The role of p19 C-H-Ras protein in metastasis and proliferative pathways

Abstract

H-Ras es un protooncogen que codifica para dos proteínas por splicing alternativo la p19 y la p21 (1,2). La p21 presenta mayor tamaño y se localiza en la membrana en donde realiza la función de GTPasa que activa múltiples vías de señalización (3-8) en cambio la p19 de menor tamaño, atraviesa la membrana nuclear, en donde forma complejos proteicos con otras proteínas y desde ahí regula múltiples vías de señalización (9-10). Las mutaciones en H-Ras inducen a la carcinogénesis y han sido frecuentemente detectadas en los tumores de los pacientes (30%) con melanoma, cáncer oral, de riñón y vejiga

Para nuestros experimentos evaluamos las siguientes líneas celulares: 1) Células HeLa transfectadas en forma transiente, y que sobreexpresaban las proteínas H-Ras de nuestro interés en forma separada (vectores PRK5); 2) Fibroblastos embrionarios de ratón knock-out H-Ras (-/-) o bien doble knock-out H-Ras (-/-), N-Ras (-/-), transfectados de manera estable con los vectores pEGFP-p19 ó pEGFP-p21 y 3) líneas mutantes de fibroblastos obtenidas de los tumores extraídos por cirugía de los pacientes con Síndrome de Costello, el cuál es un raro desorden congénito causado por la activación en la línea germinal del oncogen H-Ras que afecta tanto a la proteína p19 como a la p21 (11-13).

Nuestros resultados mostraron que las proteínas H-Ras promueven el incremento en la expresión de los miRNAs evaluados, y en forma diferencial, la p19 incrementa la expresión de miR-206 y promueve un estado de quiescencia celular en la fase G0/G1 provocando una disminución en la proliferación celular, la capacidad invasiva y la capacidad formadora de tumores; además la sobreexpresión de p19 incrementa la expresión de la proteína NM23H1 la cuál confiere protección contra el daño producido al DNA por las especies reactivas de oxígeno.

En cuanto a las líneas mutantes de los fibroblastos de los pacientes con Síndrome de Costello, detectamos que 1) la mutación G12S posee mayor capacidad invasiva que la G12A, y 2) una disminución en la expresión de miR-206, la cuál ha sido relacionada recientemente con la aparición de rabdomyosarcoma en los pacientes por lo que se ha propuesto como un marcador del pronóstico de la enfermedad.

REFERENCIAS 1. Cohen J.B., Broz S.D., and Levinson A.D. (1989). Expression of the H-Ras proto-oncogene is controlled by alternative splicing. Cell. 58: 461-472

2. Guil S, de La Iglesia N, Fernández-Larrea J, Cifuentes D, Ferrer JC, Guinovart JJ, Bach-Elias M. (2003a). Alternative splicing of the human proto-oncogene c-H-ras renders a new Ras family protein that trafficks to cytoplasm and nucleus.Cancer Res. (2003 a) 1;63(17):5178-87

3. Malumbres M., and Barbacid M. (2003). Ras oncogenes: the first 30 years. Nature Reviews 3,7-13.

4. Rodriguez-Viciana P., Sabatier C., and McCormick F. (2004) Signalling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulated. Mol. Cell. Biol. 24(11):4943-4954.

5. Mitin N., Rossman L. K., and Der C. J. (2005). Signaling interplay in Ras superfamily function. Current Biology15 (14): R563 - R574.

6. Malaney S., and Daly R.J. (2001). The Ras signalling pathway in mammary tumorigenesis and metastasis. J. Mammary Gland Biol Neoplasia. 6(1):101-113

7. Downward, J. (2002). Targeting Ras signalling pathways in cancer therapy. Nat. Rev. Cancer 3: 11-22

8. Colicelli J. (2004). Human Ras superfamily proteins and related GTPases. Sci.Signal 250: re13

9. Camats-Malet., Calin G.A., Heesom, K.J., Liu cG., Volinia S., Croce M., Ladomery M., and Bach-Elias M. (2008b). P19 activates telomerase, regulates expression of proteins of the tuberous sclerosis (TSC) pathway and upregulate miRNA’s expression. Submitted to Plos One.

10. Camats-Malet M. (2008a). Mecanismes de Senyalitzacio intracellular regulats per la proteina p19 H Ras. Tesis de Doctorat. Departament de Bioquimica I Biologia Molecular. Unitat de Ciencies.Universitat Autonoma de Barcelona.

11. Costello, J.M. (1977). A new syndrome: mental subnormality and nasal papillomata. Aust Paediat J. 13: 114-118.

12. Gripp K.W., Innes A.M., Axelrad M.E., Gillan T.L., Parboosingh J.S., Davies C., Leonard N.J., Doyle D., Catalano S., Nicholson l., Stabley D., and Sol-Church K. (2008). Costello syndrome associated with novel germline H-Ras mutations: An attenuated phenotype? American Journal of Medical Genetics Part A. 146 A:683-690

13. Gripp KW, Lin A.E, Stabley D., Nicholson L., Scott Jr. C.I., Doyle D., Aoki Y., Matsubara Y., Zachai E.H., Lapunzina P., Gonzalez-Meneses, A., Holbrook J., Agresta C.A., Gonzalesz I.L and Sol-Church K. (2006). HRAS mutation analysis in Costello Syndrome: Genotype and Phenotype correlation. Am J. Med. Genet.A 140 (1):1-7

Ras is an evolutionary and conserved family of genes present in many organisms, in humans, Ras is conformed by three members called N-Ras, K-Ras and H-Ras located on chromosomes 1, 11 and 12 respectively (1, 2). Ras proteins act as a molecular switch, activating many signalling pathways through phosphorylation of GTPases; so their punctual mutations promote a constitutive activation in their GTPase function that fosters carcinogenesis, loss of adhesion and invasion of malignant cells (3-9). In the case of H-Ras, 30% of the malignant tumours analyzed have showed mutations and they were frequently detected in melanoma, thyroid, oral, bladder, and kidney cancers (10-14).

H-Ras gene renders two different proteins by their alternative splicing called p19 and p21 (15, 16); even though both proteins have the same origin, they differ in their size function and localization. P19 and p21 are similar in their first 150 aminoacids, but they differ in the C-terminal amino acid sequences, meanwhile p21 contains 152-165 residues that confer the GTPase function, p19 lacks of it. Nevertheless p19 protein is smaller than p21, is able to cross the nuclear membrane and then p19 can bind with other proteins as such as RACK1, PKCβII, p73 and NSE (neuron specific enolase), (17,18) forming protein complexes; which suggest that p19 protein indirectly orchestrates multiple cell functions from the nucleus (19).

For the experimental development of this PhD thesis we decided to overexpress p19, p21, or their mutant protein variants transient and ectopically in HeLa cells. Three mutations were analyzed in these assays: Q61L, G12S, and G12A, the first of them induces a constitutive activation of GTPase activity and G12S, G12A are both a frequent mutations observed in Costello Syndrome, a rare congenital disorder caused by germ-line activation of H-Ras oncogenes that affects both p19 and p21(20-22). We also analyzed the contribution of p19, or p21 proteins in knock-out H-Ras (-/-) and double knock-out for H-Ras (-/-), N-Ras (-/-) murine embryonic fibroblasts (MEFs); these cell lines have the advantage that they do not show gene redundancy in their expression, so in other words this means that the absence of one member of Ras proteins does not cause that other Ras protein assume its functions. We also analyzed mutated fibroblasts obtained from tumours of Costello Syndrome patients in order to determinate the contribution of G12S and G12A mutations in this syndrome.

A general increment in the miRNA expression profile was detected when p19, p21 and their mutant variants were overexpressed in HeLa cells, even though further experiments in our knock-out H-Ras (-/-) and double knock-out H-Ras (-/-), N-Ras (-/-) MEFs transfected with pEGFP-p19 or pEGFP-p21, we detected a differential expression of miR-206 and miR-342 when p19 and p21 were expressed respectively. In addition, miR-206 was consistently downregulated in our mutated fibroblasts of Costello Syndrome patients which is agree with recent reports that have correlated the misregulation of miR-206 with the development of rhabdomyosarcoma in these patients.

In other hand, overexpression of p21 (G12S) protein in HeLa cells showed the highest rate of invasion, followed by p21, p19 (G12S) and p19 proteins; in further cotransfection assays (p19/p21 (G12S) proteins); p19 was able to diminished the invasion and in mutated fibroblasts of Costello Syndrome patients, the highest invasion capacity rate was conferred by G12S mutation.

P19 protein showed a low rate proliferation in double knock-out H-Ras (-/-), N-Ras (-/-) MEFs, further analyses of cytometry revealed that p19 induces a quiescent arrest in G0/G1 phase cell cycle. The capacity of forming colonies was also evaluated in clonogenic anchorage agar assays in which the presence of (G12S) mutation in p19 and p21 proteins contributed to the formation of bigger and more number of colonies.

Additionally, overexpression of p19 protein (pRK5-p19 vector) in HeLa cells also conferred protection against reactive oxygen species emission overexpressing NM23H1 protein, this effect was also detected in high ROS emission environment.

REFERENCES:

1. Lowy D.R., and Willumsen B.M. (1993). Function and regulation of Ras. Ann. Rev. Biochem. 62:851-891.

2. Wennerber K., Rossman K.L., and Der C.L. (2005).The Ras superfamily at glance. J. Cell Sci.118:843-846.

3. Mori K., Hata M., Neya S., Hoshino T. (2002) A study on the role of Mg+” in a Ras protein by MD simulation. CBIJ. 2 (4): 147-155

4. Cullen P. J., and Lockyer P.J. (2006) Integration of calcium and Ras signalling. Nat. Rev. 3: 339-344.

5. Rehman H., and Bos J. (2004) Thumbs up for inactivation. Nature. 249: 138-139 Ricarte-Filho JC, Fuziwara CS, Yamashita AS, Rezende E, da-Silva MJ, Kimura ET.(2009). Effects of let-7 microRNA on Cell Growth and Differentiation of Papillary Thyroid Cancer. Transl Oncol. 200. (4):236-41.

6. Campbel S.L., Khosravi-Far R., Rossman K.L., Clark G.J. and Der C.J. (1998). Increasing complexity of Ras signaling. Oncogene. 17:1395-1413. Cancer Lett. 2008 Oct 18;270(1):10-8. doi: 10.1016/j.canlet.2008.03.035. Epub 2008 May 23. Review.

7. Malumbres M., and Barbacid M. (2003). Ras oncogenes: the first 30 years. Nature Reviews 3,7-13.

8. Rodriguez-Viciana P., Sabatier C., and McCormick F. (2004) Signalling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulated. Mol. Cell. Biol. 24(11):4943-4954.

9. Mitin N., Rossman L. K., and Der C. J. (2005). Signaling interplay in Ras superfamily function. Current Biology15 (14): R563 - R574.

10. Malaney S., and Daly R.J. (2001). The Ras signalling pathway in mammary tumorigenesis and metastasis. J. Mammary Gland Biol Neoplasia. 6(1):101-113

11. Downward, J. (2002). Targeting Ras signalling pathways in cancer therapy. Nat. Rev. Cancer 3: 11-22

12. Colicelli J. (2004). Human Ras superfamily proteins and related GTPases. Sci.Signal 250: re13 13. Castro P., Soares P., Gusmao L., Seruca R., Sobrinho-Simoes. (2006). H-RAS 81 polymorphism is significantly associated with aneuploidy in follicular tumors of the thyroid. Oncogene. 25: 4620-4627 14. Castellano E, Santos E.(2011). Functional specificity of ras isoforms: so similar but so different.Genes Cancer. 2011 Mar;2(3):216-31. doi: 10.1177/1947601911408081.

15. Cohen J.B., Broz S.D., and Levinson A.D. (1989). Expression of the H-Ras proto-oncogene is controlled by alternative splicing. Cell. 58: 461-472

16. Guil S, de La Iglesia N, Fernández-Larrea J, Cifuentes D, Ferrer JC, Guinovart JJ, Bach-Elias M. (2003a). Alternative splicing of the human proto-oncogene c-H-ras renders a new Ras family protein that trafficks to cytoplasm and nucleus.Cancer Res. (2003 a) 1;63(17):5178-87 17. Jeong MH., Bae J., Kim WH., Yoo SM., Kim JW., Son PI, Choi KH. (2006). P19 ras interacts with and activates p73 by involving the MDM2 protein. The Journal of Biological Chemistry. 281(13):8707-8715 18. Camats-Malet., Calin G.A., Heesom, K.J., Liu cG., Volinia S., Croce M., Ladomery M., and Bach-Elias M. (2008b). P19 activates telomerase, regulates expression of proteins of the tuberous sclerosis (TSC) pathway and upregulate miRNA’s expression. Submitted to Plos One.

19. Camats-Malet M. (2008a). Mecanismes de Senyalitzacio intracellular regulats per la proteina p19 H Ras. Tesis de Doctorat. Departament de Bioquimica I Biologia Molecular. Unitat de Ciencies.Universitat Autonoma de Barcelona.

20. Costello, J.M. (1977). A new syndrome: mental subnormality and nasal papillomata. Aust Paediat J. 13: 114-118.

21. Gripp K.W., Innes A.M., Axelrad M.E., Gillan T.L., Parboosingh J.S., Davies C., Leonard N.J., Doyle D., Catalano S., Nicholson l., Stabley D., and Sol-Church K. (2008). Costello syndrome associated with novel germline H-Ras mutations: An attenuated phenotype? American Journal of Medical Genetics Part A. 146 A:683-690

22. Gripp KW, Lin A.E, Stabley D., Nicholson L., Scott Jr. C.I., Doyle D., Aoki Y., Matsubara Y., Zachai E.H., Lapunzina P., Gonzalez-Meneses, A., Holbrook J., Agresta C.A., Gonzalesz I.L and Sol-Church K. (2006). HRAS mutation analysis in Costello Syndrome: Genotype and Phenotype correlation. Am J. Med. Genet.A 140 (1):1-7