Progression Of Malignant Mesothelioma Research Articles

Abstract 4723: Macrophages foster neoplastic progression of malignant mesothelioma and limit response to chemotherapy.

Abstract We, and others, have previously reported that macrophages infiltrating mammary adenocarcinomas foster late-stage cancer progression1-3, and that targeted therapies diminishing macrophage presence in tumors, and improve response to standard chemotherapy (CTX) resulting in slowed primary tumor growth, reduced pulmonary metastasis and increased overall survival (OS) of tumor-bearing mice4,5. These experimental studies correlate with clinical findings indicating that high densities of CD68+ myeloid cells correlate with reduced OS in patients with breast cancer6. Malignant mesotheliomas (MM), a rare cancer that can develop as a result of prior asbestos exposure, is highly resistant to conventional cytotoxic therapy7, and in the pleural space is associated with significant densities of macrophages that also correlate with decreased OS8. To investigate a functional role for macrophages in fostering neoplastic progression of MM, and/or in limiting response to CTX, we investigated the efficacy of several antagonists targeting the colony stimulating factor-1 (CSF1)/CSF-1 receptor (CSF1-R) CSF-1R signaling cascade to reveal the functional significance of macrophages in MM, and to determine if this class of agents exhibited efficacy as monotherapy, or in combination with CTX, to improve OS of tumor-bearing mice. Using a syngeneic orthotopic mouse model for MM, our results indicate that reducing the presence of macrophages in MM significantly slows early, as well as late-stage MM growth, and improves response to standard-of-care CTX. These experimental findings indicate a protumoral role for macrophages in MM, and support clinical investigation of targeted therapies, in combination with CTX, for treatment of patients with MM. 1. Lin et al., J Exp Med 193, 727-740 (2001). 2. DeNardo et al., Cancer Cell 16, 91-102 (2009). 3. Gocheva et al., Genes Dev 24, 241-255 (2010). 4. DeNardo et al., Cancer Discov 1, 54-67 (2011). 5. Shree et al., Genes and Development in press(2011). 6. Campbell et al., Breast Cancer Res Treat 128, 703-711 (2011). 7. Sugarbaker et al., Expert Rev Respir Med 4, 363-372 (2010). 8. Burt et al., Cancer 117, 5234-5244 (2011). The authors acknowledge generous support from the NIH/NCI, Dept of Defense (DoD) Era of Hope Scholar Award Expansion Award, Susan G. Komen Fndt, Breast Cancer Research Fndt, and an Investigator-Initiated Research Award in Mesothelioma from the DoD. Citation Format: Collin Blakely, Tina Bose, Melissa Wheeler, Raphael Bueno, David Sugarbaker, David Jablons, V. Courtney Broaddus, Lisa Coussens. Macrophages foster neoplastic progression of malignant mesothelioma and limit response to chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4723. doi:10.1158/1538-7445.AM2013-4723

MiRNAs signature in microdissected tissues and primary cultured cells of malignant mesotelioma.

e21031 Background: Patients diagnosed for malignant mesothelioma (MM) have poor prognosis and show poor response to conventional chemotherapy. As a consequence, there is a compelling need to identify new targets for the management of MM patients. A growing body of evidence suggests that miRNAs play crucial roles in tumorigenesis of many cancer types. However, their role in MM progression, diagnosis and prognosis is not clear and exhaustive. Methods: miRNA profiling was analyzed in a total of 7 MM compared to 2 normal mesothelium (NM) micro-dissected paraffine embedded tissue specimens and, furthermore, in 6 human cultured primary cell lines (3 MM and 3 NM). The differently expressed miRNAs in tissues and cultured cells were validated by real time PCR. Independent samples T-test was used to determine statistical significance between samples. P <0.05 was considered significant. Results: The miRNA analysis shows that 5 miRNAs are strongly de-regulated in MM compared to NM samples: 2 of them are hypo-expresse...

Inactivation of Merlin in malignant mesothelioma cells and the Hippo signaling cascade dysregulation

Malignant mesothelioma (MM) is an aggressive tumor arising primarily from pleural or peritoneal cavities, which is caused by asbestos exposure after long latency. One of the most frequently mutated genes detected in MM cells is the neurofibromatosis type 2 (NF2) tumor suppressor gene which is located at chromosome 22q12. The NF2 gene encodes Merlin, an ERM (Ezrin/Radixin/Moesin) protein. The underphosphorylated form of Merlin is active and acts as a tumor suppressor by regulating several distinct cellular signaling pathways. One of the downstream pathways regulated by Merlin is the Hippo signaling pathway, which is conserved from Drosophila to mammalian cells and plays important roles in organ size control and cancer development. Recent studies have identified alterations of the components in the Hippo signaling cascade in MM cells, including overexpression of Yes-associated protein (YAP) and inactivation of large tumor suppressor homolog 2 (LATS2). Dysregulation of the Merlin-Hippo signaling cascade is one of the frequent and key events of MM cell development and/or progression. Thus, a strategy to normalize this signaling cascade may be the rationale for developing a new target therapy against MM.

Abstract LB-352: Functional significance of Zic1 and hsa-miR-23a over-expression in malignant mesothelioma

Abstract Background: Epigenetic inactivation of tumour suppressor genes plays a crucial role in the progression of malignant mesothelioma (MM). Previous research has shown epigenetic gene silencing, through DNA methylation or through control of mRNA expression by microRNAs, to be important in MM. Our previous work indicated that Zic1 silencing in MM lines is only partially explained through DNA hypermethylation. Interestingly, miRNA microarray analysis revealed that hsa-miR-23a is over-expressed in MM cell lines, and that this miRNA has target sites in the 3′UTR of the Zic1 mRNA. The aim of the current study was to investigate the functional significance of Zic1 silencing and hsa-miR-23a over-expression in MM. Methods: We first confirmed DNA hypermethylation of Zic1 in human MM specimens using MSP, MS-HRM and COBRA. The expression level of hsa-miR-23a was also confirmed in MM using qPCR. To investigate the functional significance of Zic1 silencing, Zic1 was re-expressed in MM cell lines and effects on proliferation, migration and growth in soft agar were assessed. The relationship between hsa-miR-23a and Zic1 expression was investigated by the use of an antagonist of hsa-miR-23a. Results: Our results indicated that Zic1 is hypermethylated and ectopic expression of Zic1 inhibited cell migration and cell proliferation in MM cell lines. Hsa-miR-23a overexpression in MM is closely linked to the expression of Zic1. Our results suggest that Zic1 is a functional tumour suppressor gene that is silenced by a combination of hypermethylation and hsa-miR-23a-mediated inhibition in MM. These findings suggest that hsa-miR-23a is a potential therapeutic target for MM deserving further pre-clinical study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-352. doi:10.1158/1538-7445.AM2011-LB-352

In Vivo Imaging of Human Malignant Mesothelioma Grown Orthotopically in the Peritoneal Cavity of Nude Mice

Malignant mesothelioma (MM) causes significant morbidity and mortality in patients. With increasing efforts devoted to developing therapeutics targeting mesothelioma, a xenograft mouse model with in vivo tumor imaging is especially desired for evaluating anti-tumor therapies. In the present study, we fluorescently labeled the NCI-H226 human mesothelioma cell line by a lentiviral vector harboring a luciferase-GFP (Luc/GFP) fusion gene driven by the RNA polymerase II promoter. After single-cell cloning by flow cytometry, a clone (named LMB-H226-GL) that stably expresses high levels of Luc/GFP was obtained. The in vivo tumorigenicity of Luc/GFP-labeled LMB-H226-GL was determined by using intraperitoneal injections of the cells in nude mice. LMB-H226-GL was found to be able to consistently form solid tumors in the peritoneum of mice. Tumor growth and aggressive progression could be quantitated via in vivo bioluminescence imaging. The model exhibited the pathological hallmarks consistent with the clinical progression of MM in terms of tumor growth and spread inside the peritoneal cavity. To evaluate the in vivo efficacy of drugs targeting mesothelioma, we treated mice with SS1P, a recombinant immunotoxin currently evaluated in Phase II clinical trials for treatment of mesothelioma. All the tumor-bearing mice had a significant response to SS1P treatment. Our results showed that this is a well-suited model for mesothelioma, and may be useful for evaluating other novel agents for mesothelioma treatment in vivo.

Role of viral induced vascular endothelial growth factor (VEGF) production in pleural effusion and malignant mesothelioma

Vascular endothelial growth factor (VEGF) plays a key role in formation of pleural effusions and in tumorigenesis and progression of malignant mesothelioma. Mesothelial cells (MC) express the viral receptors Toll-like receptor 3 (TLR3), RIG-I and MDA5. Activation of these receptors by viral RNA exemplified by poly(I:C) RNA leads to a time- and dose-dependent increase of mesothelial VEGF synthesis. To show the specific effect of viral receptors knockdown experiments with siRNA for TLR3, RIG-I and MDA5 were performed. This finding of viral induced mesothelial VEGF synthesis may indicate a novel link between viral infections and formation of pleural effusions and progression of malignant mesothelioma.

Genomic events associated with progression of pleural malignant mesothelioma

Pleural malignant mesothelioma (MM) is an aggressive cancer with a very long latency and a very short median survival. Little is known about the genetic events that trigger MM and their relation to poor outcome. The goal of our study was to characterize major genomic gains and losses associated with MM origin and progression and assess their clinical significance. We performed Representative Oligonucleotide Microarray Analysis (ROMA) on DNA isolated from tumors of 22 patients who recurred at variable interval with the disease after surgery. The total number of copy number alterations (CNA) and frequent imbalances for patients with short time (<12 months from surgery) and long time to recurrence were recorded and mapped using the Analysis of Copy Errors algorithm. We report a profound increase in CNA in the short-time recurrence group with most chromosomes affected, which can be explained by chromosomal instability associated with MM. Deletions in chromosomes 22q12.2, 19q13.32 and 17p13.1 appeared to be the most frequent events (55-74%) shared between MM patients followed by deletions in 1p, 9p, 9q, 4p, 3p and gains in 5p, 18q, 8q and 17q (23-55%). Deletions in 9p21.3 encompassing CDKN2A/ARF and CDKN2B were characterized as specific for the short-term recurrence group. Analysis of the minimal common areas of frequent gains and losses identified candidate genes that may be involved in different stages of MM: OSM (22q12.2), FUS1 and PL6 (3p21.3), DNAJA1 (9p21.1) and CDH2 (18q11.2-q12.3). Imbalances seen by ROMA were confirmed by Affymetrix genome analysis in a subset of samples.

Erionite and asbestos differently cause transformation of human mesothelial cells

Malignant mesothelioma (MM) is an aggressive tumor associated with environmental or occupational exposure to asbestos fibers. Erionite is a fibrous zeolite, morphologically similar to asbestos and it is assumed to be even more carcinogenic. Onset and progression of MM has been suggested as the result of the cooperation between asbestos and other cofactors, such as SV40 virus infection. Nevertheless, several cases of MM were associated with environmental exposure to erionite in Turkey, where SV40 was never isolated in MM specimens. We show here that erionite is poorly cytotoxic, induces proliferating signals and high growth rate in human mesothelial cells (HMC). Long term exposure to erionite, but not to asbestos fibers, transforms HMC in vitro, regardless of the presence of SV40 sequences, leading to foci formation in cultured monolayers. Cells derived from foci display constitutive activation of Akt, NF-kappaB and Erk1/2, show prolonged survival and a deregulated cell cycle, involving cyclin D1 and E overexpression. Our results reveal that erionite is able per se to turn HMC into transformed highly proliferating cells and disclose the carcinogenic properties of erionite, prompting for a careful evaluation of environmental exposure to these fibers. The genetic predisposition to the effect of erionite is a separate subject for investigation.

Induction of Stem Cell Factor/c-Kit/Slug Signal Transduction in Multidrug-resistant Malignant Mesothelioma Cells

Malignant mesothelioma (MM) is strongly resistant to conventional chemotherapy by unclear mechanisms. We and others have previously reported that cytokine- and growth factor-mediated signal transduction is involved in the growth and progression of MM. Here, we identified a pathway that involves stem cell factor (SCF)/c-Kit/Slug in mediating multidrug resistance of MM cells. When we compared gene expression profiles between five MM cells and their multidrug-resistant (MM DX) sublines, we found that MM DX cells expressed both SCF and c-Kit and had higher mRNA levels of Slug. Knockdown of c-Kit or Slug expression with their respective small interfering RNA sensitized MM DX cells to the induction of apoptosis by different chemotherapeutic agents, including doxorubicin, paclitaxel, and vincristine. Transfection of c-Kit in parental MM cells in the presence of SCF up-regulated Slug and increased resistance to the chemotherapeutic agents. Moreover, MM cells expressing Slug showed a similar increased resistance to the chemotherapeutic agents. These results indicate that induction of Slug by autocrine production of SCF and c-Kit activation plays a key role in conferring a broad spectrum chemoresistance on MM cells and reveal a novel signal transduction pathway for pharmacological or genetic intervention of MM patients.

The potential role of Interleukin-8 in human malignant mesothelioma progression

The potential role of Interleukin-8 in human malignant mesothelioma progression

Molecular deletion of 9p sequences in non‐small cell lung cancer and malignant mesothelioma

Previously we have reported non-random cytogenetic abnormalities involving the short arm of chromosome 9 (9P) in the majority of primary non-small cell lung cancer (NSCLC) patient samples, which indicated loss of DNA sequences. In another lung tumor, pleural malignant mesothelioma (MM), cytogenetic changes also include apparent deletions of 9p. To define the location and extent of deletions of 9p in NSCLC and MM, Southern blot analyses on six NSCLC and five MM cell lines using molecular probes to 9p loci (IFNA, IFNB1, D9S3, and D9S19) were performed, and DNA dosage was determined by densitometry. Our data demonstrated reduced dosage of 9p sequences in three of six NSCLC and four of five MM lines. A homozygous deletion of D9S3 was found in one NSCLC and one MM cell line. The region of common loss overlapped the D9S3 locus and was flanked by the IFNB1 and D9S19 loci. IFNB as previously been localized to 9p22, and the D9S3 and D9S19 loci have been mapped in this study by in situ hybridization to 9p21 and 9p13, respectively. We hypothesize the existence of one or more tumor suppressor genes on 9p with a role in the development or progression of NSCLC and MM.