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1.  Metformin, an Adjunct Antineoplastic Therapy, Divergently Modulates Tumor Metabolism and Proliferation, Interfering with Early Response Prediction Using 18F-FDG PET Imaging 
Over the last several years epidemiological data has emerged which suggests that the anti-diabetic drug metformin (MET), an AMP-activated protein kinase (AMPK) activator, improves progression free survival in multiple cancers; more than 30 clinical trials are underway to confirm this finding. We postulated that the role of AMPK as a central cellular energy sensor would result in opposite effects on glucose uptake and proliferation, suggesting different roles for 18F-FDG and 18F-FLT in assessing its effectiveness as an anti-neoplastic agent.
Colon cancer cell lines HT29 (human) and MC26 (murine) were treated for 24 or 72hrs with a range of MET (0–10mM). Western blotting was used to study the activation of AMPK after MET treatment. Glucose uptake and cell proliferation were measured by cell retention studies with either 18F-FDG or 18F-FLT. EdU (a thymidine analogue) and Annexin-Propidium Iodine flow cytometry were performed to determine cell cycle S-phase and apoptotic changes. In vivo 18F-FDG and 18F-FLT PET images were acquired before and 24hrs after MET treatment on HT29 tumor bearing mice.
After 24hrs of MET incubation, phosphorylated AMPK increased several fold in both cell lines while total AMPK was unchanged. In cell retention studies, 18F-FDG uptake increased whereas 18F-FLT retention decreased significantly in both cell lines. Cells in S-phase decreased 36% in HT29 and 33% in MC26 cells following MET therapy. Apoptosis increased 10.5× and 5.8×, in HT29 and MC26 cells, respectively after 72hrs of incubation with MET. PET imaging showed increased 18F-FDG uptake (mean SUV: 0.71±0.03 and 1.29±0.11 pre and post MET therapy, p<0.05) and decreased 18F-FLT uptake (mean SUV: 1.18±0.05 and 0.89±0.01 pre and post MET therapy, p<0.05) in HT29 tumor bearing mice.
MET, through activation of the AMPK pathway, exerts a dose dependent increase in tumor glucose uptake while decreasing cell proliferation in human and murine colon cancer cells. Thus, changes in 18F-FDG uptake post MET treatment may be misleading; 18F-FLT imaging is a promising alternative that correlates with tumor response.
PMCID: PMC3703242  PMID: 23376854
Metformin; AMPK; Tumor metabolic imaging; Proliferation imaging
2.  Opposing effects of androgen deprivation and targeted therapy on prostate cancer prevention 
Cancer discovery  2012;3(1):44-51.
Prostate cancer is an ideal target for chemoprevention. To date, chemoprevention clinical trials with 5α-reductase inhibitors (5-ARI) have yielded encouraging yet ultimately confounding results. Using a pre-clinical mouse model of high-grade prostatic intraepithelial neoplasia (HG-PIN) induced by PTEN loss, we observed unprecedented deteriorating effects of androgen deprivation, where surgical castration or MDV3100 treatment accelerated disease progression of the otherwise stable HG-PIN to invasive castration-resistant prostate cancer (CRPC). As an alternative, targeting the PI3K signaling pathway via either genetic ablation of PI3K components or pharmacological inhibition of PI3K pathway reversed the PTEN loss-induced HG-PIN phenotype. Finally, concurrent inhibition of PI3K and MAPK pathways was effective in blocking the growth of PTEN-null CRPC. Together, these data have revealed the potential adverse effects of anti-androgen chemoprevention in certain genetic contexts (such as PTEN loss) while demonstrating the promise of targeted therapy in the clinical management of this complex and prevalent disease.
PMCID: PMC3546223  PMID: 23258246
3.  A co-clinical approach identifies mechanisms and potential therapies for androgen deprivation resistance in prostate cancer 
Nature genetics  2013;45(7):747-755.
Here we report an integrated analysis that leverages data from treatment of genetic mouse models of prostate cancer along with clinical data from patients to elucidate new mechanisms of castration resistance. We show that castration counteracts tumor progression in a Pten-loss driven mouse model of prostate cancer through the induction of apoptosis and proliferation block. Conversely, this response is bypassed upon deletion of either Trp53 or Lrf together with Pten, leading to the development of castration resistant prostate cancer (CRPC). Mechanistically, the integrated acquisition of data from mouse models and patients identifies the expression patterns of XAF1-XIAP/SRD5A1 as a predictive and actionable signature for CRPC. Importantly, we show that combined inhibition of XIAP, SRD5A1, and AR pathways overcomes castration resistance. Thus, our co-clinical approach facilitates stratification of patients and the development of tailored and innovative therapeutic treatments.
PMCID: PMC3787876  PMID: 23727860
4.  The Ubiquitin Specific Protease USP2a Prevents Endocytosis-Mediated EGFR Degradation 
Oncogene  2012;32(13):10.1038/onc.2012.188.
Ubiquitination of EGFR is required for down-regulation of the receptor by endocytosis. Impairment of this pathway results in constitutively active EGFR, which is associated with carcinogenesis, particularly in lung cancer. We previously demonstrated that the deubiquitinating enzyme USP2a has oncogenic properties. Here we show a new role for USP2a as a regulator of EGFR endocytosis. USP2a localizes to early endosomes and associates with EGFR, stabilizing the receptor, which retains active downstream signaling. HeLa cells transiently expressing catalytically active but not mutant USP2a show increased plasma membrane-localized EGFR, as well as decreased internalized and ubiquitinated EGFR. Conversely, USP2a silencing reverses this phenotype. Importantly, USP2a prevents the degradation of mutant in addition to wild type EGFR. Finally, we observed that USP2a and EGFR proteins are coordinately over-expressed in non-small cell lung cancers. Taken together, our data indicate that USP2a antagonizes EGFR endocytosis and thus amplifies signaling activity from the receptor. Our findings suggest that regulation of deubiquitination could be exploited therapeutically in cancers over-expressing EGFR.
PMCID: PMC3866888  PMID: 22710717
USP2a; DUB; EGFR; endocytosis
5.  Blood and tissue biomarkers in prostate cancer: state of the art 
The Urologic clinics of North America  2010;37(1):131-Contents.
PMCID: PMC3784983  PMID: 20152526
prostate cancer; tissue biomarkers; blood biomarkers; molecular pathology
Whether the genomic rearrangement TMPRSS2:ERG has prognostic value in prostate cancer is unclear.
Among men with prostate cancer in the prospective Physicians’ Health and Health Professionals Follow-Up Studies, we identified rearrangement status by immunohistochemical assessment of ERG protein expression. We used Cox models to examine associations of ERG overexpression with biochemical recurrence and lethal disease (distant metastases or cancer-specific mortality). In a meta-analysis including 47 additional studies, we used random effects models to estimate associations between rearrangement status and outcomes.
The cohort consisted of 1,180 men treated with radical prostatectomy between 1983 and 2005. During a median follow-up of 12.6 years, 266 men experienced recurrence, and 85 men developed lethal disease. We found no significant association between ERG overexpression and biochemical recurrence (HR: 0.99; 95% CI: 0.78-1.26) or lethal disease (HR: 0.93; 95% CI: 0.61-1.43). The meta-analysis of prostatectomy series included 5,074 men followed for biochemical recurrence (1,623 events), and 2,049 men followed for lethal disease (131 events). TMPRSS2:ERG was associated with stage at diagnosis (RR≥T3 vs. T2: 1.23; 95% CI: 1.16-1.30) but not with biochemical recurrence (RR: 1.00; 95% CI: 0.86-1.17) or lethal disease (RR: 0.99; 95% CI: 0.47-2.09).
These results suggest that TMPRSS2:ERG, or ERG overexpression, is associated with tumor stage but does not strongly predict recurrence or mortality among men treated with radical prostatectomy.
This is the largest prospective cohort study to examine associations of ERG overexpression and lethal prostate cancer among men treated with radical prostatectomy.
PMCID: PMC3671609  PMID: 22736790
TMPRSS2:ERG; prostate cancer; gene fusion; biomarker; prognosis
7.  SMAD4–dependent barrier constrains prostate cancer growth and metastatic progression 
Nature  2011;470(7333):269-273.
Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression1. Here,we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP–SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment con-firmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.
PMCID: PMC3753179  PMID: 21289624
8.  Characterization of TMPRSS2-ERG fusion high-grade prostatic intraepithelial neoplasia and potential clinical implications 
More than 1,300,000 prostate needle biopsies are performed annually in the U.S. with up to 16% incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN has low predictive value for identifying prostate cancer (PCA) on subsequent needle biopsies in PSA screened populations. In contemporary series, PCA is detected in about 20% of repeat biopsies following a diagnosis of HGPIN. Further, discrete histological subtypes of HGPIN with clinical implication in management have not been characterized. The TMPRSS2-ERG gene fusion that has recently been described in PCA has also been demonstrated to occur in a subset of HGPIN. This may have significant clinical implications given that TMPRSS2-ERG fusion PCA is associated with a more aggressive clinical course.
Experimental Design
In this study we assessed a series of HGPIN lesions and paired PCA for the presence of TMPRSS2-ERG gene fusion.
Fusion positive HGPIN was observed in 16% of the 143 number of lesions, and in all instances the matching cancer shared the same fusion pattern. 60% of TMPRSS2-ERG fusion PCA had fusion negative HGPIN.
Given the more aggressive nature of TMPRSS2-ERG PCA, the findings of this study raise the possibility that gene fusion positive HGPIN lesions are harbingers of more aggressive disease. To date, pathological, molecular and clinical parameters do not help stratify which men with HGPIN are at increased risk for a cancer diagnosis. Our results suggest that the detection of isolated TMPRSS2-ERG fusion HGPIN would improve the positive predictive value of finding TMPRSS2-ERG fusion PCA in subsequent biopsies.
PMCID: PMC3717517  PMID: 18519767
9.  Common polymorphisms in the adiponectin and its receptor genes, adiponectin levels and the risk of prostate cancer 
Adiponectin, an insulin-sensitizing adipokine, is inversely associated with adiposity and prostate cancer risk and progression. However, the role of genetic variation in the adiponectin (ADIPOQ) and receptor genes (ADIPOR1/R2) in prostate cancer is largely unknown.
In a nested case-control study of 1286 cases and 1267 controls within the Physicians' Health Study, we evaluated 29 common single nucleotide polymorphisms (SNPs) in ADIPOQ (n=13), ADIPOR1(n=5) and ADIPOR2(n=11) in relation to the risk of prostate cancer. In subgroups, we also evaluated the association of genotype and circulating adiponectin levels (n=951) and prostate tumor expression of insulin receptor (IR) and insulin-like growth factor 1 (IGF-1R) receptor (n=181).
Among the 12 tagging polymorphisms in ADIPOQ, four (rs266729, rs182052, rs822391, rs2082940) were significantly associated (p<0.05) with overall prostate cancer risk, with no significant difference by tumor grade or clinical stage. Two of the risk SNPs (rs266729, rs182052) plus four other SNPs (rs16861209, rs17366568, rs3774261, rs7639352) were also associated with plasma adiponectin levels and three of these (rs1686109, rs17366568, rs3774261) were also significantly associated with IR expression in prostate tumor tissue. One additional SNP was associated with IGF1-R tumor tissue expression (rs16861205). None of the 16 variants in ADIPOR1/R2 were related to cancer risk or circulating adiponectin levels.
Common variants in the adiponectin gene were associated with prostate cancer risk, plasma adiponectin levels, and IR or IGF-1R expression in the prostate tumor.
These genotype-phenotype associations support the biological relevance of adiponectin for prostate carcinogenesis, particularly in earlier stages of development.
PMCID: PMC3700351  PMID: 21960694
10.  EZH2 Oncogenic Activity in Castration Resistant Prostate Cancer Cells is Polycomb-Independent 
Science (New York, N.Y.)  2012;338(6113):1465-1469.
Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. Here we report that the oncogenic function of EZH2 in castration-resistant prostate cancer (CRPC) is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a co-activator for critical transcription factors including the androgen receptor (AR). This functional switch is dependent on phosphorylation of EZH2, and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have significant therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.
PMCID: PMC3625962  PMID: 23239736
12.  The ubiquitin-specific protease USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer 
Cell Cycle  2012;11(6):1123-1130.
The deubiquitinating enzyme USP2a has shown oncogenic properties in many cancer types by impairing ubiquitination of FASN, MDM2, MDMX or Aurora A. Aberrant expression of USP2a has been linked to progression of human tumors, particularly prostate cancer. However, little is known about the role of USP2a or its mechanism of action in bladder cancer. Here, we provide evidence that USP2a is an oncoprotein in bladder cancer cells. Enforced expression of USP2a caused enhanced proliferation, invasion, migration and resistance to several chemotherapeutic reagents, while USP2a loss resulted in slower proliferation, greater chemosensitivity and reduced migratory/invasive capability compared with control cells. USP2a, but not a catalytically inactive mutant, enhanced proliferation in immortalized TRT-HU1 normal human bladder epithelial cells. USP2a bound to cyclin A1 and prevented cyclin A1 ubiquitination, leading to accumulation of cyclin A1 by a block in degradation. Enforced expression of wild-type USP2a, but not an inactive USP2a mutant, resulted in cyclin A1 accumulation and increased cell proliferation. We conclude that USP2a impairs ubiquitination and stabilizes an important cell cycle regulator, cyclin A1, raising the possibility of USP2a targeting as a therapeutic strategy against bladder tumors in combination with chemotherapy.
PMCID: PMC3335918  PMID: 22370483
USP2a; cyclin A1; bladder cancer; cisplatin resistance; deubiquitination
13.  ERG induces androgen receptor-mediated regulation of SOX9 in prostate cancer 
The Journal of Clinical Investigation  2013;123(3):1109-1122.
Fusion of the androgen receptor-regulated (AR-regulated) TMPRSS2 gene with ERG in prostate cancer (PCa) causes androgen-stimulated overexpression of ERG, an ETS transcription factor, but critical downstream effectors of ERG-mediating PCa development remain to be established. Expression of the SOX9 transcription factor correlated with TMPRSS2:ERG fusion in 3 independent PCa cohorts, and ERG-dependent expression of SOX9 was confirmed by RNAi in the fusion-positive VCaP cell line. SOX9 has been shown to mediate ductal morphogenesis in fetal prostate and maintain stem/progenitor cell pools in multiple adult tissues, and has also been linked to PCa and other cancers. SOX9 overexpression resulted in neoplasia in murine prostate and stimulated tumor invasion, similarly to ERG. Moreover, SOX9 depletion in VCaP cells markedly impaired invasion and growth in vitro and in vivo, establishing SOX9 as a critical downstream effector of ERG. Finally, we found that ERG regulated SOX9 indirectly by opening a cryptic AR-regulated enhancer in the SOX9 gene. Together, these results demonstrate that ERG redirects AR to a set of genes including SOX9 that are not normally androgen stimulated, and identify SOX9 as a critical downstream effector of ERG in TMPRSS2:ERG fusion–positive PCa.
PMCID: PMC3582143  PMID: 23426182
14.  Effect of dietary polyunsaturated fatty acids on castration-resistant Pten-null prostate cancer 
Carcinogenesis  2011;33(2):404-412.
A common treatment of advanced prostate cancer involves the deprivation of androgens. Despite the initial response to hormonal therapy, eventually all the patients relapse. In the present study, we sought to determine whether dietary polyunsaturated fatty acid (PUFA) affects the development of castration-resistant prostate cancer. Cell culture, patient tissue microarray, allograft, xenograft, prostate-specific Pten knockout and omega-3 desaturase transgenic mouse models in conjunction with dietary manipulation, gene knockdown and knockout approaches were used to determine the effect of dietary PUFA on castration-resistant Pten-null prostate cancer. We found that deletion of Pten increased androgen receptor (AR) expression and Pten-null prostate cells were castration resistant. Omega-3 PUFA slowed down the growth of castration-resistant tumors as compared with omega-6 PUFA. Omega-3 PUFA decreased AR protein to a similar extent in tumor cell cytosolic and nuclear fractions but had no effect on AR messenger RNA level. Omega-3 PUFA treatment appeared to accelerate AR protein degradation, which could be blocked by proteasome inhibitor MG132. Knockdown of AR significantly slowed down prostate cancer cell proliferation in the absence of androgens. Our data suggest that omega-3 PUFA inhibits castration-resistant prostate cancer in part by accelerating proteasome-dependent degradation of the AR protein. Dietary omega-3 PUFA supplementation in conjunction with androgen ablation may significantly delay the development of castration-resistant prostate cancer in patients compared with androgen ablation alone.
PMCID: PMC3271270  PMID: 22159221
15.  Serum Lipid Profiles and Cancer Risk in the Context of Obesity: Four Meta-Analyses 
Journal of Cancer Epidemiology  2013;2013:823849.
The objective here was to summarize the evidence for, and quantify the link between, serum markers of lipid metabolism and risk of obesity-related cancers. PubMed and Embase were searched using predefined inclusion criteria to conduct meta-analyses on the association between serum levels of TG, TC, HDL, ApoA-I, and risk of 11 obesity-related cancers. Pooled relative risks (RRs) and 95% confidence intervals were estimated using random-effects analyses. 28 studies were included. Associations between abnormal lipid components and risk of obesity-related cancers when using clinical cutpoints (TC ≥ 6.50; TG ≥ 1.71; HDL ≤ 1.03; ApoA-I ≤ 1.05 mmol/L) were apparent in all models. RRs were 1.18 (95% CI: 1.08–1.29) for TC, 1.20 (1.07–1.35) for TG, 1.15 (1.01–1.32) for HDL, and 1.42 (1.17–1.74) for ApoA-I. High levels of TC and TG, as well as low levels of HDL and ApoA-I, were consistently associated with increased risk of obesity-related cancers. The modest RRs suggest serum lipids to be associated with the risk of cancer, but indicate it is likely that other markers of the metabolism and/or lifestyle factors may also be involved. Future intervention studies involving lifestyle modification would provide insight into the potential biological role of lipid metabolism in tumorigenesis.
PMCID: PMC3563167  PMID: 23401687
16.  MYC Is Activated by USP2a-Mediated Modulation of MicroRNAs in Prostate Cancer 
Cancer discovery  2012;2(3):236-247.
Ubiquitin-specific protease 2a (USP2a) is overexpressed in almost half of human prostate cancers and c-Myc is amplified in one third of these tumor types. Transgenic MYC expression drives invasive adenocarcinomas in the murine prostate. We show that overexpression of USP2a downregulates a set of microRNAs that collectively increase MYC levels by MDM2 deubiquitination and subsequent p53 inactivation. By establishing MYC as a target of miR-34b/c, we demonstrate that this cluster functions as a tumor suppressor in prostate cancer cells. We identify a distinct mRNA signature that is enriched for MYC-regulated transcripts and transcription factor binding sites in USP2a overexpressing prostate cancer cells. We demonstrate that these genes are associated with an invasive phenotype in human prostate cancer and that the proliferative and invasive properties of USP2a overexpressing cells are MYC-dependent. These results highlight an unrecognized mechanism of MYC regulation in prostate cancer and suggest alternative therapeutic strategies in targeting MYC.
PMCID: PMC3523361  PMID: 22585994
17.  A Synthetic Form of Frizzled 8-Associated Antiproliferative Factor Enhances p53 Stability through USP2a and MDM2 
PLoS ONE  2012;7(12):e50392.
Frizzled 8-associated Antiproliferative Factor (APF) is a sialoglycopeptide urinary biomarker of interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic condition of unknown etiology with variable symptoms that generally include pelvic and/or perineal pain, urinary frequency, and urgency. We previously reported that native human APF suppresses the proliferation of normal bladder epithelial cells through a mechanism that involves increased levels of p53. The goal of this study was to delineate the regulatory mechanism whereby p53 expression is regulated by APF. Two APF-responsive cell lines (T24 bladder carcinoma cells and the immortalized human bladder epithelial cell line, TRT-HU1) were treated with asialo-APF (as-APF), a chemically synthesized form of APF. Biochemical analysis revealed that as-APF increased p53 levels in two ways: by decreasing ubiquitin specific protease 2a (USP2a) expression leading to enhanced ubiquitination of murine double minute 2 E3 ubiquitin ligase (MDM2), and by suppressing association of p53 with MDM2, thus impairing p53 ubiquitination. Biological responses to as-APF were suppressed by increased expression of wild type, but not mutant USP2a, which enhanced cell growth via upregulation of a cell cycle mediator, cyclin D1, at both transcription and protein levels. Consistent with this, gene silencing of USP2a with siRNA arrested cell proliferation. Our findings suggest that APF upregulates cellular p53 levels via functional attenuation of the USP2a-MDM2 pathway, resulting in p53 accumulation and growth arrest. These data also imply that targeting USP2a, MDM2, p53 and/or complex formation by these molecules may be relevant in the development of novel therapeutic approaches to IC/PBS.
PMCID: PMC3516501  PMID: 23236372
18.  Prognostic Determinants in Prostate Cancer 
Cancer journal (Sudbury, Mass.)  2011;17(6):429-437.
Clinical outcomes in prostate cancer are heterogeneous and given the high prevalence of the disease, there is a pressing need to identify clinically useful markers of prognosis. Many clinical, pathologic, molecular, and genetic factors have been investigated in this capacity though relatively few are routinely used. With a growing understanding of the molecular pathogenesis of prostate cancer, there is the potential that the next generation of makers will prove sufficiently robust to guide the optimal management of men with prostate cancer. Here, we review the various clinical and molecular prognostic determinants in prostate cancer.
PMCID: PMC3240856  PMID: 22157287
prognosis; prostate; gene expression signatures; immunohistochemistry
19.  Utility of multispectral imaging in automated quantitative scoring of immunohistochemistry 
Journal of clinical pathology  2012;65(6):496-502.
Automated scanning devices and image analysis software provide a means to overcome the limitations of manual semiquantitative scoring of immunohistochemistry. Common drawbacks to automated imaging systems include an inability to classify tissue type and an inability to segregate cytoplasmic and nuclear staining.
Immunohistochemistry for the membranous marker α-catenin, the cytoplasmic marker stathmin and the nuclear marker Ki-67 was performed on tissue microarrays (TMA) of archival formalin-fixed paraffin-embedded tissue comprising 471 (α-catenin and stathmin) and 511 (Ki-67) cases of prostate adenocarcinoma. These TMA were quantitatively analysed using two commercially available automated image analysers, the Ariol SL-50 system and the Nuance system from CRi. Both systems use brightfield microscopy for automated, unbiased and standardised quantification of immunohistochemistry, while the Nuance system has spectral deconvolution capabilities.
Overall concordance between scores from both systems was excellent (r=0.90; 0.83–0.95). The software associated with the multispectral imager allowed accurate automated classification of tissue type into epithelial glandular structures and stroma, and a single-step segmentation of staining into cytoplasmic or nuclear compartments allowing independent evaluation of these areas. The Nuance system, however, was not able to distinguish reliably between tumour and non-tumour tissue. In addition, variance in the labour and time required for analysis between the two systems was also noted.
Despite limitations, this study suggests some beneficial role for the use of a multispectral imaging system in automated analysis of immunohistochemistry.
PMCID: PMC3437674  PMID: 22447914
20.  DNA Fragmentation Simulation Method (FSM) and Fragment Size Matching Improve aCGH Performance of FFPE Tissues 
PLoS ONE  2012;7(6):e38881.
Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.
PMCID: PMC3376148  PMID: 22719973
21.  Vitamin D Receptor Protein Expression in Tumor Tissue and Prostate Cancer Progression 
Journal of Clinical Oncology  2011;29(17):2378-2385.
Data suggest that circulating 25-hydroxyvitamin D [25(OH)D] interacts with the vitamin D receptor (VDR) to decrease proliferation and increase apoptosis for some malignancies, although evidence for prostate cancer is less clear. How VDR expression in tumor tissue may influence prostate cancer progression has not been evaluated in large studies.
Patients and Methods
We examined protein expression of VDR in tumor tissue among 841 patients with prostate cancer in relation to risk of lethal prostate cancer within two prospective cohorts, the Physicians' Health Study and Health Professionals Follow-Up Study. We also examined the association of VDR expression with prediagnostic circulating 25(OH)D and 1,25-dihydroxyvitamin D levels and with two VDR single nucleotide polymorphisms, FokI and BsmI.
Men whose tumors had high VDR expression had significantly lower prostate-specific antigen (PSA) at diagnosis (P for trend < .001), lower Gleason score (P for trend < .001), and less advanced tumor stage (P for trend < .001) and were more likely to have tumors harboring the TMPRSS2:ERG fusion (P for trend = .009). Compared with the lowest quartile, men whose tumors had the highest VDR expression had significantly reduced risk of lethal prostate cancer (hazard ratio [HR], 0.17; 95% CI, 0.07 to 0.41). This association was only slightly attenuated after adjustment for Gleason score and PSA at diagnosis (HR, 0.33; 95% CI, 0.13 to 0.83) or, additionally, for tumor stage (HR, 0.37; 95% CI, 0.14 to 0.94). Neither prediagnostic plasma vitamin D levels nor VDR polymorphisms were associated with VDR expression.
High VDR expression in prostate tumors is associated with a reduced risk of lethal cancer, suggesting a role of the vitamin D pathway in prostate cancer progression.
PMCID: PMC3107752  PMID: 21537045
22.  mRNA Expression Signature of Gleason Grade Predicts Lethal Prostate Cancer 
Journal of Clinical Oncology  2011;29(17):2391-2396.
Prostate-specific antigen screening has led to enormous overtreatment of prostate cancer because of the inability to distinguish potentially lethal disease at diagnosis. We reasoned that by identifying an mRNA signature of Gleason grade, the best predictor of prognosis, we could improve prediction of lethal disease among men with moderate Gleason 7 tumors, the most common grade, and the most indeterminate in terms of prognosis.
Patients and Methods
Using the complementary DNA–mediated annealing, selection, extension, and ligation assay, we measured the mRNA expression of 6,100 genes in prostate tumor tissue in the Swedish Watchful Waiting cohort (n = 358) and Physicians' Health Study (PHS; n = 109). We developed an mRNA signature of Gleason grade comparing individuals with Gleason ≤ 6 to those with Gleason ≥ 8 tumors and applied the model among patients with Gleason 7 to discriminate lethal cases.
We built a 157-gene signature using the Swedish data that predicted Gleason with low misclassification (area under the curve [AUC] = 0.91); when this signature was tested in the PHS, the discriminatory ability remained high (AUC = 0.94). In men with Gleason 7 tumors, who were excluded from the model building, the signature significantly improved the prediction of lethal disease beyond knowing whether the Gleason score was 4 + 3 or 3 + 4 (P = .006).
Our expression signature and the genes identified may improve our understanding of the de-differentiation process of prostate tumors. Additionally, the signature may have clinical applications among men with Gleason 7, by further estimating their risk of lethal prostate cancer and thereby guiding therapy decisions to improve outcomes and reduce overtreatment.
PMCID: PMC3107753  PMID: 21537050
23.  Differential expression of S6K2 dictates tissue-specific requirement for S6K1 in mediating aberrant mTORC1 signaling and tumorigenesis 
Cancer research  2011;71(10):3669-3675.
The S6K1 and S6K2 kinases are considered important mTOR signaling effectors, yet their contribution to tumorigenesis remains unclear. Aberrant mTOR activation is a frequent event in cancer, that commonly results from heterozygous loss of PTEN. Here, we show for the first time a differential protein expression between S6K1 and S6K2 in both mouse and human tissues. Additionally, the inactivation of S6k1 in the context of Pten heterozygosity (Pten+/-) suggests a differential requirement for this protein across multiple tissues. This tissue-specificity appears to be governed by the relative protein expression of S6k2. Accordingly, we find that deletion of S6k1 markedly impairs Pten+/- mediated adrenal tumorigenesis, specifically due to low expression of S6k2. Concomitant observation of low S6K2 levels in the human adrenal gland supports the development of S6K1-inhibitors for treatment of PTEN loss driven pheochromocytoma.
PMCID: PMC3096749  PMID: 21444676
24.  The SETDB1 histone methyltransferase is recurrently amplified in and accelerates melanoma 
Nature  2011;471(7339):513-517.
The most common mutation in melanoma, BRAF(V600E), activates the BRAF serine/threonine kinase and causes excessive MAPK pathway activity1,2. BRAF(V600E)mutations are also present in benign melanocytic nevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumors. Such changes include recurrent copy number variations that result in the amplification of oncogenes4,5. For certain amplifications, the large number of genes in the interval has precluded an understanding of cooperating oncogenic events. Here, we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to significantly accelerate melanoma formation in the zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-Seq) and gene expression analyses revealed target genes, including Hox genes, that are transcriptionally dysregulated in response to elevated SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.
PMCID: PMC3348545  PMID: 21430779
25.  Transgenic Expression of Polyomavirus Middle T Antigen in the Mouse Prostate Gives Rise to Carcinoma▿† 
Journal of Virology  2011;85(11):5581-5592.
The middle T (MT) antigen of polyomavirus has provided fundamental insights into the regulation of mammalian cell growth in vitro and important animal models for the analysis of tumor induction. The mouse mammary tumor virus (MMTV)-MT model of breast cancer has been important for probing the cellular signaling pathways in mammary tumorigenesis. MT itself has no intrinsic enzymatic activity but, rather, transforms by binding to and activating key intracellular signaling molecules, phosphatidylinositol 3-kinase (PI3-kinase) being the best studied of these. Thus, MT mimics a constitutively activated receptor tyrosine kinase (RTK). Our recent work suggests that MT signaling, like that of RTKs, is often quite dependent on cellular context in vitro. Here, we examine contextual effects on signaling in animal models as well. In this study, we generated transgenic mice in which MT is expressed in the mouse prostate under the control of an (ARR)2-Probasin promoter. All male transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) in the ventral and dorsal/lateral prostate as early as 8 weeks of age. Notably, during the course of tumor development over time, invasive cancer, reactive stroma, and infiltration of inflammatory cells were seen. Transcriptional profiling analyses show regulation of multiple pathways, with marked upregulation of both the NF-κB and inflammatory pathways. Comparison of expression profiles of our MT prostate model with those from an MMTV-MT breast model (23) shows both tissue-specific and tissue-independent MT effects. The signature of genes regulated by MT in a tissue-independent manner may have prognostic value.
PMCID: PMC3094993  PMID: 21411524

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