Jacek Biesiada

VHL-Regulated MiR-204 Suppresses Tumor Growth through Inhibition of LC3B-Mediated Autophagy in Renal Clear Cell Carcinoma

The von Hippel-Lindau tumor-suppressor gene (VHL) is lost in most clear cell renal cell carcinomas (ccRCC). Here, using human ccRCC specimens, VHL-deficient cells, and xenograft models, we show that miR-204 is a VHL-regulated tumor suppressor acting by inhibiting macroautophagy, with MAP1LC3B (LC3B) as a direct and functional target. Of note, higher tumor grade of human ccRCC was correlated with a concomitant decrease in miR-204 and increase in LC3B levels, indicating that LC3B-mediated macroautophagy is necessary for RCC progression. VHL, in addition to inducing endogenous miR-204, triggered the expression of LC3C, an HIF-regulated LC3B paralog, that suppressed tumor growth. These data reveal a function of VHL as a tumor-suppressing regulator of autophagic programs.

Rotavirus VP8*: Phylogeny, Host Range, and Interaction with Histo-Blood Group Antigens

ABSTRACT The distal portion of rotavirus (RV) VP4 spike protein (VP8*) is implicated in binding to cellular receptors, thereby facilitating viral attachment and entry. While VP8* of some animal RVs engage sialic acid, human RVs often attach to and enter cells in a sialic acid-independent manner. A recent study demonstrated that the major human RVs (P[4], P[6], and P[8]) recognize human histo-blood group antigens (HBGAs). In this study, we performed a phylogenetic analysis of RVs and showed further variations of RV interaction with HBGAs. On the basis of the VP8* sequences, RVs are grouped into five P genogroups (P[I] to P[V]), of which P[I], P[IV], and P[V] mainly infect animals, P[II] infects humans, and P[III] infects both animals and humans. The sialic acid-dependent RVs (P[1], P[2], P[3], and P[7]) form a subcluster within P[I], while all three major P genotypes of human RVs (P[4], P[6], and P[8]) are clustered in P[II]. We then characterized three human RVs (P[9], P[14], and P[25]) in P[III] and observed a new pattern of binding to the type A antigen which is distinct from that of the P[II] RVs. The binding was demonstrated by hemagglutination and saliva binding assay using recombinant VP8* and native RVs. Homology modeling and mutagenesis study showed that the locations of the carbohydrate binding interfaces are shared with the sialic acid-dependent RVs, although different amino acids are involved. The P[III] VP8* proteins also bind the A antigens of the porcine and bovine mucins, suggesting the A antigen as a possible factor for cross-species transmission of RVs. Our study suggests that HBGAs play an important role in RV infection and evolution.

TRPM3 and miR-204 establish a regulatory circuit that controls oncogenic autophagy in clear cell renal cell carcinoma

Autophagy promotes tumor growth by generating nutrients from the degradation of intracellular structures. Here we establish, using shRNAs, a dominant-negative mutant, and a pharmacologic inhibitor, mefenamic acid (MFA), that the Transient Receptor Potential Melastatin 3 (TRPM3) channel promotes the growth of clear cell renal cell carcinoma (ccRCC) and stimulates MAP1LC3A (LC3A) and MAP1LC3B (LC3B) autophagy. Increased expression of TRPM3 in RCC leads to Ca(2+) influx, activation of CAMKK2, AMPK, and ULK1, and phagophore formation. In addition, TRPM3 Ca(2+) and Zn(2+) fluxes inhibit miR-214, which directly targets LC3A and LC3B. The von Hippel-Lindau tumor suppressor (VHL) represses TRPM3 directly through miR-204 and indirectly through another miR-204 target, Caveolin 1 (CAV1).

Survey of public domain software for docking simulations and virtual screening.

Progress in functional genomics and structural studies on biological macromolecules are generating a growing number of potential targets for therapeutics, adding to the importance of computational approaches for small molecule docking and virtual screening of candidate compounds. In this review, recent improvements in several public domain packages that are widely used in the context of drug development, including DOCK, AutoDock, AutoDock Vina and Screening for Ligands by Induced-fit Docking Efficiently (SLIDE) are surveyed. The authors also survey methods for the analysis and visualisation of docking simulations, as an important step in the overall assessment of the results. In order to illustrate the performance and limitations of current docking programs, the authors used the National Center for Toxicological Research (NCTR) oestrogen receptor benchmark set of 232 oestrogenic compounds with experimentally measured strength of binding to oestrogen receptor alpha. The methods tested here yielded a correlation coefficient of up to 0.6 between the predicted and observed binding affinities for active compounds in this benchmark.

Rational design of small molecule inhibitors targeting the Ras GEF, SOS1.

Ras GTPases regulate intracellular signaling involved in cell proliferation. Elevated Ras signaling activity has been associated with human cancers. Ras activation is catalyzed by guanine nucleotide exchange factors (GEFs), of which SOS1 is a major member that transduces receptor tyrosine kinase signaling to Ras. We have developed a rational approach coupling virtual screening with experimental screening in identifying small-molecule inhibitors targeting the catalytic site of SOS1 and SOS1-regulated Ras activity. A lead inhibitor, NSC-658497, was found to bind to SOS1, competitively suppress SOS1-Ras interaction, and dose-dependently inhibit SOS1 GEF activity. Mutagenesis and structure-activity relationship studies map the NSC-658497 site of action to the SOS1 catalytic site, and define the chemical moieties in the inhibitor essential for the activity. NSC-658497 showed dose-dependent efficacy in inhibiting Ras, downstream signaling activities, and associated cell proliferation. These studies establish a proof of principle for rational design of small-molecule inhibitors targeting Ras GEF enzymatic activity.

Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult

The Developmental Origins of Health and Disease (DOHaD) Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr -/- and in utero TCDD-exposed Ahr +/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr -/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease.

Clustering of maternal–fetal clinical conditions and outcomes and placental lesions

OBJECTIVE To identify by an inductive statistical analysis mutually similar and clinically relevant clinicoplacental clusters. STUDY DESIGN Twenty-nine maternofetal and 49 placental variables have been retrospectively analyzed in a 3382 case clinicoplacental database using a hierarchical agglomerative Ward dendrogram and multidimensional scaling. RESULTS The exploratory cluster analysis identified 9 clinicoplacental (macerated stillbirth, fetal growth restriction, placenta creta, acute fetal distress, uterine hypoxia, severe ascending infection, placental abruption, and mixed etiology [2 clusters]), 5 purely placental (regressive placental changes, excessive extravillous trophoblasts, placental hydrops, fetal thrombotic vasculopathy, stem obliterative endarteritis), and 1 purely clinical (fetal congenital malformations) statistically significant clusters/subclusters. The clusters of such variables like clinical umbilical cord compromise, preuterine and postuterine hypoxia, gross umbilical cord or gross chorionic disk abnormalities did not reveal statistically significant stability. CONCLUSION Although clinical usefulness of several well-established placental lesions has been confirmed, claims about high predictability of others have not.

Relation of placental diagnosis in stillbirth to fetal maceration and gestational age at delivery.

Abstract Aim: To study the relation of retention of dead fetus resulting in its maceration and gestational age at delivery to placental diagnosis. Methods: Some 75 clinicoplacental phenotypes have been retrospectively analyzed in 520 consecutive stillbirths, 329 macerated and 191 nonmacerated, and at three gestational age interval cohorts (330 second trimester, 102 preterm third trimester, and 88 term). Chi-square and clustering methods (Ward dendrograms and multidimensional scaling) were used for statistical analysis. Results: Maternal diabetes mellitus, induction of labor, fetal growth restriction, various umbilical cord abnormalities, and placental clusters of sclerotic/hemosiderotic chorionic villi were more common in macerated stillbirths, while clinicoplacental signs and symptoms of ascending infection and placental abruption, i.e., retroplacental hematoma, premature rupture of membranes, and acute chorioamnionitis in nonmacerated stillbirths. Placental abnormalities were less common in the second trimester, other than the acute chorioamnionitis. Patterns of chronic hypoxic placental injury were common in preterm third trimester, while signs of in-utero hypoxia (abnormal cardiotocography, meconium, and histological erythroblastosis of fetal blood) in term pregnancy. In addition to classical statistics, the clustering analyses added new information to placental investigation of cause of stillbirth. Conclusions: Macerated third trimester stillbirths have multifactorial etiology more likely than the second trimester stillbirths and the likely stasis-induced fetal thrombotic vasculopathy secondary to occult umbilical cord compromise should be sought in placental investigation in such cases. Nonmacerated stillbirths are associated with ascending infection and placental abruption.

von Hippel-Lindau–Dependent Patterns of RNA Polymerase II Hydroxylation in Human Renal Clear Cell Carcinomas

Purpose: We have previously shown that von Hippel-Lindau (VHL) regulates ubiquitylation and proline 1465 hydroxylation of the large subunit of RNA polymerase II, Rpb1, in human renal clear cell carcinoma (RCC) cell lines. Here, our goal was to determine the effect of this VHL function and the status of P1465 hydroxylation in human RCC tumors. Experimental Design: Primary human tumors and matched normal kidney samples were probed for expression levels of the large subunit of RNA polymerase II (Rpb1), Rpb1 hydroxylated on P1465 [Rpb1(OH)], Rpb1 phosphorylated on Ser5 [Rpb1(S5P)], and proline hydroxylases PHD1, PHD2, and PHD3. Results from RCC tumors were categorized according to the status of VHL gene. Mechanistic analysis was performed in orthotopic xenograft model using 786-O RCC cells with wild-type (WT) VHL and knockdown of PHD2, characterized by high levels of Rpb1(OH) and PHD1. Results: Levels of Rpb1(OH), PHD1, and PHD2 were significantly higher in RCC tumors compared with normal kidneys. RCC tumors with WT VHL had higher levels of Rpb1(OH) and PHD1 and lower levels of PHD2 than tumors with VHL gene alterations. Levels of Rpb1(OH) significantly correlated with levels of PHD1 in tumors and normal kidneys. Knockdown of PHD2 in 786-O VHL(+) cells resulted in a more malignant phenotype in orthotopic xenografts and higher expression of specific cell cycle regulators (CDC25A, cyclin-dependent kinase 2, CCNA2) compared with VHL(−) RCC cells. Conclusions: Elevated PHD1 concomitant with decreased PHD2 are causatively related to Rpb1 hydroxylation and oncogenesis in human RCC tumors with WT VHL gene. Thus, P1465-hydroxylated Rpb1 and PHD1 represent attractive drug targets for new RCC treatments. Clin Cancer Res; 16(21); 5142–52. ©2010 AACR.

Sensitivity and specificity of finding of multinucleate trophoblastic giant cells in decidua in placentas from high-risk pregnancies

This is a retrospective analysis of sensitivity and specificity of clustered placental basal plate multinucleate trophoblastic giant cells for various clinical conditions and placental lesions associated with fetal and placental hypoxia. Selected clinical and placental parameters of 375 consecutive cases of placentas with clusters of multinucleate trophoblastic giant cell (at least 3 cells with at least 3 nuclei) in the decidua (study group) were compared with all remaining 2674 placentas concurrently studied (control group) in 20-week-or-more high-risk pregnancies. Multinucleate trophoblastic giant cell was found in 12.3% of placentas. The study group had statistically significantly more cases of preeclampsia, abnormal Dopplers, induction of labor, and cesarean sections, with its placentas lighter and with more common other hypoxic lesions than in the control-group placentas. The multinucleate trophoblastic giant cell prevalence negatively correlated with gestational age (R = -0.56), peaking at the turn of the second and the third trimesters of pregnancy and declining afterward, and most strongly correlated with the excessive amount of extravillous trophoblasts in the chorionic disc (R = +0.33). The sensitivity of multinucleate trophoblastic giant cells was, on average, 3 times lower than the specificity, the latter averaging greater than 90%. In conclusion, finding of multinucleate trophoblastic giant cells is not exclusively limited to uteroplacental malperfusion of preeclampsia but is also seen in other types of high-risk pregnancy and in association with other placental hypoxic lesions and patterns. Multinucleate trophoblastic giant cells most likely reflect a premature fusion of extravillous trophoblasts because of several factors, likely including also hypoxia. Being highly specific, finding the multinucleate trophoblastic giant cells is unlikely to give a false-positive result and therefore has high value in retrospectively explaining the perinatal morbidity and mortality.