Molecular and Functional Analysis of Sunitinib-Resistance Induction in Human Renal Cell Carcinoma Cells

Abstract

Resistance in clear cell renal cell carcinoma (ccRCC) against sunitinib is a multifaceted process encompassing numerous molecular aberrations. This induces clinical complications, reducing the treatment success. Understanding these aberrations helps us to select an adapted treatment strategy that surpasses resistance mechanisms, reverting the treatment insensitivity. In this regard, we investigated the dominant mechanisms of resistance to sunitinib and validated an optimized multidrug combination to overcome this resistance. Human ccRCC cells were exposed to single or chronic treatment with sunitinib to obtain three resistant clones. Upon manifestation of sunitinib resistance, morphometric changes in the cells were observed. At the molecular level, the production of cell membrane and extracellular matrix components, chemotaxis, and cell cycle progression were dysregulated. Molecules enforcing the cell cycle progression, i.e., cyclin A, B1, and E, were upregulated. Mass spectrometry analysis revealed the intra- and extracellular presence of N-desethyl sunitinib, the active metabolite. Lysosomal sequestration of sunitinib was confirmed. After treatment with a synergistic optimized drug combination, the cell metabolic activity in Caki-1-sunitinib-resistant cells and 3D heterotypic co-cultures was reduced by >80%, remaining inactive in non-cancerous cells. These results demonstrate geno- and phenotypic changes in response to sunitinib treatment upon resistance induction. Mimicking resistance in the laboratory served as a platform to study drug responses.

Integrated mRNA and miRNA Transcriptomic Analyses Reveals Divergent Mechanisms of Sunitinib Resistance in Clear Cell Renal Cell Carcinoma (ccRCC)

“…Although the downregulation of members of the miR-17~92 cluster is consistent with the up-regulation of CCND1 observed in Caki-1 and A498-resistant clones [48], the same miRNAs are also down-regulated in 786-O-resistant clones, suggesting a different regulatory mechanism for this cell line, perhaps through the direct targeting by HIF2α [49], which is down-regulated at mRNA and protein levels. Intriguingly, although CCND1 was strongly up-regulated (8-10-fold) in resistant clones of Caki-1 cells, cyclin D1 protein was down-regulated, as has recently been described [35], suggesting post-transcriptional regulation.…”

“…The generated resistant clones had a significantly higher amount of IC 50 values greater than 10 µM (average 15.6 µM). Although several studies have investigated miRNA expression in response to sunitinibtreatment, the vast majority have looked at expression after a single dose [22,24,25], rather than prolonged exposure, which could be argued to more accurately reflect acquired resistance [21,23,35].…”

“…Proteoglycans are major components of the extracellular matrix and play important roles in many facets of cancer, including proliferation, adhesion, angiogenesis, and metastasis [39]. Recently, Rausch et al described the morphometric changes that occur in sunitinib-resistant clones, in which the authors linked the changes of more than 70 genes to cell adhesion, including many proteoglycans [35].…”

Drug Repurposing to Identify a Synergistic High-Order Drug Combination to Treat Sunitinib-Resistant Renal Cell Carcinoma

“…Sample preparation and LC-HRMS/MS analysis was performed following the same procedure as previously described by Rausch et al [50]. The MS data were converted from RAW (Thermo) standard data format to .mzXML format using the MSConvert software, part of the ProteoWizard package [51].…”

“…The three consecutive search rounds led to identifying an optimized cell-type-specific drug combination (Figures S4 and S5) in Caki-1 sunitinib-naïve (SN) and sunitinib-resistant (-SR) cells [50]. This combination consisted of Rapta-C, erlotinib, metformin and parthenolide, and was further abbreviated as ODC REMP (Figure 1B).…”