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Multiregion Quantification of Extracellular Signal-regulated Kinase Activity in Renal Cell Carcinoma.

ABSTRACT: To personalize treatment for renal cell carcinoma (RCC), it would be ideal to confirm the activity of druggable protein pathways within individual tumors. We have developed a high-resolution nanoimmunoassay (NIA) to measure protein activity with high precision in scant specimens (eg, fine needle aspirates [FNAs]). Here, we used NIA to determine whether protein activation varied in different regions of RCC tumors. Since most RCC therapies target angiogenesis by inhibiting the vascular endothelial growth factor (VEGF) receptor, we quantified phosphorylation of extracellular signal-regulated kinase (ERK), a downstream effector of the VEGF signaling pathway. In 90 ex vivo FNA biopsies sampled from multiple regions of 38 primary clear cell RCC tumors, ERK phosphorylation differed among patients. In contrast, within individual patients, we found limited intratumoral heterogeneity of ERK phosphorylation. Our results suggest that measuring ERK in a single FNA may be representative of ERK activity in different regions of the same tumor. As diagnostic and therapeutic protein biomarkers are being sought, NIA measurements of protein signaling may increase the clinical utility of renal mass biopsy and allow for the application of precision oncology for patients with localized and advanced RCC. PATIENT SUMMARY: In this report, we applied a new approach to measure the activity of extracellular signal-regulated kinase (ERK), a key cancer signaling protein, in different areas within kidney cancers. We found that ERK activity varied between patients, but that different regions within individual kidney tumors showed similar ERK activity. This suggests that a single biopsy of renal cell carcinoma may be sufficient to measure protein signaling activity to aid in precision oncology approaches.

SUBMITTER: Hoerner CR

PROVIDER: S-EPMC6697234 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimTo study the expression and phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2 in multidrug resistant (MDR) hepatocellular carcinoma (HCC) cells.MethodsMDR HCC cell lines, HepG2/adriamycin (ADM) and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein (P-gp) and multidrug resistant protein 1 (MRP1) expression levels. ERK1 and ERK2 mRNA expression levels were measured by quantitative real-time PCR (QRT-PCR). Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.ResultsMTT assay showed that HepG2/ADM and SMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells (8.92% +/- 0.22% vs 0.88% +/- 0.05%, P < 0.001) and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells (7.37% +/- 0.26% vs 1.74% +/- 0.25%, P < 0.001). However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.ConclusionERK1 and ERK2 activities are down-regulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells.

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Multiregion Quantification of Extracellular Signal-regulated Kinase Activity in Renal Cell Carcinoma.

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