Among patients with unstable angina or myocardial infarction without ST-segment elevation, prasugrel did not significantly reduce the frequency of the primary end point, as compared with clopidogrel, and similar risks of bleeding were observed. (Funded by Eli Lilly and Daiichi Sankyo; TRILOGY ACS ClinicalTrials.gov number, NCT00699998.).

We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation, revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores, suggesting that macropinocytosis can supply nutrients other than amino acids. Nontransformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically. As PTEN-deficient prostate cancer cells proliferate in low-nutrient environments by scavenging necrotic debris and extracellular protein via macropinocytosis, blocking macropinocytosis by inhibiting AMPK, RAC1, or PI3K may have therapeutic value, particularly in necrotic tumors and in combination with therapies that cause nutrient stress. .

No abstract

Triple-negative breast cancer (TNBC) is notoriously aggressive with high metastatic potential, which has recently been linked to high rates of fatty acid oxidation (FAO). Here we report the mechanism of lipid metabolism dysregulation in TNBC through the prometastatic protein, CUB-domain containing protein 1 (CDCP1). We show that a "low-lipid" phenotype is characteristic of breast cancer cells compared with normal breast epithelial cells and negatively correlates with invasiveness in 3D culture. Using coherent anti-Stokes Raman scattering and two-photon excited fluorescence microscopy, we show that CDCP1 depletes lipids from cytoplasmic lipid droplets (LDs) through reduced acyl-CoA production and increased lipid utilization in the mitochondria through FAO, fueling oxidative phosphorylation. These findings are supported by CDCP1's interaction with and inhibition of acyl CoA-synthetase ligase (ACSL) activity. Importantly, CDCP1 knockdown increases LD abundance and reduces TNBC 2D migration in vitro, which can be partially rescued by the ACSL inhibitor, Triacsin C. Furthermore, CDCP1 knockdown reduced 3D invasion, which can be rescued by ACSL3 co-knockdown. In vivo, inhibiting CDCP1 activity with an engineered blocking fragment (extracellular portion of cleaved CDCP1) lead to increased LD abundance in primary tumors, decreased metastasis, and increased ACSL activity in two animal models of TNBC. Finally, TNBC lung metastases have lower LD abundance than their corresponding primary tumors, indicating that LD abundance in primary tumor might serve as a prognostic marker for metastatic potential. Our studies have important implications for the development of TNBC therapeutics to specifically block CDCP1-driven FAO and oxidative phosphorylation, which contribute to TNBC migration and metastasis.he transmembrane glycoprotein, CUB-domain containing protein 1 (CDCP1), is a driver of migration and invasion in multiple forms of carcinoma, including renal (1, 2), ovarian (3, 4), prostate (5), pancreatic (6, 7), colon (8-12), and triple-negative breast (TNBC) (13, 14) carcinomas, among others. Furthermore, CDCP1's role in tumor metastasis was confirmed in vivo in lung (15, 16), ovarian (17), prostate (5), and colon (9) cancers. Although CDCP1's role in TNBC metastasis has not been established to date, high CDCP1 expression has been validated as a prognostic marker of poor survival in TNBC when combined with positive node status (18).Our understanding of CDCP1's upstream regulators, its mechanism of activation, and downstream signaling continues to expand (recently reviewed in ref. 19). Studies by others (5) and our group (14) have demonstrated that CDCP1 cleavage is necessary for its activation, and recently, we have further shown that cleavage stimulates CDCP1 homodimerization (14). Homodimeric CDCP1 stimulates phosphorylation of protein kinase C δ (PKCδ) by Src kinase, leading to migration and invasion of TNBC cells in vitro (14). Adding to our knowledge of CDCP1's downstream signaling, we report that CDCP1 regulates lipid m...

CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in patients with relapsed/refractory B-cell malignancies; however, it is associated with toxicities including cytokine release syndrome (CRS), neurotoxicity, and impaired hematopoietic recovery. The latter is associated with high-grade cytopenias requiring extended growth factor or transfusional support, potentially leading to additional complications such as infection or hemorrhage. To date, the factors independently associated with hematologic toxicity have not been well characterized. To address this deficit, we retrospectively analyzed 173 patients who received defined-composition CD19 CAR T-cell therapy in a phase 1/2 clinical trial (https://clinicaltrials.gov; NCT01865617), with primary end points of absolute neutrophil count and platelet count at day-28 after CAR T-cell infusion. We observed cumulative incidences of neutrophil and platelet recovery of 81% and 75%, respectively, at 28 days after infusion. Hematologic toxicity was noted in a significant subset of patients, with persistent neutropenia in 9% and thrombocytopenia in 14% at last follow-up. Using debiased least absolute shrinkage selector and operator regression analysis for high-dimensional modeling and considering patient-, disease-, and treatment-related variables, we identified increased CRS severity as an independent predictor for decreased platelet count and lower prelymphodepletion platelet count as an independent predictor of both decreased neutrophil and platelet counts after CD19 CAR T-cell infusion. Furthermore, multivariable models including CRS-related cytokines identified associations between higher peak serum concentrations of interleukin-6 and lower day-28 cell counts; in contrast, higher serum concentrations of transforming growth factor-β1 were associated with higher counts. Our findings suggest that patient selection and improved CRS management may improve hematopoietic recovery after CD19 CAR T-cell therapy.

BackgroundLeucine may function as a signaling molecule to regulate metabolism. We have previously shown that dietary leucine supplementation significantly improves glucose and energy metabolism in diet-induced obese mice, suggesting that leucine supplementation could potentially be a useful adjuvant therapy for obesity and type 2 diabetes. Since the underlying cause for obesity and type 2 diabetes is multifold, we further investigated metabolic effects of leucine supplementation in obese/diabetes mouse models with different etiologies, and explored the underlying molecular mechanisms.MethodsLeucine supplementation was carried out in NONcNZO10/LtJ (RCS10) - a polygenic model predisposed to beta cell failure and type 2 diabetes, and in B6.Cg-Ay/J (Ay) - a monogenic model for impaired central melanocortin receptor signaling, obesity, and severe insulin resistance. Mice in the treatment group received the drinking water containing 1.5% leucine for up to 8 months; control mice received the tap water. Body weight, body composition, blood HbA1c levels, and plasma glucose and insulin levels were monitored throughout and/or at the end of the study period. Indirect calorimetry, skeletal muscle gene expression, and adipose tissue inflammation were also assessed in Ay mice.ResultsLeucine supplementation significantly reduced HbA1c levels throughout the study period in both RCS10 and Ay mice. However, the treatment had no long term effect on body weight or adiposity. The improvement in glycemic control was associated with an increased insulin response to food challenge in RCS10 mice and decreased plasma insulin levels in Ay mice. In leucine-treated Ay mice, energy expenditure was increased by ~10% (p < 0.05) in both dark and light cycles while the physical activity level was unchanged. The expression levels of UCP3, CrAT, PPAR-alpha, and NRF-1, which are known to regulate mitochondrial oxidative function, were significantly increased in the soleus muscle of leucine-treated Ay mice whereas the expression levels of MCP-1 and TNF-alpha and macrophage infiltration in adipose tissue were significantly reduced.ConclusionsChronic leucine supplementation significantly improves glycemic control in multiple mouse models of obesity and diabetes with distinct etiologies. The metabolic benefits of leucine supplementation are likely mediated via multiple mechanisms in different tissues, but are not necessarily dependent of weight reduction.

, "Correlating two-photon excited fluorescence imaging of breast cancer cellular redox state with seahorse flux analysis of normalized cellular oxygen consumption," J.

We present a multiphoton microscope designed for mesoscale imaging of human skin. The system is based on two-photon excited fluorescence and second-harmonic generation, and images areas of ~0.8x0.8 mm 2 at speeds of 0.8 fps (800x800 pixels; 12 frame averages) for high signal-to-noise ratio, with lateral and axial resolutions of 0.5µm and 3.3µm, respectively. The main novelty of this instrument is the design of the scan head, which includes a fast galvanometric scanner, optimized relay optics, a beam expander and high NA objective lens. Computed aberrations in focus are below the Marechal criterion of 0.07λ rms for diffraction-limited performance. We demonstrate the practical utility of this microscope by ex-vivo imaging of wide areas in normal human skin. Sterry, and A. Patzelt, "In vivo methods for the analysis of the penetration of topically applied substances in and through the skin barrier," Int.