Zaklina Kovacevic

Tuning cell cycle regulation with an iron key

Iron (Fe) is essential for cellular metabolism e.g., DNA synthesis and its depletion causes G1/S arrest and apoptosis. Considering this, Fe chelators have been shown to be effective anti-proliferative agents. In order to understand the anti-tumor activity of Fe chelators, the mechanisms responsible for G1/S arrest and apoptosis after Fe-depletion have been investigated. These studies reveal a multitude of cell cycle control molecules are regulated by Fe. These include p53, p27Kip1, cyclin D1 and cyclin-dependent kinase 2 (cdk2). Additionally, Fe-depletion up-regulates the mRNA levels of the cdk inhibitor, p21CIP1/WAF1, but paradoxically down-regulates its protein expression. This effect could contribute to the apoptosis observed after Fe-depletion. Iron-depletion also leads to proteasomal degradation of p21CIP1/WAF1 and cyclin D1 via an ubiquitin-independent pathway. This is in contrast to the mechanism in Fe-replete cells, where it occurs by ubiquitin-dependent proteasomal degradation. Up-regulation of p38 mitogen-activated protein kinase (MAPK) after Fe-depletion suggests another facet of cell cycle regulation responsible for inhibition of proliferation and apoptosis induction. Elucidation of the complex effects of Fe-depletion on the expression of cell cycle control molecules remains at its infancy. However, these processes are important to dissect for complete understanding of Fe-deficiency and the development of chelators for cancer treatment.

The iron chelators Dp44mT and DFO inhibit TGF-β-induced epithelial-mesenchymal transition via up-regulation of N-Myc downstream-regulated gene 1 (NDRG1).

Background: NDRG1 is an iron-regulated metastasis suppressor which is up-regulated by iron depletion and may be involved in the epithelial-mesenchymal transition (EMT). Results: NDRG1 is involved in the EMT through the SMAD and Wnt pathways. Conclusion: Iron chelators could inhibit the TGF-β-induced EMT via NDRG1. Significance: The results are important for understanding the molecular roles of iron in proliferation and metastasis. The epithelial-mesenchymal transition (EMT) is a key step for cancer cell migration, invasion, and metastasis. Transforming growth factor-β (TGF-β) regulates the EMT and the metastasis suppressor gene, N-myc downstream-regulated gene-1 (NDRG1), could play a role in regulating the TGF-β pathway. NDRG1 expression is markedly increased after chelator-mediated iron depletion via hypoxia-inducible factor 1α-dependent and independent pathways (Le, N. T. and Richardson, D. R. (2004) Blood 104, 2967–2975). Moreover, novel iron chelators show marked and selective anti-tumor activity and are a potential new class of anti-metabolites. Considering this, the current study investigated the relationship between NDRG1 and the EMT to examine if iron chelators can inhibit the EMT via NDRG1 up-regulation. We demonstrated that TGF-β induces the EMT in HT29 and DU145 cells. Further, the chelators, desferrioxamine (DFO) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), inhibited the TGF-β-induced EMT by maintaining E-cadherin and β-catenin, at the cell membrane. We then established stable clones with NDRG1 overexpression and knock-down in HT29 and DU145 cells. These data showed that NDRG1 overexpression maintained membrane E-cadherin and β-catenin and inhibited TGF-β-stimulated cell migration and invasion. Conversely, NDRG1 knock-down caused morphological changes from an epithelial- to fibroblastic-like phenotype and also increased migration and invasion, demonstrating NDRG1 knockdown induced the EMT and enhanced TGF-β effects. We also investigated the mechanisms involved and showed the TGF-β/SMAD and Wnt pathways were implicated in NDRG1 regulation of E-cadherin and β-catenin expression and translocation. This study demonstrates that chelators inhibit the TGF-β-induced EMT via a process consistent with NDRG1 up-regulation and elucidates the mechanism of their activity.

Novel Thiosemicarbazone Iron Chelators Induce Up-Regulation and Phosphorylation of the Metastasis Suppressor N-myc Down-Stream Regulated Gene 1: A New Strategy for the Treatment of Pancreatic Cancer

Pancreatic cancer is an aggressive neoplasm, with a mortality rate close to 100%. The most successful agent for pancreatic cancer treatment is gemcitabine, although the overall effect in terms of patient survival remains very poor. This study was initiated to evaluate a novel class of anticancer agents against pancreatic cancer. This group of compounds belongs to the dipyridyl thiosemicarbazone class that have been shown to have potent and selective activity against a range of different neoplasms in vitro and in vivo. We demonstrate for the first time in pancreatic cancer that these agents increase the expression of the growth and metastasis suppressor N-myc downstream-regulated gene 1 and its phosphorylation at Ser330 and Thr346 that is important for its activity against this tumor. In addition, these agents increased expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1, whereas decreasing cyclin D1 in pancreatic cancer cells. Together, these molecular alterations account, in part, for the pronounced antitumor activity observed. Indeed, these agents had significantly higher antiproliferative activity in vitro than the established treatments for pancreatic cancer, namely gemcitabine and 5-fluorouracil. Studies in vivo demonstrated that a novel thiosemicarbazone, namely di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone hydrochloride, completely inhibited the growth of pancreatic cancer xenografts with no evidence of marked alterations in normal tissue histology. Together, our studies have identified molecular effectors of a novel and potent antitumor agent that could be useful for pancreatic cancer treatment.

Iron chelators for the treatment of cancer

The study of iron chelators as anti-tumor agents is still in its infancy. Iron is important for cellular proliferation and this is demonstrated by observations that iron-depletion results in cell cycle arrest and also apoptosis. In addition, many iron chelators are known to inhibit ribonucleotide reductase, the iron-containing enzyme that is the rate-limiting step for DNA synthesis. Desferrioxamine is a well known chelator used for the treatment of iron-overload disease, but it has also been shown to possess anti-cancer activity. Another class of chelators, namely the thiosemicarbazones, have been shown to possess anti-cancer activity since the 1950s, although their mechanism(s) of action have only recently been more comprehensively elucidated. In fact, the redox activity of thiosemicarbazone iron complexes is thought to be important in mediating their potent cytotoxicity. Moreover, unlike typical iron chelators which simply act to deplete tumors of iron, several thiosemicarbazones (i.e., Bp44mT and Dp44mT) do not induce this effect, their anti-cancer efficacy being due to other mechanisms e.g., redox activity. Other reports have also shown that some thiosemicarbazones inhibit topoisomerase IIα, demonstrating that this class of agents have multiple molecular targets and act by various mechanisms. The most well characterized thiosemicarbazone iron chelator in terms of its assessment in humans is 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP). Observations from these clinical trials highlight the less than optimal activity of this ligand and several side effects related to its use, including myelo-suppression, hypoxia and methemoglobinemia. The mechanisms responsible for these latter effects must be elucidated and the design of the ligand altered to minimize these problems and increase efficacy. This review discusses the development of chelators as unique agents for cancer treatment.

The iron-regulated metastasis suppressor NDRG1 targets NEDD4L, PTEN, and SMAD4 and inhibits the PI3K and Ras signaling pathways.

AIMS The metastasis suppressor gene, N-myc downstream regulated gene-1 (NDRG1), is negatively correlated with tumor progression in multiple neoplasms, including pancreatic cancer. Moreover, NDRG1 is an iron-regulated gene that is markedly upregulated by cellular iron-depletion using novel antitumor agents such as the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), in pancreatic cancer cells. However, the exact function(s) of NDRG1 remain to be established and are important to elucidate. RESULTS In the current study, using gene-array analysis along with NDRG1 overexpression and silencing, we identified the molecular targets of NDRG1 in three pancreatic cancer cell lines. We demonstrate that NDRG1 upregulates neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) and GLI-similar-3 (GLIS3). Further studies examining the downstream effects of NEDD4L led to the discovery that NDRG1 affects the transforming growth factor-β (TGF-β) pathway, leading to the upregulation of two key tumor suppressor proteins, namely phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and mothers against decapentaplegic homolog-4 (SMAD4). Moreover, NDRG1 inhibited the phosphatidylinositol 3-kinase (PI3K) and Ras oncogenic pathways. INNOVATION This study provides significant insights into the mechanisms underlying the antitumor activity of NDRG1. For the first time, a role for NDRG1 is established in regulating the key signaling pathways involved in oncogenesis (TGF-β, PI3K, and Ras pathways). CONCLUSION The identified target genes of NDRG1 and their effect on the TGF-β signaling pathway reveal its molecular function in pancreatic cancer and a novel therapeutic avenue.

The metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), upregulates p21 via p53-independent mechanisms

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), has been shown to markedly reduce metastasis of numerous tumors. The current study was focused on further elucidating the molecular mechanisms behind the antitumor function of NDRG1. We have identified for the first time that NDRG1 upregulates the potent cyclin-dependent kinase inhibitor, p21. This effect was observed in three different cancer cell types, including PC3MM and DU145 prostate cancer cells and H1299 lung carcinoma cells, and occurred independently of p53. In addition, reducing NDRG1 expression using short hairpin RNA in PC3MM and DU145 cells resulted in significantly reduced p21 protein levels. Hence, p21 is closely correlated with NDRG1 expression in these latter cell types. Examining the mechanisms behind the effect of NDRG1 on p21 expression, we found that NDRG1 upregulated p21 via transcriptional and posttranscriptional mechanisms in prostate cancer cells, although its effect on H1299 cells was posttranscriptional only. Further studies identified two additional NDRG1 protein targets. The dominant-negative p63 isoform, ΔNp63, which has been found to inhibit p21 transcription, was downregulated by NDRG1. On the other hand, a truncated 50 kDa MDM2 isoform (p50(MDM2)), which may protect p21 from proteasomal degradation, was upregulated by NDRG1. The downregulation of ΔNp63 and upregulation of p50(MDM2) are potential mechanisms by which NDRG1 increases p21 expression in these cells. Additional functional studies identified that NDRG1 inhibits cancer cell migration, suggesting that p21 is a molecular player in its antimetastatic activity.

The Medicinal Chemistry of Novel Iron Chelators for the Treatment of Cancer

Cancer is one of the leading causes of death worldwide and there is an increasing need for novel anti-tumor therapeutics with greater selectivity and potency. A new strategy in the treatment of cancer has focused on targeting an essential cell metabolite, iron (Fe). Iron is vital for cell growth and metabolism, forming a crucial component of the active site of ribonucleotide reductase (RR), the rate-limiting enzyme in DNA synthesis. Cancer cells in particular require large amounts of Fe to proliferate, making them more susceptible to the Fe deficiency caused by Fe chelators. Beginning with primordial siderophores, Fe chelators have since evolved to a new generation of potent and efficient anti-cancer agents. Recently, investigations have led to the generation of novel di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) ligands that demonstrate marked and selective anti-tumor activity both in vitro and in vivo against a wide spectrum of tumors. The mechanism of action of these novel ligands includes alterations in the expression of key regulatory molecules as well as the generation of redox active Fe complexes. Interestingly, non-synthetic Fe chelators including silybin and curcumin, both of which are derived from plants, also have vast potential in the treatment of cancer. This review explores the development of novel Fe chelators for the treatment of cancer and their mechanisms of action.

The iron-regulated metastasis suppressor, Ndrg-1: Identification of novel molecular targets

A recently identified metastasis suppressor, N-myc downstream regulated gene-1 (Ndrg-1), has been shown to reduce the invasion and metastasis of breast, colon, prostate and pancreatic cancer. Among its many functions, Ndrg-1 is involved in modulating differentiation, proliferation and angiogenesis. However, knowledge of the molecular targets of Ndrg-1 is limited. The current study has focused on examining the functions of Ndrg-1 in a number of different cancer cell models including prostate, colon, lung and pancreatic cancer to elucidate the known pleiotropic nature of this protein. Furthermore, the potential gene targets of Ndrg-1 were analyzed using whole genome gene array revealing a substantial number of genes whose expression was affected by this metastasis suppressor. Significantly, Ndrg-1 up-regulated thiamine triphosphatase (Thtpa) expression in three of the four cell models. Thtpa is known to decrease the levels of the energy currency molecule, thiamine triphosphate, suggesting a potential pathway for the anti-proliferative effects of Ndrg-1. Furthermore, Ndrg-1 reduced the protein levels of cathepsin C which plays a role in invasion, indicating a potential mechanism of its anti-metastatic role in pancreatic cancer cells. These findings provide a potential link between the observed functions of Ndrg-1 and its molecular targets, further demonstrating its anti-metastatic effect.

The metastasis suppressor NDRG1 modulates the phosphorylation and nuclear translocation of β-catenin through mechanisms involving FRAT1 and PAK4

ABSTRACT N-myc downstream-regulated gene 1 (NDRG1) is a potent metastasis suppressor that has been demonstrated to inhibit the transforming growth factor &bgr; (TGF-&bgr;)-induced epithelial-to-mesenchymal transition (EMT) by maintaining the cell-membrane localization of E-cadherin and &bgr;-catenin in prostate and colon cancer cells. However, the precise molecular mechanism remains unclear. In this investigation, we demonstrate that NDRG1 inhibits the phosphorylation of &bgr;-catenin at Ser33/37 and Thr41 and increases the levels of non-phosphorylated &bgr;-catenin at the plasma membrane in DU145 prostate cancer cells and HT29 colon cancer cells. The mechanism of inhibiting &bgr;-catenin phosphorylation involves the NDRG1-mediated upregulation of the GSK3&bgr;-binding protein FRAT1, which prevents the association of GSK3&bgr; with the Axin1–APC–CK1 destruction complex and the subsequent phosphorylation of &bgr;-catenin. Additionally, NDRG1 is shown to modulate the WNT–&bgr;-catenin pathway by inhibiting the nuclear translocation of &bgr;-catenin. This is mediated through an NDRG1-dependent reduction in the nuclear localization of p21-activated kinase 4 (PAK4), which is known to act as a transporter for &bgr;-catenin nuclear translocation. The current study is the first to elucidate a unique molecular mechanism involved in the NDRG1-dependent regulation of &bgr;-catenin phosphorylation and distribution.

Targeting the metastasis suppressor, NDRG1, using novel iron chelators: regulation of stress fiber-mediated tumor cell migration via modulation of the ROCK1/pMLC2 signaling pathway.

The iron-regulated metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), is up-regulated by cellular iron depletion mediated by iron chelators and can inhibit cancer cell migration. However, the mechanism of how NDRG1 achieves this effect remains unclear. In this study, we implemented established and newly constructed NDRG1 overexpression and knockdown models using the DU145, HT29, and HCT116 cancer cell lines to investigate the molecular basis by which NDRG1 exerts its inhibitory effect on cell migration. Using these models, we demonstrated that NDRG1 overexpression inhibits cell migration by preventing actin-filament polymerization, stress fiber assembly and formation. In contrast, NDRG1 knockdown had the opposite effect. Moreover, we identified that NDRG1 inhibited an important regulatory pathway mediated by the Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)/phosphorylated myosin light chain 2 (pMLC2) pathway that modulates stress fiber assembly. The phosphorylation of MLC2 is a key process in inducing stress fiber contraction, and this was shown to be markedly decreased or increased by NDRG1 overexpression or knockdown, respectively. The mechanism involved in the inhibition of MLC2 phosphorylation by NDRG1 was mediated by a significant (P < 0.001) decrease in ROCK1 expression that is a key kinase involved in MLC2 phosphorylation. Considering that NDRG1 is up-regulated after cellular iron depletion, novel thiosemicarbazone iron chelators (e.g., di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone) were demonstrated to inhibit ROCK1/pMLC2-modulated actin-filament polymerization, stress fiber assembly, and formation via a mechanism involving NDRG1. These results highlight the role of the ROCK1/pMLC2 pathway in the NDRG1-mediated antimetastatic signaling network and the therapeutic potential of iron chelators at inhibiting metastasis.