Decades of both epidemiological research and in vitro and in vivo laboratory studies have consistently reported an association between environmental nickel exposure and lung cancer. As early as 1990, the International Agency for Research on Cancer (IARC) classified nickel compounds as Group 1 carcinogens: substances confirmed as carcinogenic to humans. Although the multiple mechanisms, including oxidative stress, epigenetic effects, and activation of signaling pathways that trigger differential gene regulation has been thought to be associated with nickel carcinogenic effect, there is no evidence that a single nickel- inducible factor can drive transformation of human bronchial cells, to the best of our knowledge. In our Preliminary Studies, we performed long noncoding RNA (lncRNA) deep sequencing using the Illumina HiSeqTM2000/2500 high throughout system to evaluate whether nickel affects the abundance of 5,929 known lncRNAs. Nickel treatment altered the levels of 24 of these lncRNAs in normal primary human bronchial epithelial cells (HBECs), while 16 of them were verified in Real-time PCR assay. With overexpression and/or knockdown analyses of the 16 lncRNAs, we further discovered that a reduced abundance of maternally-expressed gene 3 (MEG3) was sufficient for malignant transformation of HBECs. Our demonstration that MEG3 inhibition can independently transform HBECs provides the basis of our central hypothesis that MEG3 downregulation drives transformation and tumorigenecity of HBECs after nickel exposure. Here we propose to elucidate the molecular mechanisms that underlie our novel findings that MEG3 downregulation is a crucial driver for nickel-induced malignant transformation of HBECs with the following Specific Aims: 1, To test the hypothesis that p62 and C-Myc define an important MEG3-regulated axis that promotes transformation of HBECs upon MEG3 deficiency; 2, To test the hypothesis that the crosstalk between a p62/C-Myc cascade and the PHLPP/HIF-1? axis causes the MEG3-deficiency- correlated malignant transformation of HBECs; 3, To explore the biological significance of MEG3 deletion and its activated downstream molecules in lung tumoriginecity. Our novel Preliminary Findings suggest that MEG3 is downregulated by nickel exposure both in vitro and in vivo, that knockdown of MEG3 alone can transform HBECs, and that crosstalk between the putative signaling transducers downstream of MEG3 mediate the malignant transformation of HBECs caused by MEG3 deficiency. This proposal's strengths are the complementary use of cell culture models and novel conditional MEG3 knockout mouse models to examine integration of the molecular events that account for nickel-mediated lung carcinogenesis. Clarifying these issues will provide valuable insights regarding MEG3 as a prognostic biomarker and/or as a therapeutic target. Ultimately, both uses could improve clinical outcomes in lung cancer patients.
Identification of the lung carcinogenic driver and elucidation of the molecular mechanisms involved in environmental lung carcinogenesis will be of high significance in facilitating our understanding of lung cancer development and will provide valuable information needed for the design of more effective agents for prevention and therapy of lung cancers. This application will test the hypothesis that MEG3 downregulation plays a crucial role in lung carcinogenesis caused by nickel exposure. The overall goal of this proposal is to define the in vivo functions of our novel discovery of MEG3 downregulation and its upstream regulators, downstream effectors, as well as mechanisms, leading to the transformation and tumorigenicity of HBECs following nickel exposure.
