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Cancer Sci
2017 Jan 01;1081:53-60. doi: 10.1111/cas.13111.
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Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.
Taniguchi H
,
Takeuchi S
,
Fukuda K
,
Nakagawa T
,
Arai S
,
Nanjo S
,
Yamada T
,
Yamaguchi H
,
Mukae H
,
Yano S
.
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Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer.
Figure 1. Establishment of APE‐CR and #2 cell lines. (a) A925LPE3 cells were inoculated into the thoracic cavity of SCID mice. Mice were treated with crizotinib 50 mg/kg. Treatment was given daily on days 12–70. (upper). Cancer cells were collected from pleural effusions and cultured in vitro to establish the A925LPE‐CR (APE‐CR) cell line. Those cells were cloned via limiting dilution to also establish the #2 cell line. Luminescence was evaluated twice per week. Mean ± SE of total flux are shown. (photons/s/cm2/sr). (b) APE‐CR and #2 cell lines were resistant for crizotinib. A925LPE3, APE‐CR, and #2 cells (2 × 103 per well) were incubated with various concentrations of crizotinib, alectinib or ceritinib for 72 h. Cell viability was determined by MTT assay. The data shown represent the means ± SD of three independent experiments. (c) A925LPE3, APE‐CR, and #2 cells were treated with siRNAs specific for ALK (si‐ALK, respectively), or scrambled controls (SCR). (Left) Cell lysates were evaluated for protein expression by western blot 48 h later. Three independent experiments were performed, and a representative result is shown. (Right) Cell viability was determined by MTT assay 72 h later. Data represents the mean ± SD of three independent experiments. *P < 0.05 by Student's t‐test, si‐SCR versus si‐ALK.
Figure 2. The effect of combination therapy ALK‐TKI and EGFR‐TKI or anti‐EGFR antibody. (a) A925LPE3 and #2 cells (2 × 103 per well) were incubated with various concentrations of crizotinib with or without erlotinib (1 μM) or cetuximab (50 μg/mL) for 72 h. Cell viability was determined by MTT assay. The data shown represent the means ± SD of three independent experiments. (b) #2 cells (2 × 103 per well) were incubated with various concentrations of alectinib or ceritinib with or without erlotinib (1 μM) or cetuximab (50 μg/mL) for 72 h. Cell viability was determined by MTT assay. The data shown represent the means ± SD of three independent experiments. (c) A925LPE3 and #2 cells were treated with crizotinib (1 μM) and/or erlotinib (1 μM) or cetuximab (50 μg/mL) for 1 h. Cell lysates were evaluated for protein expression by western blot. Three independent experiments were performed, and a representative result is shown. (d) A925LPE3 and #2 cells were treated with siRNAs specific for EGFR (si‐EGFR, respectively), or SCR. (Left) #2 cells were treated with or without crizotinib (1 μM) for 1 h, following transfection with siRNA. Cell lysates were evaluated for protein expression by western blot. Three independent experiments were performed, and a representative result is shown. (Right) #2 cells were treated with crizotinib (1 μM) following transfection with si‐RNA. Cell viability was determined by MTT assay 72 h later. Data represents the mean ± SD. *P < 0.05 by Student's t‐test, the group treated with si‐SCR and crizotinib versus the group treated with si‐EGFR and crizotinib.
Figure 3. Increase of AREG caused the resistance for crizotinib. (a) Five EGFR ligands (AREG, β cellulin HB‐EGF, EGF, and TGF‐α) production by #2 cells. The cells were incubated in medium for 48 h and culture supernatants were harvested. The level of ligands in the supernatants was determined by ELISA. *P < 0.05 by Student t‐test, #2 versus A925LPE3. (b) #2 cells were treated with or without crizotinib (1 μM) for 72 h following transfection with siRNAs for EGFR or AREG (si‐EGFR and si‐AREG, respectively), or SCR. Cell viability was determined by MTT assay. The data shown represent the means ± SD of three independent experiments. *P < 0.05 by Student's t‐test, versus the group treated with si‐SCR and crizotinib. (c) AREG production by #2 cells were decreased after transfection with si‐AREG. The cells were incubated in medium for 48 h and culture supernatants were harvested. The level of ligand in the supernatants was determined by ELISA. Data represents the mean ± SD. *P < 0.05 by Student's t‐test, versus the group treated with si‐SCR.
Figure 4. Epidermal growth factor receptor (EGFR) inhibitor combined with crizotinib overcome resistance to crizotinib in vivo. (a) A925LPE3 cells were inoculated subcutaneously into SCID mice (n = 5, each group). Daily oral treatment with vehicle (control) or crizotinib (50 mg/kg) was given for 15 days. Mean ± SE tumor volumes are shown. *P < 0.05 by Mann–Whitney test, versus control group. (b) #2 cells were inoculated subcutaneously into SCID mice (n = 5, each group). And the mice were ramdomized into vehicle (control), erlotinib (25 mg/kg), cetuximab (1 mg/twice a week), crizotinib (50 mg/kg), crizotinib (50 mg/kg) + erlotinib (25 mg/kg), and crizotinib (50 mg/kg) + cetuximab (1 mg/twice a week) treatment groups. The treatment was given for 15 days. Tumor volume was measured using calipers on the indicated days. Mean ± SE tumor volumes are shown. *P < 0.05 by Mann–Whitney test, versus the group treated with crizotinib. (c) (d) A925LPE3 or #2 tumors were resected from the mice 3 h after administration. And the relative levels of proteins in the tumor lysates were determined by western blot analysis. (e) Quantification of proliferative cells, as determined by their Ki‐67‐positive proliferation index (percentage of Ki‐67‐positive cells). The data shown represent the means of five areas ± SD. *P < 0.05 by Student's t‐test, versus control group in A925LPE3, versus the group treated with crizotinib in #2. (f) Representative images of A925LPE3 and #2 tumors immunohistochemically stained with antibodies to human Ki‐67. Bar, 100 μm.
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