The COP9 signalosome (CSN) contains eight-subunits and is a highly conserved protein complex implicated in ubiquitin-mediated proteolysis. Jun activation domain-binding protein 1 (Jab1) is the fifth component of CSN (CSN5) with a molecular weight of 38 kDa. Jab1 overexpression is observed in many human cancers, but there is no clear evidence how Jab1 contributes to malignant transformation in human cancers. Bcr-Abl is a cytoplasmic chimeric oncoprotein produced by Philadelphia chromosome translocation and found in more than 90% of patients with chronic myelogenous leukemia (CML). Recent studies have shown that the Jab1/COP9 signalosome subcomplex is a downstream mediator of Bcr-Abl kinase and may facilitate cell-cycle progression. In this study, we found that inhibition of Bcr-Abl kinase by STI-571 downregulated 50% of full length human Jab1 promoter activity and gene expression. Promoter deletion assay indicated that the responsive element for Bcr-Abl is located between -405/-223 region of human Jab1 promoter. Treatment of LY294002 also reduced Jab-405/-223 promoter activity and Jab1 expression. Promoter mutagenesis assay and ChIP assay suggest that Bcr-Abl stimulated both £]-catenin /TCF complex and STAT1 bind to the consensus binding sites of Jab-405/-223 promoter. Taken together, Bcr-Abl oncogene may regulate Jab1 via PI3K/AKT signal pathway, £]-catenin /TCF and STAT1 transcription factors.

Endoplasmic reticulum (ER) stress is the condition that unfolded or misfolded proteins accumulated in the ER which leads to the solubility stress. ER can activate the unfolded protein response (UPR) to restore the ER homeostasis. JAB1 was originally identified as the coactivator of AP-1 transcription factor. JAB1 was then discovered to mediate the cyclin-dependent-kinase inhibitor p27kip1 nuclear exportation and degradation. Previous studies demonstrate that ER stress may affect the regulation of JAB1, but the mechanism is still unknown. In this study, we want to investigate how JAB1 is regulated in ER stress. We applied tunicamycin, a protein N-glycosylation inhibitor, as the ER stress inducer. Western blot and reverse transcription PCR revealed that treatment with tunicamycin for 48 hours in Hep3B induced ER stress and repressed JAB1 protein and mRNA expression. Serial deletion of the JAB1 promoter activity assay revealed that the region from -405 bp to -223 bp may be responsive in the tunicamycin-induced ER stress. Computational prediction suggested that there are several candidate factors may join the regulation of JAB1 in this region. Site-directed mutation of JAB1 promoter assay revealed that the tunicamycin-induced ER stress repressed JAB1 promoter activity through the sites at -342/-338 and -331/-327 in JAB1 promoter. Chromatin immunoprecipitation assay suggested that tunicamycin-induced ER stress repressed the JAB1 promoter activity through increasing the SP1 and DNMT3b binding to the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter. Methylation specific PCR showed that the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter were methylated in tunicamycin-induced ER stress. Taken together, we demonstrated that tunicamycin-induced ER stress repressed the JAB1 gene expression in Hep3B through increasing the binding of SP1 and DNMT3b to the SP1 binding sites and inducing promoter methylation to repress JAB1 expression.

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

Os tumores de glândula salivar compreendem em torno de 2 a 6,5% dos tumores de cabeça e pescoço. Entre os tumores de glândula salivar, o adenoma pleomórfico é benigno e o mais comum. O carcinoma adenoide cístico e adenocarcinoma polimorfo de baixo grau encontram-se entre os mais frequentes malignos. Jab1 é uma de muitas proteínas que afetam diversos estágios da tumorigênese sendo importante na regulação variadas vias de sinalização e/ou proteínas como p27 e AP-1, a última composta por c-jun e c-fos, que são principalmente relacionadas com o ciclo celular e proliferação celular. O objetivo desse trabalho foi avaliar a expressão imuno-histoquímica das proteínas Jab1, p27, c-jun e c-fos no adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico das glândulas salivares. Foi realizada análise imuno-histoquímica semi-quantitativa das células marcadas nos tumores de glândula salivar e glândula salivar normal de acordo com o escore 0 (células sem expressão), 1(> 0 <= 5% de células marcadas), 2 (> 5 <= 50%) and 3 (> 50%). Para Jab1, c-jun e c-fos foi considerado apenas marcação nuclear e para p27, nuclear e citoplasmática, separadamente. Os resultados foram analisados utilizando-se os testes de Kruskal-Wallis, de Mann-Whitney, do Qui-quadrado e o teste de correlação de Spearman, cujo nível de significância foi de p<0,05 e processados com o auxílio do software GraphPad Prisma 5.0. A análise estatística revelou que a expressão de Jab1 foi significante no adenoma pleomórfico e no carcinoma adenoide cístico em relação aos ductos e no adenocarcinoma polimorfo de baixo grau em relação ao carcinoma adenoide cístico (p=0,0136, 0,0001 e 0,0344, respectivamente); a expressão de p27 nuclear foi significante no adenoma pleomórfico e no adenocarcinoma polimorfo de baixo grau quando comparados ao carcinoma adenoide cístico (p=0,0074 e 0,0004, respectivamente) e a expressão citoplasmática em todos os grupos quando comparados aos ácinos; c-fos, a expressão foi significativa nos ductos ao compará-los ao adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico (p=0,0002, 0,0048 e 0,0352, respectivamente). O teste de correlação de Spearman de Jab1, p27, c-jun e c-fos em cada lesão separadamente revelou que no adenoma pleomórfico houve correlação significativa entre Jab1 e p27 (r=0,371; p=0,020) e entre c-jun e c-fos (r=0,452; p=0,004). No adenocarcinoma polimorfo de baixo grau, houve correlação entre Jab1 e p27 (r=0,494; p=0,044) e no carcinoma adenoide cístico, entre p27 e c-fos (r=0,513; p=0,035). Foi concluído que a tumorigênese do adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico parece estar associada à expressão de Jab1 e p27. / Salivary gland tumors comprise about 2 to 6.5% of the head and neck tumors. Among the salivary gland tumors, pleomorphic adenoma is the most common and benign tumor. Adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma are the most frequent malignant tumors. Jab1 is one of many proteins which affects many stages of the tumorigenesis and regulates positively and negatively several pathways and/or proteins such as p27 and AP-1, the latter composed by c-jun and c-fos, which are mostly related to cell cycle and cell proliferation. The aim of this study was to evaluate the immunoexpression of the proteins Jab1, p27, c-jun and c-fos in pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma of the salivary glands. The semi-quantitative immunohistochemical analysis was performed in salivary gland tumors and in normal salivary gland according to the score 0 (no stained cells), 1 (> 0 <= 5% of stained cells), 2 (> 5 <= 50%) and 3 (> 50%). Nuclear immunostaining alone was considered for Jab1, c-jun and c-fos proteins and cytoplasmic and nuclear staining for p27. Results were analyzed in GraphPad Prisma 5.0 software using Kruskal-Wallis, Mann-Whitney and Chi-square tests and Spearman correlation test in which significancy level was p<0,05. Statistical analysis revealed that Jab1 expression was significant in pleomorphic adenoma and adenoid cystic carcinoma in relation to ducts and in polymorphous low-grade adenocarcinoma in relation to adenoid cystic carcinoma (p=0,0136, 0,0001 e 0,0344, respectively); the p27 nuclear expression was significant in pleomorphic adenoma and in polymorphous low-grade adenocarcinoma when compared to adenoid cystic carcinoma (p=0,0074 e 0,0004, respectively) and cytoplasmic immunostaining was significant in all groups when compared to acini; c-fos expression was significant in ducts if compared to pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (p=0,0002, 0,0048 e 0,0352, respectively). Spearman correlation test to Jab1, p27, c-jun and c-fos in each lesion separately revealed significant correlation between Jab1 and p27 (r=0,371; p=0,020) and c-jun and c-fos (r=0,452; p=0,004) in pleomorphic adenoma. There was correlation between Jab1 and p27 (r=0,494; p=0,044) in polymorphous low-grade adenocarcinoma and between p27 and c-fos (r=0,513; p=0,035) in adenoid cystic carcinoma. In conclusion, tumorigenesis in pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma seems to be associated to expression of Jab1 and p27.

Os tumores de glândula salivar compreendem em torno de 2 a 6,5% dos tumores de cabeça e pescoço. Entre os tumores de glândula salivar, o adenoma pleomórfico é benigno e o mais comum. O carcinoma adenoide cístico e adenocarcinoma polimorfo de baixo grau encontram-se entre os mais frequentes malignos. Jab1 é uma de muitas proteínas que afetam diversos estágios da tumorigênese sendo importante na regulação variadas vias de sinalização e/ou proteínas como p27 e AP-1, a última composta por c-jun e c-fos, que são principalmente relacionadas com o ciclo celular e proliferação celular. O objetivo desse trabalho foi avaliar a expressão imuno-histoquímica das proteínas Jab1, p27, c-jun e c-fos no adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico das glândulas salivares. Foi realizada análise imuno-histoquímica semi-quantitativa das células marcadas nos tumores de glândula salivar e glândula salivar normal de acordo com o escore 0 (células sem expressão), 1(> 0 <= 5% de células marcadas), 2 (> 5 <= 50%) and 3 (> 50%). Para Jab1, c-jun e c-fos foi considerado apenas marcação nuclear e para p27, nuclear e citoplasmática, separadamente. Os resultados foram analisados utilizando-se os testes de Kruskal-Wallis, de Mann-Whitney, do Qui-quadrado e o teste de correlação de Spearman, cujo nível de significância foi de p<0,05 e processados com o auxílio do software GraphPad Prisma 5.0. A análise estatística revelou que a expressão de Jab1 foi significante no adenoma pleomórfico e no carcinoma adenoide cístico em relação aos ductos e no adenocarcinoma polimorfo de baixo grau em relação ao carcinoma adenoide cístico (p=0,0136, 0,0001 e 0,0344, respectivamente); a expressão de p27 nuclear foi significante no adenoma pleomórfico e no adenocarcinoma polimorfo de baixo grau quando comparados ao carcinoma adenoide cístico (p=0,0074 e 0,0004, respectivamente) e a expressão citoplasmática em todos os grupos quando comparados aos ácinos; c-fos, a expressão foi significativa nos ductos ao compará-los ao adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico (p=0,0002, 0,0048 e 0,0352, respectivamente). O teste de correlação de Spearman de Jab1, p27, c-jun e c-fos em cada lesão separadamente revelou que no adenoma pleomórfico houve correlação significativa entre Jab1 e p27 (r=0,371; p=0,020) e entre c-jun e c-fos (r=0,452; p=0,004). No adenocarcinoma polimorfo de baixo grau, houve correlação entre Jab1 e p27 (r=0,494; p=0,044) e no carcinoma adenoide cístico, entre p27 e c-fos (r=0,513; p=0,035). Foi concluído que a tumorigênese do adenoma pleomórfico, adenocarcinoma polimorfo de baixo grau e carcinoma adenoide cístico parece estar associada à expressão de Jab1 e p27. / Salivary gland tumors comprise about 2 to 6.5% of the head and neck tumors. Among the salivary gland tumors, pleomorphic adenoma is the most common and benign tumor. Adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma are the most frequent malignant tumors. Jab1 is one of many proteins which affects many stages of the tumorigenesis and regulates positively and negatively several pathways and/or proteins such as p27 and AP-1, the latter composed by c-jun and c-fos, which are mostly related to cell cycle and cell proliferation. The aim of this study was to evaluate the immunoexpression of the proteins Jab1, p27, c-jun and c-fos in pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma of the salivary glands. The semi-quantitative immunohistochemical analysis was performed in salivary gland tumors and in normal salivary gland according to the score 0 (no stained cells), 1 (> 0 <= 5% of stained cells), 2 (> 5 <= 50%) and 3 (> 50%). Nuclear immunostaining alone was considered for Jab1, c-jun and c-fos proteins and cytoplasmic and nuclear staining for p27. Results were analyzed in GraphPad Prisma 5.0 software using Kruskal-Wallis, Mann-Whitney and Chi-square tests and Spearman correlation test in which significancy level was p<0,05. Statistical analysis revealed that Jab1 expression was significant in pleomorphic adenoma and adenoid cystic carcinoma in relation to ducts and in polymorphous low-grade adenocarcinoma in relation to adenoid cystic carcinoma (p=0,0136, 0,0001 e 0,0344, respectively); the p27 nuclear expression was significant in pleomorphic adenoma and in polymorphous low-grade adenocarcinoma when compared to adenoid cystic carcinoma (p=0,0074 e 0,0004, respectively) and cytoplasmic immunostaining was significant in all groups when compared to acini; c-fos expression was significant in ducts if compared to pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (p=0,0002, 0,0048 e 0,0352, respectively). Spearman correlation test to Jab1, p27, c-jun and c-fos in each lesion separately revealed significant correlation between Jab1 and p27 (r=0,371; p=0,020) and c-jun and c-fos (r=0,452; p=0,004) in pleomorphic adenoma. There was correlation between Jab1 and p27 (r=0,494; p=0,044) in polymorphous low-grade adenocarcinoma and between p27 and c-fos (r=0,513; p=0,035) in adenoid cystic carcinoma. In conclusion, tumorigenesis in pleomorphic adenoma, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma seems to be associated to expression of Jab1 and p27.

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