CXCR3A potencia la proliferación celular a través de la transactivación del receptor tirosina quinasa RET
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2017
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es
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Universidad Andrés Bello
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Resumen
El cáncer papilar de tiroides (CPT) es la neoplasia más frecuente del sistema endocrino. El aumento de incidencia de CPT en pacientes con tiroiditis sugiere que la inflamación es un factor de riesgo del desarrollo del cáncer de tiroides, sin embargo, aún es un tema de bastante controversia. Las quimioquinas y sus receptores han sido principalmente descritas en la quimiotaxis de células inflamatorias y en la diseminación del cáncer. La señalización del receptor CXCR3 por sus ligandos CXCL9, CXCL10, y CXCL11 se ha estudiado en la inmunidad tumoral y en el inicio de la inflamación crónica de la tiroides (tiroiditis). Recientemente, se ha descrito un aumento de CXCR3 en distintos tumores sugiriendo una función pro-tumorigénica. CXCR3 presenta respuestas celulares antagónicas en parte por la presencia de dos variantes de splicing, CXCR3A (pro-tumoral) y CXCR3B (pro-apoptótica). Sin embargo, se desconoce la función de estas variantes en el desarrollo y progresión del cáncer papilar de tiroides y su relación con la inflamación. En esta tesis se propone la hipótesis de que una alteración en el perfil de expresión de las variantes de CXCR3 promueve el desarrollo y progresión del CPT. Para estudiar la correlación de CXCR3 y sus ligandos con neoplasias de tiroides, se evaluó su expresión en tumores benignos y de CPT. Para evaluar el mecanismo de señalización de CXCR3A y CXCR3B, se realizaron ensayos in vitro y se determinó su relación con la activación del receptor proto-oncogénico RET. Para ello, se transfectó la línea celular Nthy-ori-3-1 (células epiteliales normales de tiroides humana) con plásmidos de expresión para cada variante o con siRNA para CXCR3 (siRNA-CXCR3). Además se trataron las células con un antagonista de CXCR3, interferón gamma (IFN-γ), inhibidores de quinasas y/o con los ligandos CXCL10-CXCL11 y se evaluó la activación de quinasas y la proliferación celular.Los resultados mostraron un aumento de CXCL10 (condicionado a la tiroiditis) y CXCR3A en biopsias de CPT no metastásico (CPTnM) y en TPC-1 (células epiteliales derivadas de CPT humano). En Nthy-ori-3-1, un estímulo con IFN-γ aumentó los niveles de CXCL10, mientras que el antagonista de CXCR3 disminuyó la proliferación celular. La sobre-expresión de CXCR3A potenció la proliferación celular inducida por CXCL10-CXCL11 mientras que el siRNA-CXCR3 y el inhibidor de RET mostraron el efecto contrario. Finalmente, el antagonista y el siRNA-CXCR3 revirtieron la activación de RET y de AKT mediada por CXCL10-CXCL11. Estos resultados sugieren que el ambiente inflamatorio promueve la señalización de CXCR3 y la transactivación de RET a través de CXCR3A, siendo un mecanismo que contribuye al desarrollo del fenotipo celular maligno.
Papillary thyroid cancer (PTC) is the most common malignancy of the endocrine system. The increased incidence of PTC in patients with thyroiditis suggests that inflammation might be a risk factor for thyroid neoplastic transformation, however, it is still under dabate. Chemokines and their receptors have been mainly described in inflammatory cell chemotaxis and cancer dissemination. CXCR3 signaling throught its ligands (CXCL9, CXCL10 and CXCL11) plays an important role in the initial stage of autoimmune thyroiditis. Recently, CXCR3 upregulation has been reported in many human tumors suggesting a protumoral role for this receptor. CXCR3 may have divergent effects on tumor cells in part dictated by the array of two functionally CXCR3 splicing variants; CXCR3A (protumoral) and CXCR3B (proapoptotic). However, it is still unknown whether these variants have a role in PTC development or progression and their relation with inflammation. In this thesis, it was hypothesized that an alteration on CXCR3 variants expression profile could promote PTC development or progression. To study the correlation between CXCR3 and its ligands with thyroid neoplasia, their expression was evaluated in benign thyroid tumors and PTC. In order to evaluate the CXCR3A and CXCR3B signaling mechanism, we performed in vitro assays and determined their relation with the proto-oncogenic RET receptor. Thus we transfected the Nthy-ori-3-1 cell line (normal epithelial cell from human thyroid) with expression plasmids for each variant or with siRNA for CXCR3 (CXCR3-siRNA). Further we treated the cells with a CXCR3 antagonist, interferon gamma (IFN-γ), kinase inhibitors and/or the CXCL10-CXCL11 ligands and the we evaluated kinases activation and cell proliferation. Results showed an increase of CXCL10 (conditioned to thyroiditis) and CXCR3A in non-metastatic PTC (nMPTC) biopsies and in TPC-1 (human PTC derived epithelial cells). In Nthy-ori-3-1, IFN- γ incremented the CXCL10 levels, while CXCR3 antagonist lowered cell proliferation by 30%. CXCR3A overexpression potentiated CXCL10-CXCL11 induced cell proliferation while CXCR3-siRNA and RET inhibitor showed the opposite effect to a similar magnitude. Finally, the antagonist and CXCR3-siRNA reverted CXCL10-CXCL11 mediated RET and AKT activation. These results show that the inflammatory environment (IFN-γ/CXCL10) induces the RET receptor transactivation through CXCR3A increasing proliferation, potentially contributing to PTC development.
Papillary thyroid cancer (PTC) is the most common malignancy of the endocrine system. The increased incidence of PTC in patients with thyroiditis suggests that inflammation might be a risk factor for thyroid neoplastic transformation, however, it is still under dabate. Chemokines and their receptors have been mainly described in inflammatory cell chemotaxis and cancer dissemination. CXCR3 signaling throught its ligands (CXCL9, CXCL10 and CXCL11) plays an important role in the initial stage of autoimmune thyroiditis. Recently, CXCR3 upregulation has been reported in many human tumors suggesting a protumoral role for this receptor. CXCR3 may have divergent effects on tumor cells in part dictated by the array of two functionally CXCR3 splicing variants; CXCR3A (protumoral) and CXCR3B (proapoptotic). However, it is still unknown whether these variants have a role in PTC development or progression and their relation with inflammation. In this thesis, it was hypothesized that an alteration on CXCR3 variants expression profile could promote PTC development or progression. To study the correlation between CXCR3 and its ligands with thyroid neoplasia, their expression was evaluated in benign thyroid tumors and PTC. In order to evaluate the CXCR3A and CXCR3B signaling mechanism, we performed in vitro assays and determined their relation with the proto-oncogenic RET receptor. Thus we transfected the Nthy-ori-3-1 cell line (normal epithelial cell from human thyroid) with expression plasmids for each variant or with siRNA for CXCR3 (CXCR3-siRNA). Further we treated the cells with a CXCR3 antagonist, interferon gamma (IFN-γ), kinase inhibitors and/or the CXCL10-CXCL11 ligands and the we evaluated kinases activation and cell proliferation. Results showed an increase of CXCL10 (conditioned to thyroiditis) and CXCR3A in non-metastatic PTC (nMPTC) biopsies and in TPC-1 (human PTC derived epithelial cells). In Nthy-ori-3-1, IFN- γ incremented the CXCL10 levels, while CXCR3 antagonist lowered cell proliferation by 30%. CXCR3A overexpression potentiated CXCL10-CXCL11 induced cell proliferation while CXCR3-siRNA and RET inhibitor showed the opposite effect to a similar magnitude. Finally, the antagonist and CXCR3-siRNA reverted CXCL10-CXCL11 mediated RET and AKT activation. These results show that the inflammatory environment (IFN-γ/CXCL10) induces the RET receptor transactivation through CXCR3A increasing proliferation, potentially contributing to PTC development.
Notas
Tesis (Magíster en Biotecnología)
Financiamiento otorgado por el Proyecto CORFO 13CTI-21526P2 y el Proyecto Consorcio en Biomedicina CTU06 Area 2 (Dr. Hernán González).
Financiamiento otorgado por el Proyecto CORFO 13CTI-21526P2 y el Proyecto Consorcio en Biomedicina CTU06 Area 2 (Dr. Hernán González).
Palabras clave
Cáncer de Tiroides, Quimiocinas, Receptor CXCR3A