Introduction. Nuclear factor erythroid-2 related factor 2 (Nrf2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (Keap1) negatively regulates Nrf2 activity by targeting it to proteasomal degradation. We have just described a Keap1/Nrf2 axis full genetic and epigenetic characterization of SCLC cell lines that revealed unreported molecular alterations of the Keap1/Nrf2 axis. Materials and methods. The downstream effects of the genetic and epigenetic alterations of the KEAP1 gene were investigated in 12 cell lines derived from human small cell lung carcinoma in terms of modulation of the KEAP1 transcript and protein levels, nuclear accumulation of Nrf2 and enhancing transcriptional induction of xenobiotic metabolism enzymes. Additional functional analysis of short interfering RNA (siRNA) inhibition of the KEAP1 and treatment 5-aza-2′-deoxycytidine (DAC) were conducted. Results. Our analysis revealed that the Keap1 mRNA and protein level decreased significantly in genetic and epigenetic alterated SCLC cell lines compared with those without any alterations. Con-versely Nrf2 levels and protein nuclear localization were increased and these modifications were associated with a parallel increase in the expression of AKR1C1, TXN1 and NQO1 at the cellular lev-el. Silencing RNA experiments in vitro in H1184, H69V and were performed to confirm the cause-effect relation between the gain of Nrf2 and the increase in AKR1C1, TXN1 and NQO1 expression. Treatments with 5-aza-2′-deoxycytidine restored the expression of KEAP1 in SCLC cells and replace the functional Keap1/Nrf2 equilibrium. Conclusions. Our data provide new insights into the potential downstream effects of genetic and epigenetic Keap1/Nrf2 molecular deregulation in SCLCs, suggesting that the impairment of Keap1 activity actually induces the expression of cytoprotective enzymes also in small cell lung cancer cells. Validations on tissues from SCLC affected patients combined with in vitro pharmacological studies are demanded to establish new combined therapeutic strategies in targeted cancer treat-ments of this aggressive lung tumour histotype.
Abstract 3841: Effects of KEAP1 genetic and epigenetic silencing in SCLC cell lines
2015-01-01
Abstract
Introduction. Nuclear factor erythroid-2 related factor 2 (Nrf2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (Keap1) negatively regulates Nrf2 activity by targeting it to proteasomal degradation. We have just described a Keap1/Nrf2 axis full genetic and epigenetic characterization of SCLC cell lines that revealed unreported molecular alterations of the Keap1/Nrf2 axis. Materials and methods. The downstream effects of the genetic and epigenetic alterations of the KEAP1 gene were investigated in 12 cell lines derived from human small cell lung carcinoma in terms of modulation of the KEAP1 transcript and protein levels, nuclear accumulation of Nrf2 and enhancing transcriptional induction of xenobiotic metabolism enzymes. Additional functional analysis of short interfering RNA (siRNA) inhibition of the KEAP1 and treatment 5-aza-2′-deoxycytidine (DAC) were conducted. Results. Our analysis revealed that the Keap1 mRNA and protein level decreased significantly in genetic and epigenetic alterated SCLC cell lines compared with those without any alterations. Con-versely Nrf2 levels and protein nuclear localization were increased and these modifications were associated with a parallel increase in the expression of AKR1C1, TXN1 and NQO1 at the cellular lev-el. Silencing RNA experiments in vitro in H1184, H69V and were performed to confirm the cause-effect relation between the gain of Nrf2 and the increase in AKR1C1, TXN1 and NQO1 expression. Treatments with 5-aza-2′-deoxycytidine restored the expression of KEAP1 in SCLC cells and replace the functional Keap1/Nrf2 equilibrium. Conclusions. Our data provide new insights into the potential downstream effects of genetic and epigenetic Keap1/Nrf2 molecular deregulation in SCLCs, suggesting that the impairment of Keap1 activity actually induces the expression of cytoprotective enzymes also in small cell lung cancer cells. Validations on tissues from SCLC affected patients combined with in vitro pharmacological studies are demanded to establish new combined therapeutic strategies in targeted cancer treat-ments of this aggressive lung tumour histotype.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
