Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: Loss of MRE11A leads to chemotherapy resistance. (A) Scheme of the study design. (B) Pie chart showing the proportion of ESCC cases with high or low MRE11A in the TCGA ESCA database. ESCC patients were divided according to the z ‐score values into 1) ≥ 1.5, 2) ≤ −1.5, or 3) < 1.5 and > −1.5 for MRE11A mRNA expression levels. (C) MRE11A mRNA expression levels in normal adjacent esophageal epithelia ( n = 11) and primary ESCC tumors ( n = 95) in the TCGA ESCA database (* P = 0.05). (D) Representative images of normal adjacent esophageal epithelia and primary ESCC tumors showing MRE11A protein levels stained for MRE11A using IHC. Scale bars = 50 µm. Right top insets on each picture show a magnification of MRE11A staining. Scale bars = 10 µm. (E) Comparison of H ‐scores for MRE11A protein levels for normal adjacent esophageal epithelia ( n = 7) and primary ESCC tumor tissues ( n = 59) (*** P < 0.001). (F) Kaplan–Meier curves comparing OS in ESCC patients with low ( n = 34) versus high ( n = 25) MRE11A protein levels ( P < 0.01). (G) Representative images of core biopsy tissues from nonresponders (Patients 1 and 2) and responders (Patients 1 and 2) ESCC patients to NAC that were stained for MRE11A using IHC. Scale bars = 50 µm. Right top insets on each picture show a magnification of MRE11A staining. Scale bars = 10 µm. (H) Comparison of H ‐scores for MRE11A protein levels in core biopsy tissues from nonresponders ( n = 53) or responders ( n = 8) ESCC patients to NAC (*** P < 0.001). Error bars represent the mean ± SD. Statistical differences were tested using Mann–Whitney test (C), an unpaired two‐tailed t ‐test with Welch’s correction (E and H) and log‐rank test (F).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Expressing, Staining, Comparison, MANN-WHITNEY, Two Tailed Test

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: Clinicopathological features for primary ESCC patients ( n = 60). NA, not applicable.

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques:

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: Univariable and multivariable analysis for clinicopathological features in relation to OS in ESCC patients ( n = 60).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques:

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: MRE11A expression determines cisplatin resistance in ESCC cell lines. (A, B) Western blot analysis for MRE11A, UBQLN4, and β‐actin (loading control) comparing si‐Ctrl and si‐MRE11A in TE‐10 (A) and TE‐8 (B) parental cell lines. (C, D) Drug sensitivity assays comparing si‐Ctrl or si‐MRE11A in TE‐10 (C) and TE‐8 (D) parental cell lines treated with different cisplatin concentrations (** P < 0.01, *** P < 0.001). (E, F) MRE11A, UBQLN4, and β‐actin (loading control) comparing EV and MRE11A‐OV in TE‐10 (E) or TE‐8 (F) parental cell lines. (G, H) Drug sensitivity assays for TE‐10 (G) and TE‐8 (H), EV or MRE11A‐OV parental cell lines treated with different cisplatin concentrations (** P < 0.01, *** P < 0.001). (I, J) Drug sensitivity assays for TE‐10 (I) and TE‐8 (J) parental or established cisplatin‐resistant (Cis‐Res) cell lines treated with different cisplatin concentrations (* P < 0.05, *** P < 0.001). (K, L) Western blot analysis for MRE11A, UBQLN4, and β‐actin (loading control) comparing parental and cisplatin‐resistant (Cis‐Res) TE‐10 (K) or TE‐8 (L) ESCC cell lines. (M, N) Quantification of MRE11A protein levels analyzed by western blot comparing parental and cisplatin‐resistant (Cis‐Res) TE‐10 (M) or TE‐8 (N) ESCC cell lines (** P < 0.01, *** P < 0.001). Error bars represent the mean ± SD from n = 3 replicates. Statistical differences were tested using two‐way ANOVA test and post hoc Bonferroni test (C, D, G, H, I, and J) and unpaired two‐tailed t ‐test (M and N).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Expressing, Western Blot, Control, Two Tailed Test

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: High UBQLN4 leads to worse postoperative survival in ESCC patients. (A) Pie chart showing the proportion ESCC cases with high and low UBQLN4 mRNA expression levels in the TCGA ESCA database. ESCC patients were divided according to the z ‐score values into 1) ≥ 1.5, 2) ≤ −1.5, or 3) < 1.5 and > −1.5 for UBQLN4 mRNA expression levels. (B) Comparison of UBQLN4 mRNA expression levels in normal adjacent esophageal epithelia ( n = 11) and primary ESCC tumors ( n = 95) in the TCGA ESCA database (* P < 0.05). (C) The relationship between CNV and UBQLN4 mRNA expression levels was assessed in primary ESCC tumors using the TCGA ESCA database ( n = 95) (NS, not significant, * P < 0.05, *** P < 0.001). (D) Correlation between linear CNV and UBQLN4 mRNA expression levels (Spearman r = 0.579, P < 0.001). (E) Representative images of UBQLN4 protein levels in normal adjacent esophageal epithelia and primary ESCC tumors stained for UBQLN4 by IHC. Scale bars = 50 µm. Right top insets on each picture show a magnification of MRE11A staining. Scale bars = 10 µm. (F) Comparison of H ‐scores for UBQLN4 protein levels in normal adjacent esophageal epithelia ( n = 10) and primary ESCC tumors tissues ( n = 59) (*** P < 0.001). (G) H ‐scores for UBQLN4 protein levels in normal adjacent esophageal epithelia tissue ( n = 10), nonrecurrent primary ESCC ( n = 36), and recurrent primary ESCC tumors ( n = 23) (NS, not significant, *** P < 0.001). (H) Kaplan–Meier curves comparing OS in ESCC patients with low ( n = 39) versus high ( n = 20) UBQLN4 protein levels ( P < 0.01). (I) Kaplan–Meier curves comparing OS in ESCC patients with concurrent low UBQLN4 and high MRE11A ( n = 14) versus high UBQLN4 and low MRE11A ( n = 10) protein levels ( P < 0.001). (J) Representative images of core biopsy tissues from nonresponders (Patients 1 and 2) or responders (Patients 1 and 2) ESCC patients to NAC that were stained for UBQLN4 using IHC. Scale bars = 50 µm. Right top insets on each picture show a magnification of MRE11A staining Scale bars = 10 µm. (K) Comparison of H ‐scores for UBQLN4 protein levels in core biopsy tissues from nonresponders ( n = 53) and responders ( n = 8) ESCC patients to NAC (*** P < 0.001). Error bars represent the mean ± SD. Statistical differences were tested using Mann–Whitney test (B), an unpaired two‐tailed t ‐test with Welch’s correction (F and K), ordinary one‐way ANOVA test and Bonferroni post hoc test (C and G), spearman correlation (D), and log‐rank test (H and I).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Expressing, Comparison, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: UBQLN4 expression determines the sensitivity to cisplatin in ESCC cell lines. (A) Western blot analysis for UBQLN4 and β‐actin (loading control) in TE‐4 cell lines treated with si‐Ctrl or si‐UBQLN4 (pool siRNA). (B) TE‐4 cell lines were treated with si‐Ctrl or si‐UBQLN4 and cell proliferation was analyzed at indicated time points (*** P < 0.001). (C) TE‐4 cell lines were treated with si‐Ctrl or si‐UBQLN4 and analyzed for colony formation. The bar graph showed the quantification of colonies after 12 days of incubation (*** P < 0.001). (D) Drug sensitivity assays comparing si‐Ctrl and si‐UBQLN4 in TE‐4 cell lines treated with different cisplatin concentrations (* P < 0.05). (E, F) Cell proliferation assays were performed at indicated time points in TE‐8 (E) and TE‐10 (F) cell lines with EV or UBQLN4‐OV (* P < 0.05, *** P < 0.001). (G, H) Drug sensitivity assays comparing EV and UBQLN4‐OV in TE‐8 (G) and TE‐10 (H) cell lines treated with different cisplatin concentrations (*** P < 0.001). Error bars represent the mean ± SD from n = 3 replicates. Statistical differences were tested using two‐way ANOVA test and post hoc Bonferroni test (B, D, E, F, G, and H) and unpaired two‐tailed t ‐test (C).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Expressing, Western Blot, Control, Incubation, Two Tailed Test

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: UBQLN4‐OV alleviated DNA damage induced by cisplatin in ESCC cell lines. (A–D) IF staining for 53BP1 was performed in cisplatin‐treated (5 μ m , 12 h) or untreated TE‐10 EV (A), TE‐10 UBQLN4‐OV (B), TE‐8 EV (C), and TE‐8 UBQLN4‐OV (D) cell lines. Shown are 53BP1 (red), DAPI (blue), and the merged images. Scale bars: 10 µm. (E, F) Quantification of the number (#) of 53BP1 foci per cell for TE‐10 (E) and TE‐8 (F) cell lines (NS, not significant, *** P < 0.001). (G‐J) IF staining for γ‐H2AX was performed in cisplatin‐treated (5 μ m , 12 h) or untreated TE‐10 EV (G), TE‐10 UBQLN4‐OV (H), TE‐8 EV (I), and TE‐8 UBQLN4‐OV (J) cell lines. Shown are γ‐H2AX (red), DAPI (blue), and the merged images. Scale bars = 10 µm. (K‐L) Quantification of γ‐H2AX fluorescence intensity per cell for TE‐10 (K) and TE‐8 (L) cell lines (NS, not significant, ** P < 0.01, *** P < 0.001). Error bars represent the mean ± SD from n = 3 replicates. Statistical differences were tested using ordinary one‐way ANOVA test and Bonferroni post hoc test (E, F, K, and L).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Staining, Fluorescence