The many faces of p57Kip2 : Acting in apoptosis, differentiation and cytoskeleton reorganisation

p57 is a known cyclin dependent kinase inhibitor, which has been suggested to be a tumor suppressor gene. Indeed various human cancers show a reduction in p57 expression, indicating that this protein might be of relevance in tumorigenesis. Mutated forms of p57 have rarely been detected in human tumors; rather epigenetic mechanisms are involved in inactivating the expression of the gene. Inactivation of the p57 Kip2 gene correlates with disease progression and poor prognosis for the patient, indicating that reactivation of p57 in cancer cells could possible inhibit tumor cell growth. Prior research has primarily focused on p57 ́s role as cell-cycle regulator, whereas the aim of this thesis is to study its function during other cellular events such as apoptosis, cytoskeleton reorganisation and differentiation of neuronal cells. We report that selective expression of p57 sensitizes tumor cells to cell death induced by different drugs. This function is independent of its role as a cyclin dependent kinase inhibitor. Our studies also show that p57 primarily promotes the mitochondrial apoptotic pathway. In accordance, we found that Bcl-2 overexpression or VDAC inhibition were able to inhibit p57 cell death promoting effect. We also reveal that p57 is a direct target gene for p73β, which is known for its pro-apoptotic properties. Moreover, the p73β-induced p57 expression contributes to mitochondrial events related to apoptotic cell death. Furthermore, we establish that p57 expression promotes actin stress fiber formation in cells. It interacts with, and activates the actin cytoskeleton modifying enzyme, LIMKinase-1. This activation resulted into increase phosphorylation, inactivation of cofilin and in a reduction of actin protein mobile fraction, ultimately, affecting negatively cell mobility. Remodelling of actin cytoskeleton plays a key role in cell migration and has implication for invasion and metastasis, supporting the proposal that p57 can act as a tumor suppressor gene by affecting the cytoskeleton. We further investigated the role of p57 in neural stem cell differentiation. We show that p57 transiently accumulates in the nuclei of neural progenitors during early astrocyte differentiation and represses neuronal differentiation. It can interact with a subset of pro-neuronal bHLH factors, including Mash1. p57 inhibits the transcriptional activity of Mash1 and thereby represses neuronal differentiation, possibly to allow proper glial differentiation. These findings once again confirm the importance of p57 during development and differentiation. In conclusion, our work shows that p57 plays essential roles during several cellular processes besides acting as a cell-cycle regulator. This thesis contributes to the understanding of p57’s role in apoptosis, differentiation and cytoskeleton reorganisation. LIST OF PUBLICATIONS This thesis is based on the following papers, which in the text will be referred to by their roman numerals (I-IV). I. Pinelopi Vlachos, Ulrika Nyman, Nabil Hajji and Bertrand Joseph. The cell cycle inhibitor p57(Kip2) promotes cell death via the mitochondrial apoptotic pathway. Cell Death Differ. 2007,14(8):1497-507. II. Ulrika Nyman*, Pinelopi Vlachos*, Naveen Reddy Muppani and Bertrand Joseph. The p73β transcriptional target gene p57 promotes p73β-mediated mitochondrial apoptotic cell death. Submitted to Cell Death Differ. III. Pinelopi Vlachos and Bertrand Joseph. The CDK inhibitor p57Kip2 controls LIM-Kinase-1 activity and regulates actin cytoskeleton dynamics. Oncogene, In press IV. Bertrand Joseph *, Emma Andersson*, Pinelopi Vlachos , Erik Södersten, Lining Liu, Ana Teixeira and Ola Hermanson. p57Kip2 is a repressor of Mash1 activity and neuronal differentiation in neural stem cells. Cell Death Differ. 2009 16(9):1256-65. * Authors contributed equally to this work LIST OF PUBLICATIONS NOT INCLUDED IN THE THESIS I. Nabil Hajji, Karolina Wallenborg, Pinelopi Vlachos, Ulrika Nyman, Ola Hermanson and Bertrand Joseph. Combinatorial action of the HDAC inhibitor trichostatin A and etoposide induces caspase-mediated AIF-dependent apoptotic cell death in non-small cell lung carcinoma cells. Oncogene. 2008, 27(22):3134-44. II. Karolina Wallenborg*, Pinelopi Vlachos*, Sofi Eriksson, Lucas Huijbregts, Elias SJ Arnér, Bertrand Joseph, and Ola Hermanson. Red wine triggers cell death and thioredoxin reductase inhibition: effects beyond resveratrol and SIRT1. Exp Cell Res. 2009, 315(8):1360-71. III. Ulrika Nyman, Pinelopi Vlachos, Anna Cascante, Ola Hermanson, Boris Zhivotovsky and Bertrand Joseph. Protein kinase C-dependent phosphorylation regulates the cell cycleinhibitory function of the p73 carboxy terminus transactivation domain. Mol Cell Biol. 2009, 29(7):1814-25.