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Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a “proof-of-principle” that enzymatic inhibition of QSOX1 may have clinical relevancy.

ContributorsHanavan, Paul (Author) / Borges, Chad (Author) / Katchman, Benjamin (Author) / Faigel, Douglas O. (Author) / Ho, Thai H. (Author) / Ma, Chen-Ting (Author) / Sergienko, Eduard A. (Author) / Meurice, Nathalie (Author) / Petit, Joachim L. (Author) / Lake, Douglas (Author) / College of Liberal Arts and Sciences (Contributor)
Created2015-06-01
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Clear cell renal cell carcinomas (ccRCCs) harbor frequent mutations in epigenetic modifiers including SETD2, the H3K36me3 writer. We profiled DNA methylation (5mC) across the genome in cell line-based models of SETD2 inactivation and SETD2 mutant primary tumors because 5mC has been linked to H3K36me3 and is therapeutically targetable. SETD2 depleted

Clear cell renal cell carcinomas (ccRCCs) harbor frequent mutations in epigenetic modifiers including SETD2, the H3K36me3 writer. We profiled DNA methylation (5mC) across the genome in cell line-based models of SETD2 inactivation and SETD2 mutant primary tumors because 5mC has been linked to H3K36me3 and is therapeutically targetable. SETD2 depleted cell line models (long-term and acute) exhibited a DNA hypermethylation phenotype coinciding with ectopic gains in H3K36me3 centered across intergenic regions adjacent to low expressing genes, which became upregulated upon dysregulation of the epigenome. Poised enhancers of developmental genes were prominent hypermethylation targets. SETD2 mutant primary ccRCCs, papillary renal cell carcinomas, and lung adenocarcinomas all demonstrated a DNA hypermethylation phenotype that segregated tumors by SETD2 genotype and advanced grade. These findings collectively demonstrate that SETD2 mutations drive tumorigenesis by coordinated disruption of the epigenome and transcriptome,and they have important implications for future therapeutic strategies targeting chromatin regulator mutant tumors.

ContributorsTiedmann, Rochelle L. (Author) / Hlady, Ryan A. (Author) / Hanavan, Paul (Author) / Lake, Douglas (Author) / Tibes, Raoul (Author) / Lee, Jeong-Heon (Author) / Choi, Jeong-Hyeon (Author) / Ho, Thai H. (Author) / Robertson, Keith D. (Author) / College of Liberal Arts and Sciences (Contributor)
Created2015-12-05
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Background: Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scalec linical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise

Background: Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scalec linical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers.

Methods: We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNAfrom a 63 year-old man who underwent a pancreaticoduodenectomy by whole genomesequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations.

Results: We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition.

Conclusions: Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.

ContributorsDemeure, Michael J. (Author) / Craig, David W. (Author) / Sinari, Shripad (Author) / Moses, Tracy M. (Author) / Christoforides, Alexis (Author) / Dinh, Jennifer (Author) / Izatt, Tyler (Author) / Aldrich, Jessica (Author) / Decker, Ardis (Author) / Baker, Angela (Author) / Cherni, Irene (Author) / Watanabe, April (Author) / Koep, Lawrence (Author) / Lake, Douglas (Author) / Hostetter, Galen (Author) / Trent, Jeffrey M. (Author) / Von Hoff, Daniel D. (Author) / Carpten, John D. (Author) / College of Liberal Arts and Sciences (Contributor)
Created2012-07-04
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Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = – 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired β-F1-ATPase translation as an important consequence of obesity.

ContributorsTran, Lee (Author) / Hanavan, Paul (Author) / Campbell, Latoya (Author) / De Filippis, Elena (Author) / Lake, Douglas (Author) / Coletta, Dawn (Author) / Roust, Lori R. (Author) / Mandarino, Lawrence (Author) / Carroll, Chad C. (Author) / Katsanos, Christos (Author) / College of Health Solutions (Contributor)
Created2016-08-17
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Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to

Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to understand the range of aberrations that are occurring in each tumor. Because our understanding of PAC tumorigenesis is limited, evaluation of separate cases may reveal aberrations, that are less common but may provide relevant information on the disease, or that may represent viable therapeutic targets for the patient. We used next generation sequencing to assess global somatic events across 3 PAC patients to characterize each patient and to identify potential targets. This study is the first to report whole genome sequencing (WGS) findings in paired tumor/normal samples collected from 3 separate PAC patients. We generated on average 132 billion mappable bases across all patients using WGS, and identified 142 somatic coding events including point mutations, insertion/deletions, and chromosomal copy number variants. We did not identify any significant somatic translocation events. We also performed RNA sequencing on 2 of these patients' tumors for which tumor RNA was available to evaluate expression changes that may be associated with somatic events, and generated over 100 million mapped reads for each patient. We further performed pathway analysis of all sequencing data to identify processes that may be the most heavily impacted from somatic and expression alterations. As expected, the KRAS signaling pathway was the most heavily impacted pathway (P<0.05), along with tumor-stroma interactions and tumor suppressive pathways. While sequencing of more patients is needed, the high resolution genomic and transcriptomic information we have acquired here provides valuable information on the molecular composition of PAC and helps to establish a foundation for improved therapeutic selection.

ContributorsLiang, Winnie S. (Author) / Craig, David W. (Author) / Carpten, John (Author) / Borad, Mitesh J. (Author) / Demeure, Michael J. (Author) / Weiss, Glen J. (Author) / Izatt, Tyler (Author) / Sinari, Shripad (Author) / Christoforides, Alexis (Author) / Aldrich, Jessica (Author) / Kurdoglu, Ahmet (Author) / Barrett, Michael (Author) / Phillips, Lori (Author) / Benson, Hollie (Author) / Tembe, Waibhav (Author) / Braggio, Esteban (Author) / Kiefer, Jeffrey A. (Author) / Legendre, Christophe (Author) / Posner, Richard (Author) / Hostetter, Galen H. (Author) / Baker, Angela (Author) / Egan, Jan B. (Author) / Han, Haiyong (Author) / Lake, Douglas (Author) / Stites, Edward C. (Author) / Ramanathan, Ramesh K. (Author) / Fonseca, Rafael (Author) / Stewart, A. Keith (Author) / Von Hoff, Daniel (Author) / College of Liberal Arts and Sciences (Contributor)
Created2012-10-10
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Description

Introduction: Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. Tumor specific expression of QSOX1 has been reported for numerous tumor types. In this study, we investigate QSOX1 as a marker of breast tumor progression and evaluate the role of QSOX1 as it relates to breast

Introduction: Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. Tumor specific expression of QSOX1 has been reported for numerous tumor types. In this study, we investigate QSOX1 as a marker of breast tumor progression and evaluate the role of QSOX1 as it relates to breast tumor growth and metastasis.

Methods: Correlation of QSOX1 expression with breast tumor grade, subtype and estrogen receptor (ER) status was gathered through informatic analysis using the "Gene expression based Outcome for Breast cancer Online" (GOBO) web-based tool. Expression of QSOX1 protein in breast tumors tissue microarray (TMA) and in a panel of breast cancer cell lines was used to confirm our informatics analysis. To investigate malignant cell mechanisms for which QSOX1 might play a key role, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in ER+ Luminal A-like MCF7, ER+ Luminal B-like BT474 and ER- Basal-like BT549 breast cancer cell lines.

Results: GOBO analysis revealed high levels of QSOX1 RNA expression in ER+ subtypes of breast cancer. In addition, Kaplan Meyer analyses revealed QSOX1 RNA as a highly significant predictive marker for both relapse and poor overall survival in Luminal B tumors. We confirmed this finding by evaluation of QSOX1 protein expression in breast tumors and in a panel of breast cancer cell lines. Expression of QSOX1 in breast tumors correlates with increasing tumor grade and high Ki-67 expression. Suppression of QSOX1 protein slowed cell proliferation as well as dramatic inhibition of MCF7, BT474 and BT549 breast tumor cells from invading through Matrigel™ in a modified Boyden chamber assay. Inhibition of invasion could be rescued by the exogenous addition of recombinant QSOX1. Gelatin zymography indicated that QSOX1 plays an important role in the function of MMP-9, a key mediator of breast cancer invasive behavior.

Conclusions: Taken together, our results suggest that QSOX1 is a novel biomarker for risk of relapse and poor survival in Luminal B breast cancer, and has a pro-proliferative and pro-invasive role in malignant progression partly mediated through a decrease in MMP-9 functional activity.

ContributorsKatchman, Benjamin (Author) / Ocal, I. Tolgay (Author) / Cunliffe, Heather E. (Author) / Chang, Yu-Hui (Author) / Hostetter, Galen (Author) / Watanabe, April (Author) / LoBello, Janine (Author) / Lake, Douglas (Author) / College of Liberal Arts and Sciences (Contributor)
Created2013-03-28