Receptor tyrosine kinase and downstream signalling analysis in diffuse malignant peritoneal mesothelioma
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.
References (38)
- et al.
Malignant peritoneal mesothelioma – results from the International Expanded Access Program using pemetrexed alone or in combination with a platinum agent
Lung Cancer
(2009) - et al.
Diagnostic importance of 9p21 homozygous deletion in malignant mesotheliomas
Mod Pathol
(2008) - et al.
Diffuse malignant mesothelioma of the peritoneum and pleura, analysis of markers
Mod Pathol
(2004) - et al.
PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients
Ann Oncol
(2009) - et al.
Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors
Adv Enzyme Regul
(1984) - et al.
Limited efficacy of imatinib mesylate in malignant mesothelioma: a phase II trial
Lung Cancer
(2005) - et al.
Targeted therapies: the rare cancer paradigm
Mol Oncol
(2010) - et al.
Consensus statement on peritoneal mesothelioma
J Surg Oncol
(2008) - et al.
Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for malignant peritoneal mesothelioma: multi-institutional experience
J Clin Oncol
(2009) - et al.
Diffuse malignant peritoneal mesothelioma: Failure analysis following cytoreduction and hyperthermic intraperitoneal chemotherapy (HIPEC)
Ann Surg Oncol
(2009)
Diffuse malignant mesothelioma of the peritoneum: a clinicopathological study of 35 patients treated locoregionally at a single institution
Cancer
And Ladanyi M Homozygous deletion of CDKN2A and codeletion of the methylthioadenosine phosphorylase gene in the majority of pleural mesotheliomas
Clin Cancer Res
Prognostic analysis of clinicopathologic factors in 49 patients with diffuse malignant peritoneal mesothelioma treated with cytoreductive surgery and intraperitoneal hyperthermic perfusion
Ann Surg Oncol
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
Arch Pathol Lab Med
P16 loss and mitotic activity predict poor survival in patients with peritoneal malignant mesothelioma
Clin Cancer Res
Multiple mechanisms of telomere maintenance exist and differentially affect clinical outcome in diffuse malignant peritoneal mesothelioma
Clin Cancer Res
Survivin is highly expressed and promotes cell survival in malignant peritoneal mesothelioma
Cell Oncol
Multicenter phase II trial of YM155, a small-molecule suppressor of survivin, in patients with advanced, refractory, non-small-cell lung cancer
J Clin Oncol
Molecular profiling of malignant peritoneal mesothelioma identifies the ubiquitin-proteasome pathway as a therapeutic target in poor prognosis tumors
Oncogene
Cited by (25)
Translating mesothelioma molecular genomics and dependencies into precision oncology-based therapies
2020, Seminars in Cancer BiologyCitation Excerpt :Self-sufficiency in growth signaling, frequently through deregulation and activation of receptor tyrosine kinase (RTK) pathways, is a hallmark of cancer [59,60]. While genes encoding RTKs and downstream effectors including the RAS/RAF/MEK/MAPK and PIK3CA/AKT pathways are not subject to oncogenic, activating mutations in MPM, unbiased screens as well as specific interrogation of these pathways as therapeutic targets have unveiled vulnerabilities in RTK pathways [61–73]. In fact, a growth network comprised of multiple RTKs has been proposed such that combined inhibition of multiple pathways yields greater efficacy [60,61,74].
Pharmacogenomic analyzis of the responsiveness of gastrointestinal tumor cell lines to drug therapy: A transportome approach
2016, Pharmacological ResearchCitation Excerpt :Growth factor receptors, EGFR and PDGFRA, also showed significant correlations with these clusters (Figs. 2–4) although they maintain an inverse relationship. Despite these two receptors trigger similar pathways involved in proliferation, it has been reported in different cases that their gene amplification are mutually exclusive [52,53]. Most of the genes described so far have been previously reported to be somehow related to cell proliferation and survival, using a variety of experimental approaches.
Elevated PDGFRB gene copy number gain is prognostic for improved survival outcomes in resected malignant pleural mesothelioma
2014, Annals of Diagnostic PathologyCitation Excerpt :The gene sets activated by PDGFRα included C21-steroid hormone biosynthesis and in the PDGFRβ pathway contained angiogenic and epidermal growth factor receptor (EGFR) pathways [29]. PDGFRβ activation has been associated with EGFR activation, and EGFR overexpression has been reported in mesothelioma (pleural and peritoneal) tumor cells [30,31]. It is possible that the subtypes of receptors interact in a regulatory fashion and may also impose a negative feedback inhibition.
Diffuse malignant peritoneal mesothelioma: Long-term survival with complete cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC)
2013, European Journal of CancerCitation Excerpt :However, we found no mutations in a comprehensive analysis of surgical specimens from our case-series. Conversely, our results highlighted ligand-dependent activation and co-activation of EGFR and PDGFRB, and the connection between these activated tyrosine-kinases and downstream mammalian target of rapamycin (mTOR) pathway, thus suggesting the use of combined tyrosine-kinase and mTOR inhibitors in DMPM.32 A possible criticism of this study involves the definition of adequate cytoreduction.
Crocidolite asbestos-induced signal pathway dysregulation in mesothelial cells
2011, Mutation Research - Genetic Toxicology and Environmental MutagenesisCitation Excerpt :Met protein was reported to be increased in malignant mesothelial cells [20] by no significant difference between malignant mesothelial cells and benign mesothelial cells was found in our study. PDGFRB was found to be increased in malignant mesothelial cells [24] but was not detected in our study. The variability of individual proteins may be a result of different antibodies (affinity and phosphorylation sites), different cell lines (heterogeneity) as well as different methodology (Western Blot vs. IHC) used in these studies.
Coactivation of receptor tyrosine kinases in malignant mesothelioma as a rationale for combination targeted therapy
2011, Journal of Thoracic OncologyCitation Excerpt :Together, these data suggest that MET activation in MM may account for the poor response to EGFR inhibitors alone in this disease.20,21 Finally, Perrone et al.16 recently described a cross-activation of EGFR and PDGFRβ in MM tumors. Of our 13 cell lines, only the Meso 56 cell line showed evidence of PDGFRβ signaling, in the form of slight activation of PDGFRβ after treatment with rapamycin.