(M1530-10-65) Transforming Growth Factor-Beta (TGF-β) as a Potential Therapeutic Target of Methylseleninic Acid and Seleno-l-Methionine in Clear Cell Renal Cell Carcinoma

Purpose: Clear cell renal cell carcinoma (ccRCC) tumors are highly immunogenic and resistant to conventional chemotherapies. Antiangiogenic tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors have improved the way that ccRCC is treated, however, resistance to these agents eventually develops, underlining the need to identify additional ‘druggable’ targets. In the case of ccRCC, transforming growth factor-beta (TGF-β) is a key driving force in epithelial-to-mesenchymal transition of non-sarcomatoid to a dedifferentiated sarcomatoid ccRCC morphology, which results in a more aggressive phenotype. In addition to TGF-β, programmed death-ligand 1 (PD-L1) and vascular endothelial growth factor (VEGF) are also implicated in the pathogenesis of ccRCC. TGF-β, PD-L1, and VEGF are overexpressed in ccRCC cell lines with and without sarcomatoid differentiation. These biomarkers are associated with immune response evasion, drug resistance, an unstable tumor microenvironment, and increased tumor angiogenesis. Here, methylseleninic acid (MSA) was used in vitro while seleno-l-methionine (SLM) was utilized for in vivo experiments because SLM needs the action of the β-lyase enzyme to be converted to MSA. The absence of β-lyase in various cancer cell lines makes SLM inactive in vitro. The levels of expression of TGF-β, PD-L1, and VEGF in ccRCC cell lines, and in patient tumor biopsies, with and without sarcomatoid differentiation were investigated. Effects of nontoxic concentrations of MSA on cell line expression of these markers was assessed. Also, direct interaction between MSA and TGF-β was demonstrated. In vivo toxicity studies were performed with SLM as a prelude to future therapeutic studies.

Methods: The expression levels, determined using western blotting, of TGF-β, PD-L1, and VEGF were quantified in ten ccRCC cell lines, 5 with and 5 without sarcomatoid differentiation as well as in forty-seven primary ccRCC tumors, 43 with and 4 without sarcomatoid differentiation and compared to adjacent normal kidney tissues. The in vitro effect of MSA on the expression of the three biomarkers was evaluated. The interaction between MSA and TGF-β was tested by mixing recombinant TGF-β with MSA or with other proteins to check the specificity of the interaction. Then, a cellular thermal shift assay was performed to confirm the interaction. The effect of MSA treatment was then assessed in terms of its dose- and time-dependent effect on TGF-β expression. A toxicity experiment using various doses of SLM was also carried out in vivo using nude NCI mice to determine the maximum tolerated dose.

Results: Although TGF-β is expressed by all 10 cell lines, the expression level was significantly higher in cells with sarcomatoid differentiation than in cells without sarcomatoid differentiation, which is consistent with previous research. Our laboratory is the first to identify MSA as a TGF-β inhibitor. The inhibition of TGF-β was MSA dose- and time-dependent, was achieved at nontoxic concentrations and was associated with parallel time- and dose-dependent downregulation of PD-L1 and VEGF expression. Selenium concentration was determined in the filtrate of MSA alone or mixed with recombinant TGF-β or fetal bovine serum (FBS) to check the interaction between TGF-β and MSA. The findings revealed that selenium concentration was significantly higher in the MSA alone compared to MSA with recombinant TGF-β, but it was comparable to MSA and FBS, indicating that there is a specific interaction between MSA and TGF-β. This was confirmed using metformin, an inhibitor of TGF-β, and analyses of the thermodynamic stability of TGF-β with and without MSA. Regarding in vivo experiments, a toxicity study revealed that 10 mg/kg SLM was considered to be safe without causing any change in the mice’s body weight or lethality. This SLM dose will be used later in subsequent animal experiments alone or in combination with axitinib - a tyrosine kinase inhibitor- that is commonly used in the treatment of ccRCC.

Conclusion: Overexpressed TGF-β in ccRCC is a direct target of nontoxic doses of MSA/SLM. Here it was shown that MSA is an effective inhibitor of TGF-β without being cytotoxic and its inhibition is associated with the downregulation of PD-L1 and VEGF. The documented pleiotropic effects of selenium offer the potential for the development of a mechanism-based treatment modality in combination with various cytotoxic agents. In the future, the relationship between TGF-β downregulation and associated PD-L1 and VEGF downregulation will be studied by generating cell lines knocked out for TGF-β and the efficacy of SLM on tumor growth as well as TGF-β, PD-L1, and VEGF expression in animal tumor tissues will be investigated.