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FOXO3 mutation predicting gefitinib-induced hepatotoxicity in NSCLC patients through regulation of autophagy



. 2022 Sep;12(9):3639-3649.


doi: 10.1016/j.apsb.2022.02.006.


Epub 2022 Feb 15.

Affiliations

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Shaoxing Guan et al.


Acta Pharm Sin B.


2022 Sep.

Abstract

Hepatotoxicity is a common side effect for patients treated with gefitinib, but the related pathogenesis is unclear and lacks effective predictor and management strategies. A multi-omics approach integrating pharmacometabolomics, pharmacokinetics and pharmacogenomics was employed in non-small cell lung cancer patients to identify the effective predictor for gefitinib-induced hepatotoxicity and explore optional therapy substitution. Here, we found that patients with rs4946935 AA, located in Forkhead Box O3 (FOXO3) which is a well-known autophagic regulator, had a higher risk of hepatotoxicity than those with the GA or GG variant (OR = 18.020, 95%CI = 2.473 to 459.1784, P = 0.018) in a gefitinib-concentration dependent pattern. Furthermore, functional experiments identified that rs4946935_A impaired the expression of FOXO3 by inhibiting the promotor activity, increasing the threshold of autophagy initiation and inhibiting the autophagic activity which contributed to gefitinib-induced liver injury. In contrast, erlotinib-induced liver injury was independent on the variant and expression levels of FOXO3. This study reveals that FOXO3 mutation, leading to autophagic imbalance, plays important role in gefitinib-induced hepatotoxicity, especially for patients with high concentration of gefitinib. In conclusion, FOXO3 mutation is an effective predictor and erlotinib might be an appropriately and well-tolerated treatment option for patients carrying rs4946935 AA.


Keywords:

Autophagy; FOXO3; Gefitinib; Hepatotoxicity; Pharmacogenomics; Pharmacokinetics; Pharmacometabolomic.

Figures



Graphical abstract


Figure 1


Figure 1

The concentration of gefitinib/metabolites was not associated with gefitinib-induced hepatotoxicity in the general subjects (n = 180). (A) The grades of hepatotoxicity induced by gefitinib in NSCLC patients; (B–F) Neither the concentration of gefitinib nor the metabolites was associated with hepatotoxicity in the general subjects (n = 180). ns: no significant.


Figure 2


Figure 2

G>A rs4946935 was associated with gefitinib-induced hepatotoxicity. (A) Among 194 SNPs, only G>A rs4711998 in IL17, C>T rs4795896 in CCL11, G>A rs4946935 in FOXO3 and G>A rs12722604 in IL2RA were associated with gefitinib-induced hepatotoxicity; (B) G>A rs4711998, located in IL17A, was associated with gefitinib-induced hepatotoxicity; (C) C>T rs4795896, located in CCL11, was associated with gefitinib-induced hepatotoxicity; (D) G>A rs4946935, located in FOXO3, was correlated with gefitinib-induced hepatotoxicity; (E) G>A rs1272260, located in IL2RA, was correlated with gefitinib-induced hepatotoxicity; (F) G>A rs4946935, located in FOXO3, was significantly associated with gefitinib-induced hepatotoxicity by multivariate logistic regression; (G) Plasma AST/ALT level was significantly correlated with the concentration of gefitinib in FOXO3 AA carriers. ∗P < 0.05; ∗∗P < 0.01.


Figure 3


Figure 3

rs4946935_A impaired the expression of FOXO3. (A) All tag SNPs of FOXO3 in HCB; (B) G>A rs4946935 was located in intron 3 of FOXO3 on Chromosome 6; (C) FOXO3 rs4946935 was correlated to expression levels of FOXO3 in human spleen and brain caudate according to GTEx database; (D) FOXO3 rs4946935 was correlated to expression levels of FOXO3 in NSCLC patients; (E) Luciferase activity of rs4946935_ A and _G of FOXO3 reporter vectors in LO2 cells; (F–H) Overexpression of FOXO3 significantly increased the IC50 of gefitinib while knockout of FOXO3 decreased in LO2 cells. Data represent mean ± SD of three or more independent experiments; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns: no significance.


Figure 4


Figure 4

gefitinib-induced hepatotoxicity was FOXO3-dependent by inhibiting autophagy. (A) The expression of FOXO3 was correlated with autophagy-related genes in liver tissue according to GTEx dataset; (B) FOXO3 directly regulates the expression of ATG3, ATG4A, ATG5, ATG7, ATG10, ATG12, ATG14, ATG16L1, and MAP1LC3B after treatment with gefitinib in LO2 cells; (C) Overexpression of FOXO3 decreased the threshold of autophagy initiation as indicated by LC3-II/I expression level; (D) Electron micrographs of FOXO3-knock out and -overexpression LO2 cells under incubation of gefitinib; (E) Inhibition of autophagy significantly suppressed proliferation of FOXO3 overexpression hepatocytes under incubation of gefitinib; (F) Inhibition of autophagy significantly increased the cytotoxicity of gefitinib in FOXO3 overexpression hepatocytes. Data represent mean ± SD of three or more independent experiments; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns: no significance.

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