Secreted human glycyl-tRNA synthetase implicated in defense against ERK-activated tumorigenesis

Abstract

Although adaptive systems of immunity against tumor initiation and destruction are well investigated, less understood is the role, if any, of endogenous factors that have conventional functions. Here we show that glycyl-tRNA synthetase (GRS), an essential component of the translation apparatus, circulates in serum and can be secreted from macrophages in response to Fas ligand that is released from tumor cells. Through cadherin (CDH)6 (K-cadherin), GRS bound to different ERK-activated tumor cells, and released phosphatase 2A (PP2A) from CDH6. The activated PP2A then suppressed ERK signaling through dephosphorylation of ERK and induced apoptosis. These activities were inhibited by blocking GRS with a soluble fragment of CDH6. With in vivo administration of GRS, growth of tumors with a high level of CDH6 and ERK activation were strongly suppressed. Our results implicate a conventional cytoplasmic enzyme in translation as an intrinsic component of the defense against ERK-activated tumor formation.

Fig. 1.

GRS secretion can be induced by a Fas ligand signal derived from cancer cells. (A) H460 cancer cells were first seeded (0.25 × 10⁶ cells/well). After 12 h, the medium was changed with serum-free medium and the cells were cocultured with U937 monocytes (0.125 × 10⁶– ∼1 × 10⁶ cells per well) for 6 h. Then, secretion of GRS in the cultured medium was determined by TCA precipitation of proteins followed by SDS/PAGE and Western blot analysis. (B) Cocultivation of HCT116 cancer cells with U937 cells and determination of GRS secretion were determined as stated above. (C) Bone-marrow–derived macrophage (BMDM) and H460 cells were coincubated and GRS secretion was monitored. (D) The cultured medium of H460 cells was harvested after 12 h incubation and used as the conditioned medium (CM) for RAW 264.7 and U937 cells for 6 h. The secretion of GRS was determined as above. (E) H460 cells were incubated in a Transwell chamber with or without U937 cells in the insert for 6 h in serum-free medium and the secretion of GRS was analyzed. (F) U937 cells were treated with activating anti-Fas antibody (CH11 clone, 5 μg/mL) at the indicated times and the secretion of GRS was determined. (G) Neutralizing anti-Fas antibody (ZB4 clone, 0.2 μg/mL) was added to the medium of the coculture system and GRS secretion was determined as above. Molecular mass of examined proteins: GRS, 79 kDa; KRS, 72 kDa; YRS, 51 kDa; WRS, 53 kDa; and tubulin, 50 kDa.

Fig. 2.

Secreted GRS induces apoptosis in several different tumor cells. (A) Dose-dependent binding of biotinylated GRS was determined with MCF-7, HeLa, HCT116, and RAW 264.7 cell lines by capture with streptavidin-HRP. (B) RAW 264.7 and HCT116 cells were treated with the indicated concentrations of recombinant human GRS for 24 h and the cell viability was measured by the MTT assay. Adriamycin (2 μg/mL) was used as a positive control to induce cell death. (C) The effect of GRS on cell death was also monitored by sub-G1 cells, using flow cytometry. GRS (150 nM) was boiled to see whether it can inactivate the cytokine activity. (D) GRS-induced cell death was also monitored by the generation of active caspase 3. (E) Using a Transwell, U937 cells were incubated in the insert with or without HCT116 in the chamber (for 24 h) and cell viability was analyzed as above. To neutralize the effect of GRS, anti-GRS antibody was added to the culture dish. (F) Nine different cell lines were treated with the indicated concentrations of GRS and the effect on cell viability was determined by the MTT assay. Error bars give the mean ± SD from the average of three experiments. Molecular mass of examined proteins: procaspase 3, 32 kDa; and active-caspase 3, 17 kDa.

Fig. 3.

Identification of CDH6 as a potential receptor for GRS. (A) A panel of Fc-fused soluble cadherin family proteins was bound to His-tag–fused GRS or to BSA-coated plates, and complexes were detected with the IgG₁ Fc-HRP reagent. (B) To confirm the binding between cadherins and GRS, Fc-CDH2, -6, and -18 were incubated with His-GRS. Fc-CDHs were precipitated with protein A/G agarose and coprecipitated GRS was determined by immunoblotting. (C) To calculate the equilibrium dissociation constant (K_d), immobilized Fc-fused (to gold chips) cadherins were incubated with GRS (31.25 nM– ∼1000 nM). Binding of GRS to CDH6 was determined by surface plasmon resonance and expressed as resonance units (RU). (D) Five different cancer cells were determined for the expression of CDH6 by immunoblotting. (E) HCT116 cells were transfected with nonspecific siRNA controls or two differently designed siRNAs targeting CDH6 and treated with biotinylated GRS (15 nM), and the binding of GRS was analyzed by immunoblotting. (F) HCT116 cells were transfected with si-con and si-CDH6 as described above, and cell binding of GRS was measured by FACS analysis. (G) HCT116 cells were incubated with His-GRS (150 nM). To neutralize the effects of GRS, Fc-fused CDH6 (300 nM) was added and the cell viability was determined by the MTT assay. Error bars represent SD. Molecular mass of examined proteins: CDH6, 90 kDa; Fc-CDH2, 80 kDa; Fc-CDH6, 80 kDa; and Fc-CDH18, 78 kDa.

Fig. 4.

Secreted GRS induces apoptosis of hyperphosphorylated ERK cancer cells. (A) HCT116 cells were treated with the indicated concentrations of GRS for 1 h and the phosphorylation of three different MAPKs was determined by their specific antibodies. (B) Five different cancer cell lines were investigated for the phosphorylation of ERK by immunoblotting. (C) HEK293 cells expressing each of three different active Ras transfectants were treated with GRS (150 nM) and its effect on the phosphorylation of ERK was determined as above. (D) Susceptibility of different cancer cell lines to GRS-induced cell death was determined by the MTT assay. Error bars give the mean ± SD from the average of three experiments. (E) HCT116 cells were treated with one of the inhibitors for PP2A, PP2B, PTP CD45, or PTP B1 for 15 min and incubated in the absence or presence of GRS (150 nM) for 1 h and phosphorylation of ERK was then determined. The bands of p-ERK and total ERK were quantified and the ratios of p-ERK to total ERK are shown. (F) The GRS effect on the interaction of CDH6 and PP2A was determined by coimmunoprecipitation in HCT116 cells. (G) HCT 116 cells were treated with GRS and its effect on the interaction of PP2A and ERK was determined by coimmunoprecpitation. Molecular mass of examined proteins: p-ERK, 42/44 kDa; ERK, 42/44 kDa; p-p38, 38 kDa; p38, 38 kDa; p-JNK, 46 kDa; JNK, 46 kDa; Flag-RASs, 22 kDa; and PP2A, 65 kDa.

Fig. 5.

Secreted GRS induces cancer cell death in vivo. (A) HCT116 cells were s.c. injected into the BALB/c nude mice and grown for 9 d. GRS (10 and 20 μg per dose) or PBS vehicle was intratumor injected (n = 5 animals per group). Tumor volume was calculated as the longest diameter × the shortest diameter² × 0.52. (B) Photograph of two representative HCT116 xenograft tumor mice from the control (Right) and treated (Left, 20 μg GRS) groups 12 d after the treatment. (C) Tumor weight was measured on the same day. (D) The OCT compound-embedded tissues were used for immunofluorescence staining. The tissues were treated with Yo-pro (green) and analyzed by immunofluorescence microscopy. Nuclei were stained with DAPI (blue). SN12 (E) and RENCA (F) cells were incubated with indicated concentrations of biotin-GRS (B-GRS) and the cell binding was determined by immunoblotting. SN12 (G) and RENCA (H) cells were s.c. injected into the BALB/c nude mice and grown for 5 d. GRS (2 and 6 mg/kg per dose) or PBS vehicle was i.p. injected daily for 4 d (n = 5 animals per group). Tumor volume was measured. (I) Tumor weights of the control and treated mice were determined and are shown as a bar graph. (J) The phosphorylation of ERK in SN12 xenograft tissues from the control and treated mice (6 mg/kg GRS) was determined by immunoblotting. Error bars give the mean ± SD.

Fig. P1.

Schematic representation for the antitumor activity of secreted GRS molecules in the tumor–macrophage microenvironment. GRS is secreted from immune cells called macrophages by molecules like the Fas ligand that is released from tumor cells. The GRS molecule subsequently binds to a CDH6 (K-cadherin) molecule that is expressed in tumor cells. This binding of GRS releases phosphatase 2A (PP2A) molecules from CDH6, leading to modification of activated ERK, which in turn results in targeted tumor-cell death.