Integrative clinical genomics of advanced prostate cancer.

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 new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, ß-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those 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 clinically actionable information that could impact treatment decisions for these affected individuals.

Racial differences in serum chemokines in prostate cancer patients.

BACKGROUND: This study aimed to understand the differential levels of inflammatory chemokines in association with higher prostate cancer incidence and mortality in African American (AA) men than in Caucasians (CA). METHODS: The authors used a chemokine assay to simultaneously measure 40 chemokines and cytokines levels in the serum of preoperative prostate cancer patients and healthy controls of AA and CA races. Selected chemokines (CXCL2, CXCL5, and CCL23) serum level was validated in 211 serum samples from prostate cancer patients and healthy controls. Differential expression of CXCL5 and CCL23 was analyzed using immunohistochemistry in a representative cohort of prostate tumor tissues of AA and CA races. RESULTS: Race-specific comparisons from 211 serum samples showed significantly higher levels of CXCL2 (control: 3104.0 pg/mL vs. cancer: 2451.0 pg/mL) and CXCL5 (control: 5189.0 pg/mL vs. cancer: 5459.0 pg/mL) in AA men than in CAs (CXCL2; control: 1155.0 pg/mL vs. cancer: 889.3 pg/mL, and CXCL5; control: 1183.0 pg/mL vs. cancer: 977.5 pg/mL). CCL23 differed significantly within and between the races with a lower level in AA cancer cases (454.5 vs. 966.6 pg/mL) than healthy controls (740.5 vs. 1263.0 pg/mL). Patient age, prostate-specific antigen, or Gleason scores were not significantly associated with these chemokines. Immunostaining for CXCL5 and CCL23 in a representative cohort of archival prostate tissues displayed significantly higher CXCL5 in prostate tumors than in adjacent benign tissues, whereas CCL23 was nondetectable in most of the analyzed tumor tissues. CONCLUSION: Lower levels of CCL23 in AA prostate cancer patient sera and tumor tissues and high CXCL2 and CXCL5 may contribute to aggressive prostate cancer, as often seen in AA men. The disproportionate levels of serum chemokines associated with race warrant further exploration to improve equitability in precision oncology to benefit prostate cancer patients.

Integrative clinical genomics of metastatic cancer.

Metastasis is the primary cause of cancer-related deaths. Although The Cancer Genome Atlas has sequenced primary tumour types obtained from surgical resections, much less comprehensive molecular analysis is available from clinically acquired metastatic cancers. Here we perform whole-exome and -transcriptome sequencing of 500 adult patients with metastatic solid tumours of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53, CDKN2A, PTEN, PIK3CA, and RB1. Putative pathogenic germline variants were present in 12.2% of cases of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing to identify gene fusions, pathway activation, and immune profiling. Our results show that integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers.

PRAME expression in spindle cell melanoma, malignant peripheral nerve sheath tumour, and other cutaneous sarcomatoid neoplasms: a comparative analysis.

Diagnosis of spindle cell/sarcomatoid melanoma may be challenging due to frequent loss of expression of melanocytic marker(s) and histomorphologic resemblance to various mesenchymal tumours, particularly malignant peripheral nerve sheath tumour (MPNST). Overexpression of PReferentially expressed Antigen in MElanoma (PRAME) supports a diagnosis of melanoma when evaluating challenging melanocytic tumours. PRAME expression in MPNST and other cutaneous sarcomatoid neoplasms, however, has not been well characterised. We aimed to determine the utility of PRAME immunostain in distinguishing spindle cell melanoma from MPNST and other sarcomatoid mimics. PRAME expression was scored by extent (0 to 4+) and intensity (0 to 3) of staining. A strong positive correlation was observed between the extent and intensity scores (r = 0.84). An extent score of 4+, defined by staining in 76-100% of tumour cells, was seen in 56% (23/41) of spindle cell melanomas, 18% (7/38) of MPNSTs, 15% (4/27) of cutaneous sarcomatoid squamous cell carcinomas (SCCs), 33% (5/15) of poorly differentiated cutaneous angiosarcomas, 12% (4/33) of atypical fibroxanthomas (AFXs), 4% (1/25) of pleomorphic dermal sarcomas (PDSs), and none (0/16) of the high-grade cutaneous leiomyosarcomas. A significant difference was found between spindle cell melanoma and all other examined sarcomatoid neoplasms except angiosarcoma. While diffuse (and often strong) PRAME expression is more frequently observed in spindle cell melanoma than MPNST, sarcomatoid SCC, AFX, PDS, and high-grade leiomyosarcoma, its limited sensitivity and specificity caution against its use as a standalone diagnostic marker. PRAME may complement other epigenetic or lineage-specific markers and should only be used as part of an immunohistochemical panel when evaluating these sarcomatoid neoplasms.

The role of the histone H3 variant CENPA in prostate cancer.

Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth.

Use of the MyProstateScore Test to Rule Out Clinically Significant Cancer: Validation of a Straightforward Clinical Testing Approach.

PURPOSE: The MyProstateScore test was validated for improved detection of clinically significant (grade group ≥2) prostate cancer relative to prostate specific antigen based risk calculators. We sought to validate an optimal MyProstateScore threshold for clinical use in ruling out grade group ≥2 cancer in men referred for biopsy. MATERIALS AND METHODS: Biopsy naïve men provided post-digital rectal examination urine prior to biopsy. MyProstateScore was calculated using the validated, locked multivariable model including only serum prostate specific antigen, urinary prostate cancer antigen 3 and urinary TMPRSS2:ERG. The MyProstateScore threshold approximating 95% sensitivity for grade group ≥2 cancer was identified in a training cohort, and performance was measured in 2 external validation cohorts. We assessed the 1) overall biopsy referral population and 2) population meeting guideline based testing criteria (ie, prostate specific antigen 3-10, or <3 with suspicious digital rectal examination). RESULTS: Validation cohorts were prospectively enrolled from academic (977 patients, median prostate specific antigen 4.5, IQR 3.1-6.0) and community (548, median prostate specific antigen 4.9, IQR 3.7-6.8) settings. In the overall validation population (1,525 patients), 338 men (22%) had grade group ≥2 cancer on biopsy. The MyProstateScore threshold of 10 provided 97% sensitivity and 98% negative predictive value for grade group ≥2 cancer. MyProstateScore testing would have prevented 387 unnecessary biopsies (33%), while missing only 10 grade group ≥2 cancers (3.0%). In 1,242 patients meeting guideline based criteria, MyProstateScore ≤10 provided 96% sensitivity and 97% negative predictive value, and would have prevented 32% of unnecessary biopsies, missing 3.7% of grade group ≥2 cancers. CONCLUSIONS: In a large, clinically pertinent biopsy referral population, MyProstateScore ≤10 provided exceptional sensitivity and negative predictive value for ruling out grade group ≥2 cancer. This straightforward secondary testing approach would reduce the use of more costly and invasive procedures after screening with prostate specific antigen.

Characterization of the EZH2-MMSET histone methyltransferase regulatory axis in cancer.

Histone methyltransferases (HMTases), as chromatin modifiers, regulate the transcriptomic landscape in normal development as well in diseases such as cancer. Here, we molecularly order two HMTases, EZH2 and MMSET, that have established genetic links to oncogenesis. EZH2, which mediates histone H3K27 trimethylation and is associated with gene silencing, was shown to be coordinately expressed and function upstream of MMSET, which mediates H3K36 dimethylation and is associated with active transcription. We found that the EZH2-MMSET HMTase axis is coordinated by a microRNA network and that the oncogenic functions of EZH2 require MMSET activity. Together, these results suggest that the EZH2-MMSET HMTase axis coordinately functions as a master regulator of transcriptional repression, activation, and oncogenesis and may represent an attractive therapeutic target in cancer.

Photoacoustic spectral analysis at ultraviolet wavelengths for characterizing the Gleason grades of prostate cancer.

The diagnosis of aggressive prostate cancer (PCa) has relied on microscopic architectures, namely Gleason patterns, of tissues extracted through core biopsies. Technology capable of assessing the tissue architecture without tissue extraction will reduce the invasiveness of PCa diagnosis and improve diagnostic accuracy by allowing for more sampling locations. Our recently developed photoacoustic spectral analysis (PASA) has achieved quantification of tissue architectural heterogeneity interstitially. Taking advantage of the unique optical absorption of cell nuclei at ultraviolet (UV) wavelengths, this study investigated PASA at 266 nm for quantifying the tissue architecture heterogeneity in prostates. The results have shown significant differences among the normal, early cancer, and late cancer stages in mouse prostates ex vivo and in vivo (n=20, p<0.05). The study with human samples ex vivo has shown a correlation of 0.80 (n=11, p<0.05) between PASA quantification and pathologic diagnosis.

Characterization of glycine-N-acyltransferase like 1 (GLYATL1) in prostate cancer.

BACKGROUND: Recent microarray and sequencing studies of prostate cancer showed multiple molecular alterations during cancer progression. It is critical to evaluate these molecular changes to identify new biomarkers and targets. We performed analysis of glycine-N-acyltransferase like 1 (GLYATL1) expression in various stages of prostate cancer in this study and evaluated the regulation of GLYATL1 by androgen. METHOD: We performed in silico analysis of cancer gene expression profiling and transcriptome sequencing to evaluate GLYATL1 expression in prostate cancer. Furthermore, we performed immunohistochemistry using specific GLYATL1 antibody using high-density prostate cancer tissue microarray containing primary and metastatic prostate cancer. We also tested the regulation of GLYATL1 expression by androgen and ETS transcription factor ETV1. In addition, we performed RNA-sequencing of GLYATL1 modulated prostate cancer cells to evaluate the gene expression and changes in molecular pathways. RESULTS: Our in silico analysis of cancer gene expression profiling and transcriptome sequencing we revealed an overexpression of GLYATL1 in primary prostate cancer. Confirming these findings by immunohistochemistry, we show that GLYATL1 is overexpressed in primary prostate cancer compared with metastatic prostate cancer and benign prostatic tissue. Low-grade cancers had higher GLYATL1 expression compared to high-grade prostate tumors. Our studies showed that GLYATL1 is upregulated upon androgen treatment in LNCaP prostate cancer cells which harbors ETV1 gene rearrangement. Furthermore, ETV1 knockdown in LNCaP cells showed downregulation of GLYATL1 suggesting potential regulation of GLYATL1 by ETS transcription factor ETV1. Transcriptome sequencing using the GLYATL1 knockdown prostate cancer cell lines LNCaP showed regulation of multiple metabolic pathways. CONCLUSIONS: In summary, our study characterizes the expression of GLYATL1 in prostate cancer and explores the regulation of its regulation in prostate cancer showing role for androgen and ETS transcription factor ETV1. Future studies are needed to decipher the biological significance of these findings.

Neurofilament is superior to cytokeratin 20 in supporting cutaneous origin for neuroendocrine carcinoma.

AIM: Primary cutaneous neuroendocrine carcinoma, or Merkel cell carcinoma (MCC), cannot be distinguished morphologically from small-cell neuroendocrine carcinomas (SmCC) from other sites. Immunohistochemistry is required to confirm cutaneous origin, and is also used for detection of sentinel lymph node (SLN) metastases of MCC. Cytokeratin 20 (CK20) expression is commonly used for these purposes, but is negative in some MCC cases, and has unclear specificity. We evaluated immunohistochemistry for neurofilament and CK20 in MCC compared with SmCC from other sites. METHODS AND RESULTS: We evaluated neurofilament expression in 55 MCC specimens from 39 unique patients, including nine CK20-negative MCC tumours. Neurofilament expression was observed in 42 of 55 (76.4%) MCC cases, including seven of nine (77.8%) CK20-negative MCC cases. Neurofilament was expressed in nine of 12 (75%) Merkel cell polyomavirus-positive tumours and five of 10 (50%) virus-negative tumours. Compared to a standard immunohistochemical panel (cytokeratin cocktail and CK20), neurofilament was 87.5% sensitive for detecting SLN metastases. Neurofilament and CK20 expression was also assessed in 61 extracutaneous SmCC from 60 unique patients, with primary sites including lung (27), bladder (18), cervix (3), gastrointestinal tract (3), sinonasal tract (2) and other sites (7). The specificity of neurofilament and CK20 for MCC versus non-cutaneous SmCC was 96.7% and 59.0%, respectively. CONCLUSIONS: Neurofilament has superior specificity to CK20 in distinguishing MCC from non-cutaneous SmCC. Neurofilament is frequently expressed in CK20- and virus-negative MCC tumours. Limitations of neurofilament immunohistochemistry include lower sensitivity than CK20 and subtle staining in some tumours. However, our findings indicate that neurofilament is useful for excluding non-cutaneous SmCC.

A five-CpG DNA methylation score to predict metastatic-lethal outcomes in men treated with radical prostatectomy for localized prostate cancer.

BACKGROUND: Prognostic biomarkers for localized prostate cancer (PCa) could improve personalized medicine. Our group previously identified a panel of differentially methylated CpGs in primary tumor tissue that predict disease aggressiveness, and here we further validate these biomarkers. METHODS: Pyrosequencing was used to assess CpG methylation of eight biomarkers previously identified using the HumanMethylation450 array; CpGs with strongly correlated (r >0.70) results were considered technically validated. Logistic regression incorporating the validated CpGs and Gleason sum was used to define and lock a final model to stratify men with metastatic-lethal versus non-recurrent PCa in a training dataset. Coefficients from the final model were then used to construct a DNA methylation score, which was evaluated by logistic regression and Receiver Operating Characteristic (ROC) curve analyses in an independent testing dataset. RESULTS: Five CpGs were technically validated and all were retained (P < 0.05) in the final model. The 5-CpG and Gleason sum coefficients were used to calculate a methylation score, which was higher in men with metastatic-lethal progression (P = 6.8 × 10-6 ) in the testing dataset. For each unit increase in the score there was a four-fold increase in risk of metastatic-lethal events (odds ratio, OR = 4.0, 95%CI = 1.8-14.3). At 95% specificity, sensitivity was 74% for the score compared to 53% for Gleason sum alone. The score demonstrated better prediction performance (AUC = 0.91; pAUC = 0.037) compared to Gleason sum alone (AUC = 0.87; pAUC = 0.025). CONCLUSIONS: The DNA methylation score improved upon Gleason sum for predicting metastatic-lethal progression and holds promise for risk stratification of men with aggressive tumors. This prognostic score warrants further evaluation as a tool for improving patient outcomes.

The use of exome capture RNA-seq for highly degraded RNA with application to clinical cancer sequencing.

RNA-seq by poly(A) selection is currently the most common protocol for whole transcriptome sequencing as it provides a broad, detailed, and accurate view of the RNA landscape. Unfortunately, the utility of poly(A) libraries is greatly limited when the input RNA is degraded, which is the norm for research tissues and clinical samples, especially when specimens are formalin-fixed. To facilitate the use of RNA sequencing beyond cell lines and in the clinical setting, we developed an exome-capture transcriptome protocol with greatly improved performance on degraded RNA. Capture transcriptome libraries enable measuring absolute and differential gene expression, calling genetic variants, and detecting gene fusions. Through validation against gold-standard poly(A) and Ribo-Zero libraries from intact RNA, we show that capture RNA-seq provides accurate and unbiased estimates of RNA abundance, uniform transcript coverage, and broad dynamic range. Unlike poly(A) selection and Ribo-Zero depletion, capture libraries retain these qualities regardless of RNA quality and provide excellent data from clinical specimens including formalin-fixed paraffin-embedded (FFPE) blocks. Systematic improvements across key applications of RNA-seq are shown on a cohort of prostate cancer patients and a set of clinical FFPE samples. Further, we demonstrate the utility of capture RNA-seq libraries in a patient with a highly malignant solitary fibrous tumor (SFT) enrolled in our clinical sequencing program called MI-ONCOSEQ. Capture transcriptome profiling from FFPE revealed two oncogenic fusions: the pathognomonic NAB2-STAT6 inversion and a therapeutically actionable BRAF fusion, which may drive this specific cancer's aggressive phenotype.

Detection of 6 TFEB-amplified renal cell carcinomas and 25 renal cell carcinomas with MITF translocations: systematic morphologic analysis of 85 cases evaluated by clinical TFE3 and TFEB FISH assays.

Renal cell carcinomas with MITF aberrations demonstrate a wide morphologic spectrum, highlighting the need to consider these entities within the differential diagnosis of renal tumors encountered in clinical practice. Herein, we describe our experience with application of clinical fluorescence in situ hybridization (FISH) assays for detection of TFE3 and TFEB gene aberrations from 85 consecutive renal cell carcinoma cases submitted to our genitourinary FISH service. Results from 170 FISH assays performed on these tumors were correlated with available clinicopathologic findings. Ninety-eight percent of renal tumors submitted for FISH evaluation were from adult patients. Thirty-one (37%) tumors were confirmed to demonstrate MITF aberrations (21 TFE3 translocation, 4 TFEB translocation, and 6 TFEB amplification cases). Overall, renal cell carcinomas with MITF aberrations demonstrated morphologic features overlapping with clear cell, papillary, or clear cell papillary renal cell carcinomas. Renal cell carcinomas with MITF aberrations were significantly more likely to demonstrate dual (eosinophilic and clear) cytoplasmic tones (P=0.030), biphasic TFEB translocation renal cell carcinoma-like morphology (P=0.002), psammomatous calcifications (P=0.002), and nuclear pseudoinclusions (P=0.001) than renal cell carcinomas without MITF aberrations. Notably, 7/9 (78%) renal cell carcinomas exhibiting subnuclear clearing and linear nuclear array (6 of which showed high World Health Organization/International Society of Urological Pathology nucleolar grade) demonstrated TFE3 translocation, an association that was statistically significant when compared with renal cell carcinomas without MITF aberrations (P=0.009). In this cohort comprising consecutive cases, TFEB-amplified renal cell carcinomas were more commonly identified than renal cell carcinomas with TFEB translocations, and four (67%) of these previously unreported TFEB-amplified renal cell carcinomas demonstrated oncocytic and papillary features with a high World Health Organization/International Society of Urological Pathology nucleolar grade. In summary, TFE3 and TFEB FISH evaluation aids in identification and accurate classification of renal cell carcinomas with MITF aberrations, including TFEB-amplified renal cell carcinoma, which may demonstrate aggressive behavior.

The mutational landscape of lethal castration-resistant prostate cancer.

Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains and losses, including ETS gene family fusions, PTEN loss and androgen receptor (AR) amplification, which drive prostate cancer development and progression to lethal, metastatic castration-resistant prostate cancer (CRPC). However, less is known about the role of mutations. Here we sequenced the exomes of 50 lethal, heavily pre-treated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment-naive, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPCs (2.00 per megabase) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1 that define a subtype of ETS gene family fusion-negative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in approximately one-third of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Furthermore, we identified recurrent mutations in multiple chromatin- and histone-modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with the AR, which is required for AR-mediated signalling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signalling and increases tumour growth. Proteins that physically interact with the AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX (also known as KDM6A) and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signalling deregulated in prostate cancer, and prioritize candidates for future study.

Nerve Growth Factor and Selected Cytokines in Women With and Without Vulvodynia.

OBJECTIVE: The aim of the study was to assess the association between cytokines/neurokines after in vitro stimulation with Candida antigen or lipopolysaccharide (LPS) in blood samples among women with and without vulvodynia. MATERIALS AND METHODS: Women with vulvodynia and asymptomatic controls at three offices at the University of Michigan were examined clinically and completed a comprehensive survey in this cross-sectional study. Cytokine/neurokine levels were determined on blood samples using established ELISA protocols. Analysis of 48 cases and 42 ethnically matched controls included descriptive statistics (median, minimal, and maximal levels of cytokines/neurokines), overall and in cases and controls. Because of left-censored measurements, interval censored survival analysis was used to assess the association between case/control status and pain characteristics with cytokine/neurokine levels. RESULTS: Participants ranged in age from 19 to 60 years. Levels of IL1ß, IL1-RA, TNFα, IL-6, and IL-8 increased substantially after LPS stimulation, whereas no response was seen on IFNγ or nerve growth factor (NGF). Each increased after Candida antigen stimulation, although responses to Candida antigen stimulation of IL1ß, IL-6, and TNFα were less robust than after LPS. Only NGF was significantly increased in vulvodynia cases compared with controls (Exp ß (95% CI) = 2.08 [1.08-3.98]) after 24-hour Candida antigen stimulation and persisted when controlled for age, use of oral contraceptives, or history of Candida vulvovaginitis. No association between cytokine/neurokine levels and pain characteristics was found. CONCLUSIONS: Compared with that of control women, whole blood from women with vulvodynia demonstrates an enhanced production of NGF, but not of a set of inflammation-related cytokines, in response to Candida antigen stimulation.

Expression and Role of PAICS, a De Novo Purine Biosynthetic Gene in Prostate Cancer.

BACKGROUND: Our goal was to investigate de novo purine biosynthetic gene PAICS expression and evaluate its role in prostate cancer progression. METHODS: Next-generation sequencing, qRTPCR and immunoblot analysis revealed an elevated expression of a de novo purine biosynthetic gene, Phosphoribosylaminoimidazole Carboxylase, Phosphoribosylaminoimidazole Succinocarboxamide Synthetase (PAICS) in a progressive manner in prostate cancer. Functional analyses were performed using prostate cancer cell lines- DU145, PC3, LnCaP, and VCaP. The oncogenic properties of PAICS were studied both by transient and stable knockdown strategies, in vivo chicken chorioallantoic membrane (CAM) and murine xenograft models. Effect of BET bromodomain inhibitor JQ1 on the expression level of PAICS was also studied. RESULTS: Molecular staging of prostate cancer is important factor in effective diagnosis, prognosis and therapy. In this study, we identified a de novo purine biosynthetic gene; PAICS is overexpressed in PCa and its expression correlated with disease aggressiveness. Through several in vitro and in vivo functional studies, we show that PAICS is necessary for proliferation and invasion in prostate cancer cells. We identified JQ1, a BET bromodomain inhibitor previously implicated in regulating MYC expression and demonstrated role in prostate cancer, abrogates PAICS expression in several prostate cancer cells. Furthermore, we observe loss of MYC occupancy on PAICS promoter in presence of JQ1. CONCLUSIONS: Here, we report that evaluation of PAICS in prostate cancer progression and its role in prostate cancer cell proliferation and invasion and suggest it as a valid therapeutic target. We suggest JQ1, a BET-domain inhibitor, as possible therapeutic option in targeting PAICS in prostate cancer. Prostate 77:10-21, 2017. © 2016 Wiley Periodicals, Inc.

Whole-Exome Sequencing Reveals Mutations in Genes Linked to Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome in Fatal Cases of H1N1 Influenza.

BACKGROUND: Severe H1N1 influenza can be lethal in otherwise healthy individuals and can have features of reactive hemophagocytic lymphohistiocytosis (HLH). HLH is associated with mutations in lymphocyte cytolytic pathway genes, which have not been previously explored in H1N1 influenza. METHODS: Sixteen cases of fatal influenza A(H1N1) infection, 81% with histopathologic hemophagocytosis, were identified and analyzed for clinical and laboratory features of HLH, using modified HLH-2004 and macrophage activation syndrome (MAS) criteria. Fourteen specimens were subject to whole-exome sequencing. Sequence alignment and variant filtering detected HLH gene mutations and potential disease-causing variants. Cytolytic function of the PRF1 p.A91V mutation was tested in lentiviral-transduced NK-92 natural killer (NK) cells. RESULTS: Despite several lacking variables, cases of influenza A(H1N1) infection met 44% and 81% of modified HLH-2004 and MAS criteria, respectively. Five subjects (36%) carried one of 3 heterozygous LYST mutations, 2 of whom also possessed the p.A91V PRF1 mutation, which was shown to decrease NK cell cytolytic function. Several patients also carried rare variants in other genes previously observed in MAS. CONCLUSIONS: This cohort of fatal influenza A(H1N1) infections confirms the presence of hemophagocytosis and HLH pathology. Moreover, the high percentage of HLH gene mutations suggests they are risk factors for mortality among individuals with influenza A(H1N1) infection.

Depletion of SAG/RBX2 E3 ubiquitin ligase suppresses prostate tumorigenesis via inactivation of the PI3K/AKT/mTOR axis.

BACKGROUND: SAG (Sensitive to Apoptosis Gene), also known as RBX2, ROC2 or RNF7, is a RING component of CRL (Cullin-RING ligase), required for its activity. Our recent study showed that SAG/RBX2 co-operated with Kras to promote lung tumorigenesis, but antagonized Kras to inhibit skin tumorigenesis, suggesting a tissue/context dependent function of Sag. However, it is totally unknown whether and how Sag would play in prostate tumorigenesis, triggered by Pten loss. METHODS: Sag and Pten double conditional knockout mice were generated and prostate specific deletion of Sag and Pten was achieved by PB4-Cre, and their effect on prostate tumorigenesis was evaluated by H&E staining. The methods of immunohistochemistry (IHC) staining and Western blotting were utilized to examine expression of various proteins in prostate cancer tissues or cell lines. The effect of SAG knockdown in proliferation, survival and migration was evaluated in two prostate cancer cell lines. The poly-ubiquitylation of PHLPP1 and DEPTOR was evaluated by both in vivo and in vitro ubiquitylation assays. RESULTS: SAG is overexpressed progressively from early-to-late stage of human prostate cancer with the highest expression seen in metastatic lesion. Sag deletion inhibits prostate tumorigenesis triggered by Pten loss in a mouse model as a result of suppressed proliferation. SAG knockdown in human prostate cancer cells inhibits a) proliferation in monolayer and soft agar, b) clonogenic survival, and c) migration. SAG is an E3 ligase that promotes ubiquitylation and degradation of PHLPP1 and DEPTOR, leading to activation of the PI3K/AKT/mTOR axis, whereas SAG knockdown caused their accumulation. Importantly, growth suppression triggered by SAG knockdown was partially rescued by simultaneous knockdown of PHLPP1 or DEPTOR, suggesting their causal role. Accumulation of Phlpp1 and Deptor with corresponding inactivation of Akt/mTOR was also detected in Sag-null prostate cancer tissues. CONCLUSIONS: Sag is an oncogenic cooperator of Pten-loss for prostate tumorigenesis. Targeting SAG E3 ligase may, therefore, have therapeutic value for the treatment of prostate cancer associated with Pten loss.