Suppressor role of activating transcription factor 2
(ATF2) in skin cancer
Anindita Bhoumik*, Boris Fichtman*, Charles DeRossi*, Wolfgang Breitwieser†, Harriet M. Kluger‡, Sean Davis§,
Antonio Subtil¶, Paul Meltzer§, Stan Krajewski*, Nic Jones†, and Ze’ev Ronai*?
*Burnham Institute for Medical Research, La Jolla, CA 92037;†Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX,
United Kingdom; Departments of‡Medicine and¶Dermatology, Yale University School of Medicine, New Haven, CT 06520; and§Genetics Branch,
Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
Edited by Peter Erich Angel, Deutsches Krebsforschungszentrum, Heidelberg, Germany, and accepted by the Editorial Board November 16, 2007
(received for review June 27, 2007)
Activating transcription factor 2 (ATF2) regulates transcription in
response to stress and growth factor stimuli. Here, we use a mouse
model in which ATF2 was selectively deleted in keratinocytes.
Crossing the conditionally expressed ATF2 mutant with K14-Cre
mice (K14.ATF2f/f) resulted in selective expression of mutant ATF2
within the basal layer of the epidermis. When subjected to a
two-stage skin carcinogenesis protocol [7,12-dimethylbenz[a]an-
and prevalence of papilloma development compared with the WT
ATF2 mice. Consistent with these findings, keratinocytes of
K14.ATF2f/fmice exhibit greater anchorage-independent growth
compared with ATF2 WT keratinocytes. Papillomas of K14.ATF2f/f
mice exhibit reduced expression of presenilin1, which is associated
with enhanced ?-catenin and cyclin D1, and reduced Notch1
?-catenin expression were seen in samples of squamous and basal
cell carcinoma, as opposed to normal skin. Our data reveal that loss
of ATF2 transcriptional activity serves to promote skin tumor
formation, thereby indicating a suppressor activity of ATF2 in skin
?-catenin ? keratinocyte ? presenilin ? cyclin D1 ? papilloma
its heterodimerization with members of the AP1 family, including
and the Tax proteins (6–8). ATF2 target genes include AP1-
responsive genes, such as cyclin A, IFN-?, and TNF-? (9–11).
Intriguingly, ATF2 has also been implicated in the DNA damage
response, through its phosphorylation by phosphoinositide-3-
kinase-related protein kinase, including ATM (12). This phosphor-
ylation is required for intra-S phase checkpoint control and for its
colocalization with components of the Mre11–Rad50–Nbs1
(MRN) complex within DNA damage repair foci.
The role of ATF2 in stress and DNA damage response suggests
that this protein could also play a role in tumorigenesis. Consistent
suggested an important role of ATF2 in melanoma development
and progression. Nuclear localization of ATF2 coincides with poor
prognosis in melanoma patients (13). In addition, peptides derived
from the N-terminal region of ATF2 efficiently repressed ATF2
function and reduced growth and metastasis of melanoma tumor
cells in mouse models (14–17).
The role of the AP1 transcriptional complex in skin carcinogen-
esis has been addressed. Specifically, c-Jun family members were
shown to play an important role in skin cancer development
because K14-driven expression of the TAM67 dominant-negative
Jun family construct in the basal layer of the epidermis blocked
phorbol 12-tetradecanoate 13-acetate (TPA) or UV-B induced
ctivating transcription factor 2 (ATF2) is a member of the
bZIP family of transcription factors that is activated upon its
tumors in a skin carcinogenesis model (18–20). In addition, mice in
which c-Jun or JNK2 has been deleted exhibit marked reduction in
skin cancer development (21–23).
Here, we directly assessed the role of ATF2 in a mouse skin
carcinogenesis model, by using a conditional mutant of ATF2 in
keratinocytes. Unlike the oncogenic role for c-Jun and JNK2, our
work reveals that lack of ATF2 function contributes to accelerated
development of papillomas, thereby suggesting a tumor suppressor
role of ATF2 in keratinocytes.
The mouse KO of ATF2 leads to early postnatal lethality (24).
Thus, to study the function of ATF2 in the skin, we used the
Cre-loxP system for disruption of the ATF2 gene in keratinocytes.
Cre-dependent deletion of the ATF2 DNA-binding domain and a
portion of its leucine zipper results in a transcriptionally inactive
form of ATF2 (W.B. and N.J., unpublished results). Mice homozy-
gous for the loxP-flanked (floxed) ATF2 gene (ATF2f/f) were born
at the expected Mendelian ratios and presented no obvious abnor-
malities. In addition, in a number of tissues that were analyzed, the
levels of ATF2 expression were comparable between wild-type
(WT) and ATF2f/f(data not shown).
To elucidate the role of ATF2 in skin cancer, ATF2f/fmice were
crossed with keratin14-cre transgenic mice (K14-Cre). The resulting
ATF2f/f/K14-Cre (K14.ATF2f/f) mice expressed the transcriptional
mutant ATF2 gene in keratinocytes. Immunoblot analysis con-
firmed that keratinocytes prepared from WT express a 70-kDa
band corresponding to full-length ATF2, whereas keratinocytes of
the K14.ATF2f/fmice express a 55-kDa band, corresponding to
ATF2, which lacks DNA-binding and leucine zipper domains (Fig.
throughout the nucleated layers of epidermis and the dermis (Fig.
1c). Importantly, the use of the K14-Cre transgene expression is
limited to the basal layer of stratified squamous epithelia, thereby
dermis (Fig. 1c).
Disruption of ATF2 Increases Susceptibility to Skin Carcinogenesis.To
address the role of ATF2 in de novo skin carcinogenesis, the
two-stage skin carcinogenesis protocol was used (25). In this
model, tumors are initiated in epidermal keratinocytes by
research; W.B. and N.J. contributed new reagents/analytic tools; A.B., B.F., W.B., H.M.K.,
S.D., A.S., P.M., S.K., N.J., and Z.R. analyzed data; and A.B. and Z.R. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The data reported in this paper have been deposited in the Gene
Expression Omnibus (GEO) database www.ncbi.nlm.nih.gov/geo (accession no. GSE9328).
?To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
February 5, 2008 ?
vol. 105 ?
single topical application of the chemical carcinogen 7,12-
dimethylbenz[a]anthracene (DMBA) with subsequent addi-
tion of the tumor promoter TPA over a period of 8–12 weeks.
This procedure results in the development of benign papillo-
mas with a high incidence of H-Ras mutations (25, 26). Some
of these tumors progress to squamous cell carcinomas (SCCs),
which can undergo epithelial–mesenchymal transition to spin-
dle cell carcinomas. Significantly, K14.ATF2f/fmice exhibited
increased susceptibility to skin tumorigenesis with markedly
accelerated kinetics of papilloma development (Fig. 1d). In
K14.ATF2wt/wtmice (WT), papillomas started to appear ?17
weeks after DMBA treatment (Fig. 1e), and by 30 weeks,
?40% had developed tumors. This slow papilloma develop-
ment in littermate controls is attributed to their genetic
background, FVB/C57BL/6, because mice of C57BL/6 back-
ground are more resistant to chemical-induced skin cancer
(27). In contrast, skin papillomas in K14.ATF2f/fmice were
observed as early as 11 weeks after DMBA treatment, with
50% of mice having developed tumors between weeks 13 and
15. Thus, compared with WT mice, the median appearance of
skin papillomas is 5–6 weeks earlier in the ATF2 mutant
keratinocytes [average ? SE, 11.42 ? 0.36 weeks (K14.ATF2f/f;
n ? 32) vs. 17.63 ? 0.30 weeks (WT; n ? 27); P ? 0.002] (Fig.
1e). Importantly, the number of papillomas also increased in
the K14.ATF2f/fmice. By 15 weeks, K14.ATF2f/fmice devel-
oped an average number of six tumors per mouse, whereas the
WT mice had none (Fig. 1f). These findings strongly suggest
that the absence of functional ATF2 in keratinocytes confers
increased sensitivity to skin cancer development and imply that
ATF2 may elicit tumor suppressor function in the skin. Of
note, treatment of DMBA or TPA alone did not result in
papilloma development in WT or ATF2 mutant mice. This
finding implies that lack of transcriptionally active ATF2 is not
sufficient to augment initiation or promotion phases per se but
is important for the accelerated development of initiated
ATF2 Deficiency Increases Epidermal Hyperproliferation After Addi-
tion of TPA. TodeterminethepossibleeffectofATF2disruptionon
keratinocyte proliferation, possible changes in epidermal hyperpla-
sia were assessed. The number of nucleated cell layers in the
untreated epidermis of WT and K14.ATF2f/fmice was not signif-
icantly different (Fig. 2 a Top and b). After TPA treatment,
epidermal hyperplasia was induced in all genotypes, although the
number of nucleated cell layers in the K14.ATF2f/fmice was higher
10 ?g each) induced the formation of a hyperplastic epidermis
after treatment in WT mice compared with 42 ? 3.2 ?m in the
K14.ATF2f/fmice (Fig. 2 a Middle and b). The difference was even
2 a Bottom and b). These data reveal that the lack of transcription-
after exposure to a tumor promoter.
Induced DNA Synthesis in Basal Epidermal Cells of TPA-Treated
K14.ATF2f/fMice. In light of the enhanced hyperplastic responses
seen in the epidermis of K14.ATF2f/fmice, we assessed possible
strategy shows WT allele of ATF2 encompassing exons 8 and 9 (boxes) and flanking loxP sequences (arrowheads). (b) Expression of ATF2 by immunoblot in skin
extracts from K14.ATF2wt/wt(WT) and K14.ATF2f/fmice. ?-Actin was used as loading control. ATF2* indicates the fast migrating form of ATF2 released after
deletion of exons 8 and 9. An ATF2 antibody that recognizes C-terminal epitopes was used for Western blotting. (c) Immunohistochemical analysis of the
expression of ATF2 in the frozen skin sections of WT and K14.ATF2f/fmice. (Scale bars, 50 ?m.) Arrows point to the expression of ATF2 in the epidermis of WT
the percentage of mice with skin papillomas. Bars indicate SE.*, P ? 0.04, statistically different from the WT mice, as determined by Student’s t test. (f) Average
number of papillomas per mouse after DMBA/TPA treatment. Data represent an average number of skin papillomas per mouse. Bars indicate SE.*, P ? 0.04,
statistically different from WT mice, as determined by Student’s t test.
Targeted disruption of ATF2 in mouse skin increases susceptibility to papilloma formation in the two-stage chemical carcinogenesis. (a) Targeting
Bhoumik et al.
February 5, 2008 ?
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changes in the rate of keratinocyte proliferation, which could
account for the increased hyperplasia. BrdU labeling was carried
out to quantify DNA synthesis in basal keratinocytes. In WT mice,
3% 24 h after TPA treatment (Fig. 2c). Whereas K14.ATF2f/fmice
exhibited BrdU labeling that was similar to the WT animals (5%)
before TPA treatment, 24 h after TPA treatment the K14.ATF2f/f
mice showed a significant increase in the number of BrdU-labeled
cells [(32 ? 3.0%) compared with WT ATF2 mice (15 ? 3%); P ?
0.0045 (Fig. 2d)]. Further, primary cultures of K14.ATF2f/fkera-
tinocytes exhibit a marked increase in the S phase of the cell cycle
compared with the primary WT keratinocytes (Fig. 2e). These data
suggest that the lack of transcriptionally active ATF2 causes in-
creased cell proliferation in primary keratinocytes and their hyper-
proliferation in the epidermis in response to treatment with tumor
Reduced Apoptosis in Epidermal Keratinocytes of K14.ATF2f/fMice.
We next determined whether the higher incidence of papilloma
after DNA damage such as DMBA treatment. Reduced levels of
active caspase 3 were observed in DMBA-treated K14.ATF2f/fskin
compared with the skin of DMBA-treated WT mice, suggesting
increased keratinocyte survival in K14.ATF2f/fskin (Fig. 2f). This
finding implies that the absence of transcriptionally active ATF2
supports a mutator phenotype, with a consequent increase in
Reduced Levels of Presenilin1 (PS1) Coincide with Elevated ?-Catenin
Expression in the K14.ATF2f/fPapillomas.To elucidate the molecular
pathways that were modified in the absence of transcriptionally
probes showed significant differential expression between WT and
K14.ATF2f/fmice (false discovery rate, 10%) [supporting infor-
mation (SI) Table 1; raw data are available at the National Center
for Biotechnology Information Gene Expression Omnibus, acces-
sion no. GSE9328]. Genes that were down-regulated in the
K14.ATF2f/fpapillomas included PS1, which is required for the
proteolytic processing of Notch during development and Alzhei-
mer’s disease pathogenesis (28). PS1 also serves as a scaffold
protein that affects ?-catenin phosphorylation and stability inde-
pendently of the Wnt-regulated axin–CK1? complex (29).
Interestingly, PS1 KO mice that are rescued through neuronal
expression of the human PS1 transgene develop spontaneous skin
and ?-catenin/lymphoid enhancer factor-1/T cell factor (?-catenin/
LEF)-mediated signaling. Consistent with these reports, epidermis
of K14.ATF2f/fmice not only exhibited reduced levels of PS1, but
also had an elevated level of ?-catenin expression (Fig. 3a).
Consistent with the immunoblot analysis (Fig. 3a), ?-catenin
levels were found to be up-regulated in the epidermis of the
K14.ATF2f/fmice (Fig. 3b) and in papilloma samples (Fig. 3c).
?-Catenin staining was mostly localized to the membrane of the
upper layer of the epidermis in both WT and K14.ATF2f/fpapil-
lomas (Fig. 3c). Increased accumulation of cytosolic and nuclear
stained with H&E. Histological analysis shown was performed 18 and 48 h after TPA treatment. (Scale bars, 50 ?m.) (Top) Acetone-treated control skin. (b)
Quantitative analysis of the epidermal thickness (in micrometers) after TPA treatment (P ? 0.001). Bars indicate SD (n ? 10). The thickness of the epidermis (in
received three topical treatments with 10 ?g of TPA. Twenty-four hours after TPA treatment, BrdU was injected i.p., and 1 h later the dorsal skin was isolated.
Bars indicate SD (n ? 10). (e) (Upper) Cell cycle profile of primary keratinocytes isolated from WT and K14.ATF2f/fpups subjected to FACS analysis. (Lower)
Percentages of cells in G1, S, and G2/M phases (mean ? SD of three experiments). (f) Apoptosis was assessed with active caspase 3 antibody of skin from WT and
K14.ATF2f/fmice after 5 days of DMBA alone or DMBA and 18 h of TPA treatment. The TPA treatment was done for skin thickening to facilitate visualization
skin upon treatment with DMBA after 5 days, followed by TPA treatment. Arrows indicate active caspase 3-positive cells.
Epidermal hyperplasia induced by TPA treatment. (a) Dorsal skin of 8-week-old mice after three topical treatments with 10 ?g of TPA was excised and
www.pnas.org?cgi?doi?10.1073?pnas.0706057105 Bhoumik et al.
?-catenin was observed in the basal layer of the epidermis of
papillomas derived from the K14.ATF2f/fmice. Because cell pro-
liferation takes place within the basal cell layer of the epidermis,
increased accumulation of cytosolic and nuclear ?-catenin in the
increased epidermal hyperplasia and papilloma formation.
These data suggest that in the absence of transcriptionally active
increase in the expression of ?-catenin.
Elevated Cyclin D1 and c-Myc, and Reduced Notch1 Expression in
K14.ATF2f/fTissues. Because cyclin D1 and c-Myc are direct target
genes for ?-catenin/LEF signaling (31, 32), we next assessed
changes in their levels of expression. Western blot analysis reveled
that cyclin D1 protein was indeed increased in the epidermis of
K14.ATF2f/fmice (Fig. 3a) after TPA treatment. Basal levels of
c-Myc were also up-regulated in the epidermis of the K14.ATF2f/f
mice with a modest increase after TPA treatment (Fig. 3a). The
finding that cyclin D1 and c-Myc, two of the major transcriptional
K14.ATF2f/fmice shows that the increased ?-catenin protein is
functional (see also SI Fig. 5).
Because PS1 is also implicated in the activation of the Notch1-
signaling pathway (33) we analyzed changes in the expression of
processed (cleaved) Notch1. Lower levels of processed Notch1
expression were found in protein lysates prepared from the epi-
dermis (data not shown) and in keratinocytes derived from the
epidermis of K14.ATF2f/fmice compared with the WT mice (Fig.
3d). Interestingly, nuclear localization of Notch1 was detected
throughout the WT epidermis, but only in the upper layer of the
epidermis of the K14.ATF2f/fmice (Fig. 3e). These results suggest
that the epidermis of K14.ATF2f/fmice exhibits lower levels of PS1
expression, which is associated with lower levels of processed
Notch1 and higher ?-catenin expression.
Elevated EGF Receptor (EGFR), Phospho-JNK, and Phospho-c-Jun
Expression in K14.ATF2f/fkeratinocytes. Because PS1 was also im-
plicated in the negative regulation of EGFR signaling and turnover
and ATF2 mutant mice. Western blot analysis revealed elevated
levels of EGFR in K14.ATF2f/fskin treated with TPA compared
with the WT counterpart (Fig. 3a).
Increased EGFR expression is expected to result in activation of
respective downstream signaling pathways, including the activation
of JNK and c-Jun. Immunohistochemistry of TPA-treated
K14.ATF2f/fskin samples revealed increased levels of phospho-c-
Jun expression in the basal layers of the epidermis (Fig. 3g),
consistent with increased activity of its kinase, JNK (Fig. 3f). Of
These data identify changes in JNK–Jun signaling pathways and
their upstream regulator, EGFR.
Increased Anchorage-Independent Growth of Ras-Infected K14.ATF2f/f
independent growth. A soft agar assay was used to measure
anchorage-independent growth of keratinocytes derived from the
skin of WT and K14.ATF2f/fnewborn mice that were infected in
culture with a mutant Ras oncogene. A modest, albeit significant,
increase was observed in the number (but not the size) of colonies
formed in the H-RasV12-infected K14.ATF2f/fkeratinocytes com-
suggest that lack of ATF2 transcriptional activity increases the
tumorigenic potential of keratinocytes in vitro as well as in vivo.
Reduced Level and Altered Subcellular Localization of ATF2 in Skin
Cancer Tissue Microarrays. To characterize the expression of ATF2
in human skin cancer, we used a tissue microarray (TMA) con-
with reduced Notch1 and elevated
?-catenin expression in the TPA-
K14.ATF2f/fmice. (a) Epidermis of mice
of acetone or TPA (10 ?g) was isolated
24 h after the last treatment. Tissue
ting with antibodies to PS1, ?-catenin,
jected to treatment as indicated in a
and was excised, and paraffin sections
were prepared and stained with
?-catenin antibody. (Scale bar, 50 ?m.)
(c) ?-Catenin staining in papilloma sec-
tions of the K14.ATF2f/fand WT mice.
Cells exhibiting nuclear ?-catenin
staining are marked with arrows.
(Scale bar, 50 ?m.) (d) Keratinocytes
isolated from K14.ATF2f/fand WT
1-day-old pups were lysed and were
subjected to immunoblot analysis with
as loading control. (e) The dorsal skin
of 8-week-old mice treated and pre-
pared as indicated in b was immuno-
stained with antibodies to Notch1.
for phospho-JNK (pJNK1) and total JNK levels was performed as in a. Two mice for each group are shown. ?-Actin was used as loading control. (g)
Immunohistochemical analysis of the expression of phospho-c-Jun upon TPA treatment in the frozen skin sections of WT and K14.ATF2f/fmice. (Scale bars, 50
an average of three experiments (P ? 0.005).
Reduced level of PS1 coincides
Bhoumik et al.
February 5, 2008 ?
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SCC or BCC. Significantly, in contrast to the predominant nuclear
localization of ATF2 in normal skin (Fig. 4a), samples from
malignant tissues (SCC and BCC) exhibited marked reduction of
nuclear ATF2 expression (P ? 0.0003), whereas the cytoplasmic
ATF2 expression was not significantly different (P ? 0.13; Fig. 4b
and data not shown). Decreased nuclear and variable cytosolic
staining was seen in the SCC subset of specimens compared with
normal skin (Fig. 4b; P ? 0.0005 and P ? 0.194, respectively).
Similarly, analysis of the BCC subset of samples identified de-
creased nuclear ATF2 expression compared with normal skin (P ?
0.0001) and variable cytoplasmic ATF2 expression (Fig. 4b; P ?
0.116). These data indicate that ATF2 expression is either de-
creased or shifted from the nucleus to the cytosol in the majority
of human SCC and BCC samples. Because both changes would
result in reduced ATF2 transcriptional activities, these data suggest
that ATF2 function is attenuated in these skin tumors.
Consistent with the finding in BCC and SCC samples, the level
of ATF2 expression was markedly reduced in the papillomas
developed in the WT mice compared with the normal-appearing
skin (Fig. 4c). Conversely, the expression of ?-catenin increased in
these papillomas, consistent with our finding of elevated ?-catenin
expression in the K14.ATF2f/fpapillomas. Significantly, and in
was seen in samples from SCC and BCC (Fig. 4d). These finding
skin cancers, which coincides with elevated ?-catenin expression.
The present work provides genetic evidence for a suppressor role
of ATF2 in nonmalignant skin cancer. Using K14.ATF2f/fmice, we
in the absence of a transcriptionally active ATF2 in the basal layer
of the epidermis. Consistent with these finding, keratinocytes
prepared from K14.ATF2f/fmice that were infected with mutant
Ras oncogene exhibited a marked increase in their ability to grow
on soft agar compared with their WT counterpart, suggesting a
transformed phenotype in vitro. Important support for the finding
in the mouse model used here comes from the analysis of human
skin tumors; unlike the strong nuclear expression of ATF2 in
normal skin, SCC and BCC samples exhibit a significantly reduced
nuclear staining. The latter is also consistent with reduced expres-
sion of ATF2 found in papillomas developed in the WT animals,
supporting the notion that ATF2 need to be inactivated to support
skin tumor development. Although the present study did not assess
whether a lack of transcriptionally active ATF2 could contribute
BCC, where ATF2 is largely inactivated because of either lower
expression or reduced nuclear localization, support such a role. It
is of interest to note that loss of ATF2 transcriptional activities per
se, or in combination with either DMBA or TPA, is not sufficient
to promote papilloma formation, suggesting that ATF2 is contrib-
uting to changes elicited by initiating and promoting events in
keratinocytes. Among the changes observed are reduced apoptosis
of DMBA-treated skin and increased proliferation of TPA-treated
skin, as well as of primary keratinocytes. Collectively, these finding
reveal that loss of ATF2 transcriptional activities is associated with,
and contributes to, skin tumor formation.
The role of the AP1 complex in the promotion of skin carcino-
genesis has been demonstrated in mouse models in which the
activity of JNK, c-Jun, and other AP1 family members has been
inhibited (18–21, 36–40). Surprisingly, unlike the promoting func-
carcinogenesis process. The transcriptional activity of ATF2 is
required for this tumor suppressor function because the mouse
model used in this work does not produce a transcriptionally active
form of ATF2. We show that PS1 and its targets ?-catenin, EGFR,
cyclin D1, and Notch1 are regulated by ATF2, and we suggest that
their deregulation in the mutant mice is associated with the
enhanced skin tumor phenotype seen.
patients. (a and b) ATF2 protein ex-
pression levels were assessed by using
a tissue microarray that was stained
with an antibody directed against
ATF2. (a) Examples of staining in nor-
mal skin, SCC, and BCC. (Scale bars, 50
?m.) Scoring of ATF2 nuclear and cy-
tosolic localization was performed as
detailed in Materials and Methods.
Forty samples from SCC patients, 14
samples of BCC patients, and 10 nor-
mal matched and nonmatched skin
tissues were analyzed. Scores were di-
vided into four categories: 1, scores
ranging from 0 to 75; 2, scores ranging
from 76 to 150; 3, scores ranging from
151 to 225; and 4, scores ranging from
226 to 300. (b) Distribution of nuclear
ATF2 staining in normal skin, BCC, and
SCC. (c) (Left) Staining of the skin de-
rived from the WT mouse that devel-
oped papillomas with ATF2 (Upper)
and ?-catenin (Lower). (Right) Stain-
ing of the corresponding papillomas
with antibodies to ATF2 (Upper) and
?-catenin (Lower). (Scale bars, 50 ?m.)
(d) Staining of the TMA used in a with
ATF2 (Left) and ?-catenin antibody
(Right). (Scale bars, 50 ?m.)
www.pnas.org?cgi?doi?10.1073?pnas.0706057105Bhoumik et al.
Consistent with these findings is the observation that EGFR–
JNK–c-Jun–Cyclin D1, which contributes directly to skin tumor
progression (34, 35, 41, 42) are up-regulated in the absence of
be associated with loss of PS1 suppression, PS1 positively regulates
Notch1, and reduced PS1 results in lower level of Notch1 expres-
sion, seen in the K14.ATF2f/fmice tumors and tissues, as in skin
tumor models, where it has been implicated as a tumor suppressor
Consistent with our findings in the skin, ATF2 was recently
implicated in eliciting a tumor suppressor function in mammary
tumors (45). In contrast, however, previous studies suggested a
tumor-promoting role of ATF2 in melanoma (12–17). Similarly,
Notch1 functions as a tumor suppressor in mouse skin, as opposed
to its function as an oncogene in other organs (44). Thus, tissue-
dependent expression of regulatory or accessory factors (i.e., het-
erodimeric transcription factors) is expected to alter the repertoire
explain the different functions in the two tissue types is the altered
expression and subcellular localization of ATF2 in skin tumors as
opposed to melanoma. Whereas in melanoma, nuclear ATF2
expression is associated with poor prognosis (13), its nuclear
localization is significantly reduced in BCC and SCC (Fig. 4).
Decreased nuclear expression of ATF2 in human skin tumors
further implicates loss of ATF2 function in keratinocyte
Materials and Methods
Additional procedures can be found in SI Methods.
Animal Treatment and Tumor Induction Protocols. TostudythefunctionofATF2
in the skin, we used the Cre-loxP system for disruption of the ATF2 gene in
keratinocytes (40). The K14.ATF2f/fmice and their littermate controls (WT) were
of identical FVB/C57BL/6 genetic background. For tumor induction, mice were
dorsal surface 2 days after shaving. TPA (10 ?g in 200 ?l of acetone; Sigma) was
applied every week for 30 weeks beginning 1 week after initiation. The appear-
ance of lesions in each mouse was monitored and recorded every week. Dorsal
skin and/or papillomas were dissected from euthanized mice and fixed in 10%
neutral-buffered formalin for 48 h and were embedded in paraffin. Sections (5
?m) were stained with H&E for histopathological analyses. Papilloma incidence
and multiplicity were recorded weekly. Papilloma multiplicity was calculated as
the average number of skin papillomas per mouse. Papilloma incidence was
calculated as the percentage of mice with skin papillomas.
Immunohistochemistry. Skin specimens were fixed in neutral buffered formalin
solution and processed for paraffin embedding. Skin sections (5 ?m thick) were
prepared and deparaffinized with xylene. For ?-catenin, ATF2, and Notch1 im-
for 20 min in a boiling bath, followed by treatment with 3% hydrogen peroxide
(1:500; Abcam), and Notch1 (1:100; Santa Cruz Biotechnology) were allowed to
react with tissue sections at 4°C overnight. Biotinylated anti-rabbit IgG was
allowed to react for 30 min at room temperature, and diaminobenzidine was
used for the color reaction. Hematoxylin was used for counterstaining. The
control sections were treated with normal mouse serum or normal rabbit serum
instead of each antibody. For the frozen sections, phospho-c-Jun and ATF2
antibodies (1:100; Cell Signaling) were used.
mice (8 weeks old) after treatment with acetone or TPA at the indicated time
points. The thickness of the epidermis (in micrometers) was measured with an
imaging system (SlideBook; Intelligent Imaging) in 15 fields per section.
Statistical Analysis. Data are shown as the means ? SD. Unless indicated other-
wise, statistical differences were determined with one-way ANOVA.
ACKNOWLEDGMENTS. We thank Huaxi Xu (Burnham Institute for Medical
Research) for presenilin antibody, Scott Lowe (Cold Spring Harbor Laboratory,
for providing the protocols for isolating keratinocytes and DMBA/TPA skin car-
was supported by National Cancer Institute/National Institutes of Health Grant
CA099961 (to Z.R.).
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