Browsing by Subject "EGFR"

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  • Romano, Roberta; Rivellini, Cristina; De Luca, Maria; Tonlorenzi, Rossana; Beli, Raffaella; Manganelli, Fiore; Nolano, Maria; Santoro, Lucio; Eskelinen, Eeva-Liisa; Previtali, Stefano C.; Bucci, Cecilia (2021)
    The small GTPase RAB7A regulates late stages of the endocytic pathway and plays specific roles in neurons, controlling neurotrophins trafficking and signaling, neurite outgrowth and neuronal migration. Mutations in the RAB7A gene cause the autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) disease, an axonal peripheral neuropathy. As several neurodegenerative diseases are caused by alterations of endocytosis, we investigated whether CMT2B-causing mutations correlate with changes in this process. To this purpose, we studied the endocytic pathway in skin fibroblasts from healthy and CMT2B individuals. We found higher expression of late endocytic proteins in CMT2B cells compared to control cells, as well as higher activity of cathepsins and higher receptor degradation activity. Consistently, we observed an increased number of lysosomes, accompanied by higher lysosomal degradative activity in CMT2B cells. Furthermore, we found increased migration and increased RAC1 and MMP-2 activation in CMT2B compared to control cells. To validate these data, we obtained sensory neurons from patient and control iPS cells, to confirm increased lysosomal protein expression and lysosomal activity in CMT2B-derived neurons. Altogether, these results demonstrate that in CMT2B patient-derived cells, the endocytic degradative pathway is altered, suggesting that higher lysosomal activity contributes to neurodegeneration occurring in CMT2B.
  • Leopold, Anna; Pletnev, Sergei; Verkhusha, Vladislav V. (2020)
    Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLC gamma signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light. (C) 2020 Elsevier Ltd. All rights reserved.
  • Maki-Nevala, Satu; Sarhadi, Virinder Kaur; Knuuttila, Aija; Scheinin, Ilari; Ellonen, Pekka; Lagstrom, Sonja; Ronty, Mikko; Kettunen, Eeva; Husgafvel-Pursiainen, Kirsti; Wolff, Henrik; Knuutila, Sakari (2016)
    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.
  • Donner, Iikki; Katainen, Riku; Sipilä, Lauri J.; Aavikko, Mervi; Pukkala, Eero; Aaltonen, Lauri A. (2018)
    Objectives: Although the primary cause of lung cancer is smoking, a considerable proportion of all lung cancers occur in never smokers. Gender influences the risk and characteristics of lung cancer and women are over-represented among never smokers with the disease. Young age at onset and lack of established environmental risk factors suggest genetic predisposition. In this study, we used population-based sampling of young patients to discover candidate predisposition variants for lung adenocarcinoma in never-smoking women. Materials and methods: We employed archival normal tissue material from 21 never-smoker women who had been diagnosed with lung adenocarcinoma before the age of 45, and exome sequenced their germline DNA. Results and conclusion: Potentially pathogenic variants were found in eight Cancer Gene Census germline genes: BRCAI, BRCA2, ERCC4, EXT1, HNF1 A, PTCH1, SMARCB1 and TP53. The variants in TP53, BRCAI, and BRCA2 are likely to have contributed to the early onset lung cancer in the respective patients (3/21 or 14%). This supports the notion that lung adenocarcinoma can be a component of certain cancer predisposition syndromes. Fifteen genes displayed potentially pathogenic mutations in at least two patients: ABCC10, ATP7B, CACNA1S, CFTR, CLIP4, COL6A1, COL6A6, GCN1, GJB6, RYR1, SCN7A, SEC24A, SP100, TEN and USH2A. Four patients showed a mutation in COL6A1, three in CLIP4 and two in the rest of the genes. Some of these candidate genes may explain a subset of female lung adenocarcinoma.
  • Kurppa, Kari J.; Caton, Javier; Morgan, Peter R.; Ristimaki, Ari; Ruhin, Blandine; Kellokoski, Jari; Elenius, Klaus; Heikinheimo, Kristiina (2014)
  • Maki-Nevala, Satu; Sarhadi, Virinder Kaur; Ronty, Mikko; Kettunen, Eeva; Husgafvel-Pursiainen, Kirsti; Wolff, Henrik; Knuuttila, Aija; Knuutila, Sakari (2016)
    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.
  • Tuomainen, Katja; Al-Samadi, Ahmed; Potdar, Swapnil; Turunen, Laura; Turunen, Minna; Karhemo, Piia-Riitta; Bergman, Paula; Risteli, Maija; Åström, Pirjo; Tiikkaja, Riia; Grenman, Reidar; Wennerberg, Krister; Monni, Outi; Salo, Tuula (2020)
    In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma-derived matrix "Myogel" to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo-relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma-derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. However, we found a similar response to the PI3K/mTOR inhibitors under all culturing conditions. Cells grown on Myogel more closely resembled the response rates reported in EGFR-inhibitor monotherapy clinical trials. Our findings suggest that a human tumor matrix improves the predictability of in vitro anticancer drug testing compared to current 2D and MSDM methods.
  • Thunnissen, Erik; Weynand, Birgit; Udovicic-Gagula, Dalma; Brcic, Luka; Szolkowska, Malgorzata; Hofman, Paul; Smojver-Jezek, Silvana; Anttila, Sisko; Calabrese, Fiorella; Kern, Izidor; Skov, Birgit; Perner, Sven; Dale, Vibeke G.; Eri, Zivka; Haragan, Alex; Leonte, Diana; Carvallo, Lina; Prince, Spasenja Savic; Nicholson, Siobhan; Sansano, Irene; Ryska, Ales (2020)
    A questionnaire on biomarker testing previously used in central European countries was extended and distributed in Western and Central European countries to the pathologists participating at the Pulmonary Pathology Society meeting 26-28 June 2019 in Dubrovnik, Croatia. Each country was represented by one responder. For recent biomarkers the availability and reimbursement of diagnoses of molecular alterations in non-small cell lung carcinoma varies widely between different, also western European, countries. Reimbursement of such assessments varies widely between unavailability and payments by the health care system or even pharmaceutical companies. The support for testing from alternative sources, such as the pharmaceutical industry, is no doubt partly compensating for the lack of public health system support, but it is not a viable or long-term solution. Ideally, a structured access to testing and reimbursement should be the aim in order to provide patients with appropriate therapeutic options. As biomarker enabled therapies deliver a 50% better probability of outcome success, improved and unbiased reimbursement remains a major challenge for the future.
  • Merisaari, Joni; Denisova, Oxana; Doroszko, Milena; Le Joncour, Vadim; Johansson, Patrik; Leenders, William P. J.; Kastrinsky, David B.; Zaware, Nilesh; Narla, Goutham; Laakkonen, Pirjo; Nelander, Sven; Ohlmeyer, Michael; Westermarck, Jukka (2020)
    Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood-brain barrier-permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.
  • Tuononen, Katja; Sarhadi, Virinder Kaur; Wirtanen, Aino; Rönty, Mikko Juhani; Salmenkivi, Kaisa; Knuuttila, Aija; Remes, Satu; Telaranta-Keerie, Aino I.; Bloor, Stuart; Ellonen, Pekka; Knuutila, Sakari (2013)
  • Back, Nils; Kanerva, Kristiina; Kurutihallib, Vishwanatha; Yanik, Andrew; Ikonen, Elina; Mains, Richard E.; Eipper, Betty A. (2017)
    Peptidylglycine alpha-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4 h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes. (C) 2017 Elsevier GmbH. All rights reserved.
  • Brunelli, Matteo; Bria, Emilio; Nottegar, Alessia; Cingarlini, Sara; Simionato, Francesca; Calio, Anna; Eccher, Albino; Parolini, Claudia; Iannucci, Antonio; Gilioli, Eliana; Pedron, Serena; Massari, Francesco; Tortora, Giampaolo; Borze, Florentina Ioana; Knuutila, Sakari; Gobbo, Stefano; Santo, Antonio; Tondulli, Luca; Calabro, Francesco; Martignoni, Guido; Chilosi, Marco (2012)