Publisher Correction: Fast, sensitive method for trisaccharide biomarker detection in mucopolysaccharidosis type 1.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Fast, sensitive method for trisaccharide biomarker detection in mucopolysaccharidosis type 1.

Certain recessively inherited diseases result from an enzyme deficiency within lysosomes. In mucopolysaccharidoses (MPS), a defect in glycosaminoglycan (GAG) degradation leads to GAG accumulation followed by progressive organ and multiple system dysfunctions. Current methods of GAG analysis used to diagnose and monitor the diseases lack sensitivity and throughput. Here we report a LC-MS method with accurate metabolite mass analysis for identifying and quantifying biomarkers for MPS type I without the need for extensive sample preparation. The method revealed 225 LC-MS features that were >1000-fold enriched in urine, plasma and tissue extracts from untreated MPS I mice compared to MPS I mice treated with iduronidase to correct the disorder. Levels of several trisaccharides were elevated >10000-fold. To validate the clinical relevance of our method, we confirmed the presence of these biomarkers in urine, plasma and cerebrospinal fluid from MPS I patients and assessed changes in their levels after treatment.

SCD1 inhibition causes cancer cell death by depleting mono-unsaturated fatty acids.

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence.

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

Platelet expression profiling and clinical validation of myeloid-related protein-14 as a novel determinant of cardiovascular events.

BACKGROUND: Platelets participate in events that immediately precede acute myocardial infarction. Because platelets lack nuclear DNA but retain megakaryocyte-derived mRNAs, the platelet transcriptome provides a novel window on gene expression preceding acute coronary events. METHODS AND RESULTS: We profiled platelet mRNA from patients with acute ST-segment-elevation myocardial infarction (STEMI, n=16) or stable coronary artery disease (n=44). The platelet transcriptomes were analyzed and single-gene models constructed to identify candidate genes with differential expression. We validated 1 candidate gene product by performing a prospective, nested case-control study (n=255 case-control pairs) among apparently healthy women to assess the risk of future cardiovascular events (nonfatal myocardial infarction, nonfatal stroke, and cardiovascular death) associated with baseline plasma levels of the candidate protein. Platelets isolated from STEMI and coronary artery disease patients contained 54 differentially expressed transcripts. The strongest discriminators of STEMI in the microarrays were CD69 (odds ratio 6.2, P<0.001) and myeloid-related protein-14 (MRP-14; odds ratio 3.3, P=0.002). Plasma levels of MRP-8/14 heterodimer were higher in STEMI patients (17.0 versus 8.0 microg/mL, P<0.001). In the validation study, the risk of a first cardiovascular event increased with each increasing quartile of MRP-8/14 (Ptrend<0.001) such that women with the highest levels had a 3.8-fold increase in risk of any vascular event (P<0.001). Risks were independent of standard risk factors and C-reactive protein. CONCLUSIONS: The platelet transcriptome reveals quantitative differences between acute and stable coronary artery disease. MRP-14 expression increases before STEMI, and increasing plasma concentrations of MRP-8/14 among healthy individuals predict the risk of future cardiovascular events.

Gene expression profiles predict early relapse in ovarian cancer after platinum-paclitaxel chemotherapy.

PURPOSE: Women with advanced epithelial ovarian cancer are routinely treated with platinum-paclitaxel chemotherapy following cytoreductive surgery, yet only approximately 20% achieve long-term disease-free survival. We hypothesized that differences in gene expression before treatment could distinguish patients with short versus long time to recurrence after administration of platinum-paclitaxel combination chemotherapy. EXPERIMENTAL DESIGN: To test this hypothesis, gene expression profiling of 79 primary surgically resected tumors from women with advanced-stage, high-grade epithelial ovarian cancer was done using cDNA microarrays containing 30,721 genes. Supervised learning algorithms were applied in an effort to develop a binary classifier that could discriminate women at risk for early (< or =21 months) versus late (>21 months) relapse after initial chemotherapy. RESULTS: A 14-gene predictive model was developed using a set of training samples (n = 51) and subsequently tested using an independent set of test samples (n = 28). This model correctly predicted the outcome of 24 of the 28 test samples (86% accuracy) with 95% positive predictive value for early relapse. CONCLUSIONS: Predictive markers for early recurrence can be identified for platinum-paclitaxel combination chemotherapy in primary ovarian carcinoma. The proposed 14-gene model requires further validation.

Utilizing Nottingham Prognostic Index in microarray gene expression profiling of breast carcinomas.

We report a novel approach to gene expression profiling using the Nottingham Prognostic Index (NPI) to stratify 26 patients with invasive breast carcinoma. As an aggregate index of parameters reflecting metastatic potential, growth rate, and genetic instability the NPI has distinct advantages over other clinicopathologic features used to segregate breast cancer patients. As a continuous variable it offers a responsive and sensitive means of modeling a continuum of clinical aggressiveness. Using RNA extracted from 26 tumors and cDNA microarrays with 23 343 unique genetic elements, 84 genes and expressed sequence tags were identified whose expression patterns correlated with NPI. Differential expression by immunohistochemistry (IHC) was also observed for two of three genes evaluated by this method. Correlation was determined by the Spearman rank correlation method with null distribution analysis. Among the 84 genetic elements were seven previously implicated in neoplastic progression (including the two demonstrating differential expression by IHC), 11 without specific cancer association but localized to chromosomal sites whose loss or gain has been identified in cytogenetic studies of breast carcinoma, and 73 not previously associated with breast carcinoma. Collectively, the expression patterns of these 84 elements have potential to distinguish high and low NPI patient samples. These data add support to the assertion that prognostic groups of breast carcinoma are reflected in distinguishable expression profiles of a limited set of genes.