Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1ϩ vesicles surround cilia in Pkhd1 del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.
In this study, we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor α-positive (ERα+) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ERα, HER-2/Neu overexpression and loss of CD24 surface receptor (CD24 –/low). Importantly, expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover, aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5, indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT, stemness and ultimately tumor progression. Importantly, pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24+ epithelial phenotype that was coupled to ERα expression, downregulation of HER-2/Neu, inhibition of EMT and impaired self-renewal ability, resulting in the suppression of distant metastases. Taken together, our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ERα+ breast cancer cells. Moreover, this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ERα+ breast cancer patients resistant to conventional endocrine therapy.
JAK-STAT is a rational drug target in myelofibrosis (MF) given its association with JAK2/MPL mutations and aberrant inflammatory cytokine expression. We conducted a Phase 1/2 trial of CYT387, a potent JAK1/2 inhibitor, in patients with high- or intermediate-risk primary or post-polycythemia vera/essential thrombocythemia MF. Pre-planned safety and efficacy analysis has been completed for the initial 60 patients. In the dose-escalation phase (n=21), the maximum-tolerated dose was 300 mg/day based on reversible grade 3 headache and asymptomatic hyperlipasemia. Twenty-one and 18 additional patients were accrued at two biologically effective doses, 300 mg/day and 150 mg/day, respectively. Anemia and spleen responses, per International Working Group criteria, were 59% and 48%, respectively. Among 33 patients who were red cell-transfused in the month prior to study entry, 70% achieved a minimum 12-week period without transfusions (range 4.7–>18.3 months). Most patients experienced constitutional symptoms improvement. Grade 3/4 adverse reactions included thrombocytopenia (32%), hyperlipasemia (5%), elevated liver transaminases (3%) and headache (3%). New-onset treatment-related peripheral neuropathy was observed in 22% of patients (sensory symptoms, grade 1). CYT387 is well tolerated and produces significant anemia, spleen and symptom responses in MF patients. Plasma cytokine and gene expression studies suggested a broad anticytokine drug effect.
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