An activating transcription factor 5-mediated survival pathway as a target for cancer therapy?
ABSTRACT Genes that are highly expressed in cancer cells and are essential for their viability are attractive targets for the development of novel cancer therapeutics. Activating transcription factor 5 (ATF5) is an anti-apoptotic protein that is highly expressed in malignant glioma but not normal brain tissues, and is essential for glioma cell survival. Recent work has revealed an essential survival pathway mediated by ATF5 in malignant glioma; pharmacological inhibition of this pathway leads to tumor regression in mice. ATF5 is also highly expressed in a variety of other cancers, and preliminary studies have shown that the ATF5-mediated survival pathway is active in diverse human cancer cell lines. Targeting this pathway may therefore have therapeutic implications for the treatment of a wide range of cancers. In this perspective, we summarize recent advances in ATF5 research, focusing on its role in promoting cancer and its potential as a target for cancer therapy.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: Recent reports indicate that the activating transcription factor 5 (ATF5) is required for the survival of cancer cells but not for noncancer cells. However, the mechanisms by which ATF5 regulates genes and promotes cell survival are not clear. Using a cyclic amplification and selection of targets (CASTing) approach, we identified a novel ATF5 consensus DNA binding sequence. We show in C6 glioma and MCF-7 breast cancer cells that ATF5 occupies this sequence and that ATF5 activates reporter gene expression driven by this site. Conversely, reporter activity is diminished when ATF5 activity is blocked or when ATF5 expression is down-regulated by serum withdrawal. We further show that early growth response factor 1 (Egr-1), whose promoter contains two adjacent ATF5 consensus binding sites at a conserved promoter position in rat, mouse, and human, is targeted and regulated by ATF5 in C6 and MCF-7 cells. These data provide new insight on the mechanisms by which ATF5 promotes gene regulation and cancer-specific cell survival.Molecular Cancer Research 07/2009; 7(6):933-43. · 4.35 Impact Factor
- Nature Chemical Biology 01/2008; 3(12):739-44. · 12.95 Impact Factor
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ABSTRACT: Activating transcription factor-5 (ATF5) is highly expressed in malignant glioma and has a key role in promoting cell survival. Here we perform a genome-wide RNAi screen to identify transcriptional regulators of ATF5. Our results reveal an essential survival pathway in malignant glioma, whereby activation of a RAS-mitogen-activated protein kinase or phosphoinositide-3-kinase signaling cascade leads to induction of the transcription factor cAMP response element-binding protein-3-like-2 (CREB3L2), which directly activates ATF5 expression. ATF5, in turn, promotes survival by stimulating transcription of myeloid cell leukemia sequence-1 (MCL1), an antiapoptotic B cell leukemia-2 family member. Analysis of human malignant glioma samples indicates that ATF5 expression inversely correlates with disease prognosis. The RAF kinase inhibitor sorafenib suppresses ATF5 expression in glioma stem cells and inhibits malignant glioma growth in cell culture and mouse models. Our results demonstrate that ATF5 is essential in malignant glioma genesis and reveal that the ATF5-mediated survival pathway described here provides potential therapeutic targets for treatment of malignant glioma.Nature medicine 06/2010; 16(6):671-7. · 27.14 Impact Factor
Oncotarget, Advance Publications 2010
An Activating Transcription Factor 5-Mediated Survival Pathway
as a Target for Cancer Therapy
Zhi Sheng, Sara K. Evans and Michael R. Green
* Howard Hughes Medical Institute, Programs in Gene Function and Expression and Molecular Medicine, University of
Massachusetts Medical School, Worcester, MA 01605, USA
Correspondence to: Michael R. Green , e-mail: email@example.com
Keywords: ATF5, anti-apoptosis, cancer therapy, malignant glioma, survival pathway
Received: September 15, 2010, Accepted: September 28, 2010, Published: X, 2010
Copyright: C 2010 Sheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Genes that are highly expressed in cancer cells and are essential for their viability are
attractive targets for the development of novel cancer therapeutics. Activating transcription
factor 5 (ATF5) is an anti-apoptotic protein that is highly expressed in malignant glioma
but not normal brain tissues, and is essential for glioma cell survival. Recent work
has revealed an essential survival pathway mediated by ATF5 in malignant glioma;
pharmacological inhibition of this pathway leads to tumor regression in mice. ATF5 is also
highly expressed in a variety of other cancers, and preliminary studies have shown that
the ATF5-mediated survival pathway is active in diverse human cancer cell lines. Targeting
this pathway may therefore have therapeutic implications for the treatment of a wide
range of cancers. In this perspective, we summarize recent advances in ATF5 research,
focusing on its role in promoting cancer and its potential as a target for cancer therapy.
The mammalian activating transcription factor/
cyclic AMP responsive element-binding (ATF/CREB)
family of transcription factors comprises a large group
of proteins whose members have diverse roles in
development, differentiation, cellular proliferation and
apoptosis. Members of the ATF/CREB family contain
related basic leucine zipper (bZIP) domains; the basic
region is enriched with lysine and arginine residues and
is involved in DNA binding, whereas the leucine zipper
motif mediates protein-protein interactions. ATF/CREB
proteins bind as homo- or hetero-dimers to a DNA-
binding site known as the cyclic AMP responsive element
(CRE), which has the consensus sequence TGACGTCA
(reviewed in ).
ATF5, previously designated as ATFx (or ATF7
in mice), was first isolated as a binding partner of
granulocyte colony-stimulating factor (G-CSF) gene
promoter element 1-binding protein (GPE1-BP), a protein
involved in regulating G-CSF expression . Subsequent
studies have revealed that ATF5 can either homodimerize
or form heterodimers with ATF4 or CCAAT enhancer-
binding protein (C/EBP), and bind CRE elements [3,4]
or a recently-identified novel DNA motif with the
consensus sequence C(C/T)TCT(T/C)CCTTA . ATF5
has also been shown to heterodimerize with the human
T-cell leukemia virus type 1 (HTLV-1) Tax transactivator
protein and bind to the Tax-responsive element, a member
of the asymmetric CRE family of enhancer sequences .
A role for ATF5 in promoting cell survival was first
suggested from gene expression profiling analysis in murine
pro-B lymphocytic cells induced to undergo apoptosis
following withdrawal of the cytokine interleukin-3 (IL-3)
. Among the most dramatic changes observed was a
large decrease in ATF5 expression, raising the possibility
that ATF5 had a role in cell survival. Subsequent work
revealed that ATF5 plays a critical role in antagonizing
apoptosis induced by either the deprivation of IL-3 or
the expression of a pro-apoptotic protein 24p3 in murine
pro-B lymphocytes, or by growth factor withdrawal in
HeLa cells .
AtF5 ExprEssIon In cAncEr
In cancer cells, genes that induce apoptosis are
often inactivated or down-regulated, whereas anti-
apoptotic genes are frequently activated or over-
expressed. Consistent with this paradigm, a number
of studies have demonstrated that ATF5 is highly
expressed in a variety of cancer cell types, whereas it is
not detectably expressed in most normal human tissues
(the exceptions being the liver, prostate and testis, where
ATF5 is expressed at a high level [6,9]). For example, a
comparison of ATF5 protein levels between normal and
neoplastic samples using tissue microarrays revealed
that in all malignant tissues examinedincluding those
of the prostate, colon, endometrium, breast, ovary,
pancreas, gastric, and lungthe percentage of ATF5-
positive cells is significantly higher than that in normal
tissues . Similarly, a query of the Oncomine cancer
profiling database revealed that, in general, the expression
level of ATF5 is significantly higher in malignant tissues
than their normal counterpart tissues . The only
exception appears to be hepatocellular carcinoma cells,
which express lower levels of ATF5 than normal liver
cells; this discrepancy may be due to epigenetic silencing
of ATF5 in hepatocellular carcinoma cells through
promoter methylation . Notably, increased levels of
ATF5 have been observed in primary brain tumors, and
ATF5 expression is particularly high in glioblastoma, an
aggressive form of malignant glioma [10,11].
A pair of studies has provided intriguing evidence
that high ATF5 expression levels may correlate with poor
prognosis in cancer patients. In one study, a retrospective
analysis of 23 individuals with glioblastoma revealed that
patients harboring tumors expressing high levels of ATF5
had substantially shorter survival times than those with
tumors in which ATF5 expression was low or undetectable
. In another study, expression profiling in chronic
lymphocytic leukemia (CLL) patients of known clinical
outcome identified ATF5 as a gene whose significant over-
expression correlates with poor patient outcome .
IdEntIFIcAtIon oF An EssEntIAl
pAthwAy In MAlIgnAnt glIoMA:
Inhibition of ATF5 activity, using a dominant
negative form of ATF5, kills human and rat glioblastoma
cells but does not affect normal cells surrounding the
tumor, indicating ATF5 is selectively essential for the
survival of glioblastoma cells . The high expression
of ATF5 in brain tumors, combined with the fact that it is
selectively essential for glioma cell survival, make ATF5
an appealing potential therapeutic target for the treatment
of malignant glioma. However, developing effective
small-molecular inhibitors of transcription factors has
proven to be challenging .
To uncover the upstream signaling pathways that
control the expression and activity of ATF5—with the
goal of identifying more targetable proteins, such as
kinases, required for glioma cell survival—we performed
a genome-wide RNA interference (RNAi) screen for
factors that are required for transcription of the ATF5 gene
. Because loss of ATF5 function within a cell would
induce apoptosis, and therefore preclude the subsequent
identification of candidate short hairpin RNAs (shRNAs),
we developed a novel negative-selection strategy (Figure
1). This strategy was based on the ability of diphtheria
toxin (DT) to kill cells that express the DT receptor
(DTR). Mouse cells lack a functional DTR and are DT
resistant . We generated a mouse malignant glioma
GL261 cell line stably expressing the human DTR driven
by the mouse ATF5 promoter; the ATF5 promoter is
normally active in GL261 cells, which drives expression
of the DTR gene and confers susceptibility to DT. We then
used this stable cell line to screen for shRNAs that could
inactivate the ATF5 promoter and, consequently, give rise
to a DT-resistant clone. Because these shRNAs would
also inhibit expression of the endogenous ATF5 gene
and induce apoptosis, the cell line was kept alive by the
expression of ATF5 driven by a constitutive promoter. DT-
resistant clones were isolated, and positive shRNAs were
identified and then validated for their ability to inhibit
expression of the endogenous ATF5 gene.
This approach identified 12 genes as regulators
of ATF5 expression, and further analyses revealed
the upstream signaling pathways that regulate ATF5
expression in malignant glioma (summarized in Figure
2). Cell surface receptors (e.g. fibroblast growth factor
receptor (FGFR) or epidermal growth factor receptor
Figure 1: schematic summary of the genome-wide rnAi
negative-selection screen used to identify factors required
for transcription of ATF5. Diphtheria toxin (DT)-resistant
mouse malignant glioma GL261 cells stably co-expressing the human
diphtheria toxin receptor (DTR) gene driven by the mouse ATF5
promoter and ATF5 driven by the constitutive CMV promoter were
transduced with a genome-wide mouse shRNA library. DT-resistant
clones were isolated, and positive shRNAs were identified.
shRNA targeting a gene
for ATF5 expression
shRNA targeting a gene
for ATF5 expression
(EGFR)) activate downstream RAS/mitogen-activated
protein kinase (RAS/MAPK) or phosphoinositide-3-
kinase (PI3K) signaling pathways through FGFR substrate
2 (FRS2) and culminate in the activation of CREB protein
3-like 2 (CREB3L2), which in turn stimulates transcription
of ATF5. ATF5 then antagonizes apoptosis by directly up-
regulating expression of myeloid cell leukemia sequence
1 (MCL1), an anti-apoptotic B-cell leukemia/lymphoma 2
(BCL2) family member.
Pharmacological inhibitors of several components of
that ATF5-mediated survival pathway are commercially
available (see Figure 2). One of these inhibitors, sorafenib,
is a multi-kinase inhibitor whose strongest activity is
against RAF kinases and has been used to treat several
different types of cancers [16,17]. Sorafenib inhibits the
growth of human malignant glioma xenografts in mice,
which suggests it may also be used to treat malignant
glioma . Furthermore, sorafenib sensitizes human
malignant glioma xenografts to temozolomide, a
chemotherapeutic agent commonly used to treat malignant
glioma . Inhibition of other components of the ATF5-
mediated survival pathway, including FGFR (using the
specific inhibitor PD173074), EGFR (CL-387,785), RAS
(manumycin A), MEK (U0126), ERK (FR180204) and
PI3K (LY294002), also induces apoptosis in malignant
glioma cells . Further investigation will verify whether
these inhibitors may also be used to treat malignant
In addition to glioblastoma, ATF5 is also essential
for the viability of other cancer cell types. For example,
inhibition of ATF5 by a dominant negative mutant
selectively kills breast cancer cells but not normal breast
epithelial cells . Furthermore, RNAi-mediated
knockdown of ATF5 induces apoptosis in a variety of
tumor cell lines derived from lung, prostate, skin, and
ovary . Taken together these studies demonstrate that
ATF5 mediates cell survival in many cancers, and suggest
that inhibition of the ATF5-mediated survival pathway
may have therapeutic implications for the treatment of a
wide range of malignancies.
Further investigations will reveal whether the ATF5-
mediated survival pathway represents an efficacious target
for the treatment of cancers in addition to glioblastoma.
Likewise, given that ATF5 is aberrantly over-expressed in
many tumor types, it would be interesting to investigate
whether high ATF5 expression is generally linked to poor
prognosis of cancer patients, as has been observed in
glioblastoma and CLL.
Mechanistically, a further understanding of the
physiological and pathological role of ATF5 in vivo would
greatly benefit from the generation of ATF5 knock-out
mice. If homozygous deletion of the ATF5 gene does not
result in embryonic lethality, then crossing ATF5 knock-
out mice with mice bearing genetic mutations that induce
tumorigenesis would ascertain whether ATF5 is essential
for tumor formation and progression in vivo.
It is possible that ATF5 antagonizes apoptosis
through multiple targets in addition to MCL1. For example,
ATF5 has been shown to promote cell survival through
transcriptional activation of heat shock protein 27 (Hsp27)
in rat H9c2 cells . Chromatin immunoprecipitation
coupled with deep sequencing (ChIP-seq) could be used
to identify ATF5 target genes and study ATF5 function on
a genome-wide level. Identification of additional ATF5
target genes would further define the important role of
ATF5 in cancer development, and may also identify
additional potential therapeutic targets.
Finally, it is worth noting that the genome-wide
Figure 2: An essential AtF5-mediated survival pathway
in malignant glioma. Cell surface receptors (FGFR or EGFR)
activate RAS/MAPK or PI3K signaling pathways through FGFR
substrate 2 (FRS2) and culminate in the activation of CREB3L2,
which in turn stimulates transcription of ATF5. ATF5 then antagonizes
apoptosis by directly up-regulating expression of the anti-apoptotic
factor MCL1. Commercially available pharmacological inhibitors
of several components of the ATF5-mediated survival pathway are
indicated in gray.
RNAi negative-selection screening approach used to
identify components of the ATF5-mediated survival
pathway represents a general strategy that can be applied
to identify essential survival pathways in other types of
cancer cells. Such studies may reveal new regulatory
pathways that contribute to malignant transformation and
are potential therapeutic targets.
This work is supported by National Institutes of
Health grant R01CA115817 to M.R.G. M.R.G is an
investigator of the Howard Hughes Medical Institute.
conFlIct oF IntErEst stAtEMEnt
The authors declared no potential conflicts of interest
with respect to the authorship and/or publication of this
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