Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease

Biomarkers are becoming increasingly important in the clinical management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous molecules. Here we describe the development of exogenously administered 'synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiology for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: they target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that these agents can noninvasively monitor liver fibrosis and resolution without the need for invasive core biopsies and substantially improve early detection of cancer compared with current clinically used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to additional pathophysiological processes and point-of-care diagnostics.National Institutes of Health (U.S.) (Bioengineering Research Partnership R01 CA124427)Kathy and Curt Marble Cancer Research FundNational Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (F32CA159496-01

Metabolic Regulation of Invadopodia and Invasion by Acetyl-CoA Carboxylase 1 and De novo Lipogenesis

Invadopodia are membrane protrusions that facilitate matrix degradation and cellular invasion. Although lipids have been implicated in several aspects of invadopodia formation, the contributions of de novo fatty acid synthesis and lipogenesis have not been defined. Inhibition of acetyl-CoA carboxylase 1 (ACC1), the committed step of fatty acid synthesis, reduced invadopodia formation in Src-transformed 3T3 (3T3-Src) cells, and also decreased the ability to degrade gelatin. Inhibition of fatty acid synthesis through AMP-activated kinase (AMPK) activation and ACC phosphorylation also decreased invadopodia incidence. The addition of exogenous 16∶0 and 18∶1 fatty acid, products of de novo fatty acid synthesis, restored invadopodia and gelatin degradation to cells with decreased ACC1 activity. Pharmacological inhibition of ACC also altered the phospholipid profile of 3T3-Src cells, with the majority of changes occurring in the phosphatidylcholine (PC) species. Exogenous supplementation with the most abundant PC species, 34∶1 PC, restored invadopodia incidence, the ability to degrade gelatin and the ability to invade through matrigel to cells deficient in ACC1 activity. On the other hand, 30∶0 PC did not restore invadopodia and 36∶2 PC only restored invadopodia incidence and gelatin degradation, but not cellular invasion through matrigel. Pharmacological inhibition of ACC also reduced the ability of MDA-MB-231 breast, Snb19 glioblastoma, and PC-3 prostate cancer cells to invade through matrigel. Invasion of PC-3 cells through matrigel was also restored by 34∶1 PC supplementation. Collectively, the data elucidate the novel metabolic regulation of invadopodia and the invasive process by de novo fatty acid synthesis and lipogenesis

Nicotinamide phosphoribosyl transferase (Nampt) is required for de novo lipogenesis in tumor cells.

Tumor cells have increased metabolic requirements to maintain rapid growth. In particular, a highly lipogenic phenotype is a hallmark of many tumor types, including prostate. Cancer cells also have increased turnover of nicotinamide adenine dinucleotide (NAD(+)), a coenzyme involved in multiple metabolic pathways. However, a specific role for NAD(+) in tumor cell lipogenesis has yet to be described. Our studies demonstrate a novel role for the NAD(+)-biosynthetic enzyme Nicotinamide phosphoribosyltransferase (Nampt) in maintaining de novo lipogenesis in prostate cancer (PCa) cells. Inhibition of Nampt reduces fatty acid and phospholipid synthesis. In particular, short chain saturated fatty acids and the phosphatidylcholine (PC) lipids into which these fatty acids are incorporated were specifically reduced by Nampt inhibition. Nampt blockade resulted in reduced ATP levels and concomitant activation of AMP-activated protein kinase (AMPK) and phosphorylation of acetyl-CoA carboxylase (ACC). In spite of this, pharmacological inhibition of AMPK was not sufficient to fully restore fatty acid synthesis. Rather, Nampt blockade also induced protein hyperacetylation in PC-3, DU145, and LNCaP cells, which correlated with the observed decreases in lipid synthesis. Moreover, the sirtuin inhibitor Sirtinol, and the simultaneous knockdown of SIRT1 and SIRT3, phenocopied the effects of Nampt inhibition on fatty acid synthesis. Altogether, these data reveal a novel role for Nampt in the regulation of de novo lipogenesis through the modulation of sirtuin activity in PCa cells

Role of extracellular vesicles secretion in paclitaxel resistance of prostate cancer cells

Aim: The development of chemotherapy resistance is the major obstacle in the treatment of advanced prostate cancer (PCa). Extracellular vesicles (EVs) secretion plays a significant role among different mechanisms contributing to chemoresistance. Hence, inhibition of EVs release may increase the efficacy of chemotherapeutic drugs against PCa.Methods: Paclitaxel (PTX) resistant PCa cells (PC3-R and DU145-R) were treated with GW4869, a known exosome biogenesis inhibitor. EVs were isolated from the conditioned media by ExoQuick-based precipitation method and characterized for concentration and size distribution by nanoparticle tracking analysis. The effect of GW4869 treatment on the survival and growth of PCa cells was assessed by MTT, and colony formation assays in vitro, and ectopic PC3-R xenografts in male athymic nude mice in vivo. The effect of other EV biogenesis inhibitors, imipramine and dimethyl amiloride (DMA), treatment was also analyzed on the survival of PC3-R cells.Results: GW4869 (10-20 µM) treatment of PTX resistant PCa cells significantly reduced the release of small EVs (50-100 nm size range) while increasing the release of larger EVs (> 150 nm in size), and inhibited their clonogenicity. Moreover, GW4869 (5-20 µM) treatment (24-72h) significantly inhibited the survival of PC3-R cells in a dose-dependent manner. We observed a similar growth inhibition with both imipramine (5-20 µg/mL) and DMA (5-20 µg/mL) treatment in PC3-R cells. Furthermore, GW4869 treatment (IP) in mice bearing PC3-R xenografts significantly reduced the tumor weight (65% reduction, P = 0.017) compared to the vehicle-treated control mice without causing any noticeable toxicity.Conclusion: Inhibiting the release of EVs could sensitize the resistant PCa cells to chemotherapy

Pyruvate Dehydrogenase Inhibition Leads to Decreased Glycolysis, Increased Reliance on Gluconeogenesis and Alternative Sources of Acetyl-CoA in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive disease characterized by poor outcomes and therapy resistance. Devimistat is a novel agent that inhibits pyruvate dehydrogenase complex (PDH). A phase III clinical trial in AML patients combining devimistat and chemotherapy was terminated for futility, suggesting AML cells were able to circumvent the metabolic inhibition of devimistat. The means by which AML cells resist PDH inhibition is unknown. AML cell lines treated with devimistat or deleted for the essential PDH subunit, PDHA, showed a decrease in glycolysis and decreased glucose uptake due to a reduction of the glucose transporter GLUT1 and hexokinase II. Both devimistat-treated and PDHA knockout cells displayed increased sensitivity to 2-deoxyglucose, demonstrating reliance on residual glycolysis. The rate limiting gluconeogenic enzyme phosphoenolpyruvate carboxykinase 2 (PCK2) was significantly upregulated in devimistat-treated cells, and its inhibition increased sensitivity to devimistat. The gluconeogenic amino acids glutamine and asparagine protected AML cells from devimistat. Non-glycolytic sources of acetyl-CoA were also important with fatty acid oxidation, ATP citrate lyase (ACLY) and acyl-CoA synthetase short chain family member 2 (ACSS2) contributing to resistance. Finally, devimistat reduced fatty acid synthase (FASN) activity. Taken together, this suggests that AML cells compensate for PDH and glycolysis inhibition by gluconeogenesis for maintenance of essential glycolytic intermediates and fatty acid oxidation, ACLY and ACSS2 for non-glycolytic production of acetyl-CoA. Strategies to target these escape pathways should be explored in AML

Nampt activity is required for fatty acid synthesis in cancer cells

<p>(A) PCa cells were treated with FK866 (100 nM) in the absence or presence of NAD⁺, NMN, or Na for 48 hours and fatty acid synthesis was determined by the incorporation of ¹⁴C-acetate into the lipid (*p<0.0001). Expression of ACC, AceCS1, FASN, and β-actin was determined by western blot. (B) Fatty acid synthesis was measured in Snb-19 glioblastoma cells, Src-transformed 3T3 fibroblasts, and MCF-7 breast cancer cells as in (A) (*p<0.0001). (C) PC-3 cells were transfected with scrambled or Nampt-targeting siRNA (100 nM) and fatty acid synthesis was assayed 5 days post-transfection (*p<0.0001). (D) PC-3 cells were treated as indicated and the fatty acid profile was determined by GC-MS.</p

Sirtuin activity is required for lipogenesis in prostate cancer cells

<p>(A) PCa cells were treated as indicated for 48 hours and global protein acetylation was measured by western blot. (B) PCa cells were treated with DMSO (0.1%) or Sirtinol (100 µM) for 48 hours and ¹⁴C-acetate and -choline incorporation into lipid were measured (*p<0.0001). (C) PC-3 cells were treated with scrambled, SIRT1, or SIRT3 targeting siRNA (100 nM each) and fatty acid synthesis was measured. The expression levels of SIRT1, SIRT3, and β-actin were determined by western blot. (D) PC-3 and LNCaP cells were transfected with scrambled (200 nM) or the combination of SIRT1.2 and SIRT3.2 targeting siRNA (100 nM each). The incorporation of ¹⁴C-acetate and -choline into lipid were determined 5 days after transfection (*p = 0.0002). The levels of SIRT1, SIRT3, and β-actin were determined by western blot.</p

Inhibition of Nampt activates AMPK signaling

<p>(A) Prostate tumor cells were treated with vehicle, FK866 (10 nM or 100 nM), or FK866 (100 nM) plus NAD⁺, Na or NMN. After 48 hours, ATP levels were measured by luminescence and normalized to DNA content (*p<0.0001). (B) The levels of pACC and ACC were determined in cells treated with vehicle (0.1%), or FK866 (100 nM) in the absence or presence of NAD⁺ for 48 hours (100 µM). (C) PC-3 cells were treated with vehicle, FK866 (100 nM), Compound C (CC, 10 µM), or the combination of both (FK+CC) for 48 hours and pACC, ACC, pAMPK, and AMPK levels were determined by western blot. (D) PC-3 cells were treated with vehicle, FK866, Compound C, or the combination of both for 48 hours and fatty acid synthesis was measured (*p<0.0001, #p = 0.0007). Cell killing was also determined by trypan blue exclusion (*p<0.0001).</p

Nampt inhibition does not affect the phosphatidylserine (PS), phosphatidylethanolamine (PE), or phosphatidylinositol (PI) lipid classes.

<p>(A–C) PC-3 cells were treated with vehicle, FK866 (100 nM) in the absence or presence of NMN (500 µM) for 48 hours and the PS, PE, and PI profiles were determined by mass spectrometry (*p<0.05).</p

Nampt activity is required for phosphatidylcholine (PC) synthesis in cancer cells

<p>(A) PCa cells were treated for 48 hours with vehicle, or FK866 (100 nM) in the absence or presence of NAD⁺ (100 µM) and lipid synthesis was assessed by ¹⁴C-choline incorporation (*p<0.0001). (B) PC-3 cells were transfected with scrambled or Nampt-targeting siRNA (100 nM) and lipid synthesis was assayed 5 days post-transfection (*p<0.0001). (C) PC-3 cells were treated as indicated and the PC profile was determined by mass spectrometry (*p<0.05).</p