Niclosamide overcomes acquired resistance to erlotinib through suppression of STAT3 in non-small cell lung cancer

Abstract

The emergence of resistance to EGF receptor (EGFR) inhibitor therapy is a major clinical problem for patients with non-small cell lung cancer (NSCLC). The mechanisms underlying tumor resistance to inhibitors of the kinase activity of EGFR are not fully understood. Here, we found that inhibition of EGFR by erlotinib induces STAT3 phosphorylation at Tyr705 in association with increased Bcl2/Bcl-XL at both mRNA and protein levels in various human lung cancer cells. PTPMeg2 is a physiologic STAT3 phosphatase that can directly dephosphorylate STAT3 at the Tyr705 site. Intriguingly, treatment of cells with erlotinib results in downregulation of PTPMeg2 without activation of STAT3 kinases [i.e., Janus-activated kinase (JAK2) or c-Src], suggesting that erlotinib-enhanced phosphorylation of STAT3 may occur, at least in part, from suppression of PTPMeg2 expression. Because elevated levels of phosphorylated STAT3 (pSTAT3), Bcl2, and Bcl-XL were observed in erlotinib-resistant lung cancer (HCC827/ER) cells as compared with erlotinib-sensitive parental HCC827 cells, we postulate that the erlotinib-activated STAT3/Bcl2/Bcl-XL survival pathway may contribute to acquired resistance to erlotinib. Both blockage of Tyr705 phosphorylation of STAT3 by niclosamide and depletion of STAT3 by RNA interference in HCC827/ER cells reverse erlotinib resistance. Niclosamide in combination with erlotinib potently represses erlotinib-resistant lung cancer xenografts in association with increased apoptosis in tumor tissues, suggesting that niclosamide can restore sensitivity to erlotinib. These findings uncover a novel mechanism of erlotinib resistance and provide a novel approach to overcome resistance by blocking the STAT3/Bcl2/Bcl-XL survival signaling pathway in human lung cancer.

Figure 1

Inhibition of EGFR by erlotinib downregulates PTPMeg2 in association with activation of the STAT3/Bcl2/Bcl-XL pathway in lung cancer HCC827 cells. A, HCC827 cells were treated with increasing concentrations of erlotinib (Erlo) for 48h. pEGFR, PTPMeg2, pSTAT3, Bcl2, Bcl-XL, etc., were analyzed by Western blot. B, HCC827 cells were treated with erlotinib (0.1 μM) for various times. Levels of Bcl2 or Bcl-XL mRNA were analyzed by RT-PCR.

Figure 2

Depletion of PTPMeg2 from HCC827 cells leads to upregulation of pSTAT3 and Bcl2/Bcl-XL. A, PTPMeg2 shRNA or control shRNA was transfected into HCC827 cells. Expression levels of pSTAT3, Bcl-XL, Bcl2 and Mcl-1 were analyzed by Western blot. B, HCC827 cells expressing PTPMeg2 shRNA or control shRNA were treated with increasing concentrations of erlotinib for 48h. Cell growth was determined by SRB assays. Data are mean ± SD from three independent experiments.

Figure 3

Decreased PTPMeg2 and increased levels of pSTAT3 and Bcl2/Bcl-XL are associated with erlotinib resistance in human lung cancer cells. A, HCC827 cells and acquired erlotinib resistant HCC827/ER cells were treated with increasing concentrations of erlotinib (Erlo) for 48h. Cell growth was analyzed by SRB assay. B, HCC827 and HCC827/ER cells were treated with erlotinib (Erlo, 1 μM) and colony formation assay was performed as described in “Methods”. C, pSTAT3, PTPMeg2, Bcl2, Bcl-XL, etc., were analyzed by Western Blot. D, mRNA levels of Bcl2, Bcl-XL and Mcl-1 were analyzed by RT-PCR.

Figure 4

Treatment of cells with niclosamide blocks erlotinib-induced activation of STAT3/Bcl2/Bcl-XL and reverses erlotinib resistance. A, HCC827 and HCC827/ER cells were treated with erlotinib (Erlo, 0.1 μM) in the absence or presence of increasing concentrations of niclosamide (Niclo) for 48h. pSTAT3, Bcl2, Bcl-XL and Mcl-1 were analyzed by Western Blot. B, HCC827 and HCC827/ER cells were treated with various concentrations of erlotinib (Erlo), niclosamide (Niclo, 0.5 μM) or their combination for 48 h. Cell growth was evaluated by SRB assay.

Figure 5

Specific depletion of STAT3 reverses erlotinib resistance. A, STAT3 shRNA or control shRNA was transfected into HCC827 cells. Expression levels of STAT3, Bcl-XL and Bcl2 were analyzed by Western blot. B, HCC827 cells expressing STAT3 shRNA or control shRNA were treated with erlotinib (Erlo) for 48h. Cell growth was determined by SRB assays.

Figure 6

Combination of erlotinib and niclosamide overcomes acquired erlotinib resistance in vivo. A, Mice bearing HCC827 or HCC827/ER lung cancer xenografts were treated with vehicle control, erlotinib (Erlo, 40mg/kg/d), niclosamide (Niclo, 20mg/kg/d) or their combination for 32 days. Each group includes 8 mice. Tumor volume was measured once every 2 days. After 32 days, the mice were sacrificed and the tumors were removed and analyzed. Representative tumor pictures were taken. B, Active caspase 3 was analyzed in tumor tissues at the end of experiments by IHC staining and quantified as described in “Methods”. C, Expression levels of pEGFR, pSTAT3, Bcl2 and Bcl-XL from tumor tissues in various treatment groups were analyzed by Western blot.

Figure 7

QD-IHF analysis of pEGFR and pSTAT3 in tumor tissues. A and B, HCC827 or HCC827/ER xenografts were treated with vehicle control, erlotinib (Erlo, 40mg/kg/d), niclosamide (Niclo, 20mg/kg/d) or their combination for 32 days. pEGFR and pSTAT3 were analyzed in tumor tissues at the end of experiments by QD-IHF and quantified as described in “Methods”.