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Colorectal cancer (CRC) is the one of the most frequent and deadly cancer worldwide. The regulation mediated by the N6methyladenosine (m6A) demethylase protein called FTO in CRC is quite controversial. As previously studied in obesity and food intake, currently is being linked to stemness, epithelial-mesenchymal transition, chemotherapy resistance and proliferation in colon cancer cells, however, its localization and function in the tumor microenvironment (TME) and how it connects with progression remains unclear. The aim of this study was to evaluate FTO protein in tumor and healthy corresponding tissue from 22 colorectal cancer patients with different histological TNM stages (hTNM) by immunohistochemistry and multiple immunofluorescence analysis; classifying the tumor and stromal FTO localization and associating this data with clinical and histopathological features. As for content, the frequency of FTO+ cells in stromal compartments increases in more invasive stages compared to non-tumor tissue and in tumors with low desmoplasia. Regarding to localization, FTO is mostly nuclear and present in the lamina propria cells in non-tumor tissues and in immune and fibro-immune compartments in the TME, such as T cells, cancer associated fibroblasts and macrophages. In tumor cells, the percentage of FTO+ cells increase in early carcinogenic stages (hTNM I vs healthy tissue) and in moderately differentiated tumors. In addition, nuclear FTO expression increases in normal T cells and fibroblasts with a particular pattern after exposure for 24h to a colon adenocarcinoma cell-derived conditioned media, suggesting FTO activity in the TME interaction following a paracrine signal. In conclusion, the increase of the FTO protein in both tumor and stroma from CRC suggests its participation mediating TME communication. Citation Format: Glauben T. Landskron-Ramos, Antonia L. Domínguez-Beltrando, Mauricio Zambra-Rojas, Antonella Sanguineti, Gonzalo Vasquez, Marjorie De la Fuente, Daniela Simian, Silvana Valdebenito-Silva, Juan Saavedra, Solange Gouet, Ricardo Soto-Rifo, Daniela Sauma, Eliseo Eugenin, Mario Abedrapo, Marcela A. Hermoso, Marcela A. Hermoso. Novel potential role of m6A-demethylase FTO (fat mass and obesity) protein in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1251.

Introduction: Colorectal cancer (CRC) is the fourth leading cause of cancer death in Chile. Currently, treatment is surgical and adjuvance is based on chemotherapy mainly. One of the risk factors of CRC is obesity and metabolic syndrome. In search of factors that induce cell apoptosis, orexins were identified. Orexins are neuropeptides that regulate appetite, inhibit satiety and increase energy expenditure. The altered expression of orexin or its dysregulation has been associated with a wide range of human diseases such as narcolepsy, obesity, drug addiction and cancer. In rats and mice lacking orexin receptor, an increase in body mass index (BMI) was observed. Orexin Receptor 1 (OX1R) is overexpressed in CRC, but is not detected in normal colon tissue. Its activation by Orexin-A promotes apoptosis in cancer cell lines, and can be modulated by diet. Aim: To determine OX1R expression in the adenoma-carcinoma progression of CRC as possible therapeutic target, and its correlation with BMI. Material and Methods: Patients with neoplastic colorectal lesions undergoing surgical or endoscopic treatment between 2014 and 2015 were prospectively enrolled. Tissue samples of 29 patients were divided into 4 groups. Group A: control (n = 5), Group B: low-grade adenoma (n = 5), Group C: high-grade adenoma (n = 7), Group D: adenocarcinoma (n = 12). Formalin-fixed-paraffin-embedded samples were used to perform immunohistochemistry (IHC) of OX1R. Fresh tissues from Group D were used to assess protein and mRNA expression of OX1R by Western-blot and quantitative RT-PCR, respectively. Primary cultures were established from fresh tumor tissue of patients with adenocarcinoma. To compare OX1R expression levels we used the t-student test. Results: IHC expression of OX1R was detected both in the luminal membrane of colorectal epithelium and in the cytoplasm. We observed stained cells in 0-1% of the total area in Group A, B and C. In Group D, 7 samples showed staining 0-1%, 2 samples 1-10% and 3 samples 10-20%. High mRNA and protein expression was detected in stage III- IV adenocarcinoma samples compared to stage 0, I and II tumor samples. Normal adyacent tissue of CRC patients does not express OX1R. BMI is inversely associated with OX1R expression. Orexin A was able to induce apoptosis in primary cell cultures from advanced tumors in a dose dependent manner. Conclusions: OX1R is expressed scarcely in early stages of carcinogenesis. Its expression is induced significantly only in the most advanced adenocarcinomas, both at protein and mRNA levels, and is inversely associated with BMI. In primary cultures of patients with CRC, orexin A is able to induce apoptosis in a dose dependent manner. FONDECYT 1140012. Citation Format: Ana M. Wielandt, Cynthia Villarroel, Claudia Hurtado, Kento Inada, Hiroshi Kawachi, Daniela Simian, Maria T. Vial, Marcela Figueroa, Magdalena Castro, Udo Kronberg, Francisco Lopez-Kostner. Expression levels of orexin receptor 1 in different stages of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5167.

Introduction. Tumor suppressor PTEN acts as a negative regulator of the PI3K/AKT/mTOR pathway, thus controlling cell survival and proliferation. Mutations or deletions inactivating PTEN function lead to over-activation of the PI3K/AKT/mTOR pathway in cancer. PTEN gene alterations have been described in different types of tumors. In colon cancer these mutations range from 1% to 29% of the cases. The aim of this study was to determine the prevalence of PTEN mutations and/or deletions in Chilean colon cancer patients. Methods: ninety-one colon cancer patients were recruited for this study. Genomic DNA from normal and tumor tissue was obtained, and the deletion of PTEN locus was determined by PCR amplification of three STR markers flanking PTEN, and two intragenic markers. To search point mutations we amplified exons 7 and 8 by PCR, and sequenced. PTEN expression was evaluated through immunohistochemistry. Results: We observed deletion events in 9.8% (9/91) of tumors, and inactivating mutations in 5.5% (5/91). Two tumors presented a deletion in one allele and a mutation in the other. Immunohistochemistry detected a very weak expression of PTEN in 55/76 tumors. Conclusions: 15.4 % (12/91) of tumors have PTEN gene inactivated. We observed that deletion was a frequent mechanism for PTEN inactivation in Chilean colon cancer patients. Note: This abstract was not presented at the meeting. Citation Format: Gonzalo Encina, Karin Alvarez, Paulina Orellana, Ana María Wielandt, Cynthia Villarroel, Daniela Simian, Luis Contreras, Udo Kronberg, Francisco López, Pilar Carvallo. Gene mutations and deletions inactivates PTEN tumor suppressor in Chilean colon cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2230. doi:10.1158/1538-7445.AM2014-2230

Introduction: The Epidermal Growth Factor Receptor (EGFR) signaling pathway regulates key cell functions like proliferation and survival. This pathway presents two signaling cascades, RAS/RAF/MAPK and PIK3/PTEN/AKT/mTOR. Mutations in different components of the EGFR pathway have been described in different cancers. This pathway is a therapeutic target for monoclonal antibodies and tyrosine kinase inhibitors based therapies. Additionally, in advanced colorectal cancer patients, somatic KRAS mutations are response predictors for biological therapy with anti-EGFR antibody. The sensitivity of the mutations detection methods is key to a proper application of personalized therapeutic strategies. Traditionally, somatic mutations are detected by Sanger sequencing and real-time PCR. We determined the presence of mutations in specific genes of the EGFR pathway in colon cancer tumors from Chilean patients. Methods: DNA was extracted from twenty-eight fresh frozen tumor samples. DNA libraries were created using SureSelect (Agilent) designed for the analysis of EGFR, KRAS, BRAF, MAP2K1, PIK3CA, PIK3R1 and PTEN genes. Next generation sequencing was performed in MiSeq sequencer (Illumina) at Sistemas Genómicos (Spain). Results: Sequencing coverage varied between 100 and 250 readings between tumors, which assures the real presence of the changes. This technique allows detecting mutations that are present in a smaller percentage of tumoral cells, as low as 20% in the sample. Pathogenic mutations were identified in 19 out of the 28 tumors. Mutations were found in KRAS (p.Gly12Asp, p.Gly12Arg, p.Gly12Val and p.Gly12Cys), BRAF (p.Val600Glu and p.Gly606Glu), PIK3CA (p.Glu542Lys, p.Glu545Lys, p.Glu545Gln, p.Ala1035Val, p.Asp1045Val and p.His1047Arg) and PTEN (p.Lys267ArgfsX9). Five tumors presented mutations in both branches of the EGFR signaling pathway (KRAS-PIK3CA, BRAF-PIK3CA and BRAF-PTEN). Three allelic variants were identified in MAP2K1 and PIK3R1. Mutations that were identified in KRAS, BRAF and PIK3CA genes corresponded to gain of function mutations while a change found in PTEN gene corresponded to a loss of function mutation. Finally, mutations in PIK3CA and KRAS of 6 tumors were not previously detected by Sanger sequencing. Discussion: Mutations were found in 68% of analyzed tumors. Eighteen percent of the tumors presented mutations in two genes. Forty-six percent of mutations were identified in KRAS gene. Three allelic variants of unknown significance were detected which have not been described in cancer mutations nor in SPNs databases. Therefore, it is necessary to perform functional studies to determine the effect on protein function. Next generation sequencing allowed us to obtain more reliable and reproducible results, and was able to detect mutations present in a low percentage of tumoral cells in the sample, maybe due to tumoral heterogeneity or contamination with normal cells. FONDECYT 1111020 Citation Format: Karin Alvarez, Paulina Orellana, Cynthia Villarroel, Gonzalo Encina, Daniela Simian, Camila Estay, Maki Kobayashi, Hiroshi Kawashi, Udo Kronberg, Francisco Lopez, Pilar Carvallo. Next generation sequencing of the EGFR signaling pathway in colon cancer tumors from Chilean patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2014-2232