Abstract B119: Inhibition of UCHL1 blocks proliferation and metastasis in HIF-1 dependent tumor malignancy

Abstract

Abstract Hypoxia-inducible factor 1 (HIF-1) is a key transcription factor for tumor malignancy. Recently, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, has been reported to stabilize HIF-1α by releasing the pVHL complex conjugated Ub moieties. In this study, we investigated whether inhibition of UCHL1 can be an effective strategy for HIF-1-related tumor chemotherapy. First, we found that UCHL1 inhibition by siRNA or LDN57444, a well-known UCHL1 inhibitor, drastically lowered HIF-1α protein levels in UCHL1-expressing MDA-MB-436 breast cancer cells, by Western blotting analysis and immunostaining experiments. In UCHL1 non-expressing MDA-MB-231 cells, the ectopic expression of UCHL1 significantly increased HIF-1α protein expression levels, which was canceled by the treatment of LDN57444. We further analyzed whether the UCHL1 inhibitor could affect the HIF-dependent transactivation using 5HRE-luciferase reporter assay and RT-qPCR. LDN57444 dose-dependently inhibited the HIF activity and decreased the transcription of HIF downstream genes in MDA-MB-436 cells. Next we tested the effects of the UCHL1 inhibitor on tumor cell migration. LDN57444 significantly blocked cell migration in MDA-MB-436 cells in wound healing and transwell migration assays, suggesting a crucial role of the enzyme in tumor migration. We finally investigated potential roles of UCHL1 in tumor malignancy by using a 3D spheroid culture model, which has been more widely applied in the preclinical studies to provide more physiologically relevant information. Image analysis using open source image processing suites, AnaSP and ReViSP revealed that ectopic expression of UCHL1 in MDA-MB-231 cells significantly upregulated malignancy representing factors such as integrity and volume. Whole cell number of UCHL1 transfected MDA-MB-231 spheroids was also much higher than that of the mock treated spheroids when measured by 3D cell viability assay. Depletion of UCHL1 or inhibition of UCHL1 enzyme activity in MDA-MB-436 cells, on the contrary, downregulated these malignant factors. Besides, in spheroid viability staining and spheroid invasion assay, LDN57444 abolished the UCHL1 mediated cell proliferation and invasiveness as well. Moreover, the ectopic expression of UCHL1 in MDA-MB-231 cells stabilized HIF-1α protein level and promoted the expression of HIF-1 target genes in the 3D model as in the case of 2D monolayer culture. In conclusion, we clearly proved the involvement of UCHL1 in the HIF-1 induced tumor malignancy employing the 3D spheroid culture model. Our research indicates that the UHCL1-HIF-1 pathway is a promising therapeutic target for cancer chemotherapy. Citation Format: Xuebing Li, Akira Hattori, Senye Takahashi, Yoko Goto, Hiroshi Harada, Hideaki Kakeya. Inhibition of UCHL1 blocks proliferation and metastasis in HIF-1 dependent tumor malignancy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B119. doi:10.1158/1535-7163.TARG-19-B119