Introduction: The combined therapies with checkpoint inhibitor and kinase inhibitor (TKI) have recently been approved as preferred treatment options for advanced RCC patients. However, TKIs could still be the preferred treatment in RCC patients who cannot receive checkpoint inhibitors. TKIs include pazopanib, anitinib and sunitinib, and they function mainly through inhibition of tumor angiogenesis. Eventually, most patients become insensitive or develop resistance toward TKI treatment and the underlying mechanisms have not yet been fully elucidated. Our goal is to reveal the molecular pathway of TKI resistant to find a potential method to treat TKI resistant renal cancer.
Methods: The parental and TKI-resistant (Paz-R or Suni-R) ccRCC cell lines, 786-O, A498, OSRC-2, were used. Lentiviral plasmids with cDNAs of the long non-coding RNA (lncRNA)-ECVSR, and Hif2-a were transfected into 293T cells to produce gene engineering tools. qPCR was used to evaluate the mRNA levels. Western blot was performed to assess the protein expressions. Vasculogenic mimicry assays (VM) were performed to detect cancer cell malignancy. Protein-DNA complexes were detected by Chromatin immunoprecipitation (ChIP) assays. Preclinical mouse RCC model was used to validate the efficacy of pazopanib administration together with PHTPP in TKI resistant RCC.
Results: Pazo-R or Suni-R renal cancer cells showed increased VM ability to counteract TKI angiogenesis suppression function both in vivo and in vitro. Mechanism dissection revealed that Pazo-R (or Suni-R) cells have an increased expression of lncRNA-ECVSR, thereby enhancing the stability of estrogen receptor-ß (ERß) mRNA. Subsequently, the increased ERß expression could then function via transcriptional up-regulation of Hif2-a. Notably, TKI resistance-increased lncRNA-ECVSR/ERß/Hif2-a signaling resulted in an increased cancer stem cell (CSC) phenotype, thereby promoting VM formation. Furthermore, the TKI resistance/lncRNA-ECVSR-increased ERß expression transcriptionally regulated lncRNA-ECVSR expression via a positive-feedback loop. Supportively, preclinical studies using mouse RCC xenografts demonstrated that combining pazopanib(or sunitinib) with the small molecule anti-estrogen PHTPP increased TKI efficacy with reduced VM formation. Collectively, the findings of this study may aid in the development of a potential biomarker and new therapy to better monitor and cure TKI resistant RCC.
Conclusions: Our study is the first to reveal that the resistance to TKI (pazopanib or sunitinib) occurs by increasing vasculogenic mimicry (VM) ability to counteract TKI angiogenesis suppression function. Furthermore, we find a potential therapy to cure TKI resistant renal cancer by blocking lncRNA-ECVSR/ERß/Hif2-apathway together with pazopanib(or sunitinib) administration.
Source of Funding: This work was partially supported by URMC Urology Department Research fund and George Whipple Professorship Endowment.
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