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[Cancer Research 61, 5248-5254, July 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Influence of ß1 Integrins on Epidermal Squamous Cell Carcinoma Formation in a Transgenic Mouse Model

{alpha}3ß1, but not {alpha}2ß1, Suppresses Malignant Conversion1

David M. Owens and Fiona M. Watt2

Imperial Cancer Research Fund, Keratinocyte Laboratory, London WC2A 3PX, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}2ß1 or {alpha}3ß1 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 {alpha}3ß1 transgenic than in {alpha}2ß1 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, {alpha}3ß1 transgenic papillomas displayed a diminished proliferative capacity and were more highly differentiated as judged by BrdUrd incorporation and keratin 10 expression, respectively, than {alpha}2ß1 transgenic and wild-type papillomas. Two proteins that associate with {alpha}3ß1 and not {alpha}2ß1 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 {alpha}2ß1 transgenic papillomas but in only 25% of {alpha}3ß1 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 {alpha}3ß1 integrin can suppress malignant conversion, and that the mechanism may involve CD81.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}2ß1, {alpha}3ß1, and {alpha}6ß4 (3) . In addition to mediating keratinocyte adhesion to extracellular matrix proteins, ß1 integrins regulate epidermal proliferation and terminal differentiation (4, 5, 6, 7, 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 {alpha}6ß4, as well as increased basal {alpha}6ß4 expression, is associated with tumor progression and invasion (11, 12, 13) . Highly undifferentiated spindle cell carcinomas exhibit up-regulated expression of the {alpha}5ß1 integrin (14) . Increased expression of {alpha}2ß1 and {alpha}3ß1 integrin has been observed in basal cell carcinomas (3 , 15) . Complete or focal loss of expression of {alpha}2ß1, {alpha}3ß1, and {alpha}6ß4 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 {alpha}2ß1 or {alpha}3ß1 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 {alpha}2ß1 or {alpha}3ß1 to a chemical carcinogenesis protocol that allows evaluation of the development of both benign (papillomas) and malignant (SCC) tumors (22 , 23) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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. {alpha}2 (founder line 1070; 49 copies/cell), {alpha}3 (founder line 1120C; 20 copies/cell), and ß1 (founder line 0840; 42 copies/cell) transgenic mouse lines previously on an F1 hybrid (CBA x C57Bl/6) genetic background (20 , 21) were backcrossed six generations onto a homogeneous C57Bl/6 background. {alpha}2ß1 and {alpha}3ß1 double transgenic mice were then generated by crossing {alpha}2 and {alpha}3 single transgenics to ß1 single transgenics.

Tumor Studies.
Female {alpha}2ß1 and {alpha}3ß1 integrin transgenic and wt littermate mice 7 weeks of age (25 animals/group) were shaved once on the dorsal surface with electric clippers. One week later all animals that did not show signs of hair regrowth received one topical application of 100 nmol (25 µg) DMBA in 200 µl of acetone or 200 µl of acetone alone. One week later, mice received thrice-weekly applications of 6 nmol (3.7 µg) TPA in 200 µl acetone or 200 µl acetone alone. C57Bl/6 mice have been shown previously to be resistant to twice-weekly applications of TPA but do develop tumors with a thrice-weekly protocol (22 , 23) . Benign and malignant skin tumors were recorded once a week for a period up to 52 weeks after the start of promotion. Student’s t test was conducted to statistically compare papilloma and malignancy formation between {alpha}2ß1 and {alpha}3ß1 transgenic mice and wt mice. The 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 {alpha}2ß1 and {alpha}3ß1 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 µl acetone or 200 µl 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 µg/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 {alpha}6 integrin (CD49f; Serotec), mouse ß1 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 {alpha}3 integrin (VM-2; Ref. 21 ), or human {alpha}2 integrin (HAS4; Ref. 20 ). Tissue sections were then incubated with a species-specific Alexa 488-conjugated (Molecular Probes) secondary antibody for {alpha}6 integrin, EMMPRIN, and ß1 integrin, or species-specific Alexa 594-conjugated (Molecular Probes) secondary antibodies for {alpha}2 and {alpha}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 µg/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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Suprabasal Keratinocyte Proliferation in {alpha}2ß1 and {alpha}3ß1 Transgenic Mouse Skin.
The previously reported sporadic hyperproliferative and inflammatory phenotype in {alpha}2ß1 and {alpha}3ß1 transgenic mouse skin is not observed in young animals either in the original F1 background (CBA x C57Bl/6) or after backcrossing onto a homogeneous C57Bl/6 background (20 , 21) . As shown in Fig. 1Citation panels A, C, and E, acetone vehicle-treated {alpha}2ß1 and {alpha}3ß1 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. {alpha}2ß1 and {alpha}3ß1 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 {alpha}2ß1 and {alpha}3ß1 transgenics exhibited BrdUrd incorporation in the suprabasal layers of the epidermis (Fig. 1, D and F)Citation , whereas no BrdUrd-positive keratinocytes were observed in the suprabasal layers of wt epidermis (Fig. 1B)Citation . The number of BrdUrd-positive keratinocytes in the basal layer did not differ between {alpha}2ß1 and {alpha}3ß1 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).



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  		Fig. 1. BrdUrd labeling. Immunofluorescent detection of BrdUrd-positive S-phase cells in wt (A and B), {alpha}2ß1 (C and D), and {alpha}3ß1 (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 (x6.5) of parts of each section. Scale bar, 100 µm.

 
{alpha}2ß1 and {alpha}3ß1 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)Citation could be extended to tumor formation, we compared the responses of transgenic and wt mouse skin to chemical carcinogenesis. {alpha}2ß1 and {alpha}3ß1 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. 2ACitation , 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 {alpha}2ß1 (1.9 papillomas/mouse) or {alpha}3ß1 transgenic mice (2.3 papillomas/mouse) and wt mice (1.6 papillomas/mouse; Fig. 2ACitation ; P = 0.637 for {alpha}2ß1 versus wt; P = 0.352 for {alpha}3ß1 versus wt). In addition, there was no difference in the incidence of papillomas (percentage of mice with papillomas) in {alpha}2ß1 and {alpha}3ß1 transgenic mice and wt mice (Fig. 2B)Citation . 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 {alpha}2ß1 (1.88 papillomas/mouse) or {alpha}3ß1 transgenic mice (2.13 papillomas/mouse) and wt mice (3.12 papillomas/mouse; P = 0.128 for {alpha}2ß1 versus wt; P = 0.164 for {alpha}3ß1 versus wt).



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  		Fig. 2. Papilloma formation. Mouse skin papilloma frequency (A) and incidence (B) in wt, {alpha}2ß1, and {alpha}3ß1 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.

 
{alpha}3ß1 Transgenic Mice Are Resistant to SCC Formation.
To determine whether aberrant integrin expression could influence malignant conversion, {alpha}2ß1 and {alpha}3ß1 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. 3ACitation , malignancies first emerged in wt mice 20 weeks after the start of promotion, whereas the first malignancies did not arise in {alpha}2ß1 and {alpha}3ß1 transgenic mice until 26 and 27 weeks, respectively. No difference in the average number malignancies/mouse was observed between {alpha}2ß1 transgenic and wt mice (P = 0.583; Fig. 3ACitation ). However, {alpha}3ß1 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)Citation . As shown in Fig. 3BCitation , {alpha}3ß1 transgenic mice also displayed a profound decrease in SCC incidence compared with {alpha}2ß1 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 {alpha}3ß1 transgenic (0.30 malignancies/mouse; P = 0.020) but not in {alpha}2ß1 (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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  		Fig. 3. SCC formation. Frequency (A) and incidence (B) of malignancies in wt, {alpha}2ß1, and {alpha}3ß1 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.

 
Phenotypic Characterization of {alpha}2ß1 and {alpha}3ß1 Transgenic and wt Skin Tumors.
Mouse skin papillomas and SCCs from every tumor-bearing animal were subjected to histopathological examination (Fig. 4)Citation . 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 {alpha}2ß1 papillomas, but somewhat lower in {alpha}3ß1 papillomas (Fig. 4, A, C, and ECitation and data not shown). All of the SCCs were well to moderately differentiated (Fig. 4, B, D, and F)Citation , and tumors from wt and transgenic mice could not be distinguished by histology.



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  		Fig. 4. Histopathological analysis of wt, {alpha}2ß1, and {alpha}3ß1 transgenic papillomas and SCCs. Formalin-fixed tumor sections were stained with H&E. wt papilloma (A) and SCC (moderately to well differentiated; B), {alpha}2ß1 papilloma (C) and SCC (moderately differentiated; D), and {alpha}3ß1 papilloma (E) and SCC (well differentiated; F). Scale bar, 100 µm.

 
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 {alpha}3ß1 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)Citation . Fig. 5ACitation illustrates BrdUrd staining in an {alpha}3ß1 transgenic papilloma graded as having incorporation confined to the basal layer, and Fig. 5BCitation shows strong suprabasal BrdUrd incorporation in a wt papilloma graded as +++. As shown in Table 1Citation , 65% of {alpha}3ß1 transgenic papillomas exhibited BrdUrd incorporation confined to the basal layer, compared with 10% of {alpha}2ß1 papillomas and 29% of wt papillomas. Just 4% of {alpha}3ß1 transgenic papillomas displayed suprabasal staining in >50% of the tumor section area (+++), compared with 30% of {alpha}2ß1 transgenic papillomas and 24% of wt papillomas (Table 1)Citation . Thus, the {alpha}3ß1 transgenic papillomas had a diminished capacity for suprabasal proliferation compared with wt and {alpha}2ß1 transgenic papillomas.


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  		Table 1 Phenotypic characterization of wt, {alpha}2ß1, and {alpha}3ß1 transgenic papillomas

 


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  		Fig. 5. Immunofluorescent detection of BrdUrd incorporation and EMMPRIN. BrdUrd labeling of {alpha}3ß1 transgenic (A) and wt (B) papillomas. BL, basal layer (arrows); SL, suprabasal layers. Untreated wt mouse skin (C), {alpha}3ß1 papilloma (D and E), and wt papilloma (F and G) stained with antibodies to EMMPRIN (C, D, and F), human {alpha}3ß1 (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 µm.

 
{alpha}2ß1 and {alpha}3ß1 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)Citation . All {alpha}3ß1 transgenic papillomas exhibited some degree of suprabasal keratin 10 expression, and the majority had positive staining in >50% of the papilloma area (Table 1)Citation . In contrast, keratin 10 was completely undetectable in 35% of wt papillomas and in 20% of {alpha}2ß1 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 ß1 integrins was similar in {alpha}3ß1 transgenic papillomas and {alpha}2ß1 transgenic and wt papillomas (data not shown). Suprabasal {alpha}6ß4 expression has been associated with papillomas at high risk for malignant conversion (12) . Although variability in the amount of suprabasal {alpha}6ß4 expression within each group was observed, no differences in the expression profile of {alpha}6ß4 existed between the groups as a whole (data not shown).

The decrease in SCC formation observed in {alpha}3ß1 transgenic mice might be attributable to an increased tendency for cells in the papillomas to undergo apoptosis. To examine this possibility, {alpha}2ß1 and {alpha}3ß1 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 {alpha}3ß1 transgenic mice show a reduced frequency of malignant conversion is that they develop a lower frequency of high-risk papillomas than {alpha}2ß1 transgenic and wt mice.

{alpha}3ß1 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 {alpha}3ß1 but not with {alpha}2ß1 (31) . To see whether differences in the expression or distribution of such proteins might account for the differences in the malignant conversion of {alpha}2ß1 and {alpha}3ß1 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 {alpha}3ß1 associated TM4SF proteins stimulates tumor cell invasion by modulating the actin cytoskeleton and stimulating production of MMP-2 (36) .

As illustrated in Fig. 5CCitation , basal keratinocytes in phenotypically normal epidermis of wt and transgenic mice exhibited intense 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 GCitation , the up-regulation of EMMPRIN in the suprabasal layers correlated with a loss of involucrin expression. In the {alpha}3ß1 transgenic papilloma shown in Fig. 5, D and ECitation , 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 {alpha}3ß1 transgenic papillomas and {alpha}2ß1 transgenic and wt papillomas beyond those reflected in the overall degree of differentiation.

CD81 expression was examined in normal and TPA-treated {alpha}2ß1 and {alpha}3ß1 transgenic and wt mouse skin and in skin papillomas and SCCs (Fig. 6Citation and Table 2Citation ). 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 {alpha}2ß1 and {alpha}3ß1 transgenic and wt mouse skin (Fig. 6ACitation and data not shown). As illustrated in Fig. 6Citation and in Table 2Citation , CD81 was constitutively up-regulated in the majority of wt and {alpha}2ß1 transgenic papillomas (Fig. 6, B and C)Citation , but most {alpha}3ß1 transgenic papillomas were CD81-negative (Fig. 6DCitation and Table 2Citation ). In contrast, CD81 up-regulation was detected in 100% of {alpha}2ß1 and {alpha}3ß1 transgenic and wt SCCs (Table 2)Citation . These results indicate that constitutive up-regulation of CD81 is associated with tumor progression and suggest that the absence of CD81 observed exclusively in {alpha}3ß1 transgenic papillomas may play a role in the suppression of SCC formation in {alpha}3ß1 transgenic mouse skin.



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  		Fig. 6. Immunofluorescent detection of CD81. A, {alpha}2ß1 transgenic epidermis after 8 applications of 6 nmol TPA. B–D: papillomas from wt (C), {alpha}2ß1 (B) and {alpha}3ß1 (D) transgenic mice. BL, basal layer (arrows); and SL, suprabasal layer. ---- in D, boundary between SL and BL. Scale bar: 50 µm.

 

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  		Table 2 Incidence of CD81 expression in papillomas and SCCs

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we examined the sensitivity of transgenic mice expressing suprabasal {alpha}2ß1 and {alpha}3ß1 integrins to chemically induced skin carcinogenesis. {alpha}2ß1 and {alpha}3ß1 transgenic and wt mice were equally sensitive to DMBA/TPA-induced papilloma formation. Wt and {alpha}2ß1 transgenic mice exhibited similar frequencies of malignant conversion of papillomas to SCCs. However, {alpha}3ß1 integrin-transgenic papillomas were resistant to SCC development.

Previously, it has been unclear why the hyperproliferative and inflammatory phenotype of {alpha}2ß1 and {alpha}3ß1 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 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 {alpha}3ß1 transgenics would be that {alpha}3ß1 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 {alpha}3ß1 transgenic papillomas compared with {alpha}2ß1 transgenic and wt papillomas. Thus apoptosis does not play a role in the failure of {alpha}3ß1 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 ß (44) , and components of the Ras signaling pathway (45) . However, with the exception of the link between {alpha}3ß1 and connexin 43 (42) , there is no evidence for a differential interaction between any of these molecules and {alpha}3ß1 or {alpha}2ß1.

The low frequency of {alpha}3ß1 transgenic SCCs was correlated with a tendency of {alpha}3ß1 papillomas to be well differentiated (keratin 10-positive) with a low incidence of suprabasal BrdUrd incorporation. The explanation for the difference in malignant conversion of {alpha}2ß1 and {alpha}3ß1 papillomas may lie with the integrins themselves or with proteins that specifically associate with them. In addition to differences in the ligand specificities of {alpha}2ß1 (collagen) and {alpha}3ß1 (laminin), {alpha}3ß1 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 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 {alpha}2ß1 and {alpha}3ß1.

Of the two {alpha}3ß1-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 {alpha}2ß1 and {alpha}3ß1 transgenics. Potentially more significant was the observation that, whereas CD81 was absent in normal epidermis and in the majority of {alpha}3ß1 papillomas, it was strongly up-regulated in {alpha}2ß1 and wt papillomas and in all transgenic and wt SCCs. Inasmuch as {alpha}3ß1 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 {alpha}3ß1 papillomas could explain why they only convert to SCCs at low frequency. What is unclear at present is whether the {alpha}3ß1 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, 16, 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 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.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 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 Research Service Award CA75638 from the National Cancer Institute (to D. M. O.). Back

2 To whom requests for reprints should be addressed, at Imperial Cancer Research Fund, Keratinocyte Laboratory, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom. Back

3 The abbreviations used are: SCC, squamous cell carcinoma; DMBA, 7,12-dimethylbenz[a]anthracene; TPA, 12-O-tetradecanoylphorbol-13-acetate; BrdUrd, bromodeoxyuridine; wt, wild-type; EMMPRIN, extracellular MMP inducer; TM4SF, transmembrane-4 superfamily; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling. Back

Received 1/ 8/01. Accepted 4/25/01.


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Top
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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