Le cancer du rein est un problème majeur de santé publique. Un diagnostic précoce améliore les chances de survie. Le diagnostic repose essentiellement sur les examens d’imagerie comme l’échographie, la tomodensitométrie et l’IRM. Ces examens sont parfois associés à la biopsie et sont couteux et parfois invasifs. De plus, l’imagerie n’est pas capable de faire la distinction entre les tumeurs bénignes et les tumeurs malignes et entre les sous-types histologiques de carcinome à cellules claires qui est le plus fréquent. Il n’existe pas dans le cancer rénal de marqueur comme la PSA dans le cancer de la prostate ou la Foetoprotéine et l’HCG dans le cancer du testicule. Le but de cette étude est concentré sur la recherche des marqueurs mARNs ou miARNs dans les liquides biologiques (sérum, plasma, urine) pour le cancer du rein à cellules claires. Nous avons montré que les petits ARNs dans le sérum et les urines et l’intégrité des ARNs dans les urines étaient des outils diagnostiques dans le cancer du rein à cellules claires. Si ces petits ARNs circulants sont validés, on peut éventuellement imaginer l’intérêt pratique en clinique comme la détection des petits ARNs circulants dans l’urine pour prédire le cancer, la classification de la tumeur pour aider le clinicien, la prédiction d’une récidive ou d’une progression soit après néphrectomie soit au cours d’un traitement médical / Kidney cancer is a major public health problem. Early diagnosis improves the chances of survival. The diagnosis is mainly based on the imaging tests such as ultrasound, CT and MRI. These tests are sometimes associated with biopsy and are expensive and sometimes invasive. In addition, the imaging test is not able to distinguish between benign and malignant tumors and between histological subtypes of renal cell carcinoma. There don’t exist tumor marker in renal cancer such as PSA in prostate cancer or Fetoprotein and HCG in testicular cancer. For this purpose, the study has focused on finding markers mRNAs and miRNAs in biological fluids (serum, plasma, urine) for clear cell renal cell carcinoma. We have shown that the small cell-free RNAs in serum and urine and the integrity of cell-free RNA in urine were diagnostic tools for clear cell renal cell carcinoma. If these circulating small RNAs are validated, we can possibly imagine the interest in clinical practice as the detection of circulating small RNAs in urine to predict cancer, the classification of the tumor to help the clinician, the prediction of recurrence and progression after nephrectomy or during medical treatment

Orientadores: João Palermo Neto, Antonio Armario Garcia / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T19:54:17Z (GMT). No. of bitstreams: 1 Queiroz_JuliadeSouza_D.pdf: 6420413 bytes, checksum: 5de0fbd4bbb6451ff5729cbdc2fc74ce (MD5) Previous issue date: 2012 / Resumo: A exposição do organismo a estressores psicossociais e ambientais altera de forma significativa o funcionamento do sistema imune. Os efeitos do estresse sobre a resposta imune têm sido atribuídos, principalmente, à ativação do eixo hipotálamo-pituitária-adrenal (HPA) com consequente aumento nos níveis de ACTH e glicocorticóides e à ativação do sistema nervoso autônomo simpático (SNAS), com liberação de catecolaminas. Nos últimos anos, a alga Chlorella vulgaris (CV) tem despertado o interesse da comunidade científica pelos seus efeitos moduladores sobre as defesas do hospedeiro imunossuprimido. Em estudos anteriores mostramos que o restabelecimento da geração de granulócitos-macrófagos nos órgãos hematopoéticos e a ativação das funções efetoras de fagócitos e linfócitos são cruciais na expressão da atividade imunomoduladora da alga. No entanto, nada se sabe sobre os efeitos da CV no sistema nervoso central em situações de estresse. Sendo assim, neste trabalho realizamos estudos pioneiros com relação ao efeito do tratamento com a alga sobre: 1) a ativação neuronal (c-fos) no córtex pré-frontal, septum lateral, núcleo da Rafe e lócus coeruleus; 2) a ativação do eixo HPA através da expressão do gen de hnCRF na região parvocelular do núcleo paraventricular do hipotálamo, mpdPVN, liberação de ACTH e de corticosterona e, 3) avaliação indireta da atividade do SNAS através dos níveis de glicose no plasma de animais estressados. Considerando-se a medula óssea ser o sítio de origem das células pluripotenciais das quais se originam as células do sistema hematopoético, e que este sistema é totalmente vulnerável ao controle neuroendócrino, avaliamos os efeitos do tratamento com CV sobre a hematopoese de animais estressados através dos 4) crescimento e diferenciação de precursores para granulócitos e macrófagos (CFU-GM); 5) presença de fatores estimuladores da formação de colônias do soro (colony stimulating activity - CSA); 6) quantificação de populações de células maduras e imaturas e 7) morte celular na população de células tronco. A regulação da produção de células hematopoéticas pelas células do estroma da medula óssea em camundongos estressados foi avaliada pela técnica de cultura líquida de longa duração de células da medula óssea (LTBMC), que consiste em um modelo ex vivo para o estudo das interações entre as células progenitoras hematopoéticas e as células do estroma. Nela avaliamos 8) o CFU-GM, níveis de IL-1? / IL-6 e quantificamos uma população madura e uma imatura. Nossos resultados mostraram que a aplicação do estressor produziu um aumento na expressão de c-fos em todas as áreas cerebrais avaliadas, assim como na expressão do gen de hnCRF na região mpdPVN. Os níveis de ACTH e corticosterona também estavam aumentados após o estresse, assim como os níveis de glicose. Na medula óssea observamos que a aplicação do estressor reduziu o número de CFU-GM, e aumentou os níveis de CSA no plasma. Houve um aumento na morte celular e redução no número de precursores hematopoéticos e de células maduras. Na LTBMC, um prejuízo na atividade funcional do estroma medular foi observado através: da redução do CFU-GM, dos níveis de IL-1? / IL-6 e do número de células imaturas e maduras. O aumento na expressão de c-fos após o estresse foi prevenida pelo tratamento com CV em todas as áreas avaliadas, com exceção da região magnocelular do PVN. O resultado mais acentuado do tratamento com CV foi observado na redução da expressão de c-fos no núcleo da Rafe e do gen para hnCRF no mpdPVN, que se encontrou em níveis semelhantes aos observados no grupo controle após o estresse. Todas as alterações hematopoéticas causadas pelo estresse foram prevenidas pelo tratamento com CV. Tomados em seu conjunto, nossos resultados mostraram que o efeito protetor da hematopoese pode ser devido a uma prevenção na ativação neuronal de áreas cerebrais relacionadas à decodificação do estressor do tipo emocional, reduzindo a amplitude de ativação do eixo HPA e do SNAS / Abstract: The exposition of the organism to psychosocial and environmental stressful stimuli alters the functioning of the immune system in a significant way. The effects of stress on the immune response are mainly attributed to the activation of the hypothalamic-pituitary axis (HPA) with consequent increment on ACTH and glucocorticoids levels and, to the activation of the autonomic nervous system, with the incremented levels of cathecholamines. In the last years, increasing interests about the algae Chlorella vulgaris (CV) has been demonstrated by the scientific community, due to its modulatory effects on the defenses of the immunosuppressed host. In previous studies we demonstrated that the reestablishment of the generation of granulocytes and macrophages in bone marrow and, the activation of effectors functions of phagocytes and lymphocytes, are crucial features about the immunomodulatory activity from the algae. However, nothing is known about the activity of CV in the central nervous system. Thus, pioneer investigation was made in this work about the effect of treatment with the CV on: 1) neuronal activation (c-fos) in pre-frontal cortex, lateral septum, Rafe nucleus and locus coeruleus; 2) activation of the HPA axis by analysis of expression of the gen to hnCRF in the parvocelular region from the paraventricular nucleus of the hypothalamus -mpdPVN and the release of ACTH and corticosterone) and, 3) glucose levels, as an indirect indicator of autonomic nervous system activity. Considering that the bone marrow is the site of origin from pluripotent cells from which all cells from the hematopoietic system are originated, and also that this system is vulnerable to the neuroendocrine control, we evaluated the effects of the treatment with CV on the hematopoiesis of stressed animals through 4) growing and differentiation of precursors to granulocytes and macrophages (CFU-GM); 5) colony stimulating activity from the serum (CSA); 6) quantification of population of mature and immature populations and 7) cell death. The interaction between stromal cells and hematopoietic progenitors in stressed mice was evaluated by the technique of long term bone marrow culture (LTBMC). In the culture we evaluated 8) the CFU-GM, levels of IL-1? / IL-6 and quantification of mature and immature population. The application of the stressor produced an increase in the expression of c-fos in all brain areas evaluated and in the expression of the gen to hnCRF in mpdPVN. Increased levels of ACTH, corticosterone and glucose found in stressed animals corroborate these findings. Reduced numbers of CFU-GM in the bone marrow and increase in plasma CSA, increased cell death in stem cell population (LSK) and decreased numbers of hematopoietic precursors and of mature cells was also observed in stressed group. In LTBMC we observed impairment on the functional activity from medullar stroma, which was observed by reduction of: CFU-GM, IL-1? / IL-6 levels and number of immature and mature cells. Treatment with CV partially prevented increase in c-fos activation caused by stress in the brain except in the magnocelular region from PVN. The more accentuated result from treatment with CV of stressed animals was observed in the expression of c-fos in the Raphe nucleus and in the expression of the gen to hnCRF in mpdPVN, where levels were similar to that observed in control group. All hematopoietic alterations observed after stress were prevented by the treatment with CV. Taken together, our results demonstrate that the protective effect of the treatment with CV on hematopoiesis of stressed animals may be due to a prevention of the neuronal activation in areas related to the decodification of the emotional stressful stimuli, reducing the amplitude of HPA axis and autonomic nervous system activity / Doutorado / Farmacologia / Doutora em Farmacologia

INTRODUÇÃO: Os múltiplos passos da carcinogênese do câncer de cólon envolvem a existência de subpopulações de células tronco tumorais (CSC), responsáveis pela transformação, crescimento e proliferação das células tumorais. As proteínas CD44 e CD166 são marcadores de CSC associados a sinalização celular, adesão, migração, metástase e resposta linfocitária. Alguns fatores podem modular a expressão CSC como a mutação KRAS. OBJETIVO: correlacionar a expressão dos marcadores CD44 e CD166 em carcinoma de cólon metastático e status do oncogene KRAS (selvagem/mutado) com as características clínico-patológicas e desfecho do paciente ao final do seguimento. MATERIAL E MÉTODOS: Foram coletadas 58 amostras de tecido tumoral de pacientes com neoplasia de cólon metastático, tratados com CapeOx no Serviço de Oncologia Clínica do HCFMRPUSP de 2003 a 2012. Foram coletadas informações do prontuário sobre status do gene KRAS, características clínico-patológicas e desfecho clínico, sendo também realizada imunohistoquímica para marcação CD44 e CD166 através da técnica de TMA. Utilizado software SPSS 17 para análise estatística e considerado valor de p<0,050 para significância dos dados. RESULTADOS: A expressão de CD44 e CD166 foi positiva em 41,4% e 43%, respectivamente, e o status KRAS mutado em 48,3%. No subgrupo kAs selvagem e nos idosos (>65 anos), houve associação entre CD44 e CD166, p=0,042 e p=0,001, respectivamente. Pacientes CD166 negativo tiveram 3 vezes mais chances de progressão de doença (p=0,02) do que CD166 positivo. Pacientes Kras mutado e CD166 negativo tiveram 8 vezes risco de progressão (p<0,01). Pacientes CD44 positivo tiveram 4 e 5 vezes mais chances de evoluir com metástases hepática e pulmonar (p<0,01) em relação aos CD44 negativo. Pacientes com a combinação KRAS mutado e CD44 positivo tiveram 7 vezes mais chance de evoluir com metástase pulmonar (p=0,02) em relação a pacientes KRAS selvagem e CD44 negativo. DISCUSSÃO: Na amostra estudada observamos a influência das expressões dos marcadores de CSC e suas combinações com o status de mutação do gene KRAS, de modo que pacientes com CD166 negativo no tumor primário apresentam um desfecho de maior recorrência e o CD44 positivo favorece a evolução para metástases pulmonar e hepática. A mutação do gene KRAS atua modulando a via do EGF influenciando o comportamento biologico do tumor e os desfechos (recidiva e metastases) diretamente relacionados com a expressão dos marcadores de CSC no cancer de colon metastatico. CONCLUSÃO: Este estudo demonstrou interação entre a expressão imuno-histoquímica dos marcadores CSC de cólon (CD166 e CD44) e o status KRAS, podendo carcterizar subgrupos de pacientes com maiores chances de evolução desfavorável e assim propor um modelo de tratamento e seguimento mais individualizado. / BACKGROUND: Colon cancer carcinogenesis has been recently correlated with specific cancer stem cell (CSC) subpopulations which are associated with transformation, growth and spread process of tumor cells. CD44 and CD166 are CSC markers correlated with cell signalization, adhesion, migration, metastasis, and lymphocyte response. Some factors as KRAS mutation could modulate CSC. OBJECTIVE: Analyze CD44 and CD166 expressions in metastatic colon carcinoma and its correlation with KRAS status, clinicopathological features, disease recurrence and patient survival. MATERIAL AND METHODS: Tissues were obtained from 58 patients with confirmed metastatic colon cancer, treated with CapeOx at FMRP-USP from 2003 to 2012. Clinical and outcomes informations and KRAS gene status were obtained from medical records. KRAS status was analyzed with RT-PCR. CD44 and CD166 were analyzed with TMA immunohistochemistry. Statistical analyses were performed using SPSS 17.0. A p-value <0,050 was considered to be statistically significant. RESULTS: CD44 and CD166 expressions were positive in 41,4% and 43%, respectively, and KRAS status was mutate in 48,3%. Wild-type KRAS in elderly patients had statistical association between CD44 and CD166, p=0,042 and p=0,001, respectively. Patients with CD166 negative had 3 fold increase in progression disease (p<0,01). Patients with CD44 positive had 4 and 5 fold increase in liver and lung metastasis (p<0,01), respectively. Patients with combined mutated KRAS and CD44 positive had 7 fold increase in lung metastasis (p=0,02) compared with wildtype KRAS and CD44 negative. DISCUSSION: In this study, the influence of markers expression of colon CSC (CD44 and CD166) and its combinations with status KRAS were proven. Patients with CD166 negative in primary colon tumor are more likely to present higher recurrence and, CD44 positive have a higher chance to develop lung and liver metastasis. KRAS mutation contributed, associated with studied CSC expressions, to cancer biological behavior and agressivness. CONCLUSIONS: This study demonstrated interaction between imunohistochemical expression of colonic CSC markers (CD166 and CD44) and KRAS gene status. Subgroups of patients with worse outcomes could be identified and this biological information contributed to personalized treatments and follow ups that should be proposed for these patients.

INTRODUÇÃO: Os múltiplos passos da carcinogênese do câncer de cólon envolvem a existência de subpopulações de células tronco tumorais (CSC), responsáveis pela transformação, crescimento e proliferação das células tumorais. As proteínas CD44 e CD166 são marcadores de CSC associados a sinalização celular, adesão, migração, metástase e resposta linfocitária. Alguns fatores podem modular a expressão CSC como a mutação KRAS. OBJETIVO: correlacionar a expressão dos marcadores CD44 e CD166 em carcinoma de cólon metastático e status do oncogene KRAS (selvagem/mutado) com as características clínico-patológicas e desfecho do paciente ao final do seguimento. MATERIAL E MÉTODOS: Foram coletadas 58 amostras de tecido tumoral de pacientes com neoplasia de cólon metastático, tratados com CapeOx no Serviço de Oncologia Clínica do HCFMRPUSP de 2003 a 2012. Foram coletadas informações do prontuário sobre status do gene KRAS, características clínico-patológicas e desfecho clínico, sendo também realizada imunohistoquímica para marcação CD44 e CD166 através da técnica de TMA. Utilizado software SPSS 17 para análise estatística e considerado valor de p<0,050 para significância dos dados. RESULTADOS: A expressão de CD44 e CD166 foi positiva em 41,4% e 43%, respectivamente, e o status KRAS mutado em 48,3%. No subgrupo kAs selvagem e nos idosos (>65 anos), houve associação entre CD44 e CD166, p=0,042 e p=0,001, respectivamente. Pacientes CD166 negativo tiveram 3 vezes mais chances de progressão de doença (p=0,02) do que CD166 positivo. Pacientes Kras mutado e CD166 negativo tiveram 8 vezes risco de progressão (p<0,01). Pacientes CD44 positivo tiveram 4 e 5 vezes mais chances de evoluir com metástases hepática e pulmonar (p<0,01) em relação aos CD44 negativo. Pacientes com a combinação KRAS mutado e CD44 positivo tiveram 7 vezes mais chance de evoluir com metástase pulmonar (p=0,02) em relação a pacientes KRAS selvagem e CD44 negativo. DISCUSSÃO: Na amostra estudada observamos a influência das expressões dos marcadores de CSC e suas combinações com o status de mutação do gene KRAS, de modo que pacientes com CD166 negativo no tumor primário apresentam um desfecho de maior recorrência e o CD44 positivo favorece a evolução para metástases pulmonar e hepática. A mutação do gene KRAS atua modulando a via do EGF influenciando o comportamento biologico do tumor e os desfechos (recidiva e metastases) diretamente relacionados com a expressão dos marcadores de CSC no cancer de colon metastatico. CONCLUSÃO: Este estudo demonstrou interação entre a expressão imuno-histoquímica dos marcadores CSC de cólon (CD166 e CD44) e o status KRAS, podendo carcterizar subgrupos de pacientes com maiores chances de evolução desfavorável e assim propor um modelo de tratamento e seguimento mais individualizado. / BACKGROUND: Colon cancer carcinogenesis has been recently correlated with specific cancer stem cell (CSC) subpopulations which are associated with transformation, growth and spread process of tumor cells. CD44 and CD166 are CSC markers correlated with cell signalization, adhesion, migration, metastasis, and lymphocyte response. Some factors as KRAS mutation could modulate CSC. OBJECTIVE: Analyze CD44 and CD166 expressions in metastatic colon carcinoma and its correlation with KRAS status, clinicopathological features, disease recurrence and patient survival. MATERIAL AND METHODS: Tissues were obtained from 58 patients with confirmed metastatic colon cancer, treated with CapeOx at FMRP-USP from 2003 to 2012. Clinical and outcomes informations and KRAS gene status were obtained from medical records. KRAS status was analyzed with RT-PCR. CD44 and CD166 were analyzed with TMA immunohistochemistry. Statistical analyses were performed using SPSS 17.0. A p-value <0,050 was considered to be statistically significant. RESULTS: CD44 and CD166 expressions were positive in 41,4% and 43%, respectively, and KRAS status was mutate in 48,3%. Wild-type KRAS in elderly patients had statistical association between CD44 and CD166, p=0,042 and p=0,001, respectively. Patients with CD166 negative had 3 fold increase in progression disease (p<0,01). Patients with CD44 positive had 4 and 5 fold increase in liver and lung metastasis (p<0,01), respectively. Patients with combined mutated KRAS and CD44 positive had 7 fold increase in lung metastasis (p=0,02) compared with wildtype KRAS and CD44 negative. DISCUSSION: In this study, the influence of markers expression of colon CSC (CD44 and CD166) and its combinations with status KRAS were proven. Patients with CD166 negative in primary colon tumor are more likely to present higher recurrence and, CD44 positive have a higher chance to develop lung and liver metastasis. KRAS mutation contributed, associated with studied CSC expressions, to cancer biological behavior and agressivness. CONCLUSIONS: This study demonstrated interaction between imunohistochemical expression of colonic CSC markers (CD166 and CD44) and KRAS gene status. Subgroups of patients with worse outcomes could be identified and this biological information contributed to personalized treatments and follow ups that should be proposed for these patients.

Abstract Apoptosis plays a crucial part in human ovarian function from fetal development to the end of reproductive potential. Failures in the regulation of ovarian apoptosis are associated with many pathological conditions such as premature ovarian insufficiency, infertility and cancer. The purpose of the present study was to analyze the factors regulating cell survival in human fetal and adult ovaries. The fetus is exposed to maternal- and placental-derived estrogens and insufficient estrogen action has destructive effects on rodent ovarian development. We detected estrogen receptors and estrogen-converting enzymes in human fetal ovaries after primordial follicle formation, indicating that estrogens participate in human fetal ovarian development, especially after folliculogenesis. The WNT4 gene is crucial for female sexual differentiation, follicle formation and oocyte survival. We detected WNT4 in follicular cells of fetal and adult human ovaries. In addition, Wnt4- knockout mice demonstrated a dramatic loss of oocytes before birth. However, no changes were detected in protein expression patterns of common apoptosis-related proteins. The results support the possible role of WNT4 in human ovarian function and strengthen previous knowledge on the antiapoptotic role of Wnt4. Apoptosis signaling is mediated by extracellular- and mitochondria-associated- pathways, ending in caspase cascade activation and fragmentation of cellular structures. In the present study we analyzed the expression of several apoptosis-related factors and detected TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries. In addition, TRAIL was found to be a potent and rapid inducer of human granulosa tumor cell (KGN) apoptosis. Lentiviral downregulation of Bok or Bcl-XL protein expression in KGN cells also resulted in significant changes in cell vulnerability to apoptosis. The results show for the first time the spatiotemporal expression patterns of TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries and suggest an important functional role of TRAIL, Bok and Bcl-XL in regulation of human ovarian apoptosis. The present study offers novel information on the expression and function of cell survival factors in human ovaries. These new findings open possibilities for future clinical research in attempts to understand and treat ovarian diseases caused by imbalanced regulatory pathways of apoptosis.

Numerous mutations contribute to tumorigenesis of cancer cells. For most of them it remains unclear whether they are driver or passenger mutations. A classic knock-out to study their function in cancer cells used to take a lot of effort. The CRISPR/Cas-system can be used as a programmable “genome editing” tool. In this work, oncogenes have been inactivated with the CRISPR/Cas-system. Considering off-targets, Streptococcus pyogenes sgRNAs can be designed for 88% of the known cancer mutations. The activity of 15 sgRNAs, targeting 13 mutations in proto-oncogenes (deletions, insertions and point mutations), has been tested with a RFP-GFP-reporter plasmid. For 13 sgRNAs, activity prediction scores correlated with measured activity. Furthermore, sgRNAs have shown preferential binding to mutated versions of targeted proto-oncogene sequence and did not induce double strand breaks in the wild type sequence. For 10 sgRNAs, the activity against their target sequence has been more than 4 times higher than against the wild type sequence. Most of those sgRNAs target insertions or deletions and fewer target point mutations. Permanent knock-out of three mutated proto-oncogenes NPM1, BRAF and PIK3CA has been achieved with a lentiviral expression of CRISPR/Cas. Accordingly, effects on proliferation and phenotype have been studied. Knock-out of NPM1 c.863_864insTCTG mutation has been studied in heterozygous mutated OCI AML3 cell line. Proliferation was strongly inhibited by the corresponding sgRNA. Cells arrested in G0/1-phase of cell cycle (77%) compared to control cells (56%), although no difference was observed for sub-G1 phase, indicating no induction of apoptosis. Cells treated with NPM1 sgRNA had 88% reduced expression of NPM1 c.863_864insTCTG mRNA as well as less cytoplasmic localization of nucleophosmin as assessed by immunostaining. The activity of sgRNA has been confirmed by deep sequencing, showing a shift of wild type to mutated allele ratio from 51:49 to 68:32. This effect was enhanced by the additional treatment with the NHEJ inhibitor SCR7. A BRAFV600E sgRNA was tested in homozygously mutated melanoma cell lines A-375 and SK MEL-28. No differences were detected in comparison to controls. However, in the CRC cell line RKO, heterozygous for BRAFV600E and PIK3CAH1047R, proliferation was inhibited through sgRNAs against either BRAF or PIK3CA. A combination of both had no synergistic effect on proliferation. Activity and specificity of the sgRNA targeting BRAF were confirmed by deep sequencing, while the PIK3CA sgRNA showed a moderate induction of double strand breaks also in the wild type allele. The relation of wild type to mutated allele of BRAF was changed from 32:68 before treatment to 51:49 afterwards. This effect can be explained by a “re mutation” to the wild type after DSB via HDR with wild type sister chromatid as template. This effect was observed for PIK3CA sgRNA to a lesser extent. In conclusion, these results show the applicability of the CRISPR/Cas-system for the inactivation of mutated proto-oncogenes.:List of tables III List of figures IV List of abbreviations V 1 Introduction 1 1.1 Cancer 1 1.2 Oncogenes 2 1.2.1 Role in cancer 2 1.2.2 Targeted therapies 3 1.2.3 NPM1 5 1.2.4 BRAF 6 1.2.5 PIK3CA 7 1.3 CRISPR/Cas-system 7 1.4 Aim and motivation 10 2 Material and Methods 11 2.1 Design of sgRNAs 11 2.2 Plasmids 11 2.3 Cell culture 12 2.4 FACS analysis 14 2.5 T7 assay 14 2.6 Cell cycle analysis 15 2.7 Immunostaining 15 2.8 Apoptosis assay 16 2.9 Quantification of mutant NPM1 transcripts 16 2.10 Deep sequencing 16 2.11 Statistical Analysis 19 3 Results 20 3.1 Design of sgRNAs targeting oncogenes 20 3.2 Evaluation of sgRNA efficacy and selectivity 23 3.3 Effects of oncogene knock-out in cancer cell lines 27 3.3.1 Targeting NPM1 in AML cells 27 3.3.2 Targeting BRAF in melanoma cells 30 3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31 4 Discussion 37 4.1 The design of sgRNAs is possible for most cancer mutations 37 4.2 sgRNAs targeting oncogenes have to be tested 37 4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37 4.3.1 NPM1 in AML cells 37 4.3.2 BRAF in melanoma cells 38 4.3.3 BRAF and PIK3CA in CRC cells 38 4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40 4.5 Concluding remarks 41 5 Original Article 43 6 Summary 47 7 Zusammenfassung 49 List of references 51 Appendix VIII / In Krebszellen tritt eine Vielzahl von Mutationen auf. Für den Großteil der Mutationen ist ungeklärt, ob es sich um krebsverursachende oder passagere Mutationen handelt. Ein gezieltes Ausschalten (Knock-out) dieser Gene zur Untersuchung ihrer Funktion in Krebszellen war bisher mit großem Aufwand verbunden. Das CRISPR/Cas-System lässt sich als programmierbares „Genome-editing“ Werkzeug einsetzen und wurde in der vorliegenden Arbeit verwendet, um gezielt mutierte Protoonkogene zu inaktivieren. Für 88% der bekannten, in Krebszellen auftretenden Mutationen lassen sich, unter Berücksichtigung von off-targets, Streptococcus pyogenes sgRNAs entwerfen. Mit Hilfe eines RFP-GFP-Reporter-Plasmides wurde die Aktivität von 15 sgRNAs gegen 13 Mutationen (Deletionen, Insertionen und Punktmutationen) in Protoonkogenen überprüft. Für 13 der sgRNAs zeigte sich eine Aktivität, die mit der Vorhersage durch den Algorithmus korrelierte. Außerdem wurde gezeigt, dass die sgRNAs spezifisch genug binden, um zwar bei der mutierten Sequenz eines Protoonkogens, jedoch nicht bei der Wildtyp-Sequenz Doppelstrangbrüche zu erzeugen. Unter den sgRNAs waren 10 mit mehr als 4-fach höherer Aktivität bei komplett übereinstimmender Zielsequenz gegenüber der Wildtyp-Sequenz. Diese spezifischen sgRNAs waren vor allem gegen Insertions- oder Deletionsmutationen gerichtet, einige auch gegen Punktmutationen. Durch permanente, lentivirale Expression von CRISPR/Cas wurden die Effekte eines Knock-out von drei mutierten Protoonkogenen, NPM1, BRAF und PIK3CA, auf das Wachstum und phänotypische Aspekte humaner Krebszelllinien untersucht. Ein Knock-out der NPM1 c.863_864insTCTG Mutation wurde in heterozygot mutierten OCI AML3 Zellen untersucht, es zeigte sich eine starke Proliferationshemmung. In der Zellzyklusanalyse trat ein G0/1-Arrest dieser Zellen (77%) im Vergleich mit Kontroll-Zellen (56%) auf, jedoch keine Unterschiede in der sub-G1-Analyse, sodass nicht von einer vermehrten Apoptose auszugehen ist. Die mit sgRNA behandelten OCI-AML3 Zellen zeigten sowohl eine um 88% verminderte NPM1 c.863_864insTCTG mRNA-Expression als auch verminderte zytoplasmatische Sublokalisation des Nucleophosmins in der Immunfärbung. Die hohe Aktivität der gRNA gegen mutiertes NPM1 wurde durch Deep Sequencing bestätigt, außerdem hat sich das Verhältnis vom Wildtyp- zu mutiertem Allel von 51:49 zu 68:32 verschoben. Dieser Effekt wurde durch Zugabe des NHEJ-Hemmstoffes SCR7 noch verstärkt. Die sgRNA gegen BRAFV600E wurde in den homozygot mutierten Melanom-Zelllinien A-375 und SK-MEL-28 getestet. Bei Proliferationsversuchen zeigten sich keine Unterschiede im Vergleich zu Kontrollzellen. In der kolorektalen Krebszelllinie RKO, die heterozygot BRAFV600E und PIK3CAH1047R ist, zeigte sich bei der Testung von sgRNAs gegen BRAF, PIK3CA und Kombination beider sgRNAs eine Wachstumshemmung. Jedoch lag kein synergistischer Effekt bei sgRNA-Kombination vor. Zudem bestätigten sich Aktivität und Spezifität der sgRNA gegen BRAF im Deep Sequencing, während die sgRNA gegen PIK3CA in mäßigem Umfang Doppelstrangbrüche im Wildtyp-Allel verursachte. Das Verhältnis vom Wildtyp- zu mutiertem BRAF Allel verschob sich von 32:68 ohne sgRNA zu 51:49 nach sgRNA-Behandlung. Eine mögliche Erklärung dieser Beobachtung ist die Rückmutation zum Wildtyp-Allel nach Doppelstrangbruch mit Hilfe homologer Rekombination durch das Wildtyp-Schwesterchromatid. Für PIK3CA konnte dieser Effekt in schwächerem Ausmaß ebenfalls beobachtet werden. Zusammengefasst zeigen diese Ergebnisse, dass das CRISPR/Cas-System zur Inaktivierung mutierter Protoonkogene genutzt werden kann.:List of tables III List of figures IV List of abbreviations V 1 Introduction 1 1.1 Cancer 1 1.2 Oncogenes 2 1.2.1 Role in cancer 2 1.2.2 Targeted therapies 3 1.2.3 NPM1 5 1.2.4 BRAF 6 1.2.5 PIK3CA 7 1.3 CRISPR/Cas-system 7 1.4 Aim and motivation 10 2 Material and Methods 11 2.1 Design of sgRNAs 11 2.2 Plasmids 11 2.3 Cell culture 12 2.4 FACS analysis 14 2.5 T7 assay 14 2.6 Cell cycle analysis 15 2.7 Immunostaining 15 2.8 Apoptosis assay 16 2.9 Quantification of mutant NPM1 transcripts 16 2.10 Deep sequencing 16 2.11 Statistical Analysis 19 3 Results 20 3.1 Design of sgRNAs targeting oncogenes 20 3.2 Evaluation of sgRNA efficacy and selectivity 23 3.3 Effects of oncogene knock-out in cancer cell lines 27 3.3.1 Targeting NPM1 in AML cells 27 3.3.2 Targeting BRAF in melanoma cells 30 3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31 4 Discussion 37 4.1 The design of sgRNAs is possible for most cancer mutations 37 4.2 sgRNAs targeting oncogenes have to be tested 37 4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37 4.3.1 NPM1 in AML cells 37 4.3.2 BRAF in melanoma cells 38 4.3.3 BRAF and PIK3CA in CRC cells 38 4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40 4.5 Concluding remarks 41 5 Original Article 43 6 Summary 47 7 Zusammenfassung 49 List of references 51 Appendix VIII