HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis

Background\ud Human homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated.\ud \ud Methods\ud Real time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively.\ud \ud Results\ud Overexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle.\ud \ud Conclusion\ud The present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies.This study was supported in part by a FAPESP grant 2010/01421-1

Aerobic exercise training enhances the in vivo cholesterol trafficking from macrophages to the liver independently of changes in the expression of genes involved in lipid flux in macrophages and aorta

Abstract\ud \ud Background\ud Regular exercise prevents and regresses atherosclerosis by improving lipid metabolism and antioxidant defenses. Exercise ameliorates the reverse cholesterol transport (RCT), an antiatherogenic system that drives cholesterol from arterial macrophages to the liver for excretion into bile and feces. In this study we analyzed the role of aerobic exercise on the in vivo RCT and expression of genes and proteins involved in lipid flux and inflammation in peritoneal macrophages, aortic arch and liver from wild type mice.\ud \ud \ud Methods\ud Twelve-week-old male mice were divided into sedentary and trained groups. Exercise training was performed in a treadmill (15 m/min, 30 min/day, 5 days/week). Plasma lipids were determined by enzymatic methods and lipoprotein profile by fast protein liquid chromatography. After intraperitoneal injection of J774-macrophages the RCT was assessed by measuring the recovery of 3H-cholesterol in plasma, feces and liver. The expression of liver receptors was determined by immunoblot, macrophages and aortic mRNAs by qRT-PCR. 14C-cholesterol efflux mediated by apo A-I and HDL2 and the uptake of 3H-cholesteryl oleoyl ether (3H-COE)-acetylated-LDL were determined in macrophages isolated from sedentary and trained animals 48 h after the last exercise session.\ud \ud \ud Results\ud Body weight, plasma lipids, lipoprotein profile, glucose and blood pressure were not modified by exercise training. A greater amount of 3H-cholesterol was recovered in plasma (24 h and 48 h) and liver (48 h) from trained animals in comparison to sedentary. No difference was found in 3H-cholesterol excreted in feces between trained and sedentary mice. The hepatic expression of scavenger receptor class B type I (SR-BI) and LDL receptor (B-E) was enhanced by exercise. We observed 2.8 and 1.7 fold rise, respectively, in LXR and Cyp7a mRNA in the liver of trained as compared to sedentary mice. Macrophage and aortic expression of genes involved in lipid efflux was not systematically changed by physical exercise. In agreement, 14C-cholestrol efflux and uptake of 3H-COE-acetylated-LDL by macrophages was similar between sedentary and trained animals.\ud \ud \ud Conclusion\ud Aerobic exercise in vivo accelerates the traffic of cholesterol from macrophages to the liver contributing to prevention and regression of atherosclerosis, independently of changes in macrophage and aorta gene expression.Fundação de Amparo à Pesquisa do Estado de Sao Paulo - FAPESP 12/04831-1 to MP, UFM and MLCCG; FAPESP 07/50387-8 to MP, 2011/15153-1 to PR, 06/52702-5 to DDFMRocco, 13/02854-7 to LS\ud Okuda, 12/19112-0 to AML, 10/50108-4 to GC, 12/18724-2 to KS, 11/04631-0 to DJG, 09/53412-9 to RS Pinto; 12/12088-7 to RTI, 14/07155 to GFConselho Nacional de Desenvolvimento Científico e Tecnológico (158314/\ud 2014-0 to DJG

SLC2A4 gene: a promising target for pharmacogenomics of insulin resistance

FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo

Increased plasma lipids in triple-negative breast cancer and impairment in HDL functionality in advanced stages of tumors

Abstract The association between plasma lipids and breast cancer (BC) has been extensively explored although results are still conflicting especially regarding the relationship with high-density lipoprotein cholesterol (HDLc) levels. HDL mediates cholesterol and oxysterol removal from cells limiting sterols necessary for tumor growth, inflammation, and metastasis and this may not be reflected by measuring HDLc. We addressed recently diagnosed, treatment-naïve BC women (n = 163), classified according to molecular types of tumors and clinical stages of the disease, in comparison to control women (CTR; n = 150) regarding plasma lipids and lipoproteins, HDL functionality and composition in lipids, oxysterols, and apo A-I. HDL was isolated by plasma discontinuous density gradient ultracentrifugation. Lipids (total cholesterol, TC; triglycerides, TG; and phospholipids, PL) were determined by enzymatic assays, apo A-I by immunoturbidimetry, and oxysterols (27, 25, and 24-hydroxycholesterol), by gas chromatography coupled with mass spectrometry. HDL-mediated cell cholesterol removal was determined in macrophages previously overloaded with cholesterol and 14C-cholesterol. Lipid profile was similar between CTR and BC groups after adjustment per age. In the BC group, lower concentrations of TC (84%), TG (93%), PL (89%), and 27-hydroxicholesterol (61%) were observed in HDL, although the lipoprotein ability in removing cell cholesterol was similar to HDL from CRT. Triple-negative (TN) BC cases presented higher levels of TC, TG, apoB, and non-HDLc when compared to other molecular types. Impaired HDL functionality was observed in more advanced BC cases (stages III and IV), as cholesterol efflux was around 28% lower as compared to stages I and II. The altered lipid profile in TN cases may contribute to channeling lipids to tumor development in a hystotype with a more aggressive clinical history. Moreover, findings reinforce the dissociation between plasma levels of HDLc and HDL functionality in determining BC outcomes

Identification of predictors of metastatical potential in paragangliomas to develop a prognostic score (PSPGL) - Supplemental data

INTRODUÇÃO: Paragangliomas (PGL) são tumores raros de localização adrenal (PGLAd) ou extra adrenal (PGLexAd). Metástases ocorrem em ~ 5% – 35% dos PGLs, sincronicamente ou muitos anos após o diagnóstico inicial. Não há preditores seguros para doença metastática. OBJETIVO: Construir um escore prognóstico para previsão do potencial metastático em PGL. O objetivo secundário foi identificar preditores prognósticos em PGL metastático (PGLM). MÉTODOS: Análise retrospectiva de dados clínicos e laboratoriais dos pacientes com PGL diagnosticados no Hospital das Clínicas - Faculdade de Medicina da Universidade de São Paulo, entre 1967 a 2019, e avaliação histológica e imuno-histoquímica (IHQ) de seus tumores. Os pacientes foram divididos em dois grupos: PGLM (com metástases) ou PGL não metastático (PGLNM – sem metástases durante seguimento ≥ 96 meses após a cirurgia). Realizadas análises uni e multivariada para identificar variáveis associadas a comportamento metastático. A partir de coeficientes obtidos na última análise foi construído um escore de risco. Para investigar preditores prognósticos nos PGLM, este grupo foi subdivido em PGLM “maligno” (PGLMm - pacientes que faleceram ≤ 72 meses após cirurgia) e PGLM “benigno” (PGLMb - pacientes que sobreviveram > 72 meses após cirurgia). Curvas de Kaplan Meier foram construídas para estimar sobrevida específica da doença (SED). RESULTADOS: Avaliados 263 pacientes com PGL (idade média 39,7 ± 16,7 anos, 56,3% sexo feminino, 82,5% PGLAd, 15,2%, PGLexAD e 2,7%, PGLAd + PGLexAd). Selecionados 35 pacientes com PGLM e 110 com PGLNM. Na análise univariada presença de sintomas, variantes patogênicas no gene SDHB, concentrações de ácido vanil mandélico e de noradrenalina (NAU) urinárias, PGLexAd, tamanho tumoral e pontuações no Pheochromocytoma of the adrenal gland scaled score (PASS) e no Grading system for adrenal phaeochromocytoma and paraganglioma (GAPP) estiveram associados ao comportamento maligno enquanto a IHQ para CHGB apresentou correlação inversa. Na análise multivariada, quatro dessas variáveis permaneceram relacionadas à doença metastática e compuseram o Escore Prognóstico de Paragangliomas (EPPGL): necrose (33 pontos), > 3 mitoses/10 CGA (28 pontos), extensão para tecido adiposo (20 pontos) e PGLexAd (19 pontos). A área sob a curva (AUC) da curva Receiver operator characteristic (ROC) foi de 0,970 e o corte de 24 pontos teve sensibilidade de 89,5% e especificidade de 91,5% para diagnóstico de PGLM. O risco de doença metastática foi alto para EPPGL ≥ 47 (>95%), intermediário para 28 ≤ EPPGL ≤ 39 (~30% a 80%), baixo para EPPGL19-20 (~10%) e praticamente nulo em pacientes com EPPGL=0. Nos PGLM, a IHQ-Ki-67 >3%. esteve relacionada à menor SED. CONCLUSÕES: O EPPGL é de fácil obtenção e mostrou boa performance na discriminação do potencial metastático. A IHQ-Ki-67 foi um bom marcador de agressividade em PGLM. Ambos podem ser ferramentas úteis na individualização do seguimento pós-operatório dos pacientes com PGL.INTRODUCTION: Paragangliomas (PGL) are rare tumors in adrenal (AdPGL) and extra-adrenal locations (exAdPGL). Metastasis are found in ~ 5%-35% of PGLs, synchronously or many years after initial diagnosis. There are no reliable predictors of metastatic disease. OBJECTIVE: To construct a prognostic score of prediction of metastatic potential in PGL. A secondary objective was to identify prognostic predictors in metastatic PGL (MPGL). METHODS: Retrospective analysis of clinical and laboratorial data of patients with PGL seen in Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, between 1967 – 2019, and tumors histological e immunohistochemical (IHC) evaluation. Patients were divided in two groups, MPGL (with metastasis) or non-metastatic PGL (NMPGL – without metastasis during follow up ≥ 96 months after surgery). Univariate and multivariate analysis were performed to identify characteristics associated to metastatic behavior. A risk score was constructed based on the multivariate analysis coefficients. To assess predictors of disease aggressiveness in MPGL, this group was subdivided in “malignant” metastatic PGL (mMPGL – patients that deceased ≤ 72 months after surgery) and “benign” malignant PGL (bMPGL – patients that survived > 72 months after surgery). Kaplan Meier curves were built to estimate disease specific survival (DSS). RESULTS Out of 263 patients with PGL (mean age 39.7 ± 16.7 years-old, 56.3% female, 82.5% AdPGL, 15.2% exAdPGL and 2.7% AdPGL + exAdPGL), 35 patients were selected for MPGL group and 110 for NMPGL group. In univariate analysis, presence of symptoms, pathogenic variants in SDHB gene, vanil mandelic acid and noradrenaline (NAU) urine concentrations, PGLexAd, tumor size and scores in Pheochromocytoma of the adrenal gland scaled score (PASS) and Grading system for adrenal phaeochromocytoma and paraganglioma (GAPP) scores were associated with malignant behavior whereas IHC for chromogranin B showed inverse correlation. In multivariate analysis, four features remained related to metastatic disease and composed the Prognostic Score of Paragangliomas (PSPGL): necrosis (33 points), > 3 mitosis/10 HPF (28 points), extension into adipose tissue (20 points) and exAdPGL (19 points). The receiver operator characteristic (ROC) curve showed an area under the curve (AUC) for PSPGL of 0.970 and the cut off of 24 points had 89.5% sensibility and 91.5% specificity to metastatic disease diagnostic. The risk of metastatic disease was high for PSPGL ≥ 47 (> 95%), intermediate for 28 ≤ PSPGL ≤ 39 (~30% to 80%), low for PSPGL 19-20 (~10%), and almost null in PPGL=0. In MPGL IHC for Ki-67 ≥ 3% was related to DSS. CONCLUSIONS: PSPGL is easy to obtain and demonstrated a good performance in predicting metastatic potential. Ki-67-IHC was a good marker of aggressiveness in MPGL. Both can be useful tools in individualizing post-surgical follow up in patients with PGL. Key words: Paraganglioma. Pheochromocytoma. Prognosis. Metastasis. Score. PASS. Histology. Survival.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP