Microscale Gene Expression Analysis of Tumor-Associated Macrophages

Macrophages, apart from being the key effector cells of the innate immune system, also play critical roles during the development and progression of various complex diseases, including cancer. Tumor-associated macrophages, infiltrate tumors during different stages of cancer progression to regulate motility, invasion, and intravasation to metastatic sites. Macrophages can exist in different polarization states associated with unique function in tumors. Since tumor-associated macrophages constitute a very small proportion of tumor cells, analysis of gene expression pattern using normal extraction buffer-based methods remains a challenging task. Therefore, it is imperative to develop low-throughput strategies to investigate transcriptional regulations from a small number of immune cells. Here, we describe an efficient, sensitive, and cost-effective approach for gene expression analysis of a small number of fluorescence-activated sorted tumor-associated macrophages. Our analyses from the different number of stable, primary, and sorted macrophages suggest 5,000 cells is an optimal number for performing quantitative, real-time PCR analysis of multiple genes. Our studies could detect expression of macrophage-specific genes from cultured primary macrophages, and FACS-sorted macrophages from different biological tissues without introducing biases in comparative gene expression ratios. In conclusion, our kit-based method for quantitative gene expression analysis from a small number of cells found in biological tissues will provide an opportunity to study cell-specific, transcriptional changes

RNA Interference Technology — Applications and Limitations

RNA interference (RNAi), an evolutionarily conserved mechanism triggered by double-stranded RNA (dsRNA), causes gene silencing in a sequence-specific manner. RNAi evolved naturally to mediate protection from both endogenous and exogenous pathogenic nucleic acids and to modulate gene expression. Multiple technological advancements and precision in gene targeting have allowed a plethora of potential applications, ranging from targeting infections in crop plants to improving health in human patients, which have been reviewed in this chapter

Structure and Function of Human DnaJ Homologue Subfamily A Member 1 (DNAJA1) and Its Relationship to Pancreatic Cancer

Pancreatic cancer has a dismal 5 year survival rate of 5.5% that has not been improved over the past 25 years despite an enormous amount of effort. Thus, there is an urgent need to identify truly novel yet druggable protein targets for drug discovery. The human protein DnaJ homologue subfamily A member 1 (DNAJA1) was previously shown to be downregulated 5- fold in pancreatic cancer cells and has been targeted as a biomarker for pancreatic cancer, but little is known about the specific biological function for DNAJA1 or the other members of the DnaJ family encoded in the human genome. Our results suggest the overexpression of DNAJA1 suppresses the stress response capabilities of the oncogenic transcription factor, c-Jun, and results in the diminution of cell survival. DNAJA1 likely activates a DnaK protein by forming a complex that suppresses the JNK pathway, the hyperphosphorylation of c-Jun, and the anti-apoptosis state found in pancreatic cancer cells. A high-quality nuclear magnetic resonance solution structure of the J-domain of DNAJA1 combined with a bioinformatics analysis and a ligand affinity screen identifies a potential DnaK binding site, which is also predicted to overlap with an inhibitory binding site, suggesting DNAJA1 activity is highly regulated

Inhibition of Geranylgeranyl Diphosphate Synthase is a Novel Therapeutic Strategy for Pancreatic Ductal Adenocarcinoma

Rab proteins play an essential role in regulating intracellular membrane trafficking processes. Rab activity is dependent upon geranylgeranylation, a post-translational modification that involves the addition of 20-carbon isoprenoid chains via the enzyme geranylgeranyl transferase (GGTase) II. We have focused on the development of inhibitors against geranylgeranyl diphosphate synthase (GGDPS), which generates the isoprenoid donor (GGPP), as anti-Rab agents. Pancreatic ductal adenocarcinoma (PDAC) is characterized by abnormal mucin production and these mucins play important roles in tumor development, metastasis and chemo-resistance. We hypothesized that GGDPS inhibitor (GGDPSi) treatment would induce PDAC cell death by disrupting mucin trafficking, thereby inducing the unfolded protein response pathway (UPR) and apoptosis. To this end, we evaluated the effects of RAM2061, a potent GGDPSi, against PDAC. Our studies revealed that GGDPSi treatment activates the UPR and triggers apoptosis in a variety of human and mouse PDAC cell lines. Furthermore, GGDPSi treatment was found to disrupt the intracellular trafficking of key mucins such as MUC1. These effects could be recapitulated by incubation with a specific GGTase II inhibitor, but not a GGTase I inhibitor, consistent with the effect being dependent on disruption of Rab-mediated activities. In addition, siRNA-mediated knockdown of GGDPS induces upregulation of UPR markers and disrupts MUC1 trafficking in PDAC cells. Experiments in two mouse models of PDAC demonstrated that GGDPSi treatment significantly slows tumor growth. Collectively, these data support further development of GGDPSi therapy as a novel strategy for the treatment of PDAC

Silibinin-mediated metabolic reprogramming attenuates pancreatic cancer-induced cachexia and tumor growth.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the US. Cancer-associated cachexia is present in up to 80% of PDAC patients and is associated with aggressive disease and poor prognosis. In the present studies we evaluated an anti-cancer natural product silibinin for its effectiveness in targeting pancreatic cancer aggressiveness and the cachectic properties of pancreatic cancer cells and tumors. Our results demonstrate that silibinin inhibits pancreatic cancer cell growth in a dose-dependent manner and reduces glycolytic activity of cancer cells. Our LC-MS/MS based metabolomics data demonstrates that silibinin treatment induces global metabolic reprogramming in pancreatic cancer cells. Silibinin treatment diminishes c-MYC expression, a key regulator of cancer metabolism. Furthermore, we observed reduced STAT3 signaling in silibinin-treated cancer cells. Overexpression of constitutively active STAT3 was sufficient to substantially revert the silibinin-induced downregulation of c-MYC and the metabolic phenotype. Our in vivo investigations demonstrate that silibinin reduces tumor growth and proliferation in an orthotopic mouse model of pancreatic cancer and prevents the loss of body weight and muscle. It also improves physical activity including grip strength and latency to fall in tumor-bearing mice. In conclusion, silibinin-induced metabolic reprogramming diminishes cell growth and cachectic properties of pancreatic cancer cells and animal models

Structure and Function of Human DnaJ Homologue Subfamily A Member 1 (DNAJA1) and Its Relationship to Pancreatic Cancer

Pancreatic cancer has a dismal 5 year survival rate of 5.5% that has not been improved over the past 25 years despite an enormous amount of effort. Thus, there is an urgent need to identify truly novel yet druggable protein targets for drug discovery. The human protein DnaJ homologue subfamily A member 1 (DNAJA1) was previously shown to be downregulated 5- fold in pancreatic cancer cells and has been targeted as a biomarker for pancreatic cancer, but little is known about the specific biological function for DNAJA1 or the other members of the DnaJ family encoded in the human genome. Our results suggest the overexpression of DNAJA1 suppresses the stress response capabilities of the oncogenic transcription factor, c-Jun, and results in the diminution of cell survival. DNAJA1 likely activates a DnaK protein by forming a complex that suppresses the JNK pathway, the hyperphosphorylation of c-Jun, and the anti-apoptosis state found in pancreatic cancer cells. A high-quality nuclear magnetic resonance solution structure of the J-domain of DNAJA1 combined with a bioinformatics analysis and a ligand affinity screen identifies a potential DnaK binding site, which is also predicted to overlap with an inhibitory binding site, suggesting DNAJA1 activity is highly regulated

MUC1 mucin stabilizes and activates hypoxia-inducible factor 1 alpha to regulate metabolism in pancreatic cancer

Aberrant glucose metabolism is one of the hallmarks of cancer that facilitates cancer cell survival and proliferation. Here, we demonstrate that MUC1, a large, type I transmembrane protein that is overexpressed in several carcinomas including pancreatic adenocarcinoma, modulates cancer cell metabolism to facilitate growth properties of cancer cells. MUC1 occupies the promoter elements of multiple genes directly involved in glucose metabolism and regulates their expression. Furthermore, MUC1 expression enhances glycolytic activity in pancreatic cancer cells. We also demonstrate that MUC1 expression enhances in vivo glucose uptake and expression of genes involved in glucose uptake and metabolism in orthotopic implantation models of pancreatic cancer. The MUC1 cytoplasmic tail is known to activate multiple signaling pathways through its interactions with several transcription factors/coregulators at the promoter elements of various genes. Our results indicate that MUC1 acts as a modulator of the hypoxic response in pancreatic cancer cells by regulating the expression/stability and activity of hypoxia-inducible factor-1α (HIF-1α). MUC1 physically interacts with HIF-1α and p300 and stabilizes the former at the protein level. By using a ChIP assay, we demonstrate that MUC1 facilitates recruitment of HIF-1α and p300 on glycolytic gene promoters in a hypoxia-dependent manner. Also, by metabolomic studies, we demonstrate that MUC1 regulates multiple metabolite intermediates in the glucose and amino acid metabolic pathways. Thus, our studies indicate that MUC1 acts as a master regulator of the metabolic program and facilitates metabolic alterations in the hypoxic environments that help tumor cells survive and proliferate under such conditions

Na mira do sucesso : estratégias de combate ao insucesso escolar de alunos estrangeiros

Trabalho de projeto de mestrado, Ciências da Educação (Área de especialização Formação de Adultos), Universidade de Lisboa, Instituto de Educação, 2017Considerando a importância da integração no contexto escolar e consequentemente no sucesso educativo, o presente trabalho projeto, desenvolvido no âmbito do Mestrado em Ciências da Educação, área de especialidade em Formação de Adultos, centra-se na integração de alunos estrangeiros no ensino português, assim como na dinamização de atividades de promoção do seu sucesso escolar. Deste modo, o relatório apresenta como principais temáticas a integração de estrangeiros em Portugal, o insucesso escolar dos alunos estrangeiros e as políticas educativas de combate a este fenómeno, culminando na definição e desenvolvimento de um projeto de combate ao insucesso escolar destinado a este público. O trabalho desenvolvido junto destes jovens nos últimos nove anos e o diagnóstico elaborado no Agrupamento de Escolas João de Barros permitiu compreender a importância de intervir junto da população estrangeira, fomentando o sucesso educativo e a integração no país de acolhimento, uma vez que a taxa de retenção destes jovens está muito acima dos seus colegas autóctones.Considering the importance of integration in the school context, and consequently in educational success, this project, developed in the scope of the master’s degree in Educational Sciences, specialization in Adult Education, focuses on foreign students’ integration at the Portuguese educational system and activities that could be developed to promote their school success. Therefore, the main themes of the report are the integration of foreigners in Portugal, the lack of academic experience from foreign students and educational policies to reduce this barrier, in order to define and develop a project to combat school failure among this cases. The work developed for the last nine years with this students, the diagnostic elaborated in João de Barros Group of Schools, allowed me to understand the importance of an intervention among foreign students, to foment educational success and integration in the host country, bearing in mind that the retention rate in those cases is far above the native students’ average

MUC1 mucin stabilizes and activates hypoxia-inducible factor 1 alpha to regulate metabolism in pancreatic cancer

Aberrant glucose metabolism is one of the hallmarks of cancer that facilitates cancer cell survival and proliferation. Here, we demonstrate that MUC1, a large, type I transmembrane protein that is overexpressed in several carcinomas including pancreatic adenocarcinoma, modulates cancer cell metabolism to facilitate growth properties of cancer cells. MUC1 occupies the promoter elements of multiple genes directly involved in glucose metabolism and regulates their expression. Furthermore, MUC1 expression enhances glycolytic activity in pancreatic cancer cells. We also demonstrate that MUC1 expression enhances in vivo glucose uptake and expression of genes involved in glucose uptake and metabolism in orthotopic implantation models of pancreatic cancer. The MUC1 cytoplasmic tail is known to activate multiple signaling pathways through its interactions with several transcription factors/coregulators at the promoter elements of various genes. Our results indicate that MUC1 acts as a modulator of the hypoxic response in pancreatic cancer cells by regulating the expression/stability and activity of hypoxia-inducible factor-1α (HIF-1α). MUC1 physically interacts with HIF-1α and p300 and stabilizes the former at the protein level. By using a ChIP assay, we demonstrate that MUC1 facilitates recruitment of HIF-1α and p300 on glycolytic gene promoters in a hypoxia-dependent manner. Also, by metabolomic studies, we demonstrate that MUC1 regulates multiple metabolite intermediates in the glucose and amino acid metabolic pathways. Thus, our studies indicate that MUC1 acts as a master regulator of the metabolic program and facilitates metabolic alterations in the hypoxic environments that help tumor cells survive and proliferate under such conditions

Metabolic Rewiring by Loss of Sirt5 Promotes Kras-induced Pancreatic Cancer Progression.

BACKGROUND & AIMS: SIRT5 plays pleiotropic roles via post-translational modifications, serving as a tumor suppressor, or an oncogene, in different tumors. However, the role SIRT5 plays in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) remains unknown. METHODS: Published datasets and tissue arrays with SIRT5 staining were used to investigate the clinical relevance of SIRT5 in PDAC. Furthermore, to define the role of SIRT5 in the carcinogenesis of PDAC, we generated autochthonous mouse models with conditional Sirt5 knockout. Moreover, to examine the mechanistic role of SIRT5 in PDAC carcinogenesis, SIRT5 was knocked down in PDAC cell lines and organoids, followed by metabolomics and proteomics studies. A novel SIRT5 activator was utilized for therapeutic studies in organoids and patient-derived xenografts. RESULTS: SIRT5 expression negatively regulated tumor cell proliferation and correlated with a favorable prognosis in PDAC patients. Genetic ablation of Sirt5 in PDAC mouse models promoted acinar-to-ductal metaplasia, precursor lesions, and pancreatic tumorigenesis, resulting in poor survival. Mechanistically, SIRT5 loss enhanced glutamine and glutathione metabolism via acetylation-mediated activation of GOT1. A selective SIRT5 activator, MC3138, phenocopied the effects of SIRT5 overexpression and exhibited anti-tumor effects on human PDAC cells. MC3138 also diminished nucleotide pools, sensitizing human PDAC cell lines, organoids, and patient-derived xenografts to gemcitabine. CONCLUSIONS: Collectively, we identify SIRT5 as a key tumor suppressor in PDAC, whose loss promotes tumorigenesis through increased non-canonical utilization of glutamine via GOT1, and that SIRT5 activation is a novel therapeutic strategy to target PDAC