Yes-associated protein (YAP), the nuclear effector of the Hippo pathway, is a key regulator of organ size and a candidate human oncogene located at chromosome 11q22. Since we previously reported amplification of 11q22 region in esophageal squamous cell carcinoma (ESCC), in this study we focused on the clinical significance and biological functions of YAP in this tumor. Frequent overexpression of YAP protein was observed in ESCC cells including those with a robust amplicon at position 11q22. Overexpression of the YAP protein was frequently detected in primary tumors of ESCC as well. Patients with YAP-overexpressing tumors had a worse overall rate of survival than those with non-expressing tumors, and YAP positivity was independently associated with a worse outcome in the multivariate analysis. Further analyses in cells in which YAP was either overexpressed or depleted confirmed that cell proliferation was promoted in a YAP isoform-independent but YAP expression level-dependent manner. YAP depletion inhibited cell proliferation mainly in the G(0)-G(1) phase and induced an increase in CDKN1A/p21 transcription but a decrease in BIRC5/survivin transcription. Our results indicate that YAP is a putative oncogene in ESCC and it represents a potential diagnostic and therapeutic target.

Although we have identified two putative targets, ATF3 and CENPF, for a frequently gained/amplified region around 1q32-q41 in esophageal squamous cell carcinoma (ESCC), it is possible that other amplification targets remain to be identified. In this study, we tested whether SET and MYND domain-containing protein 2 (SMYD2), located between those two genes and encoding a lysine methyltransferase for histone H3K36 and p53K370 that regulates transcription and inhibits transactivation activity, respectively, acts as a cancer-promoting gene through activation/overexpression in ESCC. Frequent overexpression of SMYD2 messenger RNA and protein was observed in KYSE150 cells with remarkable amplification at 1q32-41.1 and other ESCC cell lines (11/43 lines, 25.6%). Overexpression of SMYD2 protein was frequently detected in primary tumor samples of ESCC (117/153 cases, 76.5%) as well and significantly correlated with gender, venous invasion, the pT category in the tumor-lymph node-metastases classification and status of recurrence. Patients with SMYD2-overexpressing tumors had a worse overall rate of survival than those with non-expressing tumors, and SMYD2 positivity was independently associated with a worse outcome in the multivariate analysis. Knockdown of SMYD2 expression inhibited and ectopic overexpression of SMYD2 promoted the proliferation of ESCC cells in a TP53 mutation-independent but SMYD2 expression-dependent manner. These findings suggest that SMYD2 plays an important role in tumor cell proliferation through its activation/overexpression and highlight its usefulness as a prognosticator and potential therapeutic target in ESCC.

Metastasis is associated with poor prognosis in cancers. Exosomes, which are packed with RNA and proteins and are released in all biological fluids, are emerging as an important mediator of intercellular communication. However, the function of exosomes remains poorly understood in cancer metastasis. Here, we demonstrate that exosomes isolated by size-exclusion chromatography from a highly metastatic human oral cancer cell line, HOC313-LM, induced cell growth through the activation of ERK and AKT as well as promoted cell motility of the poorly metastatic cancer cell line HOC313-P. MicroRNA (miRNA) array analysis identified two oncogenic miRNAs, miR-342–3p and miR-1246, that were highly expressed in exosomes. These miRNAs were transferred to poorly metastatic cells by exosomes, which resulted in increased cell motility and invasive ability. Moreover, miR-1246 increased cell motility by directly targeting DENN/MADD Domain Containing 2D (DENND2D). Taken together, our findings support the metastatic role of exosomes and exosomal miRNAs, which highlights their potential for applications in miRNA-based therapeutics.

The YAP1 gene encodes a potent new oncogene and stem cell factor. However, in some cancers, the YAP1 gene plays a role of tumor suppressor. At present, the gene and its products are intensely studied and its cDNAs are used as transgenes in cellular and animal models. Here, we report 4 new potential mRNA splicing isoforms of the YAP1 gene, bringing the total number of isoforms to 8. We detected all 8 YAP1 isoforms in a panel of human tissues and evaluated the expression of the longest isoform of YAP1 (YAP1-2δ) using Real Time PCR. All YAP1 isoforms are barely detectable in human leukocytes compared to fair levels of expression found in other human tissues. We analyzed the structure of the genomic region that gave rise to alternatively spliced YAP1 transcripts in different metazoans. We found that YAP1 isoforms, which utilize exon 6 emerged in evolution with the appearance of amniotes. Interestingly, 6 YAP1 isoforms, which contain the exon 5 extension, exon 6 or both would have their leucine zipper region disrupted in the predicted protein product, compared to the intact leucine zipper found in two YAP1 (α) isoforms. This observation has direct functional ramifications for YAP1 signaling. We also propose a normalized nomenclature for the mRNA splice variants of YAP1 gene, which should aid in the characterization of signaling differences among the potential protein products of the YAP1 gene.

Epithelial-mesenchymal transition (EMT) has a major role in cancer progression, as well as normal organ development and human pathology such as organ fibrosis and wound healing. Here, we performed a gene expression array specialized in EMT of colorectal cancer (CRC). From a comprehensive gene expression analysis using epithelial-and mesenchymal-like CRC cell lines, and following the ontology (GO) analysis, SIX1 gene was identified to be an EMT-related gene in CRC. Using SW480 cells stably transfected with a SIX1 expression construct and their control counterparts, we demonstrated that SIX1 overexpression represses CDH1 expression and promotes EMT in CRC. SIX1-induced CDH1 repression and EMT in CRC cells were correlated at least in part with posttranscriptional ZEB1 activation and miR-200-family transcriptional repression. In primary tumors of CRC, in accord with the functional findings, aberrant expression of SIX1 in cancer cells was observed at the disruption of the basement membrane and at the tumor invasive front, where tumor cells underwent EMT in vivo. Taken together, SIX1 overexpression is suggested to occur in carcinogenesis, and contribute to repression of CDH1 expression and promotion of EMT partly through repression of miR-200-family expression and activation of ZEB1 in CRC.