Systematic Multi-Omics Analysis and In Vitro Experiments Suggest ITGA5 as a Potential Target for Clear Cell Renal Cell Carcinoma (ccRCC) Treatment

Research Background and Motivation

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma (RCC), accounting for about 75% of all kidney cancer cases. According to the latest cancer statistics from the United States and China, the incidence of RCC is steadily rising, and ccRCC, due to its significant immune and vascular infiltration characteristics, leads to poor prognosis for patients. Although tyrosine kinase inhibitors (TKIs) and immune checkpoint blockade (ICB) show good efficacy in the treatment of ccRCC, the lack of gene markers significantly associated with ICB efficacy in ccRCC makes it difficult to further enhance precision therapy.

Integrins play key roles in the progression of various cancers. As major receptors for extracellular matrix (ECM) and intercellular signaling, integrins are crucial in cell adhesion, proliferation, migration, and apoptosis. Especially, ITGA5 (integrin α5) forms a heterodimer with integrin β1, binds to fibronectin, activates the FAK-PI3K-AKT signaling pathway, and promotes bone metastasis, drug resistance, and angiogenesis in tumor cells. Therefore, this study aims to comprehensively explore the potential of ITGA5 as a therapeutic target in ccRCC through multi-omics analysis and in vitro experiments.

Research Methods

Data Acquisition and Processing

Study subjects included tumor and adjacent normal tissues from 232 ccRCC patients from Fudan University Shanghai Cancer Center (FUSCC) and 535 ccRCC patients from The Cancer Genome Atlas (TCGA). After data normalization, differential expression genes were screened using the Limma package (log2 fold change >1 and p-value <0.05). Additionally, somatic mutation data from TCGA-KIRC patients were obtained to assess the relationship between ITGA5 expression levels and mutation frequencies. Single-cell RNA sequencing data based on multi-omics platforms were also used to evaluate the distribution of ITGA5 among different cell types in the tumor microenvironment (TME).

In Vitro Experiments

In vitro experiments involved the selection of two ccRCC cell lines, 786-O and 769-P, for ITGA5 knockdown and overexpression, combined with cell proliferation (CCK-8), migration (scratch healing), and invasion (Transwell) assays. Transfection with small interfering RNA (siRNA) and ITGA5 overexpression plasmids was performed to explore the impact of ITGA5 on tumor cell behavior.

Bioinformatics Analysis and Machine Learning Model Construction

Multiple databases and analytical methods were utilized to predict potential functions of ITGA5, including Gene Ontology (GO), Gene Set Enrichment Analysis (GSEA), and KEGG pathway analysis. Furthermore, Lasso regression was used to screen core genes related to ITGA5 high and low expression groups, and an extreme gradient boosting (XGBoost) machine learning model was constructed. The ROC curve was applied to validate its predictive accuracy to assess the relationship between ITGA5 expression and patient prognosis.

Main Results

Expression and Prognostic Significance of ITGA5 in ccRCC

Analysis of FUSCC and TCGA data revealed that ITGA5 expression is significantly upregulated in ccRCC, particularly in VHL-mutated ccRCC samples. Kaplan-Meier survival curve analysis showed that patients in the high ITGA5 expression group had significantly lower overall and disease-free survival rates than those in the low expression group, suggesting that ITGA5 may be a negative prognostic marker. The ROC curve of the XGBoost model indicated the model’s robust predictive capability with AUCs of 0.895, 0.898, and 0.911 for 1-year, 3-year, and 5-year survival predictions, respectively.

ITGA5 Promotes Malignant Behaviors of ccRCC Cells

In vitro experiments showed that ITGA5 knockdown significantly inhibited the proliferation, migration, and invasion capabilities of 786-O cells; conversely, ITGA5 overexpression in 769-P cells enhanced these malignant phenotypes. Western blot experiments further confirmed that ITGA5 knockdown or overexpression affects the phosphorylation levels of the PI3K-AKT signaling pathway, indicating that ITGA5 may promote malignant progression of ccRCC cells through this pathway.

Regulatory Effects of ITGA5 on the Tumor Microenvironment

Using single-cell RNA sequencing data, ITGA5 was found to be highly expressed mainly in endothelial cells and macrophages in the tumor microenvironment. CIBERSORT algorithm analysis showed that ITGA5 high expression negatively correlated with reduced CD8+ T cell infiltration and positively correlated with memory CD4+ T cells, NK cells, and macrophage infiltration, suggesting that ITGA5 may induce immune evasion through an immunosuppressive TME. Additionally, GSEA analysis showed that ITGA5 high expression is significantly enriched in immune response and angiogenesis pathways, further supporting its role in immune regulation.

Drug Sensitivity Analysis and Combination Therapy Strategies

In drug sensitivity prediction, ITGA5 high expression group showed higher sensitivity to VEGFR-targeted drugs (like Axitinib, Sunitinib, Pazopanib, and Motesanib) and PARP inhibitors (like Olaparib and Rucaparib), indicating that the combined use of ITGA5 inhibitors and anti-VEGFR drugs might provide better therapeutic effects for patients with high ITGA5 expression. Meanwhile, the ITGA5 high expression group exhibited a certain degree of resistance to anti-EGFR drugs. Moreover, TIDE scores indicated that the ITGA5 high expression group poorly responded to ICB therapy, possibly due to ITGA5 expression-induced CD8+ T cell reduction.

Research Significance and Prospects

This study uses systematic multi-omics analysis and in vitro experiments to reveal the potential of ITGA5 as a therapeutic target in ccRCC. High ITGA5 expression is significantly associated with poor prognosis in patients and plays crucial roles in promoting tumor cell proliferation, migration, invasion, and immune evasion through multiple signaling pathways. Furthermore, ITGA5 may serve as a novel biomarker for sensitivity to VEGFR-targeted drugs and PARP inhibitors in ccRCC, providing new insights for precision therapy for ccRCC patients.