Pharmacological targeting of TNS3 with histone deacetylase inhibitor as a therapeutic strategy in esophageal squamous cell carcinoma

Abstract

Histone acetylation which regulates about 2-10% of genes has been demonstrated to be involved in tumorigenesis of esophageal squamous cell carcinoma (ESCC). In this study, we investigated the treatment response of ESCC to selective histone deacetylase inhibitor (HDACi) LMK-235 and potential biomarker predicting the treatment sensitivity. We identified tensin-3 (TNS3) which was highly over-expressed in ESCC as one of the down-regulated genes in response to LMK-235 treatment. TNS3 was found positively correlated with the tumor malignancy and poor prognosis in the patients. Silencing TNS3 significantly inhibited ESCC cell proliferation both in vitro and in vivo, sensitizing the treatment response to LMK-235. Our findings provide an insight into understanding the oncogenic role of TNS3 in ESCC and its clinical application for HDAC targeted therapy of ESCC.

Keywords: LMK-235; esophageal squamous cell carcinoma; histone acetylation; histone deacetylase inhibitors; tensin-3.

Figure 1

LMK-235 inhibits ESCC cells proliferation. (A) Growth curves of the five ESCC cell lines treated with LMK-235 for 48 hr. Absorbance at OD450 nm are normalized to vehicle control (0.1% DMSO; n = 5 wells/dose point). IC50 of KYSE150 is marked as the red point. (B) EdU incorporation assay of KYSE150 treated with LMK-235 (0.824 μM) for 48 hr. Scale bar = 50 μm. (C) The expressions of H3K27ac, H3K14ac, H3K9ac, HDAC4, and HDAC5 are determined by western blot in KYSE150 treated as (B). Histone H3 is used as the loading control. (D, E). Cell cycle distribution (D) and the percentage of Annexin V+ cells (E) of KYSE150 treated as (B), analyzed by flow cytometry. (A–E) Error bar denotes SEM of three replicates.

Figure 2

Putative oncogene TNS3 is mitigated by LMK-235. (A) Venn diagrams of the 56 putative tumor suppressors and 55 putative oncogenes. (B) Volcano plot exhibits the DEGs mediated by LMK-235, including the selected 10 genes. (C) Loss-of-function proliferation high-content based siRNA screen of the selected 10 genes. (D) Immunofluorescence of TNS3 in KYSE150 and TE-1, treated with LMK-235. Red: TNS3, Green: α-Tubulin, Blue: DAPI. Scale bar = 20 μm. (E) qRT-PCR analysis of TNS3 expression in Het-1A, TE-1, and KYSE150 cells treated with LMK-235 and Vorinostat. Data are normalized to GAPDH. (F) qRT-PCR analysis of TNS3 expression in EC109, KYSE150, KYSE510, TE-1, and TE-7 cells treated with LMK-235. Data are relative to vehicle control and normalized to GAPDH. (C, E, F). Error bar denotes SEM of three replicates.

Figure 3

TNS3 serves as an oncogene in ESCC based on public databases. (A) TNS3 expression in ESCC compared with the corresponding paracancerous tissues in GEO database, including GSE20347, GSE23400, and GSE44021 [–19]. (B) TNS3 expression in ESCC, the related paracancerous tissues, and related normal tissues (GSE161533). (C) TNS3 expression in EC cells (ESCC OE21 and EAC OE33) treated with MS275 and AZA (GSE57130 [23]). (D) TNS3 expression among different clinical TNM stage (left) and histopathological grade (right) of ESCC in TCGA database. (E) Correlations between TNS3 and c-Src in normal tissues based on GTEx database.

Figure 4

TNS3 expression are relevant with tumorigenic properties of ESCC. (A) Clinical features of the 153 ESCC patients. (B) Representative IHC images of TNS3 in ESCC and the normal esophageal blocks. Scale bar = 100 μm. (C) Quantified staining scores of (B) (arbitrary units). (D) Correlations of TNS3 immunostaining scores with clinical TNM stage (upper) and histopathological grade (bottom) of ESCC, respectively. (E) The five years OS rate of ESCC patients with higher and lower levels of TNS3.

Figure 5

TNS3 promotes ESCC cell proliferation. (A) EdU incorporation and (B). CCK-8 assay of KYSE150 transfected with siTNS3 (#1, #2) and siNC for 48 hr. Scale bar = 50 μm. (C) Subcutaneous tumor volumes and (D). weights of the xenograft mice model (expressed shTNS3#1, #2, shNC). Scale bar = 0.5 cm. (E) Immunostaining of TNS3 and Ki-67 in the subcutaneous tumor blocks (shTNS3 #2 and shNC). Scale bar = 100 μm. (F) Growth curves of TE-1 and TE-7 treated with LMK-235 for 48 hr post-transfection with siTNS3(#2). Absorbance at OD450 nm are normalized to vehicle control (0.1% DMSO; n = 5 wells/dose point). IC50 of cells transfected with siTNS3(#2) are marked as grey (TE-1) and red (TE-7) point, respectively. (A–D, F) Error bar denotes SEM of three replicates.