Ramteke Anand, Ting Harold, Agarwal Chapla, Mateen Samiha, Somasagara Ranganathan, Hussain Anowar, Graner Michael, Frederick Barbara, Agarwal Rajesh, Deep Gagan
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, Aurora, Colorado.
Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, India.
Mol Carcinog. 2015 Jul;54(7):554-65. doi: 10.1002/mc.22124. Epub 2013 Dec 17.
Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness.
前列腺癌（PCA）中的缺氧状态与预后不良相关；然而，缺氧促进恶性表型的确切机制仍不清楚。在此，我们分析了缺氧PCA细胞来源的外泌体在增强未处理PCA细胞的侵袭性和干性以及促进前列腺基质细胞（PrSC）中癌症相关成纤维细胞（CAF）表型方面的作用。将人PCA LNCaP和PC3细胞暴露于缺氧（1% O₂）或常氧（21% O₂）条件下，并从条件培养基中分离出在缺氧（Exo(Hypoxic)）和常氧（Exo(Normoxic)）条件下分泌的外泌体。纳米颗粒跟踪分析显示，与Exo(Normoxic)相比，Exo(Hypoxic)的平均尺寸更小。免疫印迹结果表明，与Exo(Normoxic)相比，Exo(Hypoxic)中四跨膜蛋白（CD63和CD81）、热休克蛋白（HSP90和HSP70）和膜联蛋白II的水平更高。与Exo(Hypoxic)共培养分别增加了未处理的LNCaP和PC3细胞的侵袭性和运动性。Exo(Hypoxic)还促进了LNCaP和PC3细胞的前列腺球形成，并增强了PrSC中α-SMA（一种CAF生物标志物）的表达。与Exo(Normoxic)相比，Exo(Hypoxic)表现出更高的金属蛋白酶活性和多种信号分子（TGF-β2、TNF1α、IL6、TSG101、Akt、ILK1和β-连环蛋白）水平的升高。此外，蛋白质组分析显示，与Exo(Normoxic)（62种蛋白质）相比，Exo(Hypoxic)中的蛋白质数量更多（160种蛋白质），主要与上皮黏附连接途径的重塑有关。重要的是，Exo(Hypoxic)靶向未处理PC3细胞中黏附连接蛋白的表达。这些发现表明，Exo(Hypoxic)负载有独特的蛋白质，这些蛋白质可以增强侵袭性、干性并诱导微环境变化；从而促进PCA的侵袭性。
