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Objectives: Although asbestos exposure is the most common cause of malignant mesothelioma (MM), an aggressive cancer of the pleura or peritoneum, up to 7% of patients harbor a genetic predisposition to MM. Pathogenic germline variants in the BRCA1-associated protein 1 (BAP1) gene cause a dominantly inherited tumor predisposition syndrome, BAP1-TPDS, in which MM is the second most common associated cancer. Other frequent cancers in BAP1-TPDS are uveal melanoma (UM), cutaneous melanoma and renal cell carcinoma. Additionally patients can exhibit benign skin lesions, BAP1-inactivated nevi (BIN). Most BINs arise sporadically, but patients with BAP1-TPDS may harbor multiple BINs before other tumors or as the only indication of the syndrome. Our objective was to establish the frequency of pathogenic germline BAP1 variants in Finnish patients with MM. Materials and methods: 56 DNA samples archived in the Helsinki Biobank from Finnish patients with MM were sequenced for germline BAP1 variations. Formalin fixed paraffin embedded nevi from a pathogenic variant carrier were subjected to immunohistochemistry and exome sequencing. Results: Sanger sequencing identified one patient with Finnish founder mutation c.1780_1781insT, p. (G549Vfs*49) in BAP1. The carrier was diagnosed with MM over fifteen years before the cohorts mean onset age (mean 68, range 27 to 82) although the patient had no asbestos exposure or family history of BAP1-TPDS. However, the patient had three BINs removed prior to the MM. The c.1780_1781insT is now found from five Finnish BAP1-TPDS families with unknown common ancestor. Conclusion: The frequency of pathogenic germline BAP1 variants in Finnish patients with MM is 1.8 % (95 % CI, 0.04 to 9.2), comparable to the frequency in Finnish patients with UM (1.9 %). The frequency of recurring BINs in patients with BAP1-TPDS should be studied further and genetic testing for BAP1 variants considered if the patient has >= 2 BAP1-TPDS core tumors, including BINs.

Nestekertymien sytologisten irtosolututkimusten uuden luokituksen jokaiseen kuuteen ryhmään sisältyvät sytologinen kuvaus, maligniteettiriski ja toimenpidesuositus. Järjestelmä mahdollistaa paremman tiedonkulun patologin ja kliinikon välillä. publishedVersion

Desmoid tumors (DT) are nonmalignant mesenchymal neoplasms with a high recurrence rate. Tumors concentrate into the small bowel mesentery and the abdominal wall when associated with familial adenom...

Background and objectives Desmoid tumors are soft-tissue tumors originating from myofibroblasts with a tendency to recur after surgery. High expression of proliferation markers is associated with shortened progression-free and/or overall survival in many neoplasms, including soft-tissue sarcomas. We investigated the prognostic role of cyclin A and Ki67 in desmoid tumors by immunohistochemistry. Methods The study included 76 patients with desmoid tumor operated at Helsinki University Hospital between 1987 and 2011. A tissue micro array (TMA) was constructed and the TMA sections were immunostained with cyclin A and Ki67 antibodies. A computer-assisted image analysis was performed. Results Cyclin A expression was evaluable in 74 and Ki67 in 70 patients. Cyclin A immunopositivity varied from 0% to 9.9%, with a mean of 1.9%. Cyclin A expression correlated significantly with Ki67. Cyclin A expression was associated with recurrence-free survival (HR 1.9, 95% CI = 1.1-3.2, P = .02), as were positive margin (HR 6.0, 95% CI = 1.6-22.5, P = .008) and extremity location (HR 5.3, 95% CI = 1.7-16.8, P = 0.005). Ki67 immunopositivity varied from 0.33% to 13.8%, with a mean of 4.6%, but had no significant prognostic impact (HR 1.1, P = .2). Conclusions Our study indicates that cyclin A may be a new prognostic biomarker in surgically treated desmoid tumors.

The histologic manifestation of idiopathic pulmonary fibrosis (IPF) is usual interstitial pneumonia (UIP), which is a good prognostic determinant of survival compared with other histologic interstitial lung disease patterns. According to the current international guidelines, the histologic features of suspected IPF/UIP are divided into 4 categories: UIP, probable UIP, possible UIP, and not UIP pattern. Four pulmonary pathologists who were blinded to clinicoradiologic information reevaluated 50 surgical lung biopsies (83.3%), 6 lung explant (10.0%), and 4 autopsy samples (6.7%) from the FinnishIPF registry (N=60) using the current diagnostic guidelines. Additional histologic features atypical for UIP were also evaluated and compared with clinicora-diologic information. The interobserver agreement of pathologists was examined by Cohen kappa (κ) coefficient; the survival of the patients was estimated with Kaplan-Meier curves. The histologic reevaluation indicated that 38 of 60 patients (63.3%) had definite UIP. Inflammation was the most common additional histologic finding (15/60, 25.0%). The interobserver agreement on histologic diagnosis ranged from slight (κ=0.044) to substantial (κ=0.779). The interobserver agreement varied extensively with regard to the presence of giant cells. The observed histologic features displayed no association with radiologic patterns or survival. Definite UIP and honeycombing findings in high-resolution computed tomography correlated with poor prognosis. A high level of interobserver variability was observed between pathologists, even in this well-defined cohort of IPF patients, which highlights the importance of multidisciplinary decision making in IPF diagnostics and stresses the need for a reassessment of the histologic criteria.

// Miao Yin 1, 2 , Mira Tissari 1, 2 , Jenni Tamminen 1, 2 , Irene Ylivinkka 1 , Mikko Ronty 3 , Pernilla von Nandelstadh 4 , Kaisa Lehti 4, 5, 6 , Marko Hyytiainen 4 , Marjukka Myllarniemi 7 and Katri Koli 1, 2 1 Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland 2 Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland 3 Department of Pathology, University of Helsinki and Fimlab Laboratories, Pathology, Tampere, Finland 4 Research Programs Unit, Genome Scale Biology, University of Helsinki, Helsinki, Finland 5 K. Albin Johansson Foundation, Finnish Cancer Institute, Helsinki, Finland 6 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Solna, Sweden 7 Department of Pulmonary Medicine, University of Helsinki and Helsinki University Hospital, Heart and Lung Center, Helsinki, Finland Correspondence to: Katri Koli, email: katri.koli@helsinki.fi Keywords: gremlin, mesothelioma, invasion Received: June 07, 2017 Accepted: September 21, 2017 Published: October 06, 2017 ABSTRACT Malignant mesothelioma originates from mesothelial cells and is a cancer type that aggressively invades into the surrounding tissue, has poor prognosis and no effective treatment. Gremlin-1 is a cysteine knot protein that functions by inhibiting BMP-pathway activity during development. BMP-independent functions have also been described for gremlin-1. We have previously shown high gremlin-1 expression in mesothelioma tumor tissue. Here, we investigated the functions of gremlin-1 in mesothelioma cell migration and invasive growth. Gremlin-1 promoted mesothelioma cell sprouting and invasion into three dimensional collagen and Matrigel matrices. The expression level of gremlin-1 was linked to changes in the expression of SNAI2, integrins, matrix metalloproteinases (MMP) and TGF-β family signaling - all previously associated with a mesenchymal invasive phenotype. Small molecule inhibitors of MMPs completely blocked mesothelioma cell invasive growth. In addition, inhibitors of TGF-β receptors significantly reduced invasive growth. This was associated with reduced expression of MMP2 but not SNAI2, indicating that gremlin-1 has both TGF-β pathway dependent and independent mechanisms of action. Results of in vivo mesothelioma xenograft experiments indicated that gremlin-1 overexpressing tumors were more vascular and had a tendency to send metastases. This suggests that by inducing a mesenchymal invasive cell phenotype together with enhanced tumor vascularization, gremlin-1 drives mesothelioma invasion and metastasis. These data identify gremlin-1 as a potential therapeutic target in mesothelioma.

Background and objectivesEstrogen receptor signaling and cyclin D1 have a major role in tumor cell proliferation in breast cancer. Desmoid tumors are rare neoplasms that may respond to endocrine treatment. The present study aimed to investigate the expression levels and the clinical relevance of estrogen receptor beta (ER beta) and cyclin D1 in desmoid tumors. MethodsThis study consists of 83 patients with a surgically treated desmoid tumor. ER beta and cyclin D1 expression was examined by immunohistochemistry in tissue microarrays. Cyclin A and Ki67 were studied in our previous work. ResultsMedian ER beta expression was 10.8%. ER beta expression correlated with expression of the proliferation antigens Ki67 (r(p)=0.35, P=0.003), cyclin D1 (r(p)=0.34, P=0.004), and cyclin A (r(p)=0.34, P=0.004). ER beta immunoexpression showed a trend towards predictive impact for recurrence as a continuous variable. Further explorative analysis indicated that very high ER beta expression was related to high risk of relapse (hazard ratio [HR] 2.6; P=0.02).Median cyclin D1 expression was 15.6%. High cyclin D1 expression was associated with high Ki67 and cyclin A expression. Cyclin D1 was not associated with time to recurrence. ConclusionsER beta and cyclin D1 immunopositivity correlated with high proliferation in desmoid tumors. High ER beta expression might be predictive for postoperative recurrence.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by irreversible airflow limitation. Cigarette smoking represents the main risk factor, but the specific mechanisms of COPD are not completely understood. Our aim was to identify COPD-specific proteomic changes involved in disease onset and severity. A comparative proteomic analysis of 51 lung tissues from nonsmokers, smokers, smokers with mild to moderate (stage I–II) COPD, severe to very severe COPD (stage III–IV), and patients with α-1-antitrypsin deficiency (AATD) and idiopathic pulmonary fibrosis (IPF) was performed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Selected COPD-specific changes were validated by immunoblotting and further by ELISA in 120 induced sputum and plasma samples from nonsmokers, smokers, and patients with COPD (stage I–III). Altogether 82 altered proteins were identified comprising COPD-, AATD-, and IPF-specific, overlapping, and unspecific changes. Cathepsin D (CTSD), dihydropyrimidinase-related protein 2 (DPYSL2), transglutaminase 2 (TGM2), and tripeptidyl-peptidase 1 (TPP1) were validated as COPD-specific. TGM2 was not associated with smoking and correlated with COPD severity in lung tissue. TGM2 levels in sputum and plasma were elevated in patients with COPD (stage II–III) and correlated with lung function. In conclusion, new proteins related to COPD onset and severity could be identified with TGM2 being a novel potential diagnostic and therapeutic target for COPD. Further studies in carefully characterized cohorts are required to validate the identified changes.

Malignant mesothelioma is an aggressive cancer with poor prognosis. It is characterized by prominent extracellular matrix, mesenchymal tumor cell phenotypes and chemoresistance. In this study, the ability of pirfenidone to alter mesothelioma cell proliferation and migration as well as mesothelioma tumor microenvironment was evaluated. Pirfenidone is an anti-fibrotic drug used in the treatment of idiopathic pulmonary fibrosis and has also anti-proliferative activities. Mesothelioma cell proliferation was decreased by pirfenidone alone or in combination with cisplatin. Pirfenidone also decreased significantly Transwell migration/invasion and 3D collagen invasion. This was associated with increased BMP pathway activity, decreased GREM1 expression and downregulation of MAPK/ERK and AKT/mTOR signaling. The canonical Smad-mediated TGF-β signaling was not affected by pirfenidone. However, pirfenidone blocked TGF-β induced upregulation of ERK and AKT pathways. Treatment of mice harboring mesothelioma xenografts with pirfenidone alone did not reduce tumor proliferation in vivo. However, pirfenidone modified the tumor microenvironment by reducing the expression of extracellular matrix associated genes. In addition, GREM1 expression was downregulated by pirfenidone in vivo. By reducing two major upregulated pathways in mesothelioma and by targeting tumor cells and the microenvironment pirfenidone may present a novel anti-fibrotic and anti-cancer adjuvant therapy for mesothelioma.

Desmoid tumors are soft-tissue tumors originating from myofibroblasts with a tendency to recur after surgery. High expression of proliferation markers is associated with shortened progression-free and/or overall survival in many neoplasms, including soft-tissue sarcomas. We investigated the prognostic role of cyclin A and Ki67 in desmoid tumors by immunohistochemistry.The study included 76 patients with desmoid tumor operated at Helsinki University Hospital between 1987 and 2011. A tissue micro array (TMA) was constructed and the TMA sections were immunostained with cyclin A and Ki67 antibodies. A computer-assisted image analysis was performed.Cyclin A expression was evaluable in 74 and Ki67 in 70 patients. Cyclin A immunopositivity varied from 0% to 9.9%, with a mean of 1.9%. Cyclin A expression correlated significantly with Ki67. Cyclin A expression was associated with recurrence-free survival (HR 1.9, 95% CI = 1.1-3.2, P = .02), as were positive margin (HR 6.0, 95% CI = 1.6-22.5, P = .008) and extremity location (HR 5.3, 95% CI = 1.7-16.8, P = 0.005). Ki67 immunopositivity varied from 0.33% to 13.8%, with a mean of 4.6%, but had no significant prognostic impact (HR 1.1, P = .2).Our study indicates that cyclin A may be a new prognostic biomarker in surgically treated desmoid tumors.

Background Desmoid tumors (aggressive fibromatosis) are rare soft tissue tumors which frequently recur after surgery. Desmoid tumors arise from musculoaponeurotic tissue in the extremities, head and neck, abdominal wall, or intraabdominally. Our aim was to examine the outcome of radiotherapy of desmoid tumors in a single institution series. Patients and methods We evaluated 41 patients with desmoid tumors treated with 49 radiotherapies between 1987 and 2012. Radiologic images for response evaluation were reassessed and responses to treatment registered according to RECIST criteria 1.1. For patients with local failures radiation dose distribution was determined in each local failure volume using image co-registration. Recurrences were classified as in-target, marginal, or out-oftarget. Prognostic factors for radiotherapy treatment failure were evaluated. Results Radiotherapy doses varied from 20-63Gy (median 50 Gy) with a median fraction size of 2 Gy. The objective response rate to definitive radiotherapy was 55% (12/22 patients). Median time to response was 14 months. A statistically significant dose-response relation for definitive and postoperative radiotherapy was observed both in univariate (p-value 0.002) and in multivariate analysis (p-value 0.02) adjusted for potential confounding factors. Surgery before radiotherapy or surgical margin had no significant effect on time to progression. Nine of 11 (82%) local failures were classified as marginal and two of 11 (18%) in-target. None of the recurrences occurred totally out-of-target. Conclusions Radiotherapy is a valuable option for treating desmoid tumors. Radiotherapy dose appears to be significantly associated to local control.

Screening of anaplastic lymphoma tyrosine kinase (ALK) gene fusions in non-small cell lung cancer (NSCLC) patients enables the identification of the patients likely to benefit from ALK-targeted therapy. Our aim was to assess the prevalence of ALK fusion in Finnish NSCLC patients, which has not been reported earlier, and to study the presence of ALK fusion in relation to clinicopathological characteristics and other driver gene mutations. A total of 469 formalin-fixed paraffin-embedded tumor tissue specimens from Finnish NSCLC patients were screened for ALK fusion by immunohistochemistry (IHC). For confirmation of IHC results, fluorescence in situ hybridization (FISH) was conducted for 171 specimens. Next-generation sequencing was performed for all ALK-positive specimens to characterize the association of ALK fusion with mutations in targeted regions of 22 driver genes. Of the 469 tumors screened, 11 (2.3%) harbored an ALK fusion, including nine adenocarcinomas and two large cell carcinomas. The IHC results for all 11 ALK-positive and 160 random ALK-negative specimens were confirmed by FISH. ALK fusion was significantly associated with never/ex-light smoking history (P

Non-small cell lung carcinoma (NSCLC) is the most common subtype of lung cancer. The oncogenic potential of receptor tyrosine kinases (RTKs) is widely known and they are potential targets for tailored therapy. Ephrin receptors (Ephs) form the largest group of RTKs. Nevertheless, Ephs are not widely studied in NSCLC so far. The aim of our study was to investigate novel mutations of Eph genes (EPHA1–8, EPHB1–4, EPHB6) and their association with clinically relevant mutations in BRAF, EML4-ALK, EGFR, INSR, KDR, KRAS, MET, PDGFRA, PDGFRB, PIK3, PTEN, RET, and TP53 in NSCLC patients. Targeted resequencing was conducted on 81 formalin-fixed, paraffin-embedded NSCLC tumor specimens. We analyzed missense and nonsense mutations harbored in the coding regions of the selected genes. We found 18 novel mutations of Ephs in 20% (16 of 81) of the patients. Nearly half of these mutations occurred in the protein kinase domain. The mutations were not mutually exclusive with other clinically relevant mutations. Our study shows that Ephs are frequently mutated in NSCLC patients, and occur together with other known mutations relevant to the pathogenicity of NSCLC. © 2013 Wiley Periodicals, Inc.

Objectives: Non-small cell lung cancer (NSCLC) is a common cancer with a poor prognosis. The aim of this study was to screen Finnish NSCLC tumor samples for common cancer-related mutations by targeted next generation sequencing and to determine their concurrences and associations with clinical features. Materials and methods: Sequencing libraries were prepared from DNA isolated from formalin-fixed, paraffin-embedded tumor material of 425 patients using the AmpliSeq Colon and Lung panel covering mutational hot spot regions of 22 cancer genes. Sequencing was performed with the Ion Torrent Personal Genome Machine (PGM). Results: Data analysis of the hot spot mutations revealed mutations in 77% of the patients, with 7% having 3 or more mutations reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Two of the most frequently mutated genes were TP53 (46%) and KRAS (25%). KRAS codon 12 mutations were the most recurrently occurring mutations. EGFR mutations were significantly associated with adenocarcinoma, female gender and never/light-smoking history; CTNNB1 mutations with light ex-smokers, PlIC3CA and TP53 mutations with squamous cell carcinoma, and KRAS with adenocarcinoma. TP53 mutations were most prevalent in current smokers and ERBB2, ERBB4, PIK3CA, NRAS, NOTCH1, FBWX7, PTEN and STK11 mutations occurred exclusively in a group of ever-smokers, however the association was not statistically significant. No mutation was found that associated with asbestos exposure. Conclusion: Finnish NSCLC patients have a similar mutation profile as other Western patients, however with a higher frequency of BRAF mutations but a lower frequency of STK11 and ERBB2 mutations. Moreover, TP53 mutations occurred frequently with other gene mutations, most commonly with KRAS, MET, EGFR and PIK3CA mutations. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Idiopathic pulmonary fibrosis (IPF) is characterized by activation and injury of epithelial cells, the accumulation of connective tissue and changes in the inflammatory microenvironment. The bone morphogenetic protein (BMP) inhibitor protein gremlin-1 is associated with the progression of fibrosis both in human and mouse lung. We generated a transgenic mouse model expressing gremlin-1 in type II lung epithelial cells using the surfactant protein C (SPC) promoter and the Cre-LoxP system. Gremlin-1 protein expression was detected specifically in the lung after birth and did not result in any signs of respiratory insufficiency. Exposure to silicon dioxide resulted in reduced amounts of lymphocyte aggregates in transgenic lungs while no alteration in the fibrotic response was observed. Microarray gene expression profiling and analyses of bronchoalveolar lavage fluid cytokines indicated a reduced lymphocytic response and a downregulation of interferon-induced gene program. Consistent with reduced Th1 response, there was a downregulation of the mRNA and protein expression of the anti-fibrotic chemokine CXCL10, which has been linked to IPF. In human IPF patient samples we also established a strong negative correlation in the mRNA expression levels of gremlin-1 and CXCL10. Our results suggest that in addition to regulation of epithelial-mesenchymal crosstalk during tissue injury, gremlin-1 modulates inflammatory cell recruitment and anti-fibrotic chemokine production in the lung.

Chronic obstructive pulmonary disease/emphysema (COPD/emphysema) is characterized by chronic inflammation and premature lung aging. Anti-aging sirtuin 1 (SIRT1), a NAD+-dependent protein/histone deacetylase, is reduced in lungs of patients with COPD. However, the molecular signals underlying the premature aging in lungs, and whether SIRT1 protects against cellular senescence and various pathophysiological alterations in emphysema, remain unknown. Here, we showed increased cellular senescence in lungs of COPD patients. SIRT1 activation by both genetic overexpression and a selective pharmacological activator, SRT1720, attenuated stress-induced premature cellular senescence and protected against emphysema induced by cigarette smoke and elastase in mice. Ablation of Sirt1 in airway epithelium, but not in myeloid cells, aggravated airspace enlargement, impaired lung function, and reduced exercise tolerance. These effects were due to the ability of SIRT1 to deacetylate the FOXO3 transcription factor, since Foxo3 deficiency diminished the protective effect of SRT1720 on cellular senescence and emphysematous changes. Inhibition of lung inflammation by an NF-κB/IKK2 inhibitor did not have any beneficial effect on emphysema. Thus, SIRT1 protects against emphysema through FOXO3-mediated reduction of cellular senescence, independently of inflammation. Activation of SIRT1 may be an attractive therapeutic strategy in COPD/emphysema.

A prospective diagnostics and treatment protocol for extremity and trunk wall soft tissue sarcoma (STS) was introduced by the Scandinavian Sarcoma Group in 1986 and it was also widely adopted in Finland. We have updated the protocol and made it more detailed at the Helsinki University Central Hospital. We retrospectively compared diagnostics and treatment of STS in a nationwide population-based material to this protocol with special emphasis on local control.Data for 219 patients with an STS of extremity or trunk wall diagnosed during 1998-2001 was retrieved from the nationwide Finnish Cancer Registry. Histologic review was performed. Treatment centres were divided into high-, intermediate- and low-volume centres based on the number of patients with final surgery during the study period.Significantly more patients were operated with a preoperative histological or cytological diagnosis at high-volume centres. No preoperative diagnosis was a strong predictor for the patient to undergo more than one operation (p0.0001). Wide surgical margin was achieved more often at high-volume centres, but in all centre categories a considerable percentage of patients with inadequate surgical margin did not receive adjuvant radiation therapy. Local control at five years was 82% at high-volume centres, 61% at intermediate-volume centres treating highest percentage of deep tumours and 69% at low-volume centres (p = 0.046). Local control improved as the number of patients operated (surgical volume of the centre) increased.The present quality-control study is the first nationwide population-based study to assess diagnostics and treatment of STS. When referred to a specialised sarcoma centre even patients with inadequate surgery can achieve good local control. STS is a rare cancer and its treatment should be centralised in Finland, which has 5.4 million inhabitants and approximately 100 new STSs of extremities and trunk wall annually.

Motivation: Genome-wide measurement of transcript levels is an ubiquitous tool in biomedical research. As experimental data continues to be deposited in public databases, it is becoming important to develop search engines that enable the retrieval of relevant studies given a query study. While retrieval systems based on meta-data already exist, data-driven approaches that retrieve studies based on similarities in the expression data itself have a greater potential of uncovering novel biological insights. Results: We propose an information retrieval method based on differential expression. Our method deals with arbitrary experimental designs and performs competitively with alternative approaches, while making the search results interpretable in terms of differential expression patterns. We show that our model yields meaningful connections between biological conditions from different studies. Finally, we validate a previously unknown connection between malignant pleural mesothelioma and SIM2s suggested by our method, via real-time polymerase chain reaction in an independent set of mesothelioma samples. Availability: Supplementary data and source code are available from http://www.ebi.ac.uk/fg/research/rex. Contact: samuel.kaski@aalto.fi Supplementary Information: Supplementary data are available at Bioinformatics online.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. However, the exact mechanisms leading to COPD and its progression are still poorly understood. In this study, induced sputum was analyzed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry to identify proteins involved in COPD pathogenesis. The comparison of nonsmokers, smokers, and smokers with moderate COPD revealed 15 changed proteins with the majority, including polymeric immunoglobulin receptor (PIGR), being elevated in smokers and subjects with COPD. PIGR, which is involved in specific immune defense and inflammation, was further studied in sputum, lung tissue, and plasma by Western blot, immunohistochemistry/image analysis, and/or ELISA. Sputum PIGR was characterized as glycosylated secretory component (SC). Lung PIGR was significantly elevated in the bronchial and alveolar epithelium of smokers and further increased in the alveolar area in mild to moderate COPD. Plasma PIGR was elevated in smokers and smokers with COPD compared to nonsmokers with significant correlation to obstruction. In conclusion, new proteins in smoking-related chronic inflammation and COPD could be identified, with SC/PIGR being one of the most prominent not only in the lung but also in circulating blood.

The purpose of this study was to examine pirfenidone (PFD) as a potential new therapy for malignant pleural mesothelioma. PFD is an anti-fibrotic drug used to treat idiopathic pulmonary fibrosis (IPF). PFD reduces fibrous tissue formation e.g. by downregulating the production of fibroblasts and procollagens and inhibiting TGFß, TNFα and PDGF pathways. Since mesothelioma shares characteristics with IPF alongside with the activation of PI3K/Akt/mTOR, EGF, PDGF, HGF, VEGF, FGF and TGF-β pathways, we assessed the effect of PFD on mesothelioma cells in vitro and in vivo. In vitro studies were carried out using mesothelioma cell lines (JL-1, H2052, AB12) and primary mesothelioma cells. Cells treated with PFD and/or other agents (cisplatin, pemetrexed, MK-2206, PD98059, MG132) were subjected to cell viability assays, Transwell migration assays and 3D collagen or Matrigel growth and invasion assays. TGFβ/BMP, MAPK/Erk and Akt pathway activities were studied using luciferase reporter assays and human phospho-kinase array and validated with ELISA, RT-qPCR and Western blotting. For in vivo xenograft studies mice were injected subcutaneously with primary human mesothelioma cells expressing luciferase marker. Mice were treated with pirfenidone daily for 60 days and tumor growth was followed by bioluminescent imaging. Proliferation was studied by IHC. Differential gene expression in treated vs. untreated tumors was analyzed using RNA sequencing. We demonstrated that PFD treatment reduced significantly mesothelioma cell proliferation, migration and invasive growth in 3D in a concentration-dependent manner. The inhibitory effect of PFD increased when combined with cisplatin treatment in vitro. We also observed reduction of Akt and MAPK/Erk pathway activities and increase in BMP signaling in PFD treated tumor cells. Preliminary analyses of in vivo experiment revealed differential expression of multiple genes in pirfenidone treated vs. untreated tumors associated with a shift to more anti-fibrotic tumor microenvironment in PFD treated tumors. Expression profiles are currently under closer investigation. We identified pirfenidone as an inhibitor of mesothelioma invasive growth and showed that it has anti-fibrotic effects to tumor microenvironment. Since mesotheliomas are often inherently chemoresistant in fibrotic microenvironment, the use of pirfenidone could aid in overcoming chemoresistance by loosening ECM barrier for better drug penetration into the tumor tissue. Based on the current research we suggest that pirfenidone should be subjected to more intensive study as potential adjuvant-therapy for malignant mesotheliomas. Citation Format: Chang Li, Veronika Rezov, Emmi I. Joensuu, Ville Vartiainen, Mikko Rönty, Miao Yin, Marjukka Myllärniemi, Katri Koli. Pirfenidone as a new potential therapy for malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-014.

Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can be induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin's poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin-rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src.

Background: Bactericidal/permeability-increasing (BPI) fold-containing protein BPIFB1 (formerly known as L PLUNC1 ) was one of the proteins elevated in induced sputum from smokers and patients with chronic obstructive pulmonary disease (COPD) in our previous proteomic screening. Aim: To further evaluate the association of sputum BPIFB1 levels with smoking and changes of lung function during the progression of COPD. Methods: Sputum BPIFB1 was characterized by two-dimensional gel electrophoresis and mass spectrometry. The expression of BPIFB1 and BPIFA1 (SPLUNC1) in COPD was investigated by immunoblotting and immunohistochemistry using sputum and lung tissue samples. BPIFB1 levels in induced sputum from non-smokers (n=31), smokers (n=169) and patients with COPD (n=52) were assessed with an ELISA-based method, and repeated for a subset of samples (n=74) after a 4-year follow-up. The longitudinal changes in lung function during the 4-year follow-up period were compared with the baseline sputum BPIFB1 levels. Results: Secreted and glycosylated BPIFB1 was significantly elevated in sputum of patients with COPD when compared with that of smokers and non-smokers. Sputum BPIFB1 levels correlated with pack-years and lung function as measured by FEV 1 % predicted and FEV 1 /FVC at baseline and after the 4-year follow-up in all participants. The changes in lung function over 4 years were significantly associated with BPIFB1 levels in smokers with COPD. Conclusions Higher sputum concentrations of BPIFB1 were associated with greater progression of airflow limitation over time, especially in smokers with COPD. BPIFB1 may be involved in the pathogenesis of smoking-related lung diseases.

A previous study involving a proteomic screen of induced sputum from smokers and patients with chronic obstructive pulmonary disease (COPD) demonstrated elevated levels of bactericidal/permeability-increasing fold-containing protein B1 (BPIFB1). The aim of the present study was to further evaluate the association of sputum BPIFB1 levels with smoking and longitudinal changes in lung function in smokers with COPD. Sputum BPIFB1 was characterized by two-dimensional gel electrophoresis and mass spectrometry. The expression of BPIFB1 in COPD was investigated by immunoblotting and immunohistochemistry using sputum and lung tissue samples. BPIFB1 levels were also assessed in induced sputum from nonsmokers ( n = 31), smokers ( n = 169), and patients with COPD ( n = 52) via an ELISA-based method. The longitudinal changes in lung function during the 4-year follow-up period were compared with the baseline sputum BPIFB1 levels. In lung tissue samples, BPIFB1 was localized to regions of goblet cell metaplasia. Secreted and glycosylated BPIFB1 was significantly elevated in the sputum of patients with COPD compared with that of smokers and nonsmokers. Sputum BPIFB1 levels correlated with pack-years and lung function as measured by forced expiratory volume in 1 s (FEV1) % predicted and FEV1/FVC (forced vital capacity) at baseline and after the 4-year follow-up in all participants. The changes in lung function over 4 years were significantly associated with BPIFB1 levels in current smokers with COPD. In conclusion, higher sputum concentrations of BPIFB1 were associated with changes of lung function over time, especially in current smokers with COPD. BPIFB1 may be involved in the pathogenesis of smoking-related lung diseases.

The purpose of this study was to clarify the role of BMP inhibitor gremlin 1 in invasion and migration in mesothelioma. Primary mesothelioma cells isolated from patient pleural fluid as well as mesothelioma cell lines were used for in vitro studies. Cells were transfected with siRNAs or transduced with lentiviral expression vectors. Invasive growth was analyzed in 3D Matrigel or collagen I matrices. mRNA expressions were analyzed using a commercial PCR array and quantitative RT-PCR. Migration assays were performed using scratch wound assay or transwell migration assay with fibronectin or collagen coating. TGF-β and BMP signaling activity was measured with luciferase reporter assays. For in vivo mouse xenograft experiment cells were additionally transduced to express a luciferase marker. Subcutaneous cell injection with Matrigel matrix was performed in the flank of nude mice. Malignant mesothelioma is an aggressive cancer that develops from mesothelial cells, most often in the pleural lining of the lung. We have previously shown that the BMP inhibitor protein gremlin 1 is highly expressed in mesothelioma and induces a mesenchymal and chemoresistant phenotype in mesothelioma cells. Since mesothelioma tumors are locally very invasive and has poor prognosis, we analyzed the role of gremlin 1 in mesothelioma cell migration and invasive growth. We found that mesothelioma cells expressing gremlin 1 showed invasive sprouting when tumor cell spheroids were imbedded into 3D collagen matrix. Silencing of gremlin 1 expression significantly reduced invasive growth. In addition, cells overexpressing gremlin 1 gained more invasive phenotype. This was associated with increased mRNA expression levels of Slug and matrix metalloproteinases (MMP) as well as reduced expression of E-cadherin. The cells were more migratory and exhibited increased expression of certain integrins, especially the αv subunit. Gremlin 1 induced invasive growth was dependent on MMP activity and associated with increased TGF-β activity. Intrapleural injection of mesothelioma cells overexpressing gremlin 1 isolated from a patient with epithelioid mesothelioma, produced tumors in 2/4 mice over 5 months after injection. Mice with cells transduced with vector only did not develop tumors (0/4). When cells were injected subcutaneously together with Matrigel gremlin 1 overexpressing tumors appeared more slowly, but exhibited comparable luciferase signal 2.5 months after injection. However, gremlin 1 tumors showed more vascularization and in contrast to control tumors some also developed metastases (2/6 mice). As conclusion, we identified gremlin 1 as an important regulator of mesothelioma invasive growth and chemoresistance. Blocking gremlin 1 function may overcome drug resistance and reduce invasive behavior of mesothelioma. Citation Format: Miao Yin, Mira Tissari, Emmi I. Joensuu, Jenni Tamminen, Irene Ylivinkka, Mikko Rönty, Kaisa Lehti, Marko Hyytiäinen, Marjukka Myllärniemi, Katri Koli. Gremlin-1 is an important regulator of invasiveness in malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1894. doi:10.1158/1538-7445.AM2017-1894

Lung carcinoids are rare neuroendocrine tumors of the lung. Very little is known about the genetic background of these tumors. We applied Ion Torrent Ampliseq next-generation technology to study hotspot mutations of 22 lung cancer-related genes from typical and atypical lung carcinoid tumors. DNA isolated from 25 formalin-fixed, paraffin-embedded carcinoid tumors were amplified to prepare barcoded libraries covering 507 mutations included in 90 amplicons. The libraries were pooled, purified, enriched, and sequenced on ion personal genome machine. The sequences were aligned and checked for known and novel variations using Torrent Suite Software v.4.0.2. One out of 25 patients had mutations in the targeted regions sequenced. This patient had mutations in BRAF, SMAD4, PIK3CA, and KRAS. All these mutations were confirmed as somatic and are previously known mutations. In summary, mutations in genes commonly mutated in non-small-cell lung cancer are not common in lung carcinoids.

Screening of anaplastic lymphoma tyrosine kinase (ALK) gene fusions in non-small cell lung cancer (NSCLC) patients enables the identification of the patients likely to benefit from ALK-targeted therapy. Our aim was to assess the prevalence of ALK fusion in Finnish NSCLC patients, which has not been reported earlier, and to study the presence of ALK fusion in relation to clinicopathological characteristics and other driver gene mutations. A total of 469 formalin-fixed paraffin-embedded tumor tissue specimens from Finnish NSCLC patients were screened for ALK fusion by immunohistochemistry (IHC). For confirmation of IHC results, fluorescence in situ hybridization (FISH) was conducted for 171 specimens. Next-generation sequencing was performed for all ALK-positive specimens to characterize the association of ALK fusion with mutations in targeted regions of 22 driver genes. Of the 469 tumors screened, 11 (2.3%) harbored an ALK fusion, including nine adenocarcinomas and two large cell carcinomas. The IHC results for all 11 ALK-positive and 160 random ALK-negative specimens were confirmed by FISH. ALK fusion was significantly associated with never/ex-light smoking history (P0.001) and younger age (P=0.004). Seven ALK-positive tumors showed additional mutations; three in MET, one in MET and CTNNB1, two in TP53, and one in PIK3CA. Our results show that ALK fusion is an infrequent alteration in Finnish NSCLC patients. Although the majority of ALK-positive cases were adenocarcinomas, the fusion was also seen in large cell carcinomas. Further studies are needed to elucidate the clinical significance of the coexistence of ALK fusion with MET, TP53, CTNNB1, and PIK3CA mutations.

Introduction: Among the driver gene mutations in non–small-cell lung cancer, mutations in epidermal growth factor receptor (EGFR) are the most important because of their predictive role in selecting patients eligible for targeted therapy. Our aim was to study EGFR mutations in a Finnish non–small-cell lung cancer cohort of 528 patients. Methods: Mutation testing was conducted on DNA extracted from paraffin-embedded, formalin-fixed tumor material using the following real-time polymerase chain reaction-based kits: Therascreen EGFR PCR Kit and cobas EGFR Mutation Test. Results: EGFR mutation frequency was 11.4% and all positive cases were adenocarcinomas, of which a majority had an acinar predominant pattern. Mutations were seen significantly more often in females and never-smokers than in males and smokers. The most frequent mutations were L858R in exon 21 and deletions in exon 19. Overall survival of the patients, not treated with EGFR inhibitor, did not differ between EGFR mutation-positive and EGFR mutation-negative patients. Conclusion: EGFR mutation profile in this Finnish non–small-cell lung cancer cohort resembles in many respect with that of other Western European cohorts, even though the overall frequency of mutations is slightly higher. We show the occurrence of EGFR mutations in patients with occupational asbestos exposure and also in those diagnosed with chronic obstructive pulmonary disease who have not been often investigated before.

Background: Activins are members of the TGF-ß superfamily of growth factors. First, we identified by expression array screening that activin-B and follistatin are upregulated in human idiopathic pulmonary fibrosis (IPF). Next, we wanted to clarify their specific role in lung fibrosis formation. Methods: We used specific antibodies for activin-A and -B subunits and follistatin to measure and localize their levels in idiopathic pulmonary fibrosis and control lung biopsies. To inhibit activin signaling, we used soluble activin type IIB receptor fused to the Fc portion of human IgG1 (sActRIIB-Fc) in two different mouse models of pulmonary fibrosis. Results: Activin-B and follistatin mRNA levels were elevated in the human IPF lung. Immunoreactivity to activin-A, -B and follistatin localized predominantly to the hyperplastic, activated alveolar epithelium, but was also seen in inflammatory cells. Mice treated with sActRIIB-Fc showed increased skeletal muscle mass and a clear reduction in alveolar cell counts in bronchoalveolar lavage fluid, but no significant antifibrotic effect in the lung was observed. Conclusions: The upregulation of activin-B and follistatin in IPF is a novel finding. Our results indicate that activin inhibition is not an efficient tool for antifibrotic therapy, but could be useful in reducing alveolar cellular response to injury. Activin-B and follistatin levels may be useful as biomarkers of IPF. peerReviewed

Activation of transforming growth factor (TGF)-β is a key event in the progression of fibrosis in human lung tissue. Idiopathic pulmonary fibrosis (IPF) in West Highland white terriers (WHWTs) shares histopathological features of human usual interstitial pneumonia (UIP), the histopathological counterpart of IPF and non-specific interstitial pneumonia (NSIP). The aim of the present immunohistochemical study was to investigate TGF-β signalling activity and its known extracellular matrix (ECM) regulatory proteins, latent TGF-β binding protein (LTBP)-1 and fibrillin-2, in lung tissue of WHWTs with IPF and healthy WHWTs and to compare these with findings in human UIP and NSIP. P-Smad2 immunoreactivity, indicating TGF-β signalling activity, was increased in WHWTs with IPF relative to healthy WHWTs and expression was localized predominantly in the altered alveolar epithelium, as seen in both UIP and NSIP. Increased peribronchial and perivascular LTBP-1 immunoreactivity was seen in WHWTs with IPF compared with controls, possibly indicating the importance of the small airways in the canine disease. Alveolar LTPB-1 immunolabelling in diseased WHWTs was seen mainly in the altered alveolar epithelium, resembling more closely the labelling in UIP than in NSIP. Alveolar interstitial fibrillin-2 immunoreactivity, which is up-regulated in the lungs of people with UIP, was also detected in the lungs of WHWTs with IPF and people with NSIP. However, no significant difference was seen between WHWTs with IPF and control WHWTs. The results suggest that increased TGF-β signalling and expression of the ECM regulatory proteins LTBP-1 and fibrillin-2 are part of the molecular pathophysiology of canine IPF.

Epithelioid sarcoma (ES) is a rare malignant soft tissue tumor. ES can be classified into proximal, distal, and fibroma-like subtypes. These tumors show both mesenchymal and epithelial immunophenotypes. Microscopically, the proximal type ES is usually characterized by nodules of spindle and epithelioid cells growing in granuloma-like pattern often presenting with central necrosis. Immunohistochemically these tumors are vimentin, pancytokeratin, and usually EMA (80%) positive. CD34 (50%) and CD99 (25%) may be positive, and occasionally SMA and S-100 immunopositivity has been reported. No specific genetic alterations have been found in ES. As far as we know, this is the first case in the literature to present ES in gingival mucosa.

Contractile myofibroblasts are responsible for remodeling of extracellular matrix during wound healing; however, their continued activity results in various fibrocontractive diseases. Conversion of fibroblasts into myofibroblasts is induced by transforming growth factor-beta1 (TGF-beta1) and is hallmarked by the neo-expression of alpha-smooth muscle actin (alpha-SMA), a commonly used myofibroblast marker. Moreover, myofibroblast differentiation and acquisition of the contractile phenotype involves functionally important alterations in the expression of actin-organizing proteins. We investigated whether myofibroblast differentiation is accompanied by changes in the expression of palladin, a cytoskeletal protein that controls stress fiber integrity. Palladin is expressed as several isoforms, including major 3Ig (90 kDa) and 4Ig (140 kDa) forms that differ in their N-terminal sequence. Expression of the 4Ig isoform is strongly induced in fibroblast stress fibers upon TGF-beta1 treatment preceding alpha-SMA upregulation. TGF-beta1 induced upregulation of palladin is mediated both by Smad and mitogen-activated protein kinase pathways. Furthermore, palladin 4Ig-isoform is co-expressed with alpha-SMA in vivo in experimental rat wounds and in human myofibroblast-containing lesions. Taken together these results identify palladin 4Ig as a novel marker of myofibroblast conversion in vitro and in vivo. They also provide for the first time information about the signaling cascades involved in the regulation of palladin expression.

Palladin and alpha-actinin are major components of stress fiber dense bodies, cardiomyocyte Z-discs and neuronal synapses. They function as structural molecules and cytoskeletal regulators but also as docking sites to other proteins. Both antisense and transient overexpression experiments have shown that palladin plays an important role in the regulation of actin cytoskeleton. ArgBP2 is a multi-domain scaffolding protein which shares both the tissue distribution and subcellular localization with palladin. ArgBP2 is directly linked to intracellular signaling cascades by its interaction with Abl family kinases, Pyk2 and the ubiquitin ligase Cbl. It has several actin associated binding partners and has been shown to regulate cytoskeletal dynamics. Here, we show by in vivo and in vitro methods that palladin's amino-terminal poly-proline sequences directly interact with the first carboxy-terminal SH3 domain of ArgBP2. We further demonstrate a direct interaction between alpha-actinin and the amino-terminal segment of ArgBP2. Immunoprecipitation and targeting assays suggest that a three-way complex of the proteins occurs in vivo. The interactions provide an explanation to the previously observed Z-disc-specific localization of ArgBP2 and indicate interplay between signaling adaptors and structural proteins of the Z-disc.

Activin A and B, members of the TGF-β superfamily, are regulators of differentiation, inflammation and wound healing. Dimeric activins bind to transmembrane activin type I and type II receptors and induce signaling through activation of the canonical Smad2/3 pathway as well as through activation of MAPK pathways. Depending on the tissue type activins can promote or inhibit cellular growth and/or migration. Activin A has recently been reported to have growth promoting properties in malignant mesothelioma (MM). MM is a rare and aggressive tumor originating most commonly from pleural mesothelial cells. It is asbestos exposure related malignancy that takes decades to develop. MM tumors are highly invasive and extremely resistant to conventional cancer therapy. Novel diagnostic markers and drug targets are urgently needed. We have characterized the expression of activins, follistatins and activin receptors in mesothelioma tumors and cultured mesothelioma cells. In addition, we have identified signaling pathways induced in mesothelioma cells by activin treatment. Expression of activins and receptors in mesothelioma tumors were analyzed by immunohistochemistry. Activins, follistatins and receptors in primary mesothelioma cells and in mesothelioma cell lines were analyzed at the level of mRNA and protein expression. Activation of Smad2/3 and Smad1/5/8 signaling pathways were analyzed by luciferase reporter assays and Western blotting. Activation of MAPK/JNK and MAPK/ERK pathways were analyzed by Western blotting. Immortalized but nonmalignant Met5A cells served as a control cell line. Activin A and B were strongly expressed in mesothelioma tumor tissue. Cultured primary mesothelioma cells and mesothelioma cell lines also expressed higher levels of activin A and B compared to Met5A cells. All activin receptors (ALK3, ALK4, ALK7, ACVR2A, ACVR2B) were found expressed in cultured mesothelioma cells. ACVR2A and ALK7 were significantly overexpressed in all mesothelioma cells. Majority of the mesothelioma cell lines had attenuated Smad2/3 activation upon activin stimulation. In Met5A and H28 cells there was activation of Smad2/3 as well as MAPK/JNK pathways. On the contrary, activation of MAPK/ERK pathway was detected in cells displaying attenuated Smad2/3 activation.Our results show that activin A and B are overexpressed in MM. This together with upregulation of specific activin receptors points to a role for activins in MM progression. Furthermore, we observed that mesothelioma cells have altered responses to activin stimulation, namely attenuation of canonical Smad2/3 signaling and increase in MAPK/ERK signaling. This is likely to have an impact on mesothelioma cell behavior and tumor progression. Citation Format: Jenni A. Tamminen, Mikko Rönty, Eva Sutinen, Arja Pasternack, Olli Ritvos, Marjukka Myllärniemi, Katri Koli. Overexpression of activin A and B as well as altered activation of canonical and non-canonical signaling pathways in malignant mesothelioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3449. doi:10.1158/1538-7445.AM2014-3449

39 Background: Molecular targeted tyrosine kinase inhibitor (TKI) treatments have made it crucial to perform diagnostic tests of multiple molecular targets. In lung carcinoma there are close to ten clinically relevant gene mutations, copy number alterations and/or gene fusions, such as ALK, EGFR, ERBB2, KRAS, BRAF, MET, PTEN, PI3KCA, ROS1 and RET. Presently, several different tests are utilized, requiring a high amount of tumor material and long turnaround time. Next generation sequencing or targeted deep sequencing (TDS) has opened a new era for rapid genome-wide analyses of mutations, copy number alterations and gene fusions. Our aimwas to 1) prove feasibility for applying TDS to FFPE samples, 2) compare mutations detected by prevalent methods & TDS, and 3) mine novel clinically and biologically relevant genes in lung and colorectal carcinoma. Methods: For TDS, we selected 192 lung carcinoma and colorectal carcinoma related genes and microRNA genes, focusing on the EGFR and ALK pathways. In total, 98 FFPE specimens were studied. Agilent SureSelect system and Illumina sequencing was adopted for the analysis. For diagnostic validation the following genes were selected: EGFR, KRAS, BRAF, PTEN, PI3K, RET and ALK. TDS results were confirmed by PCR, FISH and IHC. Results: We focused on the genes selected for diagnostic validation. Successful results were obtained from all specimens. The results from TDS correlated significantly with those obtained from PCR, FISH, and IHC. Importantly, TDS revealed novel mutations not detected by targeted PCR. Conclusions: An enormous advantage of TDS is that multiple mutation screening can be achieved in one analysis (saving time and material), and most importantly, provides enormous amounts of novel information, for example understanding mechanisms for drug resistance. This study was supported by Finnish Academy, Sigrid Jusélius Foundation, Finnish Cancer Organizations, the special governmental subsidy research funds appropriated to the Helsinki and Uusimaa Hospital District (HUS EVO), Pfizer Oy, AstraZeneca AS, Lab21 Ltd, Abbott Molecular Inc.

Palladin is a novel component of stress fiber dense regions. Antisense and transient overexpression studies have indicated an important role for palladin in the regulation of actin cytoskeleton. Palladin colocalizes and coimmunoprecipitates with alpha-actinin, a dense region component, but the molecular details and functional significance of the interaction have not been studied. We show here a direct association between the two proteins and have mapped the binding site within a short sequence of palladin and in the carboxy-terminal calmodulin domain of alpha-actinin. Using transfection-based targeting assays, we show that palladin is involved in targeting of alpha-actinin to specific subcellular foci indicating a functional interplay between the two actin-associated proteins.

Background KL-6 is a high-molecular-weight glycoprotein classified as a human MUC1 mucin. It was hypothesized that KL-6 could be detectable in the circulating blood and especially in airway secretions in lung diseases associated with mucus production such as chronic obstructive pulmonary disease (COPD). Additional aims of this study were to investigate whether the levels of KL-6 in plasma and sputum are related to ageing and smoking history. Methods The concentrations of KL-6 in plasma and induced sputum supernatants from young and/or middle aged/elderly non-smokers, smokers and patients with COPD were assayed by ELISA (n = 201). The subjects were classified into five groups according to age, smoking status and presence of COPD. In addition, KL-6 expression in control and diseased lung i.e. samples from patients with COPD (n = 28), were analyzed by immunohistochemistry and digital image analysis. Results The plasma levels of KL-6 increased with age both in non-smokers and smokers. Among middle aged/elderly subjects, plasma KL-6 levels in all smokers regardless of COPD were significantly higher than in non-smokers, whereas sputum levels of KL-6 were significantly higher in COPD compared not only to non-smokers but also to smokers. KL-6 was more prominently expressed in the bronchiolar/alveolar epithelium in COPD than in the control lungs. Plasma and sputum KL-6 levels correlated inversely with obstruction and positively with smoking history and ageing. The linear multiple regression analysis confirmed that age and cigarette smoking had independent effects on plasma KL-6. Conclusions KL-6 increases with ageing and chronic smoking history, but prospective studies will be needed to elucidate the significance of KL-6 in chronic airway diseases.

Actin-containing microfilaments control cell shape, adhesion, and contraction. In striated muscle, α-actinin and other Z-disk proteins coordinate the organization and functions of actin filaments. In smooth muscle and nonmuscle cells, periodic structures termed dense bodies and dense regions, respectively, are thought to serve functions analogous to Z-discs. We describe here identification and characterization of human palladin, a protein expressed mainly in smooth muscle and nonmuscle and distributed along microfilaments in a periodic manner consistent with dense regions/bodies. Palladin contains three Ig-domains most homologous to the sarcomeric Z-disk protein myotilin. The N terminus includes an FPPPP motif recognized by the Ena-Vasp homology domain 1 domain in Ena/vasodilatator-stimulated phosphoprotein (VASP)/Wiscott-Aldrich syndrome protein (WASP) protein family. Cytoskeletal proteins with FPPPP motif target Ena/VASP/WASP proteins to sites of actin modulation. We identified palladin in a yeast two-hybrid search as an ezrin-associated protein. An interaction between palladin and ezrin was further verified by affinity precipitation and blot overlay assays. The interaction was mediated by the α-helical domain of ezrin and by Ig-domains 2–3 of palladin. Ezrin is typically a component of the cortical cytoskeleton, but in smooth muscle cells it is localized along microfilaments. These cells express palladin abundantly and thus palladin may be involved in the microfilament localization of ezrin. Palladin expression was up-regulated in differentiating dendritic cells (DCs), coinciding with major cytoskeletal and morphological alterations. In immature DCs, palladin localized in actin-containing podosomes and in mature DCs along actin filaments. The regulated expression and localization suggest a role for palladin in the assembly of DC cytoskeleton.

Background Asbestos is a carcinogen linked to malignant mesothelioma (MM) and lung cancer. Some gene aberrations related to asbestos exposure are recognized, but many associated mutations remain obscure. We performed exome sequencing to determine the association of previously known mutations (driver gene mutations) with asbestos and to identify novel mutations related to asbestos exposure in lung adenocarcinoma (LAC) and MM. MethodsExome sequencing was performed on DNA from 47 tumor tissues of MM (21) and LAC (26) patients, 27 of whom had been asbestos-exposed (18 MM, 9 LAC). In addition, 9 normal lung/blood samples of LAC were sequenced. Novel mutations identified from exome data were validated by amplicon-based deep sequencing. Driver gene mutations in BRAF, EGFR, ERBB2, HRAS, KRAS, MET, NRAS, PIK3CA, STK11, and ephrin receptor genes (EPHA1-8, 10 and EPHB1-4, 6) were studied for both LAC and MM, and in BAP1, CUL1, CDKN2A, and NF2 for MM. ResultsIn asbestos-exposed MM patients, previously non-described NF2 frameshift mutation (one) and BAP1 mutations (four) were detected. Exome data mining revealed some genes potentially associated with asbestos exposure, such as MRPL1 and SDK1. BAP1 and COPG1 mutations were seen exclusively in MM. Pathogenic KRAS mutations were common in LAC patients (42 %), both in non-exposed (n = 5) and exposed patients (n = 6). Pathogenic BRAF mutations were found in two LACs. ConclusionBAP1 mutations occurred in asbestos-exposed MM. MRPL1, SDK1, SEMA5B, and INPP4A could possibly serve as candidate genes for alterations associated with asbestos exposure. KRAS mutations in LAC were not associated with asbestos exposure.

Effects of nitric oxide (NO) on gastric wound healing were investigated in primary rabbit gastric epithelial cell cultures. We analyzed the speed of cell migration, proliferation, and apoptosis after creating a round wound on the cell cultures. The monolayers were incubated with or without the NO donor sodium nitroprusside, oxatriazolimine 1,2,3,4-oxatriazolium, 5amino-3-(3,4-dichlorophenylchloride), or the peroxynitrite generator 3-morpholinosydnomine- N-ethylcarbamide. The possible role of cGMP as a second messenger of NO was investigated with 8-bromo-cGMP. The role of O[Formula: see text]· was evaluated using diethyldithiocarbamate and pyrogallol. The effects of superoxide dismutase and allopurinol were also investigated. NO inhibited the speed of cell migration and proliferation and induced cell apoptosis in a dose- and time-dependent manner. The effects were augmented with O[Formula: see text]· generators and ameliorated by O[Formula: see text]· scavengers, whereas cGMP had no significant effect on wound healing. NO donors retard gastric wound healing by inhibiting migration and proliferation and inducing cell apoptosis. These effects do not seem to be mediated via cGMP, but O[Formula: see text]· or peroxynitrites may be involved.