Prognostic impact of Schlafen 11 in bladder cancer patients treated with platinum-based chemotherapy

Abstract

The utility of Schlafen 11 (SLFN11) expression as a predictive biomarker for platinum-based chemotherapy has been established for cancers from different histologies. However, the therapeutic relevance of SLFN11 in bladder cancer (BC) is unknown. Here, we examined the clinicopathologic significance of SLFN11 expression across 120 BC cases by immunohistochemistry. We divided the cases into two cohorts, one including 50 patients who received adjuvant or neoadjuvant platinum-based chemotherapy, and the other including 70 BC patients treated by surgical resection without chemotherapy. In the cohort of 50 BC cases treated with platinum-based chemotherapy, the SLFN11-positive group (n = 25) showed significantly better overall survival than the SLFN11-negative group (n = 25, P = .012). Schlafen 11 expression correlated significantly with the expression of luminal subtype marker GATA3. Multivariate analyses identified SLFN11 expression as an independent prognostic predictor (odds ratio, 0.32; 95% confidence interval, 0.11-0.91; P = .033). Conversely, in the cohort of 70 BC cases not receiving platinum-based chemotherapy, the SLFN11-positive group (n = 29) showed significantly worse overall survival than the SLFN11-negative group (n = 41, P = .034). In vitro analyses using multiple BC cell lines confirmed that SLFN11 KO rendered cells resistant to cisplatin. The epigenetic modifying drugs 5-azacytidine and entinostat restored SLFN11 expression and resensitized cells to cisplatin and carboplatin in SLFN11-negative BC cell lines. We conclude that SLFN11 is a predictive biomarker for BC patients who undergo platinum-based chemotherapy and that the combination of epigenetic modifiers could rescue refractory BC patients to platinum derivatives by reactivating SLFN11 expression.

Keywords: SLFN11; biomarker; bladder cancer; chemotherapy; cisplatin.

FIGURE 1

Immunohistochemical expression of Schlafen 11 (SLFN11) in bladder cancer (BC) and validation of the association between SLFN11 expression and clinical course. A, Representative immunohistochemical images of SLFN11 in BC. Scale bars, 200 µm (left) and 50 µm (right). B, Distribution of the immunohistochemical expression of SLFN11 in 120 BC cases, with 5% used as the cut‐off value. C, Correlation of the expression of SLFN11 protein with overall survival (OS) of 120 patients with BC. D, Correlation of the expression of SLFN11 protein with OS of 50 patients with unresectable locally advanced or metastatic BC treated with platinum‐based chemotherapy. E, Correlation of the expression of SLFN11 protein with OS of 70 patients with local BC treated by surgical resection without chemotherapy

FIGURE 2

Representative images of H&E and immunohistochemical staining. Images of H&E, Schlafen 11 (SLFN11), p53, GATA3, cytokeratin 5/6, and programmed death ligand 1 (PD‐L1) staining in bladder cancer cells. Scale bars, 50 µm

FIGURE 3

Inactivation of Schlafen 11 (SLFN11) induces resistance to cisplatin and inhibits cell death under replication stress in bladder cancer (BC) cell lines. A, Correlation between SLFN11 mRNA expression and cisplatin sensitivity among 849 cell lines, which includes 15 BC cell lines in the Genomics of Drug Sensitivity in Cancer (GDSC) cancer cell line database. Pearson correlation (r) = .36, P = 1.1e‐27. B, Correlation between SLFN11 mRNA expression and cisplatin sensitivity among 15 BC cell lines with the GDSC cancer cell line database. r = .47, P = .075. C, Western blot analysis of SLFN11 in BC cell lines and β‐actin as a loading control. D, Western blot analysis of SLFN11 KO cells generated by CRISPR‐Cas9 gene editing technology. β‐Actin was used as a loading control. E, Immunohistochemical images of parent and SLFN11 KO cells in T24 or UM‐UC13 cell lines. Scale bars, 100 µm. F, Dose‐dependent effects of cisplatin on the viability of T24 or UM‐UC13 cell lines with parent and SLFN11 KO cells. G, Cell growth curves of the indicated cell lines under normal conditions (NT) or treated with the indicated concentrations of cisplatin for 4 h and released into a drug‐free medium. *P < .05; **P < .01. act, drug activity (−log₁₀ [IC₅₀M]); exp, mRNA expression (log₂); MGH, Massachusetts General Hospital; GDSC, Genomics of Drug Sensitivity in Cancer

FIGURE 4

Epigenetic modulators reactivate Schlafen 11 (SLFN11) expression and sensitize bladder cancer cells to platinum agents. A, Western blot analysis of RT112 (left) and 253JBV (right) cell lines treated with the indicated concentrations of 5‐aza‐2′‐deoxycytidine (5‐aza) or entinostat for 2 d. β‐Actin was used as a loading control. B, Representative immunohistochemical images of SLFN11 in RT112 (upper) and 253JBV (lower) cell lines treated with the indicated concentrations of 5‐aza or entinostat for 2 d. Scale bars, 100 µm. C, Dose‐dependent effects of cisplatin or carboplatin on the viability of RT112 (upper) and 253JBV (lower) cell lines treated with the indicated concentrations of 5‐aza or entinostat