Dysregulated fibronectin trafficking by Hsp90 inhibition restricts prostate cancer cell invasion

Abstract

The molecular chaperone Hsp90 is overexpressed in prostate cancer (PCa) and is responsible for the folding, stabilization and maturation of multiple oncoproteins, which are implicated in PCa progression. Compared to first-in-class Hsp90 inhibitors such as 17-allylamino-demethoxygeldanamycin (17-AAG) that were clinically ineffective, second generation inhibitor AUY922 has greater solubility and efficacy. Here, transcriptomic and proteomic analyses of patient-derived PCa explants identified cytoskeletal organization as highly enriched with AUY922 treatment. Validation in PCa cell lines revealed that AUY922 caused marked alterations to cell morphology, and suppressed cell motility and invasion compared to vehicle or 17-AAG, concomitant with dysregulation of key extracellular matrix proteins such as fibronectin (FN1). Interestingly, while the expression of FN1 was increased by AUY922, FN1 secretion was significantly decreased. This resulted in cytosolic accumulation of FN1 protein within late endosomes, suggesting that AUY922 disrupts vesicular secretory trafficking pathways. Depletion of FN1 by siRNA knockdown markedly reduced the invasive capacity of PCa cells, phenocopying AUY922. These results highlight a novel mechanism of action for AUY922 beyond its established effects on cellular mitosis and survival and, furthermore, identifies extracellular matrix cargo delivery as a potential therapeutic target for the treatment of aggressive PCa.

Figure 1

The Hsp90 inhibitor, AUY922, selectively alters cellular pathways involved in the cytoskeleton and ECM. (A) KEGG pathways determined by over-representation analysis of differentially expressed genes identified through RNA-seq analysis of AUY922 treated PDEs (48 h: 500 nM) using the InnateDB database. A barcode plot and heatmap of RNA-seq gene expression data from regulation of actin cytoskeleton pathway for individual explants is shown, and significant changes in gene expression were validated in independent explants by qRT-PCR (ANXA2 & AURKB shown). (B) KEGG pathways determined by the Database for Annotation, Visualization and Integrated Discovery (DAVID) using differentially expressed proteins identified through proteomic analysis of AUY922 treated PDEs 48 h: 500 nM, n = 12). Proteins identified by 2D-DIGE proteomics analysis were validated in independent PDE samples by Western blot. ERK1 was used as loading control. Full-length blots are presented in Supplementary Figure 5.

Figure 2

Hsp90 inhibition markedly influences cell morphology, growth, motility and invasive capacity. (A) Morphology of LNCaP and PC-3 cells cultured +/− AUY922 (25 nM) or 17-AAG (50 nM) for 48 h in the Incucyte live cell analysis system. (B) Cell confluency was measured in the same experiment. (C) Motility of LNCaP and PC-3 cells cultured +/− AUY922 (25 nM) or 17-AAG (50 nM) over a 48 h incubation in an Incuyte live cell analysis system, with 50 cells tracked per treatment. (D) PC-3 cells were cultured with either IC50 or 2x IC50; AUY922 (25 and 50 nM) or 17-AAG (50 and 100 nM) or vehicle (DMSO) for 48 h before harvesting for a 3D invasion assay using 24-well Falcon BioCoat Matrigel invasion chambers. Migrated cells remaining on the bottom surface were fixed in formalin and stained with crystal violet. Five random fields for each membrane were photographed using the Axio Scope A1 Fluorescent Microscope (Zeiss), and the number of migrated cells was counted manually and presented as percent of control cells ± SEM.

Figure 3

AUY922 induces expression of fibronectin, resulting in its cytosolic accumulation in punctate compartments. (A) Western blot analysis of the effect of 17-AAG or AUY922 on Hsp70, fibronectin (FN1) and cytoskeletal proteins, in LNCaP and PC-3 cells. Cells were treated with DMSO control or the indicated doses of AUY922, or 17-AAG and collected at 48 h. Full-length blots are presented in Supplementary Figure 6. To examine intracellular localization of FN1, we used (B) cellular fractionation of LNCaP cells treated as indicated for 48 h (W = whole cell lysate, C = cytoplasmic fraction, N = nuclear fraction; full length blots are presented in Supplementary Figure 6), and (C) fluorescence microscopy of LNCaP cells treated for with AUY922 (25 nM) for 48 h and probed for FN1 (antibody obtained from Professor Jane Sottile, Rochester University) followed by secondary ALEXA 488 with images taken using Leica SP8 confocal microscope at 40x magnification. (D) The number and distribution of FN1 particles in cultured LNCaP cells with AUY922 (25 nM) for 48 h was quantified by automated image analysis (CellProfiler) of fluorescence microscopy images captured with an INCell 2200 automated imaging system (GE Healthcare) at 20x magnification.

Figure 4

Fibronectin is localized to late endosome compartments which require intact tubulin microtubules to traffic to the plasma membrane. (A) To examine inhibitor effects on secretion of FN1, an ELISA assay was performed on tissue culture media (HPC-1) collected from LNCaP cells treated with AUY922 (25 nM) for 48 h and controlled for cell number. Date represents mean ± SEM of  4 independent experiments. (B) Model illustrating cytosolic vesicle and compartment trafficking to the plasma membrane along microtubule networks. (C) Co-location of FN1 with vesicle markers including endosomes (EEA1, Appl1, Rab5, Rab7), lysosome (LIMP2, LAMP1), endoplasmic reticulum (PDI) and Golgi (Tgn46) was examined by fluorescence microscopy in LNCaP cells treated with DMSO for 48 h and probed for vesicle markers (red) and FN-1 (green) and DAPI mount media (blue). Images were taken using Leica SP8 confocal microscope at 63x magnification.

Figure 5

AUY922 does not affect microtubule polymerization in prostate cancer cells. (A) AUY922, 17-AAG and control compounds at the indicated doses were incubated with purified tubulin protein at 37 °C, and polymerization was monitored using a FLUOstar Omega plate reader at 340 nm every 1 min for 60 min at 37 °C. (B) Representative images of LNCaP cells treated with AUY922 (31 or 250 nM), nocodazole (NOC) (250 nM) or vehicle control (DMSO) for 48 h and subjected to fluorescence microscopy of α-tubulin (green) and DNA (blue) using an INCell 2200 automated imaging system at 40x magnification. Cells treated with DMSO or AUY922 showed intact microtubule network, while cells treated with the microtubule destabilizing drug NOC displayed depolymerized microtubules. Scale bar = 5 µm. (C) AUY922 (15–500 nM) did not affect α-tubulin polymer mass, while NOC (250 nM) and paclitaxel (200 nM) reduced and increased, respectively, α-tubulin polymer mass, as shown by quantitative immunofluorescence microscopy of the cellular α-tubulin mean intensity as depicted in (B) (expressed as fold-change relative to DMSO control, n = 3, mean ± SD).

Figure 6

AUY922 and specific knockdown of FN1 both suppress prostate cancer cell invasion. (A) Cells were cultured with either AUY922 (25, 50 nM), or vehicle (DMSO) for 48 h before harvesting for the invasion assay. (B) PC-3 cells were transfected with 2 independent siRNAs against FN1 (Si-1, Si-2), or non-targeting siRNA as a negative control for 48 h before harvesting for the invasion assay. (C) PC-3 cells were treated with pUR4 (500 nM) or the control peptide III-11C (500 nM) for 48 h before harvesting for the invasion assay. 3D invasion assays were conducted in PC-3 cells using 24-well Falcon BioCoat Matrigel invasion chambers. Treated or siRNA-transfected cells were seeded into the upper chamber of the Transwell at a density of 1x 10⁵ cells/well in serum-free medium. The bottom chamber was filled with RPMI-1640 medium containing 5% FBS and inserts incubated at 37 °C for 48 h. Migrated cells remaining on the bottom surface were fixed in formalin and stained with crystal violet. Five random fields for each membrane were photographed using the Axio Scope A1 Fluorescent Microscope (Zeiss), and the number of migrated cells was counted manually and presented as percent of control cells ± SEM.