International Research Network on Lung Cancer epigenetic- Colaborativo con la Universidad Andrés Bello de Chile

Lung cancer is the leading cause of cancer related deaths worldwide. There are two major forms of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCL) which accounts for the remaining 80% of lung cancers. The initiation and progression of lung cancer is a result of the accumulation of a combination of permanent alterations, including point mutations, deletions, translocation and/or amplifications as well as dynamic epigenetic alterations, which are influenced by environmental factors. (1). Epigenetic changes refer to the sum of heritable alterations of the chromatin, which is a complex of proteins and DNA in the nucleus of the cell and influences DNA-dependent processes such as transcription ( 2). Chromatin mediated regulation of transcription involves DNA methylation, histone modifications, nucleosome remodeling and regulation via small noncoding RNAs ( 3). Epigenetics modifications observed in lung carcinogenesis include aberrant DNA methylation, histone modifications, and regulation by noncoding RNAs (ncRNA) (3). These alterations can occur in defined nuclear positions and chromosome domains after exposure to environmental risk factors as smoking, drugs and chronic inflammation. Cancer cells have an aberrant methylation signature; they present global hypomethylation (Lander, 2001) and hypermethylation of tumor suppressor genes (TSGs) (Rauch, 2008, Lander 2001, Gaudet, 2003). Global hypomethylation is frequent in NSCLC and is associated with genomic instability (Gupta 1997) and aberrant overexpression of oncogenic gene isoforms (Holt 1999). A large number of aberrantly methylated genes have also been identified in lung cancer, either by high-throughput methods (Kalluri 2009, Le Bras 2012)) or target-based approaches (Richardson 2012, Xiao 2010, Shing 2010). Furthermore, methylation has been described as an early event in lung tumorigenesis. A well-studied example of early hypermethylation is that of P16, contributing significantly to its transcriptional silencing [Fu 2011, Yang 2004, Tran 2012). Additional examples include H-cadherin (De Craene 2013), R ASSF1A (Vant 2011), APC (Mulshine 2003, Marshall 2013) and DAPK1 (Siegel 2013). Histone deacetylases (HDACs) are overexpressed in lung cancer (sasaki 2004, Nakagawa 2007, Bartling 2005. HDACs catalyze the removal of acetyl groups on the histone tail, resulting in a transcriptionally inactive heterochromatic state (Esteller 2008). Likewise, SIN3A (part of an HDAC repressor complex) is downregulated in NSCLC (Suzuki, 2008). Crosstalk occurs, mediated by proteins with methyl-binding domains that can recruit histone modifying enzymes (MBDs, KAISO, MeCP2), such as HDAC, that couples closely DNA methylation and histone modifications (Brzezianska 2013). Relative to normal lung, lung cancer undergoes H4K5/H4K8 hyperacetylation, H4K12/H4K16 hypoacetylation, and H4K20me3 (Vann Den, 2008). Lower global levels of H4K20me3 can be detected in precursor lesions and is particularly common in squamous cancers(Vann Den, 2008) . Other posttranslational modifiers that have been reported as overexpressed in lung cancer relative to normal lung tissue include polycomb group genes (PcGs), which are crucial epigenetic regulators of stem cell (and cancer stem cell) survival and pluripotency. Specifically, upregulation has been observed for PcGs that form the polycomb repressive complexes (PRCs; such as PRC1 and PRC2) (Cao, 2012). These complexe controls gene expression through histone H3 lysine 27 trimethylation or by interacting with DNMTs to signal transcriptional repression (Cao, 2012). Although, EZH2, is expressed constitutively in normal lung tissue (Takawa, 2011, Wan 2013). Upregulation of PRC genes has been associated with lung cancer proliferation, survival, and epithelial-mesenchymal transition (Cao, 2012 Takawa, 2011, Wan 2013). As discussed previously, promoter hypermethylation can be an early event in lung carcinogenesis and, as such, may have utility in early detection of the disease. Lung cancer mortality could be reduced significantly with earlier detection of the disease. However, only about 15% of lung tumors are localized at diagnosis, with the majority presenting at an advanced stage (2 ). Five-year survival for lung cancer is markedly better for early-stage patients, with a less than 10% 5-year survival for advanced-stage patients vs greater than 70% for early-stage patients. All these antecedents that justify the importance of implementing research projects to evaluate the role of epigenetic mechanisms in the etiology of lung cancer. Expanding our understanding of how epigenetic events contribute to the genesis of lung cancer and how they can be translated into clinically relevant biomarkers and therapeutic targets will enhance our ability to diagnostic and manage lung cancer properly. Since april 2015 the Institute of Human Genetics (Colombia), with the involvement of Martin Montecino ( UNAB Chile) as a consultant, has been developing the project ¿Regulación epigenética del gen Runx2 en pacientes con lesiones sospechosas de cáncer de pulmón¿ with ID 006276. This project is supported by the Vice-rectory of research at the Pontificia Universidad Javeriana and San Ignacio Hospital of Bogotá- Colombia. Transcription factor Runx2 is essential for osteoblast lineage commitment as it controls the expression of several key bone-phenotypic genes (Lian 2004). Transcription of Runx2 is regulated by two promoters P1 and P2 which control the expression of the isoforms Runx2-II/p57 and Runx2-I/p56, respectively (Lian 2004). It has been described that stimulation of pluripotent mesenchymal cells with BMP2 (Bone Morphogenetic Protein 2) induces the expression of the isoform Runx2-II/p57 (Lian 2004). This skeletal transcription factor is aberrantly expressed at high levels in breast and prostate tumors and cells that aggressively metastasize to the bone environment (Pratap 2006). In cancer cells, Runx2 activates expression of bone matrix and adhesion proteins, matrix metalloproteinases and angiogenic factors that have long been associated with metastasis. In addition, Runx2 mediates the responses of cells to signaling pathways hyperactive in tumors, including BMP/TGFbeta and other growth factor signals (Baniwal, 2010). This skeletal transcription factor is aberrantly expressed at high levels in breast and prostate tumors and cells that aggressively metastasize to the bone environment. In cancer cells, Runx2 activates expression of bone matrix and adhesion proteins, matrix metalloproteinases and angiogenic factors that have long been associated with metastasis. In addition, Runx2 mediates the responses of cells to signaling pathways hyperactive in tumors, including BMP/TGFbeta and other growth factor signals (Pratap 2006). To begin defining the contribution of Runx2 in lung tumor cells, we have addressed the mRNA expression patterns exhibited by this transcription factor in the A-549 and MRC5 human cells lines. Our preliminary results in human cells lines indicate that the Runx2 expression was up regulated in lung tumor cell line (A549) (Fig1). These results should be evaluated in tumor cells of patients with diagnosis of NSCLC. After obtaining these results we will evaluate epigenetic mechanisms that up regulate the expression in lung tumor cells. So far our team has received two tumor samples from human patients diagnosed with lung adenocarcinoma, which have been doing the establishment of primary cultures (Fig.2 ). Fig 1. Runx2 mRNA expression was up regulated in lung tumor cell line A549. MRC5 cells (derived from normal lung tissue) and A549 cells (lung tumor cell line) were cultured and in MEM medium with 20% serum fetal bovine. Runx2 mRNA levels were measured by qRT-PCR three days after the seeding. Values were normalized against GAPDH mRNA. Fig. 2. Morphology of outgrowth clumps of patient lung cancer tissue 6 days after the seeding. A,B patient ID 2516; C, D Patient ID 2521. As mentioned above, the lung cancer because of its characteristics is a pathology that requires the search for early molecular markers that allow us to better understand the disease and thus improve prognosis and diagnosis. Thus the creation and strengthening of an international network in the area of epigenetics and lung cancer will increase the quality of research. Recently it has been shown that collaboration can be beneficial through the sharing of resources, ideas, expertise, and institutions. This was demonstrated in a study where examined the impact of collaboration on publication significance and quality of research in the United States and the United Kingdom, world leaders in stem cell research (Luo, 2011). This study was an initial investigation of the impact of international collaboration on publications in stem cell research. The results indicated that international collaborations, on average, significantly increased best practice methodology in research and article and citation rates for the UK and US investigators (Luo, 2011). All raised above background, justifying the importance of creating and strengthening international network in lung cancer epigenetics among research centers : IGH and FONDAP . We are confident that the establishment of this network will improve the quantity and quality of research in this area that requires urgent research to improve the prognosis and early diagnosis of lung cancer.