HSP990 induces downregulation of specific protein of TRAP1 network and inhibits cell proliferation. A-B. Mitochondrial (A) and total cell (B) lysates from HT29 cells were separated by SDS-PAGE and immunoblotted with anti-sorcin and anti-VDAC (A) or anti-eEF1G, anti-eIF4A, anti-eIF4E and anti-GAPDH (B) antibodies. C-D. Inhibition of cell proliferation (C) and induction of apoptosis (D) in HT29 cells treated with increasing concentrations of AUY922 and HSP990 for indicated time points. C. *p<0.0001 versus cells treated with the same concentration of AUY922. D. *p=0.005; **p<0.0001; {degree sign}p=0.006 versus control cells.
ARTICLE ABSTRACT
Human BRAF-driven tumors are aggressive malignancies with poor clinical outcome and lack of sensitivity to therapies. TRAP1 is a HSP90 molecular chaperone deregulated in human tumors and responsible for specific features of cancer cells, i.e., protection from apoptosis, drug resistance, metabolic regulation, and protein quality control/ubiquitination. The hypothesis that TRAP1 plays a regulatory function on the BRAF pathway, arising from the observation that BRAF levels are decreased upon TRAP1 interference, was tested in human breast and colorectal carcinoma in vitro and in vivo. This study shows that TRAP1 is involved in the regulation of BRAF synthesis/ubiquitination, without affecting its stability. Indeed, BRAF synthesis is facilitated in a TRAP1-rich background, whereas increased ubiquitination occurs upon disruption of the TRAP1 network that correlates with decreased protein levels. Remarkably, BRAF downstream pathway is modulated by TRAP1 regulatory activity: indeed, TRAP1 silencing induces (i) ERK phosphorylation attenuation, (ii) cell-cycle inhibition with cell accumulation in G0–G1 and G2–M transitions, and (iii) extensive reprogramming of gene expression. Interestingly, a genome-wide profiling of TRAP1-knockdown cells identified cell growth and cell-cycle regulation as the most significant biofunctions controlled by the TRAP1 network. It is worth noting that TRAP1 regulation on BRAF is conserved in human colorectal carcinomas, with the two proteins being frequently coexpressed. Finally, the dual HSP90/TRAP1 inhibitor HSP990 showed activity against the TRAP1 network and high cytostatic potential in BRAF-mutated colorectal carcinoma cells. Therefore, this novel TRAP1 function represents an attractive therapeutic window to target dependency of BRAF-driven tumors on TRAP1 translational/quality control machinery. Cancer Res; 74(22); 6693–704. ©2014 AACR.
