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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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In vivo effects of afatinib on xenografts mice(A)(a) Left, effects of afatinib on tumor size in H358 xenografts. Afatinib inhibited the growth of H358 tumors by > 80%. Right, effects on body weight of H358 xenograft mice. (b) Left, effects of afatinib on tumor size in H460 xenograft mice. Right, effects on body weight of H460 xenograft mice. Mice were treated with vehicle, or oral afatinib at 20 mg/kg daily for 3 weeks (n = 8 each). Data are mean ± SD; *, p < 0.05. (B) Western blot analysis of CIP2A, p-AKT and AKT in H358 (Left) and H460 (Right) tumors. (C) The activities of PP2A in H358 (Left) and H460 (Right) tumors. mean ± SD, n = 8 for each condition.**p <0.01. (D) Representative immunohistochemical patterns of CIP2A and p-AKT in xenografts tumors.
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Figure 5: In vivo effects of afatinib on xenografts mice(A)(a) Left, effects of afatinib on tumor size in H358 xenografts. Afatinib inhibited the growth of H358 tumors by > 80%. Right, effects on body weight of H358 xenograft mice. (b) Left, effects of afatinib on tumor size in H460 xenograft mice. Right, effects on body weight of H460 xenograft mice. Mice were treated with vehicle, or oral afatinib at 20 mg/kg daily for 3 weeks (n = 8 each). Data are mean ± SD; *, p < 0.05. (B) Western blot analysis of CIP2A, p-AKT and AKT in H358 (Left) and H460 (Right) tumors. (C) The activities of PP2A in H358 (Left) and H460 (Right) tumors. mean ± SD, n = 8 for each condition.**p <0.01. (D) Representative immunohistochemical patterns of CIP2A and p-AKT in xenografts tumors.

Mentions: To determine whether or not the in vitro effects of afatinib on the sensitive H358 cells and the resistant H460 cells could be reproduced in vivo, mice were implanted with H358 and H460 xenografts. No apparent differences in body weight or toxicity were found (Figure 5A). Treatment with afatinib significantly inhibited H358 xenografts tumor growth by nearly 80% compared to the controls (Figure 5A-a, left), but not in the H460 xenografts (Figure 5A-b left). To correlate the clinical implications in NSCLC with the mechanism identified in vitro, the effects of afatinib on the CIP2A-PP2A-AKT pathway in these tumor were examined by Western blot and PP2A activity assay. Overall, there were significant decreases in CIP2A, p-AKT and Elk-1(Figure 5B) and an enhanced PP2A activity (Figure 5C) in the H358 tumors treated with afatinib (Figure 5B, 5C left), whereas no significant changes were observed in the control (vehicle) or H460 tumors (Figure 5B, 5C right). Immunohistochemical analysis of the tumor specimens demonstrated strong cytoplasmic staining of CIP2A and p-AKT in the vehicles of H358 and H460 cells (Figure 5D). This staining revealed weaker expression under afatinib treatment in the H358 cells (Figure 5D left), but not in the afatinib-resistant H460 cells (Figure 5D right). Taken together, these results confirmed that afatinib increased PP2A activity to repress p-AKT via CIP2A to inhibit tumor growth in this NSCLC xenografts model.


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)

In vivo effects of afatinib on xenografts mice(A)(a) Left, effects of afatinib on tumor size in H358 xenografts. Afatinib inhibited the growth of H358 tumors by > 80%. Right, effects on body weight of H358 xenograft mice. (b) Left, effects of afatinib on tumor size in H460 xenograft mice. Right, effects on body weight of H460 xenograft mice. Mice were treated with vehicle, or oral afatinib at 20 mg/kg daily for 3 weeks (n = 8 each). Data are mean ± SD; *, p < 0.05. (B) Western blot analysis of CIP2A, p-AKT and AKT in H358 (Left) and H460 (Right) tumors. (C) The activities of PP2A in H358 (Left) and H460 (Right) tumors. mean ± SD, n = 8 for each condition.**p <0.01. (D) Representative immunohistochemical patterns of CIP2A and p-AKT in xenografts tumors.
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Figure 5: In vivo effects of afatinib on xenografts mice(A)(a) Left, effects of afatinib on tumor size in H358 xenografts. Afatinib inhibited the growth of H358 tumors by > 80%. Right, effects on body weight of H358 xenograft mice. (b) Left, effects of afatinib on tumor size in H460 xenograft mice. Right, effects on body weight of H460 xenograft mice. Mice were treated with vehicle, or oral afatinib at 20 mg/kg daily for 3 weeks (n = 8 each). Data are mean ± SD; *, p < 0.05. (B) Western blot analysis of CIP2A, p-AKT and AKT in H358 (Left) and H460 (Right) tumors. (C) The activities of PP2A in H358 (Left) and H460 (Right) tumors. mean ± SD, n = 8 for each condition.**p <0.01. (D) Representative immunohistochemical patterns of CIP2A and p-AKT in xenografts tumors.
Mentions: To determine whether or not the in vitro effects of afatinib on the sensitive H358 cells and the resistant H460 cells could be reproduced in vivo, mice were implanted with H358 and H460 xenografts. No apparent differences in body weight or toxicity were found (Figure 5A). Treatment with afatinib significantly inhibited H358 xenografts tumor growth by nearly 80% compared to the controls (Figure 5A-a, left), but not in the H460 xenografts (Figure 5A-b left). To correlate the clinical implications in NSCLC with the mechanism identified in vitro, the effects of afatinib on the CIP2A-PP2A-AKT pathway in these tumor were examined by Western blot and PP2A activity assay. Overall, there were significant decreases in CIP2A, p-AKT and Elk-1(Figure 5B) and an enhanced PP2A activity (Figure 5C) in the H358 tumors treated with afatinib (Figure 5B, 5C left), whereas no significant changes were observed in the control (vehicle) or H460 tumors (Figure 5B, 5C right). Immunohistochemical analysis of the tumor specimens demonstrated strong cytoplasmic staining of CIP2A and p-AKT in the vehicles of H358 and H460 cells (Figure 5D). This staining revealed weaker expression under afatinib treatment in the H358 cells (Figure 5D left), but not in the afatinib-resistant H460 cells (Figure 5D right). Taken together, these results confirmed that afatinib increased PP2A activity to repress p-AKT via CIP2A to inhibit tumor growth in this NSCLC xenografts model.

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