Shikonin as a therapeutic agent in renal cell carcinoma: insights from TEK-related causal association with glaucoma

Abstract

Introduction: Renal cell carcinoma (RCC) is a lethal malignancy with rising incidence, while glaucoma, a chronic eye disease, shares systemic mechanisms such as oxidative stress and inflammation with cancers. This study aimed to investigate the causal link between glaucoma and RCC and explore molecular intersections to identify novel therapeutic targets.

Methods: A two-step Mendelian randomization (MR) analysis using genetic data from the NHGRI-EBI GWAS Catalog and FinnGen database was performed, supplemented by NHANES data. Gene expression analysis (GSE53757, E-MTAB-1980) identified glaucoma-related genes in RCC. Molecular docking and functional assays evaluated shikonin's effects on TEK and AKT/mTOR signaling.

Results: MR revealed a significant causal relationship between glaucoma and RCC. TEK, a glaucoma-related gene, was downregulated in RCC tissues and correlated with advanced tumor stage and metastasis. Shikonin and acetylshikonin upregulated TEK expression, inhibited RCC cell proliferation/migration, and suppressed AKT/mTOR phosphorylation.

Discussion: These findings support a role for glaucoma-associated genes in RCC development and progression, highlighting shikonin as a promising therapeutic agent targeting this molecular axis.

Keywords: Mendelian randomization (MR); NHANES; TEK; gene expression; glaucoma; renal cell carcinoma (RCC); shikonin.

FIGURE 1

The progression of the MR study and its three principal assumptions.

FIGURE 2

Ocular and glaucoma-RCC association via MR analysis. (A) Forest plot depicting the results of five MR methods for evaluating the causal associations between multiple ocular diseases. Glaucoma was significantly associated with an increased risk of RCC compared with other eye diseases. (B) Forest plot illustrating the results of five MR methods for assessing the causal relationship between glaucoma and RCC across multiple independent datasets. This validates the robustness of the glaucoma - RCC association by leveraging replication in diverse genetic backgrounds.

FIGURE 3

The forest plot of subgroup analysis visualized the robust association between glaucoma and UC across each subgroup (males, aged >65, races except Non - Hispanic Black, education ≥9th–11th grade, and subgroups with/without hypertension, overweight, or smoking; p < 0.05). Race exhibited an interaction effect (p < 0.05), that demographic factors modulate the impact of glaucoma on RCC risk.

FIGURE 4

Glaucoma-related gene expression in RCC of GSE53757. (A) Heat map showed expression patterns of glaucoma-related genes. Rows = genes, columns = samples; color gradients from red to blue reflected expression levels from high to low, and there was a clear clustering between RCC and normal tissues. (B) Volcano plot showed the statistical significance and magnitude of expression changes of glaucoma-related genes in RCC and normal kidney tissues. Genes above the horizontal dashed line had a P value<0.05, indicating significant differential expression. Green dots indicated downregulated genes in RCC, and red dots indicated upregulated genes. (C) Forest plot showed that LRP2, TEK and FOXC1 were downregulated, while COL1A2, TBK1, COL1A1 and MYOC were upregulated in RCC tissues.

FIGURE 5

Prognostic impact of glaucoma-related genes on RCC patients in E-MTAB-1980. (A) Consensus matrix from NMF clustering, illustrating the stability of assigning RCC patients in E-MTAB-1980. To high-risk and low-risk groups. (B) The survival curve analysis showed high-risk patients had significantly shorter overall survival compared to low-risk patients (p < 0.001). The number of patients alive and under observation at each time pointis shown, reflecting the differential impact of the disease on survival in the two groups. (C) Violin plots showed that LRP2, TEK and OPTN were significantly downregulated in high-risk groups, while TBK1 and MYOC were upregulated. (D) Cox regression analysis in E-MTAB-1980 showed that TEK and LRP2 were protective factors against RCC progression, while MYOC and TBK1 were risk factors.

FIGURE 6

TEK as a tumor suppressor in RCC. (A) In the E-MTAB-1980. Dataset, lower TEK expression correlated with advanced tumor stages (T3-T4), lymph node metastasis, higher histologic grades (G3-G4), distant metastasis, late clinical stages (III-IV), and sarcomatoid components. (B) TEK mRNA/protein levels were lower in RCC cell lines (786-O, 769-P) than in normal HK2 cells. (C) RT-qPCR and Western blot confirmed successful TEK overexpression in 786-O and 769-P cells. (D) MTT assays showed that cell proliferation decreased after TEK overexpressed. (E) Clone formation assays showed that TEK overexpressed cell proliferation decreased. (F) Scratch assays showed that cell metastasis ability decreased after TEK overexpression. (G) Transwell assays showed that TEK overexpressed cell metastasis and invasion ability decreased.

FIGURE 7

Shikonin and Acetylshikonin inhibit RCC via TEK activation. (A) HIT-index database predicted potential TEK-binding plant compounds, including pyridine and shikonin. (B) Pyridine (C0344) and shikonin, which has two configurations (C1106 and C0401), were identified, and the 2D/3D structures were retrieved from PubChem. (C) Molecular docking showed that pyridine had a low, while shikonin exhibited high binding affinities to TEK. (D) Shikonin and acetylshikonin are primary bioactive components of Lithospermum erythrorhizon. (E) Molecular docking also revealed that acetyshikonin had a high binding affinity to TEK. (F) Treatment of RCC cells with shikonin and acetyshikonin upregulated TEK expression. (G) MTT assay demonstrated significant inhibition of cell proliferation after treatment with shikonin and acetyshikonin. (H) Transwell migration assay revealed 1.0 μM shikonin and 2.5 μM acetyshikonin reduced cell migration.

FIGURE 8

Shikonin regulates TEK and AKT/mTOR pathway in RCC. (A) In 786-O and 769-Pcells, shikonin upregulated TEK, with no significant change in total AKT levels, but decreased phosphorylated AKT and mTOR, suggesting shikonin suppresses the AKT/mTOR cascade via TEK. GAPDH ensured equal protein loading. (B) Schematic diagram shows that shikonin acts on TEK, which then inhibits AKT/mTOR signaling pathway, which is key to RCC growth.