Publications by authors named "Michael J Slifker"

23 Publications

  • Page 1 of 1

Analysis of differential neonatal lethality in cystathionine β-synthase deficient mouse models using metabolic profiling.

FASEB J 2021 Jun;35(6):e21629

Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, USA.

Cystathionine beta-synthase (CBS) is a key enzyme of the trans-sulfuration pathway that converts homocysteine to cystathionine. Loss of CBS activity due to mutation results in CBS deficiency, an inborn error of metabolism characterized by extreme elevation of plasma total homocysteine (tHcy). C57BL6 mice containing either a homozygous null mutation in the cystathionine β-synthase (Cbs ) gene or an inactive human CBS protein (Tg-G307S Cbs ) are born in mendelian numbers, but the vast majority die between 18 and 21 days of age due to liver failure. However, adult Cbs null mice that express a hypomorphic allele of human CBS as a transgene (Tg-I278T Cbs ) show almost no neonatal lethality despite having serum tHcy levels similar to mice with no CBS activity. Here, we characterize liver and serum metabolites in neonatal Cbs , Tg-G307S Cbs , and Tg-I278T Cbs mice at 6, 10, and 17 days of age to understand this difference. In serum, we observe similar elevations in tHcy in both Tg-G307S Cbs and Tg-I278T Cbs compared to control animals, but methionine is much more severely elevated in Tg-G307S Cbs mice. Large scale metabolomic analysis of liver tissue confirms that both methionine and methionine-sulfoxide are significantly more elevated in Tg-G307S Cbs animals, along with significant differences in several other metabolites including hexoses, amino acids, other amines, lipids, and carboxylic acids. Our data are consistent with a model that the neonatal lethality observed in CBS-null mice is driven by excess methionine resulting in increased stress on a variety of related pathways including the urea cycle, TCA cycle, gluconeogenesis, and phosphatidylcholine biosynthesis.
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http://dx.doi.org/10.1096/fj.202100302RDOI Listing
June 2021

Overall tumor genomic instability: an important predictor of recurrence-free survival in patients with localized clear cell renal cell carcinoma.

Cancer Biol Ther 2020 05 1;21(5):424-431. Epub 2020 Mar 1.

Genomics Facility, Fox Chase Cancer Center, Philadelphia, PA, USA.

Measurement of a tumor's overall genomic instability has gathered recent interest over the identification of specific genomic imbalances, as it may provide a more robust measure of tumor aggressiveness. Here we demonstrate the association of tumor genomic instability in the prediction of disease recurrence in patients with clinically localized clear cell renal cell carcinoma (ccRCC). Genomic copy number analysis was performed using SNP-based microarrays on tumors from 103 ccRCC patients. The number of copy number alterations (CNAs) for each tumor was calculated, and a genomic imbalance threshold (GIT) associated with high stage and high-grade disease was determined. Cox proportional hazards regression analyzes were performed to assess the effect of GIT on recurrence-free survival adjusting for known confounders. In the cohort, copy number losses in chromosome arms 3p, 14q, 6q, 9p, and 1p and gains of 5q and 7p/q were common. CNA burden significantly increased with increasing stage ( < .001) and grade ( < .001). The median CNA burden associated with patients presenting with advanced stage (IV) and high-grade (III/IV) tumors was ≥9, defining the GIT. On regression analysis, GIT was a superior predictor of recurrence (Hazard Ratio 4.44 [CI 1.36-14.48], = .01) independent of stage, with similar results adjusting for grade. These findings were confirmed using an alternative measure of genomic instability, weighted Genomic Integrity Index. Our data support a key role for genomic instability in ccRCC progression. More importantly, we have identified a GIT (≥ 9 CNAs) that is a superior and independent predictor of disease recurrence in high-risk ccRCC patients.
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http://dx.doi.org/10.1080/15384047.2020.1721251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515487PMC
May 2020

ZBP1/DAI Drives RIPK3-Mediated Cell Death Induced by IFNs in the Absence of RIPK1.

J Immunol 2019 09 29;203(5):1348-1355. Epub 2019 Jul 29.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA 19111;

Receptor-interacting protein kinase 1 (RIPK1) regulates cell fate and proinflammatory signaling downstream of multiple innate immune pathways, including those initiated by TNF-α, TLR ligands, and IFNs. Genetic ablation of results in perinatal lethality arising from both RIPK3-mediated necroptosis and FADD/caspase-8-driven apoptosis. IFNs are thought to contribute to the lethality of -deficient mice by activating inopportune cell death during parturition, but how IFNs activate cell death in the absence of RIPK1 is not understood. In this study, we show that Z-form nucleic acid binding protein 1 (ZBP1; also known as DAI) drives IFN-stimulated cell death in settings of RIPK1 deficiency. IFN-activated Jak/STAT signaling induces robust expression of ZBP1, which complexes with RIPK3 in the absence of RIPK1 to trigger RIPK3-driven pathways of caspase-8-mediated apoptosis and MLKL-driven necroptosis. In vivo, deletion of either or core IFN signaling components prolong viability of mice for up to 3 mo beyond parturition. Together, these studies implicate ZBP1 as the dominant activator of IFN-driven RIPK3 activation and perinatal lethality in the absence of RIPK1.
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http://dx.doi.org/10.4049/jimmunol.1900216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702065PMC
September 2019

Inactivation of Cooperates with Losses of and to Drive the Development of Pleural Malignant Mesothelioma in Conditional Mouse Models.

Cancer Res 2019 08 31;79(16):4113-4123. Epub 2019 May 31.

Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

Pleural malignant mesothelioma is a therapy-resistant cancer affecting the serosal lining of the thoracic cavity. Mutations/deletions of , and are the most frequent genetic lesions in human malignant mesothelioma. We introduced various combinations of these deletions in the pleura of conditional knockout (CKO) mice, focusing on the contribution of loss. While homozygous CKO of , or alone gave rise to few or no malignant mesotheliomas, inactivation of cooperated with loss of either or to drive development of malignant mesothelioma in approximately 20% of double-CKO mice, and a high incidence (22/26, 85%) of malignant mesotheliomas was observed in (triple)-CKO mice. Malignant mesothelioma onset was rapid in triple-CKO mice, with a median survival of only 12 weeks, and malignant mesotheliomas from these mice were consistently high-grade and invasive. Adenoviral-Cre treatment of normal mesothelial cells from CKO mice, but not from mice with knockout of one or any two of these genes, resulted in robust spheroid formation , suggesting that mesothelial cells from mice have stem cell-like potential. RNA-seq analysis of malignant mesotheliomas from triple-CKO mice revealed enrichment of genes transcriptionally regulated by the polycomb repressive complex 2 (PRC2) and others previously implicated in known Bap1-related cellular processes. These data demonstrate that somatic inactivation of , and results in rapid, aggressive malignant mesotheliomas, and that deletion of contributes to tumor development, in part, by loss of PRC2-mediated repression of tumorigenic target genes and by acquisition of stem cell potential, suggesting a potential avenue for therapeutic intervention. SIGNIFICANCE: Combinatorial deletions of , and result in aggressive mesotheliomas, with loss contributing to tumorigenesis by circumventing PRC2-mediated repression of oncogenic target genes.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-4093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697648PMC
August 2019

Estrogen metabolism in the human lung: impact of tumorigenesis, smoke, sex and race/ethnicity.

Oncotarget 2017 Dec 1;8(63):106778-106789. Epub 2017 Nov 1.

Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

Previous data from this group demonstrate that the murine lung metabolizes estrogen. Production of the putative carcinogen 4-hydroxyestrogen (4-OHE) is elevated within the lungs of female vs. male mice and accelerated by tobacco smoke. The goal of this study was to determine if the human lung metabolizes estrogen and evaluate the impact of tumor formation, smoke, sex and race/ethnicity on metabolism. Urine and lung tissue (normal, tumor) were obtained from 49 non-small cell lung cancer patients. Healthy postmenopausal Caucasian ( 19) and Chinese ( 20) American women (never-smokers) donated urine. Quantitative RT-PCR analyses indicate that multiple estrogen synthesis and metabolism genes are expressed in human bronchoalveolar cells. Estrogen and its metabolites were measured in lung tissue and urine using liquid chromatography/tandem mass spectrometry. Wilcoxon rank tests were used for statistical comparisons. E, E, E and estrogen metabolites 2-OHE, 2-OHE, 4-OHE, 4-OHE, 2-OME and 2-OME were detected at higher levels in tumor vs. adjacent normal tissue and in women vs. men ( < 0.05). The proportion of 4-OHEs was higher in tumors than in normal lung tissue ( < 0.05), and elevated in normal tissue from current- vs. never-smoking women ( 0.006); similar trends were observed in urine. The proportion of 4-OHEs in the urine of postmenopausal Chinese American women was 1.8-fold higher than that of Caucasian women ( = 0.015). These data indicate that estrogen metabolites are present in the human lung. A shift towards 4-hydroxylation during lung tumorigenesis may contribute to the risk conferred by smoking, sex or race/ethnicity.
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http://dx.doi.org/10.18632/oncotarget.22269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739773PMC
December 2017

NSD1- and NSD2-damaging mutations define a subset of laryngeal tumors with favorable prognosis.

Nat Commun 2017 11 24;8(1):1772. Epub 2017 Nov 24.

Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA.

Squamous cell carcinomas of the head and neck (SCCHN) affect anatomical sites including the oral cavity, nasal cavity, pharynx, and larynx. Laryngeal cancers are characterized by high recurrence and poor overall survival, and currently lack robust molecular prognostic biomarkers for treatment stratification. Using an algorithm for integrative clustering that simultaneously assesses gene expression, somatic mutation, copy number variation, and methylation, we for the first time identify laryngeal cancer subtypes with distinct prognostic outcomes, and differing from the non-prognostic laryngeal subclasses reported by The Cancer Genome Atlas (TCGA). Although most common laryngeal gene mutations are found in both subclasses, better prognosis is strongly associated with damaging mutations of the methyltransferases NSD1 and NSD2, with findings confirmed in an independent validation cohort consisting of 63 laryngeal cancer patients. Intriguingly, NSD1/2 mutations are not prognostic for nonlaryngeal SCCHN. These results provide an immediately useful clinical metric for patient stratification and prognostication.
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http://dx.doi.org/10.1038/s41467-017-01877-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701248PMC
November 2017

MHC-I Genotype Restricts the Oncogenic Mutational Landscape.

Cell 2017 Nov 26;171(6):1272-1283.e15. Epub 2017 Oct 26.

Department of Medicine, Division of Medical Genetics, University of California San Diego, La Jolla, CA 92093, USA; Bioinformatics and Systems Biology Program, University of California San Diego, La Jolla, CA 92093, USA; Moores Cancer Center, University of California San Diego, La Jolla, CA 92093, USA; Cancer Cell Map Initiative (CCMI), University of California San Diego, La Jolla, CA 92093, USA.

MHC-I molecules expose the intracellular protein content on the cell surface, allowing T cells to detect foreign or mutated peptides. The combination of six MHC-I alleles each individual carries defines the sub-peptidome that can be effectively presented. We applied this concept to human cancer, hypothesizing that oncogenic mutations could arise in gaps in personal MHC-I presentation. To validate this hypothesis, we developed and applied a residue-centric patient presentation score to 9,176 cancer patients across 1,018 recurrent oncogenic mutations. We found that patient MHC-I genotype-based scores could predict which mutations were more likely to emerge in their tumor. Accordingly, poor presentation of a mutation across patients was correlated with higher frequency among tumors. These results support that MHC-I genotype-restricted immunoediting during tumor formation shapes the landscape of oncogenic mutations observed in clinically diagnosed tumors and paves the way for predicting personal cancer susceptibilities from knowledge of MHC-I genotype.
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http://dx.doi.org/10.1016/j.cell.2017.09.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711564PMC
November 2017

miR-6883 Family miRNAs Target CDK4/6 to Induce G Phase Cell-Cycle Arrest in Colon Cancer Cells.

Cancer Res 2017 12 23;77(24):6902-6913. Epub 2017 Oct 23.

Fox Chase Cancer Center, Philadelphia, Pennsylvania.

CDK4/6 targeting is a promising therapeutic strategy under development for various tumor types. In this study, we used computational methods and The Cancer Genome Atlas dataset analysis to identify novel miRNAs that target CDK4/6 and exhibit potential for therapeutic development in colorectal cancer. The 3'UTR of CDK4/6 mRNAs are targeted by a family of miRNAs, which includes miR-6883-5p, miR-149*, miR-6785-5p, and miR-4728-5p. Ectopic expression of miR-6883-5p or miR-149* downregulated CDK4 and CDK6 levels in human colorectal cancer cells. RNA-seq analysis revealed an inverse relationship between the expression of CDK4/6 and miR-149* and intronic miRNA-6883-5p encoding the clock gene PER1 in colorectal cancer patient samples. Restoring expression of miR-6883-5p and miR-149* blocked cell growth leading to G-G phase cell-cycle arrest and apoptosis in colorectal cancer cells. CDK4/6 targeting by miR-6883-5p and miR-149* could only partially explain the observed antiproliferative effects. Notably, both miRNAs synergized with the frontline colorectal cancer chemotherapy drug irinotecan. Further, they resensitized mutant p53-expressing cell lines resistant to 5-fluorouracil. Taken together, our results established the foundations of a candidate miRNA-based theranostic strategy to improve colorectal cancer management. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-1767DOI Listing
December 2017

A New Role for ERα: Silencing via DNA Methylation of Basal, Stem Cell, and EMT Genes.

Mol Cancer Res 2017 02 15;15(2):152-164. Epub 2016 Nov 15.

Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania.

Resistance to hormonal therapies is a major clinical problem in the treatment of estrogen receptor α-positive (ERα) breast cancers. Epigenetic marks, namely DNA methylation of cytosine at specific CpG sites (5mCpG), are frequently associated with ERα status in human breast cancers. Therefore, ERα may regulate gene expression in part via DNA methylation. This hypothesis was evaluated using a panel of breast cancer cell line models of antiestrogen resistance. Microarray gene expression profiling was used to identify genes normally silenced in ERα cells but derepressed upon exposure to the demethylating agent decitabine, derepressed upon long-term loss of ERα expression, and resuppressed by gain of ERα activity/expression. ERα-dependent DNA methylation targets (n = 39) were enriched for ERα-binding sites, basal-up/luminal-down markers, cancer stem cell, epithelial-mesenchymal transition, and inflammatory and tumor suppressor genes. Kaplan-Meier survival curve and Cox proportional hazards regression analyses indicated that these targets predicted poor distant metastasis-free survival among a large cohort of breast cancer patients. The basal breast cancer subtype markers LCN2 and IFI27 showed the greatest inverse relationship with ERα expression/activity and contain ERα-binding sites. Thus, genes that are methylated in an ERα-dependent manner may serve as predictive biomarkers in breast cancer.

Implications: ERα directs DNA methylation-mediated silencing of specific genes that have biomarker potential in breast cancer subtypes. Mol Cancer Res; 15(2); 152-64. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308451PMC
February 2017

Lack of global epigenetic methylation defects in CBS deficient mice.

J Inherit Metab Dis 2017 01 21;40(1):113-120. Epub 2016 Jul 21.

Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA.

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.
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http://dx.doi.org/10.1007/s10545-016-9958-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300059PMC
January 2017

RIPK3 Is Largely Dispensable for RIG-I-Like Receptor- and Type I Interferon-Driven Transcriptional Responses to Influenza A Virus in Murine Fibroblasts.

PLoS One 2016 8;11(7):e0158774. Epub 2016 Jul 8.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.

The kinase RIPK3 is a key regulator of cell death responses to a growing number of viral and microbial agents. We have found that influenza A virus (IAV)-mediated cell death is largely reliant on RIPK3 and that RIPK3-deficient mice are notably more susceptible to lethal infection by IAV than their wild-type counterparts. Recent studies demonstrate that RIPK3 also participates in regulating gene transcription programs during host pro-inflammatory and innate-immune responses, indicating that this kinase is not solely an inducer of cell death and that RIPK3-driven transcriptional responses may collaborate with cell death in promoting clearance of IAV. Here, we carried out DNA microarray analyses to determine the contribution of RIPK3 to the IAV-elicited host transcriptional response. We report that RIPK3 does not contribute significantly to the RLR-activated transcriptome or to the induction of type I IFN genes, although, interestingly, IFN-β production at a post-transcriptional step was modestly attenuated in IAV-infected ripk3-/- fibroblasts. Overall, RIPK3 regulated the expression of <5% of the IAV-induced transcriptome, and no genes were found to be obligate RIPK3 targets. IFN-β signaling was also found to be largely normal in the absence of RIPK3. Together, these results indicate that RIPK3 is not essential for the host antiviral transcriptional response to IAV in murine fibroblasts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158774PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938532PMC
August 2017

A global profile of gene promoter methylation in treatment-naïve urothelial cancer.

Epigenetics 2014 May 12;9(5):760-73. Epub 2014 Feb 12.

Cancer Epigenetics Program; Fox Chase Cancer Center; Philadelphia PA, USA.

The epigenetic alteration of aberrant hypermethylation in the promoter CpG island of a gene is associated with repression of transcription. In neoplastic cells, aberrant hypermethylation is well described as a mechanism of allele inactivation of particular genes with a tumor suppressor function. To investigate the role of aberrant hypermethylation in the biology and progression of urothelial cancer, we examined 101 urothelial (transitional cell) carcinomas (UC), broadly representative of the disease at presentation, with no prior immunotherapy, chemotherapy or radiotherapy, by Infinium HM27 containing 14,495 genes. The genome-wide signature of aberrant promoter hypermethylation in UC consisted of 729 genes significant by a Wilcoxon test, hypermethylated in a CpG island within 1 kb of the transcriptional start site and unmethylated in normal urothelium from aged individuals. We examined differences in gene methylation between the two main groups of UC: the 75% that are superficial, which often recur but rarely progress, and the 25% with muscle invasion and poor prognosis. We further examined pairwise comparisons of the pathologic subgroups of high or low grade, invasive or non-invasive (pTa), and high grade superficial or low grade superficial UC. Pathways analysis indicated over-representation of genes involved in cell adhesion or metabolism in muscle-invasive UC. Notably, the TET2 epigenetic regulator was one of only two genes more frequently methylated in superficial tumors and the sole gene in low grade UC. Other chromatin remodeling genes, MLL3 and ACTL6B, also showed aberrant hypermethylation. The Infinium methylation value for representative genes was verified by pyrosequencing. An available mRNA expression data set indicated many of the hypermethylated genes of interest to be downregulated in UC. Unsupervised clustering of the most differentially methylated genes distinguished muscle invasive from superficial UC. After filtering, cluster analysis showed a CpG Island Methylator Phenotype (CIMP)-like pattern of widespread methylation in 11 (11%) tumors. Nine of these 11 tumors had hypermethylation of TET2. Our analysis provides a basis for further studies of hypermethylation in the development and progression of UC.
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http://dx.doi.org/10.4161/epi.28078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063835PMC
May 2014

MicroRNA expression signatures of stage, grade, and progression in clear cell RCC.

Cancer Biol Ther 2014 Mar 18;15(3):329-41. Epub 2013 Dec 18.

Cancer Epigenetics Program; Fox Chase Cancer Center; Philadelphia, PA USA; Kidney Cancer Keystone Program; Fox Chase Cancer Center; Philadelphia, PA USA.

Clear cell RCC is the most common, and more likely to metastasize, of the three main histological types of RCC. Pathologic stage is the most important prognostic indicator and nuclear grade can predict outcome within stages of localized RCC. Epithelial tumors are thought to accumulate a series of genetic and epigenetic changes as they progress through well-defined clinical and histopathological changes. MicroRNAs (miRNAs) are involved in the regulation of mRNA expression from many human genes and miRNA expression is dysregulated in cancer. To better understand the contribution of dysregulated miRNA expression to the progression and biology of ccRCC, we examined the differences in expression levels of 723 human miRNAs through a series of analyses by stage, grade, and disease progression status in a large series of 94 ccRCC. We found a consistent signature that included significant upregulation of miR-21-5p, 142-3p, let-7g-5p, let-7i-5p and 424-5p, as well as downregulation of miR-204-5p, to be associated with ccRCC of high stage, or high grade, or progression. Discrete signatures associated with each of stage, grade, or progression were also identified. The let-7 family was significantly downregulated in ccRCC compared with normal renal parenchyma. Expression of the 6 most significantly differentially expressed miRNAs between ccRCC was verified by stem-loop qRT-PCR. Pathways predicted as targets of the most significantly dysregulated miRNAs included signaling, epithelial cancers, metabolism, and epithelial to mesenchymal transition. Our studies help to further elucidate the biology underlying the progression of ccRCC and identify miRNAs for potential translational application.
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http://dx.doi.org/10.4161/cbt.27314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3974834PMC
March 2014

Genome-wide promoter methylome of small renal masses.

PLoS One 2013 24;8(10):e77309. Epub 2013 Oct 24.

Cancer Epigenetics Program and Kidney Keystone Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.

The majority of renal cell carcinoma (RCC) is now incidentally detected and presents as small renal masses (SRMs) defined as ≤ 4 cm in size. SRMs are heterogeneous comprising several histological types of RCC each with different biology and behavior, and benign tumors mainly oncocytoma. The varied prognosis of the different types of renal tumor has implications for management options. A key epigenetic alteration involved in the initiation and progression of cancer is aberrant methylation in the promoter region of a gene. The hypermethylation is associated with transcriptional repression and is an important mechanism of inactivation of tumor suppressor genes in neoplastic cells. We have determined the genome-wide promoter methylation profiles of 47 pT1a and 2 pT1b clear cell, papillary or chromophobe RCC, 25 benign renal oncocytoma ≤ 4 cm and 4 normal renal parenchyma specimens by Infinium HumanMethylation27 beadchip technology. We identify gene promoter hypermethylation signatures that distinguish clear cell and papillary from each other, from chromophobe and oncocytoma, and from normal renal cells. Pairwise comparisons revealed genes aberrantly hypermethylated in a tumor type but unmethylated in normal, and often unmethylated in the other renal tumor types. About 0.4% to 1.7% of genes comprised the promoter methylome in SRMs. The Infinium methylation score for representative genes was verified by gold standard technologies. The genes identified as differentially methylated implicate pathways involved in metabolism, tissue response to injury, epithelial to mesenchymal transition (EMT), signal transduction and G-protein coupled receptors (GPCRs), cancer, and stem cell regulation in the biology of RCC. Our findings contribute towards an improved understanding of the development of RCC, the different biology and behavior of histological types, and discovery of molecular subtypes. The differential methylation signatures may have utility in early detection and particularly differential diagnosis for prognostic stratification as well as identify novel gene and pathway targets for therapeutic intervention.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077309PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811999PMC
August 2014

Germline Mutations in Mtap Cooperate with Myc to Accelerate Tumorigenesis in Mice.

PLoS One 2013 26;8(6):e67635. Epub 2013 Jun 26.

Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, Unites States of America.

Objective: The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (Mtap(lacZ)) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten(+/-) .

Methods: Mtap Eμ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of Mtap(lacZ/+) and Mtap (+/+) mice.

Results: Survival in both models was significantly decreased in Mtap(lacZ/+) compared to Mtap(+/+) mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap(+/+) mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc Mtap(lacZ/+) and Eµ-myc Mtap(+/+) animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap(+/+) and Mtap(lacZ/+) animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer.

Conclusion: Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067635PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694069PMC
October 2017

Correction of cystathionine β-synthase deficiency in mice by treatment with proteasome inhibitors.

Hum Mutat 2013 Aug 13;34(8):1085-93. Epub 2013 May 13.

Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

Cystathionine beta-synthase (CBS) deficiency is an inborn error of metabolism characterized by extremely elevated levels of plasma total homocysteine. The vast majority of CBS-deficient patients have missense mutations located in the CBS gene that result in the production of either misfolded or unstable protein. Here, we examine the effect of proteasome inhibitors on mutant CBS function using two different mouse models of CBS deficiency. These mice lack mouse CBS and express a missense mutant human CBS enzyme (either p.I278T or p.S466L) that has less than 5% of normal liver CBS activity, resulting in a 10-30-fold elevation in plasma homocysteine levels. We show that treatment of these mice with two different proteasome inhibitors can induce liver Hsp70, Hsp40, and Hsp27, increase levels of active CBS, and lower plasma homocysteine levels to within the normal range. However, response rates varied, with 100% (8/8) of the p.S466L animals showing correction, but only 38% (10/26) of the p.I278T animals. In total, our data show that treatment with proteostasis modulators can restore significant enzymatic activity to mutant misfolded CBS enzymes and suggests that they may be useful in treating certain types of genetic diseases caused by missense mutations.
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http://dx.doi.org/10.1002/humu.22335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3941476PMC
August 2013

Estrogen metabolism within the lung and its modulation by tobacco smoke.

Carcinogenesis 2013 Apr 31;34(4):909-15. Epub 2012 Dec 31.

Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

Although estrogen and the enzymes responsible for its metabolism have been detected within the lung, the ability of this tissue to metabolize estrogen has not been demonstrated previously. The goal of this study was to characterize the profile of estrogen metabolites within the murine lung and to determine the effect of tobacco smoke exposure on metabolite levels. Use of liquid chromatography-tandem mass spectrometry led to the detection of three estrogens (E1, E2 and E3) and five estrogen metabolites (2-OHE1, 4-OHE1, 4-OHE2, 2-OMeE1 and 2-OMeE2) within the perfused lung, with 4-OHE1 being the most abundant species. Levels of 4-OHEs, carcinogenic derivatives produced primarily by cytochrome P450 1B1 (Cyp1b1), were 2-fold higher in females than males. Deletion of Cyp1b1 in females led to a dramatic reduction (21-fold) in 4-OHEs, whereas levels of 2-OHE1 and the putative protective estrogen metabolite 2-OMeE2 were increased (2.4- and 5.0-fold, respectively) (P = 0.01). Similar quantitative differences in estrogen metabolite levels were observed between Cyp1b1 null and wild-type males. Exposure of female mice to tobacco smoke for 8 weeks (2h per day, 5 days per week) increased the levels of 4-OHE1 (4-fold) and 2-OHE2 (2-fold) within the lung while reducing the total concentration of 2-OMeEs to 70% of those of unexposed controls. These data suggest that tobacco smoke accelerates the production of 4-OHEs within the lung; carcinogenic metabolites that could potentially contribute to lung tumor development. Thus, inhibition of CYP1B1 may represent a promising strategy for the prevention and treatment of lung cancer.
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http://dx.doi.org/10.1093/carcin/bgs402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616670PMC
April 2013

Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time.

Proc Natl Acad Sci U S A 2011 Nov 19;108(47):18879-86. Epub 2011 Oct 19.

Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E(2)) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E(2) paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E(2)-induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E(2)-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E(2) in 5C cells compared with both WS8 and 2A cells and hence, were associated with E(2)-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E(2) inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E(2)-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E(2)-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E(2) interacted to superadditively induce apoptosis. Therefore, these data indicate that E(2) induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.
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http://dx.doi.org/10.1073/pnas.1115188108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3223472PMC
November 2011

NF-kappaB protects cells from gamma interferon-induced RIP1-dependent necroptosis.

Mol Cell Biol 2011 Jul 16;31(14):2934-46. Epub 2011 May 16.

Immune Cell Development and Host Defense Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

Interferons (IFNs) are cytokines with well-described immunomodulatory and antiviral properties, but less is known about the mechanisms by which they promote cell survival or cell death. Here, we show that IFN-γ induces RIP1 kinase-dependent necroptosis in mammalian cells deficient in NF-κB signaling. Induction of necroptosis by IFN-γ was found to depend on Jak1 and partially on STAT1. We also demonstrate that IFN-γ activates IκB kinase β (IKKβ)-dependent NF-κB to regulate a transcriptional program that protects cells from necroptosis. IFN-γ induced progressive accumulation of reactive oxygen species (ROS) and eventual loss of mitochondrial membrane potential in cells lacking the NF-κB subunit RelA. Whole-genome microarray analyses identified sod2, encoding the antioxidant enzyme manganese superoxide dismutase (MnSOD), as a RelA target and potential antinecroptotic gene. Overexpression of MnSOD inhibited IFN-γ-mediated ROS accumulation and partially rescued RelA-deficient cells from necroptosis, while RNA interference (RNAi)-mediated silencing of sod2 expression increased susceptibility to IFN-γ-induced cell death. Together, these studies demonstrate that NF-κB protects cells from IFN-γ-mediated necroptosis by transcriptionally activating a survival response that quenches ROS to preserve mitochondrial integrity.
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http://dx.doi.org/10.1128/MCB.05445-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133390PMC
July 2011

The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells.

Cancer Res 2010 Feb 19;70(3):1184-94. Epub 2010 Jan 19.

Fox Chase Cancer Center; Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA.

The G protein-coupled receptor GPR30 binds 17beta-estradiol (E(2)) yet differs from classic estrogen receptors (ERalpha and ERbeta). GPR30 can mediate E(2)-induced nongenomic signaling, but its role in ERalpha-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERalpha-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E(2) and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca(2+) mobilization studies, GPR30, but not ERalpha, mediated E(2)-induced Ca(2+) responses because E(2), 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca(2+) increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E(2)-induced and G-1-induced Ca(2+) mobilization, but ERalpha depletion did not. Interestingly, GPR30-coupled Ca(2+) responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G(1) phase. Thus, GPR30 antagonizes growth of ERalpha-positive breast cancer and may represent a new target to combat this disease.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-3068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879282PMC
February 2010

Mouse models of cystathionine beta-synthase deficiency reveal significant threshold effects of hyperhomocysteinemia.

FASEB J 2009 Mar 5;23(3):883-93. Epub 2008 Nov 5.

Division of Population Science, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111, USA.

Untreated cystathionine beta-synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 microM), with thrombosis as the major cause of morbidity. Treatment with vitamins and diet leads to a dramatic reduction in thrombotic events, even though patients often still have severe elevations in tHcy (>80 microM). To understand the difference between extreme and severe hyperhomocysteinemia, we have examined two mouse models of CBS deficiency: Tg-hCBS Cbs(-/-) mice, with a mean serum tHcy of 169 microM, and Tg-I278T Cbs(-/-) mice, with a mean tHcy of 296 microM. Only Tg-I278T Cbs(-/-) animals exhibited strong biological phenotypes, including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in mean survival time. Metabolic profiling of serum and liver reveals that Tg-I278T Cbs(-/-) mice have significantly elevated levels of free oxidized homocysteine but not protein-bound homocysteine in serum and elevation of all forms of homocysteine and S-adenosylhomocysteine in the liver compared to Tg-hCBS Cbs(-/-) mice. RNA profiling of livers indicate that Tg-I278T Cbs(-/-) and Tg-hCBS Cbs(-/-) mice have unique gene signatures, with minimal overlap. Our results indicate that there is a clear pathogenic threshold effect for tHcy and bring into question the idea that mild elevations in tHcy are directly pathogenic.
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http://dx.doi.org/10.1096/fj.08-120584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2653989PMC
March 2009

Patient characteristics compete with dose as predictors of acute treatment toxicity in early phase clinical trials.

Clin Cancer Res 2004 Jul;10(14):4645-51

Departments of Biostatistics and Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

Purpose: The purpose of this study was to identify patient characteristics that may be risk factors or markers of susceptibility to adverse treatment effects in cancer Phase I and II clinical trials.

Patients And Methods: A total of 459 patients enrolled in 23 therapeutic Phase I and II studies at the Fox Chase Cancer Center were included in the analysis. Patient-specific characteristics, medical and treatment history, doses of experimental agents, and graded toxicities were extracted from case report forms. We developed a novel summary measure, the toxicity index (TI), to better discriminate patients on the basis of their overall toxicity experiences. Mixed model ANOVA was used to model TI on the basis of data from all trials using a specific agent. Generalized estimating equations in the context of binary logistic regression were used to model dose-limiting toxicity.

Results: Seventeen pretreatment factors, including performance status, alkaline phosphatase, total bilirubin, serum creatinine, and tobacco use, emerged as significant predictors of toxicity as defined by dose-limiting toxicity or TI. Unexpectedly, dose was not always a predictor of toxicity. Even for values within the normal range, the TI identified serum bilirubin and alkaline phosphatase as predictors of toxicity after treatment with docetaxel and alkaline phosphatase as a predictor for toxicity after treatment with irinotecan.

Conclusions: Independent of dose, certain pretreatment characteristics, including measures of organ function that are in the normal range, were found to be predictors of treatment toxicity. Because of its sensitivity to differences in overall toxicity, the TI should prove to be a useful tool for identifying predictors of chemotherapy-related toxicity.
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http://dx.doi.org/10.1158/1078-0432.CCR-03-0535DOI Listing
July 2004

Hardy-Weinberg equilibrium diagnostics.

Theor Popul Biol 2002 Nov;62(3):251-7

Department of Biostatistics, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA.

We propose two diagnostics for the statistical assessment of Hardy-Weinberg equilibrium. One diagnostic is the posterior probability of the complement of the smallest highest posterior density credible region that includes points in the parameter space consistent with the hypothesis of equilibrium. The null hypothesis of equilibrium is to be rejected if this probability is less than a pre-selected critical level. The second diagnostic is the proportion of the parameter space occupied by the highest posterior density credible region associated with the critical level. These Bayesian diagnostics can be interpreted as analogues of the classical types I and II error probabilities. They are broadly applicable: they can be computed for any hypothesis test, using samples of any size generated according to any distribution.
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http://dx.doi.org/10.1006/tpbi.2002.1613DOI Listing
November 2002