The Defender against apoptotic cell death (DAD1) is a negative regulator of programmed cell death that was initially identified in the temperature sensitive tsBN7 cell line. It has been shown to be an essential subunit of oligosaccharyltransferase and is localized to the Endoplasmic reticulum (ER) in a normal physiological state. However, our data suggests that DAD1 localizes outside of the cell and alters the apoptotic signaling via FAS ligand to give cancer cells a survival advantage. Although the mechanism is poorly understood, increased expression of DAD1 has been associated with increasing Gleason score in prostate cancer (PCa) patient tumors. Based on the aforementioned evidence, our study attempts to unravel cellular localization and the underlying mechanisms by which DAD1 mediates prostate cancer cell survival, and explore its potential as a biomarker in prostate cancer. As evidenced by qRTPCR, immunocytochemistry, immunohistochemistry, Co-ip, ELISA, and immuno-blot analysis, cancer cells down regulate the expression of the binding partner of DAD1 responsible for retention of DAD1 in the ER, which allows DAD1 to exit the ER and be exocytosed. The exocytosed DAD1 binds to FAS L and prevents apoptotic signaling. Treatment with DAD1 antibody induces significantly higher cell death in prostate cancer cells compared to the non-tumorigenic cells. Combination of DAD1 antibody with currently used chemotherapeutic agents like Docetaxel and Doxorubicin can be used to achieve higher cell death at a lower dose of these drugs to minimize side effects. Further, our immunohistochemistry data in tumor microarray suggests that DAD1 could serve as a potential biomarker marker in PCa. In addition to the tissue, we also examined the expression of DAD1 in prostate cancer patient serum samples using sandwich ELISA; our results indicate DAD1 is a more sensitive and specific prognostic marker for prostate cancer compared to PSA. Our data suggests that localization of DAD1 outside of the cells is crucial to the survival of PCa cells and this phenomenon can be exploited to specifically target prostate cancer cells in therapy and serve as a biomarker in prostate cancer. / 1 / Nobel Bhasin

Submitted by Pós Imunologia (ppgimicsufba@gmail.com) on 2017-07-31T18:57:58Z No. of bitstreams: 1 ANAQUE DISSERTAÇÃO - Versão Final - 21.03.2017.pdf: 2518048 bytes, checksum: 7c8b38757c1fbbd07024d5cfe3bd76ad (MD5) / Approved for entry into archive by Edvaldo Souza (edvaldosouza@ufba.br) on 2017-08-01T20:30:58Z (GMT) No. of bitstreams: 1 ANAQUE DISSERTAÇÃO - Versão Final - 21.03.2017.pdf: 2518048 bytes, checksum: 7c8b38757c1fbbd07024d5cfe3bd76ad (MD5) / Made available in DSpace on 2017-08-01T20:30:58Z (GMT). No. of bitstreams: 1 ANAQUE DISSERTAÇÃO - Versão Final - 21.03.2017.pdf: 2518048 bytes, checksum: 7c8b38757c1fbbd07024d5cfe3bd76ad (MD5) / CNPQ / A asma alérgica é uma morbidade de relevante impacto mundial que afeta milhões de indivíduos. É uma doença complexa que envolve a combinação de fatores ambientais e genéticos que são determinantes de seu desenvolvimento. Muitos genes têm sido reportados por estarem envolvidos com asma e atopia, dentre eles os genes DAD1 e OXA1L, presentes no cromossomo 14, que podem também estar envolvidos a essas condições. O gene DAD1 é considerado um regulador negativo da apoptose e OXA1L é reportado por seu envolvimento na biogênese e fosforilação oxidativa mitocondrial. Não existem dados na literatura que demonstrem variantes nesses genes associados com essas morbidades. Desta forma, sabendo-se da importância da apoptose e biogênese mitocondrial na reposta inflamatória da asma e alergia, polimorfismos nesses genes podem estar associados como fatores de risco ou proteção para seu desenvolvimento. Portanto, o objetivo deste estudo é avaliar se polimorfismos em DAD1 e OXA1L estão associados com asma e marcadores de atopia na população do SCAALA do inglês (Social Change Asthma and Allergy in Latin America) e qual o impacto funcional destas variantes, uma vez que estejam associadas com estes desfechos. O DNA de 1.307 indivíduos foram genotipados usando Illumina 2.5 Human Omni Bead chip. Foram realizadas análises de regressão logística a fim de avaliar a associação de variantes em DAD1 e OXA1L com asma e marcadores de alergia utilizando software PLINK 1.9 ajustados por sexo, idade, infecções helmínticas e marcadores de ancestralidade no modelo aditivo. O alelo G do SNP rs1681577 do DAD1 foi negativamente associado com asma (OR: 0.80; IC: 0.64 – 0.99; p: 0.039). Além disso, o alelo A do rs17119961 foi positivamente associado com teste cutâneo para P. americana (OR: 2.13; IC: 1.36 – 3.32; p: <0.001). O rs3811189 teve seu alelo C associado positivamente com produção espontânea de IL-13 (OR: 1.31; IC: 1.07 – 1.61; p: 0.008). Ademais, o SNP rs4981436 no OXA1L teve seu alelo C positivamente associado com asma (OR: 1.41; IC: 1.08 – 1.84; p: 0.012). Por outro lado o alelo G do SNP rs8572 foi positivamente associado com teste cutâneo para D. pteronyssinus (OR: 1.33; IC: 1.05 – 1.68; p: 0.017). O rs200470407 teve seu alelo C associado positivamente com produção espontânea de IL-13 (OR: 1.32; IC: 1.07 – 1.64; p: 0.012). Verificamos também um aumento da expressão do gene DAD1 em indivíduos asmáticos comparados aos controles. Assim sendo, polimorfismos em DAD1 e OXA1L podem influenciar a ocorrência de asma e atopia em uma população latina / Allergic asthma is a morbidity with significant worldwide impact that affects Millions of individuals. It is a complex disease that involves the combination of environmental and genetic factors both related to its occurrence. Many genes have been reported to be involved with asthma and atopy, and among them, DAD1 and OXA1L genes , located at chromosome 14 may also be involved in such conditions. The DAD1 gene is considered to be a negative regulator of apoptosis and OXA1L is reported for its involvement in mitochondrial biogenesis as well as its oxidative phosphorylation. There are no data in the literature that previously have already reported variants in such these genes with these morbidities. Thus, along with the importance of apoptosis and mitochondrial biogenesis in the inflammatory response of asthma and allergy, polymorphisms in these genes may be associated as risk or protective factors for their development. Therefore, the aim of this study was to evaluate whether polymorphisms in DAD1 and OXA1L are associated with asthma and markers of atopy in population SCAALA (Social Change Asthma and Allergy in Latin America) and what is the functional role of these variants once associated with such outcomes. The DNA of 1,307 individuals was genotyped using the Illumina 2.5 Human Omni Bead chip. Logistic regression analyzes were performed to evaluate the association of variants in DAD1 and OXA1L with asthma and allergy markers using PLINK 1.9 software adjusted for sex, age, helminth infections and ancestral markers in the additive model. The G allele of the SNP rs1681577 of DAD1 was negatively associated with asthma (OR: 0.80; CI: 0.64 - 0.99; p: 0.039). In addition, the A allele of rs17119961 was positively associated with skin prick test for P. americana (OR: 2.13; CI: 1.36 - 3.32; p: <0.001). The C allele for rs3811189 was positively associated with spontaneous IL-13 production (OR: 1.31; CI: 1.07 - 1.61, p: 0.008). In addition, the C allele of SNP rs4981436 in OXA1L was positively associated with asthma (OR: 1.41; CI: 1.08 - 1.84; p: 0.012). On the other hand, the G allele of the SNP rs8572 was positively associated with the skin prick test for D. pteronyssinus (OR: 1.33; CI: 1.05 - 1.68; p: 0.017). The C allele of rs200470407 was positively associated with spontaneous production of IL-13 (OR: 1.32; CI: 1.07 - 1.64; p: 0.012). In addition, DAD1 gene expression was up-regulated in asthmatic individuals compared to controls. Thus, polymorphisms in DAD1 and OXA1L may be related to allergy and asthma in Latin America