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Regulation of malignant cell transformation by the stress-activated kinase {p38α [p38-alpha] [Elektronische Ressource] / Ignacio Dolado Pérez

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184 pages
The Joint Ph.D. Programme of the European Molecular Biology Laboratory (EMBL) and the Faculty of Natural Sciences of the Ruprecht-Karls University of Heidelberg Ignacio Dolado Pérez Regulation of malignant cell transformation by the stress-activated kinase p38 α Ph.D. Thesis Heidelberg, Germany June 2007 Dissertation submitted to the Joint Ph.D. Programme of the European Molecular Biology Laboratory (EMBL) and the Faculty of Natural Sciences of the University Ruprecht-Karls of Heidelberg (Germany), for the degree of Doctor of Natural Sciences Diploma chemist: Ignacio Dolado Pérez Born in: Alicante, Spain Regulation of malignant cell transformation by the stress-activated kinase p38 α Referees: Dr. Mathias Treier, Developmental Biology Programme, EMBL, Heidelberg, Germany Prof. Dr. Claus R. Bartram, Institut für Humangenetik, Heidelberg, Germany This thesis work was carried out at the European Molecular Biology Laboratory (EMBL) in Heidelberg (Germany) from 2002-2004 and at the Spanish Cancer Center (CNIO) in Madrid (Spain) from 2004-2006; under the supervision of Dr. Ángel R. Nebreda. To my mother, who demonstrated incredible bravery in fighting cancer and always inspired my life. We miss you terribly.
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The Joint Ph.D. Programme of
the European Molecular Biology Laboratory (EMBL) and
the Faculty of Natural Sciences of the Ruprecht-Karls University of Heidelberg







Ignacio Dolado Pérez


Regulation of malignant cell transformation
by the stress-activated kinase p38 α


Ph.D. Thesis











Heidelberg, Germany
June 2007








Dissertation
submitted to the
Joint Ph.D. Programme of the European Molecular Biology Laboratory (EMBL) and the Faculty
of Natural Sciences of the University Ruprecht-Karls of Heidelberg (Germany), for the degree of
Doctor of Natural Sciences


















Diploma chemist: Ignacio Dolado Pérez
Born in: Alicante, Spain













Regulation of malignant cell transformation
by the stress-activated kinase p38 α



















Referees:
Dr. Mathias Treier, Developmental Biology Programme, EMBL, Heidelberg, Germany
Prof. Dr. Claus R. Bartram, Institut für Humangenetik, Heidelberg, Germany This thesis work was carried out at the European Molecular Biology Laboratory (EMBL) in
Heidelberg (Germany) from 2002-2004 and at the Spanish Cancer Center (CNIO) in Madrid
(Spain) from 2004-2006; under the supervision of Dr. Ángel R. Nebreda.





To my mother,

who demonstrated incredible bravery
in fighting cancer and always inspired
my life.

We miss you terribly.











“ Cuando nací, un agosto
ardiente calcinaba las casas.
Al romper en llanto, sintió
mi madre un nudo en la garganta.
Del beso que posó sobre mi cara,
aún noto la tibieza de sus labios.
De sus brazos, que acogieron mi cuerpo,
con ternura, todavía los siento [...]

Y me estremezco ante un beso,
y me quebranto ante un llanto.
Por eso quisiera que mis hijos
amaran como yo,
como mi madre lo hizo ayer
con sus besos y abrazos.
Con esos besos y abrazos
que aún llevo impresos en
los pliegues de mi piel.”


Maria del Carmen Llavador. Palabras CONTENTS
TABLE OF CONTENTS


TABLE OF CONTENTS ............................................................................................................. I
ACKNOWLEDGEMENTS ...................................................................................................... III
SUMMARY ................................................................................................................................ IV
ZUSAMMENFASSUNG (SUMMARY IN GERMAN) .......................................................... V
RESUMEN (SUMMARY IN SPANISH) ................................................................................ VI
PUBLICATIONS ..................................................................................................................... VII
CONTRIBUTIONS TO PUBLICATIONS ......................................................................... VIII
ABBREVIATIONS ................................................................................................................... IX
INTRODUCTION ....................................................................................................................... 1
1. MALIGNANT TRANSFORMATION, TUMORIGENESIS AND CANCER ...................... 2
A) Concept definition .............................................................................................................. 2
B) Traits of the cancer cell and how to analyze them technically .......................................... 2
B.1) Immortalization .......................................................................................................... 3
B.2) Independence of extracellular growth signals ............................................................ 4
B.3) Independence of anti-growth signals ......................................................................... 6
B.4) Apoptosis evasion ...................................................................................................... 7
B.5) Sustained angiogenesis .............................................................................................. 8
B.6) Invasion and metastasis ............................................................................................. 9
C) Cancer at the molecular level ........................................................................................... 11
C.1) Cancer is an evolving and dynamic genetic disease ................................................ 11
C.2) Target cells in carcinogenesis .................................................................................. 13
C.3) Cancer as a systemic disease: beyond oncogenes and tumor suppressors .............. 15
C.4) The double-edged role of free radicals in cancer ..................................................... 16
C.4.1) The chemical nature of free radicals ............................................................... 16
C.4.2) Cancer as a disease of free radicals overload .................................................. 18
C.4.3) Free radicals in the clinics: a word of caution ................................................ 20
C.5) Detoxification enzymes in cancer: cleaner is not always better .............................. 21
C.5.1) Antioxidant versus detoxification enzymes .................................................... 21
C.5.2) Detoxification enzymes and cancer: a matter of equilibrium ......................... 23
D) Human cancer in the clinics: current status and future perspectives ............................... 25
2. MITOGEN-ACTIVATED PROTEIN KINASES ................................................................. 28
E) The mitogen-activated protein kinase (MAPK) family. A brief overview ...................... 28
F) Signal transduction by p38 MAPK .................................................................................. 31
F.1) Upstream activators .................................................................................................. 31
F.2) Downstream targets .................................................................................................. 32
iCONTENTS
F.3) Regulation of gene expression by p38 MAPK ......................................................... 33
F.3.1 Transcriptional regulation ........................................................................... 33
F.3.2 Translational regulation .............................................................................. 37
G) p38 MAPK roles in cancer .............................................................................................. 37
G.1 Tumor-suppressive roles: proliferation, differentiation and survival ....................... 38
G.2 Oncogenic activities: inflammation, invasion, angiogenesis and migration ............. 41
H) p38 MAPK as a putative target in cancer therapy? ......................................................... 43
OUTLOOK ................................................................................................................................. 44
BIBLIOGRAPHY ...................................................................................................................... 45
APPENDIX.. 59
• DOLADO I., NEBREDA A.R.
Regulation of tumorigenesis by p38 α MAP kinase.
In: Nebreda A.R. and Posas F. (Ed.): SAPKs - Stress-Activated Protein Kinases.
Springer-Verlag, Top Curr Genet. 19 (in press).................................................................... A1

• DOLADO I., SWAT A., AJENJO N., DE VITA G., CUADRADO A., NEBREDA A.R.
p38α MAP kinase as a sensor of reactive oxygen species in tumorigenesis.
Cancer Cell 2007 Feb;11(2):191-205.................................................................................... A2

• ALFONSO P., DOLADO I., SWAT A., NUNEZ A., CUADRADO A., NEBREDA A.R.,
CASAL J.I.
Proteomic analysis of p38 α mitogen-activated protein kinase-regulated changes in membrane
fractions of RAS-transformed fibroblasts.
Proteomics 2006 Apr;6 Suppl 1:S262-271............................................................................ A3

• FAUST D., DOLADO I., CUADRADO A., OESCH F., WEISS C., NEBREDA A.R.,
DIETRICH C.
p38α MAPK is required for contact inhibition.
Oncogene 2005 Nov 24;24(53):7941-7945........................................................................... A4

• ROUSSEAU S., DOLADO I., BEARDMORE V., SHAPIRO N., MARQUEZ R.,
NEBREDA A.R., ARTHUR S.C., TESSIER-LAVIGNE M., GAESTEL M., CUENDA A.,
COHEN P.
CXCL12 and C5a trigger cell migration via a PAK1/2-p38 α MAPK-MAPKAP-K2-HSP27
pathway.
Cell Signal. 2006 Nov;18(11):1897-1905.............................................................................. A5

• CUADRADO A., LAFARGA V., CHEUNG P., DOLADO I., LLANOS S., COHEN P.,
NEBREDA A.R.
A new p38 MAP kinase-regulated transcriptional coactivator that stimulates p53-dependent
apoptosis.
EMBO J. 2007 Apr 18;26(8):2115-2126............................................................................... A6
iiACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS

I still recall when I had my first interview with Angel at the EMBL, in particular when he asked
me the frightening question: “what would you like to do for your PhD in my laboratory?”. Truly,
I did not know.
What I knew was that I wanted to shift from chemistry into biology, in particular into the cancer
field, although I had no clue on how to undertake such step.
Luckily, just a few minutes earlier I had accidentally met Gustavo, a PhD student in Angel´s lab
at that time, who casually gave me an overview of the projects in the lab and told me about an
intriguing protein called p38 that, as he said: “it looks like it might be a tumor suppressor”. This
sounded fascinating.
So, I told Angel: “well, I would like to work with this tumor suppressor, this...p38, isn´t it?”.
And that is how it all started.

So, first of all, I am particularly grateful to my supervisor, Angel Nebreda, who has given me the
opportunity and the means during these years to learn how “big science” works and has guided
me to become a better scientist. My most sincere “thank you” as well for being so available in
the lab, for the “millions” of fruitful discussions that we have shared through these years, and for
always depositing your trust on me.

I would like to acknowledge the members of my Thesis Advisory Committee Juerg Müller,
Pernille Rørth and Claus R. Bartram, for helping me all through my PhD with their invaluable
scientific recommendations. I am especially grateful to Claus R. Bartram and Mathias Treier that
they agreed to review my thesis.

I am grateful to Manuel Serrano and Marcos Malumbres for providing so many useful reagents
and also to those with whom I have collaborated during my PhD, namely Simon Rousseau,
Cornelia Dietrich, Patricia Alfonso, Antonio Núñez, Ignacio Casal, Aneta Swat, Ana Cuadrado,
Nuria Ajenjo, and Gabriella de Vita. I am particularly in debt with Emma Black, who taught me
all the basics of cell and molecular biology at the beginning of my PhD. Thank you as well to
Iván del Barco, Ana Cuadrado and Stephan Tenbaum for reading and helping me edit the thesis
manuscript.

I would also like to express my gratitude to those with whom I have spent most of my time
during these years, my colleagues. I want to specially acknowledge Andrés Gaytan, Carmelo
López, Bea Herreros, Luis Vacs, Gustavo Gutiérrez, Bernadett Papp, Marcelo Viegas, Marlene
Rau and Hannes Simader. Thank you my friends, you made it worth!

Finally, but most importantly, I wish to acknowledge my family, in particular my mother, who
“forced” me to go abroad and pursuit this PhD despite her illness. It grieves me now without
measure that we cannot celebrate it together. Thank you mum for always being so strong.
I feel so lucky though as I will DO celebrate it with my beloved Aneta and Juanca.
Aneta, I cannot praise you enough, I would not have accomplished it all without your help and
support. Thank you for your love, your patience and for sharing your life with me.
Thank you so much as well to you, Juanca, my brother and best friend. I can never refrain a
proud smile when I think of you. Thank you for always being so encouraging and enthusiastic.
I am so happy to have you both by my side. You are the ultimate motivation in my life.
iiiSUMMARY
THESIS SUMMARY
Cancer is a dynamic process that requires the stepwise deregulation of mechanisms affecting
various cellular traits. During my PhD, I have characterized how the stress-activated p38 α
MAPK signaling pathway regulates the processes of cellular migration, proliferation, and
survival in the context of oncogene-induced malignant transformation, which recapitulates the
mechanisms of cancer initiation at the cellular level.
My studies have been mostly based on the use of human and mouse cultured cells, which I have
analyzed using both biochemical and cell biological approaches. In particular, the development
of p38a-deficient cell lines and the application of retrovirally-based gene expression techniques
have been very useful. The implementation of tools to measure the intracellular levels of reactive
oxygen species (ROS) within living cells has also been key for my work.
I have found that p38 α regulates the process of malignant transformation at various levels. First,
p38α negatively regulates cell cycle progression induced by mitogenic signals in both
exponentially proliferating and confluent cells. Oncogene-expressing cells proliferate faster in
the absence of p38 α, which may be accounted for by the negative effect of p38 α on cyclin D1
expression. Similarly, p38 α controls the process of cell-cell contact-inhibition, which requires
Kip1p27 accumulation and triggers G1-phase cell cycle arrest upon cell confluence. The process
of contact inhibition is likely to involve uncharacterized membrane-associated signaling events.
Accordingly, I have found that p38 α regulates the membrane composition of oncogene-
transformed cells. In addition to its negative role in cell proliferation, I have shown that p38 α can
interfere with the process of malignant transformation by sensing oxidative stress and inducing
apoptosis. Thus, p38 α becomes activated when oncogene-expressing cells accumulate high
levels of carcinogenic ROS and, in turn, induces the elimination of the transformed cells by
apoptosis. Interestingly, I have found that human cancer cell lines that contain high ROS levels
have developed a mechanism to by-pass this p38 α function. Finally, in contrast to its anti-
proliferative and pro-apoptotic roles, I have found that p38 α is an important mediator of
cytokine-induce cell migration, a process that is thought to be important for cancer cell
metastasis.
iv

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