Molecular mechanisms regulating BMI1 in lung cancer

Lung cancer is one of the most common cause of cancer death and can be classified in: Small Cell Lung Cancer (15% of the cases) and Non Small Cell Lung Cancer (NSCLC, 85% of the cases). NSCLC is a very heterogeneous disease, at both the cellular and molecular level. This great variability hampers development of therapeutic treatments. Almost 30% of NSCLCs are driven by activating mutations in KRAS, which result in high aggressive course of the disease and poor response to treatments. BMI1 is a component of the epigenetic complex PRC1, that leads to ubiquitination of histone H2A. It is an oncogene overexpressed in 75% of NSCLC, including the mutant KRAS molecular subtype, and has a key role in cancer development and maintenance. BMI1 is positively correlated with tumour growth, invasion and metastasis. For these reasons, BMI1 represents a potential new therapeutic target: its inhibition appears to be a promising treating approach for tumours such as lung cancer. Pharmacological inhibition of BMI1 in NSCLC cells via a chemical compound named PTC-209, leads to growth arrest and accumulation of cells in G0 phase. A miRNA-mediated mechanism of action has been hypothesized. Sequencing of microRNAs in NSCLC cells treated with PTC-209, allowed the identification of miRNAs modulated by the treatment. Through bioinformatics analysis, a subset of upregulated miRNAs predicted to bind to control sequences of BMI1 was identified. Specifically, mir192, one of the identified miRNAs, was tested in NSCLC lines and showed downregulation of BMI1, reduction in cell growth, and accumulation of cells in G0 phase, resembling the effect of anti-BMI1 treatment. The aim of this project is to investigate the molecular mechanisms that regulate BMI1 expression in lung cancer and to specifically evaluate at the single cell level the microRNA that downregulates BMI1 and causes decrease in tumour growth. Our hypothesis is that PTC-209, by upregulating mir192, negatively affects BMI1 expression. Since mir-mediated regulatory circuitry are rather complex, we will also verify whether a regulatory loop exists between BMI1 and mir192. In addition, since PTC-209 was originally identified as a compound able to downregulate BMI1 activity by acting on its 3’UTR, we will scan the entire 3’UTR to identify which fragments are necessary to mediate response to PTC-209. For this project, three differentially modified version of the cell line A549, a NSCLC line mutated in KRAS, will be utilized. 1. Inducible A549 cells to overexpress mir192: The human pre-mir192 and also a non-targeting sequence as negative control, have been cloned into a doxycycline inducible lentiviral vector (Dharmacon™ shMIMIC Inducible Lentiviral microRNA), to generate a doxycycline inducible system to conditionally upregulate mir192 or the negative control sequence. The cloned sequence is under control of the mCMV promoter, which we identified as the optimal promoter for A549 cells, when adopting SMARTvector™ Lentiviral shRNAs. Stable clones integrating either the mir192 or the negative control vectors, have been generated by puromycin selection. Subsequently, cells will be modulated through doxycycline treatment and selected through a fluorescent reporter gene (GFP). It will be possible to upregulate mir192 and consequently analyse BMI1 levels by immunofluorescence through confocal microscopy and to evaluate the effects of mir192 overexpression on cell growth through cell cycle analysis by Flow Cytometry. 2. A549 cells in which BMI1 has been transiently knocked down: A549 cells have been transfected with a siRNA against BMI1 mRNA (or a scramble siRNA, as control) to study the regulatory circuitry occurring between BMI1 and mir192. 3. A549 cells expressing luciferase under control of the human BMI1 3’UTR: To identify which region/s of the BMI1 3’UTR is/are utilized by PTC-209 to cause BMI1 downregulation, A549 will be transfected with four different luciferase constructs containing the entire BMI1 3’UTR (1949 bp) or a truncated form (containing 1364 bp of 3’ UTR, 644 bp, or a deletion construct ∆94-959 bp) in which luciferase will constitute the read out for BMI1 activation. Once we identify the relevant regions necessary for BMI1 expression, we will scan them through bioinformatics programs to identify which regulatory elements they may contain and predict the cognate protein or non-coding factors