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. 2022 Dec 21;6(1):93.
doi: 10.1038/s41698-022-00336-x.

The deubiquitinase USP10 protects pancreatic cancer cells from endoplasmic reticulum stress

Udayan Bhattacharya #  1 , Elangovan Thavathiru #  1 , Fiifi Neizer-Ashun  2 , Chao Xu  3 , Zoran Gatalica  4 , Shailendra Kumar Dhar Dwivedi  1 , Anindya Dey  1 , Priyabrata Mukherjee  4   5 , Resham Bhattacharya  6   7   8   9
Affiliations

The deubiquitinase USP10 protects pancreatic cancer cells from endoplasmic reticulum stress

Udayan Bhattacharya et al. NPJ Precis Oncol. .

Abstract

The ubiquitin-specific peptidase 10 (USP10) plays a context-specific, pro or anti-tumorigenic role in different malignancies. However, the role of USP10 in pancreatic cancer remains unclear. Our protein and RNA level analysis from archived specimens and public databases show that USP10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and expression correlates with poor overall patient survival. Phenotypically, silencing USP10 decreased viability, clonal growth and invasive properties of pancreatic cancer cells. Mechanistically, silencing USP10 upregulated BiP and induced endoplasmic reticulum (ER) stress that led to an unfolded protein response (UPR) and upregulation of PERK, IRE1α. Decreased cell viability of USP10 silenced cells could be rescued by a chemical chaperone that promotes protein folding. Our studies suggest that USP10 by protecting pancreatic cancer cells from ER stress may support tumor progression.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Expression and significance of USP10 in pancreatic cancer.
(a–d) Expression of USP10 in pancreatic tissues: Normal and Pancreatic cancer patients (Ductal tissues: Normal, n = 29; cancerous T1 n = 4; T2 n = 12, T3 n = 17 & Stromal tissues: normal (n = 26) and cancerous, T1 n = 4; T2 n = 12; T3 n = 15) stained by immunohistochemistry (IHC) to assess the expression levels of USP10 protein. Data were analyzed by Linear Mixed Model. The H-score for ductal tissue in normal sample is estimated to be 93.95, 85.49, and 52.77 units less than the H-score in T1, T2, and T3 sample respectively (p-value 0.0001, <0.0001, and 0.0001). There is no significant difference between pairwise comparisons of T1, T2, and T3. Similarly, for stroma, the H-score in normal sample is estimated to be 55.90, 30.76, and 40.51 units less than the H-score in T1, T2, and T3 sample respectively (p-value 0.0002, 0.0023, and <0.0001). There is no significant difference between pairwise comparisons of T1, T2, and T3. e Levels of USP10 mRNA in normal pancreas (GTEx), pancreatic cancer cell lines (CCLE) and pancreatic cancer tissues (ICGC and TCGA). Data represent mean ± SD. Using Kruskal-Wallis rank sum test, the FPKM/expression was significantly higher in TCGA, ICGC, and CCLE compared to GTEx (p-value <2.2e−16). f Kaplan-Meier overall survival curves in USP10 low and high expression pancreatic cancer cases (p = 0.02). The proportion survival is plotted versus time (since diagnosis in month). Kaplan-Meier curves with a log-rank test where P value < 0.05 is considered significant.
Fig. 2
Fig. 2. Cell line expression and cellular phenotypes of USP10 silencing.
a Immunoblot showing expression of USP10 in pancreatic cancer cell lines along with normal HPDEC cells. The numbers denote densitometric quantitation of USP10, normalized with respect to α-Tubulin by NIH Image J. b–e Anchorage-independent clonal potential evaluated in control and USP10 silenced pancreatic cancer cell lines and HPDEC. Percent clonal growth along with SD is plotted and compared to their respective siCTL which was set to 100%. Student’s t-test was performed for statistical analysis and P < 0.05 was considered significant; Experiments were repeated independently at least 3 times. (g–j) AsPc-1 and MiaPaCa-2 cells were transfected with either non-target control siRNA (siCTL) or siRNA targeting USP10 (siUSP10). 72 h post-transfection, cells were fixed and stained with DAPI (blue), FITC-gelatin (green), and phalloidin (Tubulin, red). Relative FITC-gelatin matrix degradation i.e., ability to migrate and invade, in siCTL and siUSP10 transfected cells is quantitated; Cells that showed FITC-gelatin degradation were counted and this was set to 1 in the control group. Similarly, in the USP10 silenced group, cells that showed gelatin degradation were counted and expressed relative to the control; >100 cells were evaluated, Data represent mean ± SD; Student’s t-test was performed for statistical analysis and *P value < 0.05. (Scale Bar = 10 μm).
Fig. 3
Fig. 3. USP10 deubiquitinates ribosomal subunits.
a, b Monoubiquitination changes of RPS2, RPS3, and RPS10 after USP10 depletion was evaluated by immunoblotting. α Tubulin was used as loading control and efficient USP10 inhibition was confirmed by immunoblotting. c Control and USP10 silenced cells were incubated with 10 μg/ml of puromycin 48 h or 72 h post-transfection for 10 min prior to harvest. For negative control, cells were treated with cycloheximide (30 μg/ml, 60 min) prior to puromycin incubation. Global protein synthesis was evaluated by anti-puromycin antibody. The numbers denote densitometric quantitation of individual lanes, normalized with respect to α-Tubulin by NIH Image J.
Fig. 4
Fig. 4. Silencing USP10 induces ER stress and unfolded protein response.
a Immunoblotting for markers of UPR after 72 h USP10 silencing. b Quantitation of fluorescent intensity of BiP-ERSE-TdTomato reporter in siCTL, siUSP10, and tunicamycin treated cells. c AsPc-1 and MiaPaCa-2 were transfected with control or USP10 siRNA and treated with indicated doses of TUDCA for 48 h and evaluated for UPR markers by immunoblotting (d, e) and quantified by densitometry analysis using Image J and normalized to their respective α-tubulin levels and then compared to siCTL which was set to 1 (f, g) Quantitation of cell viability after similar treatments in c by MTS. Data represent mean ± SD; Ordinary one-way ANOVA was performed for statistical analysis and ****P < 0.001 and ****P < 0.0001. A post-hoc Tukey test was performed for comparisons between the groups. Experiments were repeated independently at least 3 times.
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