S6Ks isoforms contribute to viability, migration, docetaxel resistance and tumor formation of prostate cancer cells

Abstract

Background: The S6 Kinase (S6K) proteins are some of the main downstream effectors of the mammalian Target Of Rapamycin (mTOR) and act as key regulators of protein synthesis and cell growth. S6K is overexpressed in a variety of human tumors and is correlated to poor prognosis in prostate cancer. Due to the current urgency to identify factors involved in prostate cancer progression, we aimed to reveal the cellular functions of three S6K isoforms-p70-S6K1, p85-S6K1 and p54-S6K2-in prostate cancer, as well as their potential as therapeutic targets.

Methods: In this study we performed S6K knockdown and overexpression and investigated its role in prostate cancer cell proliferation, colony formation, viability, migration and resistance to docetaxel treatment. In addition, we measured tumor growth in Nude mice injected with PC3 cells overexpressing S6K isoforms and tested the efficacy of a new available S6K1 inhibitor in vitro.

Results: S6Ks overexpression enhanced PC3-luc cell line viability, migration, resistance to docetaxel and tumor formation in Nude mice. Only S6K2 knockdown rendered prostate cancer cells more sensitive to docetaxel. S6K1 inhibitor PF-4708671 was particularly effective for reducing migration and proliferation of PC3 cell line.

Conclusions: These findings demonstrate that S6Ks play an important role in prostate cancer progression, enhancing cell viability, migration and chemotherapy resistance, and place both S6K1 and S6K2 as a potential targets in advanced prostate cancer. We also provide evidence that S6K1 inhibitor PF-4708671 may be considered as a potential drug for prostate cancer treatment.

Keywords: Cancer; S6K; mTOR.

Fig. 1

S6Ks increase cell viability in PC3-luc cells. Western blotting analyses of S6K isoforms expression modulation in human prostate cancer cell line PC3-luc transduced with a lentiviruses pLKO.1-shRNAs against S6K1 and S6K2 and b with retroviruses carrying pBABE construction. S6K1 antibody detects both p85-S6K1 and p70-S6K1 proteins. c Relative cell viability in PC3-luc cells with knockdown of S6Ks isoforms and d in PC3-luc cells overexpressing S6Ks isoforms. Cells were seeded in 96-well plates at a density of 10⁴ cells/well. After 24, 48 and 72 h, cells were treated with MTT (12 mM) for 4 h and absorbance was measured at 570 nm. *p < 0.05, **p < 0.01, ***p < 0,001, n = 3

Fig. 2

S6Ks increase migration of PC3-luc cells. Cells were seeded in 24-well plates at a density of 5x104 cells/well and incubated until confluence. A scratch was made in the cells monolayer with a pipette tip and cells were washed and incubated in serum free media. The scratch area was measured at 0 (time of scratch), 24 and 48 h. a Migration assay in PC3-luc cells with knockdown of S6Ks isoforms. a, b Scratch relative area of PC3-luc cells overexpressing S6Ks isoforms. c, d Representative images of scratch area taken at 0, 24 and 48 h of PC3-luc cells with S6K knockdown and overexpression, respectively. *p < 0.05, **p < 0.01, ***p < 0,001, n = 3

Fig. 3

p54-S6K2 knockdown increases cell death in response to docetaxel in PC3-luc cells. Cells were seeded in 24-well plates at a density of 8x104 cells/well. After 24 h, cells were subjected to serum free media conditions for 24 h and then treated for 48 h with docetaxel (30 nM). Living cells were counted and expressed as percentages. Results presented are means of two independent experiments. Each experiment was performed in triplicates. SMF: serum free media; DTX: docetaxel. *p < 0.05, **p < 0.01, ***p < 0,001, n = 2

Fig. 4

S6K isoforms increase tumor growth in Nude mice. a Tumor growth curves of Nude mice injected subcutaneously with PC3-luc cells overexpressing S6K isoforms (p70-S6K1, p85-S6K1 and p54-S6K2) or GFP control. Tumors measurements began 14 days after injection and proceeded until day 44. b Tumor growth curves of Nude mice injected subcutaneously with PC3-luc cells with knockdown for S6K1 and S6K2 isoforms or shRNA control. Tumors measurements began 11 days after injection and proceeded until day 39. * p < 0,05, ** p < 0,01 e *** p < 0,001; n = 7

Fig. 5

S6K1 inhibitor PF47086701 inhibits cell proliferation and migration of prostate cancer cells. a Western blotting analysis of PF47086701 efficiency. b Proliferation assay in DU145 and PC3 cell lines treated with PF47086701. c Scratch relative area of DU145 and PC3 cells treated with PF47086701. *p < 0.05, **p < 0.01, ***p < 0,001, n = 3