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

Abstract

The iron-regulated metastasis suppressor, N-myc downstreamregulated 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 actinfilament 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.

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways

“…We also previously demonstrated that NDRG1 inhibits a number of EGFR downstream pathways, including PI3K, RAS, etc. (6,8,(17)(18)(19), leading to inhibition of cell migration and invasion (4,20). Together, these results indicate that NDRG1 is a highly promising therapeutic target in cancer cells.…”

“…This occurs due to the ability of these ligands to bind and sequester intracellular iron leading to activation of HIF1␣-dependent and -independent mechanisms (1,24). Furthermore, this response to these agents has been shown to occur in both PANC-1 and HT-29 cells (3,4,20). These agents were also demonstrated to broadly inhibit a number of oncogenic pathways via NDRG1-dependent mechanisms, including TGF-␤, WNT, PI3K, etc.…”

“…The potential of these novel compounds is underscored by their marked ability to inhibit tumor cell metastasis in vivo (18) and overcome cancer cell multidrug resistance mediated by P-glycoprotein in vitro and in vivo (23,26). Interestingly, the anti-metastatic effects of these agents were demonstrated to be dependent on their ability to up-regulate NDRG1 in vitro and in vivo (4,18,20), further establishing this metastasis suppressor as an important molecular target for the treatment of cancer.…”

“…This was important to assess, as the EGFR/HER2, EGFR/HER3, and in particular the HER2/HER3 heterodimers are potent acti-vators of a number of oncogenic downstream signaling pathways (30,(55)(56)(57)(58)(59)(60). Considering the ability of NDRG1 to inhibit multiple oncogenic signaling pathways in both PANC-1 and HT-29 cells (4,6,8,17,20), we examined whether NDRG1 overexpression in these cells was able to modulate the formation of the EGFR/HER2, EGFR/HER3, and HER2/HER3 heterodimers using co-immunoprecipitation.…”

“…and HT-29 Cells-Considering that multiple cell signaling pathways are markedly affected by the metastasis suppressor, NDRG1, in both PANC-1 pancreatic and HT-29 colon cancer cells (4,8,17,20), these latter cell types were utilized to examine whether NDRG1 affected EGFR expression and activation. This was important to establish, as EGFR is a key regulator of numerous oncogenic downstream signaling pathways (31,43) that were previously found to be inhibited by NDRG1 (4,6,8,(17)(18)(19).…”