Transcriptional Repressor DAXX Promotes Prostate Cancer Tumorigenicity via Suppression of Autophagy.

The DAXX transcriptional repressor was originally associated with apoptotic cell death. However, recent evidence that DAXX represses several tumor suppressor genes, including the DAPK1 and DAPK3 protein kinases, and is up-regulated in many cancers argues that a pro-survival role may predominate in a cancer context. Here, we report that DAXX has potent growth-enhancing effects on primary prostatic malignancy through inhibition of autophagy. Through stable gene knockdown and mouse subcutaneous xenograft studies, we demonstrate that DAXX promotes tumorigenicity of human ALVA-31 and PC3 prostate cancer (PCa) cells in vivo. Importantly, DAXX represses expression of essential autophagy modulators DAPK3 and ULK1 in vivo, revealing autophagy suppression as a mechanism through which DAXX promotes PCa tumorigenicity. Furthermore, DAXX knockdown increases autophagic flux in cultured PCa cells. Finally, interrogation of the Oncomine(TM) database suggests that DAXX overexpression is associated with malignant transformation in several human cancers, including prostate and pancreatic cancers. Thus, DAXX may represent a new cancer biomarker for the detection of aggressive disease, whose tissue-specific down-regulation can serve as an improved therapeutic modality. Our results establish DAXX as a pro-survival protein in PCa and reveal that, in the early stages of tumorigenesis, autophagy suppresses prostate tumor formation.

Daxx represses expression of a subset of antiapoptotic genes regulated by nuclear factor-kappaB.

Daxx is a nuclear protein that localizes to PML oncogenic domains, sensitizes cells to apoptosis, and functions as a transcriptional repressor. We found that Daxx represses the expression of several antiapoptotic genes regulated by nuclear factor-kappaB, including cIAP2, in human tumor cell lines. Daxx interacts with RelB and inhibits RelB-mediated transcriptional activation of the human cIAP2 gene promoter. Daxx also forms complexes with RelB while bound to its target sites in the cIAP2 promoter, as shown by electrophoretic mobility shift assays and chromatin immunoprecipitation experiments. Using cells from daxx-/- mouse embryos, we observed that levels of the corresponding murine c-IAP mRNA and protein are increased in cells lacking Daxx. Conversely, c-IAP mRNA and protein levels were reduced in relB-/- cells. Taken together, these observations provide a mechanism that links two previously ascribed functions of Daxx: transcriptional repression and sensitization to apoptosis.

Recurrent MLK4 Loss-of-Function Mutations Suppress JNK Signaling to Promote Colon Tumorigenesis.

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers, including frequently in colorectal cancer, where their function and pathobiological importance have been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant-negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain tumorigenic phenotypes, we reconstituted its signaling axis in colon cancer cells harboring MLK4-inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3, and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor-suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.

The DAXX co-repressor is directly recruited to active regulatory elements genome-wide to regulate autophagy programs in a model of human prostate cancer.

While carcinoma of the prostate is the second most common cause of cancer death in the US, current methods and markers used to predict prostate cancer (PCa) outcome are inadequate. This study was aimed at understanding the genome-wide binding and regulatory role of the DAXX transcriptional repressor, recently implicated in PCa. ChIP-Seq analysis of genome-wide distribution of DAXX in PC3 cells revealed over 59,000 DAXX binding sites, found at regulatory enhancers and promoters. ChIP-Seq analysis of DNA methyltransferase 1 (DNMT1), which is a key epigenetic partner for DAXX repression, revealed that DNMT1 binding was restricted to a small number of DAXX sites. DNMT1 and DAXX bound close to transcriptional activator motifs. DNMT1 sites were found to be dependent on DAXX for recruitment by analyzing DNMT1 ChIP-Seq following DAXX knockdown (K/D), corroborating previous findings that DAXX recruits DNMT1 to repress its target genes. Massively parallel RNA sequencing (RNA-Seq) was used to compare the transcriptomes of WT and DAXX K/D PC3 cells. Genes induced by DAXX K/D included those involved in autophagy, and DAXX ChIP-Seq peaks were found close to the transcription start sites (TSS) of autophagy genes, implying they are more likely to be regulated by DAXX. In conclusion, DAXX binds active regulatory elements and co-localizes with DNMT1 in the prostate cancer genome. Given DAXX's putative regulatory role in autophagy, future studies may consider DAXX as a candidate marker and therapeutic target for prostate cancer.

Cancer-associated loss-of-function mutations implicate DAPK3 as a tumor-suppressing kinase.

Cancer kinome sequencing studies have identified several protein kinases predicted to possess driver (i.e., causal) mutations. Using bioinformatic applications, we have pinpointed DAPK3 (ZIPK) as a novel cancer-associated kinase with functional mutations. Evaluation of nonsynonymous point mutations, discovered in DAPK3 in various tumors (T112M, D161N, and P216S), reveals that all three mutations decrease or abolish kinase activity. Furthermore, phenotypic assays indicate that the three mutations observed in cancer abrogate the function of the kinase to regulate both the cell cycle and cell survival. Coexpression of wild-type (WT) and cancer mutant kinases shows that the cancer mutants dominantly inhibit the function of the WT kinase. Reconstitution of a non-small cell lung cancer cell line that harbors an endogenous mutation in DAPK3 (P216S) with WT DAPK3 resulted in decreased cellular aggregation and increased sensitivity to chemotherapy. Our results suggest that DAPK3 is a tumor suppressor in which loss-of-function mutations promote increased cell survival, proliferation, cellular aggregation, and increased resistance to chemotherapy.

Daxx represses RelB target promoters via DNA methyltransferase recruitment and DNA hypermethylation.

The apoptosis-modulating protein Daxx functions as a transcriptional repressor that binds to and suppresses the activity of nuclear factor-kappaB member RelB, among other transcription factors. The mechanism by which Daxx represses RelB target genes remains elusive. In this report, we demonstrate that Daxx controls epigenetic silencing of RelB target genes by DNA methylation. Daxx potently represses the RelB target genes dapk1, dapk3, c-flip, and birc3 (ciap2) at both the mRNA and protein levels. Recruitment of Daxx to target gene promoters, and its ability to repress them, is RelB-dependent, as shown by experiments using relB(-/-) cells. Importantly, methylation of target promoters is decreased in daxx(-/-) cells compared with daxx(+/+) cells, and stable transfection of daxx(-/-) cells with Daxx restores DNA methylation. Furthermore, Daxx recruits DNA methyl transferase 1 (Dnmt1) to target promoters, resulting in synergistic repression. The observation that Daxx functions to target DNA methyltransferases onto RelB target sites in the genome provides a rare example of a gene-specific mechanism for epigenetic silencing. Given the documented role of several of the RelB-regulated genes in diseases, particularly cancer, the findings have implications for developing therapeutic strategies based on epigenetic-modifying drugs.

p21-activated kinase 1 (PAK1) interacts with the Grb2 adapter protein to couple to growth factor signaling.

A variety of intracellular signaling pathways are linked to cell surface receptor signaling through their recruitment by Src homology 2 (SH2)/SH3-containing adapter molecules. p21-activated kinase 1 (PAK1) is an effector of Rac/Cdc42 GTPases that has been implicated in the regulation of cytoskeletal dynamics, proliferation, and cell survival signaling. In this study, we describe the specific interaction of PAK1 with the Grb2 adapter protein both in vitro and in vivo. We identify the site of this interaction as the second proline-rich SH3 binding domain of PAK1. Stimulation of the epidermal growth factor receptor (EGFR) in HaCaT cells enhances the level of EGFR-associated PAK1 and Grb2, although the PAK1-Grb2 association is itself independent of this stimulation. A cell-permeant TAT-tagged peptide encompassing the second proline-rich SH3 binding domain of PAK1 simultaneously blocked Grb2 and activated EGFR association with PAK1, in vitro and in vivo, indicating that Grb2 mediates the interaction of PAK1 with the activated EGFR. Blockade of this interaction decreased the epidermal growth factor-induced extension of membrane lamellae. Thus Grb2 may serve as an important mechanism for linking downstream PAK signaling to various upstream pathways.