Bruce Woolcock

Protein Profiling of Microdissected Prostate Tissue Links Growth Differentiation Factor 15 to Prostate Carcinogenesis

Identification of proteomic alterations associated with early stages in the development of prostate cancer may facilitate understanding of progression of this highly variable disease. Matched normal, high-grade prostatic intraepithelial neoplasia (hPIN) and prostate cancer cells of predominantly Gleason grade 3 were procured by laser capture microdissection from serial sections obtained from snap-frozen samples dissected from 22 radical prostatectomy specimens. From these cells, protein profiles were generated by surface-enhanced laser desorption/ionization-time of flight mass spectrometry. A 24-kDa peak was observed at low or high intensity in profiles of prostate cancer cells in 19 of 27 lesions and at low intensity in 3 of 8 hPIN lesions but was not detectable in matched normal cells. SDS-PAGE analysis of prostate cancer and matched normal epithelium confirmed expression of a prostate cancer-specific 24-kDa protein. Mass spectrometry and protein data-based analysis identified the protein as the dimeric form of mature growth differentiation factor 15 (GDF15). The increased expression of mature GDF15 protein in prostate cancer cells cannot be explained on the basis of up-regulation of GDF15 mRNA because reverse transcription-PCR analysis showed similar amounts of transcript in normal, hPIN, and prostate cancer cells that were obtained by laser capture microdissection in the same set of serial sections from which the protein profiles were obtained. Our findings suggest that early prostate carcinogenesis is associated with expression of mature GDF15 protein.

The Genetic Map of Xiphophorus Fishes Represented by 24 Multipoint Linkage Groups

Hybrids between distinct Xiphophorus species have been utilized for over 70 years to study melanoma and other neoplasms that can develop spontaneously in hybrid offspring. Genetic linkage mapping has proven to be important in delineating genomic areas that harbor oncogenes and tumor suppressors. Within this report, two parallel backcrosses have been utilized to generate a genetic linkage map for Xiphophorus fishes. Isozyme/allozyme, RFLP and PCR-based mapping techniques, including AP-PCR/RAPDs and microsatellite loci were utilized. The derived linkage map provides a total of 403 mapped polymorphisms distributed among 24 linkage groups, representative of 24 acro- and telocentric chromosome pairs. Genomic coverage is approximately one marker per 5.8 cM. Detailed genotypic analysis of the utilized hybrids revealed two areas of the genome that show significant segregation distortion. Loci within the linkage group harboring the sex determining locus (LG 24) and an autosomal linkage group (LG 21) show highly significant deviations from Mendelian expectations. This phenomenon is not present in a hybrid cross that utilizes a different backcross hybrid progenitor species. The derived map with sequence-tagged markers provides a framework for physical map generation, large-scale genomic sequencing and will further enable cross-genome comparisons of vertebrate genomes.

DNA fingerprinting tags novel altered chromosomal regions and identifies the involvement of SOX5 in the progression of prostate cancer

Identification of genomic alterations associated with the progression of prostate cancer may facilitate the better understanding of the development of this highly variable disease. Matched normal, premalignant high‐grade prostatic intraepithelial neoplasia and invasive prostate carcinoma cells were procured by laser capture microdissection (LCM) from human radical prostatectomy specimens. From these cells, comparative DNA fingerprints were generated by a modified PCR‐based technique called scanning of microdissected archival lesion (SMAL)‐PCR. Recurrent polymorphic fingerprint fragments were used in tagging altered chromosomal regions. Altered regions were found at cytobands 1p31.3, 1q44, 2p23.1, 3p26.3, 3q22.3, 4q22.3, 4q35.2, 5q23.2, 8q22.3, 8q24.13, 9q21.3, 9q22.32, 10q11.21, 11p13, 12p12.1, 13q12.1, 16q12.2 and 18q21.31. Candidate genes in the surrounding area that may possibly harbor mutations that change normal prostatic cells to progress into their tumor stages were proposed. Of these fragments, a 420 bp alteration, absent in all 26 normal samples screened, was observed in 2 tumors. This fragment was cloned, sequenced and localized to chromosome 12p12.1. Within this region, candidate gene sex determining region Y‐box 5 (SOX5) was proposed. Further studies of SOX5 in cell lines, xenografts and human prostate specimens, at both the RNA and protein levels, found overexpression of the gene in tumors. This overexpression was then subsequently found by fluorescent in situ hybridization to be caused by amplification of the region. In conclusion, our results suggest LCM coupled with SMAL‐PCR DNA fingerprinting is a useful method for the screening and identification of chromosomal regions and genes associated with cancer development. Further, overexpression of SOX5 is associated with prostate tumor progression and early development of distant metastasis.

Minimum altered regions in early prostate cancer progression identified by high resolution whole genome tiling path BAC array comparative hybridization.

Carcinoma of the prostate (CaP) is a serious health problem. The altered molecular mechanisms that lead to this disease are poorly understood.

Allele-Specific Marker Generation and Linkage Mapping on the Xiphophorus Sex Chromosomes

There is great interest in the sex chromosomes of Xiphophorus fishes because both WY/YY and XX/XY sex-determining mechanisms function in these species, with at least one taxon possessing all three types of sex chromosomes, and because in certain interspecific hybrids melanoma arises as a consequence of inheritance of the sex-linked macromelanophore determining locus (MDL). Representational difference analysis (RDA) has been used to clone two sequences from the sex-determining region of X. maculatus, including a cholinergic receptor, nicotinic, delta polypeptide (CHRND) orthologue. Allele-specific assays for these sequences, as well as for the sex-linked XMRK1 and XMRK2 genes, were developed to distinguish W, X, and Y chromosomes derived from a X. maculatus (XX/XY) strain and a X. helleri (WY/YY) strain. Linkage mapping localized these markers to linkage group (LG) 24. No recombinants were observed between XMRK2 and MDL, confirming a role for XMRK2 in macromelanophore development. Although the master sex-determining (SD) locus certainly resides on Xiphophorus LG 24, autosomal loci are probably involved in sex determination as well, as indicated by the abnormal sex ratios in the backcross hybrids that contrast theoretical predictions based on LG 24 genotyping. Marker development and allelic discrimination on the Xiphophorus sex chromosomes should prove highly useful for studies that utilize this genus as an animal model.

Abstract 3941: Recurrent IL4R mutations in primary mediastinal large B cell lymphoma

Introduction: Primary mediastinal large B cell lymphoma (PMBCL) is a distinct subtype of aggressive B cell lymphoma that arises from thymic medullary B cells and characteristically presents as a mass in the anterior mediastinum. A proportion of PMBCL patients suffer from refractory or relapsing disease, and subsequent salvage therapies are frequently ineffective. Development of targeted therapies is impeded by the lack of knowledge about the mutational landscape in the lymphoma genomes and mutation-associated phenotypes. We recently reported somatic mutations in the transcriptome of 7 PMBCL patients and 3 cell lines by next-generation sequencing and found a hotspot mutation in exon 8 of IL4R (Gunawardana et al, Nat Genet, 2014). Interleukin (IL)-4 is a type II cytokine that regulates immune responses through binding to a receptor complex consisting of the IL4 receptor alpha (IL4Rα) chain and IL13Rα or the common gamma chain (γc). Activation of IL4R by IL-4 and/or IL-13 initiates intracellular signal transduction mediated by the phosphorylation of JAnus Kinase-Signal Transducer and Activation of Transcription (JAK-STAT) pathway. We hypothesize that constitutively active JAK-STAT observed in PMBCL is in part due to gain-of-function IL4R mutations signaling through this oncogenic pathway. Methods and samples: A sequencing cohort consisting of fresh-frozen pretreatment lymph node biopsies from 62 PMBCL cases were selected from the BCCA tissue archive. DNA from PMBCL tumors and 3 PMBCL-derived cell lines were extracted for IL4R exonic PCR amplification and Sanger sequencing or high-throughput sequencing on an Illumina MiSeq instrument. WT and mutant (I242N) IL4R cDNA were cloned into mammalian expression vectors and expressed in the lymphoma cell line DEV and in engineered HEK293 cells expressing STAT6. Supernatant from cultured cells were used to measure the activity of STAT6-dependent expression of secreted embryonic alkaline phosphatase (SEAP). Extracted RNA and protein from transfected cells were used for qRT-PCR and Western blotting, respectively. Results: Somatic IL4R mutations were found in 18 of 65 (28%) cases confirming a hotspot mutation (I242N) in exon 8 in 11 of 18 (61%) mutated cases. Ectopic expression of the mutant in HEK293 cells showed increased SEAP levels compared to WT (534% vs. 100%) indicating STAT6 activation independent of cytokine stimulation. Introduction of the mutant into DEV cells showed IL4 independent hyperphosphorylation of JAK2, STAT5 and STAT6 proteins and upregulation of the B cell activation marker CD23 (963% vs. 100% in WT). Conclusions: IL4R is frequently mutated in PMBCL with a recurrent hotspot affecting the transmembrane domain of the protein. Functional analyses characterize these mutations as gain-of-function leading to constitutive activation of the JAK-STAT pathway and downstream target genes. These data suggest IL4R mutations as novel driver alterations in PMBCL and may provide a rational therapeutic target. Citation Format: Jay Gunawardana, Tessa Van Tol, Katina Mak, David Twa, Elizabeth Chavez, Bruce Woolcock, Robert Kridel, Anja Mottok, Shannon Healy, Adele Telenius, Merrill Boyle, Susana Ben-Neriah, Stacy Hung, Christoffer Hother, Randy Gascoyne, Christian Steidl. Recurrent IL4R mutations in primary mediastinal large B cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3941. doi:10.1158/1538-7445.AM2015-3941