Study of radiotherapy resistance associated to EGFR/MAPK signaling pathway and its application in the rational design of a treatment with simvastatin in combination with cetuximab and radiotherapy (in experimental models of carcinoma)

Abstract

1. Generation and characterization of a radioresistant cell line

In the first part of this project of Thesis, we subjected culture cells to fractionated radiation and clonal selection, to obtain a modified cohort of cells. The aim of this study was to develop an isogenic resistant cell line which could be used to identify molecular changes associated with acquired resistance to radiation and tumour aggressiveness in cancer.

We developed the stable isogenic resistant A431-R cell line from parental A431 cells presented a diminution of the radiosensitivity (decreases in SF2 and α-component of LQ model). These changes were associated with an increased efficiency in DNA repair. We demonstrated that A431-R cells acquired higher cloning efficiency and faster growth and migration ability; these properties were observed to be associated with remarkable baseline levels of relevant oncoproteins and elevated angiogenic capabilities.

2. Development of a technique to irradiate mice bearing xenografts

In the second part of this project of Thesis, we have reached four objectives during the design procedure described to administer fractionated radiotherapy to immunodeficient mice in the Radiotherapy Department of a Hospital. First, we have minimized irradiation of healthy tissues around the tumour. Second, we have fulfilled the standard irradiation principles for human treatments. Third, we reduced time and increase efficiency by irradiating several mice at a time. Finally, our procedure was easy to reproduce and repeat on a daily basis by only two people. We demonstrated the feasibility of fractionated irradiation using immunodeficient mice to evaluate the role of radiotherapy on experimental tumours simulating a clinical setting.

3. Simvastatin sensitizes to radiotherapy plus cetuximab

In the third part of this project of Thesis, we pre-clinically explored whether a treatment regime involving the addition of simvastatin to XRT and C225 merits further research. We have shown that the addition of simvastatin significantly decreased proliferation and clonogenic survival of cells treated with XRT and C225. Moreover, we used an experimental model with tumor cells derived from squamous cell carcinoma of that suggests that simvastatin may increase the antitumor effect of XRT plus C225— at doses and fractions of XRT that mimic doses administered in the clinical setting.

The addition of simvastatin was associated with an increase in apoptosis and a decrease in the levels of activated ERK1/2, AKT, and STAT3 oncoproteins, a set of observations that provide support to the higher anti-tumor effects produced by the triple treatment.

An important consideration is that when we combined simvastatin with XRT + C225 in the treatment of the radioresistant generated A431-R, we observed that the initial resistance of these cells to the treatment is in part reverted. In fact, cell migration, cell proliferation and cell survival were decreased when simvastatin was combined to the treatment with XRT+C225.

In addition, tumour growth was slowed down when simvastatin was added to XRT+C225 treatment. The model A431-WT/A431-R we have generated can be a useful tool to examine new treatments directed to revert radioresistance produced from a prior treatment with radiotherapy. Treatment effects can be studied in both cell sublines and comparison with the parental cells could give important informatio

1. Generación de una línea celular con fenotipo radiorresitente

Para el estudio de los posibles mecanismos implicados en la resistencia a radioterapia, se generó una línea celular resistente a radioterapia y se caracterizó:

1) Los tumores originados de las células resistentes mostraban un tamaño mayor al finalizar la radioterapia, mayor retraso del crecimiento y menor tasa de recrecimiento. 2) Aumento de la capacidad de migrar y reparar la herida. 3) Aumento de EGFR fosforilado basal y de AKT y ERK1/2. 4) Disminución de pERK1/2 tras irradiación (adquisición de una autosuficiencia de señales prosupervivencia). 5) Aumento de la secreción de VEGF en respuesta a radiación en las células radioresistentes (mayor angiogénesis asociada a tumor para resistir in vivo a la radioterapia.

En conjunto, las cambios fenotípicos adquiridos son compatibles con la ganancia de un fenotipo agresivo asociados a la aparición de radiorresistencia.

2. Descripción de la metodología utilizada para irradiar ratones Debido a la falta de literatura existente sobre métodos para la irradiación de xenoimplantes tumorales, se diseñó un protocolo técnico adaptado a los recursos, sobre el que puede destacarse que:

1) Minimiza la irradiación de tejidos sanos. 2) Permite la irradiación según dosis y fraccionamientos estándar para el tratamiento humano. 3) Reduce el tiempo y aumenta la eficiencia de los experimentos irradiando varios ratones simultáneamente. 4) Es fácilmente reproducible. 5) Se puede exportar a otros centros para investigación en oncología radioterápica que dispongan de un acelerador lineal.

3. Estudio de la combinación de simvastatina con cetuximab y radioterapia En este estudio se ha explorado preclínicamente si un régimen de tratamiento que implique la adición de simvastatina al tratamiento de cetuximab y radioterapia merecería mayor atención. Los hallazgos experimentales sugieren que la simvastatina podría potenciar la respuesta antitumoral de la combinación concomitante de cetuximab y radioterapia.

1) Disminución de la proliferación. 2) Reducción de la tasa de migración celular. 3) Reduccuión de la supervivencia celular clonogénica. 4) Los tumores crecieron más despacio. La tasa de crecimiento de los tumores tratados con la estatina fue significativamente menor. 5) Incremento del número de células células apoptóticas in vitro e in vivo 6) Reducción de los niveles las proteínas ERK1/2, AKT y STAT3 en su forma activa fosforilada sin alterar los niveles totales.