Limits...
Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

Chao TT, Wang CY, Chen YL, Lai CC, Chang FY, Tsai YT, Chao CH, Shiau CW, Huang YC, Yu CJ, Chen KF - Oncotarget (2015)

Bottom Line: The effects of CIP2A on afatinib-induced apoptosis were confirmed by overexpression and knockdown of CIP2A expression in the sensitive and resistant cells, respectively.The apoptotic effect of afatinib in sensitive cells was associated with downregulation of CIP2A, promotion of PP2A activity and decrease in AKT phosphorylation.Afatinib suppressed CIP2A at the gene transcription level by reducing the promoter binding activity of Elk-1.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Center, Cardinal Tien Hospital, School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan.

ABSTRACT
Afatinib has anti-tumor effect in non-small cell lung carcinoma (NSCLC) with epidermal growth factor receptor (EGFR) mutation. We found afatinib can also induce apoptosis in NSCLC cells without EGFR mutation through CIP2A pathway. Four NSCLC cell lines (H358 H441 H460 and A549) were treated with afatinib to determine their sensitivity to afatinib-induced cell death and apoptosis. The effects of CIP2A on afatinib-induced apoptosis were confirmed by overexpression and knockdown of CIP2A expression in the sensitive and resistant cells, respectively. Reduction of Elk-1 binding to the CIP2A promoter and suppression of CIP2A transcription were analyzed. In vivo efficacy of afatinib against H358 and H460 xenografts tumors were also determined in nude mice. Afatinib induced significant cell death and apoptosis in H358 and H441 cells, but not in H460 or A549 cells. The apoptotic effect of afatinib in sensitive cells was associated with downregulation of CIP2A, promotion of PP2A activity and decrease in AKT phosphorylation. Afatinib suppressed CIP2A at the gene transcription level by reducing the promoter binding activity of Elk-1. Clinical samples showed that higher CIP2A expression predicted a poor prognosis and Elk-1 and CIP2A expressions were highly correlated. In conclusion, afatinib induces apoptosis in NSCLC without EGFR mutations through Elk-1/CIP2A/PP2A/AKT pathway.

Show MeSH

Related in: MedlinePlus

Elk-1 regulated CIP2A in NSCLC cells by afatinib(A) Left, H358 cells were treated with 100 μg/ml cycloheximide (CHX) in the presence or absence of afatinib for the indicated length of time. Middle and Right, H358 and H460 cells treated with afatinib at 10 μM or 20 μM for the designated incubation time. Afatinib inhibited CIP2A mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. *, p < 0.05, **, p < 0.01, vs. no afatinib. (B) Effects of afatinib on CIP2A promoter activity. Left, H358 and H460 cells were co-transfected with CIP2A reporter constructs (−1 to −2000bp) and renilla luciferase vectors for 48 h then treated with 10 μM afatinib for an additional 24 h. Afatinib decreased CIP2A luciferase activity in H358 cells, but not in H460 cells. Right, H358 cells were transfected CIP2A reporter various lengths of constructs and renilla luciferase vector for 48 h and then treated with 2 μM or 10 μM afatinib for an additional 24 h. Cell lysates were prepared for analysis of luciferase activity. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (C) Chromatin immunoprecipitation assays of the CIP2A promoter. H358 and H460 cells were treated with 2 μM or 10 μM afatinib for 24 h and processed for ChIP assay. Soluble chromatin was immunoprecipitated with specific Elk-1 or IgG (negative control) antibodies. Immunoprecipitates were subjected to PCR with primer pair specific to CIP2A promoter (−16 to −213 bp) and RPL30 (internal control). The gel shown is representative of three independent experiments. (D) H358 and H460 cells treated with afatinib at 2 μM or 10 μM for the indicated incubation times. Afatinib inhibited Elk-1 mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of Elk1 (HA-Elk1) restored the effect of afatinib on CIP2A expression and protected the effect of afatinib-induced apoptosis in H358 cells using Western blotting and FACS. H358 cells overexpressing Elk-1 were treated with 10 μM afatinib for 24 h. Open arrow is endogenous of Elk1 and close arrow is exogenous of Elk1. (F) Knockdown of Elk-1 enhanced apoptosis in H460 cells by afatinib. Protein levels of Elk1 expressed in lower panel. Data are means ± SD. **, p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385843&req=5

Figure 4: Elk-1 regulated CIP2A in NSCLC cells by afatinib(A) Left, H358 cells were treated with 100 μg/ml cycloheximide (CHX) in the presence or absence of afatinib for the indicated length of time. Middle and Right, H358 and H460 cells treated with afatinib at 10 μM or 20 μM for the designated incubation time. Afatinib inhibited CIP2A mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. *, p < 0.05, **, p < 0.01, vs. no afatinib. (B) Effects of afatinib on CIP2A promoter activity. Left, H358 and H460 cells were co-transfected with CIP2A reporter constructs (−1 to −2000bp) and renilla luciferase vectors for 48 h then treated with 10 μM afatinib for an additional 24 h. Afatinib decreased CIP2A luciferase activity in H358 cells, but not in H460 cells. Right, H358 cells were transfected CIP2A reporter various lengths of constructs and renilla luciferase vector for 48 h and then treated with 2 μM or 10 μM afatinib for an additional 24 h. Cell lysates were prepared for analysis of luciferase activity. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (C) Chromatin immunoprecipitation assays of the CIP2A promoter. H358 and H460 cells were treated with 2 μM or 10 μM afatinib for 24 h and processed for ChIP assay. Soluble chromatin was immunoprecipitated with specific Elk-1 or IgG (negative control) antibodies. Immunoprecipitates were subjected to PCR with primer pair specific to CIP2A promoter (−16 to −213 bp) and RPL30 (internal control). The gel shown is representative of three independent experiments. (D) H358 and H460 cells treated with afatinib at 2 μM or 10 μM for the indicated incubation times. Afatinib inhibited Elk-1 mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of Elk1 (HA-Elk1) restored the effect of afatinib on CIP2A expression and protected the effect of afatinib-induced apoptosis in H358 cells using Western blotting and FACS. H358 cells overexpressing Elk-1 were treated with 10 μM afatinib for 24 h. Open arrow is endogenous of Elk1 and close arrow is exogenous of Elk1. (F) Knockdown of Elk-1 enhanced apoptosis in H460 cells by afatinib. Protein levels of Elk1 expressed in lower panel. Data are means ± SD. **, p < 0.01.

Mentions: To examine the mechanism by which afatinib inhibits CIP2A expression, we investigated whether afatinib affected CIP2A protein degradation. After protein translation was blocked by cycloheximide, the rate of CIP2A degradation did not change significantly with or without afatinib in H358 cells (Figure 4A, left). We next investigated whether afatinib affected CIP2A gene transcription. These results showed that the mRNA levels of CIP2A decreased in a time- and dose-dependent manner in the sensitive H358 cells but not in the resistant H460 cells (Figure 4A, right). To further explore the inhibition of CIP2A transcription by afatinib, the sensitive H358 cells were transfected with CIP2A promoter luciferase constructs. Afatinib significantly down-regulated the CIP2A promoter activity in a dose-dependent manner in the sensitive H358 cells (Figure 4B). However, afatinib did not alter the luciferase activity in the resistant H460 cells. Among the various lengths of CIP2A promoter regions (-1~ -62 bp, -1~ -150 bp, -1~ -300 bp, -1~ -400 bp, -1~ -1000 bp and -1~ -2000 bp), we observed that each length of the promoters was significantly depressed by increasing the dose of afatinib except for -1~ -62 bp. The putative Elk-1-binding site existed in the -62~ -150 bp element. We therefore performed ChIP assay to evaluate the binding affinity between the transcription factor of Elk-1 and CIP2A promoter. The interaction of Elk-1 with the CIP2A promoter was abolished in a dose-dependent manner in the sensitive H358 cells but not in the resistant H460 cells (Figure 4C). Furthermore, we explored the effect of afatinib on transcription factor of Elk-1, and found that both mRNA (Figure 4D) and protein levels (Figure 4E) of Elk-1 were reduced by afatinib. Moreover, the ectopic expression of Elk-1 also restored CIP2A expression (Figure 4E, left) and partially protected the cells from apoptotic death induced by afatinib in H358 cells (Figure 4E, right). On the other hand, deprivation of Elk-1 increased apoptosis by afatinib in H460 cells (Figure 4F). These results suggest that afatinib may inhibit CIP2A expression by affecting the DNA binding ability of Elk-1 via debasing Elk-1 in the sensitive H358 cells.


Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

Chao TT, Wang CY, Chen YL, Lai CC, Chang FY, Tsai YT, Chao CH, Shiau CW, Huang YC, Yu CJ, Chen KF - Oncotarget (2015)

Elk-1 regulated CIP2A in NSCLC cells by afatinib(A) Left, H358 cells were treated with 100 μg/ml cycloheximide (CHX) in the presence or absence of afatinib for the indicated length of time. Middle and Right, H358 and H460 cells treated with afatinib at 10 μM or 20 μM for the designated incubation time. Afatinib inhibited CIP2A mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. *, p < 0.05, **, p < 0.01, vs. no afatinib. (B) Effects of afatinib on CIP2A promoter activity. Left, H358 and H460 cells were co-transfected with CIP2A reporter constructs (−1 to −2000bp) and renilla luciferase vectors for 48 h then treated with 10 μM afatinib for an additional 24 h. Afatinib decreased CIP2A luciferase activity in H358 cells, but not in H460 cells. Right, H358 cells were transfected CIP2A reporter various lengths of constructs and renilla luciferase vector for 48 h and then treated with 2 μM or 10 μM afatinib for an additional 24 h. Cell lysates were prepared for analysis of luciferase activity. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (C) Chromatin immunoprecipitation assays of the CIP2A promoter. H358 and H460 cells were treated with 2 μM or 10 μM afatinib for 24 h and processed for ChIP assay. Soluble chromatin was immunoprecipitated with specific Elk-1 or IgG (negative control) antibodies. Immunoprecipitates were subjected to PCR with primer pair specific to CIP2A promoter (−16 to −213 bp) and RPL30 (internal control). The gel shown is representative of three independent experiments. (D) H358 and H460 cells treated with afatinib at 2 μM or 10 μM for the indicated incubation times. Afatinib inhibited Elk-1 mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of Elk1 (HA-Elk1) restored the effect of afatinib on CIP2A expression and protected the effect of afatinib-induced apoptosis in H358 cells using Western blotting and FACS. H358 cells overexpressing Elk-1 were treated with 10 μM afatinib for 24 h. Open arrow is endogenous of Elk1 and close arrow is exogenous of Elk1. (F) Knockdown of Elk-1 enhanced apoptosis in H460 cells by afatinib. Protein levels of Elk1 expressed in lower panel. Data are means ± SD. **, p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385843&req=5

Figure 4: Elk-1 regulated CIP2A in NSCLC cells by afatinib(A) Left, H358 cells were treated with 100 μg/ml cycloheximide (CHX) in the presence or absence of afatinib for the indicated length of time. Middle and Right, H358 and H460 cells treated with afatinib at 10 μM or 20 μM for the designated incubation time. Afatinib inhibited CIP2A mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. *, p < 0.05, **, p < 0.01, vs. no afatinib. (B) Effects of afatinib on CIP2A promoter activity. Left, H358 and H460 cells were co-transfected with CIP2A reporter constructs (−1 to −2000bp) and renilla luciferase vectors for 48 h then treated with 10 μM afatinib for an additional 24 h. Afatinib decreased CIP2A luciferase activity in H358 cells, but not in H460 cells. Right, H358 cells were transfected CIP2A reporter various lengths of constructs and renilla luciferase vector for 48 h and then treated with 2 μM or 10 μM afatinib for an additional 24 h. Cell lysates were prepared for analysis of luciferase activity. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (C) Chromatin immunoprecipitation assays of the CIP2A promoter. H358 and H460 cells were treated with 2 μM or 10 μM afatinib for 24 h and processed for ChIP assay. Soluble chromatin was immunoprecipitated with specific Elk-1 or IgG (negative control) antibodies. Immunoprecipitates were subjected to PCR with primer pair specific to CIP2A promoter (−16 to −213 bp) and RPL30 (internal control). The gel shown is representative of three independent experiments. (D) H358 and H460 cells treated with afatinib at 2 μM or 10 μM for the indicated incubation times. Afatinib inhibited Elk-1 mRNA in a dose- and time-dependent manner, especially in H358 cells. Data are mean ± SD. n = 3 for each time point. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of Elk1 (HA-Elk1) restored the effect of afatinib on CIP2A expression and protected the effect of afatinib-induced apoptosis in H358 cells using Western blotting and FACS. H358 cells overexpressing Elk-1 were treated with 10 μM afatinib for 24 h. Open arrow is endogenous of Elk1 and close arrow is exogenous of Elk1. (F) Knockdown of Elk-1 enhanced apoptosis in H460 cells by afatinib. Protein levels of Elk1 expressed in lower panel. Data are means ± SD. **, p < 0.01.
Mentions: To examine the mechanism by which afatinib inhibits CIP2A expression, we investigated whether afatinib affected CIP2A protein degradation. After protein translation was blocked by cycloheximide, the rate of CIP2A degradation did not change significantly with or without afatinib in H358 cells (Figure 4A, left). We next investigated whether afatinib affected CIP2A gene transcription. These results showed that the mRNA levels of CIP2A decreased in a time- and dose-dependent manner in the sensitive H358 cells but not in the resistant H460 cells (Figure 4A, right). To further explore the inhibition of CIP2A transcription by afatinib, the sensitive H358 cells were transfected with CIP2A promoter luciferase constructs. Afatinib significantly down-regulated the CIP2A promoter activity in a dose-dependent manner in the sensitive H358 cells (Figure 4B). However, afatinib did not alter the luciferase activity in the resistant H460 cells. Among the various lengths of CIP2A promoter regions (-1~ -62 bp, -1~ -150 bp, -1~ -300 bp, -1~ -400 bp, -1~ -1000 bp and -1~ -2000 bp), we observed that each length of the promoters was significantly depressed by increasing the dose of afatinib except for -1~ -62 bp. The putative Elk-1-binding site existed in the -62~ -150 bp element. We therefore performed ChIP assay to evaluate the binding affinity between the transcription factor of Elk-1 and CIP2A promoter. The interaction of Elk-1 with the CIP2A promoter was abolished in a dose-dependent manner in the sensitive H358 cells but not in the resistant H460 cells (Figure 4C). Furthermore, we explored the effect of afatinib on transcription factor of Elk-1, and found that both mRNA (Figure 4D) and protein levels (Figure 4E) of Elk-1 were reduced by afatinib. Moreover, the ectopic expression of Elk-1 also restored CIP2A expression (Figure 4E, left) and partially protected the cells from apoptotic death induced by afatinib in H358 cells (Figure 4E, right). On the other hand, deprivation of Elk-1 increased apoptosis by afatinib in H460 cells (Figure 4F). These results suggest that afatinib may inhibit CIP2A expression by affecting the DNA binding ability of Elk-1 via debasing Elk-1 in the sensitive H358 cells.

Bottom Line: The effects of CIP2A on afatinib-induced apoptosis were confirmed by overexpression and knockdown of CIP2A expression in the sensitive and resistant cells, respectively.The apoptotic effect of afatinib in sensitive cells was associated with downregulation of CIP2A, promotion of PP2A activity and decrease in AKT phosphorylation.Afatinib suppressed CIP2A at the gene transcription level by reducing the promoter binding activity of Elk-1.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Center, Cardinal Tien Hospital, School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan.

ABSTRACT
Afatinib has anti-tumor effect in non-small cell lung carcinoma (NSCLC) with epidermal growth factor receptor (EGFR) mutation. We found afatinib can also induce apoptosis in NSCLC cells without EGFR mutation through CIP2A pathway. Four NSCLC cell lines (H358 H441 H460 and A549) were treated with afatinib to determine their sensitivity to afatinib-induced cell death and apoptosis. The effects of CIP2A on afatinib-induced apoptosis were confirmed by overexpression and knockdown of CIP2A expression in the sensitive and resistant cells, respectively. Reduction of Elk-1 binding to the CIP2A promoter and suppression of CIP2A transcription were analyzed. In vivo efficacy of afatinib against H358 and H460 xenografts tumors were also determined in nude mice. Afatinib induced significant cell death and apoptosis in H358 and H441 cells, but not in H460 or A549 cells. The apoptotic effect of afatinib in sensitive cells was associated with downregulation of CIP2A, promotion of PP2A activity and decrease in AKT phosphorylation. Afatinib suppressed CIP2A at the gene transcription level by reducing the promoter binding activity of Elk-1. Clinical samples showed that higher CIP2A expression predicted a poor prognosis and Elk-1 and CIP2A expressions were highly correlated. In conclusion, afatinib induces apoptosis in NSCLC without EGFR mutations through Elk-1/CIP2A/PP2A/AKT pathway.

Show MeSH
Related in: MedlinePlus