The overall goal of this research is to enhance the investigators understanding of the pathways involved in lung cancer, and to identify new biomarkers and/or therapeutic targets. By comparing gene expression between normal lung tissue and tumors growing in lung-specific C/EBPa KO mice, the investigators have identified the Bmi-1 proto-oncogene as being abnormally upregulated in C/EBPa-deleted tumors. Subsequently, the investigators have validated this observation in human lung cancer, implicating the investigators KO mice are an effective discovery tool for lung cancer research. Through similar approaches, the investigators have already identified (Sonic Hedgehog, SHH), and plan to identify other pathways which are abnormally regulated in C/EBPa-/- tumors. In parallel, the investigators will proceed to define the clinical relevance of the SHH pathway and the other newly-discovered molecular aberrations, by analyzing their expression and correlate it to C/EBPa expression on the samples of patients with NSCLC at NUHS. If the investigators preliminary data on Bmi-1 will be confirmed, this proto-oncogene may generate useful correlates that could be used in diagnosis and treatment of lung cancer, as well as identify new prognostic/predictive markers in lung cancer. Similarly, SHH pathway-components may behave as potential biomarkers and therapeutic tools for C/EBPa-related lung cancers. This proposal seeks to test the hypothesis that pathways which are dysregulated in lung tumors growing in a lung-specific C/EBPa KO model can be utilized as discovery tools to identify genes involved in human lung cancer pathogenesis.
Study Type
OBSERVATIONAL
Nationa University Hospital
Singapore, Singapore
RECRUITINGDefine the C/EBPa-Bmi-1 axis in human lung cancer
Tissue microarrays will be created from the lung tumours. Only resection specimens will be used for TMA construction, and small, limited biopsies will not be used. Tissue punches of 1.5mm in diameter will be cut from the blocks (2 punches per tumour, 1 punch for normal lung). Hence the rest of the tumour block is not used, preserving most of the tumour tissue in the tissue blocks, for future diagnostic use.
Validate the relevance of the C/EBPa-SHH pathway in human NSCLC
We will stain the human lung cancer specimens with antibodies against Gli1 (the major effector of the SHH pathway), Patched-1, Patched-2 and Smoothened (the receptors of the pathway), Sonic, Indian and Desert Hedgehog (the ligands of the pathway), and Cyclin D, Cyclin E, and Myc (target genes of the pathway).Interestingly, the SHH pathway has been recently associated to primary cilia (PC), where its specific role is highly controversial. Genetic defects affecting PC result in a myriad of pathological instances, including lung pathologies
Identify new downstream targets of C/EBPa in murine lung cancer and test their involvement in human pathogenesis
RNA microarray analysis will be performed by Dr. Levantini at the BIDMC (Tenen lab, Boston), to compare gene expression between C/EBPa-/- pulmonary tumors and the neighboring normal murine tissue. The mouse MOE430A gene chip from Affymetrix will be utilized, as recently published (Basseres et al., MCB 2006). Single cell suspension will be depleted by FACS for endothelial and hematopoietic markers (CD45, Ter119, CD31). Purified populations will then undergo cRNA synthesis followed by hybridization to the Affymetrix gene chips. The top 10,000 genes will be selected based on their ranking.
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