Pablo Letelier

miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in gastric cancer through regulation of the PI3K/AKT/mTOR pathway

BackgroundGastric cancer (GC) is a deadly malignancy worldwide. In the past, it has been shown that cellular signaling pathway alterations play a crucial role in the development of GC. In particular, deregulation of the PI3K/AKT/mTOR pathway seems to affect multiple GC functions including growth, proliferation, metabolism, motility and angiogenesis. Targeting alterations in this pathway by microRNAs (miRNAs) represents a potential therapeutic strategy, especially in inhibitor-resistant tumors. The objective of this study was to evaluate the expression of 3 pre-selected miRNAs, miR-101-2, miR-125b-2 and miR-451a, in a series of primary GC tissues and matched non-GC tissues and in several GC-derived cell lines, and to subsequently evaluate the functional role of these miRNAs.MethodsTwenty-five primary GC samples, 25 matched non-GC samples and 3 GC-derived cell lines, i.e., AGS, MKN28 and MKN45, were included in this study. miRNA and target gene expression levels were assessed by quantitative RT-PCR and western blotting, respectively. Subsequently, cell viability, clone formation, cell death, migration and invasion assays were performed on AGS cells.ResultsmiR-101-2, miR-125b-2 and miR-451a were found to be down-regulated in the primary GC tissues and the GC-derived cell lines tested. MiRNA mimic transfections significantly reduced cell viability and colony formation, increased cell death and reduced cell migration and invasion in AGS cells. We also found that exogenous expression of miR-101-2, miR-125b-2 and miR-451a decreased the expression of their putative targets MTOR, PIK3CB and TSC1, respectively.ConclusionsOur expression analyses and in vitro functional assays suggest that miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in primary GCs as well as in GC-derived AGS cells.

Immunohistochemical Expression of Phospho-mTOR Is Associated With Poor Prognosis in Patients With Gallbladder Adenocarcinoma

CONTEXT Advanced gallbladder carcinoma (GBC) is a highly fatal disease with poor prognosis and few therapeutic alternatives. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a central role in cell growth and homeostasis. Its regulation is frequently altered in various tumors and is an attractive target for cancer therapy; however, its status in GBC remains unclear. OBJECTIVE To characterize immunohistochemical expression and prognostic significance of phospho-mTOR in advanced gallbladder carcinoma. DESIGN Phospho-mTOR expression was examined by immunohistochemistry in tissue microarrays containing 128 advanced GBCs and 99 cases of chronic cholecystitis, which were divided into 2 groups according to the presence or absence of metaplasia. To evaluate the association of the level of phospho-mTOR expression with clinical variables and patient survival, the advanced GBCs were classified as having low or high expression. Statistical analysis was performed by using a significance level of P < .05, and Kaplan-Meier curves were constructed for survival analysis. RESULTS Immunostaining for phospho-mTOR was positive in 82 of 128 tumors (64.1%) and in 24% of chronic cholecystitis cases (16% nonmetaplasia and 32% with metaplasia) (P < .001). Survival analysis indicated that a high phospho-mTOR immunohistochemical expression was associated with poorer prognosis in patients with advanced GBC (P = .02). CONCLUSIONS Metaplasia is a common finding in chronic cholecystitis and is considered a precursor lesion of dysplasia. Our results suggest that the activation of mTOR occurs very early during the development of GBC, contributing to the carcinogenesis process. Phospho-mTOR expression is correlated with poor survival, supporting the potential of mTOR for targeted therapy.

Emerging Role of miRNAs in the Drug Resistance of Gastric Cancer

Gastric cancer is the third leading cause of cancer mortality worldwide. Unfortunately, most gastric cancer cases are diagnosed in an advanced, non-curable stage and with a limited response to chemotherapy. Drug resistance is one of the most important causes of therapy failure in gastric cancer patients. Although the mechanisms of drug resistance have been broadly studied, the regulation of these mechanisms has not been completely understood. Accumulating evidence has recently highlighted the role of microRNAs in the development and maintenance of drug resistance due to their regulatory features in specific genes involved in the chemoresistant phenotype of malignancies, including gastric cancer. This review summarizes the current knowledge about the miRNAs’ characteristics, their regulation of the genes involved in chemoresistance and their potential as targeted therapies for personalized treatment in resistant gastric cancer.

DNA promoter methylation as a diagnostic and therapeutic biomarker in gallbladder cancer.

Gallbladder cancer is an infrequent neoplasia with noticeable geographical variations in its incidence around the world. In Chile, it is the main cause of death owing to cancer in women over 40 years old, with mortality rates up to 16.5 per 100,000 cases. The prognosis is poor with few therapeutic options; in advanced cases there is only a 10% survival at 5 years.Several studies mention the possible role of DNA methylation in gallbladder carcinogenesis. This epigenetic modification affects tumor suppressor genes involved in regulation pathways, cell cycle control, cell adhesion and extracellular matrix degradation, in a sequential and cumulative way. Determining DNA methylation patterns would allow them to be used as biomarkers for the early detection, diagnosis, prognosis and/or therapeutic selection in gallbladder cancer.

Immunohistochemical expression of vascular endothelial growth factor A in advanced gallbladder carcinoma.

Gallbladder cancer (GBC) is a highly fatal disease with poor prognosis and few therapeutic alternatives. The molecular mechanisms involved in the pathogenesis of GBC remain poorly understood. The vascular endothelial growth factor A (VEGF-A) is a potent proangiogenic agent involved in the carcinogenesis of many human tumors and is an attractive target for cancer therapy. We characterized VEGF-A expression in advanced GBC and its relation to clinicopathologic features. VEGF-A expression was examined by immunohistochemistry in tissue microarrays containing 224 advanced gallbladder carcinomas and 39 chronic cholecystitis. The cases were classified as low or high expression to evaluate the association of VEGF-A expression level with clinicopathologic variables. The Kaplan-Meier method was used to estimate survival as a function of time, and survival differences were analyzed by the log-rank test. High expression of VEGF-A was observed in 81% (183/224) of tumors and 5.1% (2/39) of chronic cholecystitis (P<0.0001). The VEGF-A expression had a significant relationship with histologic grade and TNM stage (P<0.05). Moreover, 5-year survival analysis indicated that high expression of VEGF-A is associated with a poor prognosis in patients with advanced GBC (P=0.0116). Our results indicate that VEGF-A is highly expressed in GBC and correlates with poor prognosis, suggesting that VEGF-A expression could be used as a biomarker for predicting malignant behavior and for identifying a subset of patients who may benefit from anti-VEGF-A therapies.

Global and gene-specific DNA methylation pattern discriminates cholecystitis from gallbladder cancer patients in Chile

Aim The aim of the study was to evaluate the use of global and gene-specific DNA methylation changes as potential biomarkers for gallbladder cancer (GBC) in a cohort from Chile. Material & methods DNA methylation was analyzed through an ELISA-based technique and quantitative methylation-specific PCR. Results Global DNA Methylation Index (p = 0.02) and promoter methylation of SSBP2 (p = 0.01) and ESR1 (p = 0.05) were significantly different in GBC when compared with cholecystitis. Receiver curve operator analysis revealed promoter methylation of APC, CDKN2A, ESR1, PGP9.5 and SSBP2, together with the Global DNA Methylation Index, had 71% sensitivity, 95% specificity, a 0.97 area under the curve and a positive predictive value of 90%. Conclusion Global and gene-specific DNA methylation may be useful biomarkers for GBC clinical assessment.

Efectos de la Inhibición de C-Flipl en Líneas Celulares de Cáncer de Cuello Uterino

Cuando las variantes Short y Raji de la proteina Cellular FLICE-like inhibitory protein (c-FLIP) se encuentran sobrexpresadas son capaces de inhibir la apoptosis, mientras la funcion de la isoforma Long (c-FLIPL), depende de la concentracion de esta molecula en las celulas. El objetivo de este estudio fue determinar los efectos de la inhibicion de c-FLIPL en lineas celulares de cancer de cuello uterino. Para realizar el estudio fueron utilizadas SiHa, C-4I y C-33A, lineas celulares de cancer cervical. La expresion de c-FLIPL en estas lineas fue establecida mediante PCR en tiempo real y western blot. Posteriormente la expresion de c-FLIPL fue inhibida, mediante transfecion transiente con siRNA complementario al mRNA mensajero de c.-FLIPL. Los efectos de esta inhibicion en la viabilidad celular, proliferacion y apoptosis fue comparada con celulas transfectadas con un siRNA control (scrambled). Una vez reprimido c-FLIPL, las lineas celulares SiHa y C-4I presentaron un aumento de la viabilidad celular (P<0,05). Para evaluar la proliferacion celular se utilizo inmunocitoquimica de los marcadores Ki-67 y PCNA. Las celulas SiHa transfectadas con siRNA c-FLIPL, mostraron una elevada expresion de Ki-67 (P<0,0001), mientras que las celulas C-33A con c-FLIPL inhibido mostraron una menor expresion de PCNA (P<0,01). Las tres lineas celulares con c-FLIPL reprimido mostraron un mayor nivel de apoptosis que las celulas control. Estos resultados sugieren que c-FLIPL puede tener efectos en la proliferacion y apoptosis de lineas celulares de cancer de cuello uterino.

Abstract A31: Reprimo, a potential tumor suppressor gene TP53-dependent, modulates negatively cell migration and invasion in the MDA-MB-231 breast cancer cell line

Background: Reprimo, a highly glycosylated protein, is a new downstream effector of p53-induced cell cycle arrest at the G2/M checkpoint, and a putative tumor suppressor gene frequently silenced via methylation of its promoter region in several malignances. The aim of this study was to characterize the epigenetic inactivation and its biological function in BC cell lines. Methods: The correlation between Reprimo methylation and loss of mRNA expression was assessed in six breast cancer cell lines by methylation specific PCR (MSP), 5-Aza-2′-deoxycytidine treatment and qRT-PCR assays. MDA-MB-231 cells were chosen to investigate the phenotypic effect of Reprimo in cell proliferation, cell cycle, cell death, cell migration and invasion. Results: In the cancer methylome system (CMS) (web-based system for visualizing and analyzing genome-wide methylation data of human cancers), the CpG island region of RPRM (1,1Kb) was hypermethylated in breast cancer compared to normal breast tissue. Downregulation of RPRM mRNA by methylation was confirmed in MDA-MB-231 and BT-20 cell lines. In addition, overexpression of RPRM in MDA-MB-231 cells resulted in decreased rates of cell migration, wound healing and invasion in vitro (P Conclusion: Taken together, these data suggest that RPRM is involved in decreased cell migration and invasion in vitro, acting as a potential tumor suppressor gene in the MDA-MB-231 cell line. Citation Format: Kurt Buchegger, Tamara Viscarra, Carmen Gloria Ili, Ismael Riquelme, Pablo Letelier, Alejandro Corvalan, Priscilla Brebi, Tim Hui-Ming Huang, Juan Carlos Roa. Reprimo, a potential tumor suppressor gene TP53-dependent, modulates negatively cell migration and invasion in the MDA-MB-231 breast cancer cell line. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A31.

Abstract 3907: Expression of vascular endothelial growth factor a (VEGF-A) in advanced gallbladder cancer.

Gallbladder cancer (GBC) is a highly fatal disease with poor prognosis and few therapeutic alternatives. Molecular mechanisms involved in GBC pathogenesis remain poorly understood. The Vascular Endothelial Growth Factor A (VEGF) is a potent proangiogenic agent involved in the carcinogenesis of many human tumors and it is an attractive target for cancer therapy. The aim of this study was characterize VEGF expression in advanced gallbladder cancer and determine it9s relationships with clinicopathologic features and utility as a prognostic factor. VEGF expression was examined by immunohistochemistry (IHC) in tissue microarrays containing 224 advanced gallbladder carcinomas and 39 cases of chronic cholecystitis (CC). The advanced GBC were classified as low or high expression to evaluate the association of VEGF expression level with clinical variables. Statistical analysis was performed using univariate analyses with a significance level of P Citation Format: Pablo Letelier, Kurt Buchegger, Carmen Ili, Patricia Garcia, Priscilla Brevi, Alejandra Sandoval, Pamela Leal, Ismael Riquelme, Juan Carlos Roa. Expression of vascular endothelial growth factor a (VEGF-A) in advanced gallbladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3907. doi:10.1158/1538-7445.AM2013-3907