Syntaxin 6-mediated exosome secretion regulates enzalutamide resistance in prostate cancer.

Prostate cancer (PCa) deaths are typically the result of metastatic castration-resistant PCa (mCRPC). Recently, enzalutamide (Enz), an oral androgen receptor inhibitor, was approved for treating patients with mCRPC. Invariably, all PCa patients eventually develop resistance against Enz. Therefore, novel strategies aimed at overcoming Enz resistance are needed to improve the survival of PCa patients. The role of exosomes in drug resistance has not been fully elucidated in PCa. Therefore, we set out to better understand the exosome's role in the mechanism underlying Enz-resistant PCa. Results showed that Enz-resistant PCa cells (C4-2B, CWR-R1, and LNCaP) secreted significantly higher amounts of exosomes (2-4 folds) compared to Enz-sensitive counterparts. Inhibition of exosome biogenesis in resistant cells by GW4869 and dimethyl amiloride strongly decreased their cell viability. Mechanistic studies revealed upregulation of syntaxin 6 as well as its increased colocalization with CD63 in Enz-resistant PCa cells compared to Enz-sensitive cells. Syntaxin 6 knockdown by specific small interfering RNAs in Enz-resistant PCa cells (C4-2B and CWR-R1) resulted in reduced cell number and increased cell death in the presence of Enz. Furthermore, syntaxin 6 knockdown significantly reduced the exosome secretion in both Enz-resistant C4-2B and CWR-R1 cells. The Cancer Genome Atlas analysis showed increased syntaxin 6 expressions associated with higher Gleason score and decreased progression-free survival in PCa patients. Importantly, IHC analysis showed higher syntaxin 6 expression in cancer tissues from Enz-treated patients compared to Enz naïve patients. Overall, syntaxin 6 plays an important role in the secretion of exosomes and increased survival of Enz-resistant PCa cells.

Exosome proteomic analyses identify inflammatory phenotype and novel biomarkers in African American prostate cancer patients.

African American men face a stark prostate cancer (PCa)-related health disparity, with the highest incidence and mortality rates compared to other races. Additional and innovative measures are warranted to reduce this health disparity. Here, we focused on the identification of a novel serum exosome-based "protein signature" for potential use in the early detection and better prognosis of PCa in African American men. Nanoparticle tracking analyses showed that compared to healthy individuals, exosome concentration (number/ml) was increased by ~3.2-fold (P Ë‚ 0.05) in the sera of African American men with PCa. Mass spectrometry-based proteomic analysis of serum exosomes identified seven unique and fifty-five overlapping proteins (up- or downregulated) in African Americans with PCa compared to healthy African Americans. Furthermore, ingenuity pathway analyses identified the inflammatory acute-phase response signaling as the top pathway associated with proteins loaded in exosomes from African American PCa patients. Interestingly, African American PCa E006AA-hT cells secreted exosomes strongly induced a proinflammatory M2-phenotype in macrophages and showed calcium response on sensory neurons, suggesting a neuroinflammatory response. Additionally, proteomic analyses showed that the protein Isoform 2 of Filamin A has higher loading (2.6-fold) in exosomes from African Americans with PCa, but a lesser loading (0.6-fold) was observed in exosomes from Caucasian men with PCa compared to race-matched healthy individuals. Interestingly, TCGA and Taylor's dataset as well as IHC analyses of PCa tissue showed a lower Filamin A expression in tissues of PCa patients compared with normal subjects. Overall, these results support the usefulness of serum exosomes to noninvasively detect inflammatory phenotype and to discover novel biomarkers associated with PCa in African American men.

Delitpyrones: α-Pyrone Derivatives from a Freshwater Delitschia sp.

In research focused on the discovery of new chemical diversity from freshwater fungi, a peak library was built and evaluated against a prostate cancer cell line, E006AA-hT, which was derived from an African American, as this population is disproportionately affected by prostate cancer. The chemical study of the bioactive sample accessioned as G858 (Delitschia sp.) led to the isolation of eight new α-pyrone derivatives (1:  - 7: , and 11: ), as well as the new 3S*,4S*-7-ethyl-4,8-dihydroxy-3,6-dimethoxy-3,4-dihydronaphthalen-1(2H)-one (15: ). In addition, the known compounds 5-(3-S-hydroxybutyl)-4-methoxy-6-methyl-2H-pyran-2-one (8: ), 5-(3-oxobutyl)-4-methoxy-6-methyl-2H-pyran-2-one (9: ), pyrenocine I (10: ), 5-butyl-6-(hydroxymethyl)-4-methoxy-2H-pyran-2-one (12: ), sporidesmin A (13: ), 6-ethyl-2,7-dimethoxyjuglone (14: ), artrichitin (16: ), and lipopeptide 15G256ε (17: ) were also obtained. The structures of the new compounds were elucidated using a set of spectroscopic (NMR) and spectrometric (HRMS) methods. The absolute configuration of the most abundant member of each subclass of compounds was assigned through a modified Mosher's ester method. For 15: , the relative configuration was assigned based on analysis of 3 J values. Compounds 1, 2, 5:  - 14, 16: , and 17: were evaluated against the cancer cell line E006AA-hT under hypoxic conditions, where compound 13: inhibited cell proliferation at a concentration of 2.5 µM.

Author Correction: Hypoxia-induced exosome secretion promotes survival of African-American and Caucasian prostate cancer cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Exosomes secreted by placental stem cells selectively inhibit growth of aggressive prostate cancer cells.

The current paradigm in the development of new cancer therapies is the ability to target tumor cells while avoiding harm to noncancerous cells. Furthermore, there is a need to develop novel therapeutic options against drug-resistant cancer cells. Herein, we characterized the placental-derived stem cell (PLSC) exosomes (PLSCExo) and evaluated their anti-cancer efficacy in prostate cancer (PCa) cell lines. Nanoparticle tracking analyses revealed the size distribution (average size 131.4 ±â€¯0.9 nm) and concentration of exosomes (5.23 × 1010±1.99 × 109 per ml) secreted by PLSC. PLSCExo treatment strongly inhibited the viability of enzalutamide-sensitive and -resistant PCa cell lines (C4-2B, CWR-R1, and LNCaP cells). Interestingly, PLSCExo treatment had no effect on the viability of a non-neoplastic human prostate cell line (PREC-1). Mass spectrometry (MS) analyses showed that PLSCExo are loaded with 241 proteins and mainly with saturated fatty acids. Further, Ingenuity Pathway Analysis analyses of proteins loaded in PLSCExo suggested the role of retinoic acid receptor/liver x receptor pathways in their biological effects. Together, these results suggest the novel selective anti-cancer effects of PLSCExo against aggressive PCa cells.

Hypoxia-induced exosome secretion promotes survival of African-American and Caucasian prostate cancer cells.

African American men in the United States have higher mortality due to prostate cancer (PCa) compared to other races. One reason for this disparity is the lack of in-depth understanding of the PCa biology in African Americans. For example, hypoxia in prostate tumor microenvironment is associated with adverse prognosis; still, no hypoxia-related studies have been reported in African Americans. Here, we compared African-American and Caucasian PCa cells for exosome secretion under normoxic (21% O2) and hypoxic (1% O2) conditions. All cell lines showed higher exosome secretion under hypoxia but it was clearly more prominent in African-American PCa cells. Further, under hypoxia, Rab5 (a biomarker for early endosome) was clustered in perinuclear region; and CD63 (a biomarker for exosomes and multivesicular endosomes) showed greater co-localization with actin cytoskeleton especially in African American PCa cells. Importantly, exosome biogenesis inhibitors GW4869 (10-20 µM) or DMA (10-20 µg/ml) significantly decreased cell viability and clonogenicity in PCa cells. Interestingly, we also observed higher level of lactic acid loaded in exosomes secreted under hypoxia. Overall, under chronic hypoxia, PCa cells secrete more exosomes as a survival mechanism to remove metabolic waste.

Exosomal microRNA profiling to identify hypoxia-related biomarkers in prostate cancer.

Hypoxia and expression of hypoxia-related biomarkers are associated with disease progression and treatment failure in prostate cancer (PCa). We have reported that exosomes (nanovesicles of 30-150 nm in diameter) secreted by human PCa cells under hypoxia promote invasiveness and stemness in naïve PCa cells. Here, we identified the unique microRNAs (miRNAs) loaded in exosomes secreted by PCa cells under hypoxia. Using TaqMan® array microRNA cards, we analyzed the miRNA profile in exosomes secreted by human PCa LNCaP cells under hypoxic (ExoHypoxic) and normoxic (ExoNormoxic) conditions. We identified 292 miRNAs loaded in both ExoHypoxic and ExoNormoxic. The top 11 miRNAs with significantly higher level in ExoHypoxic compared to ExoNormoxic were miR-517a, miR-204, miR-885, miR-143, miR-335, miR-127, miR-542, miR-433, miR-451, miR-92a and miR-181a; and top nine miRNA with significantly lower expression level in ExoHypoxic compared to ExoNormoxic were miR-521, miR-27a, miR-324, miR-579, miR-502, miR-222, miR-135b, miR-146a and miR-491. Importantly, the two differentially expressed miRNAs miR-885 (increased expression) and miR-521 (decreased expression) showed similar expression pattern in exosomes isolated from the serum of PCa patients compared to healthy individuals. Additionally, miR-204 and miR-222 displayed correlated expression patterns in prostate tumors (Pearson R = 0.66, p < 0.0001) by The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) genomic dataset analysis. Overall, the present study identified unique miRNAs with differential expression in exosomes secreted from hypoxic PCa cells and suggests their potential usefulness as a biomarker of hypoxia in PCa patients.

Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity.

Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1-5 µg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47(phox)). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity.

Hypoxia-Induced Signaling Promotes Prostate Cancer Progression: Exosomes Role as Messenger of Hypoxic Response in Tumor Microenvironment.

Prostate cancer (PCA) is the leading malignancy in men and the second leading cause of cancer-related deaths. Hypoxia (low O2 condition) is considered an early event in prostate carcinogenesis associated with an aggressive phenotype. In fact, clinically, hypoxia and hypoxia-related biomarkers are associated with treatment failure and disease progression. Hypoxia-inducible factor 1 (HIF-1) is the key factor that is activated under hypoxia, and mediates adaptation of cells to hypoxic conditions through regulating the expression of genes associated with angiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis, survival, proliferation, metabolism, sternness, hormone-refractory progression, and therapeutic resistance. Besides HIF-1, several other signaling pathways including PI3K/Akt/mTOR, NADPH oxidase (NOX), Wnt/b-catenin, and Hedgehog are activated in cancer cells under hypoxic conditions, and also contribute in hypoxia-induced biological effects in HIF-1-dependent and -independent manners. Hypoxic cancer cells cause extensive changes in the tumor microenvironment both local and distant, and recent studies have provided ample evidence supporting the crucial role of nanosized vesicles "exosomes" in mediating hypoxia-induced tumor microenvironment remodeling. Exosomes' role has been reported in hypoxia-induced angiogenesis, sternness, activation of cancer-associated fibroblasts (CAFs), and EMT. Together, existing literature suggests that hypoxia plays a predominant role in PCA growth and progression, and PCA could be effectively prevented and treated via targeting hypoxia/hypoxia-related signaling pathways.