The N‐terminal transactivation domain of the glucocorticoid receptor mediates apoptosis of human small cell lung cancer cells

Abstract

Small cell lung cancer (SCLC) is an aggressive disease with a poor prognosis. These cancers are deficient in glucocorticoid receptor (GR) expression, and therefore, resistant to glucocorticoids. Overexpression of the GR both in vivo and in vitro leads to apoptotic cell death suggesting that loss of GR is favorable for cancer growth. Indeed, the GR promoter is silenced in SCLC cells by methylation. We now show that treatment of the SCLC cell line (DMS79) cells with the demethylating agent, 5-aza-2'-deoxycytidine (5-aza), results in significant endogenous re-expression of both GRα and the ligand-independent GR-P. The GR gene has a complex promoter region comprising nine alternative promoters, the proximal seven of which lie within a CpG island. The endogenous re-expression seen is attributed to the constitutive promoters 1B and 1C and 1J but predominantly 1F, which we show to be heavily methylated in SCLC cells. Flow cytometric analysis using the apoptotic marker, Annexin V, shows that this endogenous re-expression is sufficient to drive the SCLC cells to apoptosis. Apoptotic induction is specific to GR re-expression as cotreatment with 5-aza and the GR antagonist, RU486 prevented apoptosis. Of the three functional GR domains (the DNA binding domain, ligand binding domain, and transactivation domain), we identified that the transactivation domain is essential for apoptosis in SCLC. The discovery that endogenous re-expression of the GR in SCLC cells is sufficient to induce apoptotic cell death by reversing a cancer-driven DNA methylation effect may lead to the development of novel adjunct therapies.

Glucocorticoid receptors in lung cancer: new perspectives

“…A recent report has extended these observations to show that the GR appears downregulated in many cancers including liver, prostate, colon and breast (Matthews et al 2015). In SCLC, GR downregulation occurs due to methylation of the GR promoter (Kay et al 2011, Singh et al 2014. Others have shown that in ovarian cancer, BRCA1 is a potential regulator of the GR, possibly through phosphorylation of Ser211 and entry of GR into the nucleus.…”

“…Furthermore, re-expression by retroviral over-expression both in vitro and in xenograft resulted in apoptotic induction, prompting us to postulate that loss of GR expression may play a role in tumourigenesis and to speculate that a threshold level of the GR is required for GR activity in SCLC cells (Sommer et al 2007(Sommer et al , 2010. We later showed that the reduction in GR is due to silencing by methylation (Kay et al 2011, Singh et al 2014. Unpublished preliminary data show that, although heterogenous, there are distinct differences in the methylation profile of the GR promoter in patients diagnosed with NSCLC and SCLC.…”

The tobacco carcinogen NNK drives accumulation of DNMT1 at the GR promoter thereby reducing GR expression in untransformed lung fibroblasts

“…Its promoter region comprises nine alternative promoters, the proximal seven of which (1B-1F, 1H and 1J) are located in a ~3.1 kb CpG island 8 , 9 . We have previously shown that GR expression in SCLC cell lines is reduced due to aberrant methylation of the proximal promoters, 1D and 1E, with extensive methylation of promoters 1F and 1C 10 , 11 . Treatment with a demethylating agent leads to re-expression of GRα (the major functional translational isoform) protein 10 , mediated by promoters 1B, 1C and 1J, but predominantly 1F 11 .…”

“…We have previously shown that GR expression in SCLC cell lines is reduced due to aberrant methylation of the proximal promoters, 1D and 1E, with extensive methylation of promoters 1F and 1C 10 , 11 . Treatment with a demethylating agent leads to re-expression of GRα (the major functional translational isoform) protein 10 , mediated by promoters 1B, 1C and 1J, but predominantly 1F 11 . Importantly, the GR is an authentic tumour suppressor gene (TSG) 12 and restoration of endogenous GR expression drives the SCLC cells to apoptosis 11 .…”

“…Treatment with a demethylating agent leads to re-expression of GRα (the major functional translational isoform) protein 10 , mediated by promoters 1B, 1C and 1J, but predominantly 1F 11 . Importantly, the GR is an authentic tumour suppressor gene (TSG) 12 and restoration of endogenous GR expression drives the SCLC cells to apoptosis 11 . Loss of GR expression thus confers a survival advantage to the cells.…”