Mikko Rönty

SIRT1 protects against emphysema via FOXO3-mediated reduction of premature senescence in mice

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.

Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS.

The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next‐generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real‐time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin‐fixed, paraffin‐embedded (FFPE) tumor tissue specimens from 81 non‐small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real‐time PCR. Moreover, targeted NGS revealed seven nonsynonymous single‐nucleotide variations and one insertion‐deletion variation in EGFR not detectable by the real‐time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

Molecular analysis of the interaction between palladin and α-actinin

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 α‐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 α‐actinin. Using transfection‐based targeting assays, we show that palladin is involved in targeting of α‐actinin to specific subcellular foci indicating a functional interplay between the two actin‐associated proteins.

Identification and Validation of Human Papillomavirus Encoded microRNAs

We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.

Targeted Resequencing Reveals ALK Fusions in Non-Small Cell Lung Carcinomas Detected by FISH, Immunohistochemistry, and Real-Time RT-PCR: A Comparison of Four Methods

Anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements occur in a subgroup of non-small cell lung carcinomas (NSCLCs). The identification of these rearrangements is important for guiding treatment decisions. The aim of our study was to screen ALK gene fusions in NSCLCs and to compare the results detected by targeted resequencing with results detected by commonly used methods, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and real-time reverse transcription-PCR (RT-PCR). Furthermore, we aimed to ascertain the potential of targeted resequencing in detection of ALK-rearranged lung carcinomas. We assessed ALK fusion status for 95 formalin-fixed paraffin-embedded tumor tissue specimens from 87 patients with NSCLC by FISH and real-time RT-PCR, for 57 specimens from 56 patients by targeted resequencing, and for 14 specimens from 14 patients by IHC. All methods were performed successfully on formalin-fixed paraffin-embedded tumor tissue material. We detected ALK fusion in 5.7% (5 out of 87) of patients examined. The results obtained from resequencing correlated significantly with those from FISH, real-time RT-PCR, and IHC. Targeted resequencing proved to be a promising method for ALK gene fusion detection in NSCLC. Means to reduce the material and turnaround time required for analysis are, however, needed.

Sputum Proteomics Identifies Elevated PIGR levels in Smokers and Mild-to-Moderate COPD

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.

Soft tissue sarcoma - a population-based, nationwide study with special emphasis on local control

Abstract Background. 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. Methods. 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. Results. 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 (p < 0.0001). 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. Conclusion. 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.

Ageing and long-term smoking affects KL-6 levels in the lung, induced sputum and plasma

BackgroundKL-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.MethodsThe 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.ResultsThe 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.ConclusionsKL-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.

Data-driven information retrieval in heterogeneous collections of transcriptomics data links SIM2s to malignant pleural mesothelioma

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.

Lung tissue proteomics identifies elevated transglutaminase 2 levels in stable chronic obstructive pulmonary disease.

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.