[CANCER RESEARCH 61, 5248 –5254, July 1, 2001]
Influence of ␤1 Integrins on Epidermal Squamous Cell Carcinoma Formation in a
Transgenic Mouse Model: ␣3␤1, but not ␣2␤1, Suppresses Malignant Conversion1
David M. Owens and Fiona M. Watt2
Imperial Cancer Research Fund, Keratinocyte Laboratory, London WC2A 3PX, United Kingdom
ABSTRACT
Although aberrant integrin expression has been documented in many
epithelial tumors, little is known about how integrins influence neoplastic
progression. To examine this issue, transgenic mice in which the ␣2␤1 or
␣3␤1 integrin was expressed in the suprabasal epidermal layers via the
involucrin promoter were subjected to skin carcinogenesis. Equal numbers of benign squamous papillomas were observed in transgenic and
wild-type animals. However, the frequency of conversion of papillomas to
malignant squamous cell carcinomas was much lower in ␣3␤1 transgenic
than in ␣2␤1 transgenic and wild-type mice. No differences were observed
in apoptosis or in the expression of endogenous integrins in transgenic and
wild-type papillomas. However, ␣3␤1 transgenic papillomas displayed a
diminished proliferative capacity and were more highly differentiated as
judged by BrdUrd incorporation and keratin 10 expression, respectively,
than ␣2␤1 transgenic and wild-type papillomas. Two proteins that associate with ␣3␤1 and not ␣2␤1 are extracellular matrix metalloproteinase
inducer and CD81. Extracellular matrix metalloproteinase inducer expression correlated inversely with the degree of differentiation in normal
epidermis and in transgenic and wild-type papillomas. Up-regulation of
CD81 was observed in 100% of wild-type and 88% of ␣2␤1 transgenic
papillomas but in only 25% of ␣3␤1 transgenic papillomas. CD81 was
undetectable in untreated epidermis and strongly expressed in all transgenic and wild-type squamous cell carcinomas. Our results demonstrate
that the ␣3␤1 integrin can suppress malignant conversion, and that the
mechanism may involve CD81.
INTRODUCTION
Integrins are cell surface receptors that mediate cell-extracellular
matrix and cell-cell adhesion events and are capable of transducing
signals that are implicated in a wide variety of cellular responses (1,
2). In the epidermis, the major keratinocyte integrins are ␣2␤1, ␣3␤1,
and ␣6␤4 (3). In addition to mediating keratinocyte adhesion to
extracellular matrix proteins, ␤1 integrins regulate epidermal proliferation and terminal differentiation (4 – 8).
Integrin expression is normally restricted to keratinocytes in the
basal layer of the epidermis. However, integrin expression is altered
when the epidermis is damaged and in benign and neoplastic diseases.
Suprabasal integrin expression has been observed during wound healing and in psoriatic epidermis (9, 10). SCCs3 exhibit variable patterns
of integrin expression, including normal expression, loss of expression, and overexpression, and variation is observed both within and
between individual tumors. In squamous papillomas and SCC, suprabasal expression of ␣6␤4, as well as increased basal ␣6␤4 expression,
is associated with tumor progression and invasion (11–13). Highly
undifferentiated spindle cell carcinomas exhibit up-regulated expression of the ␣5␤1 integrin (14). Increased expression of ␣2␤1 and
␣3␤1 integrin has been observed in basal cell carcinomas (3, 15).
Complete or focal loss of expression of ␣2␤1, ␣3␤1, and ␣6␤4 has
also been observed in SCC (16, 17). Hence, both loss of expression
and overexpression of keratinocyte integrins have been observed
in SCC.
Various in vitro models have been established to examine the
significance of alterations in keratinocyte integrin expression in SCC
(13, 18, 19). The behavior of SCC lines has been analyzed before and
after introduction of specific integrin subunits. Although such studies
clearly demonstrate that abnormal integrin expression contributes to
the failure of SCC cells to differentiate normally, they suffer from the
limitation that it is not possible to study the role of integrins in tumor
development. To overcome this difficulty, we have made use of a
transgenic mouse model that targets the expression of human integrin
subunits to the suprabasal layers of the epidermis via the involucrin
promoter (20, 21). Mice expressing suprabasal ␣2␤1 or ␣3␤1 exhibit
sporadic epidermal hyperplasia and skin inflammation, which are
indicative of the benign hyperproliferative disease psoriasis (20, 21).
However, no spontaneous epidermal tumors have been observed in
these lines. To determine whether suprabasal integrin expression can
alter the sensitivity of keratinocytes to malignant conversion, we have
subjected transgenic mouse skin expressing ␣2␤1 or ␣3␤1 to a
chemical carcinogenesis protocol that allows evaluation of the development of both benign (papillomas) and malignant (SCC) tumors
(22, 23).
MATERIALS AND METHODS
Reagents. DMBA was purchased from Argos Chemicals. TPA was purchased from LC Laboratories. Acetone was purchased from BDH Chemicals.
BrdUrd was purchased from Sigma Chemical Co. Citra Plus antigen retrieval
solution was purchased from BioGenex (San Ramon, CA). Automation buffer
was purchased from Biomeda. Fluorescein apoptosis detection system was
purchased from Promega.
Animals. All transgenic and wt mice were generated and maintained in the
Imperial Cancer Research Fund Animal Containment unit. Animals were kept
on a 12 h light/dark cycle and fed R20 rodent chow (Special Dietary Services,
Essex, United Kingdom) and water ad libidum. ␣2 (founder line 1070; 49
copies/cell), ␣3 (founder line 1120C; 20 copies/cell), and ␤1 (founder line
0840; 42 copies/cell) transgenic mouse lines previously on an F1 hybrid
(CBA ⫻ C57Bl/6) genetic background (20, 21) were backcrossed six generations onto a homogeneous C57Bl/6 background. ␣2␤1 and ␣3␤1 double
transgenic mice were then generated by crossing ␣2 and ␣3 single transgenics
to ␤1 single transgenics.
Tumor Studies. Female ␣2␤1 and ␣3␤1 integrin transgenic and wt litterReceived 1/8/01; accepted 4/25/01.
mate mice 7 weeks of age (25 animals/group) were shaved once on the dorsal
The costs of publication of this article were defrayed in part by the payment of page
surface with electric clippers. One week later all animals that did not show
charges. This article must therefore be hereby marked advertisement in accordance with
signs of hair regrowth received one topical application of 100 nmol (25 ␮g)
18 U.S.C. Section 1734 solely to indicate this fact.
1
This research was supported by the Imperial Cancer Research Fund and by National
DMBA in 200 ␮l of acetone or 200 ␮l of acetone alone. One week later, mice
Research Service Award CA75638 from the National Cancer Institute (to D. M. O.).
received thrice-weekly applications of 6 nmol (3.7 ␮g) TPA in 200 ␮l acetone
2
To whom requests for reprints should be addressed, at Imperial Cancer Research
or 200 ␮l acetone alone. C57Bl/6 mice have been shown previously to be
Fund, Keratinocyte Laboratory, 44 Lincoln’s Inn Fields, London WC2A 3PX, United
resistant to twice-weekly applications of TPA but do develop tumors with a
Kingdom.
3
The abbreviations used are: SCC, squamous cell carcinoma; DMBA, 7,12-dimeththrice-weekly protocol (22, 23). Benign and malignant skin tumors were
ylbenz[a]anthracene; TPA, 12-O-tetradecanoylphorbol-13-acetate; BrdUrd, bromoderecorded once a week for a period up to 52 weeks after the start of promotion.
oxyuridine; wt, wild-type; EMMPRIN, extracellular MMP inducer; TM4SF, transmemStudent’s t test was conducted to statistically compare papilloma and maligbrane-4 superfamily; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end
nancy formation between ␣2␤1 and ␣3␤1 transgenic mice and wt mice. The
labeling.
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SUPRABASAL ␣3␤1 EXPRESSION INHIBITS SCC FORMATION
experiment was repeated with a second group of animals, and the results
obtained in the first and second experiments were the same.
Short-Term TPA Treatment. Female ␣2␤1 and ␣3␤1 transgenic and wt
littermate mice 7 weeks of age (five animals/group) were shaved once on the
dorsal surface with electric clippers and 1 week later received either 1, 4, 8, or
12 applications of 6 nmol TPA in 200 ␮l acetone or 200 ␮l acetone alone at
a frequency of three applications per week. All skin sections were harvested
24 h after the final TPA treatment.
Tissue Harvesting. One h before sacrifice, all mice received a single i.p.
injection of 100 mg/kg BrdUrd. Skin tumors and dorsal skin sections were
harvested, and portions of each tumor or skin section were fixed in 10%
neutral-buffered formalin or frozen in liquid nitrogen-cooled isopentane after
embedding in OCT medium. All formalin-fixed tumor sections were stained
with H&E and graded as either a squamous papilloma or a SCC (24).
BrdUrd Labeling. To examine BrdUrd incorporation, formalin-fixed skin
and tumor sections were processed as described previously (25). Briefly, tissue
sections were deparaffinized and incubated in 2 M HCl for 30 min at 37°C,
dipped in borate buffer for 3 min, and then digested in 0.01% trypsin for 3 min
at 37°C. Sections were blocked in 10% normal goat serum for 20 min and then
probed with antibodies for BrdUrd (Becton Dickinson) for 1 h. Sections were
then incubated with a species-specific Alexa 488-conjugated (Molecular
Probes) secondary antibody, after which sections were counterstained with 1
␮g/ml propidium iodide. Fluorescence was observed using a Zeiss Axiophot
fluorescent microscope.
Immunofluorescence. For mouse keratin 10 staining, formalin-fixed tumor sections were deparaffinized and microwaved in Citra Plus antigen retrieval solution for 2 min 30 s and allowed to cool in the antigen retrieval
solution for an additional 15 min. Sections were blocked in 10% normal goat
serum for 20 min and then probed with antibodies for mouse keratin 10
(Babco) for 1 h. Sections were then incubated with a species-specific Alexa
488-conjugated (Molecular Probes) secondary antibody, and fluorescence was
observed using a Zeiss Axiophot fluorescent microscope.
Frozen skin and tumor sections were fixed in acetone at ⫺20°C for 10 min
prior to a 10% normal goat serum block and then probed with antibodies for
mouse ␣6 integrin (CD49f; Serotec), mouse ␤1 integrin (MB1.2; courtesy of
Bosco M. Chan, University of Western Ontario, London, Ontario, Canada;
Ref. 26), rat EMMPRIN (CE9; courtesy of James Bartles, Northwestern
University Medical School, Chicago, IL; Ref. 27), mouse CD81 (Eat2; courtesy of Shoshana Levy, Stanford University Medical Center, Stanford, CA;
Ref. 28), mouse involucrin (29), human ␣3 integrin (VM-2; Ref. 21), or human
␣2 integrin (HAS4; Ref. 20). Tissue sections were then incubated with a
species-specific Alexa 488-conjugated (Molecular Probes) secondary antibody
for ␣6 integrin, EMMPRIN, and ␤1 integrin, or species-specific Alexa 594conjugated (Molecular Probes) secondary antibodies for ␣2 and ␣3 integrin
and involucrin, or FITC-conjugated antihamster (PharMingen) for CD81. After
a 20 min secondary antibody incubation, sections were washed and mounted in
Gelvatol. Immunofluorescence was visualized using a Zeiss Axiophot fluorescent microscope.
TUNEL Assay. Mouse papilloma and skin sections were processed for
detection of apoptotic cells according to the manufacturer’s protocol. Deparaffinized formalin-fixed papilloma and skin sections were incubated in 0.85%
NaCl followed by PBS and then fixed in 4% paraformaldehyde, permeabilized
with 20 ␮g/ml Proteinase K for 10 min, and fixed again in 4% paraformaldehyde. Sections were incubated with terminal deoxynucleotidyl transferase in
the presence of fluorescein-12-dUTP at 37°C for 1 h. Sections were counterstained with 1 mg/ml propidium iodide and examined under a Zeiss Axiophot
fluorescent microscope.
RESULTS
Suprabasal Keratinocyte Proliferation in ␣2␤1 and ␣3␤1
Transgenic Mouse Skin. The previously reported sporadic hyperproliferative and inflammatory phenotype in ␣2␤1 and ␣3␤1 transgenic mouse skin is not observed in young animals either in the
original F1 background (CBA ⫻ C57Bl/6) or after backcrossing onto
a homogeneous C57Bl/6 background (20, 21). As shown in Fig. 1
panels A, C, and E, acetone vehicle-treated ␣2␤1 and ␣3␤1 transgenic
mouse skin displayed a similar labeling index to wt mouse skin, with
all BrdUrd-positive S-phase cells residing in the basal layer. However,
the transgenics showed a markedly different response to TPA from wt
mice. ␣2␤1 and ␣3␤1 transgenic and wt mouse skin was treated with
1, 4, 8, or 12 applications of 6 nmol TPA, and skin sections were
analyzed for BrdUrd incorporation. After only a single application of
TPA, before the onset of significant hyperplasia, both ␣2␤1 and ␣3␤1
transgenics exhibited BrdUrd incorporation in the suprabasal layers of
the epidermis (Fig. 1, D and F), whereas no BrdUrd-positive keratinocytes were observed in the suprabasal layers of wt epidermis
(Fig. 1B). The number of BrdUrd-positive keratinocytes in the basal
layer did not differ between ␣2␤1 and ␣3␤1 transgenic and wt mouse
skin after any of the TPA treatment regimens. The suprabasal BrdUrd
labeling in transgenic epidermis was also present after 4, 8, and 12
applications of TPA, whereas only basal BrdUrd incorporation was
observed in wt mice (data not shown).
␣2␤1 and ␣3␤1 Transgenic Mice and wt Mice Are Equally
Sensitive to Papilloma Formation. To determine whether the acute
phenotypic response to short-term TPA treatment observed in transgenic mouse skin (Fig. 1) could be extended to tumor formation, we
compared the responses of transgenic and wt mouse skin to chemical
carcinogenesis. ␣2␤1 and ␣3␤1 transgenic and wt littermate female
mice were initiated with a single dose of 100 nmol DMBA. One week
later, mice were treated with 6 nmol TPA three times/week for
25 weeks. As shown in Fig. 2A, papillomas first emerged in all groups
8 weeks after the start of TPA promotion, and maximum papilloma
responses reached a plateau by 20 weeks. No significant differences in
papilloma frequency were observed between ␣2␤1 (1.9 papillomas/
mouse) or ␣3␤1 transgenic mice (2.3 papillomas/mouse) and wt mice
(1.6 papillomas/mouse; Fig. 2A; P ⫽ 0.637 for ␣2␤1 versus wt;
P ⫽ 0.352 for ␣3␤1 versus wt). In addition, there was no difference
in the incidence of papillomas (percentage of mice with papillomas) in
␣2␤1 and ␣3␤1 transgenic mice and wt mice (Fig. 2B). No papillomas
were observed in transgenic or wt mice that were initiated with
DMBA and promoted with acetone vehicle or that were initiated with
acetone vehicle and promoted with TPA (data not shown). In a repeat
experiment, no significant differences in papilloma incidence or frequency were observed between ␣2␤1 (1.88 papillomas/mouse) or
␣3␤1 transgenic mice (2.13 papillomas/mouse) and wt mice (3.12
papillomas/mouse; P ⫽ 0.128 for ␣2␤1 versus wt; P ⫽ 0.164 for
␣3␤1 versus wt).
␣3␤1 Transgenic Mice Are Resistant to SCC Formation. To
determine whether aberrant integrin expression could influence malignant conversion, ␣2␤1 and ␣3␤1 transgenic and wt mice were
maintained under weekly observation for SCC formation for a period
of 52 weeks after the start of TPA promotion. As shown in Fig. 3A,
malignancies first emerged in wt mice 20 weeks after the start of
promotion, whereas the first malignancies did not arise in ␣2␤1 and
␣3␤1 transgenic mice until 26 and 27 weeks, respectively. No difference in the average number malignancies/mouse was observed between ␣2␤1 transgenic and wt mice (P ⫽ 0.583; Fig. 3A). However,
␣3␤1 transgenic mice developed 5– 6 fold fewer malignancies than wt
mice (P ⫽ 0.0005), and by 52 weeks, just three malignancies were
observed in the entire group (Fig. 3A). As shown in Fig. 3B, ␣3␤1
transgenic mice also displayed a profound decrease in SCC incidence
compared with ␣2␤1 transgenic and wt mice. H&E-stained histological sections from all malignancies were examined and graded to be
SCCs in all groups (24). Similar decreases in SCC formation in ␣3␤1
transgenic (0.30 malignancies/mouse; P ⫽ 0.020) but not in ␣2␤1
(0.61 malignancies/mouse; P ⫽ 0.231) transgenic mice compared
with wt (0.96 malignancies/mouse) mice were obtained in a repeat
experiment (data not shown).
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SUPRABASAL ␣3␤1 EXPRESSION INHIBITS SCC FORMATION
Fig. 1. BrdUrd labeling. Immunofluorescent detection of BrdUrd-positive S-phase cells in wt (A
and B), ␣2␤1 (C and D), and ␣3␤1 (E and F)
transgenic epidermis after a single treatment of
acetone (A, C, and E) or TPA (B, D, and F).
Formalin-fixed skin sections were stained with a
BrdUrd antibody (green) and then by a propidium
iodide counterstain (red). Inserts in B, D, and F
show higher magnification views (⫻6.5) of parts
of each section. Scale bar, 100 ␮m.
Phenotypic Characterization of ␣2␤1 and ␣3␤1 Transgenic
and wt Skin Tumors. Mouse skin papillomas and SCCs from every
tumor-bearing animal were subjected to histopathological examination (Fig. 4). Pedunculated and sessile papillomas were observed in all
groups of mice, with pedunculated papillomas predominating. The
extent of dysplasia and cellular atypia was similar in wt and ␣2␤1
papillomas, but somewhat lower in ␣3␤1 papillomas (Fig. 4, A, C, and
E and data not shown). All of the SCCs were well to moderately
differentiated (Fig. 4, B, D, and F), and tumors from wt and transgenic
mice could not be distinguished by histology.
Mouse skin papillomas categorized as at high risk for malignant
conversion have an increased number of suprabasal S-phase cells (12,
25) and reduced keratin 10 expression (30). To investigate whether the
reduced frequency of SCCs in ␣3␤1 transgenic mice was attributable
to a reduced proportion of high risk papillomas, we analyzed BrdUrd
incorporation and keratin 10 expression. Papillomas were examined
for basal and suprabasal BrdUrd incorporation. Suprabasal incorporation was scored according to whether it was observed in ⬍25% (⫹),
in 25–50% (⫹⫹), or in ⬎50% (⫹⫹⫹) of each papilloma section
(Table 1). Fig. 5A illustrates BrdUrd staining in an ␣3␤1 transgenic
papilloma graded as having incorporation confined to the basal layer,
and Fig. 5B shows strong suprabasal BrdUrd incorporation in a wt
papilloma graded as ⫹⫹⫹. As shown in Table 1, 65% of ␣3␤1
transgenic papillomas exhibited BrdUrd incorporation confined to the
basal layer, compared with 10% of ␣2␤1 papillomas and 29% of wt
papillomas. Just 4% of ␣3␤1 transgenic papillomas displayed supra-
basal staining in ⬎50% of the tumor section area (⫹⫹⫹), compared
with 30% of ␣2␤1 transgenic papillomas and 24% of wt papillomas
(Table 1). Thus, the ␣3␤1 transgenic papillomas had a diminished
capacity for suprabasal proliferation compared with wt and ␣2␤1
transgenic papillomas.
␣2␤1 and ␣3␤1 transgenic and wt papillomas were also examined
for expression of keratin 10. Keratin 10 is expressed in all of the
suprabasal layers of normal epidermis, and the loss of keratin 10
expression is a marker of malignant conversion (30). Papillomas were
graded according to whether they had no keratin 10 expression or
whether expression was detected in ⬍25% (⫹), 25–50% (⫹⫹), or
⬎50% (⫹⫹⫹) of the papilloma section area (Table 1). All ␣3␤1
transgenic papillomas exhibited some degree of suprabasal keratin 10
expression, and the majority had positive staining in ⬎50% of the
papilloma area (Table 1). In contrast, keratin 10 was completely
undetectable in 35% of wt papillomas and in 20% of ␣2␤1 transgenic
papillomas; and in those papillomas that did express keratin 10,
staining was mainly confined to ⬍50% of the tumor area.
Papillomas were also stained with antibodies to endogenous
(mouse) integrins (data not shown). Staining of endogenous ␤1 integrins was similar in ␣3␤1 transgenic papillomas and ␣2␤1 transgenic
and wt papillomas (data not shown). Suprabasal ␣6␤4 expression has
been associated with papillomas at high risk for malignant conversion
(12). Although variability in the amount of suprabasal ␣6␤4 expression within each group was observed, no differences in the expression
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SUPRABASAL ␣3␤1 EXPRESSION INHIBITS SCC FORMATION
membrane-localized staining for EMMPRIN, whereas suprabasal
cells had a more diffuse and cytosolic staining. In some papillomas,
intense membrane-localized staining was observed in the suprabasal
layers and this expression pattern correlated inversely with the degree
of differentiation. As shown in Fig. 5, F and G, the up-regulation of
EMMPRIN in the suprabasal layers correlated with a loss of involucrin expression. In the ␣3␤1 transgenic papilloma shown in Fig. 5, D
and E, the basal, transgene-negative layer stained intensely for
EMMPRIN, whereas the suprabasal, transgene-positive layers had
weaker, diffuse staining. In all papillomas and SCCs examined, increased EMMPRIN staining was associated with a loss of differentiation, and, as such, EMMPRIN may serve as a marker of progression
in mouse skin, as it does in human oral SCC (35). However, there
were no differences in EMMPRIN expression between ␣3␤1 transgenic papillomas and ␣2␤1 transgenic and wt papillomas beyond
those reflected in the overall degree of differentiation.
CD81 expression was examined in normal and TPA-treated ␣2␤1
and ␣3␤1 transgenic and wt mouse skin and in skin papillomas and
SCCs (Fig. 6 and Table 2). CD81 expression could not be detected by
immunofluorescence in frozen sections of normal wt or transgenic
epidermis (data not shown). However, CD81 was induced in the
suprabasal layers of TPA-treated ␣2␤1 and ␣3␤1 transgenic and wt
mouse skin (Fig. 6A and data not shown). As illustrated in Fig. 6 and
in Table 2, CD81 was constitutively up-regulated in the majority of wt
Fig. 2. Papilloma formation. Mouse skin papilloma frequency (A) and incidence (B) in
wt, ␣2␤1, and ␣3␤1 transgenic mice are shown. Female mice received a single topical
dose of DMBA (initiation). One week later, mice were promoted with topical applications
of 6 nmol TPA three times/week for 25 weeks.
profile of ␣6␤4 existed between the groups as a whole (data not
shown).
The decrease in SCC formation observed in ␣3␤1 transgenic mice
might be attributable to an increased tendency for cells in the papillomas to undergo apoptosis. To examine this possibility, ␣2␤1 and
␣3␤1 transgenic and wt papillomas were subjected to TUNEL labeling to visualize apoptotic cells. Heterogeneity existed in the numbers
of apoptotic cells between papillomas in the same group; however no
overall differences were observed in the numbers or in the location
(basal or suprabasal) of apoptotic cells between any of the papilloma
groups (data not shown).
Collectively these results suggest that the reason why ␣3␤1 transgenic mice show a reduced frequency of malignant conversion is that
they develop a lower frequency of high-risk papillomas than ␣2␤1
transgenic and wt mice.
␣3␤1 Transgenic Papillomas Exhibit Altered Expression of
CD81 but not EMMPRIN. A large number of proteins form complexes with integrins and several of these associate with ␣3␤1 but not
with ␣2␤1 (31). To see whether differences in the expression or
distribution of such proteins might account for the differences in the
malignant conversion of ␣2␤1 and ␣3␤1 transgenic papillomas, we
stained sections with antibodies to the TM4SF protein CD81 (32) and
to the immunoglobulin superfamily protein EMMPRIN (extracellular
MMP inducer; also known as CD147, basigin; Ref. 33). EMMPRIN
regulates the expression of MMPs in stromal fibroblasts (34) and is
thought to play a role in the progression of human oral SCC (35).
Ligation of CD81 and other ␣3␤1 associated TM4SF proteins stimulates tumor cell invasion by modulating the actin cytoskeleton and
stimulating production of MMP-2 (36).
As illustrated in Fig. 5C, basal keratinocytes in phenotypically
normal epidermis of wt and transgenic mice exhibited intense
Fig. 3. SCC formation. Frequency (A) and incidence (B) of malignancies in wt, ␣2␤1,
and ␣3␤1 transgenic mouse skin are shown. Female mice received a single topical dose
of 100 nmol DMBA (initiation). One week later, mice received topical applications of 6
nmol TPA three times/week for 25 weeks (promotion). Mice were observed for 52 weeks
after the start of promotion.
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SUPRABASAL ␣3␤1 EXPRESSION INHIBITS SCC FORMATION
Fig. 4. Histopathological analysis of wt, ␣2␤1, and ␣3␤1 transgenic papillomas and SCCs. Formalin-fixed tumor sections were stained with H&E. wt papilloma (A) and SCC
(moderately to well differentiated; B), ␣2␤1 papilloma (C) and SCC (moderately differentiated; D), and ␣3␤1 papilloma (E) and SCC (well differentiated; F). Scale bar, 100 ␮m.
and ␣2␤1 transgenic papillomas (Fig. 6, B and C), but most ␣3␤1
transgenic papillomas were CD81-negative (Fig. 6D and Table 2). In
contrast, CD81 up-regulation was detected in 100% of ␣2␤1 and
␣3␤1 transgenic and wt SCCs (Table 2). These results indicate that
constitutive up-regulation of CD81 is associated with tumor progression and suggest that the absence of CD81 observed exclusively in
␣3␤1 transgenic papillomas may play a role in the suppression of
SCC formation in ␣3␤1 transgenic mouse skin.
DISCUSSION
In this study we examined the sensitivity of transgenic mice expressing suprabasal ␣2␤1 and ␣3␤1 integrins to chemically induced
skin carcinogenesis. ␣2␤1 and ␣3␤1 transgenic and wt mice were
equally sensitive to DMBA/TPA-induced papilloma formation. Wt
and ␣2␤1 transgenic mice exhibited similar frequencies of malignant
conversion of papillomas to SCCs. However, ␣3␤1 integrin-transgenic papillomas were resistant to SCC development.
Previously, it has been unclear why the hyperproliferative and
inflammatory phenotype of ␣2␤1 and ␣3␤1 transgenics is sporadic,
although the transgenes are constitutively expressed in the suprabasal
epidermal layers (20, 21). We found that suprabasal proliferation, as
measured by BrdUrd incorporation, was strongly induced by a single
dose of TPA in the transgenics, whereas it was not induced in wt mice.
This suggests that the transgenes may act by increasing the sensitivity
of keratinocytes to external proliferative or inflammatory stimuli.
Although the presence of suprabasal S-phase cells in papillomas is
Table 1 Phenotypic characterization of wt, ␣2␤1, and ␣3␤1 transgenic papillomas
Formalin-fixed ␣2␤1 and ␣3␤1 transgenic and wt papilloma tissue sections were
probed with BrdUrd and keratin 10 antibodies, and positive staining was visualized by
fluorochrome detection.
BrdUrda
wt papillomas (n ⫽ 17)
␣2␤1 papillomas (n ⫽ 10)
␣3␤1 papillomas (n ⫽ 23)
Keratin 10a
Basal
⫹
⫹⫹
⫹⫹⫹
None
⫹
⫹⫹
⫹⫹⫹
29
10
65
23
20
22
24
40
9
24
30
4
35
20
0
47
30
9
12
30
35
6
20
56
a
The percentage of papillomas in each category is shown. Suprabasal BrdUrd labeling
and keratin 10 expression is as follows: ⫹, 0 –25% papilloma; ⫹⫹, 25–50% papilloma;
⫹⫹⫹, ⬎50% papilloma.
indicative of a high risk of malignant conversion (12, 25), the suprabasal proliferation induced in normal epidermis by one dose of TPA
did not correlate with an increased frequency of papilloma formation.
Thus, suprabasal S-phase cells are more indicative of tumor progression than promotion.
One potential explanation for the low frequency of SCCs in ␣3␤1
transgenics would be that ␣3␤1 papillomas had a tendency to regress as
a result of apoptosis. Induction of apoptosis through the loss of attachment to the extracellular matrix (anoikis) is mediated by integrins (37,
38), and resistance to anoikis is seen as a hallmark of malignant conversion in epithelial cell types (37). However, normal keratinocytes do not
undergo anoikis (39), and suprabasal integrin expression does not lead to
increased apoptosis in the epidermis of the integrin transgenic mice (20).
Furthermore, there were no differences in the total number or distribution
(basal or suprabasal) of TUNEL-positive keratinocytes in ␣3␤1 transgenic papillomas compared with ␣2␤1 transgenic and wt papillomas.
Thus apoptosis does not play a role in the failure of ␣3␤1 transgenic
papillomas to undergo malignant conversion.
The progression of papillomas to SCC has been characterized
phenotypically by inappropriate expression of certain growth factors,
cyclins/cyclin-dependent kinases, and cell surface receptors for
growth factors and adhesion molecules (40, 41). It is interesting that
many of these gene products are known to interact with or to be
regulated by integrins. Examples include gap junction proteins such as
connexin 43 (42), cyclin D1 (43), transforming growth factor ␤ (44),
and components of the Ras signaling pathway (45). However, with the
exception of the link between ␣3␤1 and connexin 43 (42), there is no
evidence for a differential interaction between any of these molecules
and ␣3␤1 or ␣2␤1.
The low frequency of ␣3␤1 transgenic SCCs was correlated with a
tendency of ␣3␤1 papillomas to be well differentiated (keratin 10positive) with a low incidence of suprabasal BrdUrd incorporation.
The explanation for the difference in malignant conversion of ␣2␤1
and ␣3␤1 papillomas may lie with the integrins themselves or with
proteins that specifically associate with them. In addition to differences in the ligand specificities of ␣2␤1 (collagen) and ␣3␤1 (laminin), ␣3␤1 has the distinct properties of being a transdominant inhibitor of other integrins and a suppresser of stress fiber formation (46).
However, the suprabasal integrins in the transgenic mice do not have
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SUPRABASAL ␣3␤1 EXPRESSION INHIBITS SCC FORMATION
Fig. 5. Immunofluorescent detection of BrdUrd incorporation and EMMPRIN. BrdUrd labeling of ␣3␤1 transgenic (A) and wt (B) papillomas. BL, basal layer (arrows); SL,
suprabasal layers. Untreated wt mouse skin (C), ␣3␤1 papilloma (D and E), and wt papilloma (F and G) stained with antibodies to EMMPRIN (C, D, and F), human ␣3␤1 (E) or
involucrin (G). D and E, the same section has been double-labeled. F and G, serial sections. In F, ---- demarcates a region of intense EMMPRIN staining, which corresponds to the
involucrin-negative area in G. Scale bar, 50 ␮m.
associated extracellular matrix ligands (20), and in the present experiments the presence of the transgenes did not affect expression of the
endogenous mouse integrins. Another possibility is that suprabasal
integrins influence tumorigenesis through a paracrine effect on the
underlying transgene-negative basal cells (21), and that the nature of
these signals differs between cells expressing ␣2␤1 and ␣3␤1.
Of the two ␣3␤1-associated proteins we examined, EMMPRIN was
down-regulated in differentiating cells of normal epidermis, papillomas, and SCCs; but there was no evidence that EMMPRIN distribution differed between ␣2␤1 and ␣3␤1 transgenics. Potentially more
significant was the observation that, whereas CD81 was absent in
normal epidermis and in the majority of ␣3␤1 papillomas, it was
strongly up-regulated in ␣2␤1 and wt papillomas and in all transgenic
and wt SCCs. Inasmuch as ␣3␤1 integrin-tetraspan complex activation has been shown to induce MMP secretion and tumor cell migration (36), and MMPs are invariably up-regulated in epithelial cancers
(47), the absence of CD81 in the ␣3␤1 papillomas could explain why
they only convert to SCCs at low frequency. What is unclear at
present is whether the ␣3␤1 transgene contributes directly to the
regulation of CD81 expression and, if so, by what mechanism.
Altered integrin expression has been extensively documented in
human SCC (11, 15–17), and some changes are useful prognostic
indicators (11). However, because tumor progression requires multiple genetic and epigenetic changes, the sequential order or timing of
Fig. 6. Immunofluorescent detection of CD81.
A, ␣2␤1 transgenic epidermis after 8 applications
of 6 nmol TPA. B–D: papillomas from wt (C),
␣2␤1 (B) and ␣3␤1 (D) transgenic mice. BL, basal
layer (arrows); and SL, suprabasal layer. ---- in D,
boundary between SL and BL. Scale bar: 50 ␮m.
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SUPRABASAL ␣3␤1 EXPRESSION INHIBITS SCC FORMATION
Table 2 Incidence of CD81 expression in papillomas and SCCs
Acetone-fixed ␣2␤1 and ␣3␤1 transgenic and wt papilloma and SCC frozen sections
were probed with an antimouse CD81 antibody, and positive staining was visualized by
FITC detection.
% CD81 positive tumors
wt
␣2␤1 transgenic
␣3␤1 transgenic
Papilloma
SCC
100 (7/7)
88 (7/8)
25 (2/8)
100 (7/7)
100 (3/3)
100 (6/6)
alterations in integrin expression may be critical, and early changes in
integrin expression may have more impact on the course of the disease
than the changes that characterize a mature tumor. The value of our
transgenic mouse model is that it can be used to discover how early
changes in integrin expression affect later stages in the disease.
ACKNOWLEDGMENTS
We thank Robert Rudling, Barbara Cross, and Toni Hill for their expert
technical assistance. We are grateful to James Bartles for providing the CE9
antibody, to Shoshana Levy for the Eat2 antibody, and to Bosco Chan for the
MB1.2 antibody.
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Influence of β1 Integrins on Epidermal Squamous Cell
Carcinoma Formation in a Transgenic Mouse Model: α3β1, but
not α2β1, Suppresses Malignant Conversion
David M. Owens and Fiona M. Watt
Cancer Res 2001;61:5248-5254.
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