The cyclin-dependent kinase inhibitor, p21WAF1, is a resistance factor for histone deacetylase inhibitors, and induces angiogenesis via the thioredoxin pathway

4355 Histone deacetylation at the promoter region of tumor suppressor genes is an important mechanism contributing to human cancer. HDAC inhibitors (HDACIs) are an exciting new class of anticancer compound whose mechanism of action is thought to involve de-repression of tumor suppressor gene transcription. HDACIs have been shown to induce expression of the cyclin-dependent kinase inhibitor, p21 WAF1 , in many cell types, suggesting p21 WAF1 may mediate the anticancer effects of HDACIs. Expression of p21 WAF1 can lead to cell cycle arrest and growth inhibition in normal and cancer cells. However, increasing evidence suggests p21 WAF1 can cause resistance to anticancer agents in some cancer cells through unknown mechanisms. We observed that siRNA-mediated knockdown of p21 WAF1 in MCF-7 breast cancer cells increased their sensitivity to HDACIs, and repressed hypoxia-inducible factor-1α expression under hypoxic conditions. We examined the hypothesis that p21 WAF1 expression may instead promote resistance to HDACIs, via effects on tumor angiogenesis. We found that conditioned media from cultured MCF-7 cells transfected with p21 WAF1 siRNA, induced significantly less endothelial cell migration, invasion and vascular sprouting, compared with media from scrambled siRNA-transfected cells. Liquid chromatography/mass spectrometry analysis of the conditioned media revealed that thioredoxin (Trx) secretion was significantly reduced after p21 WAF1 knockdown. The reduction in Trx secretion following p21 WAF1 knockdown was associated with increased expression of intracellular Trx binding protein-2 (TBP2), which was also up-regulated by p21 WAF1 siRNA in neuroblastoma and lung cancer cells. Consistent with these findings, media from MCF7 cells transfected with TBP2 specific siRNA alone, promoted endothelial cell invasion and vascular sprouting, while Trx knockdown blocked the pro-angiogenic effects of TBP2 siRNA transfection. Media from MCF-7 cells transfected with both TBP2 siRNA and p21 WAF1 siRNA, lost the anti-angiogenic effect of p21 WAF1 knockdown alone. TBP2 gene expression was suppressed 48 hours after treatment with HDACIs. Inhibition of p21 WAF1 expression by siRNA knockdown reversed the HDACI-induced reduction in TBP2 expression, and significantly increased HDACI-induced cytoxicity. Collectively, our data suggests that p21 WAF1 can promote tumor angiogenesis, and cancer cell resistance to HDACIs, by modulating the TBP2-Trx pathway. These findings highlight the p21 WAF1 -TBP2-Trx redox pathway as a potential drug target.