Molecular Alterations in Asbestos-Related Lung Cancer

Background: Asbestos is a well known cancer-causing mineral fibre, which has a synergistic effect on lung cancer risk in combination with tobacco smoking. Several in vitro and in vivo experiments have demonstrated that asbestos can evoke chromosomal damage and cause alterations as well as gene expression changes. Lung tumours, in general, have very complex karyotypes with several recurrently gained and lost chromosomal regions and this has made it difficult to identify specific molecular changes related primarily to asbestos exposure. The main aim of these studies has been to characterize asbestos-related lung cancer at a molecular level. Methods: Samples from asbestos-exposed and non-exposed lung cancer patients were studied using array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to detect copy number alterations (CNA) as well as microsatellite analysis to detect allelic imbalance (AI). In addition, asbestos-exposed cell lines were studied using gene expression microarrays. Results: Eighteen chromosomal regions showing differential copy number in the lung tumours of asbestos-exposed patients compared to those of non-exposed patients were identified. The most significant differences were detected at 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2 and 19p13.1-p13.3 (p<0.005). The alterations at 2p and 9q were validated and characterized in detail using AI and FISH analysis in a larger study population. Furthermore, in vitro studies were performed to examine the early gene expression changes induced by asbestos in three different lung cell lines. The results revealed specific asbestos-associated gene expression profiles and biological processes as well as chromosomal regions enriched with genes believed to contribute to the common asbestos-related responses in the cell lines. Interestingly, the most significant region enriched with asbestos-response genes was identified at 2p22, close to the previously identified region showing asbestos-related CNA in lung tumours. Additionally, in this thesis, the dysregulated biological processes (Gene Ontology terms) detected in the cell line experiment were compared to dysregulated processes identified in patient samples in a later study (Ruosaari et al., 2008a). Commonly affected processes such as those related to protein ubiquitination, ion transport and surprisingly sensory perception of smell were identified. Conclusions: The identification of specific CNA and dysregulated biological processes shed some light on the underlying genes acting as mediators in asbestos-related lung carcinogenesis. It is postulated that the combination of several asbestos-specific molecular alterations could be used to develop a diagnostic method for the identification of asbestos-related lung cancer.

Molecular profiling of malignant pleural mesothelioma and pulmonary fibrosis

Malignant mesothelioma (MM) is a rare, usually incurable, disease mainly caused by former exposure to asbestos. Even though MM has a strong etiological link, genetic factors may play a role, since not all cases can be linked to former asbestos exposure. This thesis focuses on lung diseases, mainly malignant mesothelioma (MM), and idiopathic pulmonary fibrosis (IPF), which resembles asbestosis. The specific asbestos-related pathways associated with malignant as well as non-malignant lung diseases, still need to be clarified. Since most patients diagnosed with MM or asbestosis/fibrosis have a dismal prognosis and few therapeutic options are available, early diagnosis and better understanding of the disease pathogenesis are of the utmost importance. The first objective of this thesis was to identify asbestos specific differentially expressed genes. This was approached by using high-resolution gene expression arrays, and three different human lung cell lines, as well as with three different bioinformatics approaches. Since the first study aimed to elucidate potential early changes, the second study was used to screen DNA copy number changes in MM tumour samples. This was performed using genome wide microarrays for identification of DNA copy number changes characterstic for MM. Study III focused on the role of gremlin in the regulation of bone morphogenetic protein (BMPs) in IPF. Further studies were conducted in asbestos-exposed cell cultures as well as in an asbestos-induced mouse model. Furthermore, GATA-6 was studied in MM and metastatic pleural adenocarcinoma. The GATA transcription factors are important during embryonic development, but their role in cancer is still unclear. GATA-6 is a co-factor/target of thyroid transcription factor 1 (TTF-1), which is used in differential diagnostics of pleural MM and adenocarcinoma. Bioinformatics probed the genes and biological processes ordered in terms of significance, clusters, and highly enriched chromosomal regions. The study revealed several already identified targets, produced new ideas about genes which are central for asbestos exposure, as well as provided supplementary data for researchers to check their own novel findings or ideas. The analysis revealed DNA copy number changes characteristic for MM tumors. The most common regions of loss were detected in 1p, 3p, 6q, 9p, 13, 14, and 22, and gains at 17q. The histological features in asbestosis and IPF are very similar, wherefore IPF can be studied in asbestos models. The BMP antagonist gremlin was up-regulated by asbestos exposure in human epithelial cell lines, which was also observed in Study I. The transforming growth factor (TGF) -β and BMP expression and signaling activities were measured from murine and human fibrotic lungs. BMP-7 signaling was down-regulated in response to up-regulation of gremlin, and restoration of BMP-7 signaling prevented progression of fibrosis in mice. Therefore, the study suggests that the restoration of BMP-7 signaling in fibrotic lung could potentially aid in the treatment of IPF patients. Study IV revealed that GATA-6 was strongly expressed in the majority of the MM cases, and correlated statistically significant with longer survival in subgroups of MM.

Microarrays in Lung Cancer Research: From Comparative Analyses to Verified Findings

Keuhkosyöpä on yleisimpiä syöpätauteja. Se jaetaan kahteen päätyyppiin: pienisoluiseen ja ei-pienisoluiseen keuhkosyöpään. Ei-pienisoluinen keuhkosyöpä jaetaan lisäksi alatyyppeihin, joista suurimmat ovat levyepiteeli-, adeno- ja suurisoluinen karsinooma. Keuhkosyövän tärkein riskitekijä on tupakointi, mutta muutkin työ- ja elinympäristön altisteet, kuten asbesti, voivat johtaa syöpään. Väitöstyössä tutkittiin kahdenlaisten keuhkosyöpäryhmien erityispiirteitä. Työssä kartoitettiin, onko löydettävissä muutoksia, jotka erottavat asbestikeuhkosyövät muista syövistä sekä luuytimeen varhaisessa vaiheessa leviävät keuhkosyövät leviämättömistä syövistä. Tutkimusten ensimmäisessä vaiheessa käytettiin mikrosirupohjaisia menetelmiä, jotka mahdollistavat jopa kaikkien geenien tarkastelun yhden kokeen avulla. Vertailevien mikrosirututkimusten avulla on mahdollista paikantaa geenejä tai kromosomialueita, joiden muutokset erottelevat ryhmät toisistaan. Asbestiin liittyvissä tutkimuksissa paikannettiin kuusi kromosomialuetta, joissa geenien kopiolukumäärän sekä ilmenemistason muutokset erottelivat potilaat altistushistorian mukaan. Riippumattomilla laboratoriomenetelmillä tehtyjen jatkoanalyysien avulla pystyttiin varmistamaan, että 19p-alueen häviämä oli yhteydessä asbestialtistukseen. Työssä osoitettiin myös, että 19p-alueen muutoksia voidaan indusoida altistamalla soluja asbestille in vitro. Tutkimuksessa saatiin lisäksi viitteitä asbestispesifisistä muutoksista signaalinvälitysreiteissä, sillä yhdessä toimivien geenien ilmentymisessä havaittiin eroja asbestille altistuneiden ja altistumattomien välillä. Vertailemalla luuytimeen syövän aikaisessa vaiheessa levinneiden ja leviämättömien keuhkoadenokarsinoomien muutosprofiileita toisiinsa, paikannettiin viisi aluetta, joilla geenien kopiolukumäärä- sekä ilmenemistason muutokset erottelivat ryhmät toisistaan. Jatkoanalyyseissä havaittiin, että 4q-alueen häviämää esiintyi adenokarsinoomien lisäksi levyepiteelikarsinoomiin, jotka olivat levinneet luuytimeen. Myös keuhkosyöpien aivometastaaseissa alue oli toistuvasti hävinnyt. Väitöstyön tutkimukset osoittavat, että vertailevien mikrosiruanalyysien avulla saadaan tietoa syöpäryhmien erityispiirteistä. Työssä saadut tulokset osoittavat, että 19p-alueen muutokset ovat tyypillisiä asbestikeuhkosyöville ja 4q-alueen muutokset luuytimeen aikaisessa vaiheessa leviäville keuhkosyöville.

Molecular basis for the development of diagnostic methods and specific treatment modalities for idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown aetiology and poor prognosis. IPF is characterized by alveolar epithelial damage that leads tissue remodelling and ultimately to the loss of normal lung architecture and function. Treatment has been focused on anti-inflammatory therapies, but due to their poor efficacy new therapeutic modalities are being sought. There is a need for early diagnosis and also for differential diagnostic markers for IPF and other interstitial lung diseases. The study utilized patient material obtained from bronchoalveolar lavage (BAL), diagnostic biopsies or lung transplantation. Human pulmonary fibroblast cell cultures were propagated and asbestos-induced pulmonary fibrosis in mice was used as an experimental animal model of IPF. The possible markers for IPF were scanned by immunohistochemistry, RT-PCR, ELISA and western blot. Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in tissue remodelling. Microarray studies have introduced potential markers that could serve as additional tools for the assessment of IPF and one of the most promising was MMP 7. MMP-7 protein levels were measured in the BAL fluid of patients with idiopathic interstitial lung diseases or idiopathic cough. MMP-7 was however similarly elevated in the BAL fluid of all these disorders and thus cannot be used as a differential diagnostic marker for IPF. Activation of transforming growth factor (TGF)-ß is considered to be a key element in the progression of IPF. Bone morphogenetic proteins (BMP) are negative regulators of intracellular TGF-ß signalling and BMP-4 signalling is in turn negatively regulated by gremlin. Gremlin was found to be highly upregulated in the IPF lungs and IPF fibroblasts. Gremlin was detected in the thickened IPF parenchyma and endothelium of small capillaries, whereas in non-specific interstitial pneumonia it localized predominantly in the alveolar epithelium. Parenchymal gremlin immunoreactivity might indicate IPF-type interstitial pneumonia. Gremlin mRNA levels were higher in patients with end-stage fibrosis suggesting that gremlin might be a marker for more advanced disease. Characterization of the fibroblastic foci in the IPF lungs showed that immunoreactivity to platelet-derived growth factor (PDGF) receptor-α and PDGF receptor-β was elevated in IPF parenchyma, but the fibroblastic foci showed only minor immunoreactivity to the PDGF receptors or the antioxidant peroxiredoxin II. Ki67 positive cells were also observed predominantly outside the fibroblastic foci, suggesting that the fibroblastic foci may not be composed of actively proliferating cells. When inhibition of profibrotic PDGF-signalling by imatinib mesylate was assessed, imatinib mesylate reduced asbestos-induced pulmonary fibrosis in mice as well as human pulmonary fibroblast migration in vitro but it had no effect on the lung inflammation.