Bars are the mean SEM of three independent experiments. expression of PRKD2 was able to partially restore HIF1 and secreted VEGF-A levels in hypoxic cancer cells treated with HSP90 inhibitors. Taken together, our findings indicate that signals from hypoxia and HSP90 pathways are interconnected and funneled by PRKD2 into the NF-B/VEGF-A signaling axis to promote tumor angiogenesis and tumor growth. Introduction Cancer development is a multistep process characterized by a multitude of genetic and epigenetic changes that induce resistance to proapoptotic stimuli, sustain angiogenesis, and confer insensitivity to antigrowth signals and immune surveillance (1). Rapid tumor growth often results in hypoxia, which triggers the stabilization of the transcription factor hypoxia-inducible factor-1 (HIF1), an oxygen sensor that controls the expression of multiple target genes implicated in angiogenesis, metabolism, and cell survival (2, 3). A prominent target of HIF1 is VEGF-A, which induces tumor angiogenesis by stimulating proliferation, survival, and migration of endothelial cells (4). HIF1 has been reported to physically interact with HSP90 (5, 6), which can be targeted hRad50 by small-molecule inhibitors of chaperone, a growing class of clinically utilized antitumori-genic agents. HSP90 is a highly conserved and ubiquitously expressed molecular chaperone involved in the correct folding and final maturation of a plethora of proteins, so-called HSP90 clients, in an effort to maintain cellular homeostasis (7, 8). There are more than 200 HSP90 clients known, including protein kinases, transcription factors, and steroid hormone receptors (9C11). HSP90 is recruited to its kinase clients through interactions with kinase-specific co-chaperone CDC37 (12, 13), which stabilizes the HSP90/kinase (14). In tumor cells, HSP90 aids in folding dysregulated oncoproteins Pseudoginsenoside-RT5 helping to sustain their aberrant activity. Amongst the most known client kinases of HSP90 are SRC (15), AKT (16), PDK-1 (17), and PKC (18). The latter was Pseudoginsenoside-RT5 shown to directly activate protein kinase D (PRKD) family members via phosphorylation at two critical serine residues within the activation loop of the kinase catalytic domain (19). Recently, an affinity-based proteomic screen conducted to identify cancer-specific networks coordinated by HSP90 revealed PRKD2 as a potential client for the chaperone in chronic myelogenous leukemia (CML) cells (20). The serine-threonine kinase PRKD2 and its sister isoforms PRKD1 and PRKD3 belong to the calcium/calmodulin-dependent protein kinase superfamily (21) and are activated by various stimuli, including phorbol esters, reactive oxygen species, receptor tyrosine kinases, and hypoxia Pseudoginsenoside-RT5 (22C24). PRKD2 expression and activity correlate positively with the state of dedifferentiation in lymphoma (25) and were demonstrated to be involved in myeloid leukemia by activating NF-B transcription factors (26). Furthermore, PRKD2 is involved in migration, invasion, and growth of glioblastoma and pancreatic cancer cells (27C29). We have recently identified PRKD2 as a crucial mediator of hypoxia-induced VEGF-A expression and secretion in pancreatic cancer cells (24). The aim of this study was to interrogate the contribution of PRKD2 to HSP90-mediated tumor growth and tumor angiogenesis. In addition, the involvement of PRKD2 in the regulation of hypoxia-mediated HIF1 stabilization, NF-B activation, and VEGF-A production in the context of pharmacologic inhibition of HSP90 represented a major focus of our work. We identified PRKD2 as a novel client of HSP90 and revealed its requirement for tumor viability and tumor angiogenesis during abrogation of chaperone activity and test. 0.05 was considered significant. Results Using an affinity-based proteomic assay followed by chemical precipitation and Western blotting validation, Moulick and colleagues (20) identified PRKD2 as a putative HSP90 client in K562 CML cells. To assess whether HSP90 is able to bind to PRKD2 in solid tumors, coimmunoprecipitation experiments with lung cancer (A549), breast cancer (MDA-MB-231), and pancreatic cancer (PaTu2) cells were performed (Fig. 1A). Although PRKD2 interacted with HSP90 in all three cancer cell lines (Fig. 1A), an interaction between PRKD2 and HSP27 or HSP70 chaperones could not be observed (data not shown). To investigate whether the stability of PRKD2 requires HSP90, we performed knockdown experiments using shRNAs targeting HSP90 (shHSP90) or HSP90 (shHSP90), respectively. shRNA-mediated abrogation of both HSP90 isoforms resulted in a decrease of PRKD2 protein levels in A549, MDA-MB-231, and PaTu2 cell lines (Fig. 1B and C) and this was associated with induction of apoptosis as revealed by enhanced PARP cleavage in Western blot analysis (Fig. 1B and C) Pseudoginsenoside-RT5 or TUNEL assay (Supplementary Fig. S1A). Altogether, these.