Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations. Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes BRAF and NRAS, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with BRAF mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene MGMT was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15. These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment. / QC 20110121

Malignant melanoma is the most lethal skin cancer with no effective therapeutic treatment in its metastatic stages. RAS and PI3K pathways have been shown to play a critical role in melanoma development and progression. In this study, we assessed the in vitro and in vivo inhibition potential of a BRAF inhibitor (Sorafenib, Bayer) and a PI3K/mTOR inhibitor (PI-103, PIramed-Genentech) in primary melanoma cell lines. We used primary cell lines isolated from spontaneous melanomas obtained in the UV induced HGF transgenic melanoma mouse model.Although PI-103 and sorafenib inhibited melanoma in vitro cell proliferation and viability, the inhibition of RAS pathway was more effective. The combination of the two drugs showed a synergistic effect inhibiting RAS and PI3K pathways and in vitro melanoma cell proliferation in a cell line dependent manner. However, the combined treatment of orthotopic xenographs in immunocompetent FVB mice did not cooperate blocking tumor growth. Surprisingly, the in vivo treatment with PI-103 enhanced tumor growth. Our results also revealed that PI-103 caused immunosuppression inducing thymus atrophy and upregulating the intratumoral transcriptional levels of inmunosuppressors. In addition, PI-103 induced the antiapoptotic BH3 family proteins Mcl-1, Bcl-2 and BclXL, which correlated with the lower apoptotic rate observed within the PI-103 treated tumors. These data indicates that due to melanoma heterogeneity, some precautions should be taken when using these inhibitors for treatment. Moreover, these results certainly make an argument for investigating unexpected effects of rational drug combinations on immunocompetent animal models before conducting clinical studies.

<p>Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations.</p><p>Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes <i>BRAF</i> and <i>NRAS</i>, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with <i>BRAF</i> mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene <i>MGMT</i> was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15.</p><p>These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment.</p>

Mutational analyses of cancer genomes have identified novel candidate cancer genes with hitherto unknown function in cancer. To enable phenotyping of mutations in such genes, we have developed a scalable technology for gene knock-in and knock-out in human somatic cells based on recombination-mediated construct generation and a computational tool to design gene targeting constructs. Using this technology, we have generated somatic cell knock-outs of the putative cancer genes ZBED6 and DIP2C in human colorectal cancer cells. In ZBED6-/- cells complete loss of functional ZBED6 was validated and loss of ZBED6 induced the expression of IGF2. Whole transcriptome and ChIP-seq analyses revealed relative enrichment of ZBED6 binding sites at upregulated genes as compared to downregulated genes. The functional annotation of differentially expressed genes revealed enrichment of genes related to cell cycle and cell proliferation and the transcriptional modulator ZBED6 affected the cell growth and cell cycle of human colorectal cancer cells. In DIP2C-/-cells, transcriptome sequencing revealed 780 differentially expressed genes as compared to their parental cells including the tumour suppressor gene CDKN2A. The DIP2C regulated genes belonged to several cancer related processes such as angiogenesis, cell structure and motility. The DIP2C-/-cells were enlarged and grew slower than their parental cells. To be able to directly compare the phenotypes of mutant KRAS and BRAF in colorectal cancers, we have introduced a KRASG13D allele in RKO BRAFV600E/-/-/ cells. The expression of the mutant KRAS allele was confirmed and anchorage independent growth was restored in KRASG13D cells. The differentially expressed genes both in BRAF and KRAS mutant cells included ERBB, TGFB and histone modification pathways. Together, the isogenic model systems presented here can provide insights to known and novel cancer pathways and can be used for drug discovery.

El cáncer colorrectal (CCR) permanece hoy día como una de las neoplasias con mayor incidencia y mortalidad en España. Su etiología es multifactorial, influyendo alteraciones genéticas y/o epigenéticas por un lado y factores ambientales que incluyen a la dieta y ciertos hábitos tóxicos por otro. El diagnóstico de confirmación se lleva a cabo mediante el análisis anatomopatológico de una muestra tumoral obtenida a través de una colonoscopia, mientras que para el tratamiento curativo se dispone de la opción quirúrgica asociando o no quimioterapia y/o radioterapia, en función de la localización y estadiaje tumoral. La carcinogénesis colorrectal se inicia en las células epiteliales colónicas, experimentando cambios que derivan desde una mucosa sana en la formación de pólipos. Muchas de estas neoformaciones benignas evolucionarán hacia la formación de un adenocarcinoma en caso de no ser extirpadas. Esta secuencia de acontecimientos, la denominada secuencia adenoma-carcinoma, se desarrolla a lo largo de varios años en la mayor parte de los casos. A día de hoy, se conocen 3 vías diferentes de carcinogénesis por las que tiene lugar esta secuencia adenoma-carcinoma: la vía de la inestabilidad cromosómica, la vía de inestabilidad de microsatélites y la vía serrada. Cada una posee alteraciones genéticas/epigenéticas diferentes, aunque en algunos casos se comparten. Visualizando en conjunto estas vías, existen cuatro marcadores al menos que han demostrado ejercer una influencia en la evolución tumoral: BRAF, KRAS, metilación aberrante (CIMP) e inestabilidad de microsatélites. En la presente tesis se pretende investigar si alteraciones a estos niveles puedan implicar pronósticos diferentes y respuesta a la quimioterapia. El artículo 1 divide el CCR en 5 subtipos, según combinaciones genético-moleculares en los 4 marcadores comentados que concuerden con las vías carcinogénicas conocidas. En una muestra de casi 900 pacientes, se evaluó si dichas combinaciones conferían un pronóstico y una respuesta a quimioterapia estándar diferentes. Así, el subtipo 2, perteneciente a un subgrupo de pacientes con CCR de la vía serrada con mutación en BRAF y fenotipo metilador, exhibían la tasa de supervivencia más baja al final del seguimiento. Por el contrario, los pacientes con CCR del subtipo 5, familiares con inestabilidad de microsatélites, presentaban la más alta. El subtipo 3, y principalmente el subtipo 4, es decir, los más frecuentes, mostraron una respuesta favorable a la quimioterapia. En el resto de subtipos dicho efecto no pudo ser valorado con fiabilidad por un tamaño muestral deficiente al tratarse de CCR con combinaciones genético/moleculares menos frecuentes. No obstante, a la luz de los resultados obtenidos parece pertinente dividir el CCR en subtipos con el fin de lograr un mejor manejo de estos pacientes. El artículo 2 pretende evaluar la utilidad del marcador TFAP2E para predecir una falta de respuesta a la quimioterapia, en caso de hallarse metilado, en pacientes con CCR. Se incluyeron pacientes de una cohorte observacional y de un ensayo clínico, en total casi 800 pacientes. Los resultados mostraron que, en estadío metastásico, la presencia de metilación en TFAP2E no se correlacionaba con una mejor ni peor respuesta al tratamiento quimioterápico, mostrando una supervivencia global similar frente a los pacientes con CCR sin metilación. En estadíos II y III, la cohorte observacional no mostró diferencias en términos de supervivencia libre de enfermedad al comparar pacientes con tumores metilados y no metilados en TFAP2E, tampoco al analizar ambos estadíos por separado. Únicamente se objetivó en la cohorte clínica una peor supervivencia en pacientes con CCR estadío II y metilación en dicho gen. En resumen, ambos artículos muestran que las alteraciones genéticas correspondientes a mutaciones en BRAF y KRAS, la inestabilidad de microsatélites y la metilación de ciertos genes pueden ser de gran utilidad a la hora predecir la evolución natural de un paciente con CCR y su respuesta a fármacos. De este modo, queda patente el potencial papel que determinados biomarcadores puede ejercer a la hora de tomar decisiones en el manejo de estos pacientes.

No description available.

Melanoma, a malignant neoplasm of melanocytes, is the most lethal form of skin cancer. A majority of melanomas are driven by activating mutations in the kinase BRAF, which drives cellular proliferation through constitutive stimulation of the mitogen-activated protein kinase (MAPK) signaling pathway. Intriguingly, expression of oncogenic BRAF alone in vivo is insufficient to promote melanoma; rather, its expression leads to the development of benign nevi (moles) comprised of growth-arrested melanocytes. The acquisition of additional genetic or epigenetic changes is therefore critical for melanocytes to evade arrest and drive melanomagenesis, however the identity of these changes remains incompletely understood. Here we demonstrate that expression of oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway in vitro, which acts to limit melanocyte proliferation through the inhibition of the pro-growth transcriptional co-activators YAP and TAZ. Melanocyte-specific inactivation of Hippo signaling in vivo, via deletion of the Hippo kinases Lats1/2 alone, or in conjunction with oncogenic Braf expression, potently induces melanoma development in mice. Collectively, our data reveal that the Hippo tumor suppressor pathway represents an important barrier to melanoma development, and implicates YAP and TAZ as new therapeutic targets for the treatment of human melanoma.

When activated by growth factors and mitogens, cellular receptors like EGFR become activated and transmit signals from the cell surface through the MAPK pathway into the nucleus of the cell, in order to elicit a cellular response like growth, apoptosis, proliferation, and survival. Over activation of EGFR even without receptor binding and members of the MAPK pathways like BRAF and MEK1 have been reported in tumorigenesis. We used Gateway Technology to study the recombinant protein expressions from these 3 proto-oncogenes in HEK293T and HeLa cell lines, using pLenti 6.3/V5 as the expression system. BRAF was neither detected in the HeLa nor in the HEK 293T cell lines, by mass spectrometry after transfection. Only in the HeLa cell line was EGFR identified meanwhile MEK1 was identified both in HEK293T and HeLa cell lines. However, the results showed that the proteins identified were endogenous to these cell lines and therefore, no recombinant proteins were expressed using the pLenti 6.3/V5, as the expression system.

No description available.

No description available.