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. 2020 Mar 19;9(3):755.
doi: 10.3390/cells9030755.

Cell Stress Induced Stressome Release Including Damaged Membrane Vesicles and Extracellular HSP90 by Prostate Cancer Cells

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Free PMC article

Cell Stress Induced Stressome Release Including Damaged Membrane Vesicles and Extracellular HSP90 by Prostate Cancer Cells

Takanori Eguchi et al. Cells. .
Free PMC article

Abstract

Tumor cells exhibit therapeutic stress resistance-associated secretory phenotype involving extracellular vesicles (EVs) such as oncosomes and heat shock proteins (HSPs). Such a secretory phenotype occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as a soluble form known as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in the release of EV proteins including CD9 and epithelial-to-mesenchymal transition (EMT), a key event in tumor progression. EVs derived from CRPC cells promoted EMT in normal prostate epithelial cells. Some HSP family members and their potential receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein types found by mass spectrometry. The small EVs (30-200 nm in size) were released even in a non-heated condition from the prostate cancer cells, whereas the EMT-coupled release of EVs (200-500 nm) and damaged membrane vesicles with associated HSP90α was increased after heat shock stress (HSS). GAPDH and lactate dehydrogenase, a marker of membrane leakage/damage, were also found in conditioned media upon HSS. During this stress response, the intracellular chaperone CDC37 was transcriptionally induced by heat shock factor 1 (HSF1), which activated the CDC37 core promoter, containing an interspecies conserved heat shock element. In contrast, knockdown of CDC37 decreased EMT-coupled release of CD9-containing vesicles. Triple siRNA targeting CDC37, HSP90α, and HSP90β was required for efficient reduction of this chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, we define "stressome" as cellular stress-induced all secretion products, including EVs (200-500 nm), membrane-damaged vesicles and remnants, and extracellular HSP90 and GAPDH. Our data also indicated that CDC37 is crucial for the release of vesicular proteins and tumor progression in prostate cancer.

Keywords: cell division control 37 (CDC37); cell stress response; ectosome; exosome; extracellular vesicle; heat shock protein 90 (HSP90); prostate cancer; stressome.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The pro-epithelial-to-mesenchymal transition (EMT) effect, proteome, and stress response of extracellular vesicles (EVs) released by PC-3 cells. (A) Western blot showing E-cadherin/CDH1, N-cadherin/CDH2, and GAPDH. EVs were prepared using 200-nm pore filter devices and polymer-based precipitation (PBP) method from culture media of PC-3. RWPE-1 cells were treated with the PC-3-derived EV at 5, 10, 25, or 50 µg/mL or PBS for 3 days and then lysed. (B) Scores of proteins identified by LC-MS/MS. (C) Western blot showing heat shock protein (HSP)90α and β-actin expressed in normal prostate epithelial cells, PC-3, DU-145, and PNT2 cells. The same set of protein samples was used previously [60]. (D) Western blot showing HSP90α, CD9, and β-actin in EVs and cells. PC-3 cells were stimulated with heat shock stress (HSS) for 1.5 or 3 h or non-heated (NH) and then cultured in serum-free media for 24 h to collect EVs and cell lysates.
Figure 2
Figure 2
Release of EVs and membrane damage upon HSS. PC-3 cells were stimulated with HSS for 0.5, 1.5, or 3 h or NH, cultured for 24 h in serum-free media, from which EVs were then collected, and images were taken. (A) TEM images. (a), Cup-like EVs with clear walls of a potential lipid bilayer. (b), The wall of an EV was partially thick, which may be associated with another particle. (c), Membrane deformation was also seen in some EVs. (d), Crescent shapes are reminiscent of membrane brake of EVs. Scale bar, 100 nm. (B) Column scatterplot analysis of particle size. n = 50, mean ± SD. ANOVA and Tukey’s multiple comparison test was performed for statistics. **p < 0.01, ***p < 0.001 (vs. NH); n.s., not significant. (C) Column scatterplot analysis of the rate of EV(200–500 nm) upon HSS. n = 3 (random three fields), **p < 0.01; n.s., not significant. (D) Particle diameter distribution analysis using Zetasizer. (E) Numbers of EVs(260–300 nm), EVs(300–380 nm), and EVs(380–420 nm) per mL conditioned medium. qNano analyzer was used for counting EVs. (FH) Cell morphological changes upon stress. (F) Representative images of cells. Top images were enlarged ones from the squares in the bottom images. Arrows, round-shaped cells. Arrowheads, cells with projections (or spindle shape). Scale, 100 µm. (G) Rate of cells with projections. (H) Rate of round-shaped cells.
Figure 3
Figure 3
Release of EVs and membrane damage upon HSS. PC-3 cells were stimulated with HSS for 0.5, 1.5, or 3 h or NH, cultured for 24 h in serum-free media, from which EVs were then collected. (A) Release of EVs in response to stress. Protein concentrations of EV fractions were quantified using protein assay. The ratio compared to NH is plotted. *p < 0.05, n = 5 to 7. (B) Release of lactate dehydrogenase (LDH) upon HSS. PC-3 cells in the sparse condition were stimulated with or without HSS for 3 h and then LDH release was measured. (C,D) Column scatterplot analysis of LDH release. PC-3 cells were stimulated with HSS for 0.5, 1.5, or 3 h or NH, cultured for 24 h in serum-free media. The released LDH was measured. Data from two independent experiments are shown in C and D. Statistical analysis was carried out using ANOVA and Tukey’s multiple comparisons test. n = 3, ***p < 0.001, *p < 0.05, ###p < 0.001, ####p < 0.0001.
Figure 4
Figure 4
Heat shock response and roles of CDC37 in vesicular protein release. (A) Promoter analysis of Cdc37 (−5000 to +1000) in human, mouse, rat, and fission yeast. Heat shock elements (HSE) were mapped as red boxes. (BD) Mapping of HSE in Cdc37 core promoter regions (−500 to transcription start site) in (B) human, (C) mouse, and (D) rat. HSEs were enclosed with red rectangles. (E) Alignment of HSE conserved among mammals. The HSE(−438) in human CDC37 is conserved with a reverse HSE(−446) in rat Cdc37. Another HSE(−171) in mouse Cdc37 is conserved with the HSE(−446) in rat Cdc37. (F) Schemes of human CDC37 promoter-luciferase reporter constructs. Promoter regions (500bp + UTR or 200bp + UTR) were connected with luciferase gene. UTR, 5′-untranslated region. (G) Activities of CDC37 promoter regulated by heat shock factor 1 (HSF1). The CDC37 promoter-reporter constructs were co-transfected with overexpression constructs of HSF1 or dominant-negative (DN)-HSF1 into DU-145 cells. *p < 0.05, n = 3. (H) Western blot showing CDC37, HSP90, CD9, and GAPDH altered by HSS in PC-3 cells, EVs, and non-EV fraction. PC-3 cells were stimulated with or without HSS (43 ℃ for 30 min), cultured in serum-free media for 24 h, and then cell lysate, EV, and non-EV fractions were collected.
Figure 5
Figure 5
Inhibition of proteostasis and CD9 in EVs by the knockdown of CDC37. The siRNA targeting CDC37 or non-targeting control siRNA was transfected into PC-3 cells. Cell lysate and EVs were prepared at 48 h after the transfection. (A) Western blot showing CDC37, CD9, and GAPDH in PC-3 cells. EVs were prepared using 200-nm pore filter devices and PBP method. (B) Cellular protein concentration per million cells. **p < 0.01, n = 3. (C) EV protein concentration per million cells. **p < 0.01, n = 3.
Figure 6
Figure 6
EMT properties, vesicle release, and tumorigenicity were declined by targeting CDC37 and HSP90. (A,B) PC-3 cells were transfected with siRNA targeting CDC37, HSP90α, their combination, or non-targeting control siRNA for 48 h and then cell lysates and EVs were collected. (A) Western blot showing CDC37, vimentin, E-cadherin, HSP90, and GAPDH. (B) The ratio of EV vs. cellular protein concentrations. *p < 0.05, n = 3. (C) Western blot showing CDC37 and HSP90 reduced by double or triple knockdown. siRNA targeting CDC37, HSP90α, and HSP90β or non-targeting control siRNA were transfected into PC-3 cells and then cells were lysed at 48 h post-transfection. Efficiencies of double or triple knockdown were tested. (DH) In vivo tumorigenesis declined by triple chaperone knockdown. PC-3 cells were transfected with triple siRNA combination (CDC37, HSP90α, HSP90β) or non-targeting control siRNA and then cells were subcutaneously xenografted to SCID mice. Tumor volumes at (D) day 21, (E) day 36, (F) day 49, and (G) day 63 post-xenograft periods were analyzed by scatter-plotting. Data are expressed as mean ± SD, n = 3. (H) The time-course graph of tumor volumes altered between triple chaperone knockdown vs. the control siRNA groups. Data are expressed as mean + SD, n = 3.
Figure 7
Figure 7
Graphical abstract. (A) Prostate cancer cells release EVs(50–200 nm) that contain HSP90α and transform normal epithelial cells by inducing EMT. (B) Cellular stress such as HSS activates HSF1, which induces the production of CDC37 and HSP90α via transcriptional activation (left). CDC37 is essential for proteostasis and the release of CD9-containing EVs. HSS for 1.5–3 h also triggers the production of larger EVs(200–500 nm) and membrane damage of EVs, from which HSP90α and GAPDH could be leaked. Therefore, we here define “stressome” as cell stress-induced all secretion products including EVs, membrane-damaged vesicles, and factors released from EVs and cells. Stressome may promote tumor progression.

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