ATR‐dependent ubiquitin‐specific protease 20 phosphorylation confers oxaliplatin and ferroptosis resistance

Abstract Oxaliplatin (OXA) resistance is a major clinic challenge in hepatocellular carcinoma (HCC). Ferroptosis is a kind of iron‐dependent cell death. Triggering ferroptosis is considered to restore sensitivity to chemotherapy. In the present study, we found that USP20 was overexpressed in OXA‐resistant HCC cells. High expression of USP20 in HCC was associated with poor prognosis. USP20 contributes OXA resistance and suppress ferroptosis in HCC. Pharmacological inhibition or knockdown of USP20 triggered ferroptosis and increased the sensitivity of HCC cells to OXA both in vitro and in vivo. Coimmunoprecipitation results revealed that the UCH domain of USP20 interacted with the N terminal of SLC7A11. USP20 stabilized SLC7A11 via removing K48‐linked polyubiquitination of SLC7A11 protein at K30 and K37. Most importantly, DNA damage‐induced ATR activation was required for Ser132 and Ser368 phosphorylation of USP20. USP20 phosphorylation at Ser132 and Ser368 enhanced its stability and thus conferred OXA and ferroptosis resistance of HCC cells. Our study reveals a previously undiscovered association between OXA and ferroptosis and provides new insight into mechanisms regarding how DNA damage therapies always lead to therapeutic resistance. Therefore, targeting USP20 may mitigate the development of drug resistance and promote ferroptosis of HCC in patients receiving chemotherapy.

The relative cell viability of primary HCC cells treated with the indicated dosage of erastin for 24h.(Each group contained 3 replicates).(F).Crystal violet staining of primary HCC cells treated with erastin.(G).CCK8 assay showing the response of primary HCC cells to erastin (20 μM)±ferrostatin (1 μM) for 24h.(H-J).Lipid ROS (H), GSH levels (I) and ferrous iron levels (J) were measured in primary HCC cells.(K).
Sphere formation assay of primary HCC cells.In vivo xenografts generated from primary HCC cells expressing an empty vector, USP20-WT, or USP20 C154A and treated with OXA.1×10 6 primary HCC cells were injected to the right dorsal flank of each mouse (n=6).After the tumors reached approximately 50 mm 3 , the mice were treated with OXA (5 mg/kg, twice a week).
Tumor sizes were measured every 5 days until the end of the experiment.(E).The relative cell viability of primary HCC cells treated with the indicated dosage of erastin.
(Each group contained 3 replicates).(F).Crystal violet staining of primary HCC cells treated with erastin.(G-I).Lipid ROS (G), GSH levels (H) and ferrous iron levels (I) were measured in primary HCC cells.(J).Sphere formation assay of primary HCC cells.

Figure S2 .
Figure S2.USP20 regulates the OXA resistance, ferroptosis and stem-like properties of primary HCC cells.(A).The survival percentage of primary HCC cells treated with increasing concentration of OXA for 48h.

Figure S5 .
Figure S5.USP20 enhances tumorigenic capacity of HCC stem cells.(A, B).Sphere formation assay of USP20-knockdown cells and control cells.(C, D).Depletion of USP20 decreased the expression of pluripotent factors in HCC cells as assessed by quantitative real-time PCR.(E, F).Tumorigenic cell frequency in USP20knockdown cells and control cells was determined with limiting dilution assays.Results shown are representative of 3 independent experiments.Data are represented as mean ± SD of biological triplicates.*, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

Figure S6 .
Figure S6.The deubiquitylation activity site of USP20 is necessary to regulate the OXA resistance, ferroptosis and stem-like properties in primary HCC cells.(A).The survival percentage of primary HCC cells treated with increasing concentration of OXA.(Each group contained 3 replicates).(B).Crystal violet staining of primary HCC cells treated with OXA.(C).Calcein/PI staining of primary HCC cells treated with OXA.(D).In vivo xenografts generated from primary HCC cells expressing an empty

Figure S7 .
Figure S7.ATR phosphorylates USP20.(A).Mass spectrometry analysis revealed lysine residue S132 and S368 of USP20 were phosphorylated in Huh-7 cells.(B).Mass spectrometry assay of USP20-associated proteins in Huh-7 cells was performed, and the specific interactive information between USP20 and ATR was shown.(C).Coimmunoprecipitation analysis of USP20 serine phosphorylation in Huh-7 cells that