Kurt Buchegger

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 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.

The Gene Expression Status of the PI3K/AKT/mTOR Pathway in Gastric Cancer Tissues and Cell Lines.

The PI3K/AKT/mTOR pathway plays a crucial role in the regulation of multiple cellular functions including cell growth, proliferation, metabolism and angiogenesis. Emerging evidence has shown that deregulation of this pathway has a role promoting gastric cancer (GC). The aim was to assess the expression of genes involved in this pathway by qPCR in 23 tumor and 23 non-tumor gastric mucosa samples from advanced GC patients, and in AGS, MKN28 and MKN45 gastric cancer cell lines. Results showed a slight overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1, EIF4EBP1 and EIF4E genes, and a slightly decreased PTEN and TSC1 expression. In AGS, MKN28 and MKN45 cells a significant gene overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1 and EIF4E, and a significant repression of PTEN gene expression were observed. Immunoblotting showed that PI3K-β, AKT, p-AKT, PTEN, mTOR, p-mTOR, P70S6K1, p-P70S6K1, 4E-BP1, p-4E-BP1, eIF4E and p-eIF4E proteins were present in cell lines at different levels, confirming activation of this pathway in vitro. This is the first time this extensive panel of 9 genes within PI3K/AKT/mTOR pathway has been studied in GC to clarify the biological role of this pathway in GC and develop new strategies for this malignancy.

AKT/mTOR substrate P70S6K is frequently phosphorylated in gallbladder cancer tissue and cell lines

Background Gallbladder carcinoma is a highly malignant tumor and a public health problem in some parts of the world. It is characterized by a poor prognosis and its resistance to radio and chemotherapy. There is an urgent need to develop novel therapeutic alternatives for the treatment of gallbladder carcinoma. The mammalian target of the rapamycin (mTOR) signaling pathway is activated in about 50% of human malignancies, and its role in gallbladder carcinoma has previously been suggested. In the present study, we investigated the phosphorylation status of the mTOR substrate p70S6K in preneoplastic and neoplastic gallbladder tissues and evaluated the effect of three mTOR inhibitors on cell growth and migration in gallbladder carcinoma cell lines. Methods Immunohistochemical staining of phospho-p70S6K was analyzed in 181 gallbladder carcinoma cases, classified according to lesion type as dysplasia, early carcinoma, or advanced carcinoma. Protein expression of AKT/mTOR members was also evaluated in eight gallbladder carcinoma cell lines by Western blot analysis. We selected two gallbladder carcinoma cell lines (G415 and TGBC-2TKB) to evaluate the effect of rapamycin, RAD001, and AZD8055 on cell viability, cell migration, and protein expression. Results Our results showed that phospho-p70S6K is highly expressed in dysplasia (66.7%, 12/18), early cancer (84.6%, 22/26), and advanced cancer (88.3%, 121/137). No statistical correlation was observed between phospho-p70S6K status and any clinical or pathological features, including age, gender, ethnicity, wall infiltration level, or histological differentiation (P < 0.05). In vitro treatment with rapamycin, RAD001, and AZD8055 reduced cell growth, cell migration, and phospho-p70S6K expression significantly in G-415 and TGBC-2TKB cancer cells (P < 0.001). Conclusion Our findings confirm the upregulation of this signaling pathway in gallbladder carcinoma and provide a rationale for the potential use of mTOR inhibitors as a therapeutic strategy for human gallbladder carcinoma.

The ERK/MAPK pathway is overexpressed and activated in gallbladder cancer

Gallbladder cancer (GBC) is a highly fatal disease with poor prognosis and few therapeutic alternatives. Molecular profiling has revealed that the deregulation in the ERK/MAPK signaling pathway plays a crucial role in many disease and malignancies, including GBC. The aim of this study was to measure the expression of ERK1/2 and p-ERK1/2 in a population with high GBC-related mortality, such as the Chilean population, and characterize the protein expression of this ERK/MAPK pathway in seven GBC cell lines. Immunohistochemistry (IHC) for ERK1/2 and p-ERK1/2 was performed in 123 GBC tissues and 37 chronic cholecystitis (CC) tissues. In addition, protein expression analysis by western blot for ERK1/2, p-ERK1/2, EGFR, ERBB2 and ERBB3 were performed in seven GBC cell lines (GB-d1, G415, NOZ, OCUG-1, TGBC-1, TGBC-2 and TGBC-24). A higher ERK1/2 and p-ERK1/2 expression was found in GBC tissues compared to chronic cholecystitis (CC) tissues (P<0.001). However, neither significant differences in overall survival nor significant associations with any of the clinicopathological features were found by comparing low and high expression of both ERK1/2 and p-ERK1/2. Western blot analysis of seven GBC cell lines showed that, in general, GB-d1, G415 and NOZ cells evidenced a strong expression of ERK1/2, p-ERK1/2, EGFR, ERBB2 and ERBB3. Therefore, ERK1/2 and p-ERK1/2 seem to be important in the development of GBC and GB-d1, G415 and NOZ cell lines may be used as experimental models for further in vitro and in vivo studies that help to decipher the role of MAPK/ERK pathway in gallbladder carcinogenesis.

Reprimo, a Potential p53-Dependent Tumor Suppressor Gene, Is Frequently Hypermethylated in Estrogen Receptor α-Positive Breast Cancer

Aberrant DNA methylation is a hallmark of many cancers. Currently, there are four intrinsic molecular subtypes in breast cancer (BC): Luminal A, B, Her2-positive, and triple negative (TNBC). Recently, The Cancer Genome Atlas (TCGA) project has revealed that Luminal subtypes have higher levels of genome-wide methylation that may be a result of Estrogen/Estrogen receptor α (E2/ERα) signaling pathway activation. In this study, we analyze promoter CpG-island (CGIs) of the Reprimo (RPRM) gene in breast cancers (n = 77), cell lines (n = 38), and normal breast tissue (n = 10) using a MBDCap-seq database. Then, a validation cohort (n = 26) was used to confirm the results found in the MBDCap-seq platform. A differential methylation pattern was found between BC and cell lines compared to normal breast tissue. In BC, a higher DNA methylation was observed in tissues that were ERα-positive than in ERα-negative ones; more precisely, subtypes Luminal A compared to TNBC. Also, significant reverse correlation was observed between DNA methylation and RPRM mRNA expression in BC. Our data suggest that ERα expression in BC may affect the DNA methylation of CGIs in the RPRM gene. This approach suggests that DNA methylation status in CGIs of some tumor suppressor genes could be driven by E2 availability, subsequently inducing the activation of the ERα pathway.

PO-183 Identification of differentially hypomethylated genes associated to metastasis behaviour in colorectal cancer

Introduction Colorectal cancer (CRC) is an important public health problem worldwide. In Chile, CRC is the fourth most frequent cancer and its incidence is rising. Sporadic CRC results from the accumulation of both acquired genetic and epigenetic changes that transform normal glandular epithelium into invasive adenocarcinoma. DNA methylation has an important role in colon carcinogenesis and also has been found involved in metastasis pathways of CRC. However, there are no studies that analysed whole genome in the search of specific methylated genes in metastasis of CRC, despite the next-generation sequencing platforms and methylation arrays available. Therefore, in this project it is proposed to find novel methylation markers of metastasis by comparing primary tumours and their corresponding lymph node metastasis, using a next-generation sequencing platform. The aim of this study was to identify differentially methylated genes associated with metastasis tumour behaviour in colorectal cancer. Material and methods Five paired FFPE samples of CRC primary tumour and its corresponding lymph node metastasis were analysed with genome-wide Methyl-Seq bisulfite sequencing. Five differently hypomethylated genes were selected using bioinformatics tools. Bioinformatic analysis was realised comparing colorectal primary tumour vs lymph node metastasis using methylKit tool. Results and discussions A total of 196 genes were detected as differentially methylated in their promoter region, 94 of which were hypomethylated on lymph node metastasis group. CS, RNF130, HERC6, ZNF717 and RNF216-IT1 genes presented differences over 50% in their methylation status, compared with CRC primary tumours group. According to their ontology, these genes are involved on regulation of tricarboxylic acid cycle, transcription, carbohydrate metabolic process and protein polyubiquitination. Conclusion CS, RNF130, HERC6, ZNF717 and RNF216-IT1 genes were found hypomethylated in colorectal metastasis compared to primary tumour. These genes could be implied in metastasis behaviour in colorectal cancer, but further studies are necessary to evaluate their functions.

PO-492 Establishment of new drug-resistant gastric cancer cell lines

Introduction Gastric cancer (GC) is an important public health problem in Chile, because constitutes the first cause of death for cancer in man and the third in women. Most GC patients are diagnosed at advanced stages where surgery is not effective. In these cases, the GC treatment is mainly based in chemotherapy with Cisplatin (CDDP) or 5-fluorouracil (5-FU). However, tumour cells usually develop chemotherapy resistance, increasing the rate of recurrence. The establishment of drug resistant cell lines could serve as an initial screen for agents that might modulate drug resistance in gastric cancer. Material and methods Two gastric cancer cell lines AGS and MKN-28 were treated with incremental doses of cisplatin or 5-FU to generate resistant variants (continuous growth method with increasing dose). The drug sensitivity of parental and resistant cells was determined by dose-dependent cytotoxicity curve using standard MTT assay. The inhibitory concentration 50% (IC50) values were calculated by non-linear regression test using GraphPad PRISM 5.0 software. The resistance index (RI) was determined as the ratio of the IC50 of the resistant cell line to the IC50 of parental cell line. Relative expression of resistance marker genes to CDDP or 5-FU was determined by qRT-PCR using 2-(ddCt) method. U-Mann Whitney test was used to compare groups (at statistical significance level of p<0.05). Changes in cell morphology were monitored continuously during the development of resistance clones using an inverted phase contrast microscope. The stabilisation of drug resistance of cell lines was tested after two months in drug-free medium. Results and discussions After 10 months of treatment, AGS resistant to CDDP, MKN-28 resistant to CDDP and AGS resistant to 5-FU exhibited an increase of 3.9, 2.6 and 3.4 fold of resistance, respectively. Resistance marker genes for CDDP (ABCC2 and CTR1) and 5-FU (TYMS) were differentially expressed in resistant cells compared to their parental cells. Changes in cell morphology were observed in resistant cells compared to their parental cells. The resistant phenotype was very stable and the values of IC50 and RI had no significant change after 2 months in drug-free medium. Conclusion The three drug-resistant lines selected by continuous growth method with increasing dose may serve as appropriate models for the study of mechanisms of drug resistance in GC. Further studies are necessary in order to identify the genes involved in the resistant-phenotype which could help to find new targets for GC therapy.

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.

mTOR/P70S6K signaling pathway as a potential target for advanced gallbladder cancer therapy.

242 Background: Gallbladder cancer (GBC) is a highly malignant tumor usually diagnosed at advanced stages and characterized by a poor prognosis. Effective therapeutic strategies are urgently needed to improve the prognosis of GBC patients. Our objective was to analyze the expression of mTOR/p70S6K pathway in primary tumors and GBC cell lines, and to evaluate the effect of mTOR inhibitors in in vitro and in vivo models of GBC. Methods: The expression of mTOR/p70S6K signaling pathway components was examined by immunohistochemistry in primary tumors and chronic cholecystitis (CC), and by Western blot in eight GBC cell lines. The in vitro effect of mTOR inhibitors (LY294002, Rapamycin, Everolimus and AZD8055) on cell viability and migration was assessed by MTS and Transwell chamber assays. The therapeutic effect of Rapamycin was evaluated in subcutaneous tumor models (NOD/SCID mice). Treatment started when tumor volumes had reached 100mm3. Animals (G-415 and TGBC-2TKB xenografts) were randomly divided (n=5 pe...