Login to Access Video or Poster Abstract: MP73-05
Sources of Funding: none

Introduction

ontinuous activation of hypoxia-inducible factor (HIF) is important for renal cell carcinoma (RCC) progression as well as resistance acquisition against mTOR and VEGFR inhibitors. Recently a HIF2α antagonist (PT2385) has been developed, and is currently being investigated in a phase I clinical trial for advanced or metastatic clear cell RCC (ccRCC) patients who had prior tyrosine kinase inhibitors. However, resistant mechanisms against the HIF2α antagonist seems to be a next problem in the near future. The aim of this study was to find activated signals involved in the HIF2α antagonist-resistance after acquiring sunitinib resistance.

Methods

First, we established sunitinib resistant 786-o (SU-R-786-o) cells in vivo by oral gavage treatment. Instead of the HIF2α antagonist administration, we completely knocked out the HIF2α gene expression in SU-R-786-o cells by using a recent genome editing technology, CRISPR/Cas9 system, with two different single guide RNAs against HIF2α. We identified their characteristics in cell proliferation assay and western blot. These cells were also analyzed in proteomics and RNA sequence analyses to elucidate their activated signals.

Results

By using CRISPR/Cas9 system, we succeeded in establishing HIF2α knockout sunitinib resistant 786-o (HIF2α-KO-SU-R-786-o) cells. Cell morphology was changed from spindle to round shape in HIF2α-KO-SU-R-786-o cells. In addition, cell proliferation in HIF2α-KO-SU-R-786-o cells became significantly slower than in the parental cells as well as SU-R-786-o cells (P < 0.01). After recovering cell proliferation in HIF2α-KO-SU-R-786-o cells, proteomics and RNA sequence revealed that CXCR4 expression was dramatically up-regulated in SU-R-786-o cells compared to the parental cells, however, it was decreased in HIF2α-KO-SU-R-786-o cells compared to SU-R-786-o cells. Another surprise was that serine biosynthesis pathway was accelerated in HIF2α-KO-SU-R-786-o cells compared to SU-R-786-o cells.

Conclusions

Based on the proteomics and RNA sequence analyses, we found that CXCR4 upregulation or serine biosynthesis might be critical for acquiring resistance against multi kinase inhibitors or HIF2α antagonist. Even though both drugs target angiogenesis pathway, the activated signals were altered in the different therapeutic stage by the different drugs administration. The inhibitions of serine biosynthesis pathways might be potential targeted therapy for advanced or metastatic ccRCC which shows resistance to HIF2α agonist after acquiring resistance to multi kinase inhibitors.

Funding

none