Abstract
ABSTRACTLoss of the gene von Hippel–Lindau (VHL) is associated with loss of primary cilia and is causally linked to elevated levels of Aurora kinase A (AURKA). We developed an image-based high-throughput screening (HTS) assay using a dual-labeling image analysis strategy that identifies both the cilium and the basal body. By using this strategy, we screened small-molecule compounds for the targeted rescue of cilia defects associated with VHL deficiency with high accuracy and reproducibility. Bexarotene was identified and validated as a positive regulator of the primary cilium. Importantly, the inability of an alternative retinoid X receptor (RXR) agonist to rescue ciliogenesis, in contrast to bexarotene, suggested that multiple bexarotene-driven mechanisms were responsible for the rescue. We found that bexarotene decreased AURKA expression in VHL-deficient cells, thereby restoring the ability of these cells to ciliate in the absence of VHL. Finally, bexarotene treatment reduced the propensity of subcutaneous lesions to develop into tumors in a mouse xenograft model of renal cell carcinoma (RCC), with a concomitant decrease in activated AURKA, highlighting the potential of bexarotene treatment as an intervention strategy in the clinic to manage renal cystogenesis associated with VHL deficiency and elevated AURKA expression.
Highlights
Loss or mutations in the von Hippel–Lindau (VHL) gene are most commonly associated with VHL disease and clear cell renal cell carcinoma (Kaelin, 2007)
We developed a high-throughput screening (HTS) assay to identify small molecules that could restore primary cilia in VHL-deficient cells, with the dual goal of identifying novel therapeutic targets and signaling pathways involved in aberrant ciliogenesis associated with loss of VHL
The assay was re-developed to be amenable to a 384-well plate format and was performed as detailed in the schematic shown in Fig. 1A. hTERT-RPE1 cells were transfected with siC or showed that this acute loss of VHL (siVHL), 24 h after seeding (7000 cells/ well), and were induced to ciliate by the simultaneous withdrawal of serum and treatment with either vehicle (DMSO) or compound at a dose of 10 μM for 48 h
Summary
Loss or mutations in the von Hippel–Lindau (VHL) gene are most commonly associated with VHL disease and clear cell renal cell carcinoma (ccRCC) (Kaelin, 2007). Recent advances in understanding the role of VHL in multiple molecular pathways, have resulted in the development of targeted therapies including tyrosine kinase inhibitors (TKIs), monoclonal antibodies, mammalian target of rapamycin (mTOR) inhibitors, and immune checkpoint therapy for the treatment of ccRCC (Hsieh et al, 2017). Successful, these strategies have plateaued since their. We recently identified AURKA as a direct target of the E3 ligase activity of VHL (Hasanov et al, 2017), and linked elevated AURKA to the loss of primary cilia associated with VHL deficiency (Dere et al, 2015; Hasanov et al, 2017)
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