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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.

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Validation of the CIP2A-PP2A-AKT pathway(A) Left, ectopic expression of CIP2A (CIP2A-DDK) increased p-AKT and attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing CIP2A were treated with 10 μM afatinib for 24 h. Right, knockdown of CIP2A expression by siRNA increased the sensitivity to afatinib-induced apoptosis in H460 cells. Cells were transfected with either control or CIP2A siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (B) Left, overexpression of CIP2A decreased PP2A activity in H358 cells. Right, downregulation of CIP2A by siRNA increased the activity of PP2A in H460 cells. (C) Silencing PP2Ac reduced the apoptotic effect of afatinib in H358 cells. Cells were transfected with either control or PP2Ac siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (D) Left, okadaic acid (OA), a PP2A inhibitor, increased p-AKT and inhibited the effects of afatinib on apoptosis of H358 cells. Right, forskolin, a PP2A agonist, sensitized resistant H460 cells to afatinib. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of AKT (AKT-DDK) attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing AKT were treated with 10 μM afatinib for 24 h.
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Figure 3: Validation of the CIP2A-PP2A-AKT pathway(A) Left, ectopic expression of CIP2A (CIP2A-DDK) increased p-AKT and attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing CIP2A were treated with 10 μM afatinib for 24 h. Right, knockdown of CIP2A expression by siRNA increased the sensitivity to afatinib-induced apoptosis in H460 cells. Cells were transfected with either control or CIP2A siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (B) Left, overexpression of CIP2A decreased PP2A activity in H358 cells. Right, downregulation of CIP2A by siRNA increased the activity of PP2A in H460 cells. (C) Silencing PP2Ac reduced the apoptotic effect of afatinib in H358 cells. Cells were transfected with either control or PP2Ac siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (D) Left, okadaic acid (OA), a PP2A inhibitor, increased p-AKT and inhibited the effects of afatinib on apoptosis of H358 cells. Right, forskolin, a PP2A agonist, sensitized resistant H460 cells to afatinib. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of AKT (AKT-DDK) attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing AKT were treated with 10 μM afatinib for 24 h.

Mentions: To verify the role of CIP2A/PP2A/p-AKT in mediating afatinib-induced apoptosis in the EGFR wild-type sensitive NSCLC cells, two approaches were used. First, the ectopic expression of CIP2A was evaluated in the sensitive H358 cells (Figure 3A, left), which showed that CIP2A was overexpression and partially protected the cells from apoptotic cell death induced by afatinib, and that PP2A activity was decreased (Figure 3B, left). Further, knockdown of the gene expression of CIP2A increased the sensitivity of afatinib-induced apoptosis in the resistant H460 cells (Figure 3A, right) and increased PP2A activity (Figure 3B, right). This suggested that afatinib may repress CIP2A, enhance PP2A activity, and then induce cell death. Furthermore, in order to examine the relationship between PP2A and AKT (Figure 3C), we knocked down PP2Ac expression by siRNA strategy in the sensitive H358 cells. The results showed that AKT phosphorylation increased with the deprived of PP2A and also partially protected the cells from apoptotic death induced by afatinib. In addition, with the treatment of okadaic acid, a known PP2A inhibitor, AKT phosphorylation was significantly increased in the sensitive H358 cells which reduced the afatinib-induced apoptosis (Figure 3D, left), whereas co-treatment with forskolin sensitized resistant H460 cells to afatinib-induced apoptosis and p-AKT downregulation (Figure 3D, right). We next analyzed the role of AKT, which is downstream of PP2A, in mediating the effects of afatinib. The ectopic expression of AKT in the sensitive H358 cells (Figure 3E) partially protected the cells from apoptotic death. Taken together, these results suggest that the afatinib-induced apoptotic effect is through the CIP2A-PP2A-AKT pathway in NSCLC 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)

Validation of the CIP2A-PP2A-AKT pathway(A) Left, ectopic expression of CIP2A (CIP2A-DDK) increased p-AKT and attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing CIP2A were treated with 10 μM afatinib for 24 h. Right, knockdown of CIP2A expression by siRNA increased the sensitivity to afatinib-induced apoptosis in H460 cells. Cells were transfected with either control or CIP2A siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (B) Left, overexpression of CIP2A decreased PP2A activity in H358 cells. Right, downregulation of CIP2A by siRNA increased the activity of PP2A in H460 cells. (C) Silencing PP2Ac reduced the apoptotic effect of afatinib in H358 cells. Cells were transfected with either control or PP2Ac siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (D) Left, okadaic acid (OA), a PP2A inhibitor, increased p-AKT and inhibited the effects of afatinib on apoptosis of H358 cells. Right, forskolin, a PP2A agonist, sensitized resistant H460 cells to afatinib. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of AKT (AKT-DDK) attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing AKT were treated with 10 μM afatinib for 24 h.
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Figure 3: Validation of the CIP2A-PP2A-AKT pathway(A) Left, ectopic expression of CIP2A (CIP2A-DDK) increased p-AKT and attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing CIP2A were treated with 10 μM afatinib for 24 h. Right, knockdown of CIP2A expression by siRNA increased the sensitivity to afatinib-induced apoptosis in H460 cells. Cells were transfected with either control or CIP2A siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (B) Left, overexpression of CIP2A decreased PP2A activity in H358 cells. Right, downregulation of CIP2A by siRNA increased the activity of PP2A in H460 cells. (C) Silencing PP2Ac reduced the apoptotic effect of afatinib in H358 cells. Cells were transfected with either control or PP2Ac siRNA for 48 h then exposed to 10 μM afatinib for 24 h. (D) Left, okadaic acid (OA), a PP2A inhibitor, increased p-AKT and inhibited the effects of afatinib on apoptosis of H358 cells. Right, forskolin, a PP2A agonist, sensitized resistant H460 cells to afatinib. Data are mean ± SD. n = 3 for each condition. **, p < 0.01, vs. no afatinib. (E) Ectopic expression of AKT (AKT-DDK) attenuated the effects of afatinib on apoptosis of H358 cells. H358 cells overexpressing AKT were treated with 10 μM afatinib for 24 h.
Mentions: To verify the role of CIP2A/PP2A/p-AKT in mediating afatinib-induced apoptosis in the EGFR wild-type sensitive NSCLC cells, two approaches were used. First, the ectopic expression of CIP2A was evaluated in the sensitive H358 cells (Figure 3A, left), which showed that CIP2A was overexpression and partially protected the cells from apoptotic cell death induced by afatinib, and that PP2A activity was decreased (Figure 3B, left). Further, knockdown of the gene expression of CIP2A increased the sensitivity of afatinib-induced apoptosis in the resistant H460 cells (Figure 3A, right) and increased PP2A activity (Figure 3B, right). This suggested that afatinib may repress CIP2A, enhance PP2A activity, and then induce cell death. Furthermore, in order to examine the relationship between PP2A and AKT (Figure 3C), we knocked down PP2Ac expression by siRNA strategy in the sensitive H358 cells. The results showed that AKT phosphorylation increased with the deprived of PP2A and also partially protected the cells from apoptotic death induced by afatinib. In addition, with the treatment of okadaic acid, a known PP2A inhibitor, AKT phosphorylation was significantly increased in the sensitive H358 cells which reduced the afatinib-induced apoptosis (Figure 3D, left), whereas co-treatment with forskolin sensitized resistant H460 cells to afatinib-induced apoptosis and p-AKT downregulation (Figure 3D, right). We next analyzed the role of AKT, which is downstream of PP2A, in mediating the effects of afatinib. The ectopic expression of AKT in the sensitive H358 cells (Figure 3E) partially protected the cells from apoptotic death. Taken together, these results suggest that the afatinib-induced apoptotic effect is through the CIP2A-PP2A-AKT pathway in NSCLC 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