Tumor suppressor functions of DAXX through histone H3.3 deposition pathway in pancreatic neuroendocrine tumor

Purpose: Pancreatic neuroendocrine tumors (PanNETs) have considerable malignant potential. Frequent somatic mutations and loss of DAXX protein expression have been frequently found in PanNETs. DAXX is known as a transcriptional repressor, however, molecular functions underlying loss of DAXX remain unclear in PanNETs. Experimental Design: We evaluated DAXX-knockdown (KD) and -knockout (KO) PanNET cells were analyzed for in vitro and vivo. The target genes were screened by microarray and chromatin immunoprecipitation (ChIP) assays for DAXX, histone H3.3 and H3K9me3 complex. Results: Microarray and ChIP analyses of DAXX-KD/KO identified 13 genes as the direct targets of DAXX transcriptional repressor. Among them, 5 genes were suppressed by DAXX/H3.3/H3K9me3 pathway. DAXX-KD/KO increased sphere forming activity, but it was suppressed by knockdown of the target gene. In xenograft models, tumorigenicity was significantly increased in DAXX-KO cells with high expression of the target. Clinically, higher recurrence was recognized in PanNETs with low expression of DAXX and high expression of the target than others. Conclusions: DAXX plays as a tumor suppressor and DAXX/H3.3 complex suppresses target genes by promoting H3K9me3 in PanNETs. DAXX and its target molecule might be effective biomarkers and therapeutic candidates. Two human NET cell lines QGP1 (PanNET) and NCI-H727 (bronchial NET) were obtained from the Health Science Research Resources Bank (Osaka, Japan) and the American Type Culture Collection (Manassas, VA), respectively. Microarray analysis was performed to compare gene expression patterns between DAXX-KD and DAXX-NC QGP1 cells. After uncharacterized genes were excluded, 81 and 96 genes were up- and down-regulated at >2-fold changes in DAXX-KD QGP1 cells.