Oncosome formation in prostate cancer: association with a region of frequent chromosomal deletion in metastatic disease - PubMed (original) (raw)

. 2009 Jul 1;69(13):5601-9.

doi: 10.1158/0008-5472.CAN-08-3860. Epub 2009 Jun 23.

Jayoung Kim, Martin H Hager, Matteo Morello, Wei Yang, Christopher J Lafargue, Lawrence D True, Mark A Rubin, Rosalyn M Adam, Rameen Beroukhim, Francesca Demichelis, Michael R Freeman

Affiliations

Oncosome formation in prostate cancer: association with a region of frequent chromosomal deletion in metastatic disease

Dolores Di Vizio et al. Cancer Res. 2009.

Abstract

Oncosomes have recently been described as membrane-derived microvesicles secreted by cancer cells, which transfer oncogenic signals and protein complexes across cell boundaries. Here, we show the rapid formation and secretion of oncosomes from DU145 and LNCaP human prostate cancer cells. Oncosome formation was stimulated by epidermal growth factor receptor activation and also by overexpression of membrane-targeted Akt1. Microvesicles shed from prostate cancer cells contained numerous signal transduction proteins and were capable of activating rapid phospho-tyrosine and Akt pathway signaling, and stimulating proliferation and migration, in recipient tumor cells. They also induced a stromal reaction in recipient normal cells. Knockdown of the actin nucleating protein Diaphanous Related Formin 3 (DRF3/Dia2) by RNA interference enhanced rates of oncosome formation, indicating that these structures resemble, and may be identical to, nonapoptotic membrane blebs, a feature of the amoeboid form of cell motility. Analysis of primary and metastatic human prostate tumors using 100K single nucleotide polymorphism arrays revealed a significantly higher frequency of deletion of the locus encoding DRF3 (DIAPH3) in metastatic tumors (P = 0.001) in comparison with organ-confined tumors. Fluorescence in situ hybridization confirmed increased chromosomal loss of DIAPH3 in metastatic tumors in a different cohort of patients (P = 0.006). These data suggest that microvesicles shed from prostate cancer cells can alter the tumor microenvironment in a manner that may promote disease progression. They also show that DRF3 is a physiologically relevant protein that seems to regulate this process.

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Figures

Figure 1

Figure 1. EGF induces formation of non-apoptotic membrane blebs

(A) Membrane staining with 0.5 μg/ml FITC-CTxB for 5 min, after 3 h treatment with: (−) vehicle or (+) EGF (50 ng/ml), and imaged by confocal microscopy (63X). All panels show DU145 cells, except the panel at upper right, which shows a PC-3 cell and vesicles shed (SV) into the medium. The inset shows a single, attached bleb at higher power. (B) Frames (5 sec interval) from Movies 1 and 2 acquired by real time confocal microscopy of DU145 cells expressing membrane-targeted pMEM-YFP, and treated with EGF. Panel B shows two examples (differently shaped arrows) of bleb dynamics. (C) Lysates from whole cells and SV contain endogenous Cav-1, consistent with localization of a Cav-1-GFP fusion to membrane blebs. (D) EGF does not induce apoptosis under these experimental conditions, as shown by an assay for cleaved PARP.

Figure 2

Figure 2. Blebbing is associated with cell migration, activation of signaling and results in production of shed vesicles that contain membrane proteins

(A) Bleb formation in DU145 cells treated with EGF (50 ng/ml, 12 h) in a wound healing assay. (B) (i) Bleb formation in DU145 cells treated with EGF or EGF plus gefitinib (ZD1839) (10 μM). (ii) Bleb formation in prostate normal epithelial (iPrEC) and stromal (WPMY-1) cells, in comparison with DU145, with and without EGF. iPrECa were kept in PrEGM medium, which is serum-free but contains EGF, while iPrECb were transferred to serum-free RPMI 12 h before treatment. (C) Bleb formation in LNCaP cells stably engineered to express MyrAkt1, pro-HB-EGF, or soluble (constitutively secreted) sHB-EGF. LacZ (irrelevant gene) and Vo (vector only) are negative controls. (D) Protein from whole cell lysates and SV from EGF-treated LNCaP/MyrAkt1 cells were blotted with the indicated Abs.

Figure 3

Figure 3. Shed vesicles exhibit oncosome activity

(A) Proteins identified by tandem mass spectrometry in bleb material and quantified using spectral counting. PDCD6IP (Programmed Cell Death 6 Interacting Protein), PABPC1 (Poly(A) Binding Protein, Cytoplasmic 1), HSPA8 (Heat Shock 70kDa Protein 8), ANXA6 (Annexin A 6), hnRNP-K (Heterogeneous Nuclear Ribonucleoprotein K, PKM2 (Pyruvate Kinase M2), PACSIN2 (Protein Kinase C and Casein Kinase Substrate in Neurons 2), PABPC4 (Poly(A) Binding Protein, Cytoplasmic 4), RPLPO (Ribosomal Protein Large P0-like Protein), IGSF8 (Immunoglobulin Superfamily, member 8), YWHAE (Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase Activation Protein, Epsilon Polypeptide), HSPA1L (Heat Shock 70kDa Protein 1-Like), RPL7 (Ribosomal Protein L7), HSPA1A (Heat Shock 70kDa protein 1A), S100A7 (S100 calcium binding protein A7), JUP (Junction Plakoglobin), XRCC6 (X-ray Repair Complementing defective repair in Chinese hamster cells 6). (B) LNCaP/LacZ cells were exposed to SV obtained from EGF-treated LNCaP/MyrAkt1 cells. Blotting of whole cell lysates is shown. (C) LNCaP/LacZ exposed to 20 μg of SV or vehicle from EGF-treated LNCaP/MyrAkt1 cells and assessed for p-Tyr (upper panels). The lower panels show the results of blotting with p-EGFR antibody (p-Tyr1068). (D) WPMY-1 cells were exposed to LNCaP/MyrAkt1 derived SV for the indicated times. Vimentin mRNA was quantified by qRT-PCR in recipient cells, and normalized using GAPDH. An irrelevant gene, PRKAR1A, was used as control.

Figure 4

Figure 4. DRF3 knockdown by RNA interference results in oncosome secretion

(A) Verification of DIAPH3 gene silencing by siDRF3 in DU145 cells by western blot (upper panel) and RT-PCR (lower panel). Control non-targeting siRNA was a negative control. (B) FITC-CTxB staining of DU145 cells showing blebbing in siDRF3-transfected or siRNA control cells, with or without EGF (3 h) (right panel). (C) Quantitative analysis of bleb formation in DU145 cells treated with siRNA for DRF3 or control siRNA, +/− EGF. (D) Proliferation assay (left panel) and migration assay (right panel) in DU145 cells treated with SV prepared from DU145 cells treated with siRNA control oligos, +/− EGF, or with DRF3-targeted oligos. *p<0.05.

Figure 5

Figure 5. Genomic profiling of primary and metastatic prostate cancer at the DIAPH3 locus

(A) Amplifications (red) and deletions (blue), determined by segmentation analysis of normalized signal intensities from 100K SNP arrays (see Methods), are displayed for 39 prostate cancers (x-axis; primaries and metastases are designated along the top) for the q arm of chromosome 13 (chromosomal positions indicated along the y-axis include the centromere, q-terminus, and RB1 loci). (B) DIAPH3 fluorescent in situ hybridization was performed by dual color FISH on PCa tissues. The FISH image on the left shows both red signals (DIAPH3 locus on chr13q21.2) and green signals (a stable region on chr21q22.12) in representative nuclei indicating no deletion of DIAPH3 in tumor cells. The FISH image on the right shows one red signal (DIAPH3 locus on chr13q21.2) and two green signals (a stable region on chr21q22.12) in representative nuclei. Loss of the second red signal is consistent with deletion at DIAPH3. Original magnification of FISH images, 60 X objective.

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