Induction of p21WAF1 expression via Sp1-binding sites by tamoxifen in estrogen receptor-negative lung cancer cells

Although originally synthesized as an anti-estrogen, tamoxifen (Tam) was found to be able to inhibit proliferation of estrogen receptor (ER)-negative cancer cells in vitro. However, the molecular basis of such ER-independent growth inhibition is largely unknown. We have previously demonstrated that Tam induces p21(WAF1) and p27(KIP1) expression in human lung cancer cells which lack ER-alpha and -beta. We found that Tam induced p21(WAF1) expression via transcriptional activation. In order to determine the molecular mechanism responsible for p21(WAF1) induction by Tam, we performed a deletion analysis on the p21(WAF1) promoter. The minimal region in the p21(WAF1) promoter required for Tam-activated induction mas mapped to a contiguous stretch of 10 bp located 83 bases upstream of the transcription initiation site. Our results showed that transcription factor Sp1 and Sp3 bound to this GC-rich region and mutation of Sp1-binding sites dramatically attenuated Tam-induced p21(WAF1) promoter activity. We also tried to elucidate the signaling pathway that mediated the activation of p21(WAF1) by Tam, Inhibition of mitogen-activated protein kinase pathways did not block Tam-induced p21(WAF1). Similarly, protein kinase C inhibitor calphostin C could not suppress Tam-induced p21(WAF1). Conversely, pretreatment of a specific protein kinase A inhibitor H89 significantly attenuated the induction of p21(WAF1) by Tam, Furthermore, PKA activators forskolin and dibutyryl-cAMP activated p21(WAF1) promoter activity and increased p21(WAF1) protein level in lung cancer cells. Taken together, these results demonstrate that Tam activates the p21(WAF1) promoter via Sp1-binding sites and suggest that PKA may be involved in the induction of p21(WAF1) by Tam in ER-negative Lung cancer cells.