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2.  Variable Reference Alignment: an improved peak alignment protocol for NMR spectral data with large inter-sample variation 
Analytical Chemistry  2012;84(12):5372-5379.
In an effort to address the variable correspondence problem across large sample cohorts common in metabolomic/metabonomic studies, we have developed a pre-alignment protocol that aims to generate spectral segments sharing a common target spectrum. Under the assumption that a single reference spectrum will not correctly represent all spectra of a data set, the goal of this approach is to perform local alignment corrections on spectral regions which share a common ‘most similar’ spectrum. A natural beneficial outcome of this procedure is the automatic definition of spectral segments, a feature that is not common to all alignment methods. This protocol is shown to specifically improve the quality of alignment in 1H NMR data sets exhibiting large inter-sample compositional variation (e.g. pH, ionic strength). As a proof-of-principle demonstration, we have utilized two recently developed alignment algorithms specific to NMR data, recursive segment-wise peak alignment and interval correlated shifting and applied them to two data sets comprised of 15 aqueous cell line extract and 20 human urine 1H NMR profiles. Application of this protocol represents a fundamental shift from current alignment methodologies that seek to correct misalignments utilizing a single representative spectrum, with the added benefit that it can be appended to any alignment algorithm.
PMCID: PMC3381959  PMID: 22616856
Metabolomic; alignment; NMR; urine
3.  The Mutational Landscape of Lethal Castrate Resistant Prostate Cancer 
Nature  2012;487(7406):239-243.
Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains/losses, including ETS gene fusions, PTEN loss and androgen receptor (AR) amplification, that drive prostate cancer development and progression to lethal, metastatic castrate resistant prostate cancer (CRPC)1. As less is known about the role of mutations2–4, here we sequenced the exomes of 50 lethal, heavily-pretreated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment naïve, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPC (2.00/Mb) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1, which define a subtype of ETS fusionnegative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in ~1/3 of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Further, we identified recurrent mutations in multiple chromatin/histone modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with AR, which is required for AR-mediated signaling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signaling and increases tumour growth. Proteins that physically interact with AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX, and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signaling deregulated in prostate cancer, and prioritize candidates for future study.
PMCID: PMC3396711  PMID: 22722839
4.  A Novel Kinase Inhibitor of FADD Phosphorylation Chemosensitizes through the Inhibition of NF-κB 
Molecular cancer therapeutics  2011;10(10):1807-1817.
FADD (Fas-associated protein with death domain) is a cytosolic adapter protein essential for mediating death receptor-induced apoptosis. It has also been implicated in a number of non-apoptotic activities including embryogenesis, cell-cycle progression, cell proliferation, and tumorigenesis. Our recent studies have demonstrated that high levels of phosphorylated FADD in tumor cells correlates with increased activation of the anti-apoptotic transcription factor NF-κB and is a biomarker for aggressive disease and poor clinical outcome. These findings suggest that inhibition of FADD phosphorylation is a viable target for cancer therapy. A high throughput screen using a cell-based assay for monitoring FADD-kinase activity identified NSC 47147 as a small molecule inhibitor of FADD phosphorylation. The compound was evaluated in live cells and mouse tumors for its efficacy as an inhibitor of FADD-kinase activity through the inhibition of CK1α. NSC 47147 was shown to decrease levels of phosphorylated FADD and NF-κB activity such that combination therapy lead to greater induction of apoptosis and enhanced tumor control as compared to either agent alone. The studies described here demonstrate the utility of bioluminescent cell based assays for the identification of active compounds and the validation of drug target interaction in a living subject. In addition, the presented results provide proof of principle studies as to the validity of targeting FADD-kinase activity as a novel cancer therapy strategy.
PMCID: PMC3191281  PMID: 21859840
FADD; phosphorylation; non-invasive molecular imaging; NF-κB; chemotherapy
5.  High Throughput Molecular Imaging for the Identification of FADD Kinase Inhibitors 
Journal of biomolecular screening  2010;15(9):1063-1070.
Fas-Associated protein with Death Domain (FADD) was originally reported as a pro-apoptotic adaptor molecule that mediates receptor induced apoptosis. Recent studies have revealed a potential role of FADD in NF-κB activation, embryogenesis, and cell cycle regulation and proliferation. Over-expression of FADD and its phosphorylation have been associated with the transformed phenotype in many cancers and is therefore a potential target for therapeutic intervention. In an effort to delineate signaling events that lead to FADD phosphorylation and to identify novel compounds that impinge on this pathway, we developed a cell based reporter for FADD kinase activity. The reporter assay, optimized for a high throughput screen (HTS), measures bioluminescence in response to modulation of FADD kinase activity in live cells. In addition, the potential use of the reporter cell line in the rapid evaluation of pharmacologic properties of HTS hits in mouse models has been demonstrated.
PMCID: PMC3108567  PMID: 20855560
FADD; phosphorylation; non-invasive molecular imaging; bioluminescence; kinase activity
6.  Metabolomic Profiles Delineate Potential Role for Sarcosine in Prostate Cancer Progression 
Nature  2009;457(7231):910-914.
Multiple, complex molecular events characterize cancer development and progression1,2. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of critical biomarkers for cancer invasion and disease aggressiveness. Although gene and protein expression have been extensively profiled in human tumors, little is known about the global metabolomic alterations that characterize neoplastic progression. Using a combination of high throughput liquid and gas chromatography-based mass spectrometry, we profiled more than 1126 metabolites across 262 clinical samples related to prostate cancer (42 tissues and 110 each of urine and plasma). These unbiased metabolomic profiles were able to distinguish benign prostate, clinically localized prostate cancer, and metastatic disease. Sarcosine, an N-methyl derivative of the amino acid glycine, was identified as a differential metabolite that was highly elevated during prostate cancer progression to metastasis and can be detected non-invasively in urine. Sarcosine levels were also elevated in invasive prostate cancer cell lines relative to benign prostate epithelial cells. Knockdown of glycine-N-methyl transferase (GNMT), the enzyme that generates sarcosine from glycine, attenuated prostate cancer invasion. Addition of exogenous sarcosine or knockdown of the enzyme that leads to sarcosine degradation, sarcosine dehydrogenase (SARDH), induced an invasive phenotype in benign prostate epithelial cells. Androgen receptor and the ERG gene fusion product coordinately regulate components of the sarcosine pathway. Taken together, we profiled the metabolomic alterations of prostate cancer progression revealing sarcosine as a potentially important metabolic intermediary of cancer cell invasion and aggressivity.
PMCID: PMC2724746  PMID: 19212411

Results 1-6 (6)