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1.  A proteolytic modification of AIM promotes its renal excretion 
Scientific Reports  2016;6:38762.
Apoptosis inhibitor of macrophage (AIM, encoded by cd5l) is a multi-functional circulating protein that has a beneficial role in the regulation of a broad range of diseases, some of which are ameliorated by AIM administration in mice. In blood, AIM is stabilized by association with IgM pentamers and maintains its high circulating levels. The mechanism regulating the excessive accumulation of blood AIM remains unknown, although it is important, since a constitutive increase in AIM levels promotes chronic inflammation. Here we found a physiological AIM-cleavage process that induces destabilization of AIM and its excretion in urine. In blood, IgM-free AIM appeared to be cleaved and reduced in size approximately 10 kDa. Cleaved AIM was unable to bind to IgM and was selectively filtered by the glomerulus, thereby excreted in urine. Amino acid substitution at the cleavage site resulted in no renal excretion of AIM. Interestingly, cleaved AIM retained a comparable potency with full-length AIM in facilitating the clearance of dead cell debris in injured kidney, which is a key response in the recovery of acute kidney injury. Identification of AIM-cleavage and resulting functional modification could be the basis for designing safe and efficient AIM therapy for various diseases.
PMCID: PMC5144010  PMID: 27929116
European urology  2015;68(6):1083-1088.
Men on active surveillance (AS) face repeated biopsies. Most biopsies will not show disease progression nor change management; such biopsies do not contribute to patient management, are potentially morbid, and costly.
To use a contemporary AS prospective trial to develop a tool to predict AS biopsy outcomes.
Biopsies (median 2, range 2 to 9 per patient) from 859 men participating in the Canary Prostate Active Surveillance Study with Gleason grade 6 prostate cancer (median follow-up: 35.8 [range 3.0–148.7] months) were analyzed.
Logistic regression was used to predict progression, defined as Gleason score increase from ≤ 6 to ≥ 7 or increase in percent of cores positive for cancer from < 34% to ≥ 34%. 5-fold internal cross-validation was performed to evaluate the Area-Underneath-the-receiver-operating-characteristic-Curve (AUC).
Statistically significant risk factors for progression on biopsy were prostate-specific antigen (PSA), (odds ratio [OR] 1.045, 95% confidence interval [CI], 1.028 to 1.063), percent cores positive for cancer on most recent biopsy, (OR 1.401, 95% CI=1.301–1.508), and history of at least one prior negative biopsy (OR 0.524, 95% CI=0.417–0.659). A multivariable predictive model incorporating these factors, age, and number of months since last biopsy achieved an AUC of 72.4%.
A combination of readily-available clinical measures risk-stratify patients considering active surveillance prostate biopsy. Risk of progression or upgrading can be estimated and incorporated in clinical practice.
The Canary-EDRN Active Surveillance Biopsy Risk Calculator, an online tool, can be used to guide patient decision-making regarding follow-up prostate biopsy.
PMCID: PMC4583313  PMID: 25819722
active surveillance; biopsy; progression; prostate-specific antigen
3.  The correlation between CT and duplex evaluation of autogenous vein bypass grafts and their relationship to failure 
Journal of vascular surgery  2015;62(6):1546-1554.e1.
Duplex ultrasound(DUS) for vein bypass graft(VBG) surveillance is confounded by technical and physiologic factors that that reduce the sensitivity for detecting impending graft failure. In contrast, three-dimensional computed tomographic angiography(CTA) offers high fidelity anatomic characterization of VBGs but its utility in detecting at risk grafts is unknown. The current study sought to analyze the correlation between DUS and CTA for detection of vein graft stenosis and evaluate the relationship of the observed abnormalities to VBG failure.
Consecutive lower extremity VBG patients underwent surveillance with concurrent DUS and CTA at 1-week, 1-month, 6-month and 12-months postoperatively. A standardized algorithm was used for CT reconstruction and extraction of the lumen geometries at 1 mm intervals. At each time interval, CT derived cross-sectional areas were co-registered and correlated to DUS peak systolic velocities(PSV) within six pre-designated anatomic zones and then analyzed for outcome association. Vein graft failure was defined as pathological change within a given anatomic zone resulting in thrombosis, amputation or re-intervention within the six month period following the observed time point.
Fifty-four patients were recruited and 10(18%) experienced failure within 18 months of implantation. The expected inverse relationship between CSA and PSV was only weakly correlated(Spearman rank coefficient= −0.19). Moderate elevations in PSV ratio(PSVr 2–3.5) were frequently transient, with 14 of 18 grafts(78%) demonstrating ratio reduction on subsequent imaging. A PSVr ≥ 3.5 was associated with a 67% failure rate. CT stenosis <50% was highly correlated with success(zero failures); however, high-grade(>80%) CT stenosis was more likely to succeed than fail(25%). Eighteen patients had significant discordance between CT and DUS. While 14 of these patients had CT stenosis > 70% with a PSVr < 3.5, only 2 subsequently failed. Conversely, 3 of 4 subjects with CT stenosis < 70% but PSVr > 3.5 experienced graft failure. Focused analysis of these cases using computational fluid dynamic modeling demonstrated that vein side branches, local tortuosity, regional diameter variations, and venovenostomies to be the drivers of these discrepancies.
This analysis demonstrated that a PSVr ≥ 3.5 is strongly correlated with VBG failure while the natural history of moderately elevated PSVr(2–3.5) is largely clinically benign. Although minimum stenosis on CT scan was highly predictive of success, high grade CT stenosis was infrequently associated with failure. The interaction of anatomic features with the local flow dynamics were identified as the primary confounder for a direct correlation between CT and DUS.
PMCID: PMC4659752  PMID: 26365660
4.  Truncation and constitutive activation of the androgen receptor by diverse genomic rearrangements in prostate cancer 
Nature Communications  2016;7:13668.
Molecularly targeted therapies for advanced prostate cancer include castration modalities that suppress ligand-dependent transcriptional activity of the androgen receptor (AR). However, persistent AR signalling undermines therapeutic efficacy and promotes progression to lethal castration-resistant prostate cancer (CRPC), even when patients are treated with potent second-generation AR-targeted therapies abiraterone and enzalutamide. Here we define diverse AR genomic structural rearrangements (AR-GSRs) as a class of molecular alterations occurring in one third of CRPC-stage tumours. AR-GSRs occur in the context of copy-neutral and amplified AR and display heterogeneity in breakpoint location, rearrangement class and sub-clonal enrichment in tumours within and between patients. Despite this heterogeneity, one common outcome in tumours with high sub-clonal enrichment of AR-GSRs is outlier expression of diverse AR variant species lacking the ligand-binding domain and possessing ligand-independent transcriptional activity. Collectively, these findings reveal AR-GSRs as important drivers of persistent AR signalling in CRPC.
Castration-resistant prostate cancer frequently presents with persistent androgen receptor signalling. Here, the authors find that the androgen receptor is subject to genetic rearrangements, resulting in variants with ligand-independent activity.
PMCID: PMC5141345  PMID: 27897170
5.  MUC1 Expression by Immunohistochemistry Is Associated with Adverse Pathologic Features in Prostate Cancer: A Multi-Institutional Study 
PLoS ONE  2016;11(11):e0165236.
The uncertainties inherent in clinical measures of prostate cancer (CaP) aggressiveness endorse the investigation of clinically validated tissue biomarkers. MUC1 expression has been previously reported to independently predict aggressive localized prostate cancer. We used a large cohort to validate whether MUC1 protein levels measured by immunohistochemistry (IHC) predict aggressive cancer, recurrence and survival outcomes after radical prostatectomy independent of clinical and pathological parameters.
Material and Methods
MUC1 IHC was performed on a multi-institutional tissue microarray (TMA) resource including 1,326 men with a median follow-up of 5 years. Associations with clinical and pathological parameters were tested by the Chi-square test and the Wilcoxon rank sum test. Relationships with outcome were assessed with univariable and multivariable Cox proportional hazard models and the Log-rank test.
The presence of MUC1 expression was significantly associated with extracapsular extension and higher Gleason score, but not with seminal vesicle invasion, age, positive surgical margins or pre-operative serum PSA levels. In univariable analyses, positive MUC1 staining was significantly associated with a worse recurrence free survival (RFS) (HR: 1.24, CI 1.03–1.49, P = 0.02), although not with disease specific survival (DSS, P>0.5). On multivariable analyses, the presence of positive surgical margins, extracapsular extension, seminal vesicle invasion, as well as higher pre-operative PSA and increasing Gleason score were independently associated with RFS, while MUC1 expression was not. Positive MUC1 expression was not independently associated with disease specific survival (DSS), but was weakly associated with overall survival (OS).
In our large, rigorously designed validation cohort, MUC1 protein expression was associated with adverse pathological features, although it was not an independent predictor of outcome after radical prostatectomy.
PMCID: PMC5112958  PMID: 27846218
6.  Analytic Validation of a Clinical-Grade PTEN Immunohistochemistry Assay in Prostate Cancer by Comparison to PTEN FISH 
PTEN loss is a promising prognostic and predictive biomarker in prostate cancer. Because it occurs most commonly via PTEN gene deletion, we developed a clinical-grade, automated and inexpensive immunohistochemical assay to detect PTEN loss. We studied the sensitivity and specificity of PTEN immunohistochemistry relative to 4-color fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in a multi-institutional cohort of 731 primary prostate tumors. Intact PTEN immunostaining was 91% specific for absence of PTEN gene deletion, (549/602 tumors with 2 copies of the PTEN gene by FISH showed intact expression of PTEN by immunohistochemistry) and 97% sensitive for presence of homozygous PTEN gene deletion (absent PTEN protein expression by immunohistochemistry in 65/67 tumors with homozygous deletion). PTEN immunohistochemistry was 65% sensitive for presence of hemizygous PTEN gene deletion, with protein loss in 40/62 hemizygous tumors. We reviewed the 53 cases where immunohistochemistry showed PTEN protein loss and FISH showed 2 intact copies of the PTEN gene. On re-review, there was ambiguous immunohistochemistry loss in 6% (3/53) and failure to analyze the same tumor area by both methods in 34% (18/53). Of the remaining discordant cases, 41% (13/32) revealed hemizygous (n=8) or homozygous (n=5) PTEN gene deletion that was focal in most cases (11/13). The remaining 19 cases had 2 copies of the PTEN gene by FISH, representing truly discordant cases. Our automated PTEN immunohistochemistry assay is a sensitive method for detection of homozygous PTEN gene deletions. Immunohistochemistry screening is particularly useful to identify cases with heterogeneous PTEN gene deletion in a subset of tumor glands. Mutations, small insertions or deletions and/or epigenetic or microRNA-mediated mechanisms may lead to PTEN protein loss in tumors with normal or hemizygous PTEN gene copy number.
PMCID: PMC4967011  PMID: 27174589
Prostatic carcinoma PTEN; fluorescence in situ hybridization; immunohistochemistry; radical prostatectomy; biomarker
7.  ABCC9/SUR2 in the brain: implications for hippocampal sclerosis of aging and a potential therapeutic target 
Ageing research reviews  2015;24(0 0):111-125.
The ABCC9 gene and its polypeptide product, SUR2, are increasingly implicated in human neurologic disease, including prevalent diseases of the aged brain. SUR2 proteins are a component of the ATP-sensitive potassium (“KATP”) channel, a metabolic sensor for stress and/or hypoxia that has been shown to change in aging. The KATP channel also helps regulate the neurovascular unit. Most brain cell types express SUR2, including neurons, astrocytes, oligodendrocytes, microglia, vascular smooth muscle, pericytes, and endothelial cells. Thus it is not surprising that ABCC9 gene variants are associated with risk for human brain diseases. For example, Cantu syndrome is a result of ABCC9 mutations; we discuss neurologic manifestations of this genetic syndrome. More common brain disorders linked to ABCC9 gene variants include hippocampal sclerosis of aging (HS-Aging), sleep disorders, and depression. HS-Aging is a prevalent neurological disease with pathologic features of both neurodegenerative (aberrant TDP-43) and cerebrovascular (arteriolosclerosis) disease. As to potential therapeutic intervention, the human pharmacopeia features both SUR2 agonists and antagonists, so ABCC9/SUR2 may provide a “druggable target”, relevant perhaps to both HS-Aging and Alzheimer’s disease. We conclude that more work is required to better understand the roles of ABCC9/SUR2 in the human brain during health and disease conditions.
PMCID: PMC4661124  PMID: 26226329
SUR2A; SUR2B; SUR2Ab; SUR1; neuropathology; GWAS; hippocampus; oldest-old; arteriolosclerosis; ABCC8
8.  SRRM4 Expression and the Loss of REST Activity May Promote the Emergence of the Neuroendocrine Phenotype in Castration-Resistant Prostate Cancer 
The neuroendocrine (NE) phenotype is associated with the development of metastatic castration-resistant prostate cancer (CRPC). Our objective was to characterize the molecular features of the NE phenotype in CRPC.
Experimental Design
Expression of chromogranin A (CHGA), synaptophysin (SYP), androgen receptor (AR), and prostate-specific antigen (PSA) was analyzed by immunohistochemistry (IHC) in 155 CRPC metastases from 50 patients and in 24 LuCaP prostate cancer patient-derived xenografts (PDX). Seventy-one of 155 metastases and the 24 LuCaP xenograft lines were analyzed by whole genome microarrays. REST splicing was verified by PCR.
Co-expression of CHGA and SYP in >30% of cells was observed in 22 of 155 metastases (9 patients); 11 of the 22 metastases were AR+/PSA+ (6 patients), 11/22 were AR−/PSA− (4 patients), and 4/24 LuCaP PDXs were AR−/PSA−. By IHC, of the 71 metastases analyzed by whole genome microarrays, 5 metastases were CHGA+/SYP+/AR− and 5 were CHGA+/SYP+/AR+. Only CHGA+/SYP+ metastases had a NE transcript signature. The neuronal transcriptional regulator SRRM4 transcript was associated with the NE signature in CHGA+/SYP+ metastases and all CHGA+/SYP+ LuCaP xenografts. Additionally, expression of SRRM4 in LuCaP NE xenografts correlated with a splice variant of REST that lacks the transcriptional repressor domain.
(a) metastatic NE status can be heterogeneous in the same patient, (b) the CRPC NE molecular phenotype can be defined by CHGA+/SYP+ dual positivity, (c) the NE phenotype is not necessarily associated with the loss of AR activity, and (d) the splicing of REST by SRRM4 could promote the NE phenotype in CRPC.
PMCID: PMC4609255  PMID: 26071481
Prostate cancer; Neuroendocrine; SRRM4
9.  A vector platform for the rapid and efficient engineering of stable complex transgenes 
Scientific Reports  2016;6:34365.
We describe the generation of a set of plasmid vector tools that allow the rapid generation of complex-interacting stable transgenes in immortalized and primary cells. Of particular importance is inclusion of a mechanism to monitor the activation status of regulatory pathways via a reporter cassette (using Gaussia Luciferase), with control of additional transgene expression through doxycycline de-repression. The resulting vectors can be used to assess regulatory pathway activation and are well suited for regulatory pathway crosstalk studies. The system incorporates MultiSite-Gateway cloning for the rapid generation of vectors allowing flexible choice of promoters and transgenes, and Sleeping Beauty transposase technology for efficient incorporation of multiple transgenes in into host cell DNA. The vectors and a library of compatible Gateway Entry clones are available from the non-profit plasmid repository Addgene.
PMCID: PMC5046065  PMID: 27694838
10.  Cerebral amyloid angiopathy and its co-occurrence with Alzheimer’s disease and other cerebrovascular neuropathologic changes 
Neurobiology of aging  2015;36(10):2702-2708.
We examined the relationship between cerebral amyloid angiopathy (CAA), Alzheimer’s disease neuropathologic changes (ADNC), other vascular brain pathologies, and cognition in a large multi-center autopsy sample. Data was obtained from the National Alzheimer’s Coordinating Center on autopsied subjects (N=3,976) who died between 2005 and 2012. Descriptive statistics and multivariable regression models estimated the associations between CAA and other pathologies, and between CAA severity and cognitive test scores proximal to death. CAA tended to co-occur with ADNC but a substantial minority of cases were discrepant. CAA was absent in 22% (n= 520) of subjects with frequent neuritic plaques but present in 20.9% (n=91) of subjects with no neuritic plaques. In subjects with no/sparse neuritic plaques, non-hemorrhagic brain infarcts were more common in those with CAA pathology than without (P= 0.007). In subjects without the APOE ε4 allele, CAA severity was associated with lower cognition proximal to death, factoring in other pathologies. The presence of CAA in non-AD patients may indicate a distinct cerebrovascular condition.
PMCID: PMC4562901  PMID: 26239176
Cerebral amyloid angiopathy; Alzheimer’s disease neuropathologic change; cerebrovascular disease; neuropathology; cognition
11.  Substantial inter-individual and limited intra-individual genomic diversity among tumors from men with metastatic prostate cancer 
Nature medicine  2016;22(4):369-378.
Intra-individual tumor heterogeneity may reduce the efficacy of molecularly guided systemic therapy for cancers that have metastasized. To determine whether the genomic alterations in a single metastasis provide a reasonable assessment of the major oncogenic drivers of other dispersed metastases within an individual, we analyzed multiple tumors from men with disseminated prostate cancer by whole exome sequencing, array CGH and RNA transcript profiling and compared the genomic diversity within and between individuals. In contrast to substantial heterogeneity between men, there was limited diversity comparing metastases within an individual. Numbers of somatic mutations, the burden of genomic copy number alterations, and aberrations in known oncogenic drivers were highly concordant as were metrics of androgen receptor (AR) activity and cell cycle activity. AR activity inversely associated with cell proliferation, whereas the expression of Fanconi anemia (FA) complex genes correlated with elevated cell cycle progression, E2F1 expression and RB1 loss. Men with somatic aberrations in FA complex genes or ATM exhibited significantly longer treatment response durations to carboplatin compared to men without defects in genes encoding DNA repair proteins. Collectively, these data indicate that though exceptions exist, evaluating a single metastasis provides a reasonable assessment of the major oncogenic driver alterations present in disseminated tumors within an individual, and may be useful for selecting treatments based on predicted molecular vulnerabilities.
PMCID: PMC5045679  PMID: 26928463
12.  Epigenetic signature of Gleason score and prostate cancer recurrence after radical prostatectomy 
Clinical Epigenetics  2016;8:97.
Identifying the subset of patients with clinically localized prostate cancer (PCa) at the highest risk of recurrence remains challenging, and better prognostic markers are needed. Gleason score is the best predictor of PCa aggressiveness and prognosis. In the present study, we generated an epigenetic signature based on high versus low Gleason score tumors and evaluated its ability to predict recurrence after radical prostatectomy.
Genome-wide DNA methylation data from The Cancer Genome Atlas (TCGA; no. of patients = 333) and the elastic net method were used to generate an epigenetic signature by contrasting patients with high (8–10) versus low (≤6) Gleason score tumors. The signature was then tested in a cohort of 523 patients with clinically localized disease who had radical prostatectomy. Samples taken from the primary tumor were used for DNA methylation and mRNA expression profiling. Patients were followed for PCa recurrence on average for 8 years after diagnosis.
The epigenetic signature includes 52 differentially methylated CpG sites. In the testing cohort, the signature was associated with poorer recurrence-free survival (hazard ratio per 25 % increase = 1.78; 95 % confidence interval 1.48, 2.16). The signature significantly improved the area under the curve (AUC) for PCa recurrence compared to clinical-pathological parameters alone, particularly among patients diagnosed with Gleason score 7 tumors (0.64 vs. 0.76, P = 1.34E−4). Results were comparable for patients with Gleason 3 + 4 and those with 4 + 3 tumors. Gene Set Enrichment Analysis showed that higher levels of the signature were associated with increased expression of genes related to cell cycle proliferation and decreased expression of androgen-responsive genes.
This report shows evidence that DNA methylation patterns measured in prostate tumor cells are predictive of PCa aggressiveness. The epigenetic signature may have clinical utility to improve prognostication particularly in patients with intermediate Gleason score 7 tumors.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-016-0260-z) contains supplementary material, which is available to authorized users.
PMCID: PMC5024414  PMID: 27651837
Clinically localized prostate cancer; Tumor tissue; DNA methylation; Gene expression; Risk prediction for prognosis; Genome-wide profiling; Elastic net regularization
13.  Novel human ABCC9/SUR2 brain-expressed transcripts and an eQTL relevant to hippocampal sclerosis of aging 
Journal of neurochemistry  2015;134(6):1026-1039.
ABCC9 genetic polymorphisms are associated with increased risk for various human diseases including hippocampal sclerosis of aging. The main goals of this study were 1 > to detect the ABCC9 variants and define the specific 3′ untranslated region (3′UTR) for each variant in human brain, and 2 > to determine whether a polymorphism (rs704180) associated with risk for hippocampal sclerosis of aging pathology is also associated with variation in ABCC9 transcript expression and/or splicing. Rapid amplification of ABCC9 cDNA ends (3′RACE) provided evidence of novel 3′ UTR portions of ABCC9 in human brain. In silico and experimental studies were performed focusing on the single nucleotide polymorphism, rs704180. Analyses from multiple databases, focusing on rs704180 only, indicated that this risk allele is a local expression quantitative trait locus (eQTL). Analyses of RNA from human brains showed increased ABCC9 transcript levels in individuals with the risk genotype, corresponding with enrichment for a shorter 3′ UTR which may be more stable than variants with the longer 3′ UTR. MicroRNA transfection experiments yielded results compatible with the hypothesis that miR-30c causes down-regulation of SUR2 transcripts with the longer 3′ UTR. Thus we report evidence of complex ABCC9 genetic regulation in brain, which may be of direct relevance to human disease.
PMCID: PMC4593479  PMID: 26115089
14.  Expression of Cell Cycle-regulated Genes and Prostate Cancer Prognosis in a Population-based Cohort 
The Prostate  2015;75(13):1354-1362.
Prostate cancer (PCa) is clinically and biologically heterogeneous, making it difficult to predict at detection whether it will take an indolent or aggressive disease course. Cell cycle-regulated genes may be more highly expressed in actively dividing cells, with transcript levels reflecting tumor growth rate. Here we evaluated expression of cell cycle genes in relation to PCa outcomes in a population-based cohort.
Gene expression data were generated from tumor tissues obtained at radical prostatectomy for 383 population-based patients (12.3-years average follow-up). The overall mean and individual transcript levels of 30 selected cell cycle genes was compared between patients with no evidence of recurrence (73%) and those who recurred (27%) or died (7%) from PCa.
The multivariate adjusted hazard ratio (HR) for a change from the 25th to 75th percentile of mean gene expression level (range 8.02–10.05) was 1.25 (95% CI 0.96–1.63; P = 0.10) for PCa recurrence risk, and did not vary substantially by Gleason score, TMPRSS2-ERG fusion status, or family history of PCa. For lethal PCa, the HR for a change (25th to 75th percentile) in mean gene expression level was 2.04 (95% CI 1.26–3.31; P = 0.004), adjusted for clinicopathological variables. The ROC curve for mean gene expression level alone (AUC = 0.740) did not perform as well as clinicopathological variables alone (AUC = 0.803) for predicting lethal PCa, and the addition of gene expression to clinicopathological variables did not substantially improve prediction (AUC = 0.827; P = 0.18). Higher TK1 expression was strongly associated with both recurrent (P = 6.7×10−5) and lethal (P = 6.4×10−6) PCa.
Mean expression level for 30 selected cell cycle-regulated genes was unrelated to recurrence risk, but was associated with a two-fold increase in risk of lethal PCa. However, gene expression had less discriminatory accuracy than clinical variables alone for predicting lethal events. Transcript levels for several genes in the panel were significantly overexpressed in lethal vs. non-recurrent PCa.
PMCID: PMC4992473  PMID: 25990700
cell cycle-regulated genes; gene expression; patient outcomes; population-based cohort; prostate cancer
15.  HDAC Inhibition Impedes Epithelial-Mesenchymal Plasticity and Suppresses Metastatic, Castration-Resistant Prostate Cancer 
Oncogene  2015;35(29):3781-3795.
PI3K/AKT and RAS/MAPK pathway co-activation in the prostate epithelium promotes both epithelial-mesenchymal transition (EMT) and metastatic castration-resistant prostate cancer (mCRPC), which is currently incurable. To study the dynamic regulation of the EMT process, we developed novel genetically-defined cellular and in vivo model systems from which epithelial, EMT, and mesenchymal-like tumor cells with Pten deletion and Kras activation can be isolated. When cultured individually, each population has the capacity to regenerate all three tumor cell populations, indicative of epithelial-mesenchymal plasticity. Despite harboring the same genetic alterations, mesenchymal-like tumor cells are resistant to PI3K and MAPK pathway inhibitors, suggesting that epigenetic mechanisms may regulate the EMT process, as well as dictate the heterogeneous responses of cancer cells to therapy. Among differentially expressed epigenetic regulators, the chromatin remodeling protein HMGA2 is significantly upregulated in EMT and mesenchymal-like tumors cells, as well as in human mCRPC. Knockdown of HMGA2, or suppressing HMGA2 expression with the histone deacetylase (HDAC) inhibitor LBH589, inhibits epithelial-mesenchymal plasticity and stemness activities in vitro and dramatically reduces tumor growth and metastasis in vivo through successful targeting of EMT and mesenchymal-like tumor cells. Importantly, LBH589 treatment in combination with castration prevents mCRPC development and significantly prolongs survival following castration by enhancing p53 and AR acetylation and in turn sensitizing castration-resistant mesenchymal-like tumor cells to ADT. Taken together, these findings demonstrate that cellular plasticity is regulated epigenetically, and that mesenchymal-like tumor cell populations in mCRPC that are resistant to conventional and targeted therapies can be effectively treated with the epigenetic inhibitor LBH589.
PMCID: PMC4896852  PMID: 26640144
epithelial-mesenchymal transition (EMT); castration-resistant prostate cancer (CRPC); metastasis; epigenetics; HDAC inhibitor
16.  Hippocampal Sclerosis of Aging, a Common Alzheimer’s Disease ‘Mimic’: Risk Genotypes are Associated with Brain Atrophy Outside the Temporal Lobe 
Hippocampal sclerosis of aging (HS-Aging) is a common brain disease in older adults with a clinical course that is similar to Alzheimer’s disease. Four single-nucleotide polymorphisms (SNPs) have previously shown association with HS-Aging. The present study investigated structural brain changes associated with these SNPs using surface-based analysis. Participants from the Alzheimer’s Disease Neuroimaging Initiative cohort (ADNI; n = 1,239), with both MRI scans and genotype data, were used to assess the association between brain atrophy and previously identified HS-Aging risk SNPs in the following genes: GRN, TMEM106B, ABCC9, and KCNMB2 (minor allele frequency for each is >30%). A fifth SNP (near the ABCC9 gene) was evaluated in post-hoc analysis. The GRN risk SNP (rs5848_T) was associated with a pattern of atrophy in the dorsomedial frontal lobes bilaterally, remarkable since GRN is a risk factor for frontotemporal dementia. The ABCC9 risk SNP (rs704180_A) was associated with multifocal atrophy whereas a SNP (rs7488080_A) nearby (~50 kb upstream) ABCC9 was associated with atrophy in the right entorhinal cortex. Neither TMEM106B (rs1990622_T), KCNMB2 (rs9637454_A), nor any of the non-risk alleles were associated with brain atrophy. When all four previously identified HS-Aging risk SNPs were summed into a polygenic risk score, there was a pattern of associated multifocal brain atrophy in a predominately frontal pattern. We conclude that common SNPs previously linked to HS-Aging pathology were associated with a distinct pattern of anterior cortical atrophy. Genetic variation associated with HS-Aging pathology may represent a non-Alzheimer’s disease contribution to atrophy outside of the hippocampus in older adults.
PMCID: PMC4978172  PMID: 27003218
Arteriolosclerosis; dementia; KATP; progranulin; rs5848; rs704180; rs1990622; rs9637454; SUR2; TDP-43
17.  Outcomes of active surveillance for the management of clinically localized prostate cancer in the prospective, multi-institutional Canary PASS cohort 
The Journal of urology  2015;195(2):313-320.
Active surveillance represents a strategy to address the overtreatment of prostate cancer, yet uncertainty regarding individual patient outcomes remains a concern. We evaluated outcomes in a prospective multi-center study of active surveillance.
We studied 905 men in the prospective Canary Prostate cancer Active Surveillance Study (PASS) enrolled between 2008 to 2013. We collected clinical data at study entry and at pre-specified intervals and determined associations with adverse reclassification defined as increased Gleason grade or greater cancer volume on follow-up biopsy. We also evaluated the relationships of clinical parameters with pathology findings in participants who underwent surgery after a period of active surveillance.
During a median follow-up of 28 months, 24% of participants experienced adverse reclassification, of whom 53% underwent treatment while 31% continued active surveillance. Overall, 19% of participants received treatment, 68% with adverse reclassification while 32% opted for treatment without disease reclassification. In multivariate Cox proportional hazards modeling, percent of biopsy cores with cancer, BMI, and PSA density were associated with adverse reclassification (P = 0.01, 0.04, 0.04). Of 103 participants subsequently treated by radical prostatectomy, 34% had adverse pathology, defined as primary pattern 4–5 or non-organ confined disease, including two with positive lymph nodes, with no significant relationship between risk category at diagnosis and findings at surgery (P = 0.76).
Most men remain on active surveillance at five years without adverse reclassification or adverse pathology at surgery. However, clinical factors had only modest association with disease reclassification, supporting the need for approaches that improve prediction of this outcome.
PMCID: PMC4970462  PMID: 26327354
prostatic neoplasms; prospective studies; active surveillance
18.  A multicenter study shows PTEN deletion is strongly associated with seminal vesicle involvement and extracapsular extension in localized prostate cancer 
The Prostate  2015;75(11):1206-1215.
Loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene is a promising marker of aggressive prostate cancer. Active surveillance and watchful waiting are increasingly recommended to patients with small tumors felt to be low risk, highlighting the difficulties of Gleason scoring in this setting. There is an urgent need for predictive biomarkers that can be rapidly deployed to aid in clinical decision-making. Our objectives were to assess the incidence and ability of PTEN alterations to predict aggressive disease in a multicenter study.
We used recently developed probes optimized for sensitivity and specificity in a four-color FISH deletion assay to study the Canary Retrospective multicenter Prostate Cancer Tissue Microarray (TMA). This TMA was constructed specifically for biomarker validation from radical prostatectomy specimens, and is accompanied by detailed clinical information with long-term follow-up.
In 612 prostate cancers the overall rate of PTEN deletion was 112 (18.3%). Hemizygous PTEN losses were present in 55/612 (9.0%) of cancers, whereas homozygous PTEN deletion was observed in 57/612 (9.3%) of tumors. Significant associations were found between PTEN status and pathologic stage (P<0.0001), seminal vesicle invasion (p=0.0008), extracapsular extension (p<0.0001), and Gleason score (p=0.0002). In logistic regression analysis of clinical and pathological variables, PTEN deletion was significantly associated with extracapsular extension, seminal vesicle involvement, and higher Gleason score. In the 406 patients in which clinical information was available, PTEN homozygous (p=0.009) deletion was associated with worse post-operative recurrence-free survival (number of events = 189), pre-operative prostate specific antigen (PSA) (p<0.001) and pathologic stage (p=0.03).
PTEN status assessed by FISH is an independent predictor for recurrence free survival in multivariate models, as were seminal vesicle invasion, extracapsular extension Gleason score and preoperative PSA. Furthermore, these data demonstrate that the assay can be readily introduced at first diagnosis in a cost effective manner analogous to the use of FISH for analysis of HER2/neu status in breast cancer. Combined with published research beginning 17 years ago, both the data and tools now exist to implement a PTEN assay in the clinic.
PMCID: PMC4475421  PMID: 25939393
active surveillance; Gleason score; biomarker; PI3K/PTEN/Akt pathway; Fluorescence in Situ Hybridization; Tissue Array Analysis
19.  miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1) 
Loss-of-function mutations in PINK1 and PARKIN are the most common causes of autosomal recessive Parkinson’s disease (PD). PINK1 is a mitochondrial serine/threonine kinase that plays a critical role in mitophagy, a selective autophagic clearance of damaged mitochondria. Accumulating evidence suggests mitochondrial dysfunction is one of central mechanisms underlying PD pathogenesis. Therefore, identifying regulatory mechanisms of PINK1 expression may provide novel therapeutic opportunities for PD. Although post-translational stabilization of PINK1 upon mitochondrial damage has been extensively studied, little is known about the regulation mechanism of PINK1 at the transcriptional or translational levels.
Here, we demonstrated that microRNA-27a (miR-27a) and miR-27b suppress PINK1 expression at the translational level through directly binding to the 3′-untranslated region (3′UTR) of its mRNA. Importantly, our data demonstrated that translation of PINK1 is critical for its accumulation upon mitochondrial damage. The accumulation of PINK1 upon mitochondrial damage was strongly regulated by expression levels of miR-27a and miR-27b. miR-27a and miR-27b prevent mitophagic influx by suppressing PINK1 expression, as evidenced by the decrease of ubiquitin phosphorylation, Parkin translocation, and LC3-II accumulation in damaged mitochondria. Consequently, miR-27a and miR-27b inhibit lysosomal degradation of the damaged mitochondria, as shown by the decrease of the delivery of damaged mitochondria to lysosome and the degradation of cytochrome c oxidase 2 (COX2), a mitochondrial marker. Furthermore, our data demonstrated that the expression of miR-27a and miR-27b is significantly induced under chronic mitophagic flux, suggesting a negative feedback regulation between PINK1-mediated mitophagy and miR-27a and miR-27b.
We demonstrated that miR-27a and miR-27b regulate PINK1 expression and autophagic clearance of damaged mitochondria. Our data further support a novel negative regulatory mechanism of PINK1-mediated mitophagy by miR-27a and miR-27b. Therefore, our results considerably advance our understanding of PINK1 expression and mitophagy regulation and suggest that miR-27a and miR-27b may represent potential therapeutic targets for PD.
Electronic supplementary material
The online version of this article (doi:10.1186/s13024-016-0121-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4960690  PMID: 27456084
PINK1; Mitophagy; miR-27a; miR-27b; Parkinson’s disease
20.  ErbB2 Signaling Increases Androgen Receptor Expression in Abiraterone-Resistant Prostate Cancer 
ErbB2 signaling appears to be increased and may enhance AR activity in a subset of CRPC, but agents targeting ErbB2 have not been effective. This study was undertaken to assess ErbB2 activity in abiraterone-resistant prostate cancer (PCa), and determine whether it may contribute to androgen receptor (AR) signaling in these tumors.
Experimental Design
AR activity and ErbB2 signaling were examined in the radical prostatectomy specimens from a neoadjuvant clinical trial of leuprolide plus abiraterone, and in the specimens from abiraterone-resistant CRPC xenograft models. The effect of ErbB2 signaling on AR activity was determined in two CRPC cell lines. Moreover, the effect of combination treatment with abiraterone and an ErbB2 inhibitor was assessed in a CRPC xenograft model.
We found that ErbB2 signaling was elevated in residual tumor following abiraterone treatment in a subset of patients, and was associated with higher nuclear AR expression. In xenograft models, we similarly demonstrated that ErbB2 signaling was increased and associated with AR reactivation in abiraterone-resistant tumors. Mechanistically, we show that ErbB2 signaling and subsequent activation of the PI3K/AKT signaling stabilizes AR protein. Furthermore, concomitantly treating CRPC cells with abiraterone and an ErbB2 inhibitor, lapatinib, blocked AR reactivation and suppressed tumor progression.
ErbB2 signaling is elevated in a subset of abiraterone-resistant prostate cancer patients and stabilizes AR protein. Combination therapy with abiraterone and ErbB2 antagonists may be effective for treating the subset of CRPC with elevated ErbB2 activity.
PMCID: PMC4947432  PMID: 26936914
prostate cancer; androgen receptor; castration-resistant; abiraterone; HER2; ERBB2; ERBB3; lapatinib
21.  Hippocampal Sclerosis of Aging Can Be Segmental: Two Cases and Review of the Literature 
Hippocampal sclerosis of aging (HS-Aging) is a neurodegenerative disease that mimics Alzheimer disease (AD) clinically and has a prevalence rivaling AD in advanced age. Whereas clinical biomarkers are not yet optimized, HS-Aging has distinctive pathological features that distinguish it from other diseases with “hippocampal sclerosis” pathology, such as epilepsy, cerebrovascular perturbations, and frontotemporal lobar degeneration. By definition, HS-Aging brains show neuronal cell loss and gliosis in the hippocampal formation out of proportion to AD-type pathology; it is strongly associated with aberrant TDP-43 pathology and arteriolosclerosis. Here, we describe 2 cases of “segmental” HS-Aging in which “sclerosis” in the hippocampus was evident only in a subset of brain sections by hematoxylin and eosin (H&E) stain. In these cases, TDP-43 pathology was more widespread on immunostained sections than the neuronal cell loss and gliosis seen in H&E stains. The 2 patients were cognitively intact at baseline and were tracked longitudinally over a decade using cognitive studies with at least 1 neuroimaging scan. We discuss the relevant HS-Aging literature, which indicates the need for a clearer consensus-based delineation of “hippocampal sclerosis” and TDP-43 pathologies in aged subjects.
PMCID: PMC4539239  PMID: 26083567
Biomarkers; Case study; Dementia; Frontotemporal lobar degeneration (FTLD); Hippocampal sclerosis; Neuropathology; Oldest-old
22.  ERG activates the YAP1 transcriptional program and induces the development of age-related prostate tumors 
Cancer cell  2015;27(6):797-808.
Significance of ERG in human prostate cancer is unclear because mouse prostate is resistant to ERG-mediated transformation. We determined that ERG activates the transcriptional program regulated by YAP1 of the Hippo signaling pathway and found that prostate-specific activation of either ERG or YAP1 in mice induces similar transcriptional changes and results in age-related prostate tumors. ERG binds to chromatin regions occupied by TEAD/YAP1 and transactivates Hippo target genes. In addition, in human luminal type prostate cancer cells, ERG binds to the promoter of YAP1 and is necessary for YAP1 expression. These results provide direct genetic evidence of a causal role for ERG in prostate cancer and reveal a connection between ERG and the Hippo signaling pathway.
PMCID: PMC4461839  PMID: 26058078
23.  The DHEA-sulfate depot following P450c17 inhibition supports the case for AKR1C3 inhibition in high risk localized and advanced castration resistant prostate cancer 
Prostate cancer is the second leading cause of cancer death in the United States. Treatment of localized high-risk disease and de novo metastatic disease frequently leads to relapse. These metastatic castration resistant prostate cancers (mCRPC) claim a high mortality rate, despite the extended survival afforded by the growing armamentarium of androgen deprivation, radiation and immunotherapies. Here, we review two studies of neoadjuvant treatment of high-risk localized prostate cancer prior to prostatectomy, the total androgen pathway suppression (TAPS) trial and the neoadjuvant abiraterone acetate (AA) trial. These two trials assessed the efficacy of the non-specific P450c17 inhibitor, ketoconazole and the specific P450c17 inhibitor, AA, to inhibit tissue and serum androgen levels. Furthermore, a novel and validated stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry assay was used to accurately quantify adrenal and gonadal androgens in circulation during the course of these trials. The adrenal androgens, Δ4-androstene-3,17-dione, dehydroepiandrosterone and dehydroepiandrosterone sulfate were significantly reduced in the patients receiving ketoconazole or AA compared to those who did not. However, in both trials, a significant amount of DHEA-S (~20 μg/dL) persists and thus may serve as a depot for intratumoral conversion to the potent androgen receptor ligands, testosterone (T) and 5α-dihydrotestosterone (DHT). The final step in conversion of Δ4-androstene-3,17-dione and 5α-androstanedione to T and DHT, respectively, is catalyzed by AKR1C3. We therefore present the case that in the context of the DHEA-S depot, P450c17 and AKR1C3 inhibition may be an effective combinatorial treatment strategy.
PMCID: PMC4414681  PMID: 25514466
Castration Resistant Prostate Cancer; Mass Spectrometry; AKR1C3; DHEA-sulfate; Abiraterone Acetate
24.  Identification of Novel Tau Interactions with Endoplasmic Reticulum Proteins in Alzheimer’s Disease Brain 
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is pathologically characterized by the formation of extracellular amyloid plaques and intraneuronal tau tangles. We recently identified that tau associates with proteins known to participate in endoplasmic reticulum (ER)-associated degradation (ERAD); consequently, ERAD becomes dysfunctional and causes neurotoxicity. We hypothesized that tau associates with other ER proteins, and that this association could also lead to cellular dysfunction in AD. Portions of human AD and non-demented age matched control brains were fractionated to obtain microsomes, from which tau was co-immunoprecipitated. Samples from both conditions containing tau and its associated proteins were analyzed by mass spectrometry. In total, we identified 91 ER proteins that co-immunoprecipitated with tau; 15.4% were common between AD and control brains, and 42.9% only in the AD samples. The remainder, 41.8% of the proteins, was only seen in the control brain samples. We identified a variety of previously unreported interactions between tau and ER proteins. These proteins participate in over sixteen functional categories, the most abundant being involved in RNA translation. We then determined that association of tau with these ER proteins was different between the AD and control samples. We found that tau associated equally with the ribosomal protein L28 but more robustly with the ribosomal protein P0. These data suggest that the differential association between tau and ER proteins in disease could reveal the pathogenic processes by which tau induces cellular dysfunction.
PMCID: PMC4881838  PMID: 26402096
Alzheimer’s disease; co-immunoprecipitation; endoplasmic reticulum; mass spectrometry; microsome; ribosome; tau; tauopathies
25.  Integrative clinical genomics of advanced prostate cancer 
Dan, Robinson | Van Allen, Eliezer M. | Wu, Yi-Mi | Schultz, Nikolaus | Lonigro, Robert J. | Mosquera, Juan-Miguel | Montgomery, Bruce | Taplin, Mary-Ellen | Pritchard, Colin C | Attard, Gerhardt | Beltran, Himisha | Abida, Wassim M. | Bradley, Robert K. | Vinson, Jake | Cao, Xuhong | Vats, Pankaj | Kunju, Lakshmi P. | Hussain, Maha | Feng, Felix Y. | Tomlins, Scott A. | Cooney, Kathleen A. | Smith, David C. | Brennan, Christine | Siddiqui, Javed | Mehra, Rohit | Chen, Yu | Rathkopf, Dana E. | Morris, Michael J. | Solomon, Stephen B. | Durack, Jeremy C. | Reuter, Victor E. | Gopalan, Anuradha | Gao, Jianjiong | Loda, Massimo | Lis, Rosina T. | Bowden, Michaela | Balk, Stephen P. | Gaviola, Glenn | Sougnez, Carrie | Gupta, Manaswi | Yu, Evan Y. | Mostaghel, Elahe A. | Cheng, Heather H. | Mulcahy, Hyojeong | True, Lawrence D. | Plymate, Stephen R. | Dvinge, Heidi | Ferraldeschi, Roberta | Flohr, Penny | Miranda, Susana | Zafeiriou, Zafeiris | Tunariu, Nina | Mateo, Joaquin | Lopez, Raquel Perez | Demichelis, Francesca | Robinson, Brian D. | Schiffman, Marc A. | Nanus, David M. | Tagawa, Scott T. | Sigaras, Alexandros | Eng, Kenneth W. | Elemento, Olivier | Sboner, Andrea | Heath, Elisabeth I. | Scher, Howard I. | Pienta, Kenneth J. | Kantoff, Philip | de Bono, Johann S. | Rubin, Mark A. | Nelson, Peter S. | Garraway, Levi A. | Sawyers, Charles L. | Chinnaiyan, Arul M.
Cell  2015;161(5):1215-1228.
Toward development of a precision medicine framework for metastatic, castration resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53 and PTEN were frequent (40–60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified novel genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin and ZBTB16/PLZF. Aberrations of BRCA2, BRCA1 and ATM were observed at substantially higher frequencies (19.3% overall) than seen in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides evidence that clinical sequencing in mCRPC is feasible and could impact treatment decisions in significant numbers of affected individuals.
PMCID: PMC4484602  PMID: 26000489
precision oncology; whole exome sequencing; transcriptome sequencing; prostate cancer genomics

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