Hao G. Nguyen

The A2B adenosine receptor protects against inflammation and excessive vascular adhesion

Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor-knockout/reporter gene-knock-in (A2BAR-knockout/reporter gene-knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-alpha, and a consequent downregulation of IkappaB-alpha are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.

Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

Extended Followup and Risk Factors for Disease Reclassification in a Large Active Surveillance Cohort for Localized Prostate Cancer

PURPOSE Active surveillance to manage prostate cancer provides an alternative to immediate treatment in men with low risk prostate cancer. We report updated outcomes from a long-standing active surveillance cohort and factors associated with reclassification. MATERIALS AND METHODS We retrospectively reviewed data on all men enrolled in the active surveillance cohort at our institution with at least 6 months of followup between 1990 and 2013. Surveillance consisted of quarterly prostate specific antigen testing, repeat imaging with transrectal ultrasound at provider discretion and periodic repeat prostate biopsies. Factors associated with repeat biopsy reclassification and local treatment were determined by multivariate Cox proportional hazards regression. We also analyzed the association of prostate specific antigen density and outcomes stratified by prostate size. RESULTS A total of 810 men who consented to participate in the research cohort were followed on active surveillance for a median of 60 months. Of these men 556 (69%) met strict criteria for active surveillance. Five-year overall survival was 98%, treatment-free survival was 60% and biopsy reclassification-free survival was 40%. There were no prostate cancer related deaths. On multivariate analysis prostate specific antigen density was positively associated with the risk of biopsy reclassification and treatment while the number of biopsies and time between biopsies were inversely associated with the 2 outcomes (each p <0.01). When stratified by prostate volume, prostate specific antigen density remained significantly associated with biopsy reclassification for all strata but prostate specific antigen density was only significantly associated with treatment in men with a smaller prostate. CONCLUSIONS Significant prostate cancer related morbidity and mortality remained rare at intermediate followup. Prostate specific antigen density was independently associated with biopsy reclassification and treatment while on active surveillance.

Targeting autophagy overcomes Enzalutamide resistance in castration-resistant prostate cancer cells and improves therapeutic response in a xenograft model

Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Androgen deprivation or treatment with androgen receptor (AR) signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or bicalutamide induced autophagy in androgen-dependent and in castration-resistant CaP (castration-resistant prostate cancer (CRPC)) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased light chain 3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays, we showed that inhibiting autophagy with clomipramine (CMI), chloroquine or metformin increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMP-dependent protein kinase (AMPK) activation and the suppression of mammalian target of rapamycin (mTOR) through Raptor phosphorylation (Serine 792). Furthermore, small interfering RNA targeting AMPK significantly inhibited autophagy and promoted cell death in CaP cells acutely or chronically exposed to ENZA or androgen deprivation, suggesting that autophagy is an important survival mechanism in CRPC. Lastly, in vivo studies with mice orthotopically implanted with ENZA-resistant cells demonstrated that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduced tumor growth when compared with control groups (P<0.005). In conclusion, autophagy is as an important mechanism of resistance to ARSI in CRPC. Antiandrogen-induced autophagy is mediated through the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in vitro and in vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen-resistance setting.

Deregulated Aurora-B induced tetraploidy promotes tumorigenesis

High expression of Aurora‐B has been observed in various cancers, and inhibition of this kinase has been shown to halt cellular proliferation. However, the mechanism of effect of Aurora‐B on cellular transformation has not been fully explored. Here, we show that overexpression of Aurora‐B in murine epithelial cells promotes generation of tetraploids. In search of a related mechanism, spectral karyotyping was carried out, showing premature chromatid separation (PCS). Of interest, PCS is a hallmark of Roberts syndrome, which also involves cellular polyploidy and aneu‐ ploidy. Sorted tetraploid Aurora‐B‐overexpressing cells promoted significant mammary epithelial cancers when injected into nude mice, as compared to injection of nonfractionated cells, suggesting that tetraploidy is an important mediator of Aurora‐B‐induced tumorigenesis. Comparative chromosome hybridization performed on DNA derived from tetraploid cell‐induced tumors indicates amplifications and deletions of regions throughout the genome, which include tumor‐promoting or tumorsuppressing genes, respectively. Thus, sustained expression of Aurora‐B induces tetraploidy, which, in turn, facilitates genomic instability and tumor development in a xenograft model.— Nguyen, H. G., Makitalo, M., Yang, D., Chinnappan, D., St. Hilaire, C., Ravid, K. Deregulated Aurora‐B induced tetraploidy promotes tumorigenesis. FASEBJ. 23, 2741–2748 (2009)

Tetraploidy/aneuploidy and stem cells in cancer promotion: The role of chromosome passenger proteins

While polyploidy, a state of having fully duplicated sets of chromosomes per cell, has been described in normally developing bone marrow megakaryocytes or as an adaptive response in other cell types, aneuploidy is never detected in normal cells. Tetraploidy or aneuploidy can be induced by several signals and it is highly prevalent in different forms of cancers, suggesting a role for this cell cycle state in promoting cellular transformation. Investigations suggested that loss of heterozygosity of cancer‐related genes in stem cells might contribute to genetic instability in progeny cells and to subsequent cancer development. Deregulated expression of chromosome passenger proteins, such as Aurora kinases or Survivin, is a hallmark of various cancers, and experimentally induced changes in these regulators can promote tetraploidy or aneuploidy and loss of heterozygosity. Our studies described an induction of tetraploidy/aneuploidy by a stable form of Aurora‐B, leading to acquisition of transformation properties. It is intriguing to speculate that in some cancers, tetraploidy/aneuploidy induced by deregulated expression of a mitotic regulator represents a primary event that leads to unbalanced expression of a cluster of crucial genes and to cellular transformation. J.Cell.Physiol.

Impact of Histologic Subtype on Cancer-specific Survival in Patients with Renal Cell Carcinoma and Tumor Thrombus

BACKGROUND Although different prognostic factors for patients with renal cell carcinoma (RCC) and vena cava tumor thrombus (TT) have been studied, the prognostic value of histologic subtype in these patients remains unclear. OBJECTIVE We analyzed the impact of histologic subtype on cancer-specific survival (CSS). DESIGN, SETTINGS, AND PARTICIPANTS We retrospectively analyzed the records of 1774 patients with RCC and TT who underwent radical nephrectomy and tumor thrombectomy from 1971 to 2012 at 22 US and European centers. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Multivariable ordered logistic and Cox regression models were used to quantify the impact of tumor histology on CSS. RESULTS AND LIMITATIONS Overall 5-yr CSS was 53.4% (confidence interval [CI], 50.5-56.2) in the entire group. TT level (according to the Mayo classification of macroscopic venous invasion in RCC) was I in 38.5% of patients, II in 30.6%, III in 17.3%, and IV in 13.5%. Histologic subtypes were clear cell renal cell carcinoma (cRCC) in 89.9% of patients, papillary renal cell carcinoma (pRCC) in 8.5%, and chromophobe RCC in 1.6%. In univariable analysis, pRCC was associated with a significantly worse CSS (p<0.001) compared with cRCC. In multivariable analysis, the presence of pRCC was independently associated with CSS (hazard ratio: 1.62; CI, 1.01-2.61; p<0.05). Higher TT level, positive lymph node status, distant metastasis, and fat invasion were also independently associated with CSS. CONCLUSIONS In our multi-institutional series, we found that patients with pRCC and vena cava TT who underwent radical nephrectomy and tumor thrombectomy had significantly worse cancer-specific outcomes when compared with patients with other histologic subtypes of RCC. We confirmed that higher TT level and fat invasion were independently associated with reduced CSS.

Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence.

We previously reported that the frequency of polyploid aortic vascular smooth muscle cells (VSMC) serves as a biomarker of aging. Cellular senescence of somatic cells is another marker of aging that is characterized by the inability to undergo cell division. Here, we examined whether polyploidy is associated with the development of cellular senescence in vivo. Analysis of aortic tissue preparations from young and old Brown Norway rats showed that expression of senescence markers such as p16INK4a and senescence‐associated β‐galactosidase activity are detected primarily in the old tissues. VSMC from p16INK4a knockout and control mice display similar levels of polyploid cells. Intriguingly, senescence markers are expressed in most, but not all, polyploid VSMC. Moreover, the polyploid cells exhibit limited proliferative capacity in comparison to their diploid counterparts. This study is the first to demonstrate in vivo that polyploid VSMC adopt a senescent phenotype.

Direct visualization of the endomitotic cell cycle in living megakaryocytes: Differential patterns in low and high ploidy cells

Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (≥ 8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before re-joining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.

Magnetic Resonance Imaging–Ultrasound Fusion Biopsy During Prostate Cancer Active Surveillance

BACKGROUND Fusion biopsy using multiparametric magnetic resonance imaging (MRI) and transrectal ultrasound has demonstrated favorable detection rates of high-grade prostate cancer (PCa) among previously undiagnosed men. However, the diagnostic yield among men with active surveillance (AS) remains undefined. OBJECTIVE To determine the utility of MRI-ultrasound fusion biopsy during AS by reporting rates of PCa upgrading and comparing findings with systematic biopsy. DESIGN, SETTING, AND PARTICIPANTS We identified patients with low- and intermediate-risk PCa enrolled in AS who received MRI-ultrasound fusion surveillance biopsies. All completed prostate multiparametric MRI with 3-T and endorectal coil reviewed by radiologists selecting regions of interest, and all underwent MRI-ultrasound fusion biopsy with concurrent systematic biopsy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS We report MRI-ultrasound fusion biopsy findings, rates of Gleason score (GS) upgrading to ≥3 + 4 (any upgrading) and to ≥4 + 3 (major upgrading), tumor involvement estimates using descriptive statistics, McNemars test of symmetry, and multivariate logistic regression. RESULTS AND LIMITATIONS Overall, 207 men underwent MRI-ultrasound fusion biopsy following radiologic suspicion on multiparametric MRI and met inclusion criteria. Agreement between systematic and MRI-ultrasound fusion biopsy GS was borderline statistically significant (p<0.047). In total, 83 men (40%) experienced any upgrading, including 49 (24%) on systematic sampling, 30 (14%) on MRI-targeted cores, and four (2%) on both. Among those with negative results on MRI-ultrasound fusion biopsy, seven (9%) exhibited major upgrading with systematic biopsy. MRI suspicion scores were high (4/5) for all but two patients with any upgrading and for all who experienced major upgrading. On multivariate analysis, older age was associated with higher odds of any upgrading for men with GS ≤3 + 3 on previous biopsy (odds ratio: 1.10; 95% confidence interval, 1.01-1.20; p=0.03). CONCLUSIONS MRI-ultrasound fusion biopsy resulted in upgrading otherwise undetected by systematic biopsy among a proportion of men with PCa managed with AS. However, upgrading also occurred in areas outside targeted biopsy, suggesting that systematic sampling should be offered to men with AS even with history of extended sextant biopsy. PATIENT SUMMARY This study examined the role of magnetic resonance imaging (MRI)-ultrasound fusion biopsy for men with prostate cancer managed with active surveillance (AS). In some patients, MRI-ultrasound fusion biopsy resulted in the detection of upgrade otherwise missed with systematic sampling. The findings indicate that MRI-ultrasound fusion biopsy may help with better sampling during AS.