The pathogenesis of mesothelioma is driven by a dysregulated translatome.

Funder: Department of HealthMalignant mesothelioma (MpM) is an aggressive, invariably fatal tumour that is causally linked with asbestos exposure. The disease primarily results from loss of tumour suppressor gene function and there are no 'druggable' driver oncogenes associated with MpM. To identify opportunities for management of this disease we have carried out polysome profiling to define the MpM translatome. We show that in MpM there is a selective increase in the translation of mRNAs encoding proteins required for ribosome assembly and mitochondrial biogenesis. This results in an enhanced rate of mRNA translation, abnormal mitochondrial morphology and oxygen consumption, and a reprogramming of metabolic outputs. These alterations delimit the cellular capacity for protein biosynthesis, accelerate growth and drive disease progression. Importantly, we show that inhibition of mRNA translation, particularly through combined pharmacological targeting of mTORC1 and 2, reverses these changes and inhibits malignant cell growth in vitro and in ex-vivo tumour tissue from patients with end-stage disease. Critically, we show that these pharmacological interventions prolong survival in animal models of asbestos-induced mesothelioma, providing the basis for a targeted, viable therapeutic option for patients with this incurable disease

Importancia de la fase G1 del ciclo celular en medios pobres en nitrógeno en Schizosaccharomyces pombe

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Salamanca.--2017-06-22In fission yeast, the cell size at division depends on the growth conditions (Fantes and Nurse, 1977). Cells growing in nitrogen-rich media divide with a large cell size. On the contrary, cells growing in nitrogen-poor media divide with a small cell size. Recently, our group has described that the Greatwall-Endosulfine pathway (Ppk18- Igo1) couples cell growth and the nutritional environment to the cell cycle machinery by modulating the activity of PP2A/B55 protein phosphatase, that counteracts Cdk1/cyclinB substrate phosphorylation at the onset of mitosis (Chica et al., 2016). In nitrogen-rich medium, fully active TORC1 inhibits Greatwall leading to high PP2A/B55 activity. As a consequence, cells spend a long time in G2 before they initiate mitosis. In nitrogen poor media, TORC1 activity is reduced leading to the activation of Greatwall and phosphorylation of its main target Endosulfine. Phosphorylated Endosulfine is a potent and specific inhibitor of PP2A/B55. Low activity of PP2A/B55 allows entry into mitosis with reduced Cdk/CyclinB activity and a small cell size. After division, these cells delay the cell cycle in G1 because the newly born cells are too small to initiate S-phase, as a consequence a population of cells with 1C peak is detected by flow cytometry (Carlson et al., 1999).The timing and orderly progression through the different cell cycle events are regulated by cyclin-dependent kinase (CDK) activity oscillations (Coudreuse and Nurse, 2010). Rum1 and Ste9 are both G1 negative regulators, which maintain a low CDK activity during G1 until entry into S-phase. Rum1 is a CDK inhibitor (CKI) of Cdc2-Cig2 and Cdc2-Cdc13 complexes (Moreno and Nurse, 1994; Correa-Bordes et al, 1997; Benito et al, 1998), whereas Ste9 is an activator of Anaphase Promoting Complex (APC) that promotes the degradation of Cig1, Cig2 and Cdc13 cyclins in G1 (Kitamura et al., 1998; Blanco et al., 2000; Yamano, 2000, 2004). Rum1 and Ste9 have been described as necessary for G1 arrest in minimal medium without nitrogen and for mating (Moreno and Nurse, 1994; Stern and Nurse, 1998; Kominami et al., 1998). The double mutant rum1Δ ste9Δ does not show mitotic defects although it shows higher Cdc13 levels than wild-type cells (Blanco et al., 2000). Thus, both rum1+ and ste9+ single deletion mutants do not show any phenotypes in rich media apart from being sterile and unable to arrest in G1 in minimal medium without nitrogen (MMN). In this work, we have characterized the phenotype of the double mutant rum1Δ ste9Δ in poor nitrogen medium, where the cells grow with a reduced cell size and they have to extend the G1-phase before they undergo S-phase. We also show that Rum1 and Ste9 are required to prevent genomic instability in MMPhe, where they are up regulated. In rich media, there is no apparent defect of rum1+ and ste9+ deletions, although the double mutant shows HU sensitivity. However, when the mutant cells are grown in nitrogen poor medium, they show a slow S-phase, high levels of DNA damage and cell cycle delay in G2 promoted by the activation of the DNA damage checkpoint. This phenotype is rescued by extending the G2-phase or by reducing the CDK activity.It has been previously described that Sic1, the budding yeast orthologue of Rum1, promotes origin licensing G1 by inhibiting CDK activity and that sic1Δ cells accumulate double strand breaks (DSB) and genomic instability (Lengronne and Schwob, 2002). Also Cdh1, the budding yeast Ste9 orthologue, together with Sic1 are required to prevent chromosome instability by promoting efficient origin firing (Ayuda-Duran et al., 2014). Moreover, in mouse primary MEFs, acute depletion or permanent ablation of Cdh1 caused genomic instability by slowing down DNA replication fork movement and increased origin activity. Partial inhibition of origin firing did not accelerate replication forks, suggesting that fork progression is intrinsically limited in the absence of Cdh1 (García-Higuera et al., 2008; Garzón et al., in press). Since budding yeast and mammalian cells have a long G1 phase, it makes sense that the fission yeast rum1Δ ste9Δ cells only shows endogenous DNA damage in poor-nitrogen medium, where the G1-phase is extended.To promote the G1/S transition, the CDK activates the MluI cell cycle box-binding factor (MBF) complex (Reymond et al., 1993; Banyai et al., 2016), the fission yeast functional orthologue of mammalian E2F, that activates the expression of genes involved in the initiation of DNA replication, nucleotide biogenesis and the regulation of S-phase (Bahler, 2005). The MBF complex is bound to its target promoters throughout the cell cycle and it is tightly regulated by positive and negative regulators that restrict MBF activation to the G1/S transition (Wuarin et al., 2002). Rep2 is a MBF transcriptional activator, whereas Nrm1 and Yox1 are repressors of MBF during Sphase and G2 (Baum et al., 1997; Nakashima et al., 1995; De Bruin et al., 2006; Aligianni et al., 2009). In S-phase, MBF could be activated during replication stress by down-regulation of Nrm1 (de Bruin et al., 2008) and Yox1 after phosphorylation by Cds1 (Ivanova et al., 2011, Aligianni et al., 2009, Purtill et al., 2011). By contrast, when the DNA damage checkpoint is active, Chk1 phosphorylates Cdc10, one of the subunits of MBF, and promotes its release from some target promoters repressing MBF-dependent transcription (Ivanova et al., 2013).Our data suggest that MBF-dependent transcription is impaired in the rum1Δ ste9Δ mutant both in nitrogen-rich and in nitrogen-poor medium. Although it has been described that mutants with higher MBF activity (yox1Δ or nrm1Δ) present genomic instability under normal laboratory conditions (nitrogen-rich media) (Gómez-Escoda et al., 2011; Caetano et al., 2014), we have shown that an up-regulation of MBF in MMPhe is less detrimental in nitrogen-poor medium. In fact, reduced MBF activity in rep2Δ cells growing in MMPhe generated higher levels of DNA damage. Our results indicate that a proper pulse of MBF activity at the end of G1 is particularly critical in MMPhe. A reduced dNTP pool may play a role in the genomic instability of the rum1Δ ste9Δ mutant in MMPhe. This could be caused by a defective degradation of Spd1, which prevents the assembly of an active RNR (Liu et al., 2003; Hakansson et al., 2005). Spd1 is normally targeted for degradation by the CRL4Cdt2 ubiquitin ligase at the onset of S-phase. Cdt2 is one of the targets of the MBF transcription factor (Liu et al., 2003, 2005; Holmberg et al., 2005; Hofmann and Beach, 1994). Thus, a defective MBF activation could be responsible for the reduced expression of RNR and high levels of Spd1. Consistent with this hypothesis deletion of spd1+ partially rescued the rum1Δ ste9Δ phenotype. Deletion of nrm1+ also rescued the DNA damage phenotype of the double mutant rum1Δ ste9Δ. However, this effect could be caused not only by an increase in the MBF activity, but also by an extension of G2, as it happens in nitrogen rich medium.In sum, we found that Rum1 and Ste9 are up regulated in nitrogen poor medium MMPhe. These activities are required to promote a delay in G1/S and to regulate MBF activity. In MMPhe, a delay in the MBF-dependent transcription is necessary to generate enough levels of transcripts required for S-phase.Peer reviewe

Nutritional cell cycle reprogramming reveals that inhibition of Cdk1 is required for proper MBF-dependent transcription

In nature, cells and in particular unicellular microorganisms are exposed to a variety of nutritional environments. Fission yeast cells cultured in nitrogen-rich media grow fast, divide with a large size and show a short G1 and a long G2. However, when cultured in nitrogen-poor media, they exhibit reduced growth rate and cell size and a long G1 and a short G2. In this study, we compared the phenotypes of cells lacking the highly conserved cyclin-dependent kinase (Cdk) inhibitor Rum1 and the anaphase-promoting complex/cyclosome (APC/C) activator Ste9 in nitrogen-rich and nitrogen-poor media. Rum1 and Ste9 are dispensable for cell division in nitrogen-rich medium. However, in nitrogen-poor medium they are essential for generating a proper wave of MluI cell-cycle box binding factor (MBF)-dependent transcription at the end of G1, which is crucial for promoting a successful S phase. Mutants lacking Rum1 and Ste9 showed premature entry into S phase and a reduced wave of MBF-dependent transcription, leading to replication stress, DNA damage and G2 cell cycle arrest. This work demonstrates how reprogramming the cell cycle by changing the nutritional environment may reveal new roles for cell cycle regulators.This research was funded by grants from the Ministerio de Economıa y ́ Competitividad (BFU2011-28274, BFU2014-55439-R and BFU2017-88335-R) and the Junta de Castilla y León (CSI084U16). A.R. and A.V.-B. were recipients of FPI pre-doctoral training grants and N.G.-B. received an FPU pre-doctoral training grant.Peer reviewe

The fission yeast Greatwall-ENSA(ARPP19)-PP2A pathway links the nutritional environment to mitotic entry

Resumen del trabajo presentado a la EMBO Conference on Fission Yeast: Pombe 2013; 7th International Fission Yeast Meeting celebrada en Londres (UK) del 24 al 29 de junio de 2013.Entry into mitosis in fission yeast requires the activity of the Cdc2/Cdc13 cyclin complex. The Wee1/Cdc25 switch ensures proper timing of mitotic entry by regulating Cdc2 activity. In addition to Cdc2/Cdc13, an opposing phosphatase might contribute to the switch. According to other biological models, a suitable candidate is the protein phosphatase type 2A (PP2A) that dephosphorylates Cdk1 substrates, including Cdc25 and Wee1. Using a genome wide synthetic lethal screening with wee1-50 and cdc2-3w, we have identified one of the catalytic subunits of PP2A, Ppa2, and some of its activators (Ypa1 and Ypa2). Further dissection of this pathway, including the fission yeast orthologues of Greatwall and ENSA-ARPP19 (Ppk18 and Igo1), suggests that this pathway connects the nutritional environment (in particular nitrogen) to the mitotic switch. We will present evidence that Ppk18-Igo1-PP2A may connect TOR signalling to the mitotic switch.Peer Reviewe

Nutritional control of cell size by the Greatwall-endosulfine-PP2A/B55 pathway

Trabajo presentado en el The Society of Experimental Biology Annual Meeting, celebrado en Gotemburgo (Suecia), del 3 al 6 de julio de 2017Peer reviewe

Extracellular Vesicles Isolated from Malignant Mesothelioma Cancer-Associated Fibroblasts Induce Pro-Oncogenic Changes in Healthy Mesothelial Cells.

Peer reviewed: TrueMalignant mesothelioma is an aggressive tumour of the pleura (MPM) or peritoneum with a clinical presentation at an advanced stage of the disease. Current therapies only marginally improve survival and there is an urgent need to identify new treatments. Carcinoma-associated fibroblasts (CAFs) represent the main component of a vast stroma within MPM and play an important role in the tumour microenvironment. The influence of CAFs on cancer progression, aggressiveness and metastasis is well understood; however, the role of CAF-derived extracellular vesicles (CAF-EVs) in the promotion of tumour development and invasiveness is underexplored. We purified CAF-EVs from MPM-associated cells and healthy dermal human fibroblasts and examined their effect on cell proliferation and motility. The data show that exposure of healthy mesothelial cells to EVs derived from CAFs, but not from normal dermal human fibroblasts (NDHF) resulted in activating pro-oncogenic signalling pathways and increased proliferation and motility. Consistent with its role in suppressing Yes-Associated Protein (YAP) activation (which in MPM is a result of Hippo pathway inactivation), treatment with Simvastatin ameliorated the pro-oncogenic effects instigated by CAF-EVs by mechanisms involving both a reduction in EV number and changes in EV cargo. Collectively, these data determine the significance of CAF-derived EVs in mesothelioma development and progression and suggest new targets in cancer therapy