An in vivo C. elegans model system for screening EGFR-inhibiting anti-cancer drugs

Abstract

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.

Figure 1. The development of the human oncogenic EGFR induced Muv model.

(A) LET-23 and LET-23-based chimeric receptor constructs. All constructs were designed to express each chimeric receptor from the let-23 promoter. (B) The LET-23::hEGFR-TK chimera rescues the vulvaless phenotype of the let-23(sy1) mutant. (C) A comparison of several Muv mutants and the jgIs6 transgenic strain which expresses LET-23::hEGFR-TK[L858R]. C. elegans expressing LET-23::hEGFR-TK[L858R] exhibited a larger pseudovulva compared to let-23(sa62) and let-60(n1700) Muv mutants. (D) Hoechst33342 (H33342) staining of the pseudovulval region in jgIs6 revealed many nuclei. The boxed region of the lower panel is enlarged in the right panel. (E) Expression of a 1° cell marker, CDH-3::GFP in the wild-type worm and jgIs6. CDH-3::GFP is highly expressed both in the vulva and pseudovulva of jgIs6. (F) Expression of 2° vulval cell fate markers in the lin-15 Muv mutant and jgIs6. Reporter genes controlled by promoters of egl-17 and dhs-31 were expressed in the vulva and pseudovulva. Arrowheads indicate normal vulvae and small arrows indicate pseudovulvae. Scale bars, 50 µm.

Figure 2. The Muv phenotype of jgIs6 and jgIs25 reflects similar responses as human cancers have against the anti-cancer drugs, gefitinib and elrotinib.

(A) A high dose of gefitinib (40 µM) did not affect normal embryogenesis, larval growth, or vulval formation of the wild-type C. elegans. (B) Gefitinib inhibited the Muv phenotype of jgIs6 which expresses LET-23::hEGFR-TK[L858R]. (C) Gefitinib inhibited the Muv phenotype of jgIs6 in a dose dependent manner, but did not inhibit that of jgIs25, which expresses LET-23::hEGFR-TK[T790M-L858R]. Numbers of worms counted (n) were 159, 96, 130, 85, 87, 125, 108 and 88 from left along the X-axis. (D) Erlotinib produced a similar effect as gefitinib in both jgIs6 and jgIs25 models. (n = 159, 96, 67, 110, 80, 106, 95 and 176). (E) Determination of the developmental stage of jgIs6 that is most responsive to gefitinib. As in normal vulval development, early larvae (L1–L3) responded well to gefitinib. (n = 128, 224, 134, 116, 139, 167, 99 and 66). Scale bars, 50 µm (A, B). X-axis, concentration of gefitinib (C), erlotinib (D) and worm stage of gefitinib treatment (E). Y-axis, % of worms showing the Muv phenotype (C–E). * P<0.001.

Figure 3. A pilot screen of 1,280 chemicals for EGFR-TK inhibitors using jgIs6.

(A) Screening method, including synchronization of C. elegans and liquid culture using the 96-well plate for inhibitor screen. (B) Chemical structures of gefitinib, AG1478 and U0126. (C) Both AG1478 and U0126 inhibit the Muv phenotype of jgIs6 in a dose dependent manner. (n = 295, 193, 381, 133, 254, 304 and 415 from left along the X-axis). (D) Effect of gefitinib, erlotinib, AG1478 and U0126 on jgIs25. Gefitinib, erlotinib, and AG1478 did not inhibit the Muv phenotype of jgIs25, but U0126 inhibited. (n = 81, 99, 106, 90 and 81). Chemical concentration, 5 µM. * P<0.001.

Figure 4. MEK inhibitors rescue the gefitinib-resistant Muv phenotype of jgIs25.

(A) U0126, PD0325901 (MEK inhibitor), AZD6244 (RAF[V600E] inhibitor) and WZ4002 (EGFR[T790M] inhibitor) were added to jgIs6. (n = 86, 94, 116, 64, 88, 66, 79, 110 and 98 from left along the X-axis). (B) Chemicals were added to gefitinib resistant jgIs25. MEK inhibitors, U0126 and PD0325901, inhibited the Muv phenotype of jgIs25, but the other chemicals did not. (n = 64, 100, 128, 113, 89, 64, 63, 79 and 98). (C) Excessive doses (100 µM) of chemicals were added to jgIs6 and jgIs25. Two MEK inhibitors perfectly inhibited the Muv phenotype of jgIs6 and jgIs25. WZ4002 inhibited the Muv phenotype of jgIs25 better than that of jgIs6. (n = 160, 213, 186, 312, 195, 323, 192 and 237). (D) The Muv phenotype of lin-15 was suppressed by U0126 and PD0325901; chemical concentration, 100 µM (X-axis). (n = 119, 89, 90 and 143). * P<0.001.

Figure 5. The Muv inhibitors suppressed the slow growth phenotype of jgIs6 and jgIs25.

(A) Ratios of adults 3 days after placing embryos on plate. All wild-type strains were adults, but most jgIs6 and jgIs25 transgenic strains were younger than the L4 stage. Five adults were transferred to each plate and were removed after 6 hours of egg-laying. Three days after removing P0 worms, F1 worms were observed. Four plates were counted for each strain. (n = 488, 313, 104 and 94 from left along the X-axis). * P<0.001 and ** P<0.05. (B) U0126 and PD0325901 suppressed the slow growth phenotype of jgIs6 and jgIs25. Gefitinib and AG1478 suppressed the slow growth phenotype of jgIs6, but not jgIs25. L1 stage larvae were incubated with each chemical for 4 days in 96-well plates, and they were recovered on a fresh plate for 6 hours before taking pictures. C. elegans grew slowly when cultured in 96-well plates. Control (0.5% DMSO) and chemical concentration (50 µM). Scale bar, 500 µm.