2722?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 118? ? ? Number 8? ? ? August 2008
Keratinocyte-specific Smad2 ablation results
in increased epithelial-mesenchymal transition
during skin cancer formation and progression
Kristina E. Hoot,1 Jessyka Lighthall,2 Gangwen Han,2 Shi-Long Lu,2,3 Allen Li,2 Wenjun Ju,4
Molly Kulesz-Martin,1,3 Erwin Bottinger,4 and Xiao-Jing Wang1,2,3
1Department of Cell and Developmental Biology,
2Department of Otolaryngology, and 3Department of Dermatology, Oregon Health & Science University, Portland, Oregon, USA.
4Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA.
TGF-β signaling is involved in tissue homeostasis and cancer
development (1). Ligand binding to heteromers of TGF-β type I
and type II receptors (TGF-βRI and TGF-βRII) induces TGF-βRI
to phosphorylate Smad2 and Smad3. Phosphorylated Smad2 and
Smad3 bind a co-Smad, Smad4, and the heteromeric Smad com-
plexes translocate into the nucleus to regulate transcription of
TGF-β target genes (1). Smad3 binds to the Smad-binding element
(SBE) of a target gene, and subsequently recruits Smad4 to the
same SBE. Smad2 does not bind to DNA directly but complexes
with Smad3 and Smad4 as either a coactivator or a corepressor for
Smad3 and Smad4 (2). Each of these Smads has been implicated
by in vitro studies in mediating the multiple functions of TGF-β
(3). However, increasing numbers of studies show that Smad2, -3,
and -4 are regulated differently and exhibit distinct physiologi-
cal functions in vivo (2, 3). For instance, both Smad2- and Smad4-
knockout mice are embryonic lethal due to failure in embryonic
axis patterning and endoderm specification (4) and failure of
proper endoderm and mesoderm formation (5), respectively. In
contrast, Smad3-knockout mice are viable but succumb to mucosal
immunity defects after birth (6).
In many cancer types, TGF-β signaling has a tumor suppressive
effect early in carcinogenesis but promotes invasion and metastasis
at later stages (7). Increasing evidence suggests that Smads medi-
ate both tumor suppression and promotion functions of TGF-β.
In epithelial cells, Smad2, -3, and -4 are involved in growth inhibi-
tion (2), a major tumor suppressive effect of TGF-β, and epithelial-
mesenchymal transition (EMT) (8, 9), an early and major tumor
promoting effect of TGF-β. However, genes associated with each of
these biological processes are differentially regulated by individual
Smads (1, 3). Loss of Smad2 or Smad4 at the genetic and protein
levels has been widely reported in various cancer types (2, 10). In
contrast, Smad3 mutation is found only in colon cancer at a very low
frequency (11), and Smad3 protein loss was reported only in pediat-
ric T cell acute lymphoblastic leukemia (12). In human skin cancer,
individual Smad expression patterns in squamous cell carcinomas
(SCCs) have not been documented. The roles of individual Smads in
skin carcinogenesis have been assessed mainly through genetically
engineered mouse models. Smad4 deletion in keratinocytes results in
spontaneous SCC formation (13, 14), indicating a dominant tumor
suppressive effect of Smad4 in skin carcinogenesis. Smad3-null
keratinocytes transduced with a v-ras oncogene exhibited increased
malignancy when grafted to immune-compromised mouse skin
(15). However, Smad3-knockout mice are resistant to skin chemi-
cal carcinogenesis (16, 17) due to abrogation of TGF-β1-mediated
inflammation and gene expression critical for tumor promotion
(17). The role of Smad2 in skin carcinogenesis has not been fully
explored in animal models as germline Smad2-knockout mice die
in early embryogenesis (4). In the current study, we assessed the role
and molecular mechanisms of Smad2 in skin carcinogenesis.
Nonstandard?abbreviations?used: DMBA, dimethylbenz[α]anthracene; EMT,
epithelial-mesenchymal transition; IHC, immunohistochemistry; K5, keratin 5;
K5.Cre*PR1, mutant Cre recombinase fused to a truncated progesterone receptor tar-
geted by K5 promoter; LOH, loss of heterozygosity; qRT-PCR, quantitative RT-PCR;
SBE, Smad-binding element; SCC, squamous cell carcinoma; TPA, 12-O-tetradec-
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 118:2722–2732 (2008). doi:10.1172/JCI33713.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 8 August 2008
Smad2 and Smad4 were frequently lost in human skin SCCs. We exam-
ined Smad2 expression patterns by immunohistochemistry (IHC)
in 83 human skin SCCs. Smad3, a Smad2 signaling partner, and
co-Smad, Smad4, were also examined. In comparison with the epi-
dermis in normal skin, Smad2 and Smad4 each were lost in 70% of
skin SCC samples, whereas Smad3 loss was only seen in 5% of cases
(Table 1 and Supplemental Figure 1; supplemental material avail-
able online with this article; doi:10.1172/JCI33713DS1). Addition-
ally, the incidence of Smad2 loss occurred in 100% of poorly dif-
ferentiated SCCs, which was significantly higher than Smad4 loss
in poorly differentiated SCCs (P = 0.005; Table 1).
To determine whether loss of Smad2 and Smad4 proteins in
human skin cancer occurs at the pretranslational level, we exam-
ined mRNA levels of Smad2 and Smad4 from 33 cases of poorly
differentiated human SCCs and 6 normal skin samples. In com-
parison with control skin, 31/33 (94%) of human
skin SCC samples showed at least a 50% reduction
of Smad2 mRNA, and 28/33 (85%) of these samples
showed at least a 50% reduction of Smad4 mRNA
(Figure 1A). Such high rates of Smad2 and Smad4
loss at the mRNA level prompted us to examine if
they are lost at the genetic level. Among samples
with more than 50% mRNA reduction of Smad2
and Smad4, 21 samples contained adjacent nonneo-
plastic skin in their paraffin sections. We dissected
the adjacent nonneoplastic skin in each section as
a control for each tumor sample and performed
a loss of heterozygosity (LOH) analysis for Smad2
and Smad4 (Figure 1B and Supplemental Figure 2).
We found that 14 samples (67%) exhibited LOH
at the Smad2 locus, and 12 samples (57%) exhibited LOH at the
Smad4 locus. A total of 17 samples (81%) had LOH at either the
Smad2 or Smad4 locus.
Keratinocyte-specific Smad2 deletion resulted in increased susceptibility to
skin carcinogenesis. Our previous study has revealed that Smad4 loss
in keratinocytes results in spontaneous skin SCCs (14). To deter-
mine if the high incidence of Smad2 loss in human skin SCCs also
plays a causal role in skin carcinogenesis, we generated keratino-
cyte-specific Smad2-knockout mice (Supplemental Figure 3). Mice
with RU486-controlled Cre recombinase targeted by a keratin 5
(K5) promoter (K5.Cre*PR1) were generated as previously reported
(18). We mated K5.Cre*PR1 mice with Smad2 floxed mice (Smad2f/f)
(19). Smad2 deletion in keratinocytes was achieved by topical appli-
cation of RU486. Unlike Smad4–/– epidermis, Smad2–/– mice did
not develop spontaneous tumors within 18 months. To further
assess whether Smad2 loss alters susceptibility to skin carcinogen-
Proteins of Smad2 and Smad4, but not Smad3, were lost in human skin SCCs
Total Smad loss/Total skin SCC samples
Poorly differentiated loss
The number of SCC cases with individual Smad protein loss compared with the total
number of SCC cases. Both Smad2 and Smad4 loss occurred at higher rates in poorly
differentiated SCCs versus in well-differentiated SCCs. However, Smad2 loss was more
closely correlated with poorly differentiated SCCs than Smad4 loss. AP = 0.025;
BP < 0.001 compared poorly differentiated SCCs to well-differentiated SCCs;
CP = 0.005 compared cases with Smad2 loss to cases with Smad4 loss.
Reduced mRNA and LOH of
Smad2 and Smad4 in human skin
SCCs. (A) qRT-PCR of 33 human
skin SCCs showed loss of Smad2
(31/33 samples) and Smad4 (28/33
samples) expression compared
with normal skin controls. The inset
shows the mean value for control
and skin SCC. **P < 0.001. (B) LOH
of Smad2 and Smad4 in human
SCCs. Microsatellite markers
D18S1137 and D18S555 were used
to assess Smad2 LOH, and D18S46
and D18S1110 were used to assess
Smad4 LOH. Het, heterozygos-
ity; Noninformative, adjacent tissue
2724? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 8 August 2008
esis, we applied a 2-stage chemical carcinogenesis protocol, which
gives discrete stages of benign papillomas and malignant SCCs
in normal mice (17). Littermates from K5.Cre*PR1 and Smad2f/f
breeding were divided by their genotypes (K5.Cre*PR1/Smad2f/f for
K5.Smad2–/–; K5.Cre*PR1/Smad2f/wt for K5.Smad2+/–; K5.Cre*PR1,
Smad2f/f, and Smad2f/wt for Smad2+/+) and treated with 20 μg RU486
daily for 5 days at 6 weeks of age to induce Smad2 gene deletion
in keratinocytes. Two weeks after RU486 treatment, when kerati-
nocytes in the entire epidermis were expected to be replaced with
Smad2–/– or Smad2+/– epidermal stem cells in K5.Cre*PR1/Smad2f/f
or K5.Cre*PR1/Smad2f/wt mice, respectively, we topically applied
a subcarcinogenic dose of dimethylbenz[α]anthracene (DMBA,
20 μg/mouse). One week later, we began to topically apply 12-O-
tetradecanoyl-phorbol-13-acetate (TPA, 5 μg/mouse) twice a week
for 20 weeks. K5.Smad2–/– and K5.Smad2+/– mice developed tumors
faster and had 2- to 3-fold more tumors per mouse than Smad2+/+
mice (P < 0.001; Figure 2A). Malignant conversion was also acceler-
ated in K5.Smad2–/– and K5.Smad2+/– mice (P < 0.05 compared with
Smad2+/+ mice). Notably, at each time point, more K5.Smad2–/– and
K5.Smad2+/– mice developed SCCs than Smad2+/+ mice (Figure 2B).
These results indicate Smad2-deficient epidermis is more suscep-
tible to skin tumor formation and malignant conversion.
Because K5.Smad2+/– also exhibited accelerated tumor forma-
tion and malignant progression similar to K5.Smad2–/– mice,
we examined endogenous Smad2 levels in K5.Smad2+/– tumors.
Smad2 protein was still detectable in all K5.Smad2+/– papillomas
but was lost in 60% cases of K5.Smad2+/– SCCs (Supplemental Fig-
ure 4). At this stage, approximately 45% SCC cases from Smad2+/+
mice also lost Smad2 protein as determined by Smad2 antibody
staining (Supplemental Figure 4; P < 0.05). These data suggest
haploid insufficiency of Smad2+/– keratinocytes at early stages of
skin carcinogenesis, and spontaneous Smad2 protein loss from
the remaining allele in SCCs caused them to progress to malig-
nancy, similar to Smad2–/– SCCs.
K5.Smad2–/– tumors were poorly differentiated and exhibited an
increase in EMT. To determine whether accelerated skin carcinogen-
Accelerated tumor formation and malignant conversion of skin carcinogenesis in K5.Smad2-knockout mice. (A) Kinetics of tumor formation.
Arrow indicates TPA withdrawal. The seemingly more rapid tumor regression after TPA withdrawal in Smad2+/– and Smad2–/– groups compared
with Smad2+/+ is due to necessity of euthanizing mice with a higher tumor burden. P < 0.001 compared with K5.Smad2–/– or K5.Smad2+/– and
Smad2+/+ (B) Kinetics of malignant conversion. Smad2+/– and Smad2–/– mice had higher rates of malignant conversion (P < 0.05 compared with
Smad2+/+ mice). The total number of mice of each genotype was used as a denominator for all time points through the entire course of tumor
kinetics in A and B. (C) Tumor pathology and keratin markers. H&E staining of K5.Smad2–/– tumors showed less differentiation compared with
K5.Smad2+/+ tumors. Immunofluorescence staining revealed that at the same histological stage, Smad2+/+ papillomas (Paps) expressed K1
(green) in suprabasal layers, whereas K5.Smad2–/– papillomas expressed K8 (red) and almost lost K1 in suprabasal layers. The dotted lines
delineate the basement membrane. At SCC stages, K5.Smad2–/– tumors developed spindle cell carcinomas (SPCCs) when K5.Smad2+/+ tumors
were well-differentiated SCCs. Rectangles in the bottom 2 panels denote areas of transition from SCC to SPCC. Two of these regions are
enlarged 4 times to illustrate this transition (top row, far right panels). Scale bars: 100 μm.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 8 August 2008
esis in K5.Smad2–/– mice was a result of abrogating TGF-β-induced
growth inhibition and/or apoptosis, we evaluated cell prolifera-
tion via BrdU incorporation and apoptosis via the TUNEL assay.
Apoptosis in K5.Smad2–/– nonlesional and tumor tissues did
not differ from those in K5.Smad2+/+ controls (data not shown).
Although nonlesional K5.Smad2–/– skin did not show increased
proliferation, TPA-treated K5.Smad2–/– skin exhibited increased
proliferation and epidermal hyperplasia (Supplemental Figure 5).
However, proliferation rates became comparable in tumors
between K5.Smad2–/– and WT mice (data not shown). Histological
analyses revealed that K5.Smad2–/– tumors were generally poorly
differentiated. The earliest K5.Smad2–/– papillomas lacked strati-
fied epithelial structure (Figure 2C) and exhibited loss of loricrin
and filaggrin, which are terminal differentiation markers (data
not shown), and K1, an early differentiation marker (Figure 2C),
but expressed K8 (Figure 2C), a marker of simple epithelia that
is not expressed in stratified epithelia but is usually expressed in
late-stage SCCs (20). In contrast, papillomas in WT mice showed
partial or complete loss of loricrin and filaggrin (data not shown),
without K8 expression, but retained uniform K1 expression (Fig-
ure 2C). At the SCC stage, K5.Smad2–/– SCCs were poorly differ-
entiated and often showed clusters of cells that underwent EMT
(Figure 2C), whereas most SCCs from WT mice were well differ-
entiated (Figure 2C). While only 1 out of 20 WT mice developed
an EMT-type of spindle cell carcinoma (SPCC) 50 weeks after TPA
promotion, K5.Smad2+/– and K5.Smad2–/– mice developed more
SPCCs at earlier time points (3 out of 12 K5.Smad2+/– and 5 out of
19 K5.Smad2–/– starting at 27–35 weeks).
K5.Smad2–/– tumors exhibited pathological alterations associated with
EMT. The poorly differentiated nature of Smad2–/– tumors prompted
us to examine the status of E-cadherin, an adhesion molecule criti-
cal for maintaining epithelial structure. While E-cadherin was lost
in only patchy areas of late-stage Smad2+/+ papillomas, Smad2–/–
papillomas exhibited nearly complete loss of E-cadherin (Figure 3).
In contrast, early stage Smad4–/– spontaneous SCCs retained
membrane-associated E-cadherin in most tumor cells (Figure 3).
We then examined expression patterns of Snail, a transcriptional
repressor of E-cadherin (21). Snail antibody staining, which rec-
ognizes both Snail and Slug, revealed a patchy, cytoplasmic stain-
ing pattern in WT papillomas and early stage Smad4–/– sponta-
neous SCCs (Figure 3). In contrast, Smad2–/– tumors exhibited
strong Snail staining primarily in the nucleus (Figure 3). Both
chemically induced WT SCCs and Smad4–/– spontaneous SCCs
showed reduced E-cadherin and increased Snail nuclear staining
in late stages, approximately 10–20 weeks after SCC formation
(data not shown). K5.Smad2–/– tumors also showed an increase in
mesenchymal markers, vimentin and αSMA (Supplemental Fig-
ure 6A), which are associated with motility and invasiveness (22,
23). These markers were restricted to the stroma of K5.Smad2+/+
papillomas and spontaneous Smad4–/– SCCs but were detected in
both tumor epithelia and stroma of K5.Smad2–/– papillomas. Addi-
tionally, vimentin and αSMA were present in the hyperplastic epi-
dermis adjacent to papillomas of K5.Smad2–/– mice (Supplemen-
tal Figure 6B), suggesting that EMT is a relatively early event in
K5.Smad2–/– carcinogenesis. We then examined if the EMT pheno-
type occurs in K5.Smad2–/– without exposure to a carcinogen. Sev-
enty-two hours after Smad2 was deleted in neonatal skin, a marked
loss of E-cadherin and an associated increase in nuclear Snail were
seen in Smad2–/– epidermis and hair follicles when compared with
RU486-treated WT and K5.Smad4–/– skin (Figure 3B). However,
vimentin and αSMA were not detected in K5.Smad2–/– epidermis
(data not shown). These results suggest that EMT is an early effect
of Smad2 loss, and additional insults during carcinogenesis fur-
ther enhanced Smad2 loss–associated EMT phenotype.
Smad2 loss resulted in molecular alterations of TGF-β target genes associ-
ated with EMT. To assess if Smad2 loss–associated EMT was associ-
Snail activation and E-cadherin (ECad) loss in
K5.Smad2–/– tissues. A K14 antibody was used
for counterstain (red). (A) K5.Smad2–/– papillomas
undergo EMT. When most of cells in Smad2+/+ papil-
lomas or spontaneous Smad4–/– SCCs still retained
E-cadherin staining (green), K5.Smad2–/– papillomas
show significant loss of E-cadherin (green). Arrows in
K5.Smad2+/+ image indicate patchy areas of E-cad-
herin loss, whereas arrows in K5.Smad2–/– tumors
show patchy retention of E-cadherin (top row). At
this stage, Snail staining (green) was primarily cyto-
plasmic in Smad2+/+ papillomas and spontaneous
Smad4–/– SCCs, but K5.Smad2–/– tumors displayed
nuclear Snail staining (bottom row). Scale bar: 100
μm. (B) K5.Smad2–/– pup skin 72 hours after Smad2
deletion demonstrated significant reduction of E-cad-
herin (green, upper row) with a concomitant increase
in nuclear Snail (green, bottom row). K5.Smad4–/–
pup skin 72 hours after Smad4 deletion showed no
change in E-cadherin and Snail expression patterns
from WT skin. Scale bar: 100 μm.
2726? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 8 August 2008
ated with increased TGF-β1 ligand, which plays an important role
in EMT (24), we examined TGF-β1 levels in WT and Smad2–/– skin
and tumors. We found neither elevated TGF-β1 nor alterations of
Smad3 and Smad4 in K5.Smad2–/– skin and papillomas in com-
parison with WT controls (data not shown). Elevated TGF-β1
protein was found in WT and K5.Smad2–/– SCCs at comparable
levels (Supplemental Figure 7). Consistent with this data, western
analyses showed that in comparison with WT tumors, K5.Smad2–/–
tumors did not have increased levels of pJNK, pERK, and pMAPK
(data not shown), the major non-Smad pathways of TGF-β–
induced EMT that require a higher level of TGF-β1 than that
found in WT tumors (25). Consistent with our previous observa-
tion that approximately 30%–40% of chemically induced SCC cases
exhibited reduction in Smad3 and Smad4 at late stages (26), both
WT and K5.Smad2–/– SCCs showed reduced Smad3 and Smad4 in
approximately 40% of cases (data not shown). SCCs that retained
Smad3 showed similar patterns between WT and K5.Smad2–/– mice
(Supplemental Figure 7). K5.Smad2–/– SCCs that retained Smad4
showed more nuclear staining than K5.Smad2+/+ SCCs (Supple-
mental Figure 7). To further determine whether changes in EMT-
associated proteins are the result of transcriptional deregulation
of these genes by Smad2 loss, we examined mRNA levels of these
molecules in K5.Smad2–/– and K5.Smad2+/+ papillomas, at the stage
prior to the pathological appearance of EMT cells in K5.Smad2–/–
tumors. Transcripts of K8, Snail, Slug, vimentin, and tenascin C
were all significantly upregulated in K5.Smad2–/– papillomas in
comparison with WT papillomas (Figure 4A). In contrast, tran-
scripts of E-cadherin were downregulated in Smad2–/– papillomas
in comparison with WT controls (Figure 4B). Further, increased
transcripts of Snail and Slug and decreased E-cadherin transcripts
were also detected in day 3 K5.Smad2–/– skin in comparison with
WT skin (Figure 4, C and D). No changes in expression levels of
vimentin and tenascin C were observed in K5.Smad2–/– skin (data
not shown). These data suggest that upregulation of Snail and/or
Slug and the subsequent E-cadherin reduction represent an early
effect of Smad2 loss in keratinocytes.
To assess if Smad2 regulates Snail and Slug differently from
Smad3 and Smad4 in keratinocytes, we examined expression lev-
els of Snail and Slug in cultured human HaCaT keratinocytes after
knocking down Smad2, -3, or -4. After 48 to 72 hours of transfec-
tion of HaCaT cells with siRNAs for Smad2, -3, or -4, the respective
mRNA levels were reduced by at least 70% (Supplemental Figure 8).
In mock-transfected cells, increased Snail mRNA was detected after
1 hour of TGF-β1 treatment and remained increased for 48 hours
(data not shown), with a 9-fold increase at 2 hours (Figure 4E).
After Smad2 was knocked down for 72 hours, Snail expression
was increased by 13-fold, and further increased by 24-fold after
2 hours TGF-β1 treatment (Figure 4E). In contrast, knockdown of
Smad3 did not affect baseline Snail expression but significantly
attenuated TGF-β1–induced Snail expression (Figure 4E). Knock-
ing down Smad4 abrogated both baseline and TGF-β1–induced
Snail expression (Figure 4E). Slug expression was also induced by
Altered gene expression associated with
dedifferentiation and EMT in K5.Smad2–/–
papillomas and epidermis. (A) Upregulated
mRNA expression of K8- and EMT-associ-
ated molecules in K5.Smad2–/– papillomas.
*P < 0.05 compared with Smad2+/+ papil-
lomas. (B) Downregulation of E-cadherin
in K5.Smad2–/– papillomas. All changes in
Smad2–/– papillomas are statistically sig-
nificant in comparison with Smad2+/+ papil-
lomas. *P < 0.05 compared with Smad2+/+
papillomas. (C) Upregulation of Snail and
Slug mRNA in K5.Smad2–/– epidermis.
‡P < 0.01 compared with WT skin. (D)
Downregulation of E-cadherin in K5.
Smad2–/– epidermis. ‡P < 0.01 compared
with WT skin. (E) Snail expression levels
after knocking down individual Smads
in HaCaT cells. #P < 0.05 compared with
mock transfection treatment with TGF-β1.
**P < 0.001 compared with mock transfec-
tion with TGF-β1 treatment. ††P < 0.001
compared with mock transfection without
TGF-β1. †P < 0.05 compared with mock
transfection without TGF-β1 (F) Slug expres-
sion levels after knocking down individual
Smads in HaCaT cells. †P < 0.05 compared
with mock transfection without TGF-β1.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 8 August 2008
TGF-β1 treatment, with a 2-fold increase 2 hours after TGF-β1
treatment (Figure 4F). However, none of the individual Smads
affected Slug expression levels, with or without TGF-β1 treatment
(Figure 4F). These data suggest that expression of Snail, but not
Slug, is regulated by Smads. Thus, in vivo Slug overexpression in
K5.Smad2–/– keratinocytes could be a secondary event.
Next, we assessed if Smad2 loss correlated to Snail overexpression
in human skin SCCs. Overall, Snail overexpression and E-cadherin
loss occurred at high frequencies in human skin SCCs. However,
consistent with the association of Smad2 loss with poorly differ-
entiated SCCs (Table 1), Smad2-negative SCCs (52 cases) exhib-
ited a higher incidence of Snail overexpression than in 23 cases of
Smad2-positive SCCs (90% vs. 73%; P < 0.05; Figure 5). Similarly,
the rate of E-cadherin loss occurred in 79% of Smad2-negative
SCCs versus 60% in Smad2-positive SCCs (P < 0.05; Figure 5).
Elevated Snail contributed markedly to Smad2 loss–associated EMT.
Since the data from K5.Smad2–/– mice, human keratinocytes, and
human skin SCCs revealed a correlation between Snail overexpres-
sion and Smad2 loss, we assessed whether increased Snail expression
functionally contributed to Smad2 loss–associated EMT by knock-
ing down Snail together with Smad2 in HaCaT cells (Figure 6). In
control keratinocytes, sporadic Snail nuclear staining cells were
detected and E-cadherin stained the cell membrane. After 72 hours
of Snail knockdown, Snail expression and protein was reduced by
80% (Supplemental Figure 8), and the number of Snail nuclear posi-
tive cells was markedly reduced (Figure 6). E-cadherin staining in
Snail siRNA–treated cells retained a similar pattern to control cells.
After 72 hours of Smad2 knockdown, HaCaT cells lost the typical
keratinocyte appearance, and some of them exhibited fibroblast-
like morphology (Figure 6), which correlated with increased Snail
nuclear staining and loss of membrane-associated E-cadherin (Fig-
ure 6). However, knocking down both Smad2 and Snail restored
membrane-associated E-cadherin staining and epithelial morphol-
ogy (Figure 6), suggesting that Snail is the major target of Smad2
loss and contributes to Smad2 loss–associated EMT.
Enhanced Smad4 binding to the Snail promoter in Smad2-deficient
keratinocytes. To further analyze whether Snail overexpression
induced by Smad2 loss was the result of enhanced transcriptional
activity of Smad3 and/or Smad4, we performed in vivo chromatin
immunoprecipitation (ChIP) for Smad binding to the Snail pro-
moter in neonatal WT and Smad2–/– mouse skin. Within the TGF-β–
regulatory region of the mouse Snail promoter, as identified by
previous studies (9, 27), there are 2 SBEs at –438 bp and –1,077
bp upstream of the Snail transcriptional start site (TSS). We found
that in WT skin, Smad2, -3, and -4 bound to both sites (Figure 7,
A and B) but not to intronic regions of the gene (data not shown).
Quantitative PCR on the precipitated chromatin revealed that in
Smad2–/– skin, Smad3 binding to both SBEs was at a capacity simi-
lar to that in WT skin (Figure 7, A and B), suggesting that Smad2
does not affect the affinity of Smad3 binding to the Snail pro-
moter. However, Smad4 binding to the Snail promoter increased
by 8- and 29-fold on the –438-bp and the –1,077-bp sites, respec-
tively, in K5.Smad2–/– skin compared with that in WT skin (Figure
7, A and B). Therefore, these data suggest that normally, Smad2
either competes with, or impedes Smad4 binding to the SBE at
the Smad3-binding site on the Snail promoter. To further assess
if Smad2 loss–associated increase in Smad4 binding to the Snail
promoter contributes to Snail overexpression, we knocked down
Smad4 together with knockdown of Smad2. Knocking down
Smad4 abrogated Smad2 loss–associated Snail overexpression
(Figure 7C), suggesting that increased Smad4 binding contrib-
uted to transcriptional regulation of Snail in K5.Smad2–/– kerati-
nocytes. Knockdown of Smad3 also abrogated Smad2 loss–asso-
ciated Snail overexpression, suggesting that Smad3 binding in a
complex with Smad4 is required for increased Snail transcription
in Smad2-deficient keratinocytes.
Smad2 and Smad4, but not Smad3, are frequently lost in human skin
SCCs. In the current study, we found that proteins of Smad2 or
Smad4, but not Smad3, were frequently lost in human skin SCCs.
In cases with LOH of Smad2 or Smad4, single-copy genetic loss
may contribute to at least 50% loss of their transcripts and pro-
tein in each case, as mice with heterozygous deletion of Smad2 or
Smad4 exhibited ~50% loss of transcripts and protein of these 2
molecules (see also ref. 28). Additionally, transcriptional and post-
translational modifications could contribute to further loss of
the remaining Smad2 and Smad4 transcripts and protein. Several
Smad ubiquitin-E3 ligases, which contribute to Smad protein deg-
radation, have been identified, some of which have been shown to
be overexpressed in cancer (29–32). Thus, multiple mechanisms
from the genetic to the posttranslational level could explain loss of
Smad2 and Smad4 proteins, which are among the most frequent
molecular alterations in skin cancer. Indeed, Smad2 and Smad4
Human skin SCCs with Smad2 loss correlated with E-cadherin loss and
nuclear Snail. Skin SCCs were stained for Smad2 IHC (brown, top row),
and immunofluorescence staining was performed for E-cadherin (green;
middle row) and Snail (green; bottom row). A K14 antibody was used
for immunofluorescent counterstain (red). An example of a pair SCCs
from serial sections showed that a Smad2-positive SCC retained mem-
brane-associated E-cadherin with a few Snail nuclear staining cells. In
contrast, SCC with Smad2 loss lost membrane-associated E-cadherin
but uniformly expressed Snail in the nucleus. Scale bar: 100 μm.
2728? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 8 August 2008
loss occurs more frequently than the currently known common
molecular alterations in human skin SCCs, e.g., oncogene ras
activation or loss of the p53 tumor suppressor (33). Notably, the
incidences of Smad2 and Smad4 loss in skin SCCs found in this
study are much higher than in head and neck SCCs (34). These dif-
ferences may reflect cancer etiology, which is largely attributed to
UV irradiation in skin cancer and to tobacco carcinogen exposure
in head and neck cancer.
Smad2 has a tumor suppressive effect on the skin. Unlike keratinocyte-
specific Smad4-knockout mice, which developed spontaneous skin
SCCs, K5.Smad2–/– mice developed skin tumors neither spontane-
ously nor with TPA treatment alone (without DMBA initiation,
data not shown), even though K5.Smad2–/– epidermis exhibited an
increase in proliferation in response to TPA application. Thus, the
increased proliferative potential of K5.Smad2–/– epidermis is insuf-
ficient as an initiator for skin carcinogenesis. This result is con-
sistent with Smad2 expression patterns in human skin SCCs, in
which Smad2 loss occurs only in SCCs but not in early stage actin-
ic keratosis (25). Conversely, in the presence of a DMBA-induced
H-ras mutation, a genetic alteration mimicking early stage human
skin cancer (35), K5.Smad2–/– mice still did not develop skin tumors
without TPA promotion (data not shown). Thus, Smad2 loss alone
is also insufficient to promote initiated cells for cancer develop-
ment. However, with both DMBA initiation and TPA promotion,
K5.Smad2–/– mice were more susceptible to skin tumor formation
and malignant conversion than WT mice. Although the current
study limits the assessment of the true malignant conversion rate
for each papilloma due to the necessity of euthanizing SCC-bear-
ing mice with multiple papillomas, more K5.Smad2–/– mice devel-
oped SCCs at the same time points when compared with WT mice.
Additionally, K5.Smad2–/– papillomas already harbored molecu-
lar changes seen in WT SCCs but not in papillomas, suggesting
that the malignant progression program was in place prior to the
pathological progression in K5.Smad2–/– tumors. Thus, Smad2 loss
appears to cooperate with other molecular alterations elicited by
the chemical carcinogenesis protocol to promote skin carcinogen-
esis. Interestingly, K5.Smad2+/– mice displayed tumor kinetics simi-
lar to K5.Smad2–/– mice. This observation is also consistent with a
previous report that germline Smad2 heterozygous mice exhibited
accelerated tumor formation and malignant progression in a skin
chemical carcinogenesis experiment (16). These studies suggest a
haploid insufficiency for Smad2 in tumor suppression.
Smad2 loss triggers molecular and pathological alterations associated with
EMT. Our current study reveals that in human skin cancer, Smad2
loss was associated with dedifferentiation, loss of E-cadherin, and
Snail activation. Correlated with this observation, the accompany-
ing animal study reveals that loss of Smad2 triggers pathological
and molecular alterations associated with dedifferentiation and
EMT started in nonlesional Smad2–/– epidermis. Among EMT asso-
ciated genes, Snail overexpression appears to be a major target and
mediator of Smad2 loss-induced EMT.
The effect of Smad2 loss on EMT is somewhat surprising given
that TGF-β signaling is well documented to promote EMT via
both Smad and non-Smad pathways (24). Unlike keratinocytes
with knockdown of TGF-βRII, which overexpress TGF-β1 (25,
36), Smad2 loss did not cause increased TGF-β1. However, our
data revealed a significant increase in promoter binding of Smad4
to the Snail promoter in Smad2–/– keratinocytes. It is possible that
Snail contributed to Smad2 loss–associated EMT.
Smad2 knockdown caused an increase in Snail
nuclear staining (green) compared with mock trans-
fection and loss of E-cadherin (green) membrane
staining, with more spindle-like morphology in HaCaT
keratinocytes. A K14 antibody was used for counter-
stain (red). Remaining Snail staining in Snail siRNA
transfected cells could be due to antibody cross
reaction with Slug. E-cadherin staining had a pattern
similar to mock control. Smad2 and Snail concomi-
tant knockdown resulted in reduced Snail staining in
comparison with Smad2 siRNA transfected cells and
restoration of membrane E-cadherin staining. Phase
contrast photos show epithelial morphology of the
mock-transfected or Snail siRNA–transfected cells.
Epithelial morphology was lost in Smad2 siRNA–
transfected cells, but was restored by cotransfection
with Snail siRNA. Scale bar: 20 μm.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 8 August 2008
normally Smad2 either competes with or impedes the ability of
Smad4 to bind Smad3 on the SBEs of the Snail promoter, there-
fore Smad2 loss confers more binding of Smad4 with Smad3.
Although Smad2 has been shown to activate Snail, it is likely that
Smad2 has a much weaker effect than Smad4, given the fact that
Smad2, but not Smad4, normally recruits transcriptional core-
pressors to SBEs (2). Based on this, once Smad2 is lost, the core-
pressors would not be recruited to the SBEs, and Smad3 together
with Smad4 would drive a higher level of Snail transcription. Sup-
porting this explanation, knocking down either Smad3 or Smad4
abrogated Smad2 loss–associated Snail overexpression, and a pre-
vious study showed that the combination of Smad3 and Smad4
had the highest transcriptional activity on the Snail promoter (9).
Our data also helps to explain the difference between our current
finding and a previous finding showing that a dominant-negative
form of either Smad2 or Smad3 blocked TGF-β1–induced EMT
in skin SCC cells (8). In that study, either increased Smad4 bind-
ing to the Snail promoter or the loss of corepressors should not
occur due to the presence of WT Smad2. Since Smad4–/– skin and
early stage SCCs did not undergo EMT or exhibit the associated
molecular alterations even in the presence of Smad2 and Smad3,
Smad4 appears to be indispensable for EMT at least at early stages
of skin carcinogenesis. Similar to our current finding, a previous
study shows that pancreatic ductal adenocarcinomas derived from
Smad4-null cells are more well differentiated and have less EMT
in comparison with tumors with intact Smad4 (37). Consistently,
Ju et al. (19) reported that in hepatocyte-specific Smad2-knock-
out mice, hepatocytes underwent de-differentiation and EMT,
whereas Smad3–/– hepatocytes did not. Since Smad4 was not lost
in Smad2–/– skin and in early stage tumor cells, enhanced Smad4
binding to the Snail promoter is likely the major contributor to
increased Snail expression, at least at early stages of skin carci-
nogenesis in Smad2–/– mice. When Smad4 is lost at late stages,
multiple genetic/epigenetic alterations accumulated in tumor
epithelia could be sufficient to sustain EMT and invasion. To this
end, other pathways commonly activated in late-stage skin carci-
nogenesis, e.g., AKT and NF-κB, have been shown to activate Snail
expression and EMT (38, 39).
It is worthwhile to mention that accelerated EMT does not
always contribute to malignant progression. For instance, Smad4–/–
keratinocytes did not exhibit EMT but proceeded to become spon-
taneous SCCs (13, 14). Conversely, accelerated EMT in Smad2–/–
keratinocytes was insufficient to cause spontaneous skin cancer
formation. Thus, EMT would promote tumor invasion in vivo
only when coupled with other oncogenic events. Further, all of
the EMT-associated genes upregulated in K5.Smad2–/– tumors
also have additional functions for promoting cancer invasion.
For instance, Snail and Slug regulate cell survival and apoptosis
(40–44), and Snail overexpression in the epidermis causes kerati-
nocyte hyperproliferation (45). Tenascin C has been implicated in
Snail transcription contributes to Smad2
loss–associated Snail overexpression. (A
and B) Comparative PCR from chroma-
tin immunoprecipitation (IP) showed an
increase in Smad4 binding to the Snail pro-
moter in Smad2–/– skin compared with WT
skin. Residue Smad2 binding in Smad2–/–
skin was from nonkeratinocyte population of
the whole skin. Smad3 binding to the Snail
promoter was not significantly changed in
Smad2–/– skin in comparison with WT skin.
Smad4 binding to the Snail promoter was
significantly increased in Smad2–/– skin.
*P < 0.05. (C) Dual knockdown of Smad2
and Smad3 or Smad2 and Smad4 abro-
gated Smad2 loss–associated Snail over-
expression. Smad2 knockdown (48 hours)
caused a significant increase in Snail expres-
sion. Knockdown of Smad4 alone caused a
reduction in Snail expression. Concomitant
knockdown of Smad2 and Smad3 or Smad2
and Smad4 reduced Snail expression back
to mock-transfection levels. †P < 0.05 com-
pared with mock transfection. ‡P < 0.05
compared with Smad2 siRNA treatment.
2730? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 8 August 2008
angiogenesis (46). All these functions could contribute to acceler-
ated tumor formation and progression K5.Smad2–/– mice.
In summary, we report that Smad2 and Smad4 are frequently
lost in human skin SCCs. The LOH of Smad2 and Smad4 in human
skin SCCs and the haploid insufficiency of Smad2 and Smad4 in
mouse skin carcinogenesis (see also ref. 47) suggest that in human
skin cancer, even if cancer lesions lose 1 allele of the Smad2 or
Smad4 or reduce their proteins to less than 50%, these lesions may
have lost the tumor suppressive effect of Smad2 or Smad4. On
the other hand, our study also shows that Smad2 loss–associ-
ated increase in Smad4 binding to the Snail promoter beyond a
physiological level facilitates Snail activation and EMT. Our study
prompts future research into how loss of both Smad2 and Smad4
affects skin carcinogenesis in vivo. It also remains to be determined
how Smad2 loss after tumor formation, as seen in human cancers,
affects tumor differentiation and malignant progression.
Human skin SCC collection and sample preparation. Human skin SCC and
normal skin samples were from a human SCC tissue array (US Biomax)
and were collected from surgically resected specimens between the years
2000 and 2005 from consenting patients at Oregon Health & Science
University under an Institutional Review Board–approved protocol. The
tissue array contained 75 SCCs and 4 normal skin samples graded by 2
pathologists from the vendor. We confirmed the grade for samples from
both the tissue array and the ones we collected. In total, tissues used for
IHC included 83 SCC cases and 10 normal skin samples. Tissues used
for immunofluorescence included 75 SCC cases and 4 skin samples. Tis-
sues used for quantitative RT-PCR (qRT-PCR) assays included 33 poorly
differentiated skin SCCs and 6 normal skin samples. Among these, 21
samples that contained adjacent nonneoplastic skin in paraffin sections
were microscopically dissected, and tissues scrapings from nonneoplas-
tic skin and SCCs were collected separately. For LOH analysis, DNA
from these tissue sections was extracted using the WaxFree DNA Paraf-
fin Sample DNA extraction kit (TrimGen Inc.) and used for PCR, using
primers for microsatellite repeat regions (48) (D18S555, forward 5′-FAM-
GTGCGATGGCAAAATAGATG-3′, reverse 5′-ATTTTCTAGGAAAGAGC-
TAGC-3′; D18S1137, forward 5′-FAM-TGACTATTTGCACATCTGGC-3′,
reverse 5′-GGACTTGCACGCTAATGAC-3′; D18S460, forward 5′-FAM-
CTGAAGGGTCCTTGCC-3′, reverse 5′-GCCAGCCTTGGCAGTC-3′). PCR
products were column purified (Wizard SV Gel and PCR Clean-Up System;
Promega) and analyzed using fragment length polymorphism analysis
(Applied Biosystems 3130xL and Peak Scanner Software, version 1.0). LOH
was determined using the following formula: peak height of allele 1 of
tumor divided by peak height of allele 2 of tumor, the result of which was
divided by peak height of allele 1 of adjacent skin divided by peak height of
allele 2 of adjacent skin, the result of which was greater than 1.5.
Tissue histology, tumor classification, and IHC. Skin and tumor samples
were fixed in 10% neutral-buffered formalin at 4°C overnight, embedded
in paraffin, sectioned to 6 μm thickness, and stained with H&E. Tumor
types were determined by H&E analysis, using the criteria described pre-
viously (49). IHC was performed on paraffin sections as we have previ-
ously described (25), using primary antibodies against human and mouse
Smad2 (1:200; Zymed), Smad3 (1:100; Santa Cruz Biotechnology Inc.),
and Smad4 (1:100; Santa Cruz Biotechnology Inc.). Sections were coun-
terstained with hematoxylin.
Generation of inducible and keratinocyte-specific Smad2- and Smad4-knockout
mice. The K5.Cre*PR1, the Smad2f/f line, and Smad4f/f line were generated on
a C57BL/6 background as previously reported (18, 19, 50). K5.Cre*PR1 mice
were crossed with Smad2f/f or Smad4f/f mice to generate WT, K5.Smad2f/wt,
K5.Smad2f/f, or K5.Smad4f/f genotypes. For genotyping, PCR using tail DNA
was performed with primers specific for the floxed region and the Cre
recombinase as previously reported (18, 19, 50). Cre-mediated Smad2 or
Smad4 deletion in keratinocytes was achieved with topical application of
RU486 (20 μg in 100 μl ethanol) once a day for 3–5 days at time points
specified in the Results section. Smad2 or Smad4 gene deletion was detected
by PCR performed on DNA extracted from RU486-treated skin, using dele-
tion-specific primers (19, 50).
Skin chemical carcinogenesis protocol. Eight-week-old mouse skin was shaved
and topically treated with 20 μg of DMBA (dissolved in 50 μl acetone;
Sigma-Aldrich). One week later, 5 μg of TPA (dissolved in 50 μl acetone;
Sigma-Aldrich) was applied to skin twice a week for 20 weeks. In total,
20 Smad2+/+, 19 Smad2–/–, and 12 Smad2+/– mice, with equal distribution of
genders in each group, were used in the carcinogenesis study (17).
RNA extraction and qRT-PCR. RNA was isolated from human and mouse
skin and tumors using RNAzol B (Tel-Test), as we have previously described
(51), and purified using a QIAGEN RNeasy Mini kit (QIAGEN). The qRT-
PCR was performed as we have previously described (52). Transcripts of
human Smad2 and Smad4, mouse Smad2, K8, Snail, Slug, vimentin, tenas-
cin C, and E-cadherin were examined using corresponding Taqman Assays-
on-Demand probes (Applied Biosystems). A K14 or GAPDH RNA probe
was used as an internal control. Three to nine samples from each genotype
of mice or cultured cells were used for qRT-PCR. The mean expression level
from K5.Smad2+/+ samples (unless otherwise specified) of each particular
gene being analyzed was set as 1 arbitrary unit.
Double-stain immunofluorescence. Double-stain immunofluorescence on
paraffin-embedded tissue sections was performed as we have previously
described (53). Each section was incubated overnight at 4°C with a primary
antibody together with a guinea pig antiserum against mouse K14, the
latter of which highlights the epithelial compartment of the skin (51). An
Alexa Fluor 488–conjugated (green) secondary antibody against the species
of the primary antibody (1:100–1:400; Molecular Probes) and Alexa Fluor
594–conjugated (red) anti–guinea pig secondary antibody (1:100; Molec-
ular Probes) were used to visualize the staining. The primary antibodies
included K1 (1:500; Covance), K8 (1:100; Fitzgerald), vimentin (1:200;
Sigma-Aldrich), E-cadherin (1:100; BD Bioscience), K14 (1:400; Fitzgerald),
Snail (1:200; Abcam), and αSMA (1:200; Sigma-Aldrich).
Quantitative chromatin immunoprecipitation. Four mouse back skins from
each group of WT and K5.Smad2–/– mice were homogenized on ice in 5 ml
of 1% formalin and incubated at room temperature for 30 minutes after
adding an additional 5 ml of 1% formalin to each tube. Each sample was
then diluted in 1 ml of 10X Glycine Stop Solution (Active Motif), incubated
at room temperature for 5 minutes, and then centrifuged at 1,400 g for 10
minutes at 4°C. The resulting pellet was used for ChIP Enzymatic Diges-
tion following the manufacturer’s protocol (Active Motif). Antibodies, 3 μg/
each, to Smad2 (Zymed), Smad3 (Upstate), and Smad4 (Upstate) were used
to immunoprecipitate the sheared chromatin complexes. Rabbit IgG (3 μg;
Santa Cruz Biotechnology Inc.) was used as a negative control for antibody
specificity. DNA obtained from eluted beads was used for quantitative
PCR using Power SYBR Green Master Mix (Applied Biosystems). Primers
encompassing the SBE sites of the Snail promoter (Supplemental Table 1)
were used for PCR. Positive binding was defined as antibody binding more
than 10-fold of the IgG-negative control. Difference in Ct (ΔCt) values were
obtained by normalizing IP Ct values to input values for each group. ΔΔCt
values were obtained by comparing the ΔCt values of Smad2–/– skin to WT
skin. Values are expressed as fold change based on ΔΔCt values.
HaCaT keratinocyte culture and siRNA knockdown. HaCaT keratinocytes
were cultured in DMEM with high levels of glucose with 10% FBS and
penicillin-streptomycin antibiotics. Twenty-four hours prior to siRNA
transfection, cells were switched to DMEM with low levels of glucose with
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 8 August 2008
0.2% FBS and penicillin-streptomycin antibiotics. Cells were transfected
with siRNAs for human Smad2, Smad3, Smad4, or Snail (Supplemental
Table 2) using XtremeGene siRNA Transfection Reagent (Roche) in 6-well
plates or chamber slides, at a final concentration of 50 pmol siRNA/μl
in Optimem media (Gibco). Four hours posttransfection, media was
switched to DMEM with high levels of glucose. Prior to cell harvest in
culture dishes or fixation in the chamber slides, cells were treated with or
without 10 pM TGF-β1 for period of times specified in the Results section.
Cells were harvested at 48 or 72 hours after siRNA transfection for RNA
extraction using Qiashredder and RNeasy kits (QIAGEN). Chamber slides
were fixed for 40 minutes at room temperature in 2% paraformaldehyde
and stained with antibodies specified in the Results section, using the
methods as described above.
Statistics. Significant differences between the values obtained in each assay
on samples from various genotypes were determined using the Student’s
t test and expressed as mean ± SEM, with the exception of evaluation of
human SCCs and tumor malignancy, in which a χ2 test was used. P values
of less than 0.05 were considered significant.
The authors thank Qinghong Zhang and Jiri Zavadil for sugges-
tions for the experiments. We would also like to thank an anony-
mous donation to our research related to this publication. This
work was supported by NIH grants to X.-J. Wang. K.E. Hoot and
J. Lighthall are recipients of NIH training grants.
Received for publication October 15, 2007, and accepted in revised
form May 28, 2008.
Address correspondence to: Xiao-Jing Wang, Portland VA Can-
cer Center (R&D 46), Building 103, Room F-221, 3710 SW US
Veterans Hospital Road, Portland, Oregon 97239-2999, USA.
Phone: (503) 220-8262, ext. 54273; Fax: (503) 402-2817; E-mail:
Xiao-Jing Wang’s present address is: Portland VA Cancer Center,
Portland, Oregon, USA.
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