Carcinogenesis vol.29 no.1 pp.2–8, 2008
doi:10.1093/carcin/bgm200
Advance Access publication October 4, 2007
REVIEW
NDRG1, a growth and cancer related gene: regulation of gene expression and function in
normal and disease states
Thomas P.Ellen, Qingdong Ke, Ping Zhang and Max Costa
Department of Environmental Medicine, Pharmacology and the NYU Cancer
Institute, New York University School of Medicine, New York, NY 10016,
USA
To whom correspondence should be addressed. Tel: 845 731 3514;
Fax: 845 351 2118;
Email: [email protected]
N-myc downstream-regulated gene 1 (NDRG1) is an intracellular
protein that is induced under a wide variety of stress and cell
growth-regulatory conditions. NDRG1 is up-regulated by cell differentiation signals in various cancer cell lines and suppresses
tumor metastasis. Despite its specific role in the molecular cause
of Charcot–Marie–Tooth type 4D disease, there has been more
interest in the gene as a marker of tumor progression and
enhancer of cellular differentiation. Because it is strongly upregulated under hypoxic conditions, and this condition is prevalent in solid tumors, its regulation is somewhat complex, governed
by hypoxia-inducible factor 1 alpha (HIF-1a)- and p53-dependent
pathways, as well as its namesake, neuroblastoma-derived myelocytomatosis, and probably many other factors, at the transcriptional and translational levels, and through mRNA stability. We
survey the data for clues to the NDRG1 gene’s mechanism and for
indications that the NDRG1 gene may be an efficient diagnostic
tool and therapy in many types of cancers.
Introduction: N-myc downstream regulated gene 1
N-myc downstream-regulated gene 1 (NDRG1) is a member of the
NDRG gene family, which, in turn, lies within the a/b hydrolase
superfamily, although NDRG1 presents no hydrolytic catalytic site.
NDRG1 is a 43 kD protein, composed of 394 amino acids, is highly
conserved among multicellular organisms and is a predominantly cytosolic protein expressed ubiquitously in tissues in response to cellular
stress signals (1,2). Diverse cellular stress response mechanisms have
been proposed, and in some cases shown, to involve NDRG1 (Table I).
These include mechanisms involved in cellular differentiation (3–5),
proliferation and growth arrest (4), neoplasia, tumor progression and
metastasis (6–8), heavy metal response (9–11), the hypoxia response
(12,13) and DNA damage response (2).
NDRG1’s namesake, neuroblastoma-derived myelocytomatosis
(N-Myc), as well as c-Myc, represses NDRG1 by a mechanism that
does not depend on its direct binding to the NDRG1 promoter and that
is histone deacetylase mediated (14). The other members of this family, NDRG2, NDRG3 and NDRG4, are homologous to NDRG1, sharing 53–65% identity. NDRG2 is also transcriptionally repressed by
Myc via a Miz-1-dependent interaction with the NDRG2 core promoter region, and possibly requires histone deacetylase recruitment to
the promoter (15). Regulation by Myc implicates NDRG in regulating
cell differentiation. Whereas NDRG1 is ubiquitously expressed,
NDRG2 is highly expressed in adult skeletal muscle and brain,
NDRG3 in brain and testis and NDRG4 in heart and brain, suggesting
tissue-specific functions for them. Each of these proteins contains an
a/b hydrolase fold similar to that of the human lysosomal acid lipase
(16). Three repeats of the GTRSRSHTSE sequence are found in the
C-terminal tail of NDRG1, which are not present in the other family
members. The function of these repeats has, as yet, not been
identified. Although NDRG family members share significant conservation of amino acid sequence, functional peptide motifs have not
been discovered within them.
N-Myc viral oncogene is a transcription factor that functions by
forming heterodimers with proteins of the Max subfamily via a basic
helix-loop-helix leucine zipper domain in its C-terminal domain followed by heterodimeric binding to gene promoters through recognition
of the consensus DNA sequence, CACGTG, known as an ‘E-box’ (17) by
means of a DNA-binding domain, which is also found in its C-terminal
domain. The N-terminus consists of two myc boxes (MBI and MBII)
essential for most of the protein’s functions (18), and the central region
harbors MBIII, implicated in cell transformation and apoptosis (19),
and MBIV, essential for induction of apoptosis, transformation and G2
arrest, functions reflective of loss of DNA-binding activity (20).
Although a sequence that is partially homologous with E-box has
been found in the proximal promoter of the mouse NDRG1 gene,
mutational studies showed that N-Myc binding to this NDRG1 promoter is not relevant to its regulation mechanism (14). Many target
genes have been shown to be activated by c-Myc pursuant to heterodimer binding to the E-box (21), but other studies have shown that
Myc-mediated repression of genes can occur by a mechanism that
does not involve (as in the case of NDRG1) direct binding of the Myc
protein to DNA (14). Thus, in general, Myc/Max heterodimer direct
binding to DNA at E-box elements is part of a gene-activating event
(NDRG2 repression is apparently the exception to the rule) (15),
whereas Myc-mediated transcriptional repression (as with NDRG1)
occurs by a mechanism that does not involve direct binding of the
target gene’s proximal promoter by Myc (14).
Myc expression is de-regulated in a variety of human cancers, and is
often associated with aggressive, poorly differentiated tumors. Myc
protein is a key regulatory activity in cell growth and proliferation,
cell-cycle progression, transcription, telomerase activity, metabolism,
differentiation, apoptosis, angiogenesis and cell motility. Upon deregulation, genomic instability, unchecked proliferation, growth factor independence, immune surveillance shutdown, transformation and
apoptosis can occur (22). c-Myc oncoprotein is likely to have a broad
influence on the composition of the transcriptome. c-Myc behaves as
both a global regulator of transcription and as a member of specific
regulatory groups of genes managing cell cycle regulation, metabolism, ribosome biogenesis, protein synthesis and mitochondrial function. The c-Myc target gene network is estimated by Henriksson et al.
(23) to comprise 15% of all the genes from flies to humans. Nevertheless, the categorization of downstream genes as either physiologic or
tumorigenic targets of c-Myc has not been accomplished.
Despite the lack of evidence for specific Myc targets, it is known
that NDRG1 mRNA gradually accumulates following a continuous
decrease of N-Myc/c-Myc in mouse embryonic development, and
NDRG1 expression is significantly increased in N-Myc-deficient
embryos (14). It is hypothesized that the negative regulatory effect
on general transcription is the mechanism for NDRG1 repression
in vitro (14).
NDRG1 and peripheral demyelinating neuropathy
Abbreviations: AP-1, activating protein 1; CMT, Charcot–Marie–Tooth; Egr,
early growth response; HIF-1a, hypoxia-inducible factor 1 alpha; HMSNL,
hereditary motor and sensory neuropathy-Lom; NDRG1, N-myc downstreamregulated gene 1; N-Myc, neuroblastoma-derived myelocytomatosis; VEGF,
vascular endothelial growth factor; VHL, von Hippel–Lindau.
NDRG1 was first recognized as a gene whose mutation was linked to
a demyelinating neuropathy, and whose location was mapped to human chromosome 8q24 (2,24). The disorder, which is of autosomal
recessive inheritance, is presumably related to a disturbance affecting
Ó The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
2
NDRG1, a growth and cancer related gene
Table I. Regulation of NDRG1 expression and suggested functions of the NDRG1 protein
NDRG1 function
Upstream signaling effector
NDRG1 outcome
Result
Regulation of expression
of NDRG1
Myc proteins (14)
Repressed
Cell differentiation (when NDRG1 present)
Cell stress (1,6,9)
Terminal differentiation (6)
Nickel compounds (9–11)
Cobalt compounds (11)
Hypoxia (11–13,33,62,76)
DNA damage (2)
Induced expression
Induced expression
Induced expression
Induced expression
Up-regulated
p53-dependent induced
expression
Induced expression
Cell growth arrest
Cell’s loss of proliferative potential
Unknown (hypoxia-like response)
Unknown (hypoxia-like response)
Hypoxia response
Cell growth arrest
p53 (2,53–55,74)
Induced expression
Cell growth arrest, biphasic cell cycle NDRG1
expression
Loss of proliferative potential
Up-regulated
Induced expression
Up-regulated
Cell differentiation, growth arrest
Unknown
Down-regulated metastasis-related genes
Up-regulated
Up-regulated
Housekeeping function
Unknown (perhaps differentiation)
Induced expression
HIF-1a-independent hypoxia response
Hypoxia response pathways
AP-1 (13)
VHL protein (50)
E2a-Pbx1 (54)
Cellular differentiation (3–6)
Cell stress (1,6,9)
Cell growth arrest (4)
Cell growth (2)
p53 (2,53–55,74,75)
HIF-1a-dependent induced
expression
Induced expression
Down-regulated
Induced expression
Up-regulated
Induced expression
Up-regulated
Inhibition of expression
Up-regulated
DNA damage (2)
Cell maturation (3)
HIF-1a (12,62)
AP-1 (13)
Myc proteins (14)
Androgen (34)
Neoplasia, transformation, tumor
(2,6–8,76)
Metastasis (5,7)
p53 (2,53–55,74)
Induced expression
Induced expression
Induced expression
Induced expression
Repressed
Up-regulated
Reduced expression
(but see 76)
Down-regulated
Up-regulated
Adenoma, adenocarcinoma,
carcinoma (6,7)
phosphatase and tensin
homologue (7,8)
HIF-1a (12,62)
AP-1 (13)
Gene mutation (3,24–28)
Reducing agents (1)
Reduced expression
Hypoxia response pathways
Unknown
Unknown (possible apoptotic response)
Differentiation
Cell growth arrest
Loss of proliferative potential
NDRG1-mediated growth-arrest is blocked
Biphasic cell cycle NDRG1 expression, cell growth
arrest, p53-mediated apoptosis, euploidy
maintenance
Cell growth arrest
Alter immature cells to mature phenotype
Hypoxia response
Hypoxia response
Cell differentiation (when NDRG1 present)
Cell differentiation, growth arrest
Reduced cell growth, growth arrest, differentiation
(when NDRG1 present)
Induced cell differentiation (when NDRG1 present)
Anti-oncogenic function, cell growth arrest,
p53-mediated apoptosis
Cell differentiation (when NDRG1 present)
Up-regulated
Down-regulates metastasis genes
Induced expression
Induced expression
Mutant NDRG1
Induced expression
Hypoxia response
Hypoxia response
HMSNL, Schwann cell dysfunction
Ischemic disease prevention
Vitamin D, retinoids and other
lipophilic signals (4)
Androgens (34)
Reducing agents (1,9)
phosphatase and tensin
homologue (7,8)
DNA methylation inhibition (5)
Histone deacetylation inhibiting
agents (5)
Intracellular calcium level
increase (9,13)
Hypoxia (11–13,33,38–42,62,76)
Cell function regulation
Cancer role
Disease role
Schwann cell function. The condition was named hereditary motor
and sensory neuropathy-Lom (HMSNL) after the town where some
of the initial cases were recognized as such, and had also previously
been known as Charcot–Marie–Tooth (CMT) syndrome (25,26).
Hereditary motor and sensory neuropathy is genetically heterogeneous,
and a number of types have been classified. All other types have not
been found to involve NDRG1, but, like HMSNL, involve mutationdependent disease mechanisms, and all these clinically classified disorders of the peripheral nervous system have been mapped to various
genomic locations (25,27).
The condition involving NDRG1 has been classified as CMT disease
type 4D (CMT 4D). It was shown that the symptoms of the disease are
confined to the peripheral nervous system, as NDRG1 is abundantly
expressed in the cytoplasm of Schwann cells, but not in myelin sheaths
or axons, and degeneration of neural tissue with concomitant demyelination in Ndrg/ mice, as well as in humans, is restricted to the
peripheral nerves (26). NDRG1 expression levels in peripheral nerve
are significantly in excess of those in any other tissue examined (28).
CMT or HMSN disease has been categorized into demyelinating
forms (CMT1, CMT3 and CMT4) and axonal forms (CMT2) (27).
However, the pathologies of myelinopathies and axonopathies are
often intermingled, as would be expected of conditions of Schwann
cells and neurons, two intimately associated cell types. These different classes of diseases are further subdivided into autosomaldominant demyelinating (CMT1), autosomal-recessive demyelinating
(CMT4), primary axonal neuropathies (CMT2: autosomal dominant
or recessive) and an assortment of subforms of hypomyelinating and/
or dysmyelinating pathologies, which include Dejerine–Sottas
3
T.P.Ellen et al.
syndrome (CMT3), hereditary neuropathies with liability to pressure
palsies and congenital hypomyelination. Demyelinating forms greatly
outnumber axonal forms in the human population.
The clinical phenotype of CMT4D is characterized by a neuropathy
that begins with gait disorder in the first decade of life, upper limb
involvement in the second decade and, in most cases, sensorineural
deafness in the third decade. This demyelinating neuropathy is so classified because of severe reduction in motor nerve conduction velocity
and demyelination as confirmed by nerve biopsy (25). There is lower
limb weakness in all patients, usually associated with muscle wasting.
The founder HMSNL mutation was identified as a C to T transition
in exon 7, at nucleotide position 564, that results in the replacement of
arginine by a translation-termination signal (nonsense mutation) at
codon position 148 of NDRG1 (28), leading to a shortened NDRG1
protein. The R148X mutation is found in the homozygous state in all
HMSNL (CMT4D)-affected individuals. The mutation segregates in 100%
agreement with the carrier status predicted by haplotype analysis.
Recently, it was discovered that early growth response (Egr)-1binding proteins, NAB1 and NAB2, when knocked out in mice,
reduce the expression of NDRG1 in their sciatic nerves (29). Egr-2deficient mice have the same phenotype. NABs are efficient modulators of Egr transcriptional activity, are highly conserved in evolution
and are essential for Schwann cell differentiation (30,31). It is of
interest that patients with mutations in Egr-2 have the neuropathies
congenital hypomyelination and CMT1D (32). Since NDRG1 deficiency causes CMT4D, it suggests that there may be a relationship
between NDRG1 and Egr-2 in Schwann cells.
Patterns of expression in determination of other biological roles of
NDRG1
Despite the careful work that has gone into the elucidation of the role
of NDRG1 in CMT4D disease, NDRG1 expression has been observed
in many other cellular environments and with many other apparent
roles. The conservation of NDRG1-related proteins attests to its importance in biological function, but the determination of function has
been elusive and is probably diverse and dependent on the cellular
context. NDRG1 is up-regulated in differentiating cells, and is also
induced by cellular stress (1,6,9). Conversely, its expression is inhibited under conditions of cell growth (2). The proliferation/differentiation program in many types of cells, for example epithelial cells,
is strictly controlled. A deviation from this regulation may lead to
transformation of cells and tumorigenesis. The down-regulation of the
NDRG1 gene in colon neoplasms may be caused by transformation
and progression, or may simply be the result of decreased differentiation in tumor cells (6). Whatever the answer to this turns out to be, it
is certain that NDRG1 expressed in different tissues has widely varying end effects (6,28), and the regulatory pathways controlling its
expression are apparently diverse. Thus, it has been identified in several in vitro assays, and renamed according to its perceived function in
each case, as HMSNL (24), RTP (1), Drg1 (6), rit42 (2), Ndr1 (14)
and, by our group, Cap43 (9).
As mentioned above, NDRG1 is induced by cellular stress. A first
step in understanding its biological role would be to determine its
pattern of expression. NDRG1 mRNA is ubiquitous among tissues,
including the digestive and respiratory tracts, reproductive, urinary and
immune systems, endocrine glands and skin. It is extensively present
in the epithelia, and this suggests a role for it there. However, many
tissues express the mRNA, yet contain no detectable levels of the protein (33), suggesting regulation in its translation as well as transcription.
Inducing agents include p53 (2), vitamin D, retinoic acid, phorbol
esters (4), androgenic (34) and estrogenic (35) hormones; reducing
agents such as homocysteine, mercaptoethanol and tunicamycin (1);
phosphatase and tensin homologue deleted on chromosome 10,
a known tumor suppressor (8); nickel compounds, both soluble and
insoluble; okadaic acid; calcium ionophores (9); hypoxia mimicking
agents such as deferoxamine (13), DNA methylation and histone
deacetylation inhibiting agents 5#-aza-2#-deoxycytidine and trichostatin A, respectively (5); DNA-damaging agents such as mitomycin C
4
(2); as well as an intracellular rise in Ca2þ (9) or decreased glucose
concentration (6).
Work describing induction by vitamin D that is potentiated by RXRand RAR-specific ligands (36) argues for a role for NDRG1 in cell
differentiation. Work that shows strong and early induction of mRNA
(4) argues for a major regulation that occurs at the transcriptional level.
NDRG1 expression is not tissue specific, and occurs in prostate, ovary,
kidney, brain, heart, placenta, lung, skeletal muscle, colon, testis and
small intestine tissues primarily, with lower amounts found in thymus,
spleen, pancreas, liver and peripheral blood leukocytes (33,37).
The enumeration of inducing agents of NDRG1 brings to light the
diverse regulatory pathways modulating NDRG1 transcription and
translation. For instance, hypoxia exposure leading to NDRG1 induction is hypoxia-inducible factor 1 alpha (HIF-1a) dependent, whereas
DNA damage induces NDRG1 in a p53-dependent fashion. Hypoxiainduced expression of NDRG1 is HIF-1a dependent, because induction
of NDRG1 does not occur in HIF-1a/ mouse embryo fibroblasts,
whereas it does occur in the wild-type HIF-1aþ/þ mouse embryo
fibroblasts (13). However, an increase in intracellular Ca2þ induces
NDRG1 mRNA in HIF-1a-deficient cells, providing evidence for
a HIF-1-independent signaling pathway of NDRG1 gene expression
that was found to involve activating protein 1 (AP-1)-dependent transcription (13), and presenting us with an example of divergent signaling pathways that lead to NDRG1 induction. Thus, NDRG1 expression
induced by hypoxia can be by HIF-1a-dependent or -independent
pathways.
The mechanisms by which HIF-1a and AP-1 induce NDRG1 are
unknown, and possible cis-elements mediating this gene’s regulation
have gone unexplored. In addition to HIF-1a and AP-1, hypoxiainduced gene expression has been shown to be mediated by Egr
gene 1 (Egr-1), Sp1, nuclear factor kappa B, cyclic adenosine 3#,
5#-monophosphate response element-binding protein and p53 (38–42).
Experimental work done to elucidate the mechanisms of hypoxiainduced Egr-1 expression assigns a pivotal role to the activation of
Egr-1 in the cellular response to hypoxia (38). This zinc finger transcription factor functions in rapid response to many types of stimuli,
including growth factors, hypoxia, hormones, and neurotransmitters
(43). The Egr-1 gene is seen as a central conversion hub where signaling cascades are coupled to gene expression. Egr-1 nuclear localization is enhanced during hypoxia and required for the hypoxic
induction of pro-coagulant tissue factor (also known as coagulant
factor III; 44). Conversely, hypoxia-induced Egr-1 activation has been
shown to be independent of HIF (45). Thus, as for NDRG1, Egr-1
expression induced by hypoxia can be by HIF-1a-dependent or
-independent pathways. The upstream signaling cascade of Egr-1
activation in response to oxygen deprivation was traced to members
of the protein kinase C family, leading to Raf- and MEK-dependent
activation of MAP kinases, ERK1/2, and activation of the ets factor
Elk-1 (38).
Hypoxic condition has been shown to increase the Sp1/Sp3 ratio by
depletion of Sp3 in nuclear extracts (46). The ubiquitous transcription
factor Sp1 plays a key role in maintenance of basal transcription of
housekeeping genes (for a review, see ref. 47). In a study of cyclooxygenase-2 regulation (39), nuclear localization of Sp1 in response
to hypoxia increased while that of Sp3 remained unchanged, supporting the role of Sp1 in cellular hypoxia response. Other labs have
reported experimental evidence that Sp1 can facilitate promoter activation in hypoxia in parallel with, and independently of, HIF-1a (48),
or by interaction with HIF-1a (49). Similar to AP-1, this is a transcription factor that appears to function in multiple pathways.
NDRG1 promoter assays with deletion or mutation constructs mapped an Sp1 site in the core promoter from 286 to 62 bp (50). Based
on this evidence, and coupled with the demonstration that von
Hippel–Lindau (VHL) interacts with Sp1 to down-regulate vascular
endothelial growth factor (VEGF) (51), it was hypothesized that VHL
inhibits NDRG1 expression by blocking Sp1 activity (50). Masuda
et al. (50) went on to show that exogenous VHL can reduce NDRG1
expression in VHL-deficient cells. The mechanism of such regulation
is as yet unknown.
NDRG1, a growth and cancer related gene
Pathways leading to nuclear factor kappa B activation by hypoxia
are important for investigating the regulation of nuclear factor kappa
B -binding site-containing genes, such as cyclooxygenase-2, tumor
necrosis factor a, interleukin-6 and -2, macrophage inflammatory
protein-2 and vascular cell adhesion molecule 1 (40,52).
Induction of NDRG1 can be hypoxia initiated and HIF-1a regulated (13), but the NDRG1 gene can be activated by an intracellular
Ca2þ increase, in a HIF-1a-independent signaling pathway, as well
(13). So too, although NDRG1 has been characterized as a downstream
mediator of p53-dependent, anti-oncogenic functions (2,53), was it
reported that the NDRG1 promoter is capable of mediating transcriptional induction by E2a-Pbx1 in a p53-independent manner (54). And
there lies corroborating evidence of this p53 independence in the
findings that NDRG1 induction by DNA-damaging agents in several
cell lines does not invariably require p53 (55). The role of NDRG1 in
DNA damage and growth arrest, tumor suppression, apoptosis and
hypoxia is multiple and complex, as exhibited by its varied relation
to the key regulatory factor, p53 (42,56). More on the relation of p53
in NDRG1 induction and function is addressed in the following
section.
A role as a developmental gene has been documented for NDRG1,
as N-myc-mediated repression has been found to operate during embryogenesis (14). The NDRG1 promoter is down-regulated by N-Myc,
as well as c-Myc, usually during periods of cell proliferation, such as
in tumor cells (4). Whereas it is down-regulated during these periods,
it does not appear to be up-regulated during opposing conditions, i.e.
during cell differentiation or periods in which cells cease to proliferate. It has been observed that the activity of the NDRG1 promoter is
repressed by the N-Myc/Max heterodimer (14). NDRG1 is probably
involved in a cell’s loss of proliferative potential (4). This role of
NDRG1 in cell differentiation, proposed for many tissues, may be
conserved in the peripheral nerve tissues and Schwann cells, and
may be at work in the many stages of Schwann cell differentiation
and the many and complex interactions of Schwann cell–axon communications necessary for the differentiation, survival and normal
function of both types of cells. Alternatively, NDRG1 may have multiple functions, some of which are the same or similar in both
Schwann cells and in cells of other systems, while certain functions
are cell-type specific in specialized cells (such as Schwann cells) with
little redundancy elsewhere.
NDRG1 expression increases in response to cell differentiation
signals. Ligands of nuclear transcription factors involved in cell differentiation, i.e. retinoids (RA or RAR synthetic agonists) and vitamin D,
which, alone or in combination, inhibit cell growth and, at the same
time, induce differentiation, have been shown to be inducers of
NDRG1 (4). These lipophilic signals are dependent upon nuclear
receptors such as RAR, RXR and VDR, which belong to a superfamily
of ligand inducible transcription factors, hold a great many possibilities for individualized endocrine signals among different cell types
and could present multiple pathways by which NDRG1 is regulated.
Likewise, phorbol myristate acetate, which is a strong effector of cell
growth arrest and differentiation, up-regulates NDRG1. Bone marrowderived mast cells, when co-cultured with fibroblasts in the presence
of an excess of the cytokine stem cell factor, alter their morphological
and functional properties from an immature to mature phenotype, and
at the same time have been shown to strongly induce NDRG1 (3).
NDRG1 significantly increases during differentiation of Schwann
cells compared with their proliferating counterparts (26). All these
examples point to the regulation of NDRG1 as part of the cellular
processes of growth arrest and terminal differentiation. Figure 1 summarizes the various transcription factors that have been shown to
directly or indirectly regulate the transcription of the NDRG1 gene.
Implications for an NDRG1 role in cancer
A putative p53-binding motif has been demonstrated in the proximal
promoter of the NDRG1 gene (53). In the absence of the binding
element, p53-mediated transcriptional activation was greatly diminished. p53 is a tumor suppressor whose ability to induce cell cycle
Fig. 1. Transcriptional regulation of NDRG1. Various environmental
stressors activate transcription factors to cause an enhanced mRNA and
protein level of NDRG1. Solid lines indicate regulation supported by direct
evidence such as CHIP, whereas broken lines indicate possible links of
regulation.
arrest and apoptosis is vital to its tumor-suppressing function. Besides
p53 binding and activating the NDRG1 gene, Stein et al. (53) showed
that p53-induced NDRG1 expression inhibited cell proliferation
(Table 1) but had no apoptotic effect in metastatic lung cancer cells,
suggesting that NDRG1 is involved in cell growth suppression in
metastatic tumor cells.
NDRG1 was not induced by ectopic expression of p53 in a metastatic cell line (H1299-p53; see ref. 53) but was induced in nonmetastatic cell lines (DLD-1-p53, HCT116 p53 þ/þ). In another study
(5), NDRG1 could not be expressed in the metastatic colon cancer cell
line SW620. Combining these data suggests that NDRG1 plays different roles in metastatic versus non-metastatic cells. NDRG1 suppresses cell growth and proliferation in the metastatic H1299-NDRG1
cell line, but has no effect on the non-metastatic DLD-1-NDRG1 cell
line (53). Although p53 can be shown to regulate NDRG1 in nonmetastatic cells, NDRG1 can be regulated in metastatic cells by intracellular calcium release in a p53-independent manner (53,57).
Moreover, silencing of NDRG1 expression abolished p53-mediated
apoptosis, showing that NDRG1 participates in multiple regulatory
pathways of the cell.
E-cadherin gain of function causes morphological changes in metastatic cell lines consistent with epithelial cell differentiation and reversion from invasive to non-invasive epithelial phenotype (5). Loss
of E-cadherin function is marked by metastasis progression (58).
Stein et al. (53) observed that metastatic cells that do not express
NDRG1 also fail to express E-cadherin. Conversely, high NDRG1
expression correlates with high E-cadherin expression (59,60). Thus,
it has been proposed that NDRG1 has a requisite function in the
formation of the E-cadherin–catenin complex (33).
The VHL tumor suppressor gene (VHL) has been demonstrated to
repress Sp1-mediated activation of VEGF promoter (51), and in the
same study, it was shown that VHL and Sp1 proteins directly interact.
Recently, Masuda et al. reported that, by mutating the Sp1-binding
element located in the promoter of the NDRG1 gene, they were able to
eliminate the VHL-mediated down-regulation of NDRG1 in human
renal cell carcinoma (50). From this evidence, it was hypothesized
that VHL inhibits NDRG1 expression through blocking Sp1 activity.
NDRG1 cDNA has an extended 3#-untranslated region. Perhaps, in
common with VEGF, a proximal regulatory nucleotide sequence in
the 3#-untranslated region of NDRG1 forms nucleotide–protein complexes that are up-regulated in VHL-deficient cells (61), which, if true
for the NDRG1 gene, would suggest that the protein, in this case
presumably Sp1, is targeted by VHL for degradation (61). Other
3#-untranslated region sequences that enhance special structure formations may also be relevant in any model of post-transcriptional
regulation of NDRG1.
Because NDRG1 is a hypoxia-inducible gene (62), and most, if not
all, tumor cells exhibit hypoxic conditions, regulation of NDRG1
5
T.P.Ellen et al.
induction by both HIF and by p53 suggests that NDRG1 plays a prominent role linking hypoxia response and p53-mediated responses.
Hypoxic tissues and tumors show a shift in gene expression from
oxidative phosphorylating enzymes to glycolytic ones. The switch
from the Krebs cycle to glycolysis for the generation of ATP is a hallmark of tumor malignancy (for review, see ref. 63). HIF is central to
the shift from oxidative phosphorylation to glucose metabolism. HIF1a drives the regulation of genes that participate in both the cellular
response to hypoxia (64) and tumor progression (65). HIF is a heterodimeric factor. HIF-1b (sometimes called aryl hydrocarbon receptor
nuclear translocator) is constitutively expressed, whereas HIF-1a is
hypoxia induced and possesses a very short half-life (about 5 min) in
the presence of oxygen (for review, see 66). HIF-1a is targeted for
degradation by the 2-oxoglutarate-dependent dioxygenase superfamily members prolyl hydroxylases 1, 2 and 3 (PHD1, PHD2 and
PHD3), which require molecular oxygen and thereby act as oxygen
sensors in the cell. Hydroxylation of the HIF-a chains causes their
recognition for proteosomal degradation by a ubiquitin ligase E3
complex containing VHL tumor suppressor protein. In most instances,
HIF-1a mRNA is not increased in response to hypoxia, but protein
levels are. Thus, regulation is post-translational. VHL also binds to an
asparagine hydroxylase, factor inhibiting HIF, to regulate HIF-1a via
a second pathway (67).
Under hypoxia, the oxygen-dependent proline and asparagine
hydroxylases are repressed, so that HIF-1a accumulates, translocates
to the nucleus and recruits the co-activator CBP/p300 (68,69) to
regulate transcription of target genes via binding to hypoxiaresponsive elements (HREs) in those genes’ promoters. Many of
the target genes identified participate in angiogenesis. Hypoxia limits
tumor growth, because poor vascularization leaves tumors unable
to grow and form metastases. However, our lab has shown previously
that hypoxia only slightly affects NDRG1 expression in normal
prostate epithelial cells, whereas it dramatically increased its expression in prostate cancer cells (PC-3M) (12). A number of research
groups have shown that over-expression of NDRG1 decreases tumor
growth, reduces invasion, and suppresses metastasis in a wide variety
of conditions (2,5,7). In addition, hypoxia-responsive elements
(HREs) have been identified upstream of NDRG1’s promoter, yet
NDRG1 up-regulation has been demonstrated to occur by HIF-1adependent and -independent mechanisms under conditions of iron
chelation (70).
Besides HIF-1a, it was shown in our laboratory, by using the
Ca2þ-chelating agent BAPTA-AM, that calcium is required for the
hypoxia-inducible expression of NDRG1 (57). HIF-1a is likely to be
a transacting factor in the induction of the NDRG1 gene, but it was
found, in our laboratory, that NDRG1 induction mediated by intracellular Ca2þ elevation is neither directly associated with HIF-1a induction nor does a calcium ionophore stimulate HIF-1a-dependent
transcription (13). Although BAPTA-AM reduces NDRG1 expression, it stimulates HIF-1a-dependent transcription, so there appears
to be no direct connection in the affect of Ca2þ and HIF-1a on
NDRG1 induction. Therefore, it is likely that, although Ca2þ elevation and HIF-1a are required for hypoxia-induced NDRG1 expression,
their mechanisms are somewhat different. Intracellular Ca2þ elevation
could affect NDRG1 induction by signaling pathways, such as protein
kinase C activation, as for Egr-1 (see above, Patterns of expression
in determination of other biological roles of NDRG1), or a transcriptional mechanism distinct from any involvement with HREs.
To date, the roles of HIF-1a and intracellular Ca2þ elevation are
undefined.
The AP-1 transcription factor is a heterodimer composed of one
c-Fos and one c-Jun protein (71). Ca2þ induces AP-1-dependent transcription, and VEGF has been shown to be a hypoxic gene that is
regulated by this factor (13). Deletion of two AP-1-binding sites in the
VEGF promoter abolishes transactivation by Ca2þ, but only partially
minimizes its response to hypoxia. An AP-1 response element in the
5# end of the NDRG1 gene has been found using computer analysis.
Using a down-regulated AP-1 construct, it was shown that both VEGF
and NDRG1 mRNAs could conceivably be induced by Ca2þ via
6
AP-1-mediated transcription (13). One reasonable conclusion is that
this HIF-1-independent, Ca2þ-dependent, AP-1-mediated pathway
regulates hypoxic genes and supports a fine regulation of gene expression under conditions of hypoxia, by cooperation of the HIF-1 and
AP-1 pathways.
NDRG1 is induced by nickel (9). Nickel is a known carcinogen.
NDRG1 protein is expressed at low levels in normal tissues, but, in
a variety of cancers, it is over-expressed. This differential expression
in cancer cells compared with normal cells makes the NDRG1 gene an
important cancer marker (72). Again, as NDRG1 is both hypoxia and
Ca2þ responsive, NDRG1 induction may be by HIF-1-dependent or
independent pathways. The underlying mechanism of Ni-induced
NDRG1 expression, like other hypoxic genes, involves intracellular
ascorbate depletion (73).
Different investigations have shown that NDRG1 associates with
microtubules at the centrosomes, thus implying that it functions by
participating in spindle checkpoint control in a p53-dependent manner
and maintaining euploidy (74). NDRG1 siRNA knockdown showed
that NDRG1 expression is required to maintain acetylated a-tubulin
levels, spindle fiber formation and spindle structure during cell division, thus preventing polyploidy cell populations among mitotic
cells. Interesting is the report that nickel treatment of cells enhances
a-tubulin acetylation, a marker for the presence of stable microtubules
(75). In addition, stable transfections leading to inducible gene expression and RNAi knockdown experiments have shown that NDRG1
is necessary but not sufficient for p53-mediated apoptosis (53). Exogenous NDRG1 expression in p53-deficient cell lines leads to growth
arrest, whereas NDRG1 knockdown in p53 wild-type cells has been
shown to prevent p53-mediated apoptosis, and forced expression of
NDRG1 in the lung cancer cell line H1299-p53 enhances p53mediated apoptosis (53). Taken together, these data point to a role
for NDRG1 in cell transformation through p53-dependent apoptosis
or spindle checkpoint.
The reasons for the unique tissue and cellular distribution patterns
of NDRG1 remain unknown. Guan et al. 2000 (5) demonstrated that
NDRG1 over-expression in colon tumor cells reduces metastatic potential, and proposed that suppression of colon cancer metastasis by
NDRG1 occurs by inducing colon cancer cell differentiation and
partially reversing the metastatic phenotype. In contrast, a pathological study drew the opposite conclusion, namely, that hepatocellular
carcinomas express elevated levels of NDRG1 protein, a likely tumor
marker, the over-expression of which is correlated with poor prognosis (76). Poorly differentiated hepatocellular carcinoma samples express higher levels of NDRG1 protein than those that are well
differentiated. Vascular invasiveness is also correlated with higher
expression levels of NDRG1 compared with those without.
Where Will NDRG1 Lead Us?
In summary, NDRG1 mRNA is ubiquitously expressed in human
tissues including various human cancers. Regulation is complex and
involves interactions of NDRG1 and major metabolic regulators (e.g.
HIF-1a, p53, N-Myc, c-Myc and AP-1) at the transcriptional, posttranscriptional and translational levels. Figure 1 summarizes the transcriptional regulation of NDRG1. NDRG1 has been most clearly
linked to functions of growth arrest and cellular differentiation. It
is likely that a tumor suppressor function of NDRG1 is only the
consequence of its up-regulation in differentiating cells and downregulation under conditions of cell growth (2). Similarly, that the
expression of NDRG1 has inverse correlation with degree of metastasis could be interpreted as either NDRG1 acting against metastasis
processes or that metastasis blocks the induction of NDRG1. However, its dual presence in cellular hypoxia and cancer leads to the
interesting possibility that the determination of its activities in response to these conditions will bring a new insight into the relationship between these two processes and the cell’s response to them.
Acknowledgements
Conflict of interest statement: None declared.
NDRG1, a growth and cancer related gene
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Received July 9, 2007; revised August 23, 2007; accepted August 26, 2007