Role of DNAJB6 in esophageal squamous cell carcinoma

Abstract

Esophageal cancer (EC), which is geographically diverse, has only a 10.7% five-year survival rate. One of the histologic forms, esophageal squamous cell carcinoma (ESCC), in Hong Kong accounts for 81.5% of the total EC cases and its five-year survival rate is only ~14%, due to its high frequency of metastasis. In our previous studies, functional complementation study of chromosome 9 defects led to the discovery of a novel tumor suppressor gene, Deleted in Esophageal Cancer 1 (DEC1), mapping to 9q32. DEC1 was shown to reduce tumorigenicity in a mouse model and its expression was shown to be associated with lymph node metastasis, early onset of ESCC, and familial ESCC development in a tissue microarray (TMA) study. Moreover, DNAJ (Hsp40) homologue subfamily B member 6 (DNAJB6), a molecular co-chaperone protein and the focus of the current study, was identified as a DEC1-interacting protein through a yeast two-hybrid screening. The interaction was further confirmed by the GST pull-down assay and co-localization studies. Using a TMA constructed with ESCC tissues from Hong Kong, the clinical relevance of DNAJB6 expression was demonstrated. In the present study, the role of DNAJB6 in ESCC was investigated using cell line-based in vivo and in vitro studies. DNAJB6 was shown to be down-regulated in ESCC cell lines. The two isoforms of DNAJB6 have distinct subcellular localizations, with DNAJB6a mainly localized to the nucleus and DNAJB6b diffused throughout the cell. Existence of a functional nuclear localization signal peptide and a functional nuclear export signal peptide was verified in DNAJB6a and DNAJB6b, respectively. In vitro evidence of possible DNAJB6a truncation was found. In vivo subcutaneous nude mice tumorigenicity assays showed that over-expression of DNAJB6a, but not DNAJB6b, suppresses tumor growth at the primary site, while DNAJB6a silencing enhances tumor growth. The suppressive effect of DNAJB6a depends on nuclear localization of the protein and the HPD tripeptide motif in the N-terminal J domain. In vitro function studies show that DNAJB6a over-expression impairs cell proliferation by suppressing G1/S transition. AKT1 phosphorylation is down-regulated in DNAJB6a over-expressed cells, leading to up-regulation of p27KIP1 protein expression and down-regulation of cyclin E1 protein expression, the G1/S transition promoter, in an AKT1-dependent manner. DNAJB6a silencing results in the opposite effect. Over-expression of DNAJB6b, but not DNAJB6a, instead suppresses lung colonization in an experimental metastasis assay, and prolongs survival of the mice. Silencing of DNAJB6a in immortalized normal esophageal epithelial cells initially induces a senescence-like phenotype with greatly reduced proliferation possibly due to oncogenic stress from up-regulation of AKT1 phosphorylation and cyclin E1 protein expression, but promotes EMT-like molecular alterations by up-regulating STAT3 phosphorylation and TWIST1 protein expression and resumes proliferation after prolonged culture. In summary, these results suggest that DNAJB6 plays a critical role in ESCC initiation, development, and metastasis and provides valuable insight into the understanding of ESCC tumorigenesis and metastasis. This suggests its usefulness as a biomarker candidate for detecting early ESCC tumor initiation.