Abstract: The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in stress-induced cell-fate decisions by orchestrating responses that go from cell-cycle arrest to apoptosis. We have identified a new p38 MAPK-regulated protein that we named p18(Hamlet), which becomes stabilized and accumulates in response to certain genotoxic stresses such as UV or cisplatin treatment. Overexpression of p18(Hamlet) is sufficient to induce apoptosis, whereas its downregulation reduces the apoptotic response to these DNA damage-inducing agents. We show that p18(Hamlet) interacts with p53 and stimulates the transcription of several proapoptotic p53 target genes such as PUMA and NOXA. This correlates with enhanced p18(Hamlet)-induced recruitment of p53 to the promoters. In proliferating cells, low steady-state levels of p18(Hamlet) are probably maintained by a p53-dependent negative feedback loop. Therefore, p18(Hamlet) is a new cell-fate regulator that links the p38 MAPK and p53 pathways and contributes to the establishment of p53-regulated stress responses.

Abstract: p38alpha is a stress-activated protein kinase that negatively regulates malignant transformation induced by oncogenic H-Ras, although the mechanisms involved are not fully understood. Here, we show that p38alpha is not a general inhibitor of oncogenic signaling, but that it specifically modulates transformation induced by oncogenes that produce reactive oxygen species (ROS). This inhibitory effect is due to the ROS-induced activation of p38alpha early in the process of transformation, which induces apoptosis and prevents the accumulation of ROS and their carcinogenic effects. Accordingly, highly tumorigenic cancer cell lines have developed a mechanism to uncouple p38alpha activation from ROS production. Our results indicate that oxidative stress sensing plays a key role in the inhibition of tumor initiation by p38alpha.

Abstract: p38 MAP kinases play an important role in the regulation of cellular responses to all kinds of stresses, but are also involved in many other cellular processes. We are using cell lines deficient in p38a, the most abundant and broadly expressed p38 MAPK, to investigate the role of this signalling pathway in oncogene-induced cell proliferation, apoptosis and malignant transformation. Consistent with the bibliography, we found that oncogenic H-RasV12 induces a more refringent morphology (a hallmark of cell transformation) in p38a-/- than in wild-type MEFs. HRasV12-expressing p38a-/- MEFs produce also many more colonies when grown in soft agar (measure of anchorage-independent growth) and are less adherent than HRasV12-transduced wild-type MEFs. The inhibitory effect of p38 MAPK on HRasV12-driven transformation was confirmed in NIH3T3 fibroblasts expressing the p38 MAPK activator MKK6. Interestingly, p38 MAP kinases do not appear to generally inhibit focus formation and anchorage independent growth induced by all oncogenes, but seem to be rather specific modulators of the Ras signalling pathway. We have also found that p38a-knockout cells are able to proliferate better in the presence of low serum concentrations or when transduced with oncogenes that upregulate cyclin D1 levels. Interestingly, p38a-/- cells can achieve a higher saturation density compared to wt MEFs, suggesting a role for p38a in contact inhibition. Of note, cells lacking p38a are also more resistant to apoptosis induced by oncogenic signalling. While all the above results suggest a negative role for p38 MAPK in tumourogenesis, we also found that p38a-/- cells have impaired motility, which correlates with a reduced number of focal adhesions as well as a less robust actin cytoskeleton. Our results indicate that p38 MAP kinases may modulate cancer cell-associated traits at multiple levels.

Abstract: Oncogenic Ras signaling has been long known to play an important role in tumorigenesis and human cancer. In this report, we have used the sensitive 2-D-DIGE coupled to MS for the identification of proteins differentially expressed at the cell membrane level between oncogenic H-RasV12-transformed wild-type and p38alpha-deficient mouse embryo fibroblasts (MEFs). Following trifluoroethanol solubilization, 76 proteins were found to be differentially regulated. After PMF, 63 spots containing 42 different proteins were unequivocally identified by MALDI-TOF MS coupled with database interrogation. As expected, many of them were membrane proteins. Six proteins were selected for further validation studies based on their potential functional link with malignant transformation and signal transduction. These were prohibitin (PHB), protein disulfide isomerase 3 (PDIA3), focal adhesion kinase 2 (FAK2), c-GMP dependent protein kinase 2 (KGP2), NADH-ubiquinone oxidoreductase 30 kDa subunit (NUGM) and translationally controlled tumor protein (TCTP). All these proteins were up-regulated in the membranes of H-RasV12-transformed p38alpha-/-cells, except for prohibitin, which was down-regulated. An excellent correlation was found between DIGE results and Western blot studies, indicating the reliability of the 2-D-DIGE analysis. The available evidence about the putative function of the identified proteins supports the emerging role of p38alpha as a negative regulator of tumorigenesis. Further studies are in progress to elucidate the implications of these findings in the regulation of H-Ras-induced transformation by p38alpha signaling.

Abstract: Cell migration is critical for many processes, such as angiogenesis, inflammation, development and wound healing, and is also involved in tumour progression and metastasis. Here we show that CXCL12, complement factor 5a (C5a), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF)-BB, which stimulate cell migration, also activate p38alpha MAPK. Pharmacological inhibition of this protein kinase with SB 203580 or BIRB 0796, or the genetic ablation of p38alpha MAPK, blocked cell migration induced by the aforementioned chemo-attractants. Macrophages from mice lacking one or more of the other p38 MAPK isoforms showed normal cell migration in response to C5a. We also show that the activation of p38alpha MAPK in response to CXCL12 requires the p21-activated protein kinases (PAK)-1 and PAK-2. MAPKAP-K2 is a protein kinase that is activated by p38alpha MAPK. Reducing its expression using RNA interference blocked CXCL12-induced HeLa cell migration, while macrophages from mice that do not express MAPKAP-K2 failed to migrate in response to C5a. Moreover, RNA interference against the small heat shock protein 27 (HSP27), a physiological substrate of MAPKAP-K2, blocked the CXCL12-induced cell migration. These results demonstrate a general and essential role of the PAK-p38alpha MAPK-MAPKAP-K2-HSP27 signalling pathway in mediating the effects of chemotactic stimuli on cell migration.

Abstract: Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.

Abstract: Proliferation of nontransformed cells is regulated by cell-cell contacts, which are referred to as contact-inhibition. Despite its generally accepted importance for cell cycle control, knowledge about the intracellular signalling pathways involved in contact inhibition is scarce. In the present work we show that p38alpha mitogen-activated protein kinase (MAPK) is involved in the growth-inhibitory signalling cascade of contact inhibition in fibroblasts. p38alpha activity is increased in confluent cultures of human fibroblasts compared to proliferating cultures. Time course studies show a sustained activation of p38alpha in response to cell-cell contacts in contrast to a transient activation after serum stimulation. The induction of contact inhibition by addition of glutaraldehyde-fixed cells is impaired by pharmacological inhibition of p38 as well as in p38alpha-/- fibroblasts. Further evidence for a central role of p38alpha in contact inhibition comes from the observation that p38alpha-/- fibroblasts show a higher saturation density compared to wild-type (wt) fibroblasts, which is reversed by reconstituted expression of p38alpha. In agreement with a defect in contact inhibition, p27(Kip1) accumulation is impaired in p38alpha-/- fibroblasts compared to wt fibroblasts. Hence, our work shows a new role for p38alpha in contact inhibition and provides a mechanistic basis for the recently proposed tumour suppressive function of this MAPK pathway.

Abstract: p38alpha is a stress-activated protein kinase that can suppress tumor formation by negatively regulating cell cycle progression or by inducing apoptosis. More recently, the ability of p38alpha to induce cell differentiation has also been connected to tumor suppression. Accordingly, several proteins that can potentially down-regulate the activity of p38alpha have been found over-expressed in human tumors and cancer cell lines. However, p38alpha can impinge on cancer progression by modulating other cellular responses, in addition to proliferation and differentiation, such as cell migration as well as the processes of invasion and inflammation. This could explain why, in some cancer types, p38alpha activation has been correlated with malignancy and poor prognosis rather than with tumor suppression. Here, we will review the evidence connecting p38alpha to distinct cancer traits and will discuss the mechanisms that may account for the oncogenic and tumor suppressor roles of p38alpha.

Abstract:

Abstract: 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 p38alpha 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 p38alpha-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 p38alpha regulates the process of malignant transformation at various levels. First, p38alpha 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 p38alpha, which may be accounted for by the negative effect of p38alpha on cyclin D1 expression. Similarly, p38alpha controls the process of cell-cell contact-inhibition, which requires p27Kip1 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 p38alpha regulates the membrane composition of oncogene-transformed cells. In addition to its negative role in cell proliferation, I have shown that p38alpha can interfere with the process of malignant transformation by sensing oxidative stress and inducing apoptosis. Thus, p38alpha 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 p38alpha function. Finally, in contrast to its anti-proliferative and pro-apoptotic roles, I have found that p38alpha is an important mediator of cytokine-induce cell migration, a process that is thought to be important for cancer cell metastasis.