USP33 Regulates DNA Damage Response and Carcinogenesis Through Deubiquitylating and Stabilising p53

Abstract

The de-ubiquitinase USP33 has been shown to possess either tumour-promoting or inhibitory effect on human cancer cells. However, all these findings are mainly based on in vitro cell culture models, and the in vivo evidence, which is more plausible to digest the functional role of USP33 in carcinogenic process, is still lacking. Here, we demonstrate that USP33 modulates DNA damage responses including cell cycle arrest and apoptosis induction through associating with p53. It directly interacts with p53 to mediate its de-ubiquitination and further stabilisation under DNA damage condition. Depletion of USP33 induces an enhanced level of p53 ubiquitination, which de-stabilises p53 protein leading to impaired DNA damage responses. Furthermore, USP33 silencing shows either promoted or inhibited effect on cell proliferation in human cancer cells with p53 WT and mutant background, respectively. Consistently, mice with hepatocyte-specific USP33 knockout are more sensitive to nitrosodiethylamine (DEN)-induced hepatocarcinogenesis compared to wild type mice. Thus, our in vitro and in vivo evidences illustrate that USP33 possesses anti-tumour activity via regulating p53 stability and activity.

FIGURE 1

USP33 interacts with p53. (A) Flag‐p53 interacts with endogenous USP33. (B) GFP‐USP33 interacts with endogenous p53. (C) Endogenous association between USP33 and p53. (D) USP33 interacts with p53 in vitro. (E) USP33 interacts with the N and M domain of p53.

FIGURE 2

USP33 deubiquitinates p53 and regulates its stability. (A) USP33 deubiquitinates Flag‐p53 in cells. (B) USP33 deubiquitinates p53 in cells. (C) USP33 deubiquitinates p53 in vitro. (D) USP33 reduced K48 ubiquitination level of p53. (E) Overexpression of USP33 enhances p53 protein level in cells. (F) Knocking down of USP33 decreases p53 protein level in cells. (G) Knocking down of USP33 decreases p53 stability. (H) Knocking down of USP33 decreases p53 stability can be rescued by overexpression of USP33. Data were showed as mean ± standard error of mean (SEM) from at least three independent experiments. Statistical analysis by one‐way or two‐way ANOVA. *p ≤ 0.05, **p ≤ 0.01.

FIGURE 3

USP33 regulates p53‐dependent DNA damage response. (A) DNA damage enhances p53 protein level in cells. (B) DNA damage increases the interaction between GFP‐USP33 and p53. (C) The interaction between endogenous USP33 and p53 was enhanced by CPT. (D) Knocking down of USP33 abolished the decreased of K48 ubiquitination of p53 induced by CPT. (E) Knocking down of USP33 inhibits the increases of p53 induced by DNA damage. (F) Knocking down of USP33 inhibits the increases of p21 induced by DNA damage. (G) Knocking down of USP33 inhibits cell cycle arrest. (H) Knocking down of USP33 inhibits apoptosis. (I) USP33 regulates apoptosis depend on p53. Data were showed as mean ± standard error of mean (SEM) from at least three independent experiments. Statistical analysis by one‐way or two‐way ANOVA. *p ≤ 0.05, **p ≤ 0.01.

FIGURE 4

USP33 regulates cell proliferation and DEN‐induced hepatocarcinogenesis. (A) Knocking down of USP33 by shUSP33‐1 + shUSP33‐2 enhances cell proliferation in BEL7402 cells. (B) Comparison of subcutaneous tumours induced in nude mice by subcutaneous injection with BEL7402 cells from two groups. (C) Tumour weight in nude mice injected with BEL7402 cells from two groups. (D) Knocking down of USP33 inhibits cell proliferation in PLC/PRF/5 cells. (E) Comparison of subcutaneous tumours induced in nude mice by subcutaneous injection with PLC/PRF/5 cells from three groups. (F) Tumour weight in nude mice injected with PLC/PRF/5 cells from three groups. (G) hepatocyte‐specific USP33 knockout decreases p53 level in liver tissues. (H) Number of visible liver tumours of liver tumours. (I) Representative images of liver tumours in the USP33 WT, Heterozygous (H) and HKO mice after DEN injection. (J) Representative images of H&E stained liver. Data were showed as mean ± standard error of mean (SEM) from at least three independent experiments. Statistical analysis by t‐test, one‐way or two‐way ANOVA. *p ≤ 0.05, **p ≤ 0.01.

FIGURE 5

A mechanism of regulation of USP33‐coupled p53 ubiquitination and activation in DNA damage response. USP33 deubiquitinates p53 and regulates its stability, and moreover, DNA damage can stabilise p53 through enhancing its association with USP33, leading to a strong DNA damage response (DDR). However, USP33 deficiency results in defective DDR, facilitating DEN‐induced hepatocarcinogenesis in hepatocyte‐specific USP33 KO mice.