Citations

Abstract Background/Aim: Glioma-associated oncogene 1 (GLI1) is one of the most important transcription factors in the hedgehog (Hh) signaling pathway and has been shown to be involved in embryonic development and tumor progression. The epithelial-to-mesenchymal transition (EMT) is an important cellular program which contributes to invasion and metastasis of cancer. We have previously shown that GLI1 may promote HCC metastasis via EMT. Here, we demonstrate that GLI1 upregulates SNAI1 expression, induces EMT, and promotes HCC recurrence in vivo and in vitro. Methods: Gli1 mRNA was assessed in 139 HCCs by microarray and its correlation with HCC recurrence and SNAI1 mRNA expression was analyzed. In vitro, the mRNA and protein expression of GLI1 was measured in Gli1-negative Huh7 cells transfected with GU1 and GU1-expressing SNU398 cells transfected with antisense oligonucleotides targeting GU1 (GLI1 ASO). GLI1-dependent transcriptional activity was assessed with GU reporter luciferase assay and electrophoretic mobility shift assay (EMSA). The interaction between Gli1, SNAI1 and EMT was assessed by real-time PCR, Western immunoblotting and immunofluorescence. Effects of GLI1 on proliferation, viability, migration, and invasion of tumor cells were also assessed. Results: GLI1 mRNA was detected in 74 HCC tumors and found to be upregulated in 43 (58.1 %) HCCs. Increased Gli1 mRNA in HCCs was associated with tumor recurrence after resection. There was a positive correlation between GLI1 and SNAI1 mRNA expression. In HCC cells, GLI1 overexpression increased Gli1-dependent transcriptional activity, proliferation, viability, migration, and invasion of tumor cells. GU1 also increased SNAI1 expression and induced the EMT. Opposite effects were observed in GU1-knockdown models. Conclusion: Together, these findings identify a novel mechanism mediating HCC recurrence that includes GLI1-mediated activation of EMT via upregulation of SNAI1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B20.

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish ( Takifugu obscurus , mefugu) forms a CaCO3 precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO3− secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na+/HCO3− cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li+/ nHCO3− cotransport; HCO3− independent, DIDS-insensitive transport; and increased basal intracellular Na+ accumulation. fNBCe1 is a voltage-dependent Na+/ nHCO3− cotransporter that rectifies, independently from the extracellular Na+ or HCO3− concentration, around −60 mV. Na+ removal (0Na+ prepulse) is necessary to produce the true HCO3−-elicited current. HCO3− addition results in huge outward currents with quick current decay. Kinetic analysis of HCO3− currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher Km) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = −80 mV; [HCO3−] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO3− secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence.

Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.

Abstract Purpose: Emerging data from cooperative group trials suggests that not all patients with a positive SLN require completion axillary lymph node dissection (ALND). The controversy regarding who will benefit from additional nodal clearance continues, suggesting a need for improved tools to predict non-SLN involvement. We hypothesized that the size of a SLN metastasis evaluated as a continuous variable would be an important predictor of non-SLN involvement. The goal of this study was to determine clinicopathologic factors including SLN metastasis size that predicted non-SLN involvement and to use these factors to construct an improved predictive nomogram. Methods: Using a prospective database, we identified 509 patients with invasive breast cancer from 1996 - 2007 with a positive SLN who underwent ALND. Patients receiving neoadjuvant therapy were excluded. Clinicopathologic data including age, primary tumor size, presence of multifocal disease, histology (invasive ductal vs. invasive lobular vs. mixed invasive), tumor grade, estrogen receptor, progesterone receptor, and HER2/neu status, presence of lymphovascular invasion (LVI), the number of SLNs identified, number of positive SLNs, the maximum SLN metastasis size and the presence of extranodal extension were recorded. For SLNs described only as containing isolated tumor cells, size was recorded as 0.1 mm. Univariate and multivariate logistic regression analyses were performed to identify factors predictive of positive non-SLNs. Using these variables, a nomogram was constructed and subsequently validated using an external cohort of 464 patients. Results: On univariate analysis, the following factors were predictive of positive non-SLNs: number of SLN identified (p<.001), number of positive SLN (p<.001), maximum SLN metastasis size (p<.001), extranodal extension (p<.001), primary tumor size (p=.001), LVI (p=.019) and histology (p=.034). On multivariate analysis, all factors remained significant at the 5% level except LVI. A nomogram was created using these variables. When applied to an external cohort, the nomogram was accurate and discriminating with an area under the receiver operating characteristic curve of .73 (95% CI: .68 - .77). Conclusions: SLN metastasis size is predictive for identifying additional disease in the axilla. These data have been incorporated into a nomogram that accurately predicts the likelihood of having additional, non-SLN metastasis suggesting that routine reporting of the SLN metastasis size would assist with clinical decision making regarding the need to perform a completion ALND. Figures available in online version. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD06-08.

Rationale: Activity of the large conductance Ca 2+ -activated K + (BK) channels is profoundly modulated by its β 1 subunit (BK-β 1 ). However, BK-β 1 expression is downregulated in diabetic vessels. The ubiquitin–proteasome system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-β 1 downregulation in diabetic vessels is unknown. Objective: We hypothesize that UPS facilitates vascular BK-β 1 degradation in diabetes. Methods and Results: Using patch clamp and molecular biological approaches, we found that BK-β 1 –mediated channel activation and BK-β 1 protein expression were reduced in aortas of streptozotocin-induced diabetic rats and in human coronary arterial smooth muscle cells (CASMCs) cultured in high glucose. This was accompanied by upregulation of F-box only protein (FBXO)-9 and FBXO-32 (atrogin-1), the key components of the Skp1-Cullin-F-box (SCF) type ubiquitin ligase complex. BK-β 1 expression was suppressed by the FBXO activator doxorubicin but enhanced by FBXO-9 small interfering RNA or by the proteasome inhibitor MG-132. Cotransfection of atrogin-1 in HEK293 cells significantly reduced Flag-hSlo-β 1 expression by 2.16-fold, compared with expression of Flag-hSlo-β 1 V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo-β 1 was increased by 1.91-fold, compared with that of hSlo-β 1 V146A, whereas cotransfection with atrogin-1ΔF (a nonfunctional mutant without the F-box motif) had no effect. Moreover, inhibition of Akt signaling attenuated the phosphorylation of forkhead box O transcription factor (FOXO)-3a and enhanced atrogin-1 expression, which in turn suppressed BK-β 1 protein levels in human CASMCs. Conclusions: Downregulation of vascular BK-β 1 expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-β 1 degradation.