Feasibility and performance of a novel probe panel to detect somatic DNA copy number alterations in clinical specimens for predicting prostate cancer progression.

BACKGROUND: To assess the feasibility of a novel DNA-based probe panel to detect copy number alterations (CNAs) in prostate tumor DNA and its performance for predicting clinical progression. METHODS: A probe panel was developed and optimized to measure CNAs in trace amounts of tumor DNA (2 ng) isolated from formalin-fixed paraffin-embedded tissues. Ten genes previously associated with aggressive disease were targeted. The panel's feasibility and performance were assessed in 175 prostate cancer (PCa) patients who underwent radical prostatectomy with a median 10-year follow-up, including 42 men who developed disease progression (either metastasis and/or PCa-specific death). Association with disease progression was tested using univariable and multivariable analyses. RESULTS: The probe panel detected CNAs in all 10 genes in tumor DNA isolated from either diagnostic biopsies or surgical specimens. A four-gene model (PTEN/MYC/BRCA2/CDKN1B) had the strongest association with disease progression; 64.3% of progressors and 22.5% of non-progressors had at least one CNA in these four genes, odds ratio (OR) (95% confidence interval) = 6.21 (2.77-13.87), P = 8.48E-06. The association with disease progression remained significant after adjusting for known clinicopathological variables. Among the seven progressors of the 65 patients with clinically low-risk disease, three (42.9%) had at least one CNA in these four genes. CONCLUSIONS: The probe panel can detect CNAs in trace amounts of tumor DNA from biopsies or surgical tissues at the time of diagnosis or surgery. CNAs independently predict metastatic/lethal cancer, particularly among men with clinically low-risk disease at diagnosis. If validated, this may improve current abilities to assess tumor aggressiveness.

LDL induces cholesterol loading and inhibits endothelial proliferation and angiogenesis in Matrigels: correlation with impaired angiogenesis during wound healing.

Hypercholesterolemia is a major risk factor for adverse cardiovascular outcomes, but its effect on angiogenesis and wound healing is not well understood. In this study, using a combination of mass spectrometry and laurdan two-photon imaging, we show that elevated levels of low-density lipoprotein (LDL), like those seen in hypercholesterolemic patients, lead to an increase in both free cholesterol and cholesterol esters, as well as increase in lipid order of endothelial cell membranes. Notably, these effects are distinct and opposite to the lack of cholesterol loading and the disruption of lipid order observed in our earlier studies in response to oxidized LDL (oxLDL). The same pathological level of LDL leads to a significant inhibition of endothelial proliferation and cell cycle arrest in G2/M phase, whereas oxLDL enhances endothelial proliferation in S phase of the cycle. LDL but not oxLDL suppresses the expression of vascular endothelial growth factor receptor-2 while enhancing the expression of vascular endothelial growth factor (VEGF). Furthermore, we show that aged (8-10 mo) hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice display delayed wound closure compared with age-matched C57/BL6 wild-type controls following a skin punch biopsy. The delay in wound healing is associated with a decreased expression of cluster of differentiation 31 platelet endothelial cell adhesion molecule endothelial marker and decreased angiogenesis within the wound bed. Furthermore, decreased endothelial responsiveness to the growth factors VEGF and basic fibroblast growth factor is observed in ApoE-/- mice in Matrigel plugs and in Matrigels with high levels of LDL in wild-type mice. We propose that plasma hypercholesterolemia is antiangiogenic due to elevated levels of LDL.

Proatherogenic Flow Increases Endothelial Stiffness via Enhanced CD36-Mediated Uptake of Oxidized Low-Density Lipoproteins.

OBJECTIVE: Disturbed flow (DF) is well-known to induce endothelial dysfunction and synergistically with plasma dyslipidemia facilitate plaque formation. Little is known, however, about the synergistic impact of DF and dyslipidemia on endothelial biomechanics. Our goal was to determine the impact of DF on endothelial stiffness and evaluate the role of dyslipidemia/oxLDL (oxidized low-density lipoprotein) in this process. APPROACH AND RESULTS: Endothelial elastic modulus of intact mouse aortas ex vivo and of human aortic endothelial cells exposed to laminar flow or DF was measured using atomic force microscopy. Endothelial monolayer of the aortic arch is found to be significantly stiffer than the descending aorta (4.2+1.1 versus 2.5+0.2 kPa for aortic arch versus descending aorta) in mice maintained on low-fat diet. This effect is significantly exacerbated by short-term high-fat diet (8.7+2.5 versus 4.5+1.2 kPa for aortic arch versus descending aorta). Exposure of human aortic endothelial cells to DF in vitro resulted in 50% increase in oxLDL uptake and significant endothelial stiffening in the presence but not in the absence of oxLDL. DF also increased the expression of oxLDL receptor CD36 (cluster of differentiation 36), whereas downregulation of CD36 abrogated DF-induced endothelial oxLDL uptake and stiffening. Furthermore, genetic deficiency of CD36 abrogated endothelial stiffening in the aortic arch in vivo in mice fed either low-fat diet or high-fat diet. We also show that the loss of endothelial stiffening in CD36 knockout aortas is not mediated by the loss of CD36 in circulating cells. CONCLUSIONS: DF facilitates endothelial CD36-dependent uptake of oxidized lipids resulting in local increase of endothelial stiffness in proatherogenic areas of the aorta.

Oxidized LDL signals through Rho-GTPase to induce endothelial cell stiffening and promote capillary formation.

Endothelial biomechanics is emerging as a key factor in endothelial function. Here, we address the mechanisms of endothelial stiffening induced by oxidized LDL (oxLDL) and investigate the role of oxLDL in lumen formation. We show that oxLDL-induced endothelial stiffening is mediated by CD36-dependent activation of RhoA and its downstream target, Rho kinase (ROCK), via inhibition of myosin light-chain phosphatase (MLCP) and myosin light-chain (MLC)2 phosphorylation. The LC-MS/MS analysis identifies 7-ketocholesterol (7KC) as the major oxysterol in oxLDL. Similarly to oxLDL, 7KC induces RhoA activation, MLCP inhibition, and MLC2 phosphorylation resulting in endothelial stiffening. OxLDL also facilitates formation of endothelial branching networks in 3D collagen gels in vitro and induces increased formation of functional blood vessels in a Matrigel plug assay in vivo. Both effects are RhoA and ROCK dependent. An increase in lumen formation was also observed in response to pre-exposing the cells to 7KC, an oxysterol that induces endothelial stiffening, but not to 5α,6α epoxide that does not affect endothelial stiffness. Importantly, loading cells with cholesterol prevented oxLDL-induced RhoA activation and the downstream signaling cascade, and reversed oxLDL-induced lumen formation. In summary, we show that oxLDL-induced endothelial stiffening is mediated by the CD36/RhoA/ROCK/MLCP/MLC2 pathway and is associated with increased endothelial angiogenic activity.

Inhibition of p70 S6 kinase (S6K1) activity by A77 1726 and its effect on cell proliferation and cell cycle progress.

Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor-mediated signaling pathway. In vitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 µM, respectively. Exogenous uridine partially blocked A77 1726-induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726-induced cell cycle arrest in the S phase.