Dianas 6(1) > Caballero-de-la-Torre etal

Dianas | Vol 6 Num 1 | marzo 2017 | e20170301a03

p38 MAPK renders RB resistant to inactivation by CDKs.

María Caballero de la Torre^a, Manel Joaquin, Eulàlia de Nadal, Francesc Posas

Cell Signaling Research Group.Departament de Ciències Experimentals i de la Salut (DCEXS).Universitat Pompeu Fabra (UPF). Parc de Recerca Biomèdica de Barcelona (PRBB). Doctor Aiguader, 88.; 08003 Barcelona. Spain.

a. maria.caballero@upf.edu

II Congreso de Señalización Celular, SECUAH 2017.
14-16 de marzo, 2017. Universidad de Alcalá. Alcalá de Henares, Madrid. España.
Sesión 1a, Cáncer. Best Communication Award.

Keywords: Cell cycle; Retinoblastoma; Cancer cells; Cyclin Dependent kinases; p38

Abstract

The main function of the tumor suppressor Retinoblastoma (RB) is the regulation of the G1/S phase transition. This event is critical for the proliferation of normal cells in tissues, and its inhibition is one of the most important hallmarks leading to cancer. In resting cells RB is unphosphorylated and represses E2F-mediated gene expression. Upon entry into cell cycle, Rb becomes phosphorylated at the C-term through the action of Cyclin-Cdk complexes thus leading to its inactivation and the transcription of essential genes for S phase entry. Upon stress, cells deploy different mechanisms in order to adapt and increase their chances of survival. Among these, the control of cell division is essential. It is well established that stress-activated protein kinases (SAPKs) such as p38 play a key role in cell cycle regulation. During the G1-S transition, p38 regulates essential components of the cell cycle machinery such as the cyclin-dependent kinase (CDK) inhibitors p21Cip1, p27Kip1and p57Kip2. The CDK inhibitor p57Kip2 is phosphorylated by p38 which enhances its association to CDK2-Cyclin complexes and leads to reduced CDK2 activity. As a consequence, cells transiently arrest at the G1 cell phase [1]. However, stressed p57Kip2 knockout cells are still able, albeit to a lesser extent, to delay G1, which is an indication that other mechanisms involved in cell cycle control exist. Remarkably, upon stress, p38 also inhibits the transcription of E2F-dependent genes, which are necessary for cell cycle progression. These results prompted us to question whether p38 might be controlling Rb activity. Our results show that, upon stress p38 phosphorylates the N-term of Retinoblastoma (RB), making RB insensitive to cyclin-dependent kinase (CDK)-Cyclin inactivation and increases its affinity toward the E2F transcription factor, thus repressing E2F-mediated gene expression and delaying cell-cycle progression [2,3].This novel mechanism of RB regulation represents an opportunity for developing new cancer drug treatments for tumors by developing compounds capable of promoting the association of E2F transcription factors to the N-term of RB.

1. Joaquin M, Gubern A, Posas F. A novel G1 checkpoint mediated by the p57 CDK inhibitor and p38 SAPK promotes cell survival upon stress. Cell Cycle 2012b; 11:3339-40.
2. Gubern A, Joaquin M, Marquès M, Maseres P, Garcia-Garcia J, Amat R, González-Nuñez D, Oliva B, Real FX, de Nadal E, Posas F. The N-Terminal Phosphorylation of RB by p38 Bypasses Its Inactivation by CDKs and Prevents Proliferation in Cancer Cells. Mol Cell. 2016 Oct 6;64(1):25-36.
3. Joaquin M, de Nadal E, Posas F. An RB insensitive to CDK regulation. Mol Cell Oncol. 2016 Dec 14;4(1):e1268242.

Dianas 6(1) > Arranz-Nicolás etal

Dianas | Vol 6 Num 1 | marzo 2017 | e20170301a02

Effect of Diacylglycerol Kinase Alpha Inhibitors as Immunomodulators for Cancer Treatment.

Javier Arranz-Nicolás, Antonia Ávila-Flores, Isabel Mérida

Centro Nacional de Biotecnología (CNB-CSIC).

a. jarranz@cnb.csic.es

II Congreso de Señalización Celular, SECUAH 2017.
14-16 de marzo, 2017. Universidad de Alcalá. Alcalá de Henares, Madrid. España.
Sesión 1a, Cáncer.

Abstract

In tumors, the recruitment of immune cells with suppressive capacity and the main characteristics of the tumor microenvironment (increased levels of adenosine, low oxygen and acidic pH) favor that T cells become anergic and the activation of the TCR does not occur or occurs in a deficient manner. Targeting and manipulation of the immune system constitutes thus a major priority in the treatment of cancer. The Diacylglycerol kinases (DGK) are a family of enzymes that phosphorylate diacylglycerol (DAG) transforming this lipid into phosphatidic acid (PA). In T lymphocytes DGK act as negative regulators of the immune response metabolizing the DAG generated upon T cell receptor (TCR) triggering. This lipid second messenger facilitates the activation of the Ras/ERK (extracellular signal-regulated kinase) cascade, providing a direct input for AP-1 (activator protein-1)-mediated transcription and the expression of activation markers such as CD69 or CD25. The over-activation and/or expression of specific DGK isoforms drive T lymphocytes into a hypofunctional or anergic state. Tumor infiltrating lymphocytes (TILs) show elevated expression of certain DGK isoforms. In addition, tumors express high levels of DGK, suggesting the contribution of this enzyme family to the mechanism that regulate tumor immune evasion. Targeting DGK activity could contribute to block tumor growth and enhance T cell immunity. The generation and validation of different tools to monitor the effectiveness of known and potential new DGK inhibitors is needed. Here we design and optimize robust and reproducible cell-based trials for screening processes as well as to develop reliable sensors for DGK activity. We used different strategies to validate the effects of the previously reported DGK inhibitor R59949 and test the effect over DGK activity of a structurally similar molecule previously characterized as an anti-anxiety, antidepressive and antipsychotic agent. In addition, we pursue to better understand the different DGK functions in tumors and immune system, focusing on the possible additional effects of DGK inhibitors and the mechanisms that modulate DGK expression and activity.

Dianas 6(1) > Gallego-Tamayo etal

Dianas | Vol 6 Num 1 | marzo 2017 | e20170301a01

Efecto de ^V600EBRAF sobre la autofagia en células tumorales

Beatriz Gallego Tamayo, Eva Jiménez-Mora, Antonio Chiloeches

Departamento Biología de Sistemas, Facultad de Medicina. Universidad de Alcalá, Alcalá de Henares, Madrid. España.

a. bgallego28@gmail.com, bgallego28@gmail.com

II Congreso de Señalización Celular, SECUAH 2017.
14-16 de marzo, 2017. Universidad de Alcalá. Alcalá de Henares, Madrid. España.
Sesión 1a, Cáncer

Palabras clave: autofagia; V600EBRAF; tiroides; apoptosis

Resumen

El cáncer de tiroides es el tumor endocrino más común y, aunque es altamente curativo, presenta una elevada tasa de recurrencia [1]. Una de las mutaciones más frecuentes en este tipo de tumores es la sustitución de la Valina 600 por un Glutámico (V600E) en la proteína BRAF. Esta mutación causa una hiperactivación de la vía de las ERK-MAPKs, favoreciendo la proliferación e invasividad tumoral e inhibiendo la muerte celular por apoptosis [2,3]. Sin embargo, las terapias con inhibidores de BRAF no dan los resultados esperados en el tratamiento del cáncer de tiroides, ya que causa importantes efectos adversos de lesiones de la piel asociadas a hiperproliferación llegando, en algunos casos, a producirse carcinomas de células escamosas y queratoacantomas en un 15-30% de los pacientes [4]. Debido a esto, se plantea la necesidad de utilizar terapias combinadas para aumentar la efectividad y especificidad del tratamiento en este tipo de cáncer. Por otra parte, la autofagia es un proceso celular por el cual se produce un reciclaje de los componentes celulares, mediante su incorporación a lisosomas a través de la formación de unas vesículas denominadas autofagosomas [5]. Este proceso ha adquirido gran importancia en el estudio del cáncer y se le han atribuido tanto efectos pro-tumorales, ya que actúa como método de defensa frente a situaciones de estrés celular, como anti-tumorales llevando a la muerte celular [6,7]. Por todo ello, estudiamos el papel que posee la autofagia en células tumorales tiroideas que presentan la mutación ^V600EBRAF sobre la viabilidad celular y la sensibilidad al tratamiento con PLX4720, el inhibidor de ^V600EBRAF. Para ello, en primer lugar analizamos como la inhibición de BRAF, por el tratamiento inhibitorio con PLX4720 o silenciamiento con siRNA, modula la autofagia en la línea celular derivada de un tumor anaplásico tiroideo BHT101. La eliminación de la actividad y expresión de BRAF disminuye los niveles de la proteína p62 y aumenta los de la proteína LC3-II, indicativos de activación del proceso de autofagia. Esta modulación parece llevarla a cabo a través de la regulación de AMPK y mTOR, principales reguladores del proceso autofágico [8]. Respecto a la viabilidad celular y la apoptosis, la determinamos mediante MTT y citometría de flujo, tras el tratamiento con los inhibidores de la autofagia Bafilomicina A1 o 3-metiladenina (3-MA), en las células BHT101. Nuestros resultados indican que la inhibición de la autofagia aumenta la muerte celular en estas células. Por otra parte, el tratamiento con PLX4720 junto con la inhibición de la autofagia produce mayores efectos sobre la supervivencia que cada tratamiento por separado.Todos estos datos indican que la autofagia es un proceso modulador de la supervivencia de las células tumorales tiroideas y podría ser utilizada como una diana terapéutica en los tumores tiroideos con la mutación ^V600EBRAF que no responden adecuadamente al tratamiento convencional con PLX4720.

1. Migzhao, Xing. 2013. Molecular pathogenesis and mechanisms of thyroid cancer. Nature Reviews/Cancer, 13:184-99.
2. Kam-Tsun, Tang y Chen Hsen, Lee. 2010. BRAF mutation in papillary thyroid carcinoma: pathogenic role and clinical implicaions. J Chin Med Assoc., 73 (3):113-128.
3. Karasarides M, Chiloeches A, Hayward R, Niculescu-Duvaz D, Scanlon I, Friedlos F, y cols. 2004. B‐RAF is a therapeutic target in melanoma. Oncogene, 23(37): 6292‐
4. Flaherty, K., Puzanov, I., Sosman, J., Kim, K., Ribas, A., McArthur, G., et al. 2009. Phase I study of PLX4032: Proof of concept for V600EBRAF mutation as a therapeutic target. J Clin Oncol., 27.
5. Boya P, Reggiori F, Codogno P. Emerging regulation and functions of autophagy. Nat Cell Biol. 15: 713-20.
6. Lin CI, Whang EE, Abramson MA, Jiang X, Price BD, Donner DB, Moore FD Jr, Ruan DT. 2009. Autophagy: a new target for advanced papillary thyroid cancer therapy. Sugery, 146: 1208-14.
7. Vucicevic L, Misirkic M, Janjetovic K, Vilimanovich U, Sudar E, Isenovic E, Prica M, Harhaji-Trajkovic L, Kravic-Stevovic T, Bumbasirevic V y Trajkovic V. 2011. Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway. Autophagy, 7(1): 40-50.
8. Eskelinen E. 2011. The dual role of autophagy in cancer. Current Opinion in Pharmacology, 11:294–300.