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Review
. 2021 Sep 10;10(9):2382.
doi: 10.3390/cells10092382.

The Oncogenic Signaling Disruptor, NDRG1: Molecular and Cellular Mechanisms of Activity

Jason Chekmarev  1 , Mahan Gholam Azad  1 , Des R Richardson  1   2
Affiliations
Review

The Oncogenic Signaling Disruptor, NDRG1: Molecular and Cellular Mechanisms of Activity

Jason Chekmarev et al. Cells. .

Abstract

NDRG1 is an oncogenic signaling disruptor that plays a key role in multiple cancers, including aggressive pancreatic tumors. Recent studies have indicated a role for NDRG1 in the inhibition of multiple tyrosine kinases, including EGFR, c-Met, HER2 and HER3, etc. The mechanism of activity of NDRG1 remains unclear, but to impart some of its functions, NDRG1 binds directly to key effector molecules that play roles in tumor suppression, e.g., MIG6. More recent studies indicate that NDRG1s-inducing drugs, such as novel di-2-pyridylketone thiosemicarbazones, not only inhibit tumor growth and metastasis but also fibrous desmoplasia, which leads to chemotherapeutic resistance. The Casitas B-lineage lymphoma (c-Cbl) protein may be regulated by NDRG1, and is a crucial E3 ligase that regulates various protein tyrosine and receptor tyrosine kinases, primarily via ubiquitination. The c-Cbl protein can act as a tumor suppressor by promoting the degradation of receptor tyrosine kinases. In contrast, c-Cbl can also promote tumor development by acting as a docking protein to mediate the oncogenic c-Met/Crk/JNK and PI3K/AKT pathways. This review hypothesizes that NDRG1 could inhibit the oncogenic function of c-Cbl, which may be another mechanism of its tumor-suppressive effects.

Keywords: NDRG1; c-Cbl; pancreatic cancer; thiosemicarbazone.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The RAS/RAF/MEK/ERK and PI3K/AKT pathways’ abnormal activation in PC. Following growth factor stimulation, receptor tyrosine kinase (RTK) undergoes auto-phosphorylation, which enables guanine nucleotide exchange factors (GEFs)—such as Son of Sevenless-1 (SOS1)—to bind to the receptor with the aid of adaptor protein GRB2. SOS1 interacts with RAS and catalyzes the exchange of GDP for GTP, forming the active RAS-GTP molecule. Active RAS then associates with and activates RAF, which subsequently phosphorylates MEK that in turn phosphorylates ERK. Ras-GTP can interact with and stimulate PI3K activity. PI3K also binds to active RTKs that initiate the catalytic phosphorylation of PIP2 to PIP3. PIP3 then phosphorylates AKT. The activation of both AKT and ERK leads to the activation of the downstream targets involved in cell growth, survival, and metastasis. Mutations lead to constitutively active KRAS, causing the constant stimulation of PI3K and RAF, leading to PC cell growth, survival and metastasis.
Figure 2
Figure 2
Canonical and alternative NF-κB pathways. As part of the canonical NF-κB pathway, the IκB kinase (IKK) complex is activated by phosphorylation. The IKK complex then phosphorylates the inhibitor of NF-κBα (IκBα), leading to its degradation by the proteasome. This event allows the NF-κB (RelA/c-Rel-p50) dimer to translocate to the nucleus. The alternative pathway is initiated when the IKKα homodimer is activated by phosphorylation, which phosphorylates the p100 bound to RelB. This phosphorylation leads to p100 being degraded by the proteasome to form the RelB-p52 dimer. This NF-κB (RelB-p52) dimer then translocates to the nucleus. Both RelA/c-Rel-p50 and RelB-p52 dimers act as transcription factors to up-regulate the expression of the downstream oncogenic effectors (e.g., c-myc and cyclin D1) that promote cell growth, survival, angiogenesis and metastasis.
Figure 3
Figure 3
The canonical TGF-β pathway. The TGF-β ligand binds to the TGF-β type II receptor (TβRII). This binding induces TGF-β type I receptor (TβRI) to form a hetero-tetrameric complex with TβRII. TβRI subsequently phosphorylates SMAD2 and SMAD3, which then form a complex with SMAD4. Under physiological conditions in normal cells, the resulting SMAD2/3/4 complex translocates to the nucleus, recruits co-transcriptional factors, and activates genes involved in apoptosis and cell cycle arrest, such as the cyclin-dependent kinase inhibitor, p21, and the death-associated protein kinase (DAPK). In advanced cancer, the same SMAD2/3/4 complex can instead promote the expression of SNAIL, SLUG, TWIST and ZEB, which are transcription factors known to promote the EMT.
Figure 4
Figure 4
Line drawings of the chemical structures of the metal-binding ligands described herein: (A) desferrioxamine (DFO), (B) di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), and (C) di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC).
Figure 5
Figure 5
Anti-oncogenic effects of iron and copper-binding ligands. Metal chelators can deplete tumor cell iron and copper. This effect inhibits the iron-dependent enzyme, ribonucleotide reductase, that is required for DNA synthesis, as well as the expression of cyclin D1 and CDK2, resulting in cell cycle arrest. Iron depletion also increases the expression of the potent metastasis suppressor, NDRG1. These complexes also deprive cancer cells of the copper needed for angiogenesis and other crucial cellular processes. Additionally, some ligands—such as Dp44mT and DpC—form iron and copper complexes that accumulate in lysosomes and generate ROS, which leads to lysosomal membrane permeabilization and the apoptosis of cancer cells.
Figure 6
Figure 6
Thiosemicarbazone lysosomal transport and ROS production. Both Dp44mT and DpC use the Pgp transporter to enhance their transport into lysosomes. The expression of Pgp is present on the plasma membrane, and also on endosomal and lysosomal membranes. Plasma membrane endocytosis results in the internalization of Pgp and its presence within endosomes and lysosomes, with this process resulting in the Pgp transporter pumping substrates, such as DpC and Dp44mT, into the lumen of the endosome or lysosome. Upon the entrance of these ligands into the lysosome, their ability to bind the iron and copper derived from the breakdown of intracellular constituents leads to the generation of redox active complexes that induce lysosomal membrane permeabilization and apoptosis.
Figure 7
Figure 7
Schematic representation of the structure of the N-myc downstream-regulated gene 1 (NDRG1) protein.
Figure 8
Figure 8
Inducers of NDRG1, including: HIF-1, Egr-1, AP-1, PTEN, p53, hypoxia, iron depletion, and inhibitors of NDRG1, including N-myc, c-myc, and trypsin cleavage.
Figure 9
Figure 9
Metal-binding ligands such as DFO induce NDRG1 expression via HIF-1α accumulation. (A) Normal oxygen and iron levels enable prolyl hydroxylase (PHD) to hydroxylate HIF-1α, creating a binding site for von Hippel-Lindau (VHL) protein to bind HIF-1α and activate ubiquitin E3 ligase, resulting in the proteasomal degradation of HIF-1α. (B) In contrast, during hypoxia or when metal-binding ligands deplete cellular iron, the PHD enzyme is inactivated and does not hydroxylate HIF-1α, preventing the binding of VHL. This mechanism prevents HIF-1α degradation, which consequently accumulates and forms the HIF-1 complex by binding to HIF-1β. (C) The HIF-1 complex then translocates to the nucleus and binds the hypoxia-response elements (HREs) located in the NDRG1 promoter, thus up-regulating its expression.
Figure 10
Figure 10
The Wnt/β-catenin signaling pathway and NDRG1’s effect on β-catenin localization. In the absence of the Wnt ligand, β-catenin is phosphorylated by the destruction complex (consisting of casein kinase 1α (CK1α), glycogen synthase kinase 3β (GSK3β), the tumor suppressor APC, scaffold protein AXIN, and others (not shown)) leading to its proteasomal degradation. The Wnt ligand binds to and activates the LRP5/6 and Frizzled co-receptors. The activated receptor then recruits Dishevelled (DVL), where it activates and then sequesters the “destruction complex”, which inhibits its activity, allowing non-phosphorylated β-catenin to accumulate in the cytosol and then translocate to the nucleus. Here β-catenin acts as a co-activator with the T cell factor (TCF) family of transcription factors, which up-regulate proteins such as cyclin D1 and c-myc that promote oncogenesis. NDRG1 up-regulation by iron-binding ligands such as DFO or Dp44mT inhibits β-catenin from translocating to the nucleus, preventing its transactivation activity and promoting cytosolic β-catenin localization to the membrane to form part of the adherens complex.
Figure 11
Figure 11
Targeting oncogenic Sonic Hedgehog signaling between PC cells and PSCs via NDRG1 induced by DpC. Pancreatic cancer (PC) cells have an abnormal NF-ĸB activation, which stimulates the production of Sonic Hedgehog (SHH). The secreted SHH binds to the PTCH1 membrane receptor on pancreatic stellate cells (PSCs). The binding of SHH to PTCH1 leads to the activation of the smoothened (SMO) receptor in PSCs that subsequently activates GLI1. GLI1 promotes HGF and IGF-1 transcription in PSCs, which are secreted and bind to c-Met and IGF-1Rβ receptors on PC cells, promoting their growth and migration. HGF and IGF-1 further stimulate SHH production, establishing bidirectional crosstalk between PSCs and PC cells. It was found that the DpC-induced upregulation of NDRG1 potently inhibits this bidirectional crosstalk by inhibiting SHH production by PC cells, preventing the activation of PSCs to secrete HGF and IGF-1.
Figure 12
Figure 12
Schematic drawing representing the structure of the Casitas B-lineage lymphoma (c-CBL) protein.
Figure 13
Figure 13
c-Cbl-mediated internalization, ubiquitination and degradation of activated RTKs. (A) Growth factor (GF) binding induces RTK auto-phosphorylation and recruits c-CBL to the activated receptor via the Grb2 adaptor protein. The RTK activates Src kinase, which phosphorylates c-Cbl and Sprouty2 (hSpry2), an inhibitor of c-Cbl’s E3 ligase activity. (B) Phosphorylated hSpry2 detaches from the ring finger domain (RF) and binds the terminal tyrosine kinase binding (TKB) domain of c-Cbl. (C) The RF domain of c-Cbl then recruits the E2-conjugating enzyme, which promotes the polyubiquitylation of hSpry2. (D) The polyubiquitylation of hSpry2 targets it for proteasomal degradation. (E) The TKB domain of c-Cbl is now free to bind the RTK, which enables c-Cbl to catalyze the addition of ubiquitin (Ub) molecules from E2 to the RTK. The addition of Ub leads to receptor internalization via endocytosis. (F) Ubiquitin molecules (Ub) continue to be added, and mark the RTK for lysosomal degradation.
Figure 14
Figure 14
c-Cbl enhances the PI3K/AKT pathway via PI3K recruitment. (A) RTK activation allows the p85 regulatory subunit of PI3K to bind to the phosphor-tyrosine residues of RTKs. This interaction relieves the p85 suppression of the p110 catalytic subunit, and allows PI3K to phosphorylate PIP2 to PIP3, initiating the PI3K/AKT pathway that leads to cell survival and proliferation. (B) c-Cbl’s Tyr731 residue is phosphorylated after RTK stimulation, enabling it to bind and recruit PI3K via the p85 regulatory subunit. This results in enhanced PIP2 phosphorylation and PI3K/AKT pathway activation, and thuss promotes cellular survival and proliferation.
Figure 15
Figure 15
The c-Met/Crk/JNK pathway is enhanced by GAB1 and c-Cbl by promoting JNK phosphorylation. (A) c-Met is activated by hepatocyte growth factor (HGF), leading to its autophosphorylation. The adaptor protein, Gab1, recognizes and binds to phosphotyrosine residues on activated c-Met. Then c-Met phosphorylates Gab1 and enables it to recruit Crk (via Tyr residue) to promote JNK phosphorylation. (B) Similarly, c-Cbl also recognizes and binds to the phosphotyrosine residues on activated c-Met. Subsequently, c-Cbl is phosphorylated by c-Met-induced Src kinases at either Tyr700 or Tyr774, which enables the recruitment of Crk and the enhancement of JNK phosphorylation. In addition, c-Cbl simultaneously ubiquitinates the c-Met receptor, resulting in its degradation. Both c-Cbl and GAB1 enhance JNK phosphorylation, which promotes cellular transformation.
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