The LIM-Only Protein FHL2 Mediates Ras-Induced Transformation through Cyclin D1 and p53 Pathways

et al. (2008) The LIM-Only Protein FHL2 Mediates Ras-Induced Transformation through Cyclin D1 and p53 Pathways. PLoS ONE 3(11): e3761. doi:10.1371/journal.pone.0003761 The LIM-Only Protein FHL2 Mediates Ras-Induced Transformation through Cyclin D1 and p53 Pathways Charlotte Labalette 0 Yann Noue t 0 Florence Levillayer 0 Carolina Armengol 0 Claire-Ange lique 0 Renard 0 Guillaume Soubigou 0 Tian Xia 0 Marie-Annick Buendia 0 Yu Wei 0 Terry Means, MGH/Harvard University, United States of America 0 1 Institut Pasteur, Unite d'Oncogene`se et Virologie Mole culaire, Paris, France, 2 Inserm U579, Paris, France, 3 Institut Pasteur, PT ''Puce a` AND'' , Paris , France Background: Four and a half LIM-only protein 2 (FHL2) has been implicated in multiple signaling pathways that regulate cell growth and tissue homeostasis. We reported previously that FHL2 regulates cyclin D1 expression and that immortalized FHL2-null mouse embryo fibroblasts (MEFs) display reduced levels of cyclin D1 and low proliferative activity. Methodology/Principal Findings: Here we address the contribution of FHL2 in cell transformation by investigating the effects of oncogenic Ras in FHL2-null context. We show that H-RasV12 provokes cell cycle arrest accompanied by accumulation of p53 and p16INK4a in immortalized FHL22/2 MEFs. These features contrast sharply with Ras transforming activity in wild type cell lines. We further show that establishment of FHL2-null cell lines differs from conventional immortalization scheme by retaining functional p19ARF/p53 checkpoint that is required for cell cycle arrest imposed by Ras. However, after serial passages of Ras-expressing FHL22/2 cells, dramatic increase in the levels of D-type cyclins and Rb phosphorylation correlates with the onset of cell proliferation and transformation without disrupting the p19ARF/p53 pathway. Interestingly, primary FHL2-null cells overexpressing cyclin D1 undergo a classical immortalization process leading to loss of the p19ARF/p53 checkpoint and susceptibility to Ras transformation. Conclusions/Significance: Our findings uncover a novel aspect of cellular responses to mitogenic stimulation and illustrate a critical role of FHL2 in the signalling network that implicates Ras, cyclin D1 and p53. - Funding: This work was supported in part by grants from the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer (Comite de Paris) and the Institut National du Cancer. C.L. was supported by the Ecole Normale Superieure de Lyon and Y.N. by the Canceropole Ile-de-France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Four and a half LIM-only protein 2 (FHL2) is a multi-functional protein that shuttles between focal adhesion sites and nucleus. Focal adhesions are specialized adhesive junctions where integrin receptors for extracellular matrix (ECM) are concentrated [1]. FHL2 interacts with a- and b-integrin subunits and focal adhesion kinase (FAK) [2,3] and is involved in assembly of ECM proteins on the cell surface [4]. Deregulation of genes involved in focal adhesion, ECM-receptor interaction and response to external stimuli has been evidenced in mouse embryo fibroblasts (MEF) deficient for FHL2 [5], suggesting that FHL2 plays important roles in tissue physiology and homeostasis and in the transduction of signals emitted from extracellular environment. Indeed, in response to RhoA signals or serum stimulation, FHL2 is translocated to the nucleus [6,7] where it acts as either transcription coactivator or corepressor in interaction with numerous transcription factors including the androgen receptor, AP1, CREB, PLZF, SKI, b-catenin, FOXO1, Runx2 and serum response factor (SRF) [7,8,9,10,11,12,13,14,15,16]. The ability of FHL2 to assemble multiple partners into protein complexes is attributed to its LIM-domain structure that serves as protein binding interface through double zinc finger motifs [17]. Thus, FHL2 participates in regulating expression of a large spectrum of genes involved in cell proliferation, differentiation, transformation and apoptosis. Despite absence of gross defects during development, FHL2-deficient mice harbor cardiac hypertrophy in response to b-adrenergic stimulation, osteopenia due to decreased activity of osteoblasts and wound healing defect [15,18,19]. Several lines of evidence have implicated FHL2 in the control of cell proliferation. We have recently shown that FHL2 physically occupies the cyclin D1 promoter and directly stimulates cyclin D1 transcription [5]. Accordingly, we found reduced expression levels of cyclin D1 in MEFs isolated from FHL2-null mice. After spontaneous immortalization, these cells exhibited weak proliferative capacity associated with low expression levels of all D-type cyclins and cyclin E, as well as cyclin-dependent kinase (Cdk) inhibitors, in particular p16INK4a [5,20]. It has been shown that FHL2 can inhibit the antiproliferative functions of the transcription factor E4F1 by inhibiting the transcription repressor function and p53 binding activity of E4F1 [21]. In gastric and colon cancer cell lines, suppression of FHL2 inhibited anchorage-dependent and -independent cell growth, and tumor formation in nude mice xenograft [22]. In line with its effects on cell growth, overexpression of FHL2 has been detected in various types of cancer [13,22,23,24]. Moreover, androgen-induced nuclear accumulation of FHL2 in highly malignant prostate carcinoma has been linked to disease progression and poor outcome [6,25]. In this study, we sought to gain further insight into the role of FHL2 in neoplastic transformation by investigating the effects of oncogenic Ras in a FHL2-deficient context. In primary rodent cells, activated Ras mutant induces premature senescence associated with activation of p16INK4a and p53 [26]. Cellular senescence involves an essentially irreversible cell cycle arrest that is associated with heterochromatin formation mediated by the retinoblastoma (Rb) tumor suppressor [27]. The ARF/p53 and p16INK4a/Rb pathways are clearly critical for establishing the senescence growth arrest, and primary MEFs deficient for p19ARF and p53 exhibit resistance to Ras-induced senescence and are susceptible to transformation by activated Ras alone [28]. In general, cooperation of Ras and additional immortalizing factors is required for transformation of primary rodent cells. However, oncogenic Ras alone readily transforms immortalized cells lines, since 80% of these cells contain mutant p53, whereas most others sustain biallelic loss of the INK4a/ARF locus [29]. Here, we report that H-RasV12 provokes a cell cycle arrest in spontaneously immortalized FHL2-null cells, in contrast to cell transformation in wild type (wt) counterparts. The cell growth arrest induced by Ras is controlled by p53 which remains wt after spontaneo (...truncated)