Jaime Lopez

Cellular FLICE-like inhibitory protein long form (c-FLIPL) overexpression is related to cervical cancer progression.

Cervical cancer is a leading cause of cancer deaths in women worldwide and infection by high-risk human papillomavirus types is a precursor event. The cellular FLICE-like inhibitory protein (c-FLIP) has been found to be overexpressed in several types of cancers and could be associated with cervical cancer progression because of its ability to inhibit the apoptotic process. To detect c-FLIP expression in cervical cancer, an immunohistochemical staining was performed, using tissue microarrays, on a series of 536 archival biopsy samples, including normal cervical tissues, low-grade and high-grade squamous intraepithelial lesions, and squamous cervical carcinomas. The epithelium in the normal cervix and low-grade squamous intraepithelial lesions mainly stained negatively for c-FLIP, whereas high-grade intraepithelial lesions and cancer samples showed an elevated expression of c-FLIP. A direct association was observed between the increasing grade of the lesion and the intensity of c-FLIP staining, in which the frequency of intense c-FLIP expression increased from 12.5% in the normal tissue to 82.1% in the cervical cancer tissue. An increased expression of c-FLIP may be an important factor in the progression of cervical cancer. This finding could aid in identifying patients with preneoplastic lesions at greater risk of developing cervical cancer. c-FLIP expression in cervical tissue may be a potential cervical cancer progression marker.

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.

Genotyping of human papillomavirus in cervical intraepithelial neoplasia in a high-risk population†

Infection with the human papillomavirus (HPV) is responsible for 99.7% of cervical cancers, the second most prevalent neoplasia in women worldwide and the fifth leading cause of death by cancer in this population. In Chile, the incidence rate is 14.4 cases per 100,000 women per year and it is considered a significant public health problem. The natural history of cervical cancer begins gradually from low‐grade and high‐grade squamous intraepithelial lesions to an invasive disease. In this study the frequency of HPV types was determined by HPV genotyping with reverse line blot hybridization in 200 cytobrushes of women with preneoplastic lesions in a high‐risk population. HPV DNA was found in 89% of the lesions (83.3% of low‐grade squamous intraepithelial lesions and 93.6% of high‐grade squamous intraepithelial lesions). Multiple HPV infections were found in 14.4% and 15.5% of low‐ and high‐grade lesions, respectively. HPV 16 was the most frequent genotype in single infections, followed by HPV 18. These results show that most of the preneoplastic lesions of the cervix (60%) were associated with HPV 16 and/or HPV 18, supporting the implementation of an HPV vaccination program in this high‐risk population. J. Med. Virol. 83:833–837, 2011.

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.

Abstract B28: FKBP6 gene is involved in progression of cervical cancer

Introduction: Cervical Cancer (CC) is an important heath problem in developing countries. The silencing of tumor suppressor genes (TSG) could be implied in cervical carcinogenesis. Methylation microarray showed an aberrant hypermethylation of FKBP6, a potential TSG gen in CC. The aim of this study was to characterizer FKBP6 role in cervical carcinogenesis. Materials and methods: ECT1 E6/E7 (immortalized normal squamous epithelia cell line) and three CC cell lines: SiHa, C-4I and C-33A were cultivated for experiments. FKBP6 expression was determined by qRT-PCR and western blot. Treatment with 10 μM 5-aza-2′deoxycytidine (5-aza) was performed to evaluate a possible epigenetic regulation. Transfection with pCMV6-FKBP6-GFP (FKBP6) and pCMV6-GFP (Empty) was carried out in SiHa cell line through lipofection followed by G418 selection to obtain stable transfection. Viability and clonogenic assay was performed to evaluate transfected cell behaviour. In other hand, 497 biopsy samples of the cervix were analyzed by FKBP6 immunohistochemistry (48 normal; 192 low-squamous intraepithelial lesions; 200 high- squamous intraepithelial lesions and 57 squamous cervical cancers). Results: FKBP6 mRNA and protein expression was significantly downregulated in all cervical cancer cell lines respect to ECT1 E6/E7. In western blot, also could be observed a variant of FKBP6 mainly in C-4I cell line. After treatment with 5-aza, mRNA (p Conclusions: There is a FKBP6 dysregulation in CC cell lines, and the inactivation mechanism seems to be methylation. Restored expression of FKBP6 in SiHa cell line modifies the tumor phenotype, decreasing colony formation, but increasing cell viability. These results suggest that FKBP6 could be implied in cervical progression, but more studies are necessary to evaluate FKBP6 functions in cervical cancer. Acknowledgments: FONDECYT N°3130630, Project Corfo N° 09CN14-5960, Project Corfo N°12IDL2-18157. Project FONDECYT N° 11150802, Project FONDECYT N°1115062 Citation Format: Carmen Gloria Ili, Tamara Viscarra, Juan Carlos Araya, Jaime Lopez, Barbara Mora, Javier Retamal, Susana Aedo, Enrique Bellolio, Juan Carlos Roa, Priscilla Brebi. FKBP6 gene is involved in progression of cervical cancer. [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 B28.

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.

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.

Abstract B29: ZNF516 a potential tumor suppressor gene candidate is implied in tumor progression in cervical cancer

Background: In Cervical Cancer (CC) the role of HPV is fundamental; however, not all HPV infected women will develop this disease. Therefore, other mechanisms, such as silencing of tumor suppressor genes (TSG), could be implied in cervical carcinogenesis. In a previous study, we constructed methylation microarrays, where we found promoter aberrant hypermethylation of ZNF516 gen in CC, postulating it as TSG candidate. The aim of this study was to characterizer ZNF516 role in cervical carcinogenesis and cell cycle. Materials and methods: ECT1 E6/E7 (immortalized normal squamous epithelia cell line) and three CC cell lines: SiHa, C-4I and C-33A were cultivated for experiments. ZNF516 expression was determined by qRT-PCR and western blot. Treatment with 10 μM 5-aza-2′deoxycytidine (5- aza) was performed to evaluate a possible epigenetic regulation. Transfection with pCMV6-ZNF516-GFP (ZNF516) and pCMV6-GFP (Empty) was carried out in C-33A cell line through lipofection followed by G418 selection to obtain stable transfection. Viability and clonogenic assay was performed to evaluate transfected cell behaviour. Also, immnunohistochemical analysis was performed in 509 cervical biopsy tissues (55 normal; 188 low-squamous intraepithelial lesions; 205 high- squamous intraepithelial lesions and 57 squamous cervical cancers). Results: ZNF516 mRNA expression was significantly downregulated in SiHa (p Conclusions: There is a clear ZNF516 dysregulation in CC cell lines, and the inactivation mechanism seem to be methylation. Restored expression of ZNF516 in C-33A cell line modifies the tumor phenotype, decreasing cellular viability and colony formation. These results suggest that ZNF516 could be a TSG, and its inactivation promotes CC developing. Citation Format: Carmen Gloria Ili, Tamara Viscarra, Juan Carlos Araya, Jaime Lopez, Barbara Mora, Javier Retamal, Enrique Bellolio, Susana Aedo, Juan Carlos Roa, Priscilla Brebi. ZNF516 a potential tumor suppressor gene candidate is implied in tumor progression in cervical cancer. [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 B29.