Erin K Bojanek

Matthew W Mosconi

Stephen Guter

Catalina Betancur

Carol Macmillan

Edwin H Cook

Clinical and neurocognitive issues associated with Bosch-Boonstra-Schaaf optic atrophy syndrome: A case study

Keywords: autism, Bosch-Boonstra-Schaaf optic atrophy syndrome, cognitive discrepancy, NR2F1

Nuclear receptor subfamily 2 group F member 1 (NR2F1) is an orphan receptor and transcriptional regulator that is involved in neurogenesis, visual processing and development, and cortical patterning.

Alterations in NR2F1 cause Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), a recently described autosomal dominant disorder characterized by intellectual and developmental disabilities and optic atrophy. This study describes the clinical and neurocognitive features of an individual with a de novo nonsense variant in NR2F1 (NM_005654.5:c.82C > T, p.Gln28*), identified by whole exome sequencing. The patient was diagnosed with autism spectrum disorder (ASD) and unlike most previously reported cases, he had no developmental delay, superior verbal abilities (verbal IQ = 141), and high educational attainment despite reduced non-verbal abilities (nonverbal IQ = 63). He had optic nerve hypoplasia with minimal visual impairment as well as mild dysmorphic features. Compared to both agematched individuals with ASD and healthy controls, the patient showed reductions in manual motor speed, accuracy of saccadic eye movements, and rates of successful behavioral response inhibition.

Although the majority of previously reported cases of BBSOAS have been associated with more global intellectual dysfunction, we report on a patient with selective disruption of nonverbal abilities and superior verbal abilities.

INTRODUCTION

Nuclear receptor subfamily 2 group F member 1 (NR2F1), also known as chicken ovalbumin upstream promotor-transcription factor I (COUP-TFI) in mice, is a highly conserved orphan receptor protein in the steroid/thyroid hormone receptor family that regulates transcription [START_REF] Bosch | NR2F1 mutations cause optic atrophy with intellectual disability[END_REF][START_REF] Zhou | The Nuclear Orphan Receptor COUP-TFI Is Required for Differentiation of Subplate Neurons and Guidance of Thalamocortical Axons[END_REF]. NR2F1 has two functional domains, a DNA-binding domain and a ligand-binding domain; the ligand remains unknown [START_REF] Lin | Coup d'Etat: an orphan takes control[END_REF]. The functions of NR2F1 have been extensively studied in mice, though studies of patients are sparse. In mice, COUP-TFI plays important roles in neurogenesis [START_REF] Zhou | The Nuclear Orphan Receptor COUP-TFI Is Required for Differentiation of Subplate Neurons and Guidance of Thalamocortical Axons[END_REF], visual processing and development [START_REF] Tang | COUP-TFs regulate eye development by controlling factors essential for optic vesicle morphogenesis[END_REF], and cortical patterning [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF].

In humans, alterations in NR2F1 cause Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), an autosomal dominant disorder characterized by visual impairment and optic atrophy, developmental delay and intellectual disability, hypotonia, corpus callosum thinning, and seizures [START_REF] Bosch | NR2F1 mutations cause optic atrophy with intellectual disability[END_REF][START_REF] Chen | The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotypephenotype correlations[END_REF]. Autism spectrum disorder (ASD) has been reported in 35% of affected individuals, and obsessive-compulsive disorder and attention deficit-hyperactivity disorder (ADHD) also have been documented [START_REF] Chen | The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotypephenotype correlations[END_REF].

In this report, we describe a 23-year-old male with ASD and a de novo nonsense variant in NR2F1 (c.82C>T, p.Gln28*). We completed a detailed genetic, clinical, and neurocognitive evaluation of this patient including an assessment of sensorimotor and neuropsychological functioning. We also compared the patient's functioning to age-matched individuals with ASD and controls. This report provides one of the best characterized BBSOAS patients to date and expands on the clinical features associated with alterations in NR2F1 and BBSOAS by reporting selective disturbance of nonverbal abilities and superior verbal abilities.

METHODS

Editorial policies and ethical considerations

All study procedures were approved by the local Institutional Review Board of the University of Illinois at Chicago. All participants provided written consent to participate.

Participants

The patient was 23 years old at the time of testing and was examined as part of ongoing Autism Center of Excellence studies at the University of Illinois at Chicago. Six individuals (five males) with ASD and six controls (three males) matched on age (range: 20-29 years-old) and full-scale IQ also were examined (Table 1). For ASD participants, a diagnosis was established according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR; American Psychiatric Association, 2000) criteria, and based on the Autism Diagnostic Observation Schedule, Second Edition (ADOS-2; [START_REF] Lord | Autism diagnostic observation schedule, (ADOS-2) modules 1-4[END_REF] and the Autism Diagnostic Interview-Revised (ADI-R; [START_REF] Rutter | Autism Diagnostic Interview, Revised (ADI-R)[END_REF]. No parents were available to complete the ADI-R for one participant with ASD. Control participants scored <8 on the Social Communication Questionnaire [START_REF] Rutter | Social communication questionnaire[END_REF] and were excluded for any individual or family history of psychiatric disorders or ASD.

Genetic characterization

As part of the study, participants underwent copy number variant analysis using Illumina 1M or 5M SNP microarrays. ASD participants and parents also were exome sequenced and analyzed as described previously [START_REF] Chen | Leveraging blood serotonin as an endophenotype to identify de novo and rare variants involved in autism[END_REF]. Clinical confirmation of the NR2F1 mutation was performed in the University of Chicago Genetic Services Laboratory by PCR amplification of exon one and Sanger sequencing in both forward and reverse directions.

Developmental/medical assessment

A detailed clinical evaluation was completed for the patient including medical history, psychiatric, ophthalmological, and physical evaluations. Medical records also were reviewed.

Intellectual functioning and diagnostic evaluation

Caregivers of the patient, four participants with ASD, and two controls completed the Aberrant Behavior Checklist to assess hyperactivity, irritability, lethargy, and stereotypy [START_REF] Aman | The aberrant behavior checklist: a behavior rating scale for the assessment of treatment effects[END_REF]. Caregivers of the patient and four participants with ASD completed the Vineland Adaptive Behavior Scales to assess adaptive functioning [START_REF] Sparrow | Vineland adaptive behavior scales[END_REF]. To estimate cognitive abilities, the patient was administered the Wechsler Adult Intelligence Scale, Fourth Edition (WAIS IV; [START_REF] Wechsler | WAIS-IV: Wechsler adult intelligence scale[END_REF]; patient previously completed separate IQ tests) while all other participants were administered the Wechsler Abbreviated Scales of Intelligence (WASI; [START_REF] Wechsler | Wechsler abbreviated scale of intelligence[END_REF] Table 1). At the most recent time point, the patient was administered the Wechsler Adult Intelligence Scale, Fourth Edition (WAIS IV; [START_REF] Wechsler | WAIS-IV: Wechsler adult intelligence scale[END_REF] while all other partici-pants were administered the Wechsler Abbreviated Scales of Intelli-gence (WASI; Wechsler, 1999; Table 1). At age 23, the patient also completed the California Verbal Learning Test-Second Edition (CVLT-II; [START_REF] Delis | California verbal learning test second edition (CVLT-II)[END_REF] and the Brief Visual Spatial Mem-ory Test-Revised (BVMT-R; [START_REF] Benedict | Brief visuospatial memory test-revised[END_REF] to assess his verbal and visual memory, respectively.

Neurocognitive Function

Sensorimotor behavior

To assess basic sensorimotor processes, a visually guided saccade test was administered in which participants fixated on a central cue, and targets were presented at ±12° of center [START_REF] Mosconi | Impaired inhibitory control is associated with higher-order repetitive behaviors in autism spectrum disorders[END_REF].

Saccade latency and gain (saccade amplitude: target amplitude) were examined.

In order to assess participants' ability to inhibit prepotent movements, gap and overlap conditions of an antisaccade task were administered, as previously reported [START_REF] Mosconi | Impaired inhibitory control is associated with higher-order repetitive behaviors in autism spectrum disorders[END_REF]. Participants fixated on a central target before a peripheral target appeared. They were instructed to inhibit their saccade and look in the mirror location. Saccade latencies and percent of trials where participants looked toward the target (prosaccade errors) were measured.

Oculomotor and manual motor stop-signal tests (SST) also were administered to assess participants' ability to inhibit prepotent responses during conditions in which the target response was not predictable.

SST procedures have been reported previously [START_REF] Schmitt | Inhibitory control processes and the strategies that support them during hand and eye movements[END_REF]. Briefly, each SST consisted of GO and STOP trials. During GO trials, participants fixated on a central cross-hair and were instructed to respond as quickly as possible when a peripheral target appeared. During STOP trials, a central stop-signal was presented after the target and participants were instructed not to respond. The percentage of accurate STOP trials and GO trial reaction times were examined. Participants completed a baseline task including only GO trials prior to each SST to assess how much they delayed their GO trial responses during the SST. The change in reaction time from baseline to SST GO trials was examined.

Neuropsychological function

Trails A and B were administered to assess visuomotor planning and cognitive flexibility [START_REF] Reitan | Trail making test manual for administration and scoring[END_REF], respectively. Finger tapping was administered to measure bilateral gross motor speed [START_REF] Baron | Domains and tests[END_REF], and the grooved pegboard test was administered to assess fine motor planning and speed [START_REF] Trites | Grooved pegboard test: Royal Ottawa Hospital[END_REF].

Additionally, handedness was measured using the Annett Handedness Questionnaire [START_REF] Annett | Hand preference observed in large healthy samples: classification, norms and interpretations of increased non-right-handedness by the right shift theory[END_REF].

RESULTS

Genetic characterization

Exome sequencing identified a de novo heterozygous nonsense variant in NR2F1, chr5(GRCh37):g.92920811C>T, NM_005654.5:c.82C>T, p.Gln28*, in the patient, not present in either parent [START_REF] Chen | Leveraging blood serotonin as an endophenotype to identify de novo and rare variants involved in autism[END_REF]. This variant is predicted to result in a premature stop codon with nonsensemediated decay and/or protein truncation. The variant was confirmed by targeted sequencing analysis.

No clinically relevant copy number variants were identified in the patient.

Developmental/medical assessment

The patient was born full-term weighing 3.7 kg. He had hyperbilirubinemia treated with phototherapy.

At 24 months, hand flapping and toe walking were noted. While in preschool, he received speech therapy for poor articulation and occupational therapy for fine motor skill delays with hypotonia. At age three, he was diagnosed with pervasive developmental disorder-not otherwise specified and ADHD; at age eight he was diagnosed with a nonverbal learning disability; and at age 23 he was diagnosed with ASD after being enrolled in this study. The patient met diagnostic criteria for ASD on the ADOS-2 but not the ADI-R (Table 1) and had a consensus diagnosis of ASD based on DSM-IV-TR criteria.

At age eight, EEG showed evidence of rare isolated sharp waves arising from central regions. At age 12, EEG revealed a single sharp wave arising from the left central region and a brain MRI was normal. He had cosmetic otoplasty at age 12 for prominent ears, and micrognathia was corrected by jaw expansion surgery at age 15. At age 23, his head circumference was 56 cm (31 st percentile), height was 171 cm (29 th percentile), and weight was 55.6 kg (fourth percentile). His physical exam revealed bilateral ear cupping, midface hypoplasia, a flat nasal tip, prominent lips, and generalized joint hyperextensibility.

At age 30, the NR2F1 mutation was confirmed and an MRI showed decreased size of the right optic nerve intraocular segment. Evaluation by a neuro-ophthalmologist showed that both optic nerves otherwise were normal in size, supporting a diagnosis of optic nerve hypoplasia. Retinal nerve fiber layer optical coherence tomography revealed retinal nerve fiber layer thinning in both eyes. The patient has 20/30 vision with glasses and no nystagmus.

Intellectual functioning and diagnostic evaluation

The patient graduated from college with a double major in history and political science and has had competitive employment. He lives alone with support but does not drive because he is unable to process the visual information, despite his corrected to normal vision. The patient has had a significantly higher verbal IQ than nonverbal IQ across all cognitive assessments with a difference ranging from three to five standard deviations. At the most recent assessment, he had a 5.2 standard deviation discrepancy between his verbal IQ (141) and nonverbal IQ (63) (Table 1). On the memory assessments, the patient showed average verbal memory abilities on the CVLT-II but moderate to severe impairments of visual memory across delayed recall and recognition trials on the BVMT-R. On the Vineland Adaptive Behavior Scales, the patient showed deficits in daily living skills but communication skills in the average range.

Standardized neuropsychological measures of motor ability indicated that the patient showed reduced motor speed relative to controls and ASD participants (Table S1).

Neurocognitive function

During the visually guided saccade task, the patient showed similar latencies and reduced saccade accuracy compared to controls. During the antisaccade task, the patient had similar latencies to controls but more prosaccade errors than both the control and ASD groups, on gap and overlap conditions.

During oculomotor and manual motor stop-signal tests, the patient showed reduced stopping accuracy and reaction time slowing compared to both groups (Table 2).

DISCUSSION

We document a unique clinical profile characterized by superior verbal and impaired nonverbal abilities, optic nerve hypoplasia with minimal visual impairment, ASD, mild dysmorphic features, reduced sensorimotor precision and speed, and executive dysfunction in a patient with a de novo nonsense variant in NR2F1 (p.Gln28*), occurring before the DNA-and ligand-binding domains. This sequence change is predicted to result in an abnormal transcript, which may be degraded or lead to the production of a truncated NR2F1 protein. The majority of the previously reported individuals with NR2F1 haploinsufficiency had global developmental delays and intellectual disability. However, one patient with a whole gene deletion was reported to have normal intelligence and also a higher verbal IQ (96) than nonverbal IQ (70) [START_REF] Bosch | NR2F1 mutations cause optic atrophy with intellectual disability[END_REF][START_REF] Chen | The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotypephenotype correlations[END_REF], while a boy with a nonsense variant (p.Tyr171*) was reported with borderline intellectual disability (IQ ca. 77-80;[START_REF] Park | Targeted panel sequencing identifies a novel NR2F1 mutations in a patient with Bosch-Boonstra-Schaaf optic atrophy syndrome[END_REF]. Although few reports have provided a detailed cognitive evaluation, these findings are consistent with the observation that patients with deletions or truncating variants have generally milder phenotypes than those with missense variants in the DNA binding domain [START_REF] Chen | The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotypephenotype correlations[END_REF]. Indeed, missense variants in the DNA binding domain completely abolish transcriptional activity and may exert a dominant negative effect, given that NR2F1 binds to DNA in the form of dimers [START_REF] Chen | The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotypephenotype correlations[END_REF].

The magnitude of the disparity between the patient's verbal and nonverbal abilities was consistent since age 3 years, with a verbal-nonverbal IQ difference ranging from three to five standard deviations as well as significantly impaired visual memory but intact verbal memory. This large split between verbal and nonverbal abilities is extremely rare and in contrast with reports showing a relative weakness in verbal abilities in ASD [START_REF] Shah | Why do autistic individuals show superior performance on the block design task[END_REF]. The patient's nonverbal abilities are consistent with studies documenting diagnoses of intellectual disability in patients with BBSOAS [START_REF] Bosch | NR2F1 mutations cause optic atrophy with intellectual disability[END_REF].

Impairments in nonverbal abilities also implicate dysfunctions of thalamocortical circuits. In mouse models, deficits in COUP-TFI lead to improper guidance of thalamocortical axons due to changes in cortical patterning resulting in atypical sensory processing [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF][START_REF] Zhou | The Nuclear Orphan Receptor COUP-TFI Is Required for Differentiation of Subplate Neurons and Guidance of Thalamocortical Axons[END_REF].

Our results suggest that thalamic connections within parietal and occipital cortices may contribute to reduced visuospatial ability, consistent with cerebral visual impairments and elevated rates of optic nerve hypoplasia in BBSOAS [START_REF] Bosch | NR2F1 mutations cause optic atrophy with intellectual disability[END_REF][START_REF] Chen | The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotypephenotype correlations[END_REF]. Deficits in nonverbal processing also may result from the patient's optic nerve hypoplasia [START_REF] Bertacchi | The pleiotropic transcriptional regulator COUP-TFI plays multiple roles in neural development and disease[END_REF], though he did not show visual impairment, as seen in other reported BBSOAS cases [START_REF] Chen | The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotypephenotype correlations[END_REF]. These findings are consistent with a study of heterozygous Nr2f1 deficient mice that show deficits in learning and processing visual information despite relatively unaffected visual acuity and general cognitive function (Bertacchi et al., 2019). Nr2f1 haploinsufficiency in mice led to defective optic nerve myelination and increased astrogliosis, resulting in slower optic axonal conduction velocity from the retina to higher level visual processing regions [START_REF] Bertacchi | Mouse Nr2f1 haploinsufficiency unveils new pathological mechanisms of a human optic atrophy syndrome[END_REF]. Thus, the patient's unique profile of impaired nonverbal abilities and minimal visual impairment suggests that his non-verbal deficits may reflect both the optic nerve hypoplasia and cerebral visual deficits, including alterations in thalamocortical development, abnormal optic nerve axonal conduction, and impaired visual learning and memory.

Our findings contribute to evidence implicating NR2F1 in the etiology of ASD, similar to prior studies documenting ASD in patients with BBSOAS [START_REF] Bosch | NR2F1 mutations cause optic atrophy with intellectual disability[END_REF][START_REF] Chen | The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotypephenotype correlations[END_REF][START_REF] Dimassi | Whole-exome sequencing improves the diagnosis yield in sporadic infantile spasm syndrome[END_REF].

The patient also had jaw expansion surgery for micrognathia. Micrognathia and retrognathia have been reported in several patients with BBSOAS [START_REF] Al-Kateb | NR2F1 haploinsufficiency is associated with optic atrophy, dysmorphism and global developmental delay[END_REF][START_REF] Chen | The expanding clinical phenotype of Bosch-Boonstra-Schaaf optic atrophy syndrome: 20 new cases and possible genotypephenotype correlations[END_REF][START_REF] Hino-Fukuyo | Long-term outcome of a 26-year-old woman with west syndrome and an nuclear receptor subfamily 2 group F member 1 gene (NR2F1) mutation[END_REF][START_REF] Kaiwar | Novel NR2F1 variants likely disrupt DNA binding: molecular modeling in two cases, review of published cases, genotype-phenotype correlation, and phenotypic expansion of the Bosch-Boonstra-Schaaf optic atrophy syndrome[END_REF][START_REF] Park | Targeted panel sequencing identifies a novel NR2F1 mutations in a patient with Bosch-Boonstra-Schaaf optic atrophy syndrome[END_REF] and Nr2f receptors are important in the development of the vertebrate upper jaw [START_REF] Barske | Essential role of Nr2f nuclear receptors in patterning the vertebrate upper jaw[END_REF]. We also document otoplasty and bilateral ear cupping in this patient, similar to descriptions of ear abnormalities, including prominent ears, in 16 of 24 (67%) individuals with BBSOAS . The patient's sensorimotor impairments suggest weakened modulation of the cerebellum on pontine-brainstem burst cells [START_REF] Leigh | The Neurology of Eye Movements[END_REF]. Slower reaction times implicate cerebellar-thalamofrontal circuits involved in initiating motor output consistent with studies showing motor behaviors in COUP-TFI mutation mice [START_REF] Tomassy | Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI[END_REF]. We also show a deficit of response inhibition suggesting that changes of subcortical-cortical processes may extend to prefrontal-striatal circuits involved in the development of executive functioning.

In summary, we show that a pathogenic truncating NR2F1 variant is associated with a unique profile characterized by ASD, mild dysmorphic features, reduced sensorimotor precision and speed, and executive dysfunction. This patient's unique cognitive profile of superior verbal abilities and reduced nonverbal abilities contrasts with the majority of patients with BBSOAS diagnosed with intellectual disability or developmental delay, suggesting that NR2F1 variants may confer unique risk for multiple cognitive issues and expands the phenotype of BBSOAS.

Table 2 .

 2 Sensorimotor task results for patient, individuals with ASD, and healthy controls.Data are mean (SD). ASD, autism spectrum disorder; ms, milliseconds.

	Patient	ASD	Controls
	(N=1)	(N=6)	(N=6)

ACKNOWLEDGMENTS

Data used in the preparation of this manuscript have been shared in the NIH-supported National Database for Autism Research (NDAR), collection ID 1. The University of Illinois at Chicago Autism Center of Excellence was supported by NICHD/NINDS/NIEHS through P50HD055751 (MWM, SG, EC). Sequencing services were provided by the Center for Inherited Disease Research (CIDR) through X01 HG007235. CIDR is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University, contract number HHSN268201200008I. The NICHD provided funding for the U54 Kansas Intellectual and Developmental Disabilities Research Award (HD090216-01). Additional funding was provided by the National Institutes of Mental Health through R01 MH112734-01 (MWM, EB) and K23 MH092696-05 (MWM).

Eunice Kennedy Shriver National Institute of Child Health and Human Development, Grant/ Award Number: U54 HD090216-01; National Institute of Child Health and Human Development, Grant/Award Number: P50HD055751; National Institute of Mental Health, Grant/Award Numbers: K23 MH092696-05, R01 MH112734-01; National Institutes of Health, Grant/Award Numbers: HHSN268201200008I, X01 HG007235

DATA AVAILABILITY STATEMENT

Data used in the preparation of this manuscript have been shared as NDAR study #784 (https://doi.org/10.15154/1504186).

CONFLICT OF INTEREST

None.

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of this article.


Marie-Claude Jaurand
email: marie-claude.jaurand@inserm.fr

Didier Jean

BIOMOLECULAR PATHWAYS AND MALIGNANT PLEURAL MESOTHELIOMA

Keywords: Mesothelioma, signalling pathways, targeted therapy

Although asbestos is banned in several countries, Malignant Pleural Mesothelioma (MPM) incidence is increasing worldwide, and this cancer remains a disease of concern. MPM is a very severe cancer, with no curative treatment despite the development of different kinds of therapeutic approaches. To date the treatments are based on multimodal chemotherapy and surgery. The longest survival data in patients is obtained with the combination of chemotherapy, radical surgery and radiotherapy. The development of targeted therapies offers new strategies to kill cancer cells. To be efficient, they need a precise identification of the critical targets. This objective can be reached by a deep knowledge of the molecular and physiological changes associated with the neoplastic transformation of MPM cells. One approach consists in the identification of gene mutations, epigenetic alterations, study of gene expression profiles and identification of the deregulated signalling pathways in malignant cells, with the goal to select molecules or mechanisms that could kill cancer cells or abolish tumour growth. The present knowledge on the main alterations in genes and signalling pathways indicates that MPM have recurrent mutations in a limited number of tumour suppressor genes, and oncogenic mutation in the promoter of TERT. A number of studies have emphasized the role of receptor tyrosine kinase (RTK) driven signalling, although not related to mutations in RTK. Multiple signalling pathways are altered in MPM. Transcriptomic analyses permitted to classify mesotheliomas in subgroups, according to prognosis. They showed heterogeneity of MPM, not only defined by the histological subtype, but also by molecular features. So far, targeted therapy was unsuccessful, at least partly due to the heterogeneity of MPM. Moreover, the complexity stands in the interconnection between pathways, which is a challenge to choose the most critical target for an efficient therapy. This review summarizes the main alterations identified in genes and signalling pathways in MPM, the impact on therapeutics, and discusses the future of these approaches to improve MPM outcome, especially knowing molecular and physiological characteristics of MPM to define their diversity.

BACKGROUND

Malignant pleural mesothelioma (MPM) is an uncommon and severe neoplasm of poor prognosis with a median survival of 8 to 14 months. Since the publication of the association between mesothelioma and asbestos exposure in South Africa, epidemiological and fundamental researches have been developed to define the populations at risk and evaluate the risk level, to investigate the mechanism of action of asbestos and of mesothelioma carcinogenesis, and to treat the disease [START_REF] Wagner | Diffuse pleural mesothelioma and asbestos exposure in the North Western Cape Province[END_REF].

However, pathobiological and clinical researches are difficult in the case of uncommon diseases, and MPM suffers from a deficit of medical and scientific knowledge. Nevertheless, the interest on MPM is now increasing; national tools and international studies are implemented to improve patients' care, and our knowledge on the biological features of MPM. Presently, researchers can take advantage of the new methodologies to describe the molecular characteristics, and work through networks to improve their efficiency. In the future, these advances will be beneficial to the patients and to overcome this cancer.

To date, there is no cure for this type of cancer, and the treatments do not sufficiently improve patients' survival. Chemotherapy improved the management of symptoms by a stabilisation of the disease, but increase survival of only few months. To date association of platinum-based chemotherapy combined with antifolate pemetrexed is accepted as standard treatment. The longest survival data in patients is obtained with the multimodal treatment consisting of chemotherapy, radical surgery and radiotherapy [START_REF] Haas | Malignant pleural mesothelioma: update on treatment options with a focus on novel therapies[END_REF][START_REF] Opitz | Management of malignant pleural mesothelioma-The European experience[END_REF]. Surgical resection is offered to few selected cases, depending on patient's performance status, and its interest is debated [START_REF] Opitz | Biomolecular and clinical practice in malignant pleural mesothelioma and lung cancer: what thoracic surgeons should know[END_REF][START_REF] Treasure | Extra-pleural pneumonectomy versus no extra-pleural pneumonectomy for patients with malignant pleural mesothelioma: clinical outcomes of the Mesothelioma and Radical Surgery (MARS) randomised feasibility study[END_REF].

Mesothelioma incidence varies markedly from one country to another. Asbestos is the main etiological factor of MPM, which is diagnosed 30 to 40 years after exposure [START_REF] Reid | Mesothelioma risk after 40 years since first exposure to asbestos: a pooled analysis[END_REF]. MPM is related to both occupational and environmental asbestos exposures. Although banned in several countries, asbestos still remains used in many other countries [START_REF] Frank | The global spread of asbestos[END_REF][START_REF] Leong | Asbestos in Asia[END_REF]. From a recent review mesothelioma epidemic does not show signs of attenuation [START_REF] Bianchi | Global mesothelioma epidemic: Trend and features[END_REF]. Epidemiological studies have demonstrated that exposure to other factors such as erionite fibres and non-asbestos amphiboles lead to MPM occurrence [START_REF] Jean | Thoracic Neoplasia-Mesothelioma[END_REF]. Co-exposure to asbestos and man made mineral fibres enhances the risk of MPM [START_REF] Guida | Effets sur la santé des fibres minérales artificielles[END_REF]. Although debated, the virus SV40 has been associated to the risk of MPM as a cofactor in some studies [START_REF] Carbone | Malignant mesothelioma: facts, myths, and hypotheses[END_REF][START_REF] Rascoe | Molecular pathogenesis of malignant mesothelioma[END_REF]. Moreover, MPM diagnosis is sometimes difficult, because of the lack of specific biomarkers and the similitudes with pleural metastatic carcinomas, frequently from lung and breast carcinomas [START_REF] Betta | Immunohistochemistry and molecular diagnostics of pleural malignant mesothelioma[END_REF]. Therefore, because of the natural history of MPM, and for pathological, clinical, environmental and societal reasons, mesothelioma is still a worldwide major health concern. A precise characterization of mesothelioma hallmarks is a great challenge to improve current therapies and to deliver specific and effective treatments.

Carcinogenesis is a long-term term process in which cells differentiate from normal cells by the acquirement of new phenotypes and genomic changes. Tumour cells express specific characteristics consisting in dedifferentiated morphological features, expression of specific biomarkers (miRNA, protein), chromosome alterations, allelic disequilibrium, gene mutations, and deregulation of signalling pathways. Several mechanisms can lead to tumour growth; they are evidenced by multiple mutations (mutator phenotype) or chromosomal misseggregation (aneuploidy) [START_REF] Loeb | Human cancers express mutator phenotypes: origin, consequences and targeting[END_REF]. Intratumoral heterogeneity is observed in solid cancers; it may arise from a clonal evolution from differentiated cell transformation or follow the cancer stem cell model [START_REF] Cabrera | Cancer stem cell plasticity and tumor hierarchy[END_REF][START_REF] Shackleton | Heterogeneity in cancer: cancer stem cells versus clonal evolution[END_REF]. Tumour growth is also dependent on the cell microenvironment that may produce many types of regulatory factors (cytokines, growth factors…), and interactions with stromal cells in the tumour (fibroblasts and endothelial cells, immune cells such as tumour associated macrophages and lymphocytes) [START_REF] Hanahan | Accessories to the crime: functions of cells recruited to the tumor microenvironment[END_REF]. Because of the variety of the different mechanisms by which cells can be modified and move forward in the neoplastic process, tumour cells exhibit an heterogeneity which is highlighted at the morphological, molecular and cell signalling levels. The identification of these characteristics is a basic issue to progress in the science of mesothelioma.

PATHOBIOLOGY OF MPM

Mesothelioma diagnosis

Presently, MPM is classified according to pathological features. Histologically, three major types of MPM are described: epithelioid, biphasic, or sarcomatoid, with more than ten uncommon subtypes for the epithelioid form and five for the sarcomatoid type [START_REF] Husain | Guidelines for Pathologic Diagnosis of Malignant Mesothelioma: 2012 Update of the Consensus Statement from the International Mesothelioma Interest Group[END_REF]. Biphasic MPM include both epithelial and sarcomatoid component, the latter being less than 10% of the tumour cell population [START_REF] Husain | Guidelines for Pathologic Diagnosis of Malignant Mesothelioma: 2012 Update of the Consensus Statement from the International Mesothelioma Interest Group[END_REF]. The epithelial subtype is the most common form of MPM, accounting for approximately 50-70% of cases. Histologic MPM type is associated with different median survival time of the patients, the better prognosis being for the epithelioid type. As for many other types of cancers, the diversity of histological features of MPM suggests that individual tumours have different specificities. The more recent large scale molecular studies have confirmed the MPM heterogeneity [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF].

So far, MPM diagnosis is based on pathological features and biomarkers, but the development of genetic, epigenetic and cell regulation studies in MPM will permit to better know the biology of the MPM subtypes, and will provide more precise diagnosis to define therapeutic decisions.

The main results from researches carried out during the last 10 years on gene expression profiles in MPM and primary tumours allowed advances in our knowledge of the molecular biology of malignant mesothelioma. They demonstrated that differential gene expression profiles exist between malignant mesotheliomas, most often linked to the histological subtypes [START_REF] Gordon | Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling[END_REF][START_REF] Hoang | Gene expression profiling identifies matriptase overexpression in malignant mesothelioma[END_REF]. However, recent findings based on unsupervised hierarchical clustering on transcriptomic data in MPM primary cultures and primary tumours defined two MPM subgroups, C1 and C2, closely related to prognosis and partly to histological subtypes, demonstrating that MPM occurs with different molecular profiles, gene alterations, and survival outcomes [START_REF] De Reynies | Molecular classification of malignant pleural mesothelioma: Identification of a poor prognosis subgroup linked to the epithelial-tomesenchymal transition[END_REF].

Signalling pathways in MPM

Tumour growth is controlled by numerous pathways, which are regulated by the activity of both intrinsic effectors and extrinsic factors, produced either by the tumour cells or by nearby non-tumour cells, which activate or negatively regulate signalling pathways of importance to the maintenance of cell homeostasis. These pathways can also be deregulated by alterations in key members, due to mutations in genes encoding driving proteins, gene silencing by epigenetic mechanisms, changes in gene expression of initiating factors or unscheduled gene expression. In this work, we will summarize the data showing the alteration of the pathways of importance to the evolution of the carcinogenic process in MPM, and discuss their interest to implement more precise therapies.

Gene mutations in MPM.

Numerous studies focused on gene mutations in candidate genes, oncogenes, tumour suppressor genes and key regulatory genes, accounting for mesothelioma oncogenesis. Earlier sequencing studies identified the genes most recurrently mutated in MPM as CDKN2A/2B (cyclin-dependent kinase inhibitor 2A/2B), NF2 (neurofibrin 2) and BAP1 (BRCA1 Associated Protein-1), and showed low frequency of mutation in the well-known tumour suppressor gene TP53 [START_REF] Bott | The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma[END_REF][START_REF] Jean | Syntenic relationships between genomic profiles of fiber-induced murine and human malignant mesothelioma[END_REF];. CDKN2A/2B are regulatory genes in cell cycle, NF2 is involved in several signalling pathways including the Hippo pathway and plays a role in contact inhibition of cell proliferation, and BAP1 belongs to the ubiquitin carboxy-terminal hydrolase subfamily of deubiquitinating enzymes that are involved in the removal of ubiquitin from nuclear proteins. Interestingly, all are tumour suppressor genes and no recurrent oncogenic mutation has been reported until recently. The first recurrent oncogenic mutation was identified in the TERT promoter [START_REF] Tallet | Overexpression and promoter mutation of the TERT gene in malignant pleural mesothelioma[END_REF]. Most recent studies using next-generation sequencing (NGS) assays confirmed mutation in these tumour suppressor genes and provided new information on the mutational status of a series of genes in MPM. A whole exome sequencing on DNA from 22 frozen MPM tumour samples and matched blood samples identified frequent somatic genetic alterations in CUL1 (cullin1), which encodes a core component of SCF complex (Skp, Cullin, F-box containing complex), a E3 ubiquitin-protein ligase complex that mediates the ubiquitination of proteins involved in cell cycle progression, signal transduction and transcription [START_REF] Guo | Whole exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A and CUL1 in malignant pleural mesothelioma[END_REF]. Another series of 123 formalin-fixed, paraffin-embedded tissue samples was analysed for mutations in 50 cancer genes by NGS. Frequent variations were detected in seven new genes related to signalling pathways: PDGFRA, KIT, KDR, HRAS, PIK3CA, STK11 and SMARCB1, including genes involved in the PI3K/AKT and MAPK pathways or playing a role in the P53/DNA repair pathway [START_REF] Lo Iacono | Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study[END_REF]. Patients with multiple genetic variations in PIK3CA, KIT, STK11 and TP53 correlated with shorter time to progressive disease and reduced overall survival [START_REF] Lo Iacono | Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study[END_REF]. A NGS sequencing in six MPM cell lines suggests mutation in genes involved in Hippo, MAPK, Wnt, nuclear factor kappa B, and angiogenesis pathways [START_REF] Miyanaga | Hippo pathway gene mutations in malignant mesothelioma: revealed by RNA and targeted exon sequencing[END_REF].

Receptor Tyrosine Kinases (RTKs) in MPM. RTKs are membrane receptors driving downstream cell signalling, involved in several pathways [START_REF] Lemmon | Cell signaling by receptor tyrosine kinases[END_REF][START_REF] Son | Reactive oxygen species in the activation of MAP kinases[END_REF]. From the studies carried out with other types of cancers, it is known that mutations in RTKs play a role in the neoplastic transformation of normal cells. Then, the status of RTKs has been investigated in MPM.

Epidermal growth factor receptor (EGFR). From immunohistochemical studies, EGFR was expressed in about 50% of MPM cases, suggesting an overexpression. This was not found in benign mesothelial lesion, and EGFR expression seems to be a factor negatively affecting prognosis [START_REF] Edwards | EGFR expression: associations with outcome and clinicopathological variables in malignant pleural mesothelioma[END_REF][START_REF] Enomoto | Epidermal growth factor receptor mutations in malignant pleural and peritoneal mesothelioma[END_REF][START_REF] Gaafar | Tissue and serum EGFR as prognostic factors in malignant pleural mesothelioma[END_REF][START_REF] Rena | Epidermal growth factor receptor overexpression malignant pleural mesothelioma: Prognostic correlations[END_REF]. However, the relationship between EGFR expression and patient's survival is not clear. Either no relationship or a favourable prognosis was reported [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF]. EGFR is generally not mutated in human MPM, although a few papers reported a low percentage of mutations. No EGFR gene mutation was detected from histological samples or frozen tissues of MPM [START_REF] Cortese | Common EGFR mutations conferring sensitivity to gefitinib in lung are not prevalent in human malignant mesothelioma[END_REF][START_REF] Mezzapelle | Mutation analysis of the EGFR gene and downstream signalling pathway in histologic samples of malignant pleural mesothelioma[END_REF][START_REF] Velcheti | Absence of mutations in the epidermal growth factor receptor (EGFR) kinase domain in patients with mesothelioma[END_REF], and no mutation or amplification in EGFR in and ERBB2 [START_REF] Shukuya | Identification of actionable mutations in malignant pleural mesothelioma[END_REF]. EGFR mutations were found in 6% and 16% of MPM samples [START_REF] Enomoto | Epidermal growth factor receptor mutations in malignant pleural and peritoneal mesothelioma[END_REF][START_REF] Schildgen | Low Frequency of EGFR Mutations in Pleural Mesothelioma Patients[END_REF]. A phosphorylation of EGFR was reported in 10 of 14 mesothelioma cell lines, not due to mutation in the kinase domain [START_REF] Brevet | Coactivation of receptor tyrosine kinases in malignant mesothelioma as a rationale for combination targeted therapy[END_REF]. By DNA sequencing, EGFR mutations were not detected in 34 primary cultures from MPM, in contrast to lung cancer [START_REF] Andujar | Differential mutation profiles and similar intronic TP53 polymorphisms in asbestos-related lung cancer and pleural mesothelioma[END_REF]. Interestingly, EGFR mutations in lung cancer were not linked with asbestos exposure, suggesting that EGFR mutations do not result from the effect of asbestos, consistent with the absence of mutations in MPM that is mainly due to asbestos exposure [START_REF] Andujar | Differential mutation profiles and similar intronic TP53 polymorphisms in asbestos-related lung cancer and pleural mesothelioma[END_REF].

Vascular endothelial growth factor receptors (VEGFR). MPM cells express both vascular endothelial growth factors (VEGF), and the VEGFR receptors fmsrelated tyrosine kinases (FLT1 and FLT4) and fetal liver kinase (KDR/FLK1) [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF]. Several immunohistochemical studies have demonstrated an enhanced expression of VEGF in comparison with non neoplastic specimens [START_REF] Lee | Update on the molecular biology of malignant mesothelioma[END_REF]. The results of in vitro studies using neutralizing antibodies against VEGF or VEGFR, or antisense oligonucleotides against VEGF have suggested an autocrine role of VEGF in the proliferation of MPM cells. Contradictory results were found regarding the correlation between VEGF expression and survival [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF].

Platelet-derived growth factor receptor (PDGFR). Normal human mesothelial cells express platelet-derived growth factor (PDGF) PDGF-A mRNA and protein, but these no detectable PDGF-B. In contrast, human MPM cells express high levels of both PDGF-A and PDGF-B, as well as PDGFR-B [START_REF] Gerwin | Comparison of production of transforming growth factor-beta and platelet-derived growth factor by normal human mesothelial cells and mesothelioma cell lines[END_REF]. An autocrine proliferation involving receptor and ligand has been suggested in MPM. Regulation of proliferation by PDGF may occur, either directly or indirectly, via the hyaluronan, an important component of the extracellular matrix, and CD44 pathway. PDGF-Bstimulated normal human mesothelial cells express both hyaluronan synthase and hyaluronan [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF]. Gain in copy number of PDGFR-B was reported in 40% of 88

MPM tumours, with a potential role as prognostic biomarker [START_REF] Tsao | Phase I trial of cisplatin, pemetrexed, and imatinib mesylate in chemonaive patients with unresectable malignant pleural mesothelioma[END_REF].

Mesenchymal epithelial transition factor, c-MET oncogene (MET). MET, also known as hepatocyte growth factor receptor (HGFR) transduces signals from binding its ligand, hepatocyte growth factor/scatter (HGF), leading to the activation of several signalling cascades including the MAPK and PI3K/AKT.

MET is overexpressed and activated in a majority of cases of mesothelioma when compared with normal tissues, and MET siRNA and MET inhibitor (SU11274) inhibited cell growth and migration [START_REF] Rascoe | Molecular pathogenesis of malignant mesothelioma[END_REF]. MET protein was overexpressed in 6 of 14 mesothelioma cell lines, and co-expression of phosphorylated EGFR and MET was observed in 8 of 14 (57%) cell lines [START_REF] Brevet | Coactivation of receptor tyrosine kinases in malignant mesothelioma as a rationale for combination targeted therapy[END_REF]. MET expression was detected in 82% of MPM tissue samples, and in mesothelioma cell lines, but not in normal tissue, or at low level in non-malignant mesothelial cell lines [START_REF] Jagadeeswaran | Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma[END_REF]. No mutation was found in cell lines, but 2 mutations were found among 70 MPM tumour samples [START_REF] Brevet | Coactivation of receptor tyrosine kinases in malignant mesothelioma as a rationale for combination targeted therapy[END_REF]. Mutations of MET were identified in 5 of 43 (11.6%) primary tumours and 2 of 7 (28.6%) cell lines [START_REF] Jagadeeswaran | Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma[END_REF].

Other RTKs: AXL, Insulin growth factor (IGFR); fibroblast growth factor (FGFR).

AXL belongs to the TAM family of RTKs. It is activated by binding growth factor GAS6 (growth arrest-specific 6), and regulates cell survival, cell proliferation, migration and differentiation, via the PI3K/AKT pathway. AXL is overexpressed and activated in many human cancers, including MPM with spindle cell morphology, but somatic AXL mutations were not found in MPM [START_REF] Ou | AXL regulates mesothelioma proliferation and invasiveness[END_REF][START_REF] Paccez | The receptor tyrosine kinase Axl in cancer: biological functions and therapeutic implications[END_REF].

Human MM cells express insulin growth factor (IGF) and IGFR, and IGF-1 appears to function as an autocrine growth stimulus to MPM cells [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF]. Insulinlike growth factor-binding proteins (IGFBP) form a complex with IGFR subunit and IGF, and have been shown to either inhibit or stimulate the growth-promoting effect of IGF. IGFBP can be either expressed or unexpressed in MM, modulating the aggressiveness of the MM phenotype [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF]. IGF-1 stimulation resulted in activation of PI3K and ERK1/2 pathways leading to a stimulation of the eIF4F complex and cap-dependent translation [START_REF] Jacobson | Activated 4E-BP1 represses tumourigenesis and IGF-I-mediated activation of the eIF4F complex in mesothelioma[END_REF]. Few data are available on the activity of FGFR in MPM. A study of mutations in FGFR1 and FGFR2 detected one mutation in FGFR3 from 42 MPM patients [START_REF] Shukuya | Identification of actionable mutations in malignant pleural mesothelioma[END_REF]. No amplification of FGFR1, a common feature in other cancers, was found by FISH analysis in 19 MPM tissues [START_REF] Plones | Absence of amplification of the FGFR1gene in human malignant mesothelioma of the pleura: a pilot study[END_REF]. Otherwise, FGFR1 and FGFR2 were coexpressed in three of seven MPM cell lines, and FGF2/FGFR1 may form an autocrine signal [START_REF] Marek | Nonamplified FGFR1 is a growth driver in malignant pleural mesothelioma[END_REF]. KIT is a receptor for stem cell factor or kit ligand. It activates several regulatory pathways, including PI3K and MAPK. KIT expression has been mostly studied by immunohistochemistry, showing a low percentage of positivity in MPM tumours [START_REF] Butnor | The spectrum of Kit (CD117) immunoreactivity in lung and pleural tumors: a study of 96 cases using a single-source antibody with a review of the literature[END_REF]. No expression was detected by RT-PCR in a study of 37 MPMs [START_REF] Horvai | c-Kit is not expressed in malignant mesothelioma[END_REF]. KIT has not been shown to be characteristic of MPM at the present time. Ou et al [START_REF] Ou | Targeted inhibition of multiple receptor tyrosine kinases in mesothelioma[END_REF] determined the relative levels of tyrosine phosphorylation of 42 distinct RTKs in mesothelioma cell lines established from surgical specimens. They found coordinated activation of several RTKs: EGFR, ERBB3, AXL, and MET [START_REF] Ou | Targeted inhibition of multiple receptor tyrosine kinases in mesothelioma[END_REF].

Frequent coactivation of multiple RTKs in MPM cells including EGFR, ERBB3

and MET, but also ERBB2 was also observed in another study [START_REF] Kawaguchi | Combined inhibition of MET and EGFR suppresses proliferation of malignant mesothelioma cells[END_REF][START_REF] Menges | A Phosphotyrosine Proteomic Screen Identifies Multiple Tyrosine Kinase Signaling Pathways Aberrantly Activated in Malignant Mesothelioma[END_REF].

Activity of signalling pathways in MPM

MAPK pathway. MAPK pathway comprises several signalling cascades of protein phosphorylations, which activation depends on the nature of the external stimuli [START_REF] Son | Reactive oxygen species in the activation of MAP kinases[END_REF]. In MPM, the activation and role of several MAPK, extracellular-regulated kinases (ERK1/2), Jun amino-terminal kinases/stress-activated kinases (JNK/SAPK) and MAPKp38 have been studied. A phosphorylation of ERK, JNK and p38 MAPK was observed in several studies, by immunohistochemistry or western blots [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF]. MAPK activation does not seem differentiate between benign and malignant mesothelial cells, but MAPK expression and phosphorylation were better predictive factors of outcome [START_REF] Vintman | Mitogen-activated protein kinase expression and activation does not differentiate benign from malignant mesothelial cells[END_REF]. The ERK and SAPK/JNK signalling pathways are activated in adherent 2D cultures of MPM cells, but not when cells are cultured in 3D conditions [START_REF] Daubriac | Malignant pleural mesothelioma cells resist anoikis as quiescent pluricellular aggregates[END_REF]. In 3D cultures, MPM are resistant to anoïkis (the death process due to loss of anchorage). This resistance is likely a consequence of the aggregation state of the cells in 3D conditions; it can be reversed by activating SAPK/JNK with anisomycin, according to a Bim-dependent mitochondrial pathway [START_REF] Daubriac | Malignant pleural mesothelioma cells resist anoikis as quiescent pluricellular aggregates[END_REF]. The toxicity of drugs able to interact with the MAPK pathways has been investigated in MPM cells. In vitro end points concern inhibition of proliferation and induction of apoptosis and in animals, tumour growth in immunodeficient mice (xenograft), or in syngenic mice. Activation of EGFR, MET and AXL in MPM is associated with activation of MAPK signalling cascade [START_REF] Zhou | Multipoint targeting of the PI3K/mTOR pathway in mesothelioma[END_REF]. Activation of p21-activated kinases (PAKs), which regulate signalling pathways, was investigated in MPM by IHC [START_REF] Menges | Group I p21-Activated Kinases (PAKs) Promote Tumor Cell Proliferation and Survival through the AKT1 and Raf-MAPK Pathways[END_REF]. All 15 MPM tumours were more positive in comparison with normal pleural tissue. In MPM cell lines, inhibition of PAKs reduced mesothelioma cell proliferation and survival, and RAF-MAPK signalling [START_REF] Menges | Group I p21-Activated Kinases (PAKs) Promote Tumor Cell Proliferation and Survival through the AKT1 and Raf-MAPK Pathways[END_REF]. Silencing of the RTK, AXL, by shRNA suppressed mesothelioma migration, invasiveness and cellular proliferation [START_REF] Ou | AXL regulates mesothelioma proliferation and invasiveness[END_REF]. Arsenic trioxide (As2O3) induced apoptosis and phosphorylation of ERK1/2 and JNK1/2, but not p38MAPK, after As2O3 treatment, indicating the involvement of the ERKdependent and JNK-dependent, pathway in the cell response. In contrast, As2O3 treatment did not alter phosphorylation of either AKT or SRC [START_REF] Eguchi | Arsenic trioxide induces apoptosis through JNK and ERK in human mesothelioma cells[END_REF]. The mechanism seems complex as combination of As2O3 treatment and EGFR inhibitor exerted a synergistic effect [START_REF] Kryeziu | Synergistic anticancer activity of arsenic trioxide with erlotinib is based on inhibition of EGFR-mediated DNA double-strand break repair[END_REF].

PI3K/AKT/mTOR pathway.

The phosphatidylinositol 3'-kinase(PI3K)-Akt (PI3K/AKT) signalling pathway regulates transcription, translation, proliferation, growth, and survival. It is negatively regulated by the phosphatase and tensin homolog (PTEN) and can activate many downstream targets such as Mechanistic Target Of Rapamycin (mTOR) (Figure 2). An alteration of the PI3K/AKT pathway has been reported in several studies [START_REF] De Assis | The role of key genes and pathways involved in the tumorigenesis of Malignant Mesothelioma[END_REF]. Phosphorylation of AKT protein, the active form of the protein, was demonstrated in MPM cells by IHC and western blots analyses. AKT activation was observed in 13 (62%) of the 21 mesothelioma cell lines under serum-starved conditions [START_REF] Suzuki | Activation of the PI3K-AKT pathway in human malignant mesothelioma cells[END_REF]. The relationship between AKT and mTOR was observed in tumours demonstrating an association of elevated phospho-mTOR positivity with of AKT pathway activation [START_REF] Besson | PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis[END_REF]. However, while the PI3K-Akt signalling pathway was activated in adherent MPM cells, loss of anchorage resulted in inactivation of this pathway and failed to restore apoptosis [START_REF] Daubriac | Malignant pleural mesothelioma cells resist anoikis as quiescent pluricellular aggregates[END_REF]. AKT phosphorylation and expression of downstream signalling factors, 4E-BP1, p4E-BP1, eIF-4E, peIF-4E, pS6 and FOXO3a were studied by IHC in 30 mesothelioma cases, demonstrating a positive immunophenotype of the PI3K signalling pathway [START_REF] Cedres | Exploratory analysis of activation of PTEN-PI3K pathway and downstream proteins in malignant pleural mesothelioma (MPM)[END_REF]. Correlation of IHC expression levels and progression free survival (PFS) showed that patients with concomitant low expression of pS6 and p4E-BP1 and overexpression of FOXO3a had significantly better prognosis [START_REF] Cedres | Exploratory analysis of activation of PTEN-PI3K pathway and downstream proteins in malignant pleural mesothelioma (MPM)[END_REF]. IGF stimulation of MPM cells resulted in an increased phosphorylation of AKT, S6 and 4EBP1. Moreover the fraction of side-population (SP, a potential stem cell fraction) was increased, showing that active PI3K signalling may play a role in regulating the SP phenotype in MPM cell lines [START_REF] Fischer | Inhibition of phosphoinositide-3 kinase pathway down regulates ABCG2 function and sensitizes malignant pleural mesothelioma to chemotherapy[END_REF].

Inactivation of PTEN could account for the activation of the AKT pathway. No clear-cut data are available on PTEN status in MPM. PTEN homozygous deletion has been reported in 2 of 9 of MPM cell lines, and low or no mRNA expression in 9/21 cell lines compared to expression levels in the non-tumour MeT-5A cell line [START_REF] Suzuki | Activation of the PI3K-AKT pathway in human malignant mesothelioma cells[END_REF][START_REF] Altomare | Human and mouse mesotheliomas exhibit elevated AKT/PKB activity, which can be targeted pharmacologically to inhibit tumor cell growth[END_REF]. Loss of PTEN expression was found in 62% of 206 MPM, by tissue microarray, but lower percentage (26.7%) was found in 68 mesothelioma tissues, where no relationship was found with survival [START_REF] Agarwal | PTEN protein expression in malignant pleural mesothelioma[END_REF][START_REF] Opitz | PTEN expression is a strong predictor of survival in mesothelioma patients[END_REF]. More recently, enhanced expression pAKT in comparison with normal samples, and no difference in PTEN expression was reported in tissue microarray of 213 MPM samples [START_REF] Kanteti | MET and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma[END_REF]. PTEN expression correlated with survival was an independent prognostic biomarker in patients with mesothelioma [START_REF] Opitz | PTEN expression is a strong predictor of survival in mesothelioma patients[END_REF].

The mTOR and PI3K/AKT pathways are strongly interconnected and may be considered as a single pathway [START_REF] Gao | Translational control by oncogenic signaling pathways[END_REF][START_REF] Porta | Targeting PI3K/Akt/mTOR Signaling in Cancer[END_REF][START_REF] Xu | mTOR signaling in tumorigenesis[END_REF]. The mTOR pathway is activated in mesotheliomas, and the mTOR inhibitor, rapamycin, reduces apoptosis resistance and cell spreading [START_REF] Barbone | Mammalian target of rapamycin contributes to the acquired apoptotic resistance of human mesothelioma multicellular spheroids[END_REF][START_REF] Ranzato | Spreading of mesothelioma cells is rapamycinsensitive and requires continuing translation[END_REF][START_REF] Wilson | mTOR mediates survival signals in malignant mesothelioma grown as tumor fragment spheroids[END_REF]. Targeting of PI3K/AKT pathway in MPM cell lines was carried out with inhibitors of either AKT, or dual AKT and mTOR inhibitors (GDC-0980; NVP-BEZ235). Cell viability was reduced with GDC-0980 or NVP-BEZ235 [START_REF] Kanteti | MET and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma[END_REF]. The combination of MET inhibition with ARQ 197 (Tivantinib) and PI3K/mTOR dual inhibitors enhanced reduction of cell viability [START_REF] Kanteti | MET and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma[END_REF]. ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts in mice, and the combination of the two drugs was synergistic [START_REF] Kanteti | MET and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma[END_REF]. The PI3K/mTOR inhibitor NVP-BEZ235 and PI3K inhibitor wortmannin reduced the phosphorylation of downstream target AKT, S6 and 4EBP1, decreased the SP fraction and sensitized SP cells to chemotherapy, via the regulation of ABCG2, a potential marker of stem cells [START_REF] Fischer | Inhibition of phosphoinositide-3 kinase pathway down regulates ABCG2 function and sensitizes malignant pleural mesothelioma to chemotherapy[END_REF]. Other inhibitors were tested in mesothelioma cell lines. Perifosine, an analogue of lysophospholipids tested in clinical trials for other types of cancers, that targets PI3K/AKT signalling reduced AKT activation, and also affected EGFR and MET phosphorylation in mesothelioma cells [START_REF] Zhou | Multipoint targeting of the PI3K/mTOR pathway in mesothelioma[END_REF][START_REF] Pinton | Perifosine as a potential novel anti-cancer agent inhibits EGFR/MET-AKT axis in malignant pleural mesothelioma[END_REF] studied phosphorylation of the PI3K/AKT pathway in five human mesothelioma cell lines in comparison non-neoplastic mesothelial cells. Curiously, activation of EGFR, MET and AXL activated AKT but not MAPK pathways. AKT inhibitors reduced proliferation and cell viability, but the apoptosis yield was low. Inhibition of both AKT and mTOR induced maximal inhibition, but did not abolish cell proliferation or induced strong apoptosis [START_REF] Zhou | Multipoint targeting of the PI3K/mTOR pathway in mesothelioma[END_REF]. Moreover, AKT inhibition by shRNAs enhanced mTOR activation [START_REF] Zhou | Multipoint targeting of the PI3K/mTOR pathway in mesothelioma[END_REF]. These results suggest that other control of cell survival operates in mesothelioma cells. Moreover, mTOR pathway also cross-talks with other regulatory pathways, MAPK and Hippo [START_REF] Lopez-Lago | Loss of the tumor suppressor gene NF2, encoding merlin, constitutively activates integrin-dependent mTORC1 signaling[END_REF][START_REF] Sekido | Molecular pathogenesis of malignant mesothelioma[END_REF] TSC2 can be phosphorylated by ERK or RSK [START_REF] Xu | mTOR signaling in tumorigenesis[END_REF]. Collectively, these results show that inhibitors can impact factors of different pathways or cross-talk between distinct pathways, rendering difficult to specifically target a given signalling pathway. In confluent cells, merlin inhibits the activity of the cofactor of transcription, YAP, then inhibiting cell growth (Figure 3) [START_REF] Sekido | Molecular pathogenesis of malignant mesothelioma[END_REF][START_REF] Hao | Role of extracellular matrix and YAP/TAZ in cell fate determination[END_REF][START_REF] Piccolo | The biology of YAP/TAZ: hippo signaling and beyond[END_REF][START_REF] Varelas | The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease[END_REF]. In MPM, an inactivation of the NF2 gene has been evidenced by allelic or biallelic losses and point mutations, occurring in 40-60% of the cases [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF][START_REF] Sekido | Molecular pathogenesis of malignant mesothelioma[END_REF]. Large deletion is the main type of NF2 inactivation. Genetic alterations were also described in other members of the Hippo pathway, SAV1, LATS1 and LATS2, with the higher frequency in LATS2 [START_REF] Bott | The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma[END_REF][START_REF] Miyanaga | Hippo pathway gene mutations in malignant mesothelioma: revealed by RNA and targeted exon sequencing[END_REF][START_REF] Murakami | LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma[END_REF]. Conversely, YAP seems to be overexpressed in MPM and knockdown of YAP expression inhibits proliferation of MPM cells in vitro [START_REF] Mizuno | YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle-promoting genes[END_REF].

Hippo signalling pathway. The Hippo

Other members of these pathways such as AJUBA are also deregulated at the expression level [START_REF] Tanaka | LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade[END_REF]. These findings converge to conclude to an inactivation of the Hippo pathway, in a situation where NF2 cannot regulate YAP, which in turn allows its transcriptional and oncogenic activity. Moreover, NF2 is a target of miRNAs upregulated in MPM samples [START_REF] Guled | CDKN2A, NF2, and JUN are dysregulated among other genes by miRNAs in malignant mesothelioma -A miRNA microarray analysis[END_REF]. Despite a wild-type status for NF2, merlin also appears to be present in an inactivated phosphorylated form in some MPM cells [START_REF] Thurneysen | Functional inactivation of NF2/merlin in human mesothelioma[END_REF].

Hedgehog signalling pathway. The Hedgehog pathway works with a family of secreted signalling proteins; it plays a central role in the development and for tissue homeostasis; it is also involved in control of stem cell proliferation [START_REF] Yao | Hedgehog signaling: From basic research to clinical applications[END_REF]. Its activation is initiated through binding of hedgehog (Hh) proteins (SHH: Sonic hedgehog; DHH: Desert hedgehog, and IHH: Indian hedgehog) to the receptor Patched 1 (PTCH1). PTCH1 is a negative regulator of the Hh signalling pathway via its association with Smoothened (SMO) and triggers a cascade of signalling events downstream from SMO. PTCH1 is considered to have a tumour suppressor function. Activation of the hedgehog pathway results in PTCH1 degradation and SMO release and the transduction of the hedgehog's proteins signal. This leads to the activation of the GLI family of transcription factors (GLI 1-3), through complex interactions of Costal2 (Cos2), Fused (Fu) and Suppressor of fu [Su(fu)] [START_REF] Yao | Hedgehog signaling: From basic research to clinical applications[END_REF].

Mutations of Hh signalling pathway member are infrequent in MPM [START_REF] Lim | Mutational analysis of hedgehog signaling pathway genes in human malignant mesothelioma[END_REF]. A role of the Hh signalling pathway in MPM has been suggested in several works and some members may be regarded as therapeutic targets. An overexpression of genes involved in Hh signalling, SMO and GLI1, was reported in primary cultures derived from MPM tumour samples [START_REF] Li | Gli as a novel therapeutic target in malignant pleural mesothelioma[END_REF][START_REF] Shi | Role of hedgehog signaling in malignant pleural mesothelioma[END_REF][START_REF] You | Targeting of the Hedgehog signal transduction pathway suppresses survival of malignant pleural mesothelioma cells in vitro[END_REF]. Hh pathway may cross-talk with other signalling pathways. Interestingly, inhibition also resulted in a decrease in the expression of Yap [START_REF] Shi | Role of hedgehog signaling in malignant pleural mesothelioma[END_REF]. So far, the activity of antagonists was investigated in a few studies, either alone or in association. The inhibition of SMO or GLI1 by RNA interference or Hh drug antagonists (HhAntag) inhibited cell viability, cell growth and tumour growth in xenografts, accompanied by decreased Ki-67 [START_REF] Shi | Role of hedgehog signaling in malignant pleural mesothelioma[END_REF]. Using siRNA strategy and small molecule inhibitors, vismodegib and cyclopamine, in vitro, inhibition of GLI was more efficient than that of SMO. Combination of GLI-I and pemetrexed, as well as GLI-I and vismodegib demonstrated synergistic effects in suppression of MPM proliferation [START_REF] Li | Gli as a novel therapeutic target in malignant pleural mesothelioma[END_REF]. In another study, SMO inhibitor GDC-0449 and the Gli inhibitor GANT61 reduced Hh signalling in MPM cells, as assessed by the level of GLI1 expression [START_REF] You | Targeting of the Hedgehog signal transduction pathway suppresses survival of malignant pleural mesothelioma cells in vitro[END_REF].

Other pathways: WNT; ubiquitin/proteasome system; p53 network

The WNT signalling pathway normally acts in development; it increases cell proliferation and decrease apoptosis, and is misregulated in cancer [START_REF] Logan | The Wnt signaling pathway in development and disease[END_REF]. WNT signals develop according to three different pathways: the canonical pathway, the planar cell polarity (PCP) pathway and the Wnt/Ca 2+ pathway.

An activation of WNT signalling pathway has been reported in mesothelioma [START_REF] De Assis | The role of key genes and pathways involved in the tumorigenesis of Malignant Mesothelioma[END_REF].

The protein expression of WNT1, WNT2B and WNT5A was studied in 107 MPM tumours by immunohistochemistry [START_REF] Kobayashi | Intratumoral Wnt2B expression affects tumor proliferation and survival in malignant pleural mesothelioma patients[END_REF]. The percentage of WNT2B-positive tumours was significantly higher compared to that of the other WNTs, and was significantly correlated with the expression of survivin and c-Myc. A Cox multivariate analysis demonstrated the WNT2B status to be a significant prognostic factor in MPM patients [START_REF] Kobayashi | Intratumoral Wnt2B expression affects tumor proliferation and survival in malignant pleural mesothelioma patients[END_REF]. Gene expression profiling of MM cell lines, primary MPM tumours, and normal pleural tissue has been studied to determine the expression of genes involved in the WNT signalling pathway, and downstream of WNT signalling [START_REF] Jean | Molecular changes in mesothelioma with an impact on prognosis and treatment[END_REF][START_REF] Fox | Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells[END_REF]. Numerous WNT genes (WNT1, WNT2, WNT5) and WNT-related genes (MYC, CCND1, JUN) were upregulated. WNT2 was most frequently upregulated and played a role for in cell survival [START_REF] Mazieres | Wnt2 as a new therapeutic target in malignant pleural mesothelioma[END_REF]. In another study, WNT3 and WNT5A, as well as FZD2, 4 and 6 were expressed, and several members, WNT4, FZD3, sFRP4, APC and axin2 were downregulated in comparison to mesothelial cells. Moreover, LEF1 was overexpressed in mesothelioma cells [START_REF] Fox | Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells[END_REF]. WNT signalling inhibition is dependent on several factors including the Dickkopf (DKK) gene family. REIC/Dickkopf-3 expression was lower in mesothelioma than in normal tissue, and orthotopic inoculation of REIC/Dickkopf-3-deficient cells followed by intrapleural injection of recombinant REIC/Dickkopf-3-adenovirus resulted in a strong antitumor effect [START_REF] Kashiwakura | Down-regulation of inhibition of differentiation-1 via activation of activating transcription factor 3 and Smad regulates REIC/Dickkopf-3-induced apoptosis[END_REF].

Conflicting data are reported on β-catenin localization in mesothelioma cells [START_REF] Anani | beta-catenin expression in benign and malignant pleural disorders[END_REF].

One study carried out to study miRNAs that were downregulated in MPM compared to lung adenocarcinoma identified potential regulators of WNT signalling [100]. Collectively, these results provide evidence for altered expression of members of WNT pathway and that identification of key targets could be of interest to therapeutic approaches.

The ubiquitin/proteasome system is the main system for controlled protein degradation, and a key regulator of fundamental cellular processes. It mediates proteolysis following two main steps, protein labelling for destruction, and protein degradation by the 26S proteasome [101]. This pathway degrades the damaged or unwanted proteins and prevents constant activation of specific molecular signalling pathways. The process of ubiquitination can be reversed by the action of deubiquitinases (DUB) that counteract E3 ligases. There is some indication that ubiquitin and DUB proteins may play a role in mesothelioma. Identification of proteins interacting with NF2/merlin revealed that the unphosphorylated, growthinhibiting form of NF2/merlin, accumulates in the nucleus and binds to CRL4 DCAF1 (Cul4-Roc1-DDB1-DCAF1) E3 ubiquitin ligase, suppressing NF2 activity and promoting YAP-dependent transcription activity [102,103]. Further studies would be necessary to determine whether the tumour suppressive activity of NF2/merlin is due to its localisation and/or by inhibiting the E3 ubiquitin ligase CRL4 DCAF1 in the nucleus in mesothelioma [103]. Otherwise, as discussed above, mutations were detected in CUL1, which is an essential component of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, and in BAP1 that encodes a DUB enzyme that mediates deubiquitination of histone H2A and HCFC1.

Another important issue accounting for lack of response of MPM cells to antitumoural agents stands in the cells' resistance to apoptosis. Although not mutated, P53 is expressed in MPM cells despite of the presence of negative regulators and does not seem critical in human MPM. However, P53 levels are also regulated by members in PI3K, mTOR and Hippo pathways [START_REF] Piccolo | The biology of YAP/TAZ: hippo signaling and beyond[END_REF][START_REF] Varelas | The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease[END_REF]104].

Further understanding of the implication of P53 in drug-related apoptosis of MPM cells would be of interest.

TARGETED THERAPY

RTKs targeting

Preclinical studies suggested that EGFR targeting with RTKs inhibitors such as gefitinib or erlotinib resulted in reduction of cell proliferation and tumour growth in immunosuppressed mice by down regulation of the PI3K/AKT pathway inhibition. Several clinical trials have been performed, but the results were disappointing as no benefit was found in comparison with current therapies [105,106]. Similar results were obtained with drugs targeting PDFGR such as imatinib, even when associated with chemotherapy [START_REF] Tsao | Phase I trial of cisplatin, pemetrexed, and imatinib mesylate in chemonaive patients with unresectable malignant pleural mesothelioma[END_REF]107] Synthetic lethality approach could be pertinent for mesothelioma therapy as mutations in MPM mainly concern tumour suppressor genes and not oncogenes.

The method consists in provoking cell death by the inactivation of 2 genes cells [110]. When mutated alone, cells are viable, while both inactivation are lethal. In MPM mutations mainly provoke inactivation of tumour suppressor genes and that confers viability to the cells. Targeting tumour suppressor genes by inhibitors is not pertinent, as their activity should be restored. Recently, this type of therapeutic strategy has been validated in preclinical models to NF2 mutated mesothelioma cells using inhibitor of FAK (Focal Adhesion kinase) pathway, based on the hypothesis that FAK pathway became essential to cell adhesion, and consequently survival, when NF2 is inactivated [111]. A clinical trial using FAK inhibitor is ongoing and the results should bring new information on the potential of this strategy.

Targeting the Hippo pathway is of particular interest as this pathway is altered in a large proportion of mesotheliomas [112]. It has be found that several members may be altered, and the transcriptional co-activator YAP plays a central role.

Targeting of YAP protein would be interesting. However, YAP is not only regulated by the Hippo pathway, but also by crosstalks between Hippo and other signalling pathways including WNT, TGF Hedgehog [106]. This renders difficult the targeting of pathways without a clear knowledge of the concurrent activated pathways and cross-talks. However, one could take advantage of the redundancy between pathways to target co-activated members. Regarding the multiple alterations in individual mesothelioma tumours, one may assume that a precise molecular and physiological knowledge of mesotheliomas will benefit to targeted therapies.

CONCLUSIONS

Presently, targeted therapies remain unsuccessful, except for subsets of patients.

Apart from patient status, one of the reasons likely stands in the role and importance of the targeted factors in the tumour status of mesothelioma cells, and the complexity of the mechanisms regulating the functions mesothelioma cells. So far, patients are not selected according to the characteristics of the tumour cells, except for morphological features. Moreover, the question of the nature of the target cell is discussed with regard to the existence of cancer stem cells in tumours. Mutations in PI3KCA, a gene encoding a catalytic subunit of PI3K, have been detected at very low frequency in MPM [START_REF] Mezzapelle | Mutation analysis of the EGFR gene and downstream signalling pathway in histologic samples of malignant pleural mesothelioma[END_REF][START_REF] Shukuya | Identification of actionable mutations in malignant pleural mesothelioma[END_REF]. For review see [START_REF] Gao | Translational control by oncogenic signaling pathways[END_REF][START_REF] Xu | mTOR signaling in tumorigenesis[END_REF]. For reviews, see [119,120].

Legends to Figures

Figure 4

Signalling is initiated by Wnt proteins, which bind to the cell surface receptor Frizzled (FZ) and low density lipoprotein receptor-related protein (LRP) complex.

The signal is transduced to several intracellular proteins (dishevelled, DVL;

glycogen synthase kinase-3β, GSK-3; axin; adenomatous polyposis coli, APC).

Activation of WNT signalling releases the transcription factor, β-catenin, that is normally maintained at low level through continuous proteasome-mediated degradation by the GSK-3/APC/Axin complex that allows phosphorylated βcatenin to be recognized by β-TrCP. Then, β-catenin accumulates and is translocated to the nucleus where it interacts with T-cell factor/lymphoid enhancer factor (LEF/TCF) to affect transcription. Additional non-canonical Wnt signalling pathways, have bee described (see text). For review, see [START_REF] Logan | The Wnt signaling pathway in development and disease[END_REF].

  Pathway is a regulator of organ size, development and differentiation. It is involved in negative control of cell proliferation, and also tissue regeneration and mechanotransduction. It is initiated by extracellular signals that activate NF2, also called merlin by dephosphorylation

  . Several agents synthetized to target VEGFR, such as bevacizumab, sorafenib, cediranib, demonstrated some effects in pre-clinical assays [105]. Association with cytotoxic molecule generally enhanced the cell response. Despite unsuccessful early trials of anti-VEGF therapy, new clinical trials have been carried out, in combination with chemotherapy, and the agents often targeted RTKs other than VEGFR. Only few responses were observed in these assays combining inhibitors and chemotherapy [106]. RTK analyses in MPM showed no or low rate of mutations, frequent autocrine processes and coactivation of several RTK. These findings may account for the lack of responsiveness to RTK inhibitors, and emphasize the need for a better knowledge of the mechanism of mesothelial carcinogenesis. Targeting of pathways So far, in vivo targeting of PI3K/AKT pathway in MPM patients did not entail encouraging results [106, 108]. Several trials showed low responsiveness to the mTOR inhibitor, everolimus, likely due to a compensatory upregulation of pathways. Recently, the results of a phase II trial using everolimus in 59 MPM patients who had received at least one but no more than two platinum-based chemotherapy demonstrated that everolimus has limited clinical activity in advanced MPM patients. Authors concluded that additional studies of single-agent everolimus in advanced MPM are not warranted [109].

  Schematic representation of the MAPKs pathways. Signalling occurs through three different MAPK pathways, extracellular-signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNK), or p38 MAPK. (1) ERK1/2 pathway is activated by mitogens and growth factors such as PDGF and EGF. Following receptor activation, RAS (a small GDP binding protein) is activated by exchange GDP/GTP; recruitment of other membrane components allows the phosphorylation of downstream members RAF, MEK and ERK1/2. ERK1/2 pathway activates various transcription factors (TF) such as ELK1, allowing the expression of the TF c-FOS that may dimerizes with c-JUN to form the TF complex AP-1, c-MYC or STAT3. The Ras/Raf/MEK/MAPK cascade involves members of the RAS family, which are recognized as oncogenes, such as H-RAS, K-RAS and N-RAS. Mutations in members of MAPK pathway have a very low frequency in MPM. Low frequency of mutations were found in KRAS and NRAS [37, 39, 117]). This pathway plays a role in the control of cell proliferation. (2) p38 pathway is mostly stimulated by inflammatory cytokines and in response to various cellular stresses (oxidative stress, UV radiations, hypoxia, ischemia). Several TF can be activated (ATF, ELK1, ETS1, P53) inducing the production of factors involved in immune and inflammatory responses, cell proliferation, survival, migration and gene expression. The p38a isoform negatively regulates cell cycle progression. (3) The third pathway activates JNK; it is also activated by cellular stresses (heat shock, ionizing radiation, UV radiation, oxidative stress, DNA damage, decrease in growth factors and reduction of protein synthesis). The activation of TF: c-JUN, ATF-2, JUNB, ALK1, STAT3, c-MYC, P53 induces control of cell proliferation. JNK isoforms play a role in the apoptotic response to cellular stresses. For details on activation and function of MAPKs, see review [118].

Figure 2 The 2 (

 22 Figure 2

Figure 3

 3 Figure 3

  of knowing the molecular status of targets the tumours, to avoid unfavourable targeting, and not suppress the activity of favourable factors.However, further analyses are needed to establish the exact role of these factors in the viability and survival of mesothelioma cells. The studies carried out in MPM showed that the PI3K/AKT pathway deregulation likely plays a role in the viability of MPM cells, that other pathways are relevant and that the signalling intercrossed. Antitumoural targeted strategy should take these findings into consideration. Otherwise, the heterogeneity between the mesothelioma tumours is evidenced by the data concerning morphology, mutational status of genes, activity of signalling pathways. Knowledge of the different pathways involved in mesothelioma tumour progression is necessary to identify the relevant pathway, in a given patient, and to focus on this pathway. For this purpose, preclinical models reflecting mesothelioma diversity need to be developed. New 2D and 3D models using human mesothelioma cells, and co-cultures would be of interest. As quoted

	It could be necessary to develop new antitumoural drugs [113]. It brings an
	additional level of complexity related to the intratumoural heterogeneity. Targeting
	of cytokines and growth factors receptors that activate signalling pathways with
	inhibitors has been tested in clinical trials. Some growth factors have been
	associated with poor prognosis, and may be more relevant than non-significant
	factors. EGFR does not seem to be related to prognosis; some relation might be
	suggested with expression of AXL or PDGFR-B as good prognosis [114]. VEGF
	serum level has been linked to poor prognosis, but not IHC expression of FGF or
	TGF [114]. Although some molecules in cell cycle and signalling pathways
	(P16 INK4A or MDM2) have been associated to poor prognosis, others were linked
	to better prognosis (P27 KIP1 , P21 CIP1 , AKT, PTEN) [114]. These observations
	demonstrate the interest

by Ceresoli

[115]

, only a tight integration of preclinical and clinical studies will allow achievement of a real progress in MPM patients with this therapeutic strategy

[115]

. It can be added that a better knowledge of the perturbations in the regulation of cell homeostasis in mesothelioma cells is needed. The knowledge of the genetic and epigenetic changes that disrupt cell division, growth, mobility and apoptosis in individual tumours is also necessary to progress. Network organizations gathering epidemiologists, clinicobiologists, basic researchers and therapists, founded on databases and tissue biobanks are progressing and would have strong the impact to treat this disease

[116]

.