Abstract
Heightened DJ-1 (Park7) expression is associated with a reduction in chemotherapeutic-induced cell death and poor prognosis in several cancers, whereas the loss of DJ-1 function is found in a subgroup of Parkinson disease associated with neuronal death. This study describes a novel pathway by which DJ-1 modulates cell survival. Mass spectrometry shows that DJ-1 interacts with BBS1, CLCF1, MTREF, and Cezanne/OTUD7B/Za20d1. Among these, Cezanne is a known deubiquitination enzyme that inhibits NF-κB activity. DJ-1/Cezanne interaction is confirmed by co-immunoprecipitation of overexpressed and endogenous proteins, maps to the amino-terminal 70 residues of DJ-1, and leads to the inhibition of the deubiquitinating activity of Cezanne. Microarray profiling of shRNA-transduced cells shows that DJ-1 and Cezanne regulate IL-8 and ICAM-1 expression in opposing directions. Similarly, DJ-1 enhances NF-κB nuclear translocation and cell survival, whereas Cezanne reduces these outcomes. Analysis of mouse Park7(-/-) primary cells confirms the regulation of ICAM-1 by DJ-1 and Cezanne. As NF-κB is important in cellular survival and transformation, IL-8 functions as an angiogenic factor and pro-survival signal, and ICAM-1 has been implicated in tumor progression, invasion, and metastasis; these data provide an additional modality by which DJ-1 controls cell survival and possibly tumor progression via interaction with Cezanne.
Highlights
Lung cancer is the leading cause of cancer-related mortality in the world
That work uncovered a broader role of DJ-1 in cell survival outside of the nervous system
DJ-1 has been shown to be a central molecule in cell survival by positively regulating Nrf2-dependent detoxification pathways [19], inhibiting p53-mediated cell cycle checkpoints [20] possibly via caspase-6 [51], and inducing Akt activity through the inhibition of Phosphatase and Tensin homolog (PTEN) [15]
Summary
Cell Culture, Treatments, and Plasmid Constructs—H157 cells were grown in RPMI 1640 (Invitrogen) with 10% FCS (HyClone). After H2O2 or vehicle control treatment, samples were lysed in 0.5% Triton X-100 lysis buffer and immunoprecipitated with antiFLAG M2-agarose (Sigma). Affymetrix Analysis—RNA was harvested from H157 cells transduced with DJ-1, Cezanne, or mutant control shRNA lentivirus using an RNeasy kit (Qiagen). The bacteria were lysed by sonication in the presence of hen egg lysozyme (Sigma) and Benzonase (Novagen), and the target recombinant proteins were purified using a nickel-nitrilotriacetic acid agarose (Invitrogen) column. Luciferase Reporter Assay—HEK293T cells transduced with Cezanne, DJ-1, or control shRNA-containing lentivirus were plated in 6-well dishes at 4 ϫ 105/well. Data are shown as luciferase relative light units in NF-B reporter construct containing sample normalized to a matched SV40 control. Experiments were performed in triplicate with error bars representing S.D. in Figs. 4 and 5
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