APCCDC20-mediated degradation of PHD3 stabilizes HIF-1a and promotes tumorigenesis in hepatocellular carcinoma
Authors: Qing-Hui Zhang
Shi-Hao Xiang
Wei-Qi Dai
Jue Wei
Bin Liu
Rong-Rong Jia
Yu-Gang Wang
Ling Xu
Min Shi
Journal: Cancer Letters (2021)
Discipline: CLINICAL MEDICINE
Keywords: Destruction-box; Fluorescence-based multiplex immunohistochemistry; Hypoxia-inducible factor-1; WGCNA.
Affiliations: Department of Gastroenterology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, 1111 XianXia Road, Shanghai, 200336, China., Department of Clinical Laboratory, Kunshan First People's Hospital, Jiangsu University, Kunshan, 215300, China., Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, College of Pharmacy, Jiangsu Ocean University, Lianyungang, 222005, China. Electronic address: ., Department of Gastroenterology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, 1111 XianXia Road, Shanghai, 200336, China. Electronic address: .

Abstract:  CDC20 regulates cell cycle progression by targeting key substrates for destruction, but its role in hepatocellular carcinoma (HCC) tumorigenesis remains to be explored. Here, by using weighted gene co-expression network analysis (WGCNA), we identified CDC20 as a hub gene in HCC. We demonstrated that CDC20 expression is correlated with HIF-1 activity and overall survival (OS) of clinic HCC patients. The activity of HIF-1 is regulated by the stability of HIF-1a subunit, which is hydroxylated by oxygen-dependent prolyl hydroxylase enzymes, the PHDs. In addition, we show that genetic ablation or pharmacological inhibition of CDC20 can accelerate the degradation of HIF-1a and impair VEGF secretion in HCC cells. Mechanistically, we found that CDC20 binds to the destruction-box (D-box) motif present in the PHD3 protein to promote its polyubiquitination and degradation. The depletion of endogenous PHD3 in CDC20 knockdown HCC cells greatly attenuated the decline of HIF-1a protein and restored the secretion of VEGF. In contrast, overexpression of a non-degradable PHD3 mutant significantly inhibited the proliferation of HCC cells both in vitro and in vivo. Collectively, our findings indicate that CDC20 plays a crucial role in the development of HCC by governing PHD3 protein. 

Retracted: No

PubPeer comments:
 We found some possible mis-labelling or inconsistencies in the figures: Figure 4 (I): the text states that in the presence of CDC20, ubiquitination of HIF1A was reduced, however the immunoblot does not reflect that as the CDC20 overexpressed lane has higher ubiquitin staining. ![] Figure 4 (J): shouldnu2019t the overexpression of CDC20 in control shRNA cells stabilise HIF1A, even during normoxia? HIF1A is expressed in control shRNA cells in (H), so wouldnu2019t we expect it to be expressed in CDC20 overexpressed cells too? ![] ![] Edit: images added for clarity , 
 Thank you for your interest in our work. For Figure 4i, there is indeed an unintentional mistake. We incorrectly marked this figure and after CDC20 overexpression, the ubiquitinated form of FLAG-HIF1A protein was reduced. We will contact the publisher to see if it needs to be corrected. It is a big challenge for us and even for the entire HIF community to faithfully detect endogenous HIF1A protein expression under normoxia condition by western blot. Because HIF1A is very easily to be degraded and thereby hardly to be detected, and the detection usually required skilled experimental skills. Indeed, through appropriate antibody concentration and exposure time, we detected the expression of endogenous HIF1A protein and revealed the regulation of HIF1A by CDC20 under normoxia condition, as shown in Figure 4g. However, the experimental results of the two lanes on the left of panel of Figure 4j could neither support nor oppose this conclusion, because the experimental conditions between two figures are not accurately consistent. For the two lanes on the left panel of Figure 4j, the HIF1A protein bands were vague and meaningless, whereas the HIF1A proteins in the two lanes on the right panel were clearly detected which was mainly due to the loss of VHL protein, resulting in the accumulation of endogenous HIF1A. Under this circumstance, if we still used the same exposure condition of western blot assay as in Figure 4g, there is no doubt that the signals of the two lanes on the right will cover the bands on the left. Therefore, Figure 4j is not a particularly suitable example for repeatedly revealing the regulation of CDC20 on HIF1A under normoxia. On the contrary, after losing VHL, endogenous HIF1A protein was easily to be detected, making it is more appropriate to detect the regulation of HIF1A by CDC20. Unlike HIF1A proteins, the detection of endogenous VHL protein was not too difficult, as revealed in Figure 4j. However, overexpression of CDC20 failed to further accumulate HIF1A protein in absence of VHL. Therefore, combined with the subsequent IP experiments, all these data showed that the regulation of HIF1A by CDC20 is not direct and VHL is required in this process. Limited by various experimental conditions, it is often difficult for experimental data to present perfect or ideal results. Nevertheless, we do hope that the above explanation can help readers to relieve their concerns and thanks again for your interest in our work. ,