Targeting an IL-6/JAK/STAT/PIM1 pathway in renal cell carcinoma.

Abstract

376 Background: Renal cell carcinoma (RCC) is the most common cancer of the kidney and accounts for over 430,000 new cases worldwide and 179,000 deaths each year. PIM1 overexpression is associated with poor clinical outcomes in patients with RCC. PIM1 is a constitutively active effector serine/threonine kinase with roles in tumor progression including cell proliferation, apoptosis, invasion, and migration. Yet, the mechanisms that underlie PIM1 expression and its function in RCC are not fully understood. Previous studies have implicated the cytokine IL-6 as an activator of STAT3 and stimulator of PIM1 expression in pancreatic cancer. The aim of the present study is to explore expression of PIM1 in RCC and identify potential signaling pathways that influence PIM1 expression. Methods: To evaluate the contribution of PIM1 expression in RCC we first established baseline PIM1 levels across four RCC cell lines (786-O, 769-P, CAKI-1, and RCC-4). Next, we quantified the presence of IL-6 in cell culture supernatants for each cell line using an ELISA assay. IL-6 signals through the JAK/STAT pathway, consequently we then used immunoblotting to determine whether ruxolitinib, a JAK1/2 inhibitor, would affect PIM1 expression. Finally, we examined cell viability post-ruxolitinib therapy using an MTS assay. Results: Our results show that 769-P, 786-O, and CAKI-1 but not RCC-4 cells overexpress PIM1 relative to normal renal proximal tubule epithelial cells. Results from the ELISA assay show that 786-O, CAKI-1, and to a lesser degree RCC-4 cells secrete IL-6 into their media. Interestingly, 769-P cells had little to no IL-6 present in their media. Based on these differences we sought to understand what effect inhibiting JAK may have on PIM1 expression. After treating cells with ruxolitinib, we found that 786-O and CAKI-1 cells show a time and dose dependent decrease in PIM1 levels whereas 769-P cells did not. To understand if these differences correlate with cell viability, we performed an MTS assay after treating cells with ruxolitinib. Preliminary results show that at 8hr post-treatment, CAKI-1 but not 769-P cells display a time and concentration dependent loss of cell viability. Conclusions: Our initial studies suggest that differential expression of PIM1 among RCC cell lines may be linked to autocrine IL-6 secretion. In CAKI-1 cells JAK inhibition decreases PIM1 expression and cell viability. In the context of 769-P cells with high PIM1 expression but low IL-6 secretion levels, JAK inhibition did not affect cell viability. These data suggest that in 769-P cells there exists a non-IL-6 dependent mechanism stimulating PIM1 expression. Further investigation is required to elucidate the contexts of these PIM1 regulatory pathways in order to develop IL-6/JAK/PIM1 targeted anti-cancer strategies in RCC.