Abnormal Localization and Tumor Suppressor Function of Epithelial Tissue-Specific Transcription Factor ESE3 in Esophageal Squamous Cell Carcinoma

Abstract

Esophageal cancer is one of the most common malignant cancers worldwide. The molecular mechanism of esophageal squamous cell carcinoma (ESCC) is still poorly understood. ESE3 is a member of the Ets transcription family, which is only expressed in epithelial tissues and acts as a tumor suppressor gene in prostate cancer. Our study aim was to confirm whether ESE3 is involved in the carcinogenesis of ESCC. Immunohistochemical analysis revealed that ESE3 was mainly located in cell nuclei of normal tissues and the cytoplasm in ESCC tissues. Immunofluorescence and western blot analyses of the normal esophageal cell line HEEpiC and ESCC cell lines EC9706 TE-1, KYSE150, and KYSE410 confirmed these results. pEGFP-ESE3 and pcDNA3.1-V5/HisA-ESE3 plasmids were constructed for overexpression of ESE3 in EC9706 and KYSE150 cells. The stably transfected cells showed restoration of the nuclear localization of ESE3. EC9706 cells with re-localization of ESE3 to the nucleus showed inhibition of proliferation, colony formation, migration, and invasion. To explore the possible mechanism of the differences in localization of ESE3 in normal esophageal cells and ESCC cells, ESCC cell lines were treated with the nuclear export inhibitor leptomycin B, transcription inhibitor actinomycin D, PKC inhibitor sphinganine, P38 MAPK inhibitor SB202190, and CK II inhibitor TBCA. These reagents were chosen according to the well-known mechanisms of protein translocation. However, the localization of ESE3 was unchanged after these treatments. The sequence of ESE3 cDNA in ESCC cells was identical to the standard sequence of ESE3 in the NCBI Genebank database, indicating that there was no mutation in the coding region of ESE3 in ESCC. Taken together, our study suggests that ESE3 plays an important role in the carcinogenesis of ESCC through changes in subcellular localization and may act as a tumor suppressor gene in ESCC, although the mechanisms require further study.

Fig 1. Expression and subcellular localization of ESE3 in normal human esophageal epithelial tissues and ESCC tissues.

(A) Positivity rate in normal human esophageal epithelial tissues and ESCC tissues (P>0.05). (B) Subcellular localization of ESE3 in normal esophageal epithelial cells and ESCC cells. N indicates nuclear localization, C indicates cytoplasmic localization, and N/C indicates nuclear/cytoplasmic localization (equal distribution or uncertain localization). The white bar represents normal esophageal epithelial cells, and the black bar represents ESCC cells (P<0.05). (C) Positive staining of ESE3 in the nucleus of normal esophageal epithelial cells. (D) Positive staining of ESE3 in the cytoplasm of ESCC cells.

Fig 2. Subcellular localization of ESE3 in esophageal cell lines by immunofluorescence analysis.

N indicates nuclear localization, C indicates cytoplasmic localization, and N/C indicates nuclear/cytoplasmic localization (equal distribution or uncertain localization).

Fig 3. Western blot analysis of ESE3 localization.

Nuclear and cytoplasmic protein extracts were verified by detection of lamin B1 and α-tubulin, respectively. ESE3 was found in the nuclear protein extracts of HEEpiC cells and in the cytoplasmic protein extracts of EC9706 cells.

Fig 4. Localization of overexpressed ESE3.

(A) RT-PCR analysis showed that ESE3 was overexpressed in pEGFP-ESE3- and pcdna3.1-V5/HisA-ESE3-transfected EC9706 cells. (B) Localization of ESE3 in pcdna3.1-V5/HisA-ESE3-transfected EC9706 cells by western blot analysis.

Fig 5. Localization of overexpressed ESE3 in living pEGFP-ESE3-transfected EC9706 cells.

(A) location of overexpressed ESE3-EGFP in living pEGFP-ESE3-transfected EC9706 cells by fluorescence microscope. (B) image of pEGFP-ESE3-transfected EC9706 cells by phase contrast microscope. (C) location of EGFP in living vector-transfected EC9706 cells by fluorescence microscope. (D) image of pEGFP-transfected EC9706 cells by phase contrast microscope.

Fig 6. Immunofluorescence analysis of the localization of ESE3 in pcdna3.1-V5/HisA-ESE3-transfected EC9706 cells.

Fig 7. Effects of ESE3 re-localization to the nuclei of EC9706 cells.

(A) Cell proliferation was inhibited in EC9706 cells transfected with pcdna3.1-V5/His-ESE3 in MTT assays. (B) Fewer colonies were formed by EC9706 cells transfected with pcdna3.1-V5/His-ESE3 compared with control groups. (C) Migratory or (D) invasive cells on the bottom surface were fixed, stained with hematoxylin and eosin, and counted under a microscope at 200×. Data are the means ± SD of three independent experiments (*P<0.05). (E) and (F) shows no difference in apoptosis or the cell-cycle distribution of the two groups.

Fig 8. Subcellular localization of ESE3 in EC9706 after treatment by immunofluorescence analysis.

(A), (B), (C), (D) and (E) indicate subcellular localization of ESE3 after treatment by leptomycin B, actinomycin D, sphinganine (pKC inhibitor), SB202190 (pk38 MAPK inhibitor) and TBCA (CK II inhibitor), respectively. N indicates nuclear localization, C indicates cytoplasmic localization, and N/C indicates nuclear/cytoplasmic localization (equal distribution or uncertain localization).