Orientador: Debora Aparecida Pires de Campos Zuccari / Banca: Ana Elizabete Silva / Banca: Dorotéia Rossi Silva Souza / Banca: Samuel Cos Corral / Banca: Bruno Cogliati / Resumo: O câncer de mama apresenta altas taxas de incidência e mortalidade, sendo a neoplasia mais comum entre as mulheres. O rápido crescimento do tumor resulta em hipóxia no microambiente tumoral, levando a uma cascata de eventos que induzem a angiogênese e conseqüente progressão do câncer. Assim, a identificação de agentes terapêuticos que possam inibir a angiogênese é essencial para o controle da progressão tumoral. Nesse sentido, tem sido demonstrado que a administração exógena da melatonina, um hormônio naturalmente secretado pela glândula pineal, apresenta diversos efeitos oncostáticos em diferentes tipos de câncer. Assim, o objetivo desse estudo foi avaliar a efetividade do tratamento com melatonina sobre a angiogênese do câncer de mama, em estudos in vitro e in vivo. No estudo in vitro, as linhagens de câncer de mama MCF-7 e MDA-MB-231 foram tratadas com melatonina sob condições de hipóxia mimetizadas pelo Cloreto de Cobalto (CoCl2). A viabilidade celular foi verificada pelo ensaio MTT, a expressão do fator de transcrição induzido por hipóxia (HIF-1α) e do fator de crescimento endotelial vascular (VEGF-A) foi avaliada por PCR em tempo real e imunocitoquímica. Além disso, demais proteínas envolvidas na angiogênese foram avaliadas por Protein Array. No estudo in vivo, as células MDA-MB-231 foram implantadas em camundongos nude atímicos, os quais foram tratados com melatonina (40 mg/kg) por 21 dias. O tumor foi medido semanalmente e a avaliação da angiogênese foi realizada pela técnica de tomografia computadorizada por emissão de fóton único (SPECT), com o radiotraçador Tc- 99m-HYNIC-VEGF-c, agente específico para os receptores de VEGF (VEGFR2/VEGF3). Além disso, as proteínas VEGFR2, VEGFR3, fator de fator de von Willebrand (vWF) e marcador de proliferação celular (Ki-67) foram avaliados no tecido tumoral por imuno-histoquímica, e demais proteínas angiogênicas por Protein Array. O estudo in ... / Abstract: Breast cancer has high rates of incidence and mortality, and it is the most common cancer among women. The rapid tumor growth results in hypoxia on tumor microenvironment, leading to a cascade of events that induce angiogenesis and subsequent cancer progression. Thus, the identification of therapeutic agents that can inhibit angiogenesis is essential for the control of tumor progression. Exogenous administration of melatonin, a hormone secreted by the pineal gland, has been shown several oncostatics effects on different types of cancers. The aim of this study was to evaluate the effectiveness of melatonin treatment on angiogenesis in breast cancer, in the in vitro and in vivo studies. In the in vitro study, breast cancer cell lines (MCF-7 and MDA-MB-231) were treated with melatonin under cobalt chloride (CoCl2)-induced hypoxic conditions. Cell viability was measured by MTT assay, the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF-A) was assessed by real-time PCR and immunocytochemistry. Additionally, other proteins involved in angiogenesis were evaluated by the Protein Array. In the in vivo study, the MDA-MB-231 cells were implanted in athymic nude mice, which were treated with melatonin (40 mg/kg) for 21 days. The tumor was measured weekly and evaluation of angiogenesis was performed by single-photon emission computed tomography (SPECT) with Tc-99m-HYNIC-VEGF-c, which is specific for VEGF receptors (VEGFR2/VEGF3). Moreover, VEGFR2, VEGFR3, von Willebrand factor (vWF) and cell proliferation marker (Ki-67) were evaluated in tumor tissue by immunohistochemistry, and other angiogenic proteins by Protein Array. Results from the in vitro study showed that 1 mM of melatonin under hypoxic conditions (200 μM CoCl2) led to decreased cell viability, protein levels of HIF- 1α and gene and protein expression of VEGF-A in both cell lines (p < 0.05). Among other proteins evaluated, melatonin ... / Doutor

DNA double strand breaks (DSB) are cytotoxic lesions that can lead to genome rearrangements and genomic instability, which are hallmarks of cancer. The two main DSB repair pathways are non-homologous end joining and homologous recombination (HR). While HR is generally highly accurate, it has the potential for gross chromosomal rearrangements (GCRs) that occur directly or through intermediates generated during the repair process. Whole genome sequencing of cancers has revealed numerous types of structural rearrangement signatures that are often indicative of repair mediated by sequence homology. However, it can be challenging to delineate repair mechanisms from sequence analysis of rearrangement end products from cancer genomes, or even model systems, because the same rearrangements can be generated by different pathways. Numerous studies have provided insights into the types of spontaneous GCRs that can occur in various Saccharomyces cerevisiae mutants. However, understanding the mechanism and frequency of formation of these GCR without knowledge of the initiating lesions is limited. Here, we focus on DSB-induced repair pathways that lead to GCRs. Inverted duplications occur at a surprisingly high frequency when a DSB is formed near short inverted repeats in cells deficient for the nuclease activity of Mre11. Similar to previously proposed models, the inverted duplications occur through intra-strand foldback annealing at resected inverted repeats to form a hairpin-capped chromosome that is a precursor to dicentric chromosomes. Surprisingly, we found that DNA polymerase δ proof-reading activity but not the Rad1-Rad10 nuclease is required for inverted duplication formation, suggesting a role for Pol δ in the removal of the heterologous tails formed during foldback annealing. Contrary to previous work on spontaneous inverted duplications, we find that DSB-induced inverted duplications require the Pol δ processivity subunit Pol32 and that RPA plays little role in their inhibition, suggesting that spontaneous inverted duplications arise differently than those induced by DSBs. We show that stabilization of dicentric chromosomes after breakage involves telomere capture through a strand-invasion step mediated by repeat sequences and requires Rad51. Previous work on spontaneous inverted duplications suggested that Tel1, but not Mre11-Sae2, inhibits inverted duplications that initiate from inverted repeats separated by long spacers (> 12 bp). However, we do not find evidence for this requirement. Cells with Tel1 deletion can still resolve hairpins containing loops up to 30 nt long. Furthermore, deletion of Sae2, but not Tel1, increases the frequency of inverted duplications when a DSB is induced near an inverted repeat separated by a 20 bp-long spacer. This highlights another difference between spontaneous and DSB-induced GCRs. Finally, we find that the sequence context of a DSB affects the type of GCR outcome. Inverted repeats are required for the formation of inverted duplications, as the deletion of a DSB-proximal inverted repeat significantly reduces the incidence of this type of rearrangement. Furthermore, introduction of a DSB near short telomere-like sequence is required for chromosome truncations stabilized by de novo telomere addition. The effect of the sequence context can partly explain how repair pathways can be channeled to different mutagenic outcomes. Our results highlight the importance of considering how the initiating lesion can affect the type of resulting GCRs and the mechanisms by which they occur.

Thesis (Ph.D. (Biochemistry)) -- University of Limpopo, 2021 / Breast cancer is the leading cause of female deaths in the world. Varying types of therapy options are available, yet these conventional treatments for the malignancy are known to have numerous side effects. Similar to other diseases, herbal remedies are being explored as alternative treatment options as well as starting points for development of new drugs to treat breast cancer. Bidens pilosa is a weed distributed throughout the world with known medicinal properties. Its anti-cancer activity has been established in various cancers. This study aimed to investigate the epigenetic patterns affected by a bioactive extract of B. pilosa in breast cancer. A crude methanol extract of B. pilosa was fractionated with n-hexane, chloroform, ethyl acetate, n-butanol, 65% methanol and water. Healing properties of plants are often an attribute of the presence of phenolic compounds within the plant and the sub-fractions of the methanol extract of B. pilosa were, therefore, assayed for these compounds. The water sub-fraction showed the highest content of total phenolic compounds, however, when the sub-fractions were analysed for the presence of two classes of specific phenolic compounds, the butanol sub-fraction boasted the highest concentration of flavonoids and tannins, affording it superior antioxidant activity in a quantitative DPPH assay. Distribution of the antioxidant compounds in TLC-DPPH analysis also supported this finding. Despite its high antioxidant compound content, cytotoxicity of the butanol sub-fraction in MCF-7 breast cancer cells was not impressive in the MTT viability assay. Treatment with varying concentrations of the chloroform sub-fraction resulted in a better dose- and time-dependent decrease in cell viability of MCF-7 cells than all the other sub-fractions as well as the crude methanol extract. Analysis of breast cancer genes affected by the chloroform sub-fraction on the Human Breast Cancer RT2 Profiler PCR array showed repression in BRCA1 and BRCA2, genes classified as tumour suppressors. Bisulfite pyrosequencing showed no significant modification in methylation of selected CpG islands within the promoter regions of both genes. Results of the array also showed decreased expression of CDH1 which is associated with invasiveness and aggression of tumours. Its investigated CpG island was also shown not to be differentially methylated by treatment of the cells with the chloroform sub-fraction of the extract. As a well-appreciated biomarker for breast cancer risk, BRCA1 protein expression was further investigated. Western blot analysis showed parallel findings to those of the PCR array, with down-regulation of BRCA1 within 24 hours of treatment of MCF-7 cells with the sub-fraction. Repression of the BRCA genes is strongly linked to arrest of cells at the G2/M phase of the cell division cycle, and this was therefore also assessed. Treatment of MCF-7 cells with the chloroform sub-fraction effected a dose-dependent accumulation of cells at the G2/M phase of the cell cycle as determined by flow cytometry. Results of global DNA methylation analysis showed an increase in chromosomal instability by a significantly reduced level of methylation of the genome. This hypomethylation also supports arrest of the cells at the G2/M phase of the cell cycle, as cells accumulate at this checkpoint, awaiting repair to prevent segregation of broken chromosomes during mitosis. However, the lack of BRCA1 suggests that repair proteins were not recruited to the sites of repair and the cells were consequently directed to apoptosis. Analysis of the effect of the chloroform sub-fraction of the methanol extract of B. pilosa in the Mitopotential assay showed an increase in the number of dead cells with depolarised mitochondrial membranes, alluding to the intrinsic mode of apoptotic cell death in MCF-7 cells treated with the sub-fraction. Down-regulation of BRCA1 is further associated with telomerase inactivation in cancer cells. Treatment of MCF-7 cells with the chloroform sub-fraction reduced telomerase activity within 24 hours of treatment, with an absence of activity following treatment with 100 and 125 μg/ml of the sub-fraction. This lack of telomerase activity resulted in shortened telomeres which limit proliferative ability of the cells. Characterisation of the six sub-fractions of the methanol extract of B. pilosa with GC-MS showed an abundance of fatty acids in the chloroform sub-fraction, specifically α-linolenic acid, palmitic acid and linoleic acid. Palmitic acid is alleged to play a role in down-regulation of BRCA1 and its abundance in this sub-fraction leads to the conclusion that palmitic acid may be responsible for the decreased expression of BRCA1 in MCF-7 breast cancer cells. The down-regulation results in hypomethylation of the genome leading to cell cycle arrest at the G2/M checkpoint and subsequent apoptosis as a result of this repression of BRCA1. Repression of BRCA1 also leads to inactivation of telomerase, inhibiting cell proliferation. Taken together, the observed antioxidant activity and pro-apoptotic potential attributed to epigenetic modifications validate B. pilosa as an anticancer agent. Our findings merit the plant for use in development of potential breast cancer drugs. / SAMRC and University of Limpopo (UL)

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths worldwide with a five-year survival rate of only 9%. The mevalonate (MVA) pathway, which results in synthesis of cholesterol, has been demonstrated to be overexpressed in numerous cancers, leading to its involvement in various aspects of the disease, including proliferation, invasion, and metastasis. In recent years, the MVA pathway has been implicated in PDAC as well, however the underlying mechanisms behind its upregulation in this context, as well as its role in tumor progression, remain largely unknown. An initial analysis of The Cancer Genome Atlas (TCGA) datasets using the Gene Expression Profiling Interactive Analysis (GEPIA) tool revealed one mevalonic gene in particular, SQLE—encoding squalene epoxidase (SQLE)—to be significantly overexpressed in PDAC compared to other genes within the pathway. We find that patients with higher SQLE expression profiles have significantly reduced five-year survival rates that can be further correlated to tumor stage and grade, and we demonstrate that knockdown of SQLE, a branchpoint enzyme of the cholesterol pathway, reduces tumor proliferation in both two-dimensional and three-dimensional assays. We further characterize the SQLE promoter landscape by demonstrating an interesting dynamic of gene activation between the sterol regulatory element binding protein-2 (SREBP2), BRG1-specific SWI/SNF chromatin remodelers, and mutant p53. These results highlight SQLE as a therapeutic target in PDAC and provide a better understanding of its regulation in the context of this devastating disease.

Metastasis accounts for more than 90% of cancer-related mortality, and thus, there is a compelling need for innovative therapeutic breakthroughs. TP53 mutations are detected in up to 80% of esophageal squamous cell carcinomas (ESCCs), the major subtype of esophageal cancer and one of the most lethal cancers worldwide, as well as in other SCCs. These mutations in turn correlate with poor patient prognosis and high metastatic rates. To elucidate novel mutant p53-dependent mechanisms in promoting ESCC metastasis, we generated a mouse model combining genetic and carcinogenic approaches: We treated the L2-Cre (esophageal specific promoter); LSL-Trp53R172H/-, Trp53-/- or Trp53+/+; Rosa26LSL-YFP mice with a carcinogen 4-NQO, and isolated primary and metastatic tumor cells that vary in their p53 statuses. We have shown that ESCC cells with Trp53R172H exhibit greater metastatic capabilities compared to the tumor cells harboring Trp53-/-, indicating gain-of-function (GOF) activity. Through comprehensive RNA-seq and cytokine array analyses, we identified that Colony-stimulating factor-1 (Csf-1) is significantly upregulated in a p53-R172H-dependent manner in metastatic lung lesions of ESCC. p53-R172H binds to the promoter region of Csf-1 locus in metastatic ESCC cells. Our findings demonstrate that p53-R172H-dependent Csf-1 signaling through its cognate receptor Csf-1r enhances tumor cell invasion and lung metastasis by utilizing complementary genetic and pharmacological approaches. This mechanism is mediated in part through Stat3 phosphorylation and epithelial-to-mesenchymal transition (EMT). These findings are further supported through in vivo targeting of Csf-1r. In addition, high levels of CSF-1 also correlate with mutant p53 in ESCC Tissue Microarrays (TMAs) and The Cancer Genome Atlas (TCGA) datasets. Our CUT&RUN-seq analysis on ESCC tumor cells revealed that both the Csf-1 locus and EMT-associated genes are enriched with histone 3 lysine 27 acetylation (H3K27ac). This enrichment creates a permissive environment for the interaction between Brd4 and p53-R172H, thereby regulating Csf-1 transcription. Notably, Brd4 interacts specifically with p53-R172H. Inhibiting Brd4 not only decreases tumor invasion and lung metastasis, but also reduces circulating Csf-1 levels in blood serum in vivo. Overall, our results establish a novel p53-R172H-dependent Brd4-Csf-1 signaling axis that facilitates lung metastasis in ESCC and underscores the GOF properties of p53-R172H. Our discoveries identify therapeutic vulnerabilities in metastatic ESCC, which can be applicable to other SCCs with similar transcriptomic and epigenetic profiles. These insights pave the way for developing therapeutic strategies for this difficult-to-treat disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Telomere maintenance is a critical component of genomic stability. An increasing body of evidence suggests BRCA1, a tumor suppressor gene with a variety of functions including DNA repair and cell cycle regulation, plays a role in telomere maintenance. Mutations in BRCA1 account for approximately half of all hereditary breast and ovarian cancers, and the gene is silenced via promoter methylation and loss of heterozygosity in a proportion of sporadic breast and ovarian cancers. The objective of this study was to determine whether GRN163L, a telomerase inhibitor, currently in clinical trials for the treatment of cancer, has enhanced anti-cancer activity in BRCA1 mutant breast/ovarian cancer cell lines compared to wild-type cancer cells. BRCA1 mutant cancer cells were observed to have shorter telomeres and increased sensitivity to telomerase inhibition, compared to cell lines with wild-type BRCA1. Importantly, GRN163L treatment was synergistic with DNA-damaging drugs, suggesting potential synthetic lethality of the BRCA1 cancer subtype and telomerase inhibition In a related study to examine the roles of BRCA1/2 in telomere maintenance, DNA and RNA extracted from peripheral blood were used to investigate the age-adjusted telomere lengths and telomere-related gene expression profiles of BRCA1 and BRCA2 individuals compared to individuals who developed sporadic cancer and healthy controls. BRCA1 mutation carriers and breast cancer patients showed the shortest average telomere lengths compared to the other groups. In addition, distinct genomic profiles of BRCA mutation carriers were obtained regarding overexpression of telomere-related genes compared to individuals who developed sporadic or familial breast cancer. In summary, telomerase inhibition may be a viable treatment option in BRCA1 mutant breast or ovarian cancers. These data also provides insights into further investigations on the role of BRCA1 in the biology underlying telomere dysfunction in cancer development.

Exosomes are small secreted vesicles of endocytic origin with a size range of 50-150 nm. They are secreted by many cell types and display multiple biological functions including immune-activation, immune-suppression, antigen presentation, and the shuttling of mRNA and miRNA, as well as other cargo. We have characterised the exosomes secreted from two breast cancer cell lines, MDA-MB-231 and MCF7. Exosomes secreted from both cell lines display typical markers including ALIX, Tsg101, CD9 and CD63, and were capable of inducing apoptosis of the Jurkat T cell line, indicating the potential immune-suppressive function of such tumour-derived exosomes. To further investigate the biological potential of exosomes, we loaded purified exosomes with gene specific siRNAs using electroporation, and observed the targeted inhibition of both a known component of the exosome pathway, Rab27a, and also the arthritis associated gene ERAP1, demonstrating the potential novel use of exosomes as therapeutic gene delivery vectors. We have also shown that exosomes derived from MDA-MB-231 cells and the parental cells have different lipid composition, as analysed by lipidomics study. Nanoparticle tracking analysis (NTA), which allows the rapid detection of size and concentration of nanoparticles within the size range 10 nm-1000 nm was tested for its ability to accurately measure size and concentration of exosomes and microvesicles under different conditions. NTA was capable of detecting apoptotic vesicles induced by Taxol and Curcumin treatment. Immunodepletion was used to determine the percentage of CD9 and CD63 positive vesicles. Our data suggest that NTA is a useful technique for measuring size and concentration of exosomes and microvesicles. We hypothesized that NTA could assist in the screening of agents that interfere or promote exosome release. NTA was therefore used to detect increases in exosomes secretion induced by Tamoxifen and Thimerosal treatment, and to monitor the inhibition of exosome secretion from MDA-MB-231 breast cancer cells expressing inhibitory RNA targeted for Rab27a, a component of the exosome pathway. Increases in exosome release induced by Tamoxifen and Thimerosal was detected by NTA and a significant reduction in the release of exosomes by inhibition of Rab27a expression was also observed. Treatment with the known exosomal pathway inhibitor DMA also reduced exosome release, establishing the principle of NTA as a screening technique. We further compared the siRNA targeted cells for their ability to migrate, invade and form anchorage-independent colonies, which were all significantly reduced. Supplementation with MDA-MB-231 derived exosomes restored the ability to form colonies, suggesting exosomes may contribute to metastatic lesion formation. These data suggest that the exosomal pathway is a valid target to disrupt the behaviour of tumour cells and NTA can be used to monitor its activity.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Mining the human genome for therapeutic target(s) discovery promises novel outcome. Over half of the proteins in the human genome however, remain uncharacterized. These proteins offer a potential for new target(s) discovery for diverse diseases. Additional targets for cancer diagnosis and therapy are urgently needed to help move away from the cytotoxic era to a targeted therapy approach. Bioinformatics and proteomics approaches can be used to characterize novel sequences in the genome database to infer putative function. The hypothesis that the amino acid motifs and proteins domains of the uncharacterized proteins can be used as a starting point to predict putative function of these proteins provided the framework for the research discussed in this dissertation. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

This thesis is the construct of a computational system for studying the nasopharyngeal carcinoma (NPC) using high-throughput sequencing data. The system involves several components, including discovery of gene fusion in NPC cell line, construction of Esptein-Barr virus (EBV) genome, and evaluation on contaminated sequencing data alignment approaches. We successfully discovered a gene fusion (UBR5-ZNF423) in a NPC cell line (C666-1) which was verified by lab experiments and found in 8.3% of primary tumors. It was discovered the regulation of this gene affect the growth of cancer cell. We constructed the EBV genome in C666-1. It serves as an important reference for studying this important NPC cell line, which was the only NPC cell line in the world for a long time. We also evaluated three mapping approaches. Two of them are designed to filter out potential mouse contamination reads on human sequencing data, which can originate from NPC human-in-mouse xenografts. We found that special care should always be applied to contaminated data. Although direct mapping can give acceptable results if in most cases, the combined-based approached is suggested. It can effectively reduce false positive variants and maintain good enough numbers of true positive variants. Filtering approach is an alternative to the combined-based approach that can also effectively reduce contamination when memory is not sufficient. / 本論文利用電腦有系統地研究鼻咽癌，當中的數據利用了高通量測序技術來定序。其中章節包括在鼻咽癌胞系中尋找融合基因、組建潛藏於人體可引致鼻咽癌的EB病毒基因組、還有評價幾種可處理受污染序列的序列排列方法。我們成功地在鼻咽癌胞系（C666-1）中發現出一個融合基因（UBR5-ZNF423），並在實驗中確定此成果，其中發現在原發腫瘤中有8.3%的樣本中找出此融合基因。此外，也發現這融合基因調控會影響到癌細胞的生長。C666-1鼻咽癌胞系在過往有一段很長的時間裡，都是全世界唯一的鼻咽癌胞系，因此它有非常重要的參考價值，在此研究，我們組建了在C666-1裡的EB病毒基因組，使它作為研究C666-1的參考樣本。另外，我們評價了三種處理排列的方法，其中兩種的設計能過濾部分人類序列數據當中老鼠基因組的污染，老鼠基因組的污染可以來自於異種移植，即把人類癌細腫瘤移植於老鼠身上種植，我們建議在情況許可下都使用特殊的處理方法而不是直接作序列排列。直接作序列排列數據雖然已有合理的表現，但相比之下組合基因組式序列排列方法能有效減少錯誤肯定的遺傳變異，並同時保留足夠多正確肯定的遺傳變異，所以組合基因組式序列排列方法應在情況許可下都使用它。過濾式序列排列方法也是一種特殊的處理方法，它也能有效減少錯誤肯定的遺傳變異，它對記憶體的需求比組合基因組式序列排列方法少，可在電腦的記憶體不足時使用它。 / Tso, Kai Yuen. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 112-120). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Detailed summary in vernacular field only.