During the metastatic phase, cancer cells require the dissolution of cadherin-mediated cell-cell adhesion and a dramatic re-organization of the cytoskeleton through epithelial-mesenchymal transition (EMT), thereby acquiring migratory and invasive capabilities. In most tumors, EMT is accompanied by hypoxia. However, the intracellular signaling molecule that mediates hypoxia-induced EMT remained overlooked. By utilizing the microarray database system of the Cancer Genome Atlas, we identified ubiquitin-specific protease 47 (USP47), a deubiquitinating enzyme, as a potential mediator of hypoxia-induced EMT. Immunofluorescence staining of human colorectal tissue microarrays revealed that USP47 is overexpressed in colorectal adenocarcinoma tissues compared with normal adjacent tissues. The expression of USP47 was found to be elevated in three different human colorectal cancer cell lines. The enhancement of USP47 in colorectal cancer cells under hypoxic conditions induced the disassembly of E-cadherin and promoted EMT through deubiquitination of Snail. Silencing of USP47 accelerated the proteasomal degradation of Snail and inhibited EMT. Notably, hypoxia-induced USP47 upregulation was mediated by Sox9. These results demonstrate, for the first time, the role for USP47, as a novel target of Sox9, in the regulation of EMT and metastasis of colorectal cancer cells.

The E2F members have been divided into transcription activators (E2F1-E2F3) and repressors (E2F4-E2F8). E2F8 with E2F7 has been known to play an important physiologic role in embryonic development and cell cycle regulation by repressing E2F1. However, the function of E2F8 in cancer cells is unknown. E2F8 expression was assessed by immunoblotting or immunofluorescence staining in human lung cancer (LC) cells and tissues from LC patients (n = 45). Cell proliferation, colony formation, and invasion analysis were performed to evaluate the role of E2F8 in LC. Microarray analysis was used to determine the target genes of E2F8. The regulation of E2F8 on the expression of ubiquitin-like PHD and RING domain-containing 1 (UHRF1), one of E2F8 target genes, was determined using chromatin immunoprecipitation and promoter activity assays. Human LC xenograft models were used to determine the effects of inhibiting E2F8 by siRNAs (n = 7 per group) or antisense morpholino (n = 8 per group) on tumor growth. Survival was analyzed using the Kaplan-Meier method and group differences by the Student’s t test. All statistical tests were two-sided. LC tumors overexpressed E2F8 compared with normal lung tissues. Depletion of E2F8 inhibited cell proliferation and tumor growth. E2F8 knockdown statistically significantly reduced the expression of UHRF1 (~60%-70%, P < .001), and the direct binding of E2F8 on the promoter of UHRF1 was identified. Kaplan-Meier analysis with a public database showed prognostic significance of aberrant E2F8 expression in LC (HR = 1.91 95% CI = 1.21 to 3.01 in chemo-naïve patients, P = .0047). We demonstrated that E2F8 is overexpressed in LC and is required for the growth of LC cells. These findings implicate E2F8 as a novel therapeutic target for LC treatment.

Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.

There are multiple lines of evidence supporting that chronic inflammation is linked to carcinogenesis. Nuclear factor-kappaB (NF-kappaB), a major redox-sensitive transcription factor responsible for the induction of a wide array of pro-inflammatory genes, is frequently overactivated in many tumors. Moreover, constitutive activation of IkappaB kinase (IKK), a key regulator of NF-kappaB signaling, has been implicated in inflammation-associated tumorigenesis. Piceatannol (trans-3,4,3',5'-tetrahydroxystilbene; PIC) derived from grapes, rhubarb and sugarcane exhibits immunosuppressive and antitumorigenic activities in several cell lines, but the underlying mechanisms are poorly understood. In the present study, we found that PIC inhibited migration and anchorage-independent growth of human mammary epithelial cells (MCF-10A) treated with the prototypic tumor promoter, 12-O-tetradecanoylphorbol-13-aceate (TPA). PIC treatment suppressed the TPA-induced activation of NF-kappaB and expression of cyclooxygenase-2 (COX-2) in MCF-10A cells. We speculate that an electrophilic quinone formed as a consequence of oxidation of PIC bearing the catechol moiety may directly interact with critical cysteine thiols of IKKbeta, thereby inhibiting its catalytic activity. In support of this speculation, the reducing agent dithiothreitol abrogated the inhibitory effects of PIC on TPA-induced activation of NF-kappaB signaling and expression of COX-2. In addition, the inhibitory effects of PIC on NF-kappaB activation and COX-2 induction were blunted in cells expressing mutant IKKbeta (C179A) in which cysteine 179 was replaced by alanine. In conclusion, our results show that direct modification of IKKbeta by PIC, presumably at the cysteine 179 residue, blocks NF-kappaB activation signaling and COX-2 induction in TPA-treated MCF-10A cells and also migration and transformation of these cells.

Estrogen is converted by cytochrome P450 1B1 to 4-hydroxyestradiol (4-OHE₂), a putative carcinogenic metabolite of estrogen. This catechol estrogen metabolite is oxidized further to produce a reactive quinone via semiquinone. Redox cycling between 4-OHE₂ and its quinoid generates reactive oxygen species (ROS). ROS not only causes oxidative DNA damage but also promotes neoplastic transformation of initiated cells. In the present study, 4-OHE₂ induced anchorage-independent colony formation in human mammary epithelial cells (MCF-10A). MCF-10A cells treated with 4-OHE₂ exhibited increased accumulation of intracellular ROS. The antioxidant N-acetyl-l-cysteine inhibited the neoplastic transformation induced by 4-OHE₂. ROS overproduced by 4-OHE₂ increased the nuclear translocation of nuclear factor-κB (NF-κB) and its DNA binding through induction of IκB kinase α (IKKα) and IKKβ activities. The inhibition of the IKK activities with Bay 11-7082 significantly reduced the anchorage-independent growth induced by 4-OHE₂. The 4-OHE₂–induced activation of extracellular signal-regulated kinase and Akt resulted in enhanced IKK activities and phosphorylation of IκBα, thereby inducing NF-κB activation and anchorage-independent growth of MCF-10A cells. In conclusion, ROS, concomitantly overproduced during redox cycling of 4-OHE₂, activates IKK signaling, which may contribute to neoplastic transformation of MCF-10A cells. [Cancer Res 2009;69(6):2416–24]