotherapy drugs to treat BCa. The tumor microenvironment (TME) of malignant solid tumors, which includes not only tumor cells but also normal cells such as stromal and immune cells, plays a significant role in tumor growth and progression. This suggests the feasibility of designing drugs that target these normal cells. This article explores a novel approach to identifying potential drugs for BCa immunotherapy based on an algorithm predicting the infiltration level of immune and stromal cells in the TME.

The Role of the Tumor Microenvironment in Bladder Cancer

The TME is a complex ecosystem that includes tumor cells, stromal cells, immune cells, and extracellular matrix components. These elements interact dynamically to influence tumor progression, metastasis, and response to therapy. In BCa, the TME is particularly significant because it modulates the immune response and contributes to the limited efficacy of current immunotherapies. Understanding the composition and function of the TME is essential for developing targeted therapies that can enhance the immune response against BCa.

Methodology: Analyzing the Tumor Microenvironment

To investigate the TME in BCa, level 3 gene expression data from 393 patients were obtained from The Cancer Genome Atlas (TCGA) program. Clinical variables such as gender, age, race, histology classification, tumor stage, and survival outcomes were included in the analysis. Patients with a survival time of less than one month were excluded. Immune and stromal scores were calculated to represent the infiltration levels of immune and stromal cells in the TME using a newly established algorithm. The optimal cut-off values for these scores were identified as 1895.774 for the immune score and -858.8528 for the stromal score.

Kaplan-Meier survival analysis revealed that a higher stromal score was associated with worse overall survival, while a higher immune score correlated with improved overall survival. Additionally, the stromal score was significantly associated with tumor stage, whereas the immune score was not. These findings highlight the distinct roles of stromal and immune cells in BCa progression and prognosis.

Differential Expression Genes and Prognostic Analysis

The study identified 136 differential expression genes (DEGs) between groups with high and low stromal scores and 468 DEGs in the immune groups. A Venn diagram was used to identify 562 DEGs after intersection. Kaplan-Meier survival curves further screened 123 prognostic genes out of these 562 DEGs. Among these, the Ephrin B2 gene (EFNB2) was highlighted due to its significant prognostic value.

A protein-protein interaction (PPI) network was constructed based on the 123 prognostic genes to better understand their interactions. The core module of the PPI network consisted of 34 genes, which were found to be correlated with multiple pathways, including hypoxia-induced factor 1 (HIF-1) and tumor necrosis factor signaling pathways. Enrichment analysis revealed the biological significance of these genes in BCa progression.

Validation of Prognostic Genes

The prognostic value of the 34 core genes was validated using an independent dataset (GSE31684) from the Gene Expression Omnibus, which included 78 BCa patients. External validation confirmed EFNB2 as the only prognostic gene with a significant p-value of 0.036. EFNB2 encodes the B class ephrin, which has been previously associated with TP53 mutation and worse survival in BCa.

Regulatory Mechanisms: ncRNAs and Transcription Factors

The pivot function of the core module was explored based on ncRNA-mRNA and transcription factor (TF)-mRNA interactions using the RNA Interactome Database and the Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining database. Five ncRNAs and four TFs were identified as regulators of the core module. The five ncRNAs included metastasis-associated lung adenocarcinoma transcript 1, apoptotic BCL2L1-antisense long non-coding RNA, taurine up-regulated gene 1, FOXF1 adjacent non-coding developmental regulatory RNA, and Angelman syndrome chromosome region. The four TFs were hypoxia-inducible factor 1 subunit alpha (HIF1a), interferon regulatory factor 1, RUNX family transcription factor 3, and signal transducer and activator of transcription 3.

These ncRNAs and TFs are involved in various biological processes and diseases, as detailed in the study. Their regulatory roles in the core module provide insights into the molecular mechanisms underlying BCa progression and potential therapeutic targets.

Drug Discovery Based on the Core Module

Six types of drugs potentially regulative for BCa were identified based on the core module and its correlated ncRNAs and TFs using the DrugBank database. These drugs include FG-2216, ENMD-1198, 2-Methoxyestradiol, PX-478, Carvedilol, and Emricasan. The structures and detailed information about these drugs are provided in the study.

HIF-1a, a classic regulator of tumor angiogenesis, is the target of five of these drugs (Carvedilol, FG-2216, ENMD-1198, PX-478, and 2-Methoxyestradiol). Hypoxia, a critical characteristic of the TME, leads to malignant progression and resistance to anti-cancer therapies. HIF-1a activation under hypoxic conditions orchestrates immune-suppressive effects, such as the recruitment of regulatory T cells and inhibition of CD4+ and CD8+ T cells and antigen-presenting cells. Previous studies have reported that HIF-1a is associated with an unfavorable prognosis in BCa and plays a critical role in gemcitabine resistance. The HIF-1a/multidrug resistance mutation 1 gene pathway has been shown to confer chemoresistance to cisplatin in BCa patients, highlighting HIF-1a as a potential target for BCa immunotherapy.

Emricasan, a caspase inhibitor, is an investigational drug for non-alcoholic steatohepatitis. Caspase-8, the target of Emricasan, regulates immune response, B and T lymphocyte activation, and macrophage differentiation and polarization. These functions are crucial for maintaining immune cell homeostasis and cytokine production, which are key components of the TME. Therefore, Emricasan has the potential to improve the efficacy of BCa immunotherapy.

Discordance Between mRNA and Protein Expression

The drug discovery process in this study is based on transcriptional data, while the targets of the identified drugs are usually proteins. The discordance between mRNA and protein expression is a well-known phenomenon in research. Various mechanisms, including translation rates and protein stability, have been proposed to explain this discordance. Recent studies have suggested that the secretion of proteins in the form of membrane vesicles could be a contributing factor. This understanding is essential for interpreting the findings and developing effective therapeutic strategies.

Conclusion

This study identified six potentially regulative drugs (FG-2216, Emricasan, ENMD-1198, Carvedilol, PX-478, and 2-Methoxyestradiol) and a potential immunotherapy target (EFNB2) for BCa based on the concept of the TME. The findings provide new insights into the immunotherapy of BCa and highlight the importance of targeting the TME for effective cancer treatment. The identification of these drugs and targets offers promising avenues for future research and clinical applications in BCa immunotherapy.

doi.org/10.1097/CM9.0000000000001535

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