Renal Cell Carcinoma (RCC) is the most common form of kidney cancer, with clear cell RCC (ccRCC) representing about 85% of all RCC tumors. There are limited curable treatments available for metastatic ccRCC because this disease is unresponsive to conventional targeted systemic pharmacotherapy. Exosomes (Exo) are small extracellular vesicles (EVs) secreted from cancer cells with marked roles in tumoral signaling and pharmacological resistance. Ketoconazole (KTZ) is an FDA approved anti-fungal medication which has been shown to suppress exosome biogenesis and secretion, yet its role in ccRCC has not been identified. A time-course, dose-dependent analysis revealed that KTZ selectively decreased secreted Exo in tumoral cell lines. Augmented Exo secretion was further evident by decreased expression of Exo biogenesis (Alix and nSMase) and secretion (Rab27a) markers.
FIG: Measurement of exosome secretion by qNano analysis. (A) Total number of EVs in the plasma of patients with Kidney cancer and matched controls were measured by a qNano analysis (n = 59). (B) Expression of exosome CD63, and exosome biogenesis markers Alix in the exosome of 293-T, RCC-24, 786-O, and Caki-2 cells. Exosomes were isolated by UC and filtration (0.45 μm). Full blots are presented in Supplementary Fig. (C) qNano-IZON particle quantitative analysis (NP-100 nanopore) depicting a significant decrease in exosome concentrations (50–200 nm size) in the CM of RCC-24, 786-O, and Caki-2 cells treated with KTZ compared to vehicle treated controls, but not normal kidney HEK 293-T cells. *Denotes significance at p < 0.05 compared to controls and was calculated using GraphPad Prism. (D) Inhibition of exosome secretion using the MACSQuant Analyzer 10 Flow Cytometer and qNano-IZON: The exosome in the conditioned media were isolated and analyzed by the MACSQuant Analyzer 10 Flow Cytometer as described in the “Materials and methods” section. Treatments were at concentrations of 1 μM ketoconazole compounds. Exosome from DMSO treated 786-O-CD63-GFP is used as GFP control and PBS served as a reference control. *Denotes significance at p < 0.05 compared to controls and was calculated using GraphPad Prism. Data for EVs and exosomes was captured using IZON’s Control Suite software version 184.108.40.206.
Interestingly, KTZ-mediated inhibition of Exo biogenesis was coupled with inhibition of ERK1/2 activation. Next, selective inhibitors were employed and showed ERK signaling had a direct role in mediating KTZ’s inhibition of exosomes. In sunitinib resistant 786-O cells lines, the addition of KTZ potentiates the efficacy of sunitinib by causing Exo inhibition, decreased tumor proliferation, and diminished clonogenic ability of RCC cells. Our findings suggest that KTZ should be explored as an adjunct to current RCC therapies.
Greenberg, J.W., Kim, H., Moustafa, A.A. et al. Repurposing ketoconazole as an exosome directed adjunct to sunitinib in treating renal cell carcinoma. Sci Rep 11, 10200 (2021). https://doi.org/10.1038/s41598-021-89655-w