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The Role of Integrins and Exosomes in Sunitinib Resistance in ccRCC Cell Lines
Jared P. Schober, MD, Marc Calabrese, MD, Daniel Kaufman, MD, Kevin Yang, MD, Kristian D. Stensland, MD, Juliana Kostas, None, Travis Sullivan, MS, Kimberly Rieger-Christ, Ph.D..
Lahey Hospital and Medical Center, Burlington, MA, USA.
Tyrosine kinase inhibitors (TKIs) are used as first line therapy for stage IV or unresectable clear cell renal cell carcinoma (ccRCC). While the efficacy of TKIs is attributed to downregulation of vascular endothelial growth factor and a decrease in tumor angiogenesis, tumor response is often mitigated by acquired resistance. Integrins, cell surface proteins involved in regulating cell adhesion, have been shown to promote TKI resistance in several tumor types. Exosomes are small extracellular vesicles released by cells and have been shown to horizontally transfer bioactive molecules to recipient cells thereby promoting chemoresistance. In this study we have investigated the role of integrins and exosomes in ccRCC resistance to sunitinib.
The ccRCC cell lines Caki-1 and Caki-2 were continuously exposed to increasing concentrations of sunitinib for 6-8 months until acquiring resistance. These cells were termed Caki-1/SR and Caki-2/SR. Cell viability was assessed using MTT assays. Exosomes were harvested and characterized by nanoparticle tracking analysis. Changes in the integrin expression profiles and various signaling pathways were determined using Western blot analysis of cell and exosome lysates. Alterations in adhesion were established using standard in vitro assays. Integrin inside-out signaling pathway array plates were used to compare mRNA expression levels.
Integrin expression levels were found to be variable among parental and resistant ccRCC cell lines. Gene expression analysis revealed reduced levels of COL1A1 and COL1A2, components of type I collagen, in Caki-1/SR cells. Western blot analysis demonstrated a significant decrease in beta-3 integrin protein levels in Caki-1/SR compared to Caki-1 cells. An assessment of adhesion determined Caki-1/SR cells exhibited decreased adhesion to several extracellular matrices, including collagen I, laminin, and fibronectin. On the other hand, Caki-2/SR cells demonstrated an increase in beta-1 and beta-3 integrins compared to Caki-2 cells. Altered levels of beta-1 and beta-3 integrins were observed in exosomes released by both Caki-1/SR and Caki-2/SR cells. Initial protein studies suggest downstream signaling pathways affected by the change in levels of integrin expression include FAK and NF-kB.
Our findings suggest sunitinib resistance in Caki-1 and Caki-2 ccRCC cells is associated with integrin expression. Beta-1 and Beta-3 integrin levels correlated with resistance in Caki-2 cells. Exosomal integrin levels were altered in sunitinib resistant cells. Continued investigation into
the role of integrins in sunitinib resistant cell lines, including the effect of variable integrin profiles on downstream signaling pathways and their potential transference via exosomes, will improve understanding of TKI resistance in ccRCC.
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