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Characterization of the DNA methylation patterns of chemosensitive and chemoresistant human cancer cells: Biological and clinical impact

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Universitat de Barcelona. Facultat de Medicina

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[spa]  	
La quimioresistencia es el principal limitante para el tratamiento del paciente oncológico. La comprensión de los mecanismos celulares que conducen a la quimioresistencia puede tener un impacto drástico en la utilización de los fármacos. Estos agentes destruyen las células sensibles por inducción de programas de muerte celular. La desregulación de genes implicados en estos mecanismos puede ser una manera de quimioresistencia. Genes supresores tumorales y de reparación del ADN son importantes en estos procesos. Mientras que la inactivación de los primeros lleva a la quimioresistencia, la inactivación de los segundos puede conducir a quimosensibilidad. Mecanismos como la epigenética puede ser responsables por la alteración de estos genes. Por ejemplo, la hipermetilación del ADN en los promotores puede causar el silenciamiento de genes, y/o la hipometilación global producir la activación de otros genes. Pretendemos determinar si los cambios del perfil de metilación del ADN son importantes en la adquisición de resistencia a la quimioterapia. 1º estudio: 68 pacientes con cáncer colorectal (CCR) metastásico recibieron tratamiento con dacarbazina. 3% alcanzó una respuesta parcial y 12% la estabilización de la enfermedad. El mejor resultado se asoció a la hipermetilación del gen MGMT. 2º estudio: La resistencia al oxaliplatino depende en parte de la hipermetilación del gen SRBC. Este hecho predice una supervivencia libre de progresión más corta en pacientes tratados con oxaliplatino para los que no estaba indicada la cirugía de las metástasis. 3º Estudio: Descubrimos que el gen UGCG tiene un papel en resistencia de tumores testiculares germinales al cisplatino (CDDP). Sugerimos un agente químico que podría ser utilizado re-sensibilizar este tipo de tumores refractarios al CDDP. 4º estudio: MGMT se presenta hipermetilado en los modelos de cáncer testicular sensibles al cisplatino. En pacientes la hipermetilación de este gen encontrase asociada a una mayor supervivencia global. Se demostró que la inactivación de esto gen en tumores resistentes podría re-sensibilizarlos al CDDP. El estado de metilación de los promotores génicos influye en la sensibilidad tumoral a diferentes agentes quimioterapéuticos. Ponemos en evidencia que la inhibición de MGMT y UGCG pueden ser opciones terapéuticas para la rescatar pacientes refractarios al CDDP.

# metadata.dc.description.abstract[].value
[eng] Although chemotherapeutic drugs are widely used in order to improve the cancer outcome, drug resistance remain the most unpredictable factor affecting chemotherapy and a major impediment to successful patient’s treatment. Understanding the cellular mechanisms leading to chemoresistance may dramatically impact on the way chemotherapeutic drugs are used. Then, it would allow selecting the most suitable personalized therapy. It has become increasingly clear that many chemotherapeutic agents kill susceptible cells through the induction of the physiological cell death program. Accordingly, deregulation of any gene involved in the activation or execution of the death processes may be a major mechanism of chemoresistance. Tumor suppressor and DNA repair genes were classified as important mediators of chemotherapeutic respons. While inactivation of tumor suppressor genes could lead to drug resistance, inactivation of DNA repair genes, drug metabolisms, and detoxification genes might lead to drug sensitivity. This can be due to different mechanisms like regional hypermethylation and/or global hypomethylation. The possibility that some genes conferring chemoresistance are reversibly switched on/off by DNA methylation is particularly important and may have relevant clinical implications. A very potent specific inhibitor of DNA methylation, 5-AZA, has been widely used as a demethylating agent in vitro, and is used clinically in the treatment of acute leukemia and myelodysplasia. The present Doctoral Thesis has been devoted to provide further knowledge about the cross-talk between genes promoters DNA methylation status and tumors chemosensitivity, on a more detailed understanding of the influence of its changes in resistance to dacarbazine and to platinum agents, such oxaliplatin and cisplatin. We also pretend to explore alternative therapies as an attempt for reverse tumors chemoresistance the main cause of patients dead. In order to address these goals, we studied two cancer models, colorectal and testicular germ cell tumors. 1º Study: 68 patients were enrolled in this study and treated with dacarbazine. Overall, 2% achieved partial response and 12% had stable disease. Better outcome was significantly associated with MGMT promoter hypermethylation. 2º Study: We found that oxaliplatin resistance in colon cancer cells depends on the DNA methylation-associated inactivation of SRBC gene. SRBC over-expression or depletion gives rise to sensitivity or resistance to oxaliplatin, respectively. SRBC epigenetic inactivation occurred in primary tumors from a discovery cohort of colorectal cancer patients, predicting shorter progression free survival, particularly in oxaliplatin­treated cases for which metastasis surgery was not indicated. 3º Study: We report the perpetuation of serially cisplatin-refractory orthotopic transplantable patient-derived nonseminomatous tumor grafts in mice, named orthoxenografts, as a system to investigate cisplatin refractoriness from a genetic perspective and for the preclinical development of novel targeted therapies based on overcoming cisplatin-resistance. Here we found UGCG implicated in cisplatin resistance. By quimical inactivation we were able to revert cisplatin resistance in ortoxenograt tumors. 4º Study: To explore if MGMT promoter methylation changes have a role in cisplatin chemoresistance, we study it methylation status in cisplatin sensitive and paired resistant human non-seminoma cancer cell lines, xenograft tumors and in clinical samples from metastasic patients treated with cisplatin-based chemotherapy. We found that cisplatin sensitive samples are related with MGMT promoter hypermethylation associated with its loss of expression. Clinically, the presence of MGMT promoter methylation is related with better overall survival in metastasic patients with testicular germ cell cancer. Inhibition of MGMT with O6-benzylguanine in vitro or in vivo increases the sensitivity to cisplatin and temozolomide, being this a possible chemotherapeutic approach to re-sensibilize human non-seminoma refractory tumors. We conclude that the methylation status of the gene promoters influences tumor sensitivity to different chemotherapeutic agents. We demonstrated that inhibition of MGMT and UGCG can be therapeutic ways for rescue patient’s refractory to cisplatin.

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Characterization of the DNA methylation patterns of chemosensitive and chemoresistant human cancer cells: Biological and clinical impact