Glucocorticoid receptor promoter expression and apoptosis induction in small cell lung cancer.
Lung cancer is the most common cancer worldwide and is the fourth leading cause of death in South Africa. Lung cancer is categorised into two types; non-small cell lung cancer and small cell lung cancer (SCLC). SCLC constitutes 20% of all lung cancers and is considered to be an aggressive tumour as it gains chemo-resistance and exhibits early metastasis in diagnosed patients. SCLC cells originate from the neuroendocrine cells of the bronchoepithelium and are known to secrete the neuropeptide, proopiomelanocortin (POMC). POMC undergoes proteolytic cleavage to produce the adrenocorticotropin hormone (ACTH). ACTH stimulates the production of the steroid hormone, glucocorticoid hormone (GC), through the hypothalamus-pituitary-adrenal (HPA) axis. The produced GCs mediate a negative feedback system of the HPA axis to sequester ACTH production. SCLC cells are insensitive to this negative feedback stimulus. GCs elicit their actions through the glucocorticoid receptor (GR). Studies have shown that SCLC cells have a reduced expression of GR which perpetuates the GC-insensitivity. Importantly, over-expression of exogenous GR in SCLC cells leads to cell death by apoptosis. It was postulated that SCLC cells select against GR expression for longevity. Cancer cells are known to alter/silence the expression of tumour suppressor genes by a mechanism known as methylation. Methylation occurs when the enzyme, DNA methyltransferase 1, adds a methyl group to a cytosine present in a guanine-cytosine rich region of the gene (CpG island). The GR gene has a 5’-untranslated exon 1 region that consists of eight promoter regions (1A-1J), in these promoter regions are many CpG islands that have the potential to be methylated. The first aim of this study was to determine the promoter/s utilised by SCLC cells to express the GR protein. Conventional PCR revealed that all three cell lines predominantly utilise promoters 1B and 1C for GR expression. Bioinformatic analysis revealed that these promoters contain putative CpG islands and new data suggests that the GR is silenced by methylation and that treatment with a de-methylating agent results in GR re-expression. To determine which promoter is responsible for GR re-expression after de-methylation, the SCLC cell line, DMS79, as well as two control cell lines, A549 and HEK cells, were treated with the de-methylating agent, 5-aza-2’-deoxycytidine, for 72 hours. qPCR analyses revealed that all three cell lines expressed promoters 1B and 1C with A549 cells showing no evidence of methylation. The HEK cells showed methylation in promoter 1C and not promoter 1B. The SCLC cells showed methylation in both promoter 1B and 1C, however, only promoter 1B showed a significantincrease in transcript levels. SCLC cells are induced to undergo GC-mediated apoptosis when GR expression is restored however the mechanism utilised by the GR to induce the apoptotic cascade is unknown. The GR structure is divided into three domains; ligand binding domain (LBD), DNA binding domain (DBD) and amino terminal domain (NTD). The second aim of this study was to determine the component of the GR that induces apoptosis of SCLC cells. HEK and SCLC cells were infected with empty virus and various GR construct viruses; containing either a wild-type GR, ligand binding mutant, DNA binding mutant or a transactivation mutant (NTD); for 72 hours. Both cell lines were quantified for apoptosis and cell death using microscopic analyses. In HEK cells, it was shown that apoptosis occurred in cells expressing the wild-type GR, the DNA binding mutant and transactivation mutant constructs but apoptosis was reduced in cells expressing the ligand binding viruses. This indicates that the LBD may be necessary for inducing apoptosis in HEK cells. In DMS79 cells, apoptosis occurred in cells expressing the wild-type GR, ligand binding mutant and the DNA binding mutant constructs. There was less apoptotic activity exhibited in the transactivation constructs which indicates the NTD may be necessary for apoptosis induction in these cells. The NTD of the GR is responsible for interaction with other transcription factors to mediate GR transcriptional activity and this study has shown that the transactivation domain plays a necessary role in apoptosis induction. An analysis of the various pathways the GR interacts with through the NTD domain could lead to the identification of the pathway which triggers apoptosis in SCLC cells. This discovery, together with knowledge of promoter methylation and expression may contribute to the development of new, more effective therapies for SCLC.