Elsevier

European Journal of Cancer

Volume 46, Issue 15, October 2010, Pages 2837-2848
European Journal of Cancer

Receptor tyrosine kinase and downstream signalling analysis in diffuse malignant peritoneal mesothelioma

https://doi.org/10.1016/j.ejca.2010.06.130Get rights and content

Abstract

Our aim was to assess the activation profile of EGFR, PDGFRB and PDGFRA receptor tyrosine kinases (RTK) and their downstream effectors in a series of cryopreserved diffuse malignant peritoneal mesothelioma (DMPM) surgical specimens to discover the targets for drug inhibition. We also made a complementary analysis of the cytotoxic effects of some kinase inhibitors on the proliferation of the human peritoneal mesothelioma STO cell line.

We found the expression/phosphorylation of EGFR and PDGFRB in most of the tumours, and PDGFRA activation in half. The expression of the cognate ligands TGF-α, PDGFB and PDGFA in the absence of RTK mutation and amplification suggested the presence of an autocrine/paracrine loop. There was also evidence of EGFR and PDGFRB co-activation. RTK downstream signalling analysis demonstrated the activation/expression of ERK1/2, AKT and mTOR, together with S6 and 4EBP1, in almost all the DMPMs. No KRAS/BRAF mutations, PI3KCA mutations/amplifications or PTEN inactivation were observed. Real-time polymerase chain reaction revealed the decreased expression of TSC1 c-DNA in half of the tumours. In vitro cytotoxicity studies showed the STO cell line to be resistant to gefitinib and sensitive to sequential treatment with RAD001 and sorafenib; these findings were consistent with the presence of the KRAS mutation G12D in these cells although it was not detectable in the original tumour.

Our results highlight the ligand-dependent activation and co-activation of EGFR and PDGFRB, as well as a connection between these activated RTKs and the downstream mTOR pathway, thus supporting the role of combined treatment with RTK and mTOR inhibitors in DMPM.

Introduction

Diffuse malignant peritoneal mesothelioma (DMPM) is a rare tumour that accounts for 10–20% of all forms of malignant mesothelioma. It was once regarded as a rapidly lethal disease but, over the last few years, survival has remarkably improved (up to 5 years) as a result of the introduction of intensive loco-regional treatment including cytoreductive surgery together with perioperative intraperitoneal chemotherapy in the form of intraperitoneal hyperthermic chemotherapy (HIPEC) +/- early postoperative intraperitoneal chemotherapy.1, 2 However, for those patients not amenable to curative treatment, due to advanced disease stage or deterioration of clinical conditions, prognosis remains poor and systemic treatment is still unsatisfactory.3 Furthermore, approximately 40–60% of patients recurs, but optimal management of recurrent or progressive DMPM has never been standardised.4

Clearly, there is a need to explore new therapies in management of this tumour. One of these is to try to improve our understanding of DMPM molecular and cytogenetic biology, which might make it possible to identify specific new drugs and biomarkers useful for selecting the patients who could benefit from them.

Little is known about DMPM biology, although the prognostic impact of a few biomarkers has been investigated, mainly by means of immunohistochemistry and fluorescence in situ hybridisation. It has been reported that reduced or no p16 protein expression is a frequent alteration in DMPMs and pleural mesotheliomas5, 6 but, although homozygous deletion of the 9p21 locus harbouring the p16INK4a gene significantly correlates with pleural mesothelioma (67–100%), only a small (25%) fraction of DMPMs shows 9p21 deletion.6, 7 On the contrary, EGFR overexpression seems to be significantly more frequent in DMPMs than in pleural mesotheliomas (92% versus 33%, p = 0.0004),5, 8, 9 and both pleural and peritoneal mesotheliomas are negative for c-Kit protein expression.9, 10

The prognostic significance of loss of p16 expression is controversial,3, 11 while overexpression of EGFR,8 matrix metalloprotease-2 (MMP-2) and MMP-9 is described in DMPMs5 did not correlate with prognosis. On the contrary, it has recently been reported that telomerase activity is expressed in the majority of DMPMs and that it negatively affects the clinical outcome of patients undergoing cytoreductive surgery and hyperthermic i.p. chemotherapy.12

Potentially targetable biomarkers are indicated by the overexpression of cytoprotective factors (such as survivin and the other members of the family of apoptosis protein inhibitors) in most DMPMs.13 For example, it has been found that siRNA-mediated survivin knockdown in DMPM cells enhances both spontaneous and cisplatin/doxorubicin-induced apoptosis, thus supporting the notion that survivin antagonists (such as YM-155, which is currently being tested in phase I and II trials) may provide new approaches to the treatment of DMPMs.14 Moreover, microarray analysis, revealing a distinct molecular signature between epithelial and biphasic DMPM, indicated the ubiquitin–proteasome pathway as a potential therapeutic target in biphasic tumours as it was found upregulated.15

Despite the development and present availability of a wide spectrum of drugs targeting receptor tyrosine kinases (RTKs), little is known concerning their status and downstream effectors in DMPMs. There are no published data concerning PDGFRB and PDGFRA expression in DMPMs, whereas both,16 and particularly PDGFRB,17, 18 are expressed in pleural mesotheliomas, although neither plays a diagnostic role nor affects patient outcome. Finally, a recent mutational analysis of exons 18–24 spanning the entire EGFR tyrosine kinase domain found EGFR mutations in 31% of the 29 DMPMs investigated.19

Taking advantage of the availability of a series of DMPM undergoing cytoreductive surgery and subsequent HIPEC, we assessed the deregulation of RTKs (EGFR, PDGFRB and PDGFRA) and their downstream effectors in an attempt to identify the targets susceptible to drug inhibition. This analysis was complemented by a study of the effects of some kinase inhibitors on the proliferation of a human DMPM cell line.

Section snippets

Samples and patients

We analysed DMPM specimens taken from 20 patients who were treated by cytoreductive surgery and subsequent HIPEC at Fondazione IRCCS Istituto Nazionale dei Tumori between 2007 and 2008. Their ages and gender are listed in Table 1. Seventeen DMPMs were histologically classified as epitheliod, one biphasic, one sarcomatoid and one papillary. All the diagnoses were confirmed by immunophenotyping, including immunoreactivity for calretinin (Ventana, pre-diluted), keratin 5 and 6 (clone D5/I6B4,

Biochemical analysis

Frozen surgical specimes of the 20 DMPMs were investigated for EGFR, PDGFRB and PDGFRA expression and phosphorylation status by means of immunoprecipitation and WB (Table 1). Almost all the cases showed EGFR and PDGFRB expression; EGFR and PDGFRB phosphorylation levels similar to or higher than those observed in the controls were found in, respectively, 90% and 75%. PDGFRA expression was found in 17 cases (85%), only 45% of which showed low levels of receptor activation (Fig. 1A shows some

Discussion

This study describes the activation profile of RTKs in DMPMs for the first time as assessed using a comprehensive approach based on biochemical and molecular analyses of frozen surgical specimens, complemented by FISH and immunohistochemical analyses of paired fixed material.

We found the expression and phosphorylation of both EGFR and PDGFRB in most of the tumours, and PDGFRA activation in 50%, coupled with the expression of the cognate ligands TGF-α, PDGFB and PDGFA, and the presence of EGFR

Conflict of interest statement

None declared.

Acknowledgements

The authors wish to greatly acknowledge the financial support received from Istituto Superiore Sanita’ and Associazione Italiana per la Ricerca sul Cancro.

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