The deubiquitinase (DUB) USP13 promotes Mcl-1 stabilisation in cervical cancer

Abstract

Protein ubiquitination is a critical regulator of cellular homeostasis. Aberrations in the addition or removal of ubiquitin can result in the development of cancer and key components of the ubiquitination machinery serve as oncogenes or tumour suppressors. An emerging target in the development of cancer therapeutics are the deubiquitinase (DUB) enzymes that remove ubiquitin from protein substrates. Whether this class of enzyme plays a role in cervical cancer has not been fully explored. By interrogating the cervical cancer data from the TCGA consortium, we noted that the DUB USP13 is amplified in ~15% of cervical cancer cases. We confirmed that USP13 expression was increased in cervical cancer cell lines, cytology samples from patients with cervical disease and in cervical cancer tissue. Depletion of USP13 inhibited cervical cancer cell proliferation. Mechanistically, USP13 bound to, deubiquitinated and stabilised Mcl-1, a pivotal member of the anti-apoptotic BCL-2 family. Furthermore, reduced Mcl-1 expression partially contributed to the observed proliferative defect in USP13 depleted cells. Importantly, the expression of USP13 and Mcl-1 proteins correlated in cervical cancer tissue. Finally, we demonstrated that depletion of USP13 expression or inhibition of USP13 enzymatic activity increased the sensitivity of cervical cancer cells to the BH3 mimetic inhibitor ABT-263. Together, our data demonstrates that USP13 is a potential oncogene in cervical cancer that functions to stabilise the pro-survival protein Mcl-1, offering a potential therapeutic target for these cancers.

Fig. 1. USP13 expression is upregulated in pre-malignant cervical disease and cervical cancer.

A Genomic alterations of USP13 across human cancers determined by cBioportal analysis of TCGA data. B Scatter dot plot analysis of USP13 mRNA expression against USP13 copy number alterations in cervical cancer determined by cBioportal analysis of TCGA data. Correlation was determined using Spearman’s analysis. C RT-qPCR analysis of USP13 mRNA expression in normal human keratinocytes (NHKs), HPV- C33A cells, HPV16 + SiHa and CaSKi cells and HPV18 + SW756 and HeLa cells. mRNA expression was normalized against U6 mRNA levels. D Representative western blot of USP13 expression in NHKs, C33A cells, SiHa, CaSKi, SW756 and HeLa cells. GAPDH served as a loading control. Quantification of the protein band intensities from four biological, independent repeats are shown on the right. E Representative immunohistochemical (IHC) staining of USP13 expression in cervical cancer tissues and normal cervical epithelium from a tissue microarray (TMA). Scale bars, 100 μm. Scatter dot plot analysis of USP13 expression from a larger cohort of cervical cancer cases (n = 41) and normal cervical epithelium (n = 9) is shown on the right. F Scatter dot plot of RT-qPCR analysis of USP13 mRNA expression from a panel of cervical cytology samples representing CIN lesions of increasing grade. Five samples from each clinical grade (negative (Neg) and CIN I-III) were analysed and mRNA levels were normalized to the negative samples. Samples were normalized against U6 mRNA levels. G Representative western blot of cervical cytology samples of CIN lesions of increasing grade analysed for USP13 protein expression. GAPDH served as a loading control. Scatter dot plot analysis of a larger cohort of samples (n = 15 for each grade) is shown on the right. Bars represent the mean ± standard deviation from at least three biological repeats unless otherwise stated. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test).

Fig. 2. USP13 expression is regulated by c-Jun/AP-1 activity in cervical cancer cells.

A Schematic of potential AP-1 binding sites in the USP13 promoter region. B Representative western blot of HeLa and SiHa cells after treatment with JNK-IN-8 (3 µM) or DMSO control for 48 h. Lysates were analysed for the expression of USP13, phosphorylated c-Jun (S73) and c-Jun. GAPDH was used as a loading control. C RT-qPCR analysis of USP13 mRNA expression in HeLa and SiHa cells after treatment with JNK-IN-8 (3 µM) or DMSO control for 48 h. mRNA expression was normalized against U6 mRNA levels. D Representative western blot of HeLa and SiHa cells after transfection of a pool of four specific c-Jun siRNA for 72 h. Lysates were analysed for the expression of USP13 and c-Jun. GAPDH was used as a loading control. E RT-qPCR analysis of USP13 and cJUN mRNA expression of HeLa and SiHa after transfection of a pool of four specific c-Jun siRNA for 72 h. mRNA expression was normalized against U6 mRNA levels. F Cells were treated as in B and chromatin was prepared from HeLa cells and c-Jun was immunoprecipitated using an anti-c-Jun antibody, followed by RT-qPCR using primers specific to the two putative AP-1 binding sites in the USP13 promoter. c-Jun binding is presented as percentage of input chromatin. Bars are the means ± standard deviation from at least three biological repeats. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test).

Fig. 3. USP13 expression is required for the proliferation of HPV + cervical cancer cells.

A Representative western blot of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA for 72 h. Lysates were analysed for the expression of USP13 and GAPDH was used as a loading control. B Growth curve analysis of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA for 72 h. C Colony formation assay (anchorage dependent growth) of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA for 72 h. D Soft agar assay of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA for 72 h. E Representative western blot of HeLa and SiHa cells after transfection of a FLAG-USP13 or FLAG-USP13 (C345A) for 48 h. Lysates were analysed for the expression of USP13 and GAPDH was used as a loading control. F Growth curve analysis of HeLa and SiHa cells after transfection of a FLAG-USP13 or FLAG-USP13 (C345A) for 48 h. G Colony formation assay (anchorage dependent growth) of HeLa and SiHa cells after transfection of a FLAG-USP13 or FLAG-USP13 (C345A) for 48 h. H Soft agar assay of HeLa and SiHa cells after transfection of a FLAG-USP13 or FLAG-USP13 (C345A) for 48 h. Bars are the means ± standard deviation from at least three biological repeats. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test).

Fig. 4. USP13 promotes the stability of the pro-survival protein Mcl-1.

A Representative western blot of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA for 72 h. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. B RT-qPCR analysis of USP13 and MCL1 mRNA expression of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA for 72 h. mRNA expression was normalized against U6 mRNA levels. C Representative western blot of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA for 72 h. Cells were additionally treated with 10 µM MG132 for 6 h. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. D Representative western blot of HeLa and SiHa cells after transfection of FLAG-USP13 or FLAG-USP13 (C345A). Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. E RT-qPCR analysis of USP13 and MCL1 mRNA expression of HeLa and SiHa cells after transfection of FLAG-USP13 or FLAG-USP13 (C345A). mRNA expression was normalized against U6 mRNA levels. F Representative western blot of HeLa cells after transfection of a pool of four specific USP13 siRNA for 72 h. Cells were additionally treated with 20 µM cycloheximide and harvested at the indicated time points. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. Quantification of the protein band intensities from three biological, independent repeats are shown on the right. G Representative western blot of HeLa cells after transfection of FLAG-USP13 or FLAG-USP13 (C345A). Cells were additionally treated with 20 µM cycloheximide and harvested at the indicated time points. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. Quantification of the protein band intensities from three biological, independent repeats are shown on the right. Bars are the means ± standard deviation from at least three biological repeats. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test).

Fig. 5. USP13 interacts with and deubiquitinates Mcl-1 in cervical cancer cells.

A HEK293T cells were transfected with V5-Mcl-1, Flag-USP13, or both V5-Mcl-1 and FLAG-USP13. Cells were treated with 10 µM MG132 for 6 h and either Mcl-1 or USP13 were immunoprecipitated using an anti-V5 or anti-FLAG antibody. Co-immunoprecipitated Mcl-1 or FLAG-USP13 were detected using the respective antibodies. GAPDH was used as a loading control. B Endogenous Mcl-1 and USP13 were immunoprecipitated from HeLa and SiHa cells after treatment with 10 µM MG132 using an anti-Mcl-1 or anti-USP13 antibodies. Co-immunoprecipitated Mcl-1 or USP13 were detected using the respective antibodies. GAPDH was used as a loading control. C HEK293T cells were co-transfected with V5-Mcl-1 and HA-Ubiquitin, with or without Flag-USP13 or a FLAG-USP13 mutant (C345A). Cells were treated with 10 µM MG132 for 6 h and V5-Mcl-1 was immunoprecipitated using an anti-Mcl-1 antibody. Ubiquitinated Mcl-1 was detected using an anti-HA antibody. D HEK293T cells were co-transfected with V5-Mcl-1, HA-Ubiquitin or mutant Ubiquitin (K48R or K63R), with or without Flag-USP13. Cells were treated with 10 µM MG132 for 6 h and V5-Mcl-1 was immunoprecipitated using an anti-V5 antibody. Ubiquitinated Mcl-1 was detected using an anti-HA antibody. E HeLa and SiHa cells were transfected with a pool of four specific USP13 siRNA for 72 h. Cells were treated with 10 µM MG132 for 6 h before harvesting. Mcl-1 was immunoprecipitated using an anti-Mcl-1 antibody. Ubiquitinated Mcl-1 was detected using an anti-ubiquitin antibody. GAPDH was used as a loading control.

Fig. 6. Restoration of Mcl-1 expression partially rescues the proliferation defect in USP13 depleted cervical cancer cells.

A Representative western blot of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA, or scramble siRNA, for 72 h. After 24 h, cells were transfected with hMcl-1 or control vector. Lysates were analysed for the expression of USP13, Mcl-1 and GAPDH was used as a loading control. B Growth curve analysis of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA, or scramble siRNA, for 72 h. After 24 h, cells were transfected with hMcl-1 or control vector. C Colony formation assay (anchorage dependent growth) of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA, or scramble siRNA, for 72 h. After 24 h, cells were transfected with hMcl-1 or control vector. Bars are the means ± standard deviation from at least three biological repeats. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test).

Fig. 7. USP13 and Mcl-1 expression correlate in cervical cancer.

A Representative western blot of Mcl-1 expression in normal human keratinocytes (NHKs), C33A, SiHa, CaSKi, SW756 and HeLa cells. GAPDH served as a loading control. B Representative IHC staining of Mcl-1 expression in cervical cancer tissues and normal cervical epithelium from a tissue microarray (TMA). Scale bars, 100 μm. C Scatter dot plot analysis of Mcl-1 expression from a larger cohort of cervical cancer cases (n = 41) and normal cervical epithelium (n = 9) is shown on the right. D Representative IHC staining of USP13 and Mcl-1 expression in cervical cancer tissue from two patients. Staining was performed from separate cores from the same patient samples. Scale bars, 100 μm. Correlation was determined using Spearman’s analysis and is shown on the right. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test).

Fig. 8. Pharmacological inhibition of USP13 sensitises HPV + cervical cancer cells to BH3 mimetics.

A Representative western blot of HeLa and SiHa cells after treatment with increasing doses of Spautin-1 or DMSO control for 48 h. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. B RT-qPCR analysis of MCL1 mRNA expression in HeLa and SiHa cells after treatment with Spautin-1 (10 µM) or DMSO control for 48 h. mRNA expression was normalized against U6 mRNA levels. C HeLa and SiHa cells were treated with Spautin-1 (10 µM) or DMSO control for 48 h. Cells were additionally treated with 10 µM MG132 for 6 h. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. D Representative western blot of HeLa and SiHa cells after transfection of FLAG-USP13 with or without Spautin-1 (10 µM) or DMSO control. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. E Cell viability assay (MTT) of HeLa and SiHa cells treated with increasing doses of ABT-263, with or without Spautin-1 (10 μM), for 24 h. F Synergistic activities of Spautin-1 and ABT-263 in HeLa and SiHa cells. Left: heatmaps of growth inhibition; right: Bliss synergy map. G Flow cytometric analysis of Annexin V assay in HeLa and SiHa cells after treatment with DMSO, Spautin-1 (10 μM), ABT-263 (5 μM) or both for 24 h. H Colony formation assay of HeLa and SiHa cells after treatment with DMSO, Spautin-1 (10 μM), ABT-263 (5 μM) or both for 24 h. Error bars represent the mean ± standard deviation of a minimum of three biological repeats. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).