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Purpose Although YAP1 and TAZ are believed to be equivalent downstream effectors of the Hippo pathway, differential expression of YAP1 or TAZ suggests distinct functions during cancer progression. The exact role of YAP1 and TAZ in esophageal cancer, the 6th leading cancer-related mortality in the world, remains elusive. Methods Following single or double manipulation of YAP1 or TAZ expression, we subjected these manipulated cells to proliferation, migration, invasion, and xenograft tumorigenesis assays. We used RT-qPCR and Western blotting to examine their expression in the manipulated cells with or without inhibition of transcription or translation. We also examined the impact of YAP1 or TAZ deregulation on clinical outcome of esophageal cancer patients from the TCGA database. Results We found that YAP1 functions as a tumor suppressor whereas TAZ exerts pro-tumor functions in esophageal cancer cells. We also found a significant increase in TAZ mRNA expression upon YAP1 depletion, but not vice versa, despite the downregulation of CTGF and CYR61, shared targets of YAP1 and TAZ, in xenografted tissue cells. In addition to transcriptional regulation, YAP1-mediated TAZ expression was found to occur via protein synthesis. Restored TAZ expression mitigated YAP1-mediated suppression of cellular behavior. By contrast, TAZ silencing reduced the promoting effect exerted by YAP1 depletion on cellular behaviors. The observed anti-tumor function of YAP1 was further supported by a better overall survival among esophageal cancer patients with a high YAP1 expression. Conclusion From our data we conclude that YAP1 functions as a suppressor and negatively regulates pro-tumor TAZ expression via transcriptional and translational control in esophageal cancer.

A powerful endo-chitosanase (CSN) previously described for a large scale preparation of chito-oligosaccharides (Cheng, C.-Y., and Li, Y.-K. (2000) Biotechnol. Appl. Biochem. 32, 197-203) was cloned from Aspergillus fumigatus and further identified as a member of glycosyl hydrolase family 75. We report here a study of gene expression, functional characterization, and mutation analysis of this enzyme. Gene cloning was accomplished by reverse transcription-PCR and inverse PCR. Within the 1382-bp Aspergillus gene (GenBank accession number AY190324), two introns (67 and 82 bp) and an open reading frame encoding a 238-residue protein containing a 17-residue signal peptide were characterized. The recombinant mature protein was overexpressed as an inclusion body in Escherichia coli, rescued by treatment with 5 m urea, and subsequently purified by cation exchange chromatography. A time course 1H NMR study on the enzymatic formation of chito-oligosaccharides confirmed that this A. fumigatus CSN is an inverting enzyme. Tandem mass spectrum analysis of the enzymatic hydrolysate revealed that the recombinant CSN can cleave linkages of GlcNAc-GlcN and GlcN-GlcN in its substrate, suggesting that it is a subclass I chitosanase. In addition, an extensive site-directed mutagenesis study on 10 conserved carboxylic amino acids of glycosyl hydrolase family 75 was performed. This showed that among these various mutants, D160N and E169Q lost nearly all activity. Further investigation using circular dichroism measurements of D160N, E169Q, wild-type CSN, and other active mutants showed similar spectra, indicating that the loss of enzymatic activity in D160N and E169Q was not because of changes in protein structure but was caused by loss of the catalytic essential residue. We conclude that Asp160 and Glu169 are the essential residues for the action of A. fumigatus endo-chitosanase.