Acireductone dioxygenase 1 (ADI1) is regulated by cellular iron by a mechanism involving the iron chaperone, PCBP1, with PCBP2 acting as a potential co-chaperone.

The iron-containing protein, acireductone dioxygenase 1 (ADI1), is a dioxygenase important for polyamine synthesis and proliferation. Using differential proteomics, the studies herein demonstrated that ADI1 was significantly down-regulated by cellular iron depletion. This is important, since ADI1 contains a non-heme, iron-binding site critical for its activity. Examination of multiple human cell-types demonstrated a significant decrease in ADI1 mRNA and protein after incubation with iron chelators. The decrease in ADI1 after iron depletion was reversible upon incubation of cells with the iron salt, ferric ammonium citrate (FAC). A significant decrease in ADI1 mRNA levels was observed after 14 h of iron depletion. In contrast, the chelator-mediated reduction in ADI1 protein occurred earlier after 10 h of iron depletion, suggesting additional post-transcriptional regulation. The proteasome inhibitor, MG-132, prevented the iron chelator-mediated decrease in ADI1 expression, while the lysosomotropic agent, chloroquine, had no effect. These results suggest an iron-dependent, proteasome-mediated, degradation mechanism. Poly r(C)-binding protein (PCBPs) 1 and 2 act as iron delivery chaperones to other iron-containing dioxygenases and were shown herein for the first time to be regulated by iron levels. Silencing of PCBP1, but not PCBP2, led to loss of ADI1 expression. Confocal microscopy co-localization studies and proximity ligation assays both demonstrated decreased interaction of ADI1 with PCBP1 and PCBP2 under conditions of iron depletion using DFO. These data indicate PCBP1 and PCBP2 interact with ADI1, but only PCBP1 plays a role in ADI1 expression. In fact, PCBP2 appeared to play an accessory role, being involved as a potential co-chaperone.

Pharmacological targeting and the diverse functions of the metastasis suppressor, NDRG1, in cancer.

N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that is regulated by hypoxia, metal ions including iron, the free radical nitric oxide (NO.), and various stress stimuli. This intriguing molecule exhibits diverse functions in cancer, inhibiting epithelial-mesenchymal transition (EMT), cell migration and angiogenesis by modulation of a plethora of oncogenes via cellular signaling. Thus, pharmacological targeting of NDRG1 signaling in cancer is a promising therapeutic strategy. Of note, novel anti-tumor agents of the di-2-pyridylketone thiosemicarbazone series, which exert the "double punch" mechanism by binding metal ions to form redox-active complexes, have been demonstrated to markedly up-regulate NDRG1 expression in cancer cells. This review describes the mechanisms underlying NDRG1 modulation by the thiosemicarbazones and the diverse effects NDRG1 exerts in cancer. As a major induction mechanism, iron depletion appears critical, with NO. also inducing NDRG1 through its ability to bind iron and generate dinitrosyl-dithiol iron complexes, which are then effluxed from cells. Apart from its potent anti-metastatic role, several studies have reported a pro-oncogenic role of NDRG1 in a number of cancer-types. Hence, it has been suggested that NDRG1 plays pleiotropic roles depending on the cancer-type. The molecular mechanism(s) underlying NDRG1 pleiotropy remain elusive, but are linked to differential regulation of WNT signaling and potentially differential interaction with the tumor suppressor, PTEN. This review discusses NDRG1 induction mechanisms by metal ions and NO. and both the anti- and possible pro-oncogenic functions of NDRG1 in multiple cancer-types and compares the opposite effects this protein exerts on cancer progression.

Thiosemicarbazones suppress expression of the c-Met oncogene by mechanisms involving lysosomal degradation and intracellular shedding.

Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells. NDRG1 silencing without Dp44mT and DpC up-regulated c-Met expression, demonstrating that NDRG1 modulates c-Met levels. Dp44mT and DpC up-regulated NDRG1 by an iron-dependent mechanism and decreased c-Met levels, c-Met phosphorylation, and phosphorylation of its downstream effector, GRB2-associated binding protein 1 (GAB1). However, incubation with Dp44mT and DpC after NDRG1 silencing or silencing of the receptor tyrosine kinase inhibitor, mitogen-inducible gene 6 (MIG6), decreased c-Met and its phosphorylation, suggesting NDRG1- and MIG6-independent mechanism(s). Lysosomal inhibitors rescued the Dp44mT- and DpC-mediated c-Met down-regulation in DU145 cells. Confocal microscopy revealed that lysosomotropic agents and the thiosemicarbazones significantly increased co-localization between c-Met and lysosomal-associated membrane protein 2 (LAMP2). Moreover, generation of c-Met C-terminal fragment (CTF) and its intracellular domain (ICD) suggested metalloprotease-mediated cleavage. In fact, Dp44mT increased c-Met CTF while decreasing the ICD. Dp44mT and a γ-secretase inhibitor increased cellular c-Met CTF levels, suggesting that Dp44mT induces c-Met CTF levels by increasing metalloprotease activity. The broad metalloprotease inhibitors, EDTA and batimastat, partially prevented Dp44mT-mediated down-regulation of c-Met. In contrast, the ADAM inhibitor, TIMP metallopeptidase inhibitor 3 (TIMP-3), had no such effect, suggesting c-Met cleavage by another metalloprotease. Notably, Dp44mT did not induce extracellular c-Met shedding that could decrease c-Met levels. In summary, the thiosemicarbazones Dp44mT and DpC effectively inhibit oncogenic c-Met through lysosomal degradation and metalloprotease-mediated cleavage.

Roads to melanoma: Key pathways and emerging players in melanoma progression and oncogenic signaling.

Melanoma has markedly increased worldwide during the past several decades in the Caucasian population and is responsible for 80% of skin cancer deaths. Considering that metastatic melanoma is almost completely resistant to most current therapies and is linked with a poor patient prognosis, it is crucial to further investigate potential molecular targets. Major cell-autonomous drivers in the pathogenesis of this disease include the classical MAPK (i.e., RAS-RAF-MEK-ERK), WNT, and PI3K signaling pathways. These pathways play a major role in defining the progression of melanoma, and some have been the subject of recent pharmacological strategies to treat this belligerent disease. This review describes the latest advances in the understanding of melanoma progression and the major molecular pathways involved. In addition, we discuss the roles of emerging molecular players that are involved in melanoma pathogenesis, including the functional role of the melanoma tumor antigen, p97/MFI2 (melanotransferrin).