Browsing by Author "Tapia, Julio C."

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    A Single Variant in Pri-miRNA-155 Associated with Susceptibility to Hereditary Breast Cancer Promotes Aggressiveness in Breast Cancer Cells
    (2022) Landeros, Natalia; Gonzalez-Hormazabal, Patricio; Perez-Moreno, Pablo; Tapia, Julio C.; Jara, Lilian
    Variants in genes encoding for microRNAs have been associated with their deregulation in breast cancer (BC). Sequencing of microRNAs deregulated in BC was performed using DNA from Chilean patients with a strong family history and negative for mutations in BRCA1/BRCA2. Seventeen variants were identified, three of which were selected for a case-control association study: rs376491654 (miR-335), rs755634302 (miR-497), and rs190708267 (miR-155). For rs190708267 C>T, the heterozygous T allele was detected in four BC cases and absent in controls, while homozygous TT cases were not detected. Variants were modelled in silico, cloned in a plasmid, expressed in BC cell lines, and functional in vitro assays were performed. Overexpression of the miR-155-T allele increased mature miR-155-5p levels in both BC cell lines, suggesting that its presence alters pre-miR-155 processing. Moreover, BC cells overexpressing the miR-155-T allele showed increased proliferation, migration, and resistance to cisplatin-induced death compared to miR-155-C overexpressing cells. Of note, the 3 ' UTR of APC, GSK3 beta, and PPP1CA genes, all into the canonical Wnt signaling pathway, were identified as direct targets. APC and GSK3 beta mRNA levels decreased while PP1 levels increased. These results suggest a pathogenic role of the variant rs190708267 (miR-155) in BRCA 1/2 negative BC, conferring susceptibility and promoting traits of aggressiveness.
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    Differential localization of α' and β subunits of protein kinase CK2 during rat spermatogenesis
    (2009) Alvarado-Diaz, Carlos P.; Tapia, Julio C.; Antonelli, Marcelo; Moreno, Ricardo D.
    Protein kinase CK2 is a serine/threonine kinase expressed in organisms from yeast to human and is composed of a catalytic subunit (alpha or alpha') and a regulatory subunit (beta) forming a holoenzyme with the possible subunit combinations alpha(2)beta(2), alpha'(2)beta(2), or alpha alpha'beta(2). This kinase has been shown to be involved in embryonic development and gametogenesis. We have studied the expression of the CK2 alpha' and CK2 beta subunits during the first wave of spermatogenesis and in adult testis in the rat. Western blot analyses have demonstrated that both CK2 alpha' and CK2 beta are expressed in testes from birth to adulthood. A more detailed study of the protein localization of CK2 alpha' and CK2 beta by immunohistochemistry suggests that CK2 alpha' and CK2 beta are localized in the nuclei of Sertoli cells in 5-day-old rats, whereas they appear to have a cytoplasmic localization in older animals. In adult testes, CK2 alpha' and CK2 beta subunits are present in spermatocytes. Both subunits exhibit a similar expression pattern with the highest level in spermatocytes at stages VIII-XIV. Interestingly, CK2 beta is highly expressed in spermatogonia, whereas CK2 alpha' is barely detectable. Mature epididymal spermatozoa express CK2 alpha' in the acrosome and CK2 beta in the flagellum. This new evidence therefore indicates that protein kinase CK2 has a possible role at various stages during mammalian spermatogenesis, a process that involves proliferation, meiosis, apoptosis, and differentiation. CK2 might thus emerge as a new pivotal control enzyme at various levels in mammalian spermatogenesis.
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    Effect of Melatonin on Chemoresistance Exhibited by Spheres Derived from Canine Mammary Carcinoma Cells
    (2024) Cataldo, Dania; Aravena, Guillermo; Escobar, Alejandro; Tapia, Julio C.; Peralta, Oscar A.; Torres, Cristian G.
    Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. Within the mammary tumor microenvironment, there is a cell subpopulation called cancer stem cells (CSCs), which are capable of forming spheres in vitro and resisting anti-tumor treatments, partly explaining the recurrence of some tumors. Previously, it has been described that spheres derived from canine mammary carcinoma cells CF41.Mg and REM 134 exhibit stemness characteristics. Melatonin has shown anti-tumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by stem-like neoplastic cells derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. CF41.Mg and REM 134 cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and L-glutamine. The spheres were cultured in ultra-low attachment plates in DMEM/F12 medium without fetal bovine serum and with different growth factors. The CD44(+)/CD24(-/low) phenotype was analyzed by flow cytometry. The viability of sphere-derived cells (MTS reduction) was studied in the presence of melatonin (0.1 or 1 mM), doxorubicin, mitoxantrone, and luzindole. In addition, the gene (RT-qPCR) of the multidrug resistance bombs MDR1 and ABCG2 were analyzed in the presence of melatonin. Both cell types expressed the MT1 gene, which encodes the melatonin receptor MT1. Melatonin 1 mM does not modify the CD44(+)/CD24(-/low) phenotype; however, the hormone reduced viability (p < 0.0001) only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. These effects were independent of the binding of the hormone to its receptor MT1, since, by pharmacologically inhibiting them, the effect of melatonin was not blocked. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1 was decreased in response to the hormone (p < 0.001). These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the gene expression of MDR1 and ABCG2.
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    Increase in ADAR1p110 activates the canonical Wnt signaling pathway associated with aggressive phenotype in triple negative breast cancer cells
    (2022) Morales, Fernanda; Perez, Paola; Tapia, Julio C.; Lobos-Gonzalez, Lorena; Manuel Herranz, Jose; Guevara, Francisca; Rojas de Santiago, Pamela; Palacios, Esteban; Andaur, Rodrigo; Sagredo, Eduardo A.; Marcelain, Katherine; Armisen, Ricardo
    Triple-negative breast cancer (TNBC) represents a challenge in the search for new therapeutic targets. TNBCs are aggressive and generate resistance to chemotherapy. Tumors of TNBC patients with poor prognosis present a high level of adenosine deaminase acting on RNA1 (ADAR1). We explore the connection of ADAR1 with the canonical Wnt signaling pathway and the effect of modulation of its expression in TNBC. Expression data from cell line sequencing (DepMap) and TCGA samples were downloaded and analyzed. We lentivirally generated an MDA-MB-231 breast cancer cell line that overexpress (OE) ADAR1p110 or an ADAR knockdown. Abundance of different proteins related to Wnt/beta-catenin pathway and activity of nuclear beta-catenin were analyzed by Western blot and luciferase TOP/FOP reporter assay, respectively. Cell invasion was analyzed by matrigel assay. In mice, we study the behavior of tumors generated from ADAR1p110 (OE) cells and tumor vascularization immunostaining were analyzed. ADAR1 connects to the canonical Wnt pathway in TNBC. ADAR1p110 overexpression decreased GSK-3 beta, while increasing active beta-catenin. It also increased the activity of nuclear beta-catenin and increased its target levels. ADAR1 knockdown has the opposite effect. MDA-MB-231 ADAR1 (OE) cells showed increased capacity of invasion. Subsequently, we observed that tumors derived from ADAR1p110 (OE) cells showed increased invasion towards the epithelium, and increased levels of Survivin and CD-31 expressed in vascular endothelial cells. These results indicate that ADAR1 overexpression alters the expression of some key components of the canonical Wnt pathway, favoring invasion and neovascularization, possibly through activation of the beta-catenin, which suggests an unknown role of ADAR1p110 in aggressiveness of TNBC tumors.
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    LINC00662 Promotes Aggressive Traits by Modulating OCT4 Expression through miR-335-5p in Gallbladder Cancer Cells
    (MDPI, 2024) Perez-Moreno, Pablo; Riquelme, Ismael; Bizama Soto, Carolina Del Carmen; Vergara-Gomez, Luis; Tapia, Julio C.; Brebi, Priscilla; Garcia Canete, Patricia Del Carmen; Roa Strauch, Juan Carlos Enrique
    Long non-coding RNAs (lncRNAs) are nucleotide sequences that participate in different biological processes and are associated with different pathologies, including cancer. Long intergenic non-protein-coding RNA 662 (LINC00662) has been reported to be involved in different cancers, including colorectal, prostate, and breast cancer. However, its role in gallbladder cancer has not yet been described. In this article, we hypothesize that LINC00662 has an important role in the acquisition of aggressiveness traits such as a stem-like phenotype, invasion, and chemoresistance in gallbladder cancer. Here, we show that LINC00662 is associated with larger tumor size and lymph node metastasis in patients with gallbladder cancer. Furthermore, we show that the overexpression of LINC00662 promotes an increase in CD133+/CD44+ cell populations and the expression of stemness-associated genes. LINC00662 promotes greater invasive capacity and the expression of genes associated with epithelial-mesenchymal transition. In addition, the expression of LINC00662 promotes resistance to cisplatin and 5-fluorouracil, associated with increased expression of chemoresistance-related ATP-binding cassette (ABC) transporters in gallbladder cancer (GBC) cell lines. Finally, we show that the mechanism by which LINC00662 exerts its function is through a decrease in microRNA 335-5p (miR-335-5p) and an increase in octamer-binding transcription factor 4 (OCT4) in GBC cells. Thus, our data allow us to propose LINC00662 as a biomarker of poor prognosis and a potential therapeutic target for patients with GBC.
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    Risk variants in BMP4 promoters for nonsyndromic cleft lip/palate in a Chilean population
    (2011) Suazo, José; Tapia, Julio C.; Santos Martín, José Luis; Castro, Víctor G.; Colombo, Alicia.; Blanco, Rafael.
    Abstract Background Bone morphogenetic protein 4 gene (BMP4) plays a key role during maxillofacial development, since orofacial clefts are observed in animals when this gene is conditionally inactivated. We recently reported the existence of association between nonsyndromic cleft lip/palate (NSCLP) and BMP4 polymorphisms by detecting transmission deviations for haplotypes that include a region containing a BMP4 promoter in case-parent trios. The aim of the present study was to search for possible causal mutations within BMP4 promoters (BMP4.1 and BMP4.2). Methods We analyzed the sequence of BMP4.1 and BMP4.2 in 167 Chilean NSCLP cases and 336 controls. Results We detected three novel variants in BMP4.1 (c.-5514G > A, c.-5365C > T and c.-5049C > T) which could be considered as cleft risk factors due to their absence in controls. Additionally, rs2855530 G allele (BMP4.2) carriers showed an increased risk for NSCLP restricted to males (OR = 1.52; 95% C.I. = 1.07-2.15; p = 0.019). For this same SNP the dominant genotype model showed a higher frequency of G/G+G/C and a lower frequency of C/C in cases than controls in the total sample (p = 0.03) and in the male sample (p = 0.003). Bioinformatic prediction analysis showed that all the risk variants detected in this study could create new transcription factor binding motifs. Conclusions The sex-dependent association between rs2855530 and NSCLP could indirectly be related to the differential gene expression observed between sexes in animal models. We concluded that risk variants detected herein could potentially alter BMP4 promoter activity in NSCLP. Further functional and developmental studies are necessary to support this hypothesis.Abstract Background Bone morphogenetic protein 4 gene (BMP4) plays a key role during maxillofacial development, since orofacial clefts are observed in animals when this gene is conditionally inactivated. We recently reported the existence of association between nonsyndromic cleft lip/palate (NSCLP) and BMP4 polymorphisms by detecting transmission deviations for haplotypes that include a region containing a BMP4 promoter in case-parent trios. The aim of the present study was to search for possible causal mutations within BMP4 promoters (BMP4.1 and BMP4.2). Methods We analyzed the sequence of BMP4.1 and BMP4.2 in 167 Chilean NSCLP cases and 336 controls. Results We detected three novel variants in BMP4.1 (c.-5514G > A, c.-5365C > T and c.-5049C > T) which could be considered as cleft risk factors due to their absence in controls. Additionally, rs2855530 G allele (BMP4.2) carriers showed an increased risk for NSCLP restricted to males (OR = 1.52; 95% C.I. = 1.07-2.15; p = 0.019). For this same SNP the dominant genotype model showed a higher frequency of G/G+G/C and a lower frequency of C/C in cases than controls in the total sample (p = 0.03) and in the male sample (p = 0.003). Bioinformatic prediction analysis showed that all the risk variants detected in this study could create new transcription factor binding motifs. Conclusions The sex-dependent association between rs2855530 and NSCLP could indirectly be related to the differential gene expression observed between sexes in animal models. We concluded that risk variants detected herein could potentially alter BMP4 promoter activity in NSCLP. Further functional and developmental studies are necessary to support this hypothesis.
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    The Ski Protein is Involved in the Transformation Pathway of Aurora Kinase A
    (WILEY, 2016) Rivas, Solange; Armisen, Ricardo; Rojas, Diego A.; Maldonado, Edio; Huerta, Hernan; Tapia, Julio C.; Espinoza, Jaime; Colombo, Alicia; Michea, Luis; Hayman, Michael J.; Marcelain, Katherine
    Oncogenic kinase Aurora A (AURKA) has been found to be overexpresed in several tumors including colorectal, breast, and hematological cancers. Overexpression of AURKA induces centrosome amplification and aneuploidy and it is related with cancer progression and poor prognosis. Here we show that AURKA phosphorylates in vitro the transcripcional co-repressor Ski on aminoacids Ser326 and Ser383. Phosphorylations on these aminoacids decreased Ski protein half-life. Reduced levels of Ski resulted in centrosomes amplification and multipolar spindles formation, same as AURKA overexpressing cells. Importantly, overexpression of Ski wild type, but not S326D and S383D mutants inhibited centrosome amplification and cellular transformation induced by AURKA. Altogether, these results suggest that the Ski protein is a target in the transformation pathway mediated by the AURKA oncogene. (C) 2015 Wiley Periodicals, Inc.