Prognostic multibiomarker signatures in prostate cancer (PCa) may improve patient management and provide a bridge for developing novel therapeutics and imaging methods. Our objective was to evaluate the association between expression of 33 candidate protein biomarkers and time to biochemical failure (BF) after prostatectomy.
Malignant peripheral nerve sheath tumors (MPNSTs) are genetically diverse, aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 1 syndrome. Reduced TP53 gene expression and amplification/overexpression of the epidermal growth factor receptor (EGFR) gene occur in MPNST formation. We focused on determining the cooperativity between reduced TP53 expression and EGFR overexpression for Schwann cell transformation in vitro (immortalized human Schwann cells) and MPNST formation in vivo (transgenic mice). Human gene copy number alteration data, microarray expression data, and TMA analysis indicate that TP53 haploinsufficiency and increased EGFR expression co-occur in human MPNST samples. Concurrent modulation of EGFR and TP53 expression in HSC1? cells significantly increased proliferation and anchorage-independent growth in vitro. Transgenic mice heterozygous for a Trp53-null allele and overexpressing EGFR in Schwann cells had a significant increase in neurofibroma and grade 3 PNST (MPNST) formation compared with single transgenic controls. Histological analysis of tumors identified a significant increase in pAkt expression in grade 3 PNSTs compared with neurofibromas. Array comparative genome hybridization analysis of grade 3 PNSTs identified recurrent focal regions of chromosomal gains with significant enrichment in genes involved in extracellular signal-regulated kinase 5 signaling. Collectively, altered p53 expression cooperates with overexpression of EGFR in Schwann cells to enhance in vitro oncogenic properties and tumorigenesis and progression in vivo.
The clinical course of prostate cancer (PCa) measured by biochemical failure (BF) after prostatectomy remains unpredictable in many patients, particularly in intermediate Gleason score (GS) 7 tumors, suggesting that identification of molecular mechanisms associated with aggressive PCa biology may be exploited for improved prognostication or therapy. Hyaluronan (HA) is a high molecular weight polyanionic carbohydrate produced by synthases (HAS1 through HAS3) and fragmented by oxidative/nitrosative stress and hyaluronidases (HYAL1 through HYAL4, SPAM1) common in PCa microenvironments. HA and HA fragments interact with receptors CD44 and hyaluronan-mediated motility receptor (HMMR), resulting in increased tumor aggressiveness in experimental PCa models. This study evaluated the association of HA-related molecules with BF after prostatectomy in GS7 tumors.
Genetic changes required for the formation and progression of human Schwann cell tumors remain elusive. Using a Sleeping Beauty forward genetic screen, we identified several genes involved in canonical Wnt signaling as potential drivers of benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). In human neurofibromas and MPNSTs, activation of Wnt signaling increased with tumor grade and was associated with downregulation of ?-catenin destruction complex members or overexpression of a ligand that potentiates Wnt signaling, R-spondin 2 (RSPO2). Induction of Wnt signaling was sufficient to induce transformed properties in immortalized human Schwann cells, and downregulation of this pathway was sufficient to reduce the tumorigenic phenotype of human MPNST cell lines. Small-molecule inhibition of Wnt signaling effectively reduced the viability of MPNST cell lines and synergistically induced apoptosis when combined with an mTOR inhibitor, RAD-001, suggesting that Wnt inhibition represents a novel target for therapeutic intervention in Schwann cell tumors.
Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.
Atmospheric oxygen (?20% O(2)) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines.
Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-?), given 4h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-? enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-? pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-? induced vascular pre-conditioning events that peaked at 4h and diminished over several days. Early events (4-24 h) include upregulation of inflammatory markers (nuclear factor-?B (NF?B) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-? pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-? pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.
Human CD133 (prominin-1), a cell surface glycoprotein, is used as a marker of hematopoietic and neural stem cells. Antibodies that recognize a glycosylation-dependent CD133 epitope have been extensively used for enrichment of tumor initiating cells in a variety of cancers. These currently available antibodies are restricted for use in only a subset of biological assays. We have generated a novel anti-human CD133 monoclonal antibody, using a recombinant protein consisting of highly immunogenic amino acid residues selected from the native CD133 protein as an immunogen. The antibody (identified as clone 7) specifically recognizes the CD133 protein in a variety of immunological applications including Western blot, immunofluorescence, flow cytometry and immunohistochemistry. Further, clone 7 specifically recognizes an unmodified CD133 extracellular domain, and not its glycosylated epitope. In conclusion, the specificity and usefulness in a wide range of applications suggest that clone 7 could be a valuable tool to identify CD133 positive cells as well as to target them for therapy.
Spontaneous mouse models of cancer show promise to more accurately recapitulate human disease and predict clinical efficacy. Transgenic mice or viral vectors have been required to generate spontaneous models of glioma, a lethal brain tumor, because nonviral gene transfer is typically transient. To overcome this constraint, we used the Sleeping Beauty transposable element to achieve chromosomal integration of human oncogenes into endogenous brain cells of immunocompetent mice. Genetically engineered, spontaneous brain tumors were induced with plasmid DNA in a matter of weeks in three separate mouse strains. The phenotype of tumors was influenced by the combination of oncogenes delivered, resembling human astrocytoma or glioblastoma in the majority of cases. At least five different genes can be cotransfected simultaneously including reporters, allowing measurement of tumor viability by in vivo imaging. This model can accelerate brain tumor research in a variety of ways such as generation of "humanized" models for high throughput drug screening and candidate gene validation with exceptional speed and flexibility.
Hormone receptor status determination for breast cancer is an important part of pathologists daily sign outs and many retrospective and prospective studies. In this study, we compared the estrogen receptor (ER) expression tested on tissue microarray (TMA) sections to those tested on whole sections (WS) to find out concordance and frequency of intratumoral heterogeneity. Five and one-half percent of all tumors showed discrepancy in ER expression between TMA and WS. The rate of discrepancy was lower with increasing number of cores from individual cases. In 1.4% of cases, there was discrepancy in ER expression between cores in which >1 core was available on TMA section. We concluded that TMA can be used to determine ER status with good concordance to ER determination done on WS. Whenever the size of the tumor in the block allows it, more cores should be taken to construct TMAs.
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