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. 2016 Feb;22(2):194-201.
doi: 10.1038/nm.4032. Epub 2016 Jan 18.

Blocking c-Met-mediated PARP1 phosphorylation enhances anti-tumor effects of PARP inhibitors

Yi Du  1 , Hirohito Yamaguchi  1 , Yongkun Wei  1 , Jennifer L Hsu  1 , Hung-Ling Wang  2 , Yi-Hsin Hsu  1 , Wan-Chi Lin  1 , Wen-Hsuan Yu  1   3 , Paul G Leonard  4   5 , Gilbert R Lee 4th  4   5 , Mei-Kuang Chen  1   3 , Katsuya Nakai  1 , Ming-Chuan Hsu  1 , Chun-Te Chen  1 , Ye Sun  1 , Yun Wu  6 , Wei-Chao Chang  2   7 , Wen-Chien Huang  8 , Chien-Liang Liu  8 , Yuan-Ching Chang  8 , Chung-Hsuan Chen  7 , Morag Park  9 , Philip Jones  5 , Gabriel N Hortobagyi  10 , Mien-Chie Hung  1   2   3   11
Affiliations

Blocking c-Met-mediated PARP1 phosphorylation enhances anti-tumor effects of PARP inhibitors

Yi Du et al. Nat Med. 2016 Feb.

Erratum in

Abstract

Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapeutics for many diseases, including cancer, in clinical trials. One PARP inhibitor, olaparib (Lynparza, AstraZeneca), was recently approved by the FDA to treat ovarian cancer with mutations in BRCA genes. BRCA1 and BRCA2 have essential roles in repairing DNA double-strand breaks, and a deficiency of BRCA proteins sensitizes cancer cells to PARP inhibition. Here we show that the receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907 (PARP1 pTyr907 or pY907). PARP1 pY907 increases PARP1 enzymatic activity and reduces binding to a PARP inhibitor, thereby rendering cancer cells resistant to PARP inhibition VSports手机版. The combination of c-Met and PARP1 inhibitors synergized to suppress the growth of breast cancer cells in vitro and xenograft tumor models, and we observed similar synergistic effects in a lung cancer xenograft tumor model. These results suggest that the abundance of PARP1 pY907 may predict tumor resistance to PARP inhibitors, and that treatment with a combination of c-Met and PARP inhibitors may benefit patients whose tumors show high c-Met expression and who do not respond to PARP inhibition alone. .

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Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. ROS induces the association of c-Met and PARP1
(a) Human breast cancer tissue microarray was stained with 8-OHdG-specific antibody. Representative images of 216 non-TNBC and 90 TNBC cases are shown. Bar, 100 μm. (b) Human breast cancer cell lines shown in panel (e) were stained with 8-OHdG-specific antibody (see Supplementary Fig. 1a). Quantitation of 8-OHdG is shown. (c) Human breast cancer cell lines shown in panel (e) were subjected to ELISA assay to measure 8-OHdG abundance. (d) Human breast cancer cell lines shown in panel (e) were incubated with 10 μM of DCF-DA for 30 min. Quantitation of DCF is shown. (e) Western blot showing expression of PAR, PARP1, and tubulin in lysates of the indicated human breast cancer cell lines. Blots are representative of triplicate experiments. Right, band intensity of PAR normalized to tubulin. (f) MDA-MB-231 cells were treated with or without 20 μM sodium arsenite for 18 h. Left, endogenous PARP1 and c-Met association detected by immunoprecipitation (IP) and Western blot. Right, input control. (g) Detection of PARP1 and c-Met co-localization (green signals) in MDA-MB-231 cells treated with H2O2 or sodium arsenite (As), and in those cells not treated (control) by a Duolink assay. Bar, 20μm. Representative images and quantitation of PLA signals from 50 cells and three independent experiments are shown. (h) MDA-MB-231 cells with ectopic expression of HA-tagged PARP1 were treated with 20 μM H2O2 for 30 min with or without a one-hour pre-treatment with 2 μM c-Met inhibitor crizotinib. Left, IP and Western blot analysis of cytosolic and nuclear fractions with the indicated antibodies. The treatment-to-control ratio of c-Met/PARP1 interaction is shown below. Right, input control. TNBC, triple-negative breast cancer. LAR, luminal androgen receptor; As, sodium arsenite; L, long exposure; S, short exposure. Error bars represent s.d. *P < 0.05, t-test.
Figure 2
Figure 2. c-Met regulates resistance to PARP inhibitors
(a) Left, c-Met-knockdown cells were treated with the indicated concentrations of ABT-888 for 72 h and subjected to a cell viability assay. Right, Western blot showing c-Met expression in c-Met-knockdown MDA-MB-231 cells. (b) MDA-MB-231 cells were treated with the indicated concentrations of AG014699 and crizotinib or foretinib for 8 days and subjected to clonogenic cell survival assay. Quantitation of clonogenic cells from three independent experiments is shown. (c) Left, c-Met-knockdown MDA-MB-231 cells were treated with the indicated concentrations of AG014699 for 8 days and subjected to clonogenic cell survival assay. Quantitation of clonogenic cell from three independent experiments is shown. Right, Western blot showing c-Met expression in c-Met- knockdown cells at the 3′-UTR (shMet-C) and re-expression of wild-type (Wt) and kinase dead (KD) c-Met in MDA-MB-231 cells. (d) Western blot analysis of c-Met expression in MCF-7 cells. (e) MCF-7-c-Met and vector control cells were treated with the indicated concentrations of AG014699 for 8 days and subjected to clonogenic cell survival assay. Quantitation of clonogenic cells from three independent experiments is shown. (f) Median inhibitory concentration (IC50) of PARP inhibitors in BRAC1-mutant TNBC cells (MDA-MB-436 and HCC1937). Top, Western blot showing c-Met expression. (g) c-Met-knockdown HCC1937 cells were treated with the indicated concentrations of AG014699 for 72 h and subjected to cell viability assay. Top, Western blot showing c-Met expression in c-Met-knockdown HCC1937 cells. (h) MDA-MB-436 cells with ectopic expression of c-Met were treated with the indicated concentrations of AG014699 for 72 h and subjected to cell viability assay. Top, expression of wild-type (Wt) and kinase dead (KD) c-Met in MDA-MB-436 cells. (i) Western blot showing expression of c-Met in MDA-MB-231 and MDA-MB-157 cells. (j) BRCA1- and BRCA2-knockdown MDA-MB-157 cells were treated with the indicated concentrations of AG014699 for 72 h and subjected to cell viability assay. Right, Western blot showing c-Met expression in c-Met-knockdown MDA-MB-157 cells. Error bars represent s.d. Cri, crizotinib; Ft, foretinib; ABT, ABT-888; AG, AG014699; AZD, AZD2281. *P < 0.05, rANOVA.
Figure 3
Figure 3. c-Met mediates PARP1 functions through phosphorylation of PARP1 at Y907
(a) MDA-MB-231 cells were treated with ABT-888 (50 μM), foretinib (1 μM), or the combination for 18 h. Representative images and quantitation of γ-H2AX (green) from three independent experiments are shown. Bar, 20 μm. (b) c-Met- or control-knockdown MDA-MB-231 cells as well as c-Met-knockdown MDA-MB-231 cells re-expressing wild-type (Wt) or kinase dead (KD) mutant were treated with the indicated drugs for 18 h. Representative images and quantitation of γ-H2AX (green) from three independent experiments are shown. Bar, 20 μm. (c) HEK293T cells were transfected with V5-PARP1 and Flag-c-Met expression plasmids, and the cells were treated with 10 μM H2O2 for 15 min. PARP1 was immunoprecipitated with V5 antibody, followed by Western blotting with 4G10 (anti phosphor-tyrosine antibody). (d) Left, Western blot showing expression of PARP1 and tubulin in PARP1-knockdown (shRNA targeting 3′-UTR) MDA-MB-231 cells and PARP1-knockdown MDA-MB-231 cells re-expressing PARP1 wild-type or Y907 mutant. Center, DNA damage as measured by comet assay with pre-incubation with formanidopyrimidine DNA glycosylase (Fpg) in PARP1-wild-type-, PARP1-Y907E-, PARP1-Y907F-expressing MDA-MB-231 stable cells treated with 20 μM H2O2 for 30 min. Right, quantitation of intensity of damaged DNA from three independent experiments. Bar, 100 μm. (e) Western blot showing poly-ADP ribosylation as indicated by PAR in MDA-MB-231 stable cells described in (d) treated with or without 20 μM H2O2 for 30 min. (f) MDA-MB-231 cells were treated with or without 20 μM H2O2 for 30 min or H2O2 plus 2 μM crizotinib or 1 μM foretinib pre-treatment 1h. Cell lysates were subjected to Western blot analysis using the indicated antibodies. (g) Re-expression of wild-type or mutant PARP1 in PARP1-knockdown MDA-MB-231 cells with or without c-Met knockdown were treated with 50 μM ABT-888 for 18 h. γ-H2AX (green) was detected by immunofluorescence confocal microscopy. Bar, 20 μm. Representative images and quantitation of three independent experiments are shown. (h) Stable cells described in (g) were treated with the indicated concentrations of AG014699 and subjected to clonogenic formation assay for 8 days. Representative images and quantitation of clongenic cells from three independent experiments are shown. Bar, 10 mm. Error bars represent s.d. *P < 0.05, t-test. n.s., not significant. ABT, ABT-888; Cri, crizotinib; Ft, foretinib.
Figure 4
Figure 4. Clinical relevance and potential therapeutic strategy targeting PARP1 and c-Met in TNBC
(a) Representative images of immunohistochemical staining for pY907-PARP1 and c-Met in tissue microarrays of 77 cases of breast cancer (see Supplementary Table 3). (b) The synergistic effect of inhibiting c-Met and PARP in TNBC cell lines (MDA-MB-231 and HCC1937) was measured by cell viability assay following a 72-hour treatment. Fa, fraction affected. AG, AG014699; ABT, ABT-888; Cri, crizotinib; Ft, foretinib. (c) The synergistic effect of c-Met inhibitor crizotinib and PARP inhibitor AG014699 in MDA-MB-231 cells and HCC1937 cells was measured by soft agar assay following a 4-week treatment. (d) MDA-MB-231 cells were inoculated into the mammary fat pad of nude mice (10 mice per group) on day 0. When the tumor volume reached ~50 mm3, mice were orally administered crizotinib (5 mg/kg), AG014699 (5 mg/kg), or the combination five times per week for 21 days. Tumor volume was measured at the indicated time points. (e) MDA-MB-231 cells were inoculated into the mammary fat pad of nude mice (10 mice per group) on day 0. When the tumor volume reached ~50 mm3, mice were orally administered foretinib (5 mg/kg), ABT-888 (25 mg/kg), or the combination five times per week for 26 days. Tumor volume was measured at the indicated time points. (f) TUNEL, Ki67, and γ-H2AX staining of MDA-MB-231 xenograft tumor tissues after treatment. (g) The synergistic effect of c-Met and PARP inhibition on MCF-7/vector, MCF-7/c-Met wild-type, or MCF-7/c-Met KD cells was measured by cell viability assay following a 72-hour treatment. (h) MCF-7 cells with ectopic expression of c-Met were inoculated into the mammary fat pad of nude mice (10 mice per group) on day 0. When the tumor volume reached ~50 mm3, mice were orally administered crizotinib (5 mg/kg), AG014699 (5 mg/kg), or the combination five times per week for 21 days. Tumor volume was measured at the indicated time points. (i) H1993 cells were injected subcutaneously into the right flank of female nude mice (10 mice per group) on day 0. When the tumor volume reached ~50 mm3, mice were orally administered crizotinib (5 mg/kg), AG014699 (5 mg/kg), or the combination five times per week for 21 days. Tumor volume was measured at the indicated time points. Error bars represent s.d. *P < 0.05, t-test.
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