Biochimica et Biophysica Acta 1845 (2014) 1–19

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Review

Molecular functions of the iron-regulated metastasis suppressor, NDRG1,

and its potential as a molecular target for cancer therapy
Bernard A. Fang, Žaklina Kovačević, Kyung Chan Park, Danuta S. Kalinowski, Patric J. Jansson, Darius J.R. Lane,
Sumit Sahni, Des R. Richardson ⁎
Molecular Pharmacology and Pathology Program, Discipline of Pathology and Bosch Institute, Blackburn Building (D06), The University of Sydney, Sydney, NSW 2006, Australia

a r t i c l e i n f o a b s t r a c t

Article history: N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also
Received 26 September 2013 involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination,
Received in revised form 11 November 2013 stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influ-
Accepted 13 November 2013
ence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by
Available online 21 November 2013
multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron
Keywords:
levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells
Metastasis suppressor after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Al-
N-myc down-regulated gene 1 though the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship
Thiosemicarbazone between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest
that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins,
leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this im-
portant metastasis suppressor and discusses interesting unresolved issues regarding this protein.
© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Molecular structure of NDRG1 and its interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Isoforms of NDRG1 and the potential role of trypsins in NDRG1 cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1.1. Cancer-associated trypsin-like proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3. Tissue and cellular distribution of NDRG1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4. Biological functions and regulation of NDRG1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.1. Regulation of NDRG1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2. Biological functions of NDRG1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.2.1. Embryogenesis and development in early life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.2.2. Cell growth and differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.2.3. Lipid biosynthesis and myelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.2.4. Stress response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.2.5. Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Abbreviations: Akt, protein kinase B; CPT-11, irinotecan; CMT4D, Charcot–Marie–Tooth disease, type 4D; DFO, deferoxamine (or desferrioxamine B); DpC, di-2-pyridylketone 4-
cyclohexyl-4-methyl-3-thiosemicarbazone; DpT, di-2-pyridylketone thiosemicarbazone; Dp44mT, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone; ECM, extracellular matrix;
ERG-1, v-ets avian erythroblastosis virus E26 oncogene homologue; EGR1, early growth factor-1; ETS, v-ets avian erythroblastosis virus E26 oncogene homologue 2; HDL-C, high-
density lipoprotein cholesterol; HIF-1α, hypoxia inducible factor-1α; HMSNL, hereditary motor and sensory neuropathy-Lom; Hsc70, 70-kDa heat shock cognate protein; HTE, human
tracheal epithelial cell; HUVEC, human umbilical vein endothelial cell; IL, interleukin; LDL, low-density lipoprotein; LPC, lysophosphatidylcholine; MMP, matrix metalloproteinase;
NDRG1, N-myc down-stream regulated gene 1; NF-κB, nuclear factor-κB; NIH, 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone; PAR-1, protease activated receptor; PKA, protein
kinase A; PKA, protein kinase C; PrEC, prostate epithelial cell; pMLC2, phosphorylated myosin light chain 2; PTEN, phosphatase and tensin homologue deleted on chromosome 10;
pVHL, von Hippel–Lindau protein; ROCK1, Rho-associated, coiled-coil containing protein kinase 1 (ROCK1); ROS, reactive oxygen species; SGK, serum- and glucocorticoid-induced kinase;
TAT, tumour-associated trypsinogen; TAT2, tumour-associated trypsinogen-2; TCF/LEF, T-cell factor/lymphoid enhancer-binding factor; Thtpa, thiamine triphosphatase; VEGF, vascular
endothelial growth factor
⁎ Corresponding author. Tel.: +61 2 9036 6548; fax: +61 2 9351 3429.
E-mail address: [Link]@[Link] (D.R. Richardson).

0304-419X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
[Link]
2 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19

5. NDRG1 and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

5.1. Regulatory pathways of NDRG1 in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.1.1. NDRG1 and p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.1.2. NDRG1 and PTEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.1.3. NDRG1 and myc oncoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
5.2. Functions and functional pathways of NDRG1 in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
5.2.1. NDRG1 and primary tumour growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
5.2.2. NDRG1 and metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
5.2.3. NDRG1 and angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.2.4. NDRG1, attachment and adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.2.5. The controversial role of NDRG1 in oncogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.3. NDRG1 and chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.3.1. Targeting NDRG1 by binding cellular iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
6. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1. Introduction (Fig. 1A) [51]. This group of genes is subdivided into two subfamilies,
one of which is composed of NDRG1 and NDRG3 and the other is com-
Cancer is a major public health issue, with one in four deaths in the posed of NDRG2 and NDRG4 [50]. Proteins encoded by this family
United States attributable to the disease [1]. Cancers encompass multi- share approximately 57–65% amino acid identities with each other
ple entities of complex and multifactorial diseases [2], each having its (Fig. 1B, C) [49,50] and are characterised by an NDR protein domain,
own specific aetiology and genomic, histological and clinical character- consisting of an esterase-/lipase-/thioesterase-active-site serine and
istics [3,4]. Despite recent advances, effective clinical management re- an α/β hydrolase fold of approximately 220 amino acids [50].
mains elusive due to intra-tumoural heterogeneity and therapeutic Although NDRG proteins do not possess a catalytic triad consisting of
resistance [5–11]. Therefore, novel therapies and further investigation nucleophile–acid–histidine residues that is required for function as a
into the pathophysiology of cancer are essential [12,13]. hydrolase [48,52], the proteins belong to the α/β hydrolase superfamily
Metastasis is the process by which cancer cells disseminate from a [48]. The various NDRG family member genes may have arisen from the
primary tumour site to a distal secondary tissue microenvironment convergent evolution for this specific molecular architecture, but with
[14] and therefore represents an important therapeutic target [15]. Me- distinct conformations and functions [53]. In fact, analyses indicate
tastasis markedly complicates clinical management and is a major cause that the NDRG genes arose from multiple duplication events during evo-
of death due to cancer [16]. Growing evidence has underscored the in- lution [51].
volvement of oncogenes, tumour suppressor genes and metastasis sup- NDRG1 is mapped to the long arm of chromosome 8 [54], specifically
pressor genes in the development and progression of cancer [17–20]. to the locus 8q24.2 [55] or, in agreement with the AceView database
One metastasis suppressor, namely N-myc down-stream regulated [56], 8q24.3 (Fig. 2A) [41]. Spanning 60,085-bp, NDRG1 contains 16
gene 1 (NDRG1), is of particular interest and plays an anti-oncogenic exons and 15 introns, and it encodes a 2997-bp mRNA, 1182 bp of
role in cancers of the brain [21], breast [22,23], colon and rectum which are coding (Fig. 2B) [24,55]. NDRG1 mRNA is translated into a de-
[24–28], oesophagus [29], pancreas [30] and prostate [31]. Paradoxically, duced 42,835-Da, 394-amino acid protein with a predicted pI of 5.7
NDRG1 is highly expressed in cancers of the kidney [32], liver [33–35], [24,40,42,49]. The chromosomal region in which NDRG1 is located fre-
mouth [36], skin [37] and uterine cervix [38,39], where it has been sug- quently undergoes genetic alteration in cancers, such as those of the
gested to play a role in promoting tumourigenesis. prostate [57], and can be amplified in other tumours [44]. The proto-
Human NDRG1, the inaugural member of the NDRG family, was first oncogene, c-myc, is also located in close proximity to NDRG1, at the
identified as a predominantly cytoplasmic protein encoded by the locus 8q24.21 [58] and is also frequently amplified in tumours [59,60].
ubiquitously expressed N-myc down-stream regulated gene 1 (NDRG1) Interestingly, c-myc, like N-myc, can repress NDRG1 transcription and
[24,40]. The molecule has been referred to under a number of different is implicated in causing a more metastatic phenotype [44,61].
designations, including reducing agent and tunicamycin-responsive Human NDRG1 differs from other NDRG proteins by the inclusion
protein (RTP) [40], differentiation-related gene (Drg)-1 [24], reduced of not one but three decapeptide tandem repeats [49], each of which
in tumours, 42-kDa protein (Rit42) [41], Ca2 +-associated protein 43 consists of the residues GTRSRSHTSE (Fig. 2C) [40]. In addition, other
(Cap43) [42] and protein regulated by oxygen (PROXY)-1 [43]. The NDRG proteins also lack the C-terminal two residues, Ser and Glu
current designation, “NDRG1”, has been agreed upon by a consortium of [62]. The C-terminal domain of NDRG1 is unique amongst the
NDRG1 researchers and is the currently accepted gene symbol assigned NDRG proteins, as it can act as a substrate for phosphorylation by
by the HUGO Gene Nomenclature Committee [44]. Orthologues of serum- and glucocorticoid-induced kinase (SGK)-1, which primes
NDRG1 have been identified in the mouse, as Ndr1 [45] and TDD5 [46], NDRG1 for phosphorylation by glycogen synthase kinase (GSK)-3β
and the common sunflower (Helianthus annuus L.), as sf21 [47]. Therefore, [63,64]. Specifically, SGK-1 has been confirmed to phosphorylate
NDRG1 appears to be highly conserved across a wide range of organisms, NDRG1 at Thr328, Ser330, Thr346, Thr356 and Thr366 [65,66], while GSK-
indicating its important function in the cell. 3β subsequently phosphorylates NDRG1 at Ser342, Ser352 and Ser362
[63]. Thus far, enhanced phosphorylation via increased SGK-1 activity
2. Molecular structure of NDRG1 and its interactions has been associated with severe Alzheimer's disease, but the underlying
mechanisms have not been elucidated [67]. Moreover, phosphorylation
NDRG1 is a member of the NDRG family [48], currently consisting of NDRG1 is a reversible process that has been suggested to influence
of NDRG1, NDRG2, NDRG3 and NDRG4 [49,50]. Notably, phylogenetic the localisation of NDRG1 [68,69]. Further, phosphorylation of NDRG1
analysis indicates that each NDRG family member forms a separate is putatively associated with the multiple physiological roles of
homology cluster across multiple species with possibly specific and NDRG1, including cell differentiation [70], centromere function during
functionally divergent roles relative to the other family members mitosis [71] and partitioning of daughter cells during telophase [71].
 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19 3

Fig. 1. Predicted structural relationship amongst NDRG proteins. (A) The phylogenetic tree of NDRG genes. The phylogenetic tree is visualised using TreeView [263]. Relative evolutionary
distances by the sum of horizontal branch lengths are indicated. (B) The multiple sequence alignment of NDRG proteins. Protein sequences of NDRG family members were obtained
from the UniProtKB/Swiss-Prot database [264,265] and aligned using the MUSCLE programme [266,267]. (C) The predicted protein secondary structure of NDRG family members.
These were generated using the MLRC method [268]. In this figure, helices, extended strands and random coils are shown in blue, red and pink, respectively. Adapted from [53].

In the context of cancer, phosphorylation of NDRG1 by SGK-1 at Ser330 and processes critical to metastasis, such as proliferation, adhesion
and Thr346 has been shown to be crucial for the suppression of the nu- and resistance to chemotherapeutic agents [73,74]. Hence, the phos-
clear factor (NF)-κB signalling pathway and expression of CXC cyto- phorylation of NDRG1 may play a critical role in its anti-metastatic
kines [72]. The CXC cytokines are known to mediate tumourigenesis activity.
4 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19

Fig. 2. Structure of NDRG1 gene and NDRG1 protein. (A) Chromosomal location of the NDRG1 gene. (B) Organisation of NDRG1 gene. NDRG1 contains 16 exons. Myc oncoproteins, such as N-
myc and c-myc, can repress NDRG1 at the transcription level, albeit not via direct binding with the proximal promoter of NDRG1. The exons are shown as blue boxes. (C) Structure of the
NDRG1 protein. Sites of post-translational modification and attachment are indicated. PKA, protein kinase A phosphorylation site; PKC, protein kinase C phosphorylation site; CK-II, casein
kinase II site; CAMK, calmodulin kinase II site; P, SGK1 phosphorylation site.

The decapeptide tandem repeats contained in the C-terminus do- silencing of NDRG1 expression [58]. In fact, NDRG1 was found to be
main of NDRG1 were originally speculated to be significant for the bind- hyper-methylated in breast [88], gastric [89], colon [90] and pancre-
ing of divalent metals (e.g., Ni2+ and Cu2+) to the C-terminus domain atic [58] cancer cells, leading to suppressed NDRG levels. Important-
and the formation of coordination complexes [75]. Indeed, subsequent ly, promoter methylation of NDRG1 correlated with metastasis,
studies showed that the C-terminal domain effectively binds Ni2 + lymph node invasion and poor histological grade in breast cancer
[76,77] and Cu2+ [78]. Currently, the effect of this activity is unclear, patients [88].
but it is suggested that metal-binding is important in regulating Certain evolutionary characteristics are conserved across, or shared
NDRG1 expression and its potential function in detoxification processes amongst, the NDRG family and, by way of analogy, may be of utility in
[30,79,80]. However, unlike Ni2+, it has been reported that Cu2+ does speculating upon the molecular structure of NDRG1 and its function.
not markedly induce NDRG1 expression [42]. Although the crystal structure of NDRG1 remains to be elucidated, the
Structure prediction programs [81] do not suggest the presence of crystal structure of another member of the NDRG family, NDRG2, a pu-
any trans-membrane domains [82] or amphipathic in-plane membrane tative tumour-suppressive protein, has recently been solved [62]. Nota-
anchors in NDRG1. While NDRG1 does not appear to possess a trans- bly, the predicted protein secondary and tertiary structures of NDRG1
membrane domain, signal sequence, or endoplasmic reticulum reten- and NDRG2 demonstrate their similarity (Fig. 3). NDRG2 is composed
tion sequence [40], it possesses at least seven phosphorylation sites of an α/β hydrolase fold domain and a cap-like domain [62]. The α/β
[83,84], with five calmodulin kinase II phosphorylation sites, a casein ki- hydrolase fold domain consists of a β-hairpin structure (β1–β2) and a
nase II site, five myristoylation sites, a phosphopantotheine attachment six-stranded, parallel sheet (β3–β8) surrounded by an outer solvent-
site and a tyrosine phosphorylation site [85]. The significance of these exposed layer composed of eight α-helices (α1–α5 and α11–α13)
particular phosphorylation sites on the activity of NDRG1 has been sug- and two 310 helices, whereas the cap-like domain consists of five α-
gested to affect its function in mast cells, which will be further discussed helices (α6–α10) [62]. Consistent with a previous study [48], structural
in Section 4.2.5 below. similarity search analysis of NDRG2 suggests that it belongs to the α/β
In multiple cancers [24,25,41,55], the oncoprotein N-myc represses hydrolase superfamily due to significant similarity between NDRG2
NDRG1 expression at the transcriptional level [61] via a mechanism and multiple α/β hydrolases [62]. However, compared to other α/β
independent of N-myc directly binding to the NDRG1 proximal promot- hydrolases, in NDRG2 Ser96 is substituted with Gly132; His247 with
er [45]. In fact, the 5′ end of NDRG1 contains a CpG island, to which myc Gly279; and Asp219 with Ala251 [62]. The sequence, Ser96–His247–Asp251
oncoproteins preferentially bind [86]. The binding of myc oncoproteins normally constitutes the catalytic triad conserved amongst α/β hydro-
to CpG islands suggests a potential role for methylation in the repres- lases, but this is absent in NDRG proteins and no apparent catalytic ac-
sion of NDRG1 expression [25,87] and for hyper-methylation in the tivity is consequently observed [62].
 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19 5

Table 1
Summary of NDRG1 band number after immunoblotting studies using anti-NDRG1 anti-
body.

Cell Line Type No. of Approx. masses Reference(s)

bands (kDa)

SK-N-MC Brain cancer 2 or 3 43, 44 or 43, 44, 45 [97]

MCF7 Breast cancer 1 1
2 43, 44 [152]
MDA 2 [272]
SK-BR-3 2 [272]
HeLa Cervical cancer 2 [63]
HT29 Colon cancer 2 43, 44 [94]
U973 Leukaemia 1 43 [145]
DMS-53 Lung cancer 2 43, 44 [97]
H1299 2 43, 43 [183]
MIAPaCa-2 Pancreatic cancer 2 43, 44 [96]
PANC-1 2 43, 44 [96]
CAPAN-2 2 43, 44 [96]
CFPAC-1 2 43, 44 [96]
PrEC Prostate epithelium 1 43 [95]
1 46 [93]
DU145 Prostate cancer 3 43, 43, 46 [183]
2 43, 44 [94]
2 43, 44 [95]
2 41, 46 [93]
LNCaP 2 41, 46 [93]
PC-3MM 2 43, 43 [183]
2 43, 44 [97]
2 41, 46 [93]

2.1. Isoforms of NDRG1 and the potential role of trypsins in NDRG1 cleavage

As with other proteins, the structure of NDRG1 is critical to its func-

tion, and structural alterations may result in significant changes in func-
tionality. In support of this hypothesis, isoforms of NDRG1 have
been identified in diseases, such as hereditary motor and sensory neu-
ropathy type-Lom (HMSNL) [92] and cancer [93–97]. In addition, in v-
ets avian erythroblastosis virus E26 oncogene homologue-1 (ERG)-over-
expressing prostate cancers, NDRG1 has been shown to be fused to
the coding region of ERG-1, which is suggested to disrupt NDRG1
and its protein product and may result in an increased risk of metas-
Fig. 3. Predicted protein tertiary structures of NDRG1 and NDRG2. Protein sequences of
NDRG1 and NDRG2 were obtained from the UniProtKB/Swiss-Prot database [264,265] tasis [98].
and modelled using Geno3D [269]. The models were visualised using Jmol [270,271]. In Recent immunoblotting studies using specific anti-NDRG1 antibod-
this figure, α-helices, 310 helices, β-strands, β-turns and other secondary structures are ies have revealed the presence of additional NDRG1-immunoreactive
shown in light pink, dark pink, blue and white, respectively.
bands in multiple cancer cell lines and other biological samples, under
different conditions (Table 1). These additional bands indicate the pres-
Other critical structural differences between NDRG proteins and ence of isoforms of NDRG1 that may alter the function of this protein
non-NDRG α/β hydrolases include the presence of His186 and the differ- [93]. Multiple immunoreactive forms of NDRG1 have also been detected
ent residue distribution of helix α6 in NDRG proteins [62]. His186 pre- during rat brain and kidney postnatal development [99]. In addition
vents the formation of a substrate-binding site via extension of helix to a prominent band at approximately 43-kDa in brain and kidney
α7 into the region where other α/β hydrolases possess Lys187 and a tissue, additional bands were detected at approximately 129-kDa
substrate-binding site [62]. A different residue-distribution within and 172-kDa in brain tissue and at approximately 215-kDa in kidney
helix α6, in which hydrophilic residues are inward-facing and hydro- tissue, particularly prior to postnatal day 14 [99]. These higher mo-
phobic residues are outward-facing and exposed to solvents, is respon- lecular weight species may correspond to a trimer, tetramer or
sible for the absence of a hydrophobic substrate-binding site [62]. Thus, pentamer composed of NDRG1, respectively, and it was suggested
instead of possessing enzymatic activity, NDRG2 may engage in binding that NDRG1 forms polymers to function during postnatal development
interactions, as observed with other non-enzymatic α/β hydrolases in vivo [99].
[91]. In fact, as helix α6 extends from the main body of NDRG proteins, In subsequent studies, multiple immunoreactive bands have been
the region may more easily interact with potential binding targets [62]. detected with anti-NDRG1 antibodies in numerous cell lines (Table 1).
Indeed, the point mutation L172D in NDRG2 has been shown to result in It has been suggested that these bands correspond to different phos-
a reduction in the ability of helix α6 to interact with β-catenin and phorylation states of NDRG1 or other post-translational modifications
abolition of T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) of this protein [63]. Notably, NDRG1 has been shown to be a multi-
signalling [62]. This point mutation disrupts the molecular architecture phosphorylated protein [63,83,84], and its phosphorylation has been
of helix α6 at its middle and where it is fully exposed to solvents [62]. reported to be associated with its tumour-suppressive activity as de-
Following this, it is clear that subtle differences in the molecular archi- scribed in Section 2 above [71,72]. In addition to the tabulated observa-
tecture of NDRG proteins may elicit significant differences in their bio- tions (Table 1), it is of interest that the bands of greater molecular mass,
logical activities. such as the 44-kDa and 45-kDa bands in SK-N-MC cells [97] and the
6 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19

44–46-kDa band in DU145 cells [93,95], were more intense after in- 3. Tissue and cellular distribution of NDRG1
cubation with agents that induce NDRG1 expression. As these bands
may correspond to different phosphorylation states of NDRG1, it has NDRG1 mRNA is ubiquitously expressed in human tissues
been suggested that these phosphorylation states could be involved [24,40,124–126]. However, higher mRNA levels are detectable in the
in modulating NDRG1 activity [72,96]. brain, kidney, placenta, prostate and small intestine than in other tissues
Further studies examining the multiple NDRG1 bands in immuno- [24,40,124]. Interestingly, NDRG2 mRNA is highly expressed in brain,
blots indicated that the higher Mr band was not a phosphorylated or salivary gland, skeletal muscle and spinal cord; NDRG3 mRNA is highly
glycosylated isoform of NDRG1 [93]. In fact, assessing DU145, LNCaP expressed in testis and NDRG4 mRNA is highly expressed in heart
and PC-3MM prostate cancer cells, a C-terminal antibody against [49,50]. Although structurally similar, the distinct mRNA expression
NDRG1 yielded two bands, while the N-terminal antibody yielded only patterns of NDRG genes suggest that corresponding proteins possess
one NDRG1 band [93]. In these three prostate cancer cell lines, the different but related functions in tissues or organs [50].
band of greater mass, migrating at approximately 46-kDa, was shown Further to this, three NDRG4 isoforms, NDRG4-B, NDRG4-Bvar and
to be full-length, unmodified NDRG1, which suggests that the lower NDRG4-H, all of which are encoded by the NDRG4 gene, have been
41-kDa band may be a truncated isoform of NDRG1 that lacks the N- identified [49]. NDRG4-B and NDRG4-Bvar are produced by alterna-
terminal region [93]. In addition, using primary cultures of normal tive splicing of the NDRG4 gene [49]. These NDRG4 isoforms exhibit
prostate epithelial cells (PrECs), only one NDRG1 band migrating at ap- distinct mRNA and protein expression patterns, possibly due to
proximately 46-kDa was identified, and this corresponded to the higher alternative promoter usage [49]. For instance, NDRG4-B mRNA is
Mr band in the three prostate cancer cell lines [93]. Hence, this further only detectable in brain and NDRG4-B var mRNA level is higher in
suggests that NDRG1 is truncated in prostate cancer cells when com- heart than in brain [49].
pared to normal prostate cells where this protein is intact. It was spec- In contrast to its ubiquitous mRNA expression, human NDRG1 pro-
ulated that truncated isoforms of NDRG1 may be implicated in the tein (UniProt entry: Q92597) is predominantly expressed in epithelial
altered function of NDRG1 in metastasis suppression and carcinogene- cells [41], although it is absent in specific tissues, such as blood vessels,
sis [93]. connective tissue, muscle and a majority of the nervous system, sug-
gesting roles for NDRG1 other than as a housekeeping protein [124]. In-
deed, the precise subcellular localisation of NDRG1 varies amongst cell
2.1.1. Cancer-associated trypsin-like proteases types [124]. For instance, NDRG1 is predominantly localised in the nu-
Protein sequence analysis using PSORT II [100] has predicted a cleus of prostate epithelial cells (PrECs), the plasma membrane of intes-
potential cleavage site within NDRG1 between Cys49 and Gly50 [93]. tinal and lactating breast epithelial cells and the mitochondrial inner
Further, a cleavage event at this site was predicted to yield a 5-kDa N- membrane of renal proximal tubule cells [124,127]. In mast cells,
terminal by-product [93], which is consistent with the difference in NDRG1 exhibits a punctate distribution in the cytoplasm, particularly
mass between the two immunoreactive bands detected using an anti- around secretory granules [127,128]. However, NDRG1 also translocates
body raised to the NDRG1 C-terminal. At this juncture, the proteins from the cytoplasm to the nucleus after IgE/Ag activation of mast cells,
that cleave NDRG1 at this predicted cleavage site remain unknown. suggesting possible nuclear regulatory functions in activated cells
However, protein sequence analysis using the ExPASy PeptideCutter [128]. Also in mast cells, NDRG1 has been shown to interact with the
tool [101,102] has predicted that NDRG1 can be cleaved by trypsins, 70-kDa heat shock cognate protein (Hsc70), a chaperone that regulates
such as chymotrypsin, mesotrypsin, pseudotrypsin and trypsin [93]. the translocation of NDRG1 between the cytoplasm and nucleus and is
As a link between trypsin expression and tumours has been implicated in ATP-dependent protein folding, vesicular transport and
established [103–109], it may be speculated that trypsins cleave exocytosis [129–131].
NDRG1 in cancers, thereby potentially attenuating or altering its The program PSORT II [100] predicts that NDRG1 is localised in the
functionality and metastasis-suppressing activities [110–112]. The cytoplasm, nucleus and mitochondrion, at probabilities of 47.8%, 26.1%
trypsin-like proteases, including mesotrypsin and tumour-associated and 8.7%, respectively. Further, PSORT II predicts that NDRG1 is localised
trypsinogen (TAT), that may be involved in NDRG1 cleavage are briefly in the cytoskeleton, peroxisome, plasma membrane and vacuoles, at
discussed below. probabilities of 4.3% each. Interestingly, during embryonic develop-
It is well known that mesotrypsin (also known as protease, serine, 3; ment, NDRG1 mRNA is detectable in brain [45,132] and during postnatal
PRSS3) promotes tumour growth and metastasis of human pancreatic, development in rat, NDRG1 is detectable in brain and kidney [99]. De-
breast and prostate cancers [109,113,115]. Moreover, PRSS3 expression spite NDRG1 being observed to localise in the nucleus of certain cell
in primary prostate cancer is prognostic of systemic progression after types [124], no nuclear sequence is predicted by WoLF PSORT [133] or
prostatectomy [109]. Importantly, a novel and potent inhibitor of is identifiable from its amino acid sequence [124]. The nuclear
mesotrypsin, BPTI-K15R/R17G, was demonstrated to inhibit prostate localisation of NDRG1 is speculated to involve phosphorylation or inter-
cancer cell invasion to a similar extent as PRSS3 gene silencing [109]. action with a nuclear protein, such as Hsc70, as described above [124].
In fact, the high levels of mesotrypsin in tumours can be attributed to Indeed, in response to DNA damage following low concentrations of
the pathological trypsinogen activator, cathepsin B, which is often high- actinomycin D, NDRG1 translocates from the cytoplasm to the nucleus,
ly expressed in tumours [114] and activates mesotrypsinogen where it may inhibit cell growth and promote DNA repair mechanisms
[109,113,115,116]. The carcinogenic effects of mesotrypsin are mediat- [41]. Hence, it is suggested that NDRG1 acts as a stress response gene
ed via its substrates, which include protease activated receptor (PAR)-1 [41] or potentially as a transcription factor [134].
in pancreatic cancer and CD109 in breast cancer cells [113,115]. Consistent with the translocation of NDRG1 between the cytoplasm,
Another trypsin-like protease that is often highly expressed in cancer cell membrane and nucleus, NDRG1 has been detected in these subcellu-
cells is tumour-associated trypsinogen-2 (TAT-2) [117]. TAT-2 is the lar compartments and perinuclear regions [135]. Moreover, NDRG1 has
predominant isoform shown to be expressed in ovarian neoplasms been observed to associate with cell membranes, adherens junctions, des-
[118], pancreatic cancer [119], colon carcinoma, fibrosarcoma, leukaemia mosomes and intermediate and microfilament bundles that insert into
[120], colorectal carcinoma [121] and lung neoplasms [122]. The level of adherens junctions, strongly suggesting a role for NDRG1 in cell adhesion
TAT-2 has been shown to correlate with the malignant phenotype [124]. Indeed, the maintenance of membrane-bound E-cadherin and β-
and cell invasive potential of tumours [123]. Hence, the effect of catenin by over-expression of NDRG1 supports the hypothesis that
proteases such as mesotrypsin and TAT-2 on NDRG1 may affect NDRG1 is involved in increasing cell adhesion via stabilisation of the
NDRG1 function and is an intriguing possibility that requires further adherens junction, which is composed of these two membrane proteins
investigation. [25,94].
 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19 7

Table 2
Induction of NDRG1 and its putative functions.

Induced by Function(s) Reference(s)

Terminal differentiation Loss of cell proliferative potential [25]

Cell differentiation and maturation Promotion of immature cells to become mature cells [128,170]
Cell stress Cell growth arrest [24,40,42]
DNA damage and p53 Cell growth arrest; euploidy maintenance; possible cell cycle regulation; [41,79,148–150]
p53-dependent apoptosis; down-regulation of angiogenesis
High cell density and nutritional starvation Hypoxia-like response [162]
Hypoxia, HIF-1α, hypoxia mimetics and iron chelators Hypoxia response [43,94,124,140,141,143,151,152,158,273–275]
Androgens Cell growth arrest; cell differentiation [125]
AP-1 Hypoxia response [158]
Intracellular Ca2+ HIF-1α-independent hypoxia response [42,139,158,276,277]
Co2+ and Ni2+ Possible hypoxia-like response [42,140,141,273,278]
DNA methylation inhibitors and histone Housekeeping function(s); possible cell differentiation [25]
deacetylation inhibitors
E2a-Pbx1 Possible apoptotic response [149]
Free-radical nitric oxide (NO●) Unknown [159]
Lipophilic agents Loss of cell proliferative potential [70,145]
Phorbol myristate acetate Unknown [145]
PTEN Down-regulation of angiogenesis; down-regulation of [23,43,147]
metastasis (e.g., MMP-1, -2, -13)
Reducing agents Possible ischaemic disease prevention [40,42]

Through an understanding of the expression and distribution of Three hypoxia-inducible factor (HIF)-1-binding sites have been
NDRG1, it is apparent that NDRG1 may be involved in DNA repair and identified in the non-coding sequence of NDRG1, of which one is located
cell adhesion, both of which are important to tumourigenesis and in the promoter region and two are located in the 3′ untranslated region
metastasis. Therefore, isoforms of NDRG1 may thus have significant [151]. Moreover, putative HIF-response elements identified upstream of
consequences on these processes crucial to cancer development and the NDRG1 promoter, at −1370-bp and −7503-bp, are involved in its
progression. regulation [143]. Indeed, studies have shown that hypoxia promotes
NDRG1 expression via HIF-1α-dependent and –independent mecha-
4. Biological functions and regulation of NDRG1 nisms [140], the latter of which may involve eukaryotic initiation factor
3a (eIF3a) and its ability to regulate the balance between translation
The precise role of NDRG1 remains unclear, but it appears that this and translational suppression via the formation of stress granules
protein is multi-functional (Tables 2 and 3). Putative functions include [152], Sir2-like protein 1 (SIRT1), p53-dependent mechanisms [153]
roles in cell growth, differentiation, development and stress responses. and by the transcription factor c-Jun/AP-1/JUN in response to the iron
Recent proteomic studies have suggested a link between NDRG1 and chelator, L-mimosine [154].
genes associated with immune responses [136] and with carcinogenesis Incubation with iron chelators, which mimic hypoxia, results in in-
[137]. Further, the expression of NDRG1 is induced, repressed and regu- duction of NDRG1, decreased proliferation and the induction of G1/S
lated by a diverse range of effectors (Fig. 4) [138]. arrest and apoptosis in normal and neoplastic cells [143,155–157]. Fur-
ther, hypoxic responses via HIF-1α-independent mechanisms involve
4.1. Regulation of NDRG1 intracellular Ca2+, which itself can induce NDRG1 [158]. Cellular stress
due to iron depletion has also been shown to positively regulate
In addition to the diverse mechanisms by which NDRG1 is regulated, NDRG1 at the message and protein levels via an eIF3a-dependent mech-
as outlined above in Section 2, DNA methylation inhibitors (e.g., 5′-aza- anism [152]. This activity of eIF3a could be due to its recruitment to
2′-deoxycytidine) and histone deacetylase inhibitors (e.g., trichostatin stress granules under conditions of hypoxia and iron depletion and/or
A), promote NDRG1 expression, indicating a role for DNA methylation its ability to differentially regulate mRNA translation during cellular
and histone acetylation in regulating its levels [25]. Multiple other stress [152].
agents, including Ca2 + [139] and heavy metal ions (e.g., Co2 + [140], In a recent study, the free radical messenger molecule nitric oxide
Ni2+ [42,140]), androgens [68,125], DNA-damaging agents (e.g., actino- (•NO) was shown to up-regulate NDRG1 [159]. Interestingly, •NO has
mycin D), forskolin [65], lysophosphatidylcholine [30], okadaic acid a very strong affinity for iron [160], and it is known to complex intracel-
[42,141], sulfhydryl reducing agents and tunicamycin [40], ligands of lular free iron and also prevent degradation of the HIF-1α protein [161].
the peroxisome proliferator-activated receptor (PPAR)-γ and retinoid This study further demonstrated that concomitant up-regulation of
X receptor (RXR) [25], iron chelators [96,111,142,143] and vitamins A NDRG1 and HIF-1α by •NO was required for •NO-mediated migration
and D3 [70,144,145] also promote NDRG1 expression [49,146]. Further, suppression [159].
phosphatase and tensin homologue deleted on chromosome 10 The presence of functional binding sites for early growth response
(PTEN) [23,43,147], p53 [41,79,148–150] and the von Hippel–Lindau protein (EGR)-1 and specificity protein (Sp)-1 that overlap the NDRG1
protein (pVHL) [32] have been reported to promote NDRG1 expression. promoter indicates their potential roles in regulating NDRG1 expres-
sion [80]. In fact, this was confirmed by the concomitant transcriptional
Table 3 activation of the NDRG1 promoter after specific binding of EGR1 to the
Down-regulation of NDRG1 expression and its putative functions. binding sites for EGR1 and Sp-1, and this interaction can mediate
Downregulated by Function(s) Reference(s) hypoxia-induced transcription of NDRG1 [80]. Other transcription fac-
tors, such as activator protein (AP)-1, [158] and v-ets avian erythroblas-
Myc oncoproteins Cell differentiation [45]
Reoxygenation Cell migration [231] tosis virus E26 oncogene homologue 2 (ETS) [162], are also known to
Camptothecin analogues Apoptosis [200] regulate NDRG1 expression.
Digoxin Unknown [279] The effect of protein kinases on NDRG1 expression has also been
Oestradiol Unknown [280] evaluated, but only to a limited extent. For instance, incubation of cells
von Hippel–Lindau protein (pVHL) Hypoxia response [32]
with a protein kinase C (PKC) inhibitor led to an increase in NDRG1
8 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19

Fig. 4. Biological functions and regulation of NDRG1. Transcription factors, such as AP-1/c-Jun, JNK, HIF-1, ETS-1, Egr-1 and Sp-1, and other proteins, such as PTEN, p53 and pVHL, can up-
regulate NDRG1 expression, whereas myc oncoproteins, such as N-myc and c-myc, can down-regulate NDRG1. In certain cancers, the activity of these regulators may be altered. NDRG1
can promote apoptosis, cell differentiation, cell proliferation, inflammation and other immune responses, lipid biosynthesis, myelination and cell adhesion, while inhibiting interleukin
(IL)-8 and vascular endothelial growth factor (VEGF) signalling. Through the interplay of its multiple functions, NDRG1 inhibits metastasis in certain cancers.

levels, whereas this was not observed after treatment with either a Immunohistochemistry (IHC) studies demonstrated that NDRG1
protein kinase A (PKA) inhibitor or protein tyrosine kinase inhibitor was highly expressed in the rat renal proximal convoluted tubules dur-
[69]. Therefore, it has been suggested that activation of the PKC pathway ing the first two weeks of postnatal life, reflecting the morphological
is also involved in NDRG1 expression. and functional differentiation of the proximal convoluted tubules [99].
IHC studies also revealed immunoreactivity in the pyramidal neurons
of the hippocampus, with a peak in immunoreactivity after 8 to 10 days
4.2. Biological functions of NDRG1 of postnatal life, reflecting the metabolic changes in the developing
brain [99]. Moreover, western blot studies revealed an NDRG1-
4.2.1. Embryogenesis and development in early life immunoreactive band at approximately 215-kDa in rat early postnatal
NDRG1 expression is correlated with multiple stages of differentia- life brain and kidney samples [99]. This protein was shown to disappear,
tion, including placentation [69], trophoblast differentiation [153] and while other smaller bands were shown to appear and become more
organogenesis and organ morphogenesis [163]. After treatment with pronounced from the second week of postnatal life [99]. Considering
forskolin, which elevates cAMP levels and activates PKA, NDRG1 and this, it was suggested that during postnatal development, NDRG1 may
β-human chorionic gonadotropin, a trophoblastic differentiation mark- be processed leading to alterations in its molecular mass [99]. While
er, were induced in the BeWo human choriocarcinoma cell line [69]. the aforementioned investigations underscore a developmental role
Indeed, NDRG1 is predominantly expressed in syncytiotrophoblasts for NDRG1, there is an apparent lack of studies on its role in human
and in the intermediate trophoblasts of the basal plate during the organogenesis and organ morphogenesis.
second- and third-trimester placenta [69]. Further, as NDRG1 is elevated
in trophoblasts cultured under hypoxic conditions and in placental villi 4.2.2. Cell growth and differentiation
of pregnancy with growth-restricted foetuses, NDRG1 may be involved NDRG1 is suggested to be involved in cell growth and differentiation.
in the response to hypoxic injury or other stresses in trophoblasts [164], In fact, NDRG1 expression is shown to be biphasic, peaking during the G1
as in intrauterine growth restriction or preeclampsia [165]. and G2/M phases and being lowest during the S phase [41]. In the
Furthermore, NDRG1 is implicated during the development of the kidney, over-expression of the polycystic kidney disease gene 2
urinary and reproductive organs with its expression being tightly regulat- (PKD2) induces NDRG1 via the EGR1 transcription factor [167]. Under
ed during this process [163]. Whereas silencing of NDRG1 is associated the influence of this gene, NDRG1 appears to play a pro-proliferative
with failure of pronephric morphogenesis, over-expression is associated role where it can be induced by PKD2 via its effects on the transcription
with disrupted formation of pronephric ducts, reduced size of pronephric factor, EGR1 [167]. Further, NDRG1 can associate with the microtubule
tubules and somite disorganisation in the African clawed frog (Xenopus and localise in centrosomes and spindle esters and it is required to
laevis) [163]. In addition, NDRG1 has been shown to promote specifica- maintain spindle structure in a p53-dependent manner during cell
tion of oesophagus, pancreas, stomach and duodenum progenitor cells division [168]. NDRG1 knockdown is associated with a reduction in α-
via the repression of Wnt/B-catenin signalling in Xenopus spp. embryos tubulin acetylation, disappearance of α-tubulin, changes in microtubule
[166]. structure and a failure to form dividing spindle fibres [168]. These latter
 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19 9

studies indicate a role for NDRG1 in ensuring fidelity of cell division and HMSNL sufferers, in whom NDRG1 contains the truncating R148X muta-
suppression of genomic instability. Conversely, reduced NDRG1 expres- tion, have been identified to possess decreased HDL-C levels and an in-
sion may result in genomic instability and tumourigenesis [168]. creased total cholesterol/HDL-C ratio [135], which is associated with an
NDRG1 is induced during cellular differentiation [24,55], a role that increased risk for atherosclerosis and other cardiovascular diseases
is corroborated by multiple studies [25,145,169,170]. For instance, [184].
NDRG1 has been identified as a gene that is up-regulated when
myelomonocytic cells cease to proliferate, suggesting a role for NDRG1 4.2.4. Stress response
in promoting terminal differentiation [145]. Consistent with this, NDRG1 Another role for NDRG1 has been identified in terms of the response
over-expression is associated with up-regulation of epithelial cell to cellular stress. For instance, elevated homocysteine levels which can
differentiation markers in metastatic colon cancer [25] and epider- be cytotoxic are associated with NDRG1 up-regulation [40]. Hence,
mal keratinocyte cell lines [134]. Furthermore, ligands of nuclear NDRG1 is speculated to possess cyto-protective functions. Consistent
transcription factors involved in cellular differentiation, such as with putative cytoprotective activities, an association between NDRG1
PPAR-γ [25], vitamin D [70,144,145] as well as differentiation- and responses to hypoxia have also been identified [140,151]. During
inducing isobutylmethyl xanthine [44], have all been shown to hypoxia, NDRG1 is up-regulated [140] (see Section 4.1) and attenuates
induce NDRG1 expression. NDRG1 has also been to shown to down- p53 expression, which is crucial in the induction of apoptosis in hypoxic
regulate a number of genes involved in suppression of cellular differ- trophoblasts [153]. Thus, from these studies, NDRG1 is suggested to pos-
entiation (e.g., Hod, S100a10, connective tissue growth factor [Ctgf], sess a pro-survival function. In contrast, NDRG1 was found to sensitise
ectonucleotide pyrophosphatase/phosphodiesterase family member 3 cells to p53-mediated apoptosis and caspase activation in colon cancer
[Enpp3] and secreted phosphoprotein 1 [Spp1]) [142]. Down-regulation cells [79]. Hence, the effect of NDRG1 on cell survival may be tissue-
of these genes by NDRG1 resulted in increased cellular differentiation, or cell-type specific or pleiotropic [53,112].
indicating the important role of NDRG1 in this process [142].
4.2.5. Immunity
4.2.3. Lipid biosynthesis and myelination Recently, studies have shown that NDRG is an important mediator of
NDRG1 is implicated in lipid synthesis and myelination, with its ex- the immune system and is involved in allergy and anaphylaxis, defence
pression being integral to axonal survival [171]. Although its expression against bacterial pathogens and bacterial clearance, inflammation and
is not detectable in sensory or motor neurons or their axons, NDRG1 is wound healing [51,136,138]. Given the centrality of the immune system,
highly expressed in peripheral nerves [92], Schwann cells [92] and NDRG1 may be implicated in pathologies other than those in which its
oligodendrocytes [172]. Consistent with the presence of a putative expression is dysregulated or otherwise abnormal [185]. In fact, chronic
phosphopantetheine attachment site in its structure, this expression inflammation is now regarded as an enabling characteristic of cancer
pattern suggests a potential role for NDRG1 in lipid biosynthesis, and contributes to the pathogenesis of this condition [186–191].
myelination and axonal survival [171]. In fact, truncated isoforms of Expression of NDRG1 mRNA is induced during the maturation of
NDRG1 are associated with demyelinating diseases. HMSNL, which is primitive mouse bone marrow-derived mast cells into their mature
also known as Charcot–Marie–Tooth disease, type 4D (CMT4D) [173], counterparts in connective tissue [170]. Interestingly, the expression of
is an early-onset, autosomal recessive form of Charcot–Marie–Tooth NDRG1 allows these cells to degranulate more rapidly and enhance exo-
disease, characterised by demyelination of peripheral nerves [54]. cytosis in response to extracellular stimuli [170]. Notably, NDRG1−/−
HMSNL progresses to severe morbidity and disability in adulthood and mice, or mice transfected with mutant NDRG1 where the three
its signs and symptoms include muscle weakness, gait abnormality, decapeptide tandem repeats are deleted, exhibit markedly reduced im-
myopia, bowel dysfunction, deafness and scoliosis [174–177]. A truncat- mune responses when compared to wild-type mice, indicating that the
ing nonsense mutation in NDRG1, namely R148X, has been identified as C-terminal domain is required for NDRG1 function in mast cells [170].
causative or characteristic of the disease phenotype and suggests a Further, NDRG1−/− mice exhibit decreased numbers of mast cells that
critical role for NDRG1 in myelination and axonal survival [54,171]. In- possess impaired degranulation, resulting in an attenuated immune
terestingly, mutated NDRG1 is also associated with polyneuropathies response to antigens [128]. Finally, relevant to its role in immune func-
in Alaskan Malamutes [178] and Greyhound dogs [179]. tion, NDRG1 expression has been correlated with neutrophil differentia-
Additional support for the role for NDRG1 in myelination came from tion [192]. Collectively, these studies provide evidence in support of the
studies using NDRG1−/− mice. In fact, these animals retained complicat- differentiation-associated and immunomodulatory activities of NDRG1.
ed motor skills, although they exhibited muscle weakness, particularly In vitro, NDRG1 has been shown to undergo phosphorylation at
in distal limbs and progressive demyelination of nerves, such as the sci- multiple Ser and Thr residues by calmodulin kinase II, PKA and PKC
atic nerve [180]. Consistent with previous studies, NDRG1 was shown to
be highly expressed in the cytoplasm of Schwann cells, which further
suggests a role for NDRG1 in both the physiological function of Schwann Table 4
Expression and putative functions of NDRG1 in reproductive cancers.
cells, as well as the production and maintenance of the myelin sheath
[180]. Recently, another mechanism leading to Schwann cell dysfunc- Tissue Expression and function(s)
tion has been suggested to occur after NDRG1 down-regulation [181]. Breast Low expression in cancer [22,23,281]
In these studies, NDRG1 silencing led to decreased uptake of low- Expression may be lower due to aberrant methylation [88]
density lipoprotein (LDL) due to reduced LDL receptor expression [181]. Expression higher in invasive breast cancer than in non-invasive cancer
In addition, there was down-regulation of oligodendrocyte differentia- [140,282]
Expression is associated with cell differentiation [272], reduced vascular
tion factor, Olig2 [181]. Both LDL and Olig2 are implicated in multi- invasion in vitro [22,23,283,284], SPARC down-regulation [285]
vesicular body formation, endosomal LDL receptor trafficking, lipid Cervical Low expression in cancer [39]
processing and differentiation of Schwann cells [181]. Together, these Expression higher in invasive cervical cancer than in carcinoma [38]
studies furnish evidence in favour of a role for NDRG1 in myelination. Expression may be associated with angiogenesis [39]
Endometrial High expression in cancer [214]
In addition to its putative role in myelination and axonal survival,
Silencing enhances cell proliferation, colony formation, cell migration
NDRG1 has been associated with other lipid biosynthetic pathways. and invasion [232]
NDRG1 interacts with apolipoprotein A-I and -II, two lipoprotein con- Prostate Low expression in cancer [23,31]
stituents of high-density lipoprotein cholesterol (HDL-C) [135,181]. As Expression appears to be associated with favourable clinical outcomes
HDL-C is an established preventative factor for atherosclerosis [182], Expression is associated with reduced vascular invasion [31] and
expression of E-cadherin [286]
NDRG1 may play a role in atherogenesis [53,135,137,146,183]. In fact,
10 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19

Table 5 between NDRG1 expression and cancer stage [32,35,37,38,89], which

Expression and putative functions of NDRG1 in gastrointestinal cancers. suggests that this protein plays pleiotropic roles, with its activity being
Tissue Expression and function(s) context-dependent. In addition, recent studies have identified NDRG1
isoforms [54,94–97,171], which may be relevant to alterations in its activ-
Colorectal Low expression in cancer [24–28,55,287–289]
Expression appears to be associated with favourable clinical outcomes ity and subsequent promotion or suppression of tumourigenesis or
[24–28,55,287–289] carcinogenesis.
Expression may be associated with angiogenesis via JNK activation and
an autocrine loop of IL-1 signalling [247]
5.1. Regulatory pathways of NDRG1 in cancer
Gallbladder High expression in cancer [290]
Expression higher in advanced tumours than in early tumours [290]
Liver High expression in cancer [34,140,291] In addition to the regulatory mechanisms outlined above in
High expression is associated with vascular invasion, metastasis, late Section 4.1, NDRG1 is regulated at the transcriptional and translational
tumour stage, large tumour size and high histological grade [34,35] levels by pathways involving transcription factors and proteins, such as
Silencing reduced cell proliferation, apoptosis and vascular invasion
p53, PTEN and myc oncoproteins [194]. Moreover, growing evidence
in vitro and inhibited tumour growth in vivo [33]
Oesophageal Expression lower in advanced tumours (0–I vs. II–IV) and local tumour demonstrates that NDRG1 expression is epigenetically regulated in
invasion (T1–2 vs. T3–4) [29] cancer [58,88,90,195]. These mechanisms are described in detail below.
Oral High expression in cancer [36,292]
Pancreatic Expression may be altered in cancer by epigenetic regulation [58]
5.1.1. NDRG1 and p53
Expression appears to be associated with favourable clinical outcomes
Expression is associated with cell differentiation [293] and reduced The tumour suppressor protein, p53, has been reported to induce
tumour growth in vitro and in vivo [228]; reduced production of IL-8 NDRG1 expression by binding to the NDRG1 promoter [41,79]. DNA
and VEGF-A, microvascular density and vascular invasion [30]; damage also appears to induce NDRG1 expression in a p53-dependent
reduced production of CXC cytokines, decreased inhibitor of κB manner [41,79]. However, conflicting studies in different cell lines and
signalling and induced nuclear factor-κB signalling [245]
tissues have not demonstrated a consistent association between DNA
Stomach Low expression in cancer [229]
High expression in cancer [199,235] damage and NDRG1 expression, though p53 expression may be up-
Expression higher in cytoplasm in local tumours and in nucleus in regulated [85,143]. Further, using p53-null H1299 lung cancer cells
advanced, invasive tumours [199] [143] or DLD-1 colorectal cancer cells expressing a mutant, inactive
Expression is associated with reduced stromal invasion, lymph node
form of p53 [79], it was shown that while p53 expression could be up-
metastasis and pathological stage [229]; aberrant expression of p53,
cell differentiation and tumour growth [199]; and angiogenesis [235]
regulated, NDRG1 expression did not occur. As these observations were
made in different cell lines, it is possible that p53 regulates NDRG1 expres-
sion in a cell- or tissue-specific manner. Interestingly, p53 expression was
[83]. Phosphorylation at these residues are associated with marked shown to be down-regulated by NDRG1 in human placental trophoblasts
mast cell degranulation and exocytosis [83]. Deletion of the C- [153], possibly via up-regulation of Mdm2 [196,197]. However, the in-
terminus domain results in increased phosphorylation by PKA and volvement of NDRG1 in such an autoregulatory pathway may be limited
PKC, but not by calmodulin kinase II, suggesting that the C-terminus do- to only normal human placental trophoblasts and not to cancer cells.
main masks the PKA and PKC phosphorylation sites [83]. NDRG1 has Although p53 appears to be a positive regulator of NDRG1, it is nota-
also been reported to interact with other proteins involved in mediating ble that NDRG1 is still expressed in p53−/− mice, suggesting that other
immune responses and carcinogenesis, including Hsc70 [131] and hep- effectors up-regulate NDRG1 [41]. Indeed, it is notable that other mech-
atitis C virus protein NS5A [193]. Together, these studies suggest NDRG1 anisms have been identified which induce NDRG1 expression, such as
plays a potential immunomodulatory role that is cell- or tissue-specific. HIF-1 [140,198], EGR1 [146,199–202] and PTEN [85,111].

5. NDRG1 and cancer 5.1.2. NDRG1 and PTEN

Like p53 [203], PTEN is a commonly inactivated or mutated tumour
Studies utilising a variety of methodologies have revealed an associ- suppressor protein in cancer that is implicated to inhibit oncogenesis via
ation between NDRG1 expression and cancer, with NDRG1 levels being NDRG1 [204]. Both PTEN and NDRG1 have been demonstrated to be
higher in certain cancers and lower in others (Tables 4–6). Hence, it is more highly expressed in normal breast and prostate tissue than in
clear that NDRG1 plays an important role in the promotion or inhibition matched neoplastic breast and prostate tissue [23]. Moreover, PTEN ex-
of carcinogenesis depending on factors such as the cell-type affected. pression is associated with decreased lymph node metastasis in mice
In fact, a number of studies revealed a negative correlation between with prostate cancer [205], while down-regulation of PTEN is correlated
NDRG1 expression and severity of cancer, which suggests that NDRG1 with vascular invasion and metastasis in vitro and in vivo [23,205,206].
may have a role as a tumour suppressor, metastasis suppressor, or both The tumour-suppressive activities of PTEN are mediated through its in-
[51,53,138]. However, some studies also revealed a positive correlation hibition of the phosphoinositide 3-kinase (PI3K) pathway [207], with
which it is coupled in normal physiology [207–210]. The PI3K and
Table 6 PTEN pathways constitute a negative feedback loop that is normally
Expression and putative functions of NDRG1 in non-gastrointestinal and non- tightly regulated [207–210]. However, in cancer, the pathways are
reproductive cancers. uncoupled and pro-oncogenic PI3K signalling overwhelms and reduces
Tissue Expression and function(s) tumour-suppressive PTEN signalling [207–210].
PTEN is a tumour suppressor protein that induces NDRG1 [25], and
Brain Low expression in cancer [21,140]
High expression in central neurocytoma [294] and grade IV–V glioma
immunohistochemistry studies have demonstrated similar expression
[21,140] patterns for PTEN and NDRG1 in breast and prostate cancer tissue
Expression higher in glioblastoma than in low-grade astrocytoma [234] [23]. PTEN appears to be a mediator of NDRG1 expression, as silencing
Lung High expression in cancer [140,295–297] of PTEN expression has been shown to down-regulate NDRG1 levels,
High expression is associated with production of IL-8 and VEGF-A, high
whereas over-expression of PTEN up-regulates NDRG1 in a dose- and
microvascular density and vascular invasion, [295,296]
Renal High expression in cancer [32,140] time-dependent manner [23]. Although no PTEN-binding sequence
Expression higher in nucleus in progression-free survival [298] can be identified in the promoter region of NDRG1 [211,212], a potential
Skin High expression in cancer [37,140] binding site has been identified for BPOZ, a protein involved in the PTEN
Thyroid Low expression in cancer [299] signalling pathway [147], in the promoter region of NDRG1 [211,212],
High expression in cancer [300]
indicating a possible mechanism by which PTEN regulates NDRG1 [53].
 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19 11

In addition, up-regulation of NDRG1 by PTEN has been demon- survival rates for patients with higher levels of NDRG1 expression
strated to involve PTEN phosphatase activity, which decreases in vivo [30]. In this latter study, although NDRG1 over-expression affect-
phosphatidylinositol-3,4,5-triphosphate and results in suppression of ed cell growth in vivo, it did not affect cell growth in vitro, potentially
the PI3K/protein kinase B (Akt)/mTOR pathway [23]. Conversely, Akt due to in vivo modulatory factors such as those associated with the stro-
activation was suggested to inhibit NDRG1 expression [213]. ma and angiogenesis [30].
Interestingly, loss or down-regulation of PTEN has been associated Further evidence for the role of NDRG1 in cell proliferation
with enhanced expression of NDRG1 in endometrial cancer, possibly comes from a microarray study, which showed NDRG1 dependent
via the PI3K/Akt/mTOR pathway [214,215]. Although the loss or down- up-regulation of thiamine triphosphatase (Thtpa) protein levels
regulation of PTEN is consistent with previous studies [216–218], the in prostate and pancreatic cancer cells over-expressing NDRG1 [142].
up-regulation of NDRG1 is unexpected and may suggest a possible cell- Thtpa is involved in hydrolyzing thiamine triphosphate, which is an
or tissue-specific, pro-oncogenic role in endometrial tissue [215]. The important energy currency molecule in cells [227]. Thus, increased
precise mechanisms underlying the association between PTEN and NDRG1 levels may lead to a decrease in the energy state of the cell
NDRG1 remain to be elucidated, but it is suggested that other regulatory followed by diminished cell growth.
mechanisms may be involved and further investigation is necessary. Recently, it has been suggested that NDRG1 expression in pancreatic
Recently, NDRG1 has been demonstrated to induce PTEN expression cancer may inhibit tumour growth via increasing apoptosis [228]. Ex-
in pancreatic cancer cells [111], indicating that NDRG1 and PTEN consti- pression of NDRG1 was associated with decreased tumour growth
tute a positive feedback loop. In fact, when PI3K signalling overwhelms in vitro and in vivo, and NDRG1 was demonstrated to completely inhibit
PTEN signalling, up-regulation of NDRG1 restores the coupling of the PI3K tumour growth in anchorage-independent assays [228]. These
and PTEN pathways via up-regulation of PTEN and down-regulation of observations indicate a role for NDRG1 as a tumour suppressor, but
PI3K [111], thereby resulting in reduced phosphorylated Akt levels and the underlying mechanisms responsible for this activity remain to
suppressed tumourigenesis [201]. be investigated.

5.1.3. NDRG1 and myc oncoproteins 5.2.2. NDRG1 and metastasis

The myc family consists of three oncoproteins, c-myc, L-myc and N- As described in Section 4.2.2, NDRG1 is involved in cell differentia-
myc [87]. Myc oncoproteins are commonly over-expressed in cancer tion and its expression has been negatively associated with tumour
[219,220]. Indeed, dysregulation of myc oncoproteins is strongly cor- metastasis [22,25–27,31,229–231]. Indeed, the role of NDRG1 as a me-
related with carcinogenesis, as they activate and interact with a vari- tastasis suppressor has been demonstrated in vitro [31] and in vivo
ety of oncogenic pathways [221] and transcriptionally repress [142]. NDRG1 was shown to inhibit metastasis by reducing cell–cell
NDRG1 expression [44,45]. Interestingly, N-myc expression itself and cell–matrix adhesion in AT6.1 rat prostate cancer cells [142] and
may be down-regulated by Ni2 + and retinoids, thereby resulting in to inhibit metastasis to lungs without affecting primary tumour growth
the up-regulation of NDRG1 expression [44]. In neuroblastoma cell in a SCID mouse model [31]. NDRG1 expression was also found to
lines with amplified N-myc and neuroepithelioma cell lines with ampli- inhibit cell proliferation in metastatic cell lines, but not in non-
fied c-myc, over-expression of c-myc and of N-myc were both shown to metastatic cell lines, indicating its differential functions and activities
strongly repress NDRG1 expression [44]. Indeed, as the expression of [80,138]. Further, suppression of NDRG1 was demonstrated to signifi-
these myc oncoproteins decreases during differentiation, the resultant cantly enhance cell proliferation, migration and invasion in Ishikawa
up-regulation of NDRG1 may be required for the initiation of cellular endometrial cancer cells [232]. In contrast, over-expression of NDRG1
differentiation [44]. was shown to inhibit cell proliferation and invasion of this latter cell
The repressive effect of c-myc and N-myc on NDRG1 is mediated via line [232].
the promoter region of NDRG1 at a site 52-bp from the transcription In support of a role of NDRG1 in metastasis suppression, NDRG1
start site of NDRG1 [45] and involves hetero-dimerisation of myc expression has been demonstrated to be higher in organ-confined tu-
oncoprotein with myc-associated factor X (MAX) [222–226]. However, mours compared to bone or lymph node metastases [31]. In fact, it has
the repression mediated by the myc-MAX heterodimer does not appear been suggested that NDRG1 could be used as a prognostic biomarker
to be due to direct binding [45] and may instead depend on an interac- for gastric cancer, as strong evidence indicates that it can reasonably
tion of the myc-MAX complex with general transcriptional machinery predict metastasis and early invasion [229], in addition to discerning
[45]. hypoxic regions within tumours [233,234]. On the other hand, higher
expression has been reported in the cytoplasm in local tumours and in
5.2. Functions and functional pathways of NDRG1 in cancer the nucleus in advanced metastatic cancers, and NDRG1 expression
has been associated with aberrant expression of p53, cell differentiation,
In contrast to normal cells and tissues in which NDRG1 is widely and tumour growth and angiogenesis [199,235]. These conflicting observa-
highly expressed [124], neoplastic cells and tissues have been demon- tions may be due to a multitude of factors, including disparities between
strated to express low levels of NDRG1 [51]. Hence, the functions of stage of cancer, sample selection, or experimental protocols. Hence, it
NDRG1 in primary and metastastic tumour growth as well as the asso- appears that the efficacy of NDRG1 as a prognostic biomarker for gastric
ciated processes of angiogenesis, attachment and adhesion have been cancer is currently limited and requires further study.
intensely investigated and are described in the following sections. Recently, NDRG1 has been shown to suppress metastasis by a
mechanism involving the modulation of the structural protein
5.2.1. NDRG1 and primary tumour growth actin [236]. In cancer cells, actin is polymerised to form stress fibres
NDRG1 has been demonstrated to inhibit primary tumour growth that are required for cell migration [237]. NDRG1 has been demon-
in vitro and in vivo [41]. In vitro, after over-expression of NDRG1, the strated to inhibit the Rho-associated, coiled-coil containing protein
proliferation rate of MCF7 breast and EJ bladder cancer cell lines kinase 1 (ROCK1)/phosphorylated myosin light chain 2 (pMLC2)
decreased by approximately 70% and colonies on soft agar of these cell pathway [236], which would therefore result in suppression of the as-
lines were observed to be smaller than the relative control [41]. Further, sembly and rearrangement of stress fibres from actin [238]. As this
in vivo, mice injected with EJ bladder cancer cells that over-expressed pathway has also been implicated in diverse processes, including adhe-
NDRG1 were observed to exhibit smaller tumours compared to those sion, cell motility, proliferation, differentiation, apoptosis, production of
injected with control EJ bladder cancer cells [41]. Subsequent studies proteolytic enzymes and invasion [239–241], inhibition of the ROCK1/
reported a reduction in tumour microvascular density, invasion depth pMLC2 pathway suggests another mechanism by which NDRG1 sup-
and histopathological grading, with a corresponding increase in overall presses cancer cell metastasis [236].
12 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19

5.2.3. NDRG1 and angiogenesis expression was higher in more advanced lesions, leading to speculation
Angiogenesis is the process by which new blood vessels are formed that NDRG1 is a metastasis promoter gene [35]. In more recent studies,
from pre-existing vessels and it is well known to play an important role NDRG1 was shown to be up-regulated in a variety of tumours including
in multiple diseases, including cancer [242,243]. In metastasis, angio- those of the liver, cutaneous squamous cell carcinoma, oral squamous
genesis is required for tumours to grow and invade [244] and NDRG1 cell carcinoma, cervical cancer and renal carcinoma [32,35–38]. In fact,
over-expression has been associated with decreased production of silencing NDRG1 in liver cancer reduced proliferation, invasion and
pro-angiogenic factors, such as IL-8, MMP-9 and VEGF1, leading to de- apoptosis in vitro and inhibited tumour growth in vivo [35].
creased angiogenesis in pancreatic cancer [30]. In addition, NDRG1 has However, the observed increase of NDRG1 in certain cancers can be
been demonstrated in pancreatic tumours to suppress the production attributed to the hypoxic conditions within tumours [251], as hypoxia
of CXC cytokines [72,245], which are known to mediate processes criti- also up-regulates NDRG1 levels [143]. In fact, a clinical study involving
cal to metastasis, such as proliferation, adhesion, resistance to chemo- 223 prostate cancer specimens found no conclusive correlation of
therapeutic agents and inhibition of apoptosis [73,74]. Moreover, NDRG1 with tumour stage and grading, this being due to the differential
NDRG1 has been shown to suppress angiogenesis via attenuating the response to hypoxia and androgens in the prostate epithelium of differ-
expression and phosphorylation of the inhibitor of κB kinase (IκBα) ent patients [252]. These results again indicate the tissue-specific effects
and also NF-κB signalling [245]. of NDRG1 and outline the importance of considering the tumour micro-
Another putative mechanism by which NDRG1 modulates angio- environment and hormonal factors when assessing NDRG1 function.
genesis has been recently proposed. In prostate cancer, the PI3K/
PTEN/AKT pathway has been shown to modulate angiogenesis via 5.3. NDRG1 and chemotherapy
regulation of HIF-1 and VEGF expression and that PTEN, which inhibits
PI3K and AKT, reduces angiogenesis and tumour growth [246]. Consid- NDRG1 has been suggested to be involved in modulating sensitivity
ering that NDRG1 promotes PTEN expression in prostate cancer [95], and resistance of cancer cells to chemotherapeutic agents, such as
this further supports an anti-angiogenic role for NDRG1 in this neo- irinotecan (CPT-11), a topoisomerase I inhibitor [253]. Specifically,
plasm. In contrast, expression of NDRG1 has been associated with an- over-expression of NDRG1 in the SW620 colon cancer cell line is associ-
giogenesis and low survival rates for patients with cervical cancer [39] ated with resistance to CPT-11 and repression of NDRG1 in the HCT116
and gastric cancer [235]. In cervical cancer, high NDRG1 expression is colon cancer cell line increased the sensitivity of cells to undergo apo-
correlated with high VEGF1 expression and high microvascular density ptosis after treatment with CPT-11 [253]. In support of this, tumours
[39]. In support of this role for NDRG1 as a pro-angiogenic protein, established from xenografted HCT116 and SW620 colon cancer cells
angiogenic activity and mRNA levels of angiogenesis-related proteins, that over-express NDRG1 were more resistant to CPT-11 compared to
including CXC cytokines, IL-1β, IL-6 receptor, MMP-1 and VEGF1, the vector-transfected controls in mice, indicating a role for NDRG1 in
were shown to be higher in gastric cancer cells that over-expressed resistance to chemotherapeutic agents [253]. Moreover, patients with
NDRG1 than in control cancer cells [247]. The mechanism by which low NDRG1 expression consistently appeared to remain on CPT-11-
NDRG1 promoted angiogenesis was demonstrated to involve IL-1α based chemotherapy for longer durations than patients with high
and activation of JNK/AP-1 [247]. Together, these studies again indicate NDRG1 expression [26]. Further studies are required to elucidate the
that NDRG1 possesses pleiotropic effects and that its activities depend precise role and mechanism by which NDRG1 modulates sensitivity of
upon the tumour-type. The differences in pro- and anti-angiogenic activ- cancer cells to chemotherapeutic agents [26].
ity may, in part, be due to involvement of the complex microenvironment
in which these tumours develop [51,53,142]. 5.3.1. Targeting NDRG1 by binding cellular iron
Iron is a trace element integral to multiple metabolic processes, in-
5.2.4. NDRG1, attachment and adhesion cluding DNA synthesis [157] and the metabolic demand for iron is great-
In the last decade, emerging evidence has suggested that NDRG1 er in neoplastic than in normal cells [254]. Indeed, intracellular iron
modulates metastasis via proteins including MMPs, which degrade depletion is known to result in anti-tumour activity, prompting the
extracellular matrix, and adhesion molecules, such as β-catenin and E- development of chelating agents that possess marked anti-proliferative
cadherin that form the adherens junction at the cell membrane efficacy [157,255,256]. Intracellular iron depletion by iron chelators,
[25,94,248]. In agreement with these latter studies, NDRG1 has also such as desferrioxamine (DFO) [143], 2-hydroxy-1-naphthaldehyde
been demonstrated to up-regulate β-catenin in breast cancer cells isonicotinoyl hydrazone (NIH) [143], di-2-pyridylketone 4,4-dimethyl-
[25]. Further, NDRG1 has also been shown to inhibit TGF-β-induced 3-thiosemicarbazone (Dp44mT) [143,257,258], Triapine [257] and di-2-
epithelial–mesenchymal transition and to restore β-catenin and E- pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) [96],
cadherin levels, which are suppressed by TGF-β in cancer cells [94]. more significantly affects neoplastic cells than normal cells [254].
Hence, these observations indicate that NDRG1 promotes the formation A number of iron chelators have been shown in vitro [143] and
of the adherens junction, which is critical for cell–cell adhesion, and in vivo [257] to exhibit their potent, selective anti-tumoural activity, in
therefore reduces neoplastic cell migration and invasion. However, in part, by the up-regulation of NDRG1 [143]. Further, up-regulation of
gastric cancer, down-regulation of NDRG1 led to the induction of MMP- NDRG1 via iron chelators involves depletion of intracellular iron pools
2 via MT1-MMP [249], while expression of NDRG1 was associated with and the up-regulation of HIF-1α-dependent and -independent mecha-
down-regulation of E-cadherin, vimentin and Snail [250]. These observa- nisms [143,152]. Notably, the potent anti-proliferative efficacy of chela-
tions, as for other activities of NDRG1, reflect the cell- or tissue-specific tors is due to their effects on a variety of critical molecular targets in
activities of NDRG1 [53]. addition to NDRG1, including p21CIP1/WAF1 [96], cyclin D1 [96] and the
rate-limiting enzyme of DNA synthesis, ribonucleotide reductase
5.2.5. The controversial role of NDRG1 in oncogenesis [259], all of which are involved in regulating proliferation and/or
Although the majority of studies examining NDRG1 report a tumour metastasis.
suppressive role for this protein, there are also a number of studies that A variety of chelators have been developed [255,260], with ligands of
contradict this view and demonstrate a tumourigenic role for NDRG1. In the di-2-pyridylketone thiosemicarbazone (DpT; Fig. 5) class being a
fact, in some studies, androgen receptor positive prostate cancers shows highly active series that demonstrate marked potency and selectivity
higher levels of NDRG1 compared with normal tissue [125]. These stud- in vitro and in vivo against tumour cells [257,261]. Unlike conventional
ies claim the decreased level of NDRG1 observed in other studies with iron chelators, such as desferrioxamine, these compounds do not
prostate cancer is due to loss of hormone-dependence [53]. Further- cause whole body iron depletion at optimal doses [262]. In fact, these
more, another study examining colorectal cancer found that NDRG1 agents work via a “double-punch" mechanism in which they: (1) bind
 B.A. Fang et al. / Biochimica et Biophysica Acta 1845 (2014) 1–19 13

Fig. 5. Chemical structures of di-2-pyridylketone thiosemicarbazone iron chelators. Line drawings of the general structure of the di-2-pyridylketone thiosemicarbazone ligands, together
with the structures of di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC).

and sequester iron, and (2) form redox active metal complexes, leading may lead to the development of new anti-cancer therapeutics such as
to generation of reactive oxygen species (ROS), which further potenti- the DpT group of thiosemicarbazones that markedly up-regulate this
ates their anti-cancer activity [262]. protein to inhibit metastasis in vivo [258].
Of these agents, a second-generation DpT analogue, DpC (Fig. 5),
demonstrates marked and selective anti-tumour activity in vivo using Acknowledgements
models of pancreatic [96] and lung cancer [262]. In fact, DpC possesses
greater anti-tumoural efficacy in vivo than the current gold-standard D. R. R. thanks the National Health and Medical Research Council of
chemotherapeutic agents against pancreatic cancer, namely gemcitabine Australia (NHMRC) for a Senior Principal Research Fellowship and Pro-
[96]. Moreover, unlike the former lead compound of the DpT series of ject Grants. Ž. K. and D. J. R. L. thank the NHMRC for an Australian Train-
ligands, namely Dp44mT (Fig. 5) [257], DpC does not cause cardiac ing Fellowship (Peter Doherty Biomedical Post-Doctoral Fellowship)
cardiotoxicity [96,262]. Notably, DpC was shown to markedly increase and the Cancer Institute New South Wales (CINSW) for an Early Career
NDRG1 expression and phosphorylation in pancreatic cancer cells and Development Fellowship. D. S. K. thanks the NHMRC for Project Grant
it is probable that its effect on this molecular target, as well as others Funding, and P. J. J. acknowledges the CINSW for an Early Career Devel-
(e.g., cyclin D1 and p21), is critical for its anti-tumour activity [96]. More opment Fellowship and the Prostate Cancer Foundation of Australia for
recent studies have demonstrated that DpC inhibits ROCK1/pMLC2- a Young Investigator Grant.
modulated actin-filament polymerization, stress fibre assembly and for-
mation that are critical for the migration and metastasis of tumour cells
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