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[Cancer Research 59, 2861-2868, June 1, 1999]
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[Cancer Research 59, 2861-2868, June 15, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Blocking Transforming Growth Factor ß Signaling in Transgenic Epidermis Accelerates Chemical Carcinogenesis

A Mechanism Associated with Increased Angiogenesis1

Cindy Go, Ping Li and Xiao-Jing Wang2

Departments of Otolaryngology [C. G.], Cell Biology [P. L., X-J. W.], and Dermatology [X-J. W.], Baylor College of Medicine, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the transforming growth factor ß type II receptor (TGF-ßRII) have been identified in human cancers, which suggests a causal role for the loss of TGF-ßRII in cancer development. To directly test this in vivo, we have generated transgenic mice expressing a dominant negative TGF-ßRII ({Delta}ßRII) in the epidermis, using a truncated mouse loricrin promoter (ML). ML.{Delta}ßRII transgenic mice exhibited a thickened skin due to epidermal hyperproliferation. When these mice were subjected to a standard two-stage chemical carcinogenesis protocol, they exhibited an increased sensitivity, with an earlier appearance and a 2-fold greater number of papillomas than control mice. In addition, papillomas in control mice regressed after termination of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment; whereas ML.{Delta}ßRII papillomas progressed to carcinomas. Furthermore, TPA promotion alone induced papilloma formation in ML.{Delta}ßRII mice, which suggests an initiating role for {Delta}ßRII in skin carcinogenesis. ML.{Delta}ßRII tumors also exhibited increased neovascularization and progressed to metastases, although the primary tumors were still classified as carcinoma in situ or well-differentiated carcinomas. Increased expression of vascular endothelial growth factor, an angiogenesis factor, and decreased expression of thrombospondin-1, an angiogenesis inhibitor, were also observed in ML.{Delta}ßRII tumors. The increased angiogenesis correlated with elevated endogenous TGF-ß1 in ML.{Delta}ßRII tumors. These data provide in vivo evidence that inactivation of TGF-ßRII accelerates skin carcinogenesis at both earlier and later stages, and increased angiogenesis is one of the important mechanisms of accelerated tumor growth and metastasis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The TGF-ß3 family has multiple functions in regulating cell proliferation and differentiation, tissue remodeling and repair, and immunomodulation (for reviews, see Refs. 1 and 2 ). In epithelial tissues, TGF-ß is a potent growth inhibitor, playing an important role in the maintenance of tissue homeostasis (3 , 4) . Because malignant epithelial tumors develop mechanisms of escaping from TGF-ß-induced growth inhibition (5, 6, 7) , it is believed that TGF-ß plays an important role in preventing cancer cell growth at earlier stages. In agreement with this, Glick et al. (8) reported that skin tumors devoid of TGF-ß1 are associated with a high risk for malignant conversion. They also reported that grafts of v-rasHa-initiated TGF-ß1-null keratinocytes progressed much more rapidly to SCCs than did wild-type TGF-ß1 keratinocytes initiated with v-rasHa (9) . Consistent with these observations, transgenic mice coexpressing both TGF-ß1 and TGF-{alpha} in mammary glands exhibited a reduced rate of spontaneous or chemically induced carcinomas, as compared with transgenic littermates, which only expressed TGF-{alpha} (10) . Tang et al. (11) recently reported that mice hemizygous for the TGF-ß1 gene showed accelerated chemical carcinogenesis in the liver and lung, indicating that TGF-ß1 is a tumor suppressor with haploid-insufficiency. Conversely, many reports documented high levels of TGF-ß in malignant tumors and metastases both in clinical specimens as well as those induced experimentally in animal models (5, 6, 7 , 12, 13, 14) , thus suggesting a direct promoting role for TGF-ß in late-stage carcinogenesis.

If the role of TGF-ß is switched from a tumor suppressor to a tumor promoter, this must occur after cells have lost their responsiveness to TGF-ß-induced growth inhibition. Because TGF-ß ligand is present at both early and late stages of carcinogenesis, it is possible that this role reversal occurs because of altered functions of TGF-ß receptors. Although the type I and II TGF-ß receptors (TGF-ßRI and TGF-ßRII) form tetra-heteromers for TGF-ß signaling, TGF-ßRII is considered to be the primary receptor (15) . Upon ligand binding, the intrinsic serine/threonine kinase of TGF-ßRII phosphorylates TGF-ßRI to activate its serine/threonine kinase activity (16 , 17) , and this activated receptor complex transduces TGF-ß signals (18) . Deletion of the serine/threonine kinase domain of TGF-ßRII produces a dominant negative form ({Delta}ßRII), which is able to block the growth inhibitory function of TGF-ß in vitro (19 , 20) and in transgenic animals (21, 22, 23) , which suggests that TGF-ßRII is required to mediate the growth inhibitory effect of TGF-ß. Mutations in TGF-ßRII were initially detected in TGF-ß-resistant cancer cell lines (24, 25, 26) . Similar mutations have then been reported in primary cancers of the colon (25 , 27, 28, 29, 30) , head and neck (31) , ampulla (32) , and pancreas (33) . Recently, germ-line mutations of TGF-ßRII have been identified in familial colorectal cancer (29) . These data suggest that inactivation of TGF-ßRII may be one mechanism by which epithelial tumor cells escape from TGF-ß-induced growth inhibition and progress to malignancy. To test this hypothesis in the skin, we have generated transgenic mice expressing {Delta}ßRII in the epidermis, using a truncated mouse loricrin expression vector (ML.{Delta}ßRII). ML.{Delta}ßRII mice exhibit marked hyperplasia/hyperkeratosis at birth, which suggests that the {Delta}ßRII can block TGF-ß-mediated growth inhibition in the epidermis (21) . This is further supported by the fact that primary keratinocytes isolated from the epidermis of ML.{Delta}ßRII transgenic mice were resistant to exogenous TGF-ß1-induced growth inhibition (21) . However, only a few ML.{Delta}ßRII mice have developed spontaneous papillomas, which suggests that other genetic/epigenetic events are required for the development of overt lesions.

In the present study, we have subjected ML.{Delta}ßRII mice to a chemical carcinogenesis protocol and observed an increased susceptibility compared with nontransgenic mice, with greatly accelerated benign papilloma formation, malignant conversion, and metastasis. We also provide evidence that the accelerated chemical carcinogenesis observed in mice expressing the {Delta}ßRII in the epidermis is associated with increased angiogenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemical Carcinogenesis Protocols.
Ten-week-old ML.{Delta}ßRII heterozygous mice and their nontransgenic littermates (ICR) were divided into groups for treatments with DMBA/TPA, DMBA, and TPA, respectively. The back skin of each mouse was carefully shaved two days before the treatment. Mice in the TPA group received a topical application of 5 µg of TPA (dissolved in 50 µl of acetone) once a week for 60 weeks. Mice in the DMBA group received one treatment of DMBA with either 50 µg or 250 µg (dissolved in 100 µl acetone). Mice in the DMBA/TPA group were treated with a subcarcinogenic dose of DMBA (50 µg), followed by TPA promotion beginning one week after DMBA initiation, i.e., 5 µg of TPA was applied to the initiated area of the skin once a week for 20 weeks.

Tissue Histology and Immunofluorescence.
Biopsied tumors were fixed in 10% neutral-buffered formalin at 4°C overnight, embedded in paraffin, sectioned to 6-µm thickness, and stained with H&E. Immunofluorescence analysis for keratin 13 (K13) was performed on frozen sections as described previously (34) . Vascularization was visualized by immunofluorescence analysis using a rat antimouse CD31 (PECAM-1) antibody (PharMingen), and a rabbit antimouse keratin 14 (K14) antibody. Frozen sections were washed with PBS twice for 10 min, fixed in cold acetone for 10 min, and rinsed with water. The CD31 antibody (5 µg/ml) and the K14 antibody (1:500), diluted in 12% BSA/PBS (w/v), were applied to the sections at room temperature for overnight. Sections were then washed as above and a secondary antibody, biotinylated goat antirat IgG (1:100, PharMingen) was applied for 1 h at room temperature. Tissue sections were washed again and a 1:400 dilution Streptavidin-Texas Red (Life Technologies, Inc.) and a 1:40 dilution of FITC-labeled, antirabbit IgG (Dakopatts) antibody was applied for 30 min at room temperature. Sections were then washed, air dried, coverslipped and photographed.

Microvessel Counting.
Microvessel counting was performed as described by Bolontrade et al. (35) . Briefly, each stained section was screened with a x10 eyepiece and x10 objective magnification to identify the areas of highest vascularization. The number of vessels in each tumor were counted and averaged in five areas of highest vascular density with a x10 eyepiece and x20 objective magnification (defined as the area unit). The percentage stromal area covered by vessels was determined in the same fields screened for vessel counts by computer analysis. Five to seven tumors were counted in each group. The number of vessels/area unit, as well as the percentage of stromal area covered by vessels is expressed as mean ± SD.

RPAs.
Total RNA was isolated from chemically induced tumors with RNAzol B (Tel-Test, Inc.) as described previously (34) . RPAs were performed using the RPA II kit (Ambion Inc., Austin, TX) and 32P-riboprobes. ML.{Delta}ßRII transgene expression was detected using a riboprobe specific for the {Delta}ßRII (21) . The riboprobe template of murine VEGF was prepared from the VEGF cDNA clone (provided by Dr. Georg Breier, Max-Plank Institute, Bad Nauheim, Germany), and linearized at the ClaI site within the vector, which is upstream of the start codon. The TSP-1 cDNA clone (provided by Dr. Peter J. Polverini, University of Michigan, Ann Arbor, MI) containing bp 491–822 of murine TSP-1 (in pGem7, Promega) was linearized at the 5' ClaI site and used to generate the TSP-1 riboprobe. To normalize each RNA sample for differences in loading, a [32P]GAPDH riboprobe was included in each analysis. The intensity of protected bands was determined by densitometeric scanning of X-ray films.

Analysis of the Endogenous c-rasHa, p53, and TGF-ßRII Genes for Mutations.
An analysis for mutations in the endogenous c-rasHa and p53 genes was performed under conditions described previously (34) . To screen for mutations in the endogenous TGF-ßRII gene, tumor RNA was reverse-transcribed. The resultant cDNA was amplified by PCR using the T7- or SP6-linked primers encompassing:

(a) the extracellular domain and the transmembrane domain (forward: 5'-T7-GGGGGCTCGGTCTATGACGA-3'; reverse: 5'-SP6-CCTTCCGGTGGAACGCCGTG-3'); and

(b) the cytosolic domain (forward: 5'-T7-TCATCCTGGAGGACGACCGC-3'; reverse: 5'-SP6-TTTGGTAGTGTTCAGAGAG-3').

SP6 sequence is: 5'-TCATTTAGGTGACACTATA-3'. T7 sequence is: 5'-GATAATACGACTCACTATA-3'. The PCR products were then purified through a PCR purification kit (Qiagen), and analyzed using the Mismatch II kit (Ambion, Inc., Austin, TX), as well as automated sequencing using internal primers.

Immunohistochemistry for TGF-ß1 Expression.
Tumors were fixed in 10% formalin-PBS (pH 7.4), embedded in paraffin, and sectioned on polylysine slides. The sections were deparaffinized and hydrated. The endogenous peroxidase was quenched by incubating sections in 0.6% H2O2 in methanol at room temperature for 30 min. Slides were rinsed four times with water and washed three times with Tris-buffered saline (5 min each), and the sections were treated with bovine testicular hyaluronidase [1 mg/ml in 0.1 M sodium acetate (pH 5.5)] at 37°C for 30 min. The sections were then washed as above and blocked with 5% normal goat serum at room temperature for 30 min. The TGF-ß1 antibody (LC, a gift from Dr. Kathleen C. Flanders, NIH, Bethesda, MD), which recognizes intracellular TGF-ß1 precursor, was applied to each section (5 µg/ml in Tris-buffered saline with 1% BSA) at room temperature overnight. The immune complex was detected by the avidin-biotin-peroxidase complex using Vectastain kits (Vector Lab, Burlingame, CA). Immune complexes were visualized with diaminobenzidine and counterstained with hematoxylin.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ml.{Delta}ßRII Transgenic Mice Exhibit a Higher Susceptibility to Chemically Induced Skin Carcinogenesis.
Although ML.{Delta}ßRII mice exhibited a hyperplastic skin phenotype (21) , they rarely developed spontaneous tumors. We, therefore, assessed their susceptibility to chemical carcinogenesis. DMBA was used as an initiator followed by repeated promotion with TPA. In total, 30 transgenic mice and 30 nontransgenic littermates in each line of ML.{Delta}ßRII (lines B9223 and B9273; Ref. 21 ) were treated. The results from both transgenic lines were virtually identical. ML.{Delta}ßRII mice exhibited accelerated papilloma formation, with the average appearance of papillomas by 6 weeks after DMBA initiation compared with 9 weeks in control siblings (Fig. 1A)Citation . By 10 weeks after the DMBA initiation, all of the ML.{Delta}ßRII mice developed papillomas, whereas only 70% of the nontransgenic mice developed papillomas at the termination of this study (50 weeks; Fig. 1ACitation ). Additionally, the number of the tumors in ML.{Delta}ßRII mice averaged nine papillomas/mouse at the end of the promotion stage (20 weeks, Fig. 1BCitation ), 2-fold greater than that of control siblings (four papillomas/mouse). Furthermore, papillomas in control mice were prone to regression after the termination of TPA treatment (20 weeks, Fig. 1BCitation ), and only 10% of control mice developed carcinomas by 40 weeks after DMBA initiation (Fig. 1C)Citation . However, ML.{Delta}ßRII papillomas grew autonomously and converted to SCCs as early as 25 weeks after DMBA initiation, and 90% of mice developed SCC by 40 weeks (Fig. 1C)Citation . The SCCs in ML.{Delta}ßRII mice further metastasized to inguinal lymph nodes as early as 30 weeks after DMBA initiation, and 50% of ML.{Delta}ßRII mice had already developed metastases by 40 weeks. In contrast, control siblings did not develop any metastases by 50 weeks after DMBA initiation. Previous reports using this chemical protocol revealed that metastasis generally occurs at late stages and exhibits a poorly differentiated histotype. However, metastases in ML.{Delta}ßRII mice occurred at earlier stages (Fig. 2B)Citation when the primary ML.{Delta}ßRII tumors were still classified as carcinomas in situ or well-differentiated SCC (Fig. 2A)Citation . Consistent with the differentiation stage of the primary lesions, both primary and metastatic lesions showed expression of keratin 13 (Fig. 2, C and D)Citation , an early marker for progression of skin tumors that is usually lost in late-stage SCC (36) . Only a few undifferentiated spindle cell carcinomas developed in ML.{Delta}ßRII mice by 30 to 40 weeks after DMBA initiation, which also metastasized to inguinal lymph nodes (data not shown).



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  		Fig. 1. Kinetics of chemically induced skin tumors in ML.{Delta}ßRII mice versus nontransgenic control. top and bottom panels, data points, the percentage of afflicted mice calculated against the total (30 mice) for each group. Middle panel, data points, the average number of tumors per mouse, starting with 30 mice/group, ending with 24 mice/group at 40 weeks. ML.{Delta}ßRII mice had earlier tumor appearance (top) and increased tumor numbers (middle) compared with control mice. In addition, tumors in ML.{Delta}ßRII mice persisted, whereas tumors in control mice regressed after the removal of TPA promotion. The slight decrease in tumor number at 30 weeks in the ML.{Delta}ßRII group simply reflects the necessity to kill mice with a high tumor burden or malignancy; otherwise, ML.{Delta}ßRII tumors did not regress. At 40 weeks, only one well-differentiated SCC was identified in control siblings, whereas well-to-poorly differentiated SCCs were identified in 90% of ML.{Delta}ßRII mice (bottom). The data shown were obtained with line B9273. Virtually identical results were obtained in line B9223.

 


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  		Fig. 2. H&E staining and immunofluorescence to identify stages of tumors. A, a well-differentiated SCC histotype from a ML.{Delta}ßRII mouse at 30 weeks after DMBA initiation. B, a metastatic lymph node from A, 30 weeks after DMBA initiation, displays a histotype similar to its original SCC but is surrounded by normal lymphatic tissue (top). Expression of Keratin 13 (green) is shown in suprabasal cells of the tumor (C) and its metastatic lesion (D), the same specimens as in A and B, respectively. The red color is Keratin 14, which highlights the epithelial portion of tumors but is absent in areas of lymphatic tissue (D).

 
These chemical carcinogenesis experiments suggest roles of {Delta}ßRII in carcinogenesis at both the early and late stages. To determine at which stage {Delta}ßRII influences skin carcinogenesis in vivo, DMBA and TPA were applied separately to ML.{Delta}ßRII mice. If epidermal cells only contain an initiation event, such as c-rasHa mutations, typically induced by a subcarcinogenic dose of DMBA (37) , they will not develop tumors unless a constitutive promoting stimulus, e.g., TPA-treatment, is provided (37 , 38) . ML.{Delta}ßRII mice failed to develop tumors with a subcarcinogenic DMBA application, suggesting that {Delta}ßRII did not serve as a constitutive tumor promoter. However, when the DMBA treatment was increased to 250 µg, 5 times higher than the initiating dose, 54% of ML.{Delta}ßRII mice developed papillomas within 12 months, the earliest was 1 month after the treatment (Fig. 3A)Citation . In addition, 50% of these papillomas converted to SCCs or spindle cell carcinomas. The nontransgenic siblings did not develop tumors with the same treatment, indicating that this dose of DMBA is still below a full carcinogenic dose for normal mice of this strain. Therefore, inactivation of TGF-ßRII seems to be sufficient, in combination with additional genetic insults elicited by the higher dose of DMBA, to serve as a full-strength tumor promoter.



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  		Fig. 3. Kinetics of DMBA- or TPA-induced skin tumors in ML.{Delta}ßRII mice. Data points, the percentage of mice (30 mice in each group).

 
Consistent with previous reports (37 , 38) , topical application of TPA alone did not induce tumor formation in nontransgenic mice, indicating that an initiation event is required for overt lesions to occur. However, when TPA was applied to ML.{Delta}ßRII mice, they developed papillomas as early as 4 months of weekly TPA promotion (Fig. 3B)Citation , and 50% of TPA-treated ML.{Delta}ßRII mice developed papillomas within 12 months (Fig. 3B)Citation . This result suggests that {Delta}ßRII can act as an initiation event. Although these papillomas persisted, they did not convert to SCC by the end of this study (over 18 months).

Expression of {Delta}ßRII Transgene in ML.{Delta}ßRII Tumors.
Expression of {Delta}ßRII at both RNA and protein levels in ML.{Delta}ßRII transgenic epidermis has been documented previously (21) . To determine whether the ML.{Delta}ßRII transgene was causally involved in accelerating chemically induced tumor formation and progression, RPA was used to detect transgene expression in ML.{Delta}ßRII tumors. As shown in Fig. 4Citation , ML.{Delta}ßRII transgene was strongly expressed in both papillomas and carcinomas as well as in metastatic lymph nodes.



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  		Fig. 4. ML.{Delta}ßRII transgene expression in chemically induced tumors. RPA was performed using probes for {Delta}ßRII and GAPDH. {Delta}ßRII was exclusively expressed in papillomas ({Delta}ßRII, Pap.) SCCs ({Delta}ßRII, SCC), and lymph node metastases ({Delta}ßRII, Meta.)—all from lines B9223 and B9273, respectively—of ML.{Delta}ßRII mice but not in the papilloma or SCC from a nontransgenic (NL) littermate nor in a metastasis from a p53-mutant (p53m, Meta.) transgenic mouse (34) .

 
Analysis of Chemically Induced Tumors for Endogenous c-rasHa, p53, and TGF-ßRII Mutations.
Consistent with previous reports that greater than 90% of DMBA-initiated skin tumors contain c-rasHa mutations (37) , most ML.{Delta}ßRII tumors elicited by the two-stage carcinogenesis protocol exhibited c-rasHa mutations. In nine tumors that we have analyzed, seven had an A->T transversion at codon 61 (Gln->Leu), and one possessed a C->T transition at codon 11 (Ala->Val) of the c-rasHa gene (Table 1)Citation . In four DMBA-elicited tumors, one possessed a G->A transition at codon 12 (Gly->Glu), and two had an A->T transversion at codon 61 (Gln->Leu) of the c-rasHa gene (Table 1)Citation . In addition, metastatic lesions exhibited the same c-rasHa mutations as found in the primary tumors. In contrast, in seven papillomas analyzed from TPA-treated ML.{Delta}ßRII mice, only one papilloma possessed a G->T transition at codon 12 (Gly->Glu); six tumors possessed the wild-type sequence of the entire c-rasHa gene (Table 1)Citation , further supporting the conclusion that the {Delta}ßRII may serve as an alternative initiation event.


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  		Table 1 c-rasHa mutations in ML{Delta}ßRII tumors

 
Previous chemical carcinogenesis experiments have associated p53 loss with malignant conversion in skin carcinogenesis (39 , 40) ; therefore, ML.{Delta}ßRII tumors were analyzed for mutations in the endogenous p53 gene. In total, eight papillomas, eight carcinomas, and six metastases were analyzed, and all of them were devoid of spontaneous mutations in the coding region of the p53 gene (data not shown). This result suggests that the ability of {Delta}ßRII to accelerate malignant conversion is independent of p53 mutations.

Because mutations in the endogenous TGF-ßRII have been found in various malignant tumor cells, we also analyzed ML.{Delta}ßRII tumors for mutations in the endogenous TGF-ßRII gene. All of the 12 ML.{Delta}ßRII carcinomas and 3 metastases analyzed possessed wild-type TGF-ßRII coding sequence (data not shown), which suggests that the {Delta}ßRII can accelerate skin carcinogenesis via its dominant negative effect on wild-type TGF-ßRII.

Ml.{Delta}ßRII Tumors Exhibit Increased Angiogenesis.
In general, histopathology of ML.{Delta}ßRII tumors exhibited increased vascularization in comparison with nontransgenic tumors. To easily visualize the vascular density, tumors were subjected to immunofluorescence staining using an antibody against CD31, a marker of endothelial intercellular junctions as well as platelets and leukocyte subsets (41) . Increased neovascularization was shown in ML.{Delta}ßRII papillomas and carcinomas, in comparison with tumors in nontransgenic mice (Fig. 5)Citation . Microvascular density (number of vessels/unit area) in ML.{Delta}ßRII papillomas increased to 15 ± 2.8, compared with 6.3 ± 1.9 in nontransgenic papillomas (P < 0.005). The percentage of the stromal area covered by vessels in ML.{Delta}ßRII papillomas also increased to 53 ± 24%, compared with 29 ± 12% in nontransgenic papillomas (P < 0.05). Angiogenesis was also obvious in chemically induced SCCs, with ML.{Delta}ßRII SCCs exhibiting a larger number of vessels and increased area of vascularization compared with SCCs in nontransgenic control. Microvascular density in ML.{Delta}ßRII SCCs increased to 25 ± 5.3 versus 11 ± 2.5 in nontransgenic SCCs (P < 0.005); and the percentage of the stromal area covered by vessels was 64 ± 18% in ML.{Delta}ßRII SCCs versus 36 ± 15% in nontransgenic SCCs (P < 0.05).



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  		Fig. 5. A, staining of CD31 by immunofluorescence. CD31 (red) highlights endothelial intercellular junctions. Keratin 14 (green) highlights the epithelial portion of tumors. ML.{Delta}ßRII papilloma ({Delta}ßRII, Pap.) and SCC ({Delta}ßRII, SCC) exhibit a larger numbers of vessels and an increased area of vascularization compared with a papilloma and an SCC from nontransgenic mice (NL, Pap. and NL, SCC, respectively). B, the number of vessels/area unit and percentage of area covered by vessels in ML.{Delta}ßRII and nontransgenic tumors (five to seven tumors from each group). **, P < 0.005; *, P < 0.05.

 
We then examined changes in VEGFs (a critical factor for angiogenesis and tumor cell growth; Ref. 42 ) and TSP-1 (an angiogenesis inhibitor; Ref. 43 ) by RPA. Although there were variations in VEGF and TSP-1 expression levels in nontransgenic tumors, ML.{Delta}ßRII tumors showed more consistent expression levels of VEGF and TSP-1. ML.{Delta}ßRII papillomas and SCCs exhibited a 2 to 3-fold increase in VEGF expression and a 2 to 4-fold decrease in TSP-1 expression, in comparison with nontransgenic papillomas and SCCs, respectively (these values were normalized with the intensity of the corresponding GAPDH signal, Fig. 6Citation ).



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  		Fig. 6. Changes in expression of VEGF and TSP-1 in ML.{Delta}ßRII tumors. RPAs were performed using specific riboprobes as labeled in each panel. NL, a nontransgenic control; Pap., papilloma RNA. {Delta}ßRII papillomas are from lines B9223 and B9273, as are the SCCs.

 
ML.{Delta}ßRII Tumors Exhibit Elevated Levels of Endogenous TGF-ß1.
Previous chemical carcinogenesis studies revealed that carcinogens or tumor promoters induce TGF-ß1 expression in skin tumors (44 , 45) . It is not clear whether TGF-ß1 induction is a negative feedback system for cells against a tumor promotion event. In the present study, we examined endogenous TGF-ß1 expression by immunohistochemistry using an antibody specific for intracellular TGF-ß1. TGF-ß1 was not detectable in normal epidermis (not shown) but was overexpressed in suprabasal cells of papillomas in nontransgenic mice (Fig. 7A)Citation . Interestingly, TGF-ß1 staining in ML.{Delta}ßRII papillomas was not only stronger in suprabasal cells but was also observed in proliferative cells (Fig. 7B)Citation . Carcinomas in nontransgenic mice expressed a higher level of TGF-ß1 than papillomas (Fig. 7CCitation versus 7ACitation ), with TGF-ß1 positive cells primarily located in the terminally differentiated layers (Fig. 7C)Citation . In contrast, more cell layers in ML.{Delta}ßRII carcinomas expressed TGF-ß1, including proliferative cells (Fig. 7D)Citation . The elevation of endogenous TGF-ß1 in ML.{Delta}ßRII tumors may represent a compensatory effect due to the expression of {Delta}ßRII throughout the entire epidermis. Because {Delta}ßRII is able to block TGF-ß1-induced growth inhibition in keratinocytes (21) , higher levels of TGF-ß1 in ML.{Delta}ßRII tumors may facilitate its paracrine effect on angiogenesis and thereby accelerate tumor growth and invasion.



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  		Fig. 7. Immunohistochemical staining of intracellular TGF-ß1. A, papilloma from a nontransgenic mouse; B, papilloma from a ML.{Delta}ßRII mouse; C, SCC from a nontransgenic mouse; D, SCC from a ML.{Delta}ßRII mouse.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The detection of mutations in TGF-ßRII in human cancer suggests that inhibiting TGF-ß signaling may be an important mechanism in the malignant progression of epithelial tumors. To test this in an in vivo model, ML.{Delta}ßRII transgenic mice were exposed to a chemical carcinogenesis protocol. Consistent with the previous report on transgenic mice expressing {Delta}ßRII in the basal cells of the epidermis [K5.{Delta}ßRII, (45) ], ML.{Delta}ßRII mice exhibited a rapid progression of papillomas to malignant carcinomas. Although we used a lower initiation dose of DMBA and a reduced frequency of TPA promotion in ML.{Delta}ßRII mice than used in the study of K5.{Delta}ßRII mice (45) , ML.{Delta}ßRII mice exhibited a more rapid benign papilloma appearance and a more rapid metastasis relative to nontransgenic controls than K5.{Delta}ßRII mice. This higher susceptibility of ML.{Delta}ßRII mice to chemical carcinogenesis than K5.{Delta}ßRII mice may reflect differences in the genetic backgrounds of these transgenic models (ICR for ML.{Delta}ßRII mice versus FVB/N for K5.{Delta}ßRII), although the FVB/N strain is generally more susceptible to skin chemical carcinogenesis (46) . A more likely explanation for the differences in susceptibility of these two models to chemical carcinogenesis may lie in expression patterns of the {Delta}ßRII transgene. Unlike K5.{Delta}ßRII mice, which express {Delta}ßRII in the basal compartment, our ML.{Delta}ßRII mice express {Delta}ßRII throughout the entire epidermis, including both basal (proliferative) and suprabasal (differentiated) keratinocytes (21) . The difference in the site of synthesis of {Delta}ßRII may also account for the fact that ML.{Delta}ßRII mice show significant epidermal hyperplasia (21) , whereas mice expressing {Delta}ßRII exclusively in basal keratinocytes exhibited a normal epidermis (45) . Thus, our present chemical carcinogenesis experiments demonstrate that a constitutive block in TGF-ß signaling throughout the entire epidermis accelerates skin carcinogenesis at both early and late stages.

Role of {Delta}ßRII in Both Early and Late Stages of Skin Carcinogenesis.
Mutations in TGF-ßRII have been detected in malignant cell lines and cancers (24, 25, 26, 27, 28, 29, 31 , 31, 32, 33) , suggesting that inactivation of TGF-ßRII is an important mechanism for malignant progression at late stages of carcinogenesis. Previous reports on transgenic mice expressing {Delta}ßRII in the mammary gland and lung (47) or basal epidermal keratinocytes (45) have consistently demonstrated that loss of TGF-ß responsiveness plays a late role in chemical carcinogenesis. Interestingly, we observed an accelerated benign papilloma formation in ML.{Delta}ßRII mice at a very early stage (Fig. 1, A and B)Citation . More surprisingly, we observed that TPA application alone, without DMBA initiation, induced papilloma formation in ML.{Delta}ßRII mice (Fig. 3B)Citation . These results suggest that blocking TGF-ßRII signaling can serve as an initiating event in skin carcinogenesis. Supporting this, most of the TPA-induced papillomas in ML.{Delta}ßRII mice did not exhibit c-rasHa mutations (Table 1)Citation , a common initiating event in chemical carcinogenesis (37) . Such alternative initiating events have also been described in transgenic mice expressing TGF-{alpha} in the epidermis, in which the lack of c-rasHa mutations has been documented in TPA-induced papillomas (48) . Because TPA-induced papilloma formation in ML.{Delta}ßRII mice is much slower than that elicited by the DMBA/TPA two-stage carcinogenesis protocol (Fig. 3Citation versus Fig. 1Citation ) as well as that of the TPA-treated rasHa transgenic mice (49) , the initiating role of {Delta}ßRII seems to be weaker than c-rasHa activation. Nevertheless, this novel observation suggests that blocking TGF-ßRII signaling may serve as a predisposing event in skin carcinogenesis.

In addition to a role in initiation, if {Delta}ßRII also serves as a tumor promoter, ML.{Delta}ßRII mice would develop spontaneous papillomas. To date, only a few ML.{Delta}ßRII mice have developed spontaneous papillomas, which argues against {Delta}ßRII playing a role in promotion. Amendt et al. (45) reported that K5.{Delta}ßRII mice developed carcinomas by repeated DMBA treatments. The strain of their transgenic mice is FVB/N, which develops an unusually high incidence of SCC when exposed to chemical carcinogenesis protocols (46) , and the dosage of DMBA used in their study was 4-fold higher than that used in the present study. To clarify whether {Delta}ßRII by itself is sufficient to serve as a constitutive tumor promoter, our present study used two different doses of DMBA. Under a subcarcinogenic, initiation dosage (50 µg), ML.{Delta}ßRII mice failed to develop papillomas without further TPA promotion, which suggests that {Delta}ßRII by itself is insufficient to serve as a constitutive tumor promoter. However, when ML.{Delta}ßRII mice were treated with 250 µg of DMBA, five times higher than the initiating dose, they developed papillomas (Fig. 3)Citation that further converted to carcinomas and metastases. In this case, the c-rasHa mutation elicited by DMBA (Table 1)Citation was likely to provide the initiation event, and the cooperation of {Delta}ßRII with mutated c-rasHa and/or other genetic insults provided a promoting event to achieve tumor formation and progression. Because tumor formation in response to DMBA alone was only observed in ML.{Delta}ßRII mice and not in nontransgenic mice, inactivation of TGF-ßRII seems to be sufficient—in combination with additional genetic insults elicited by the higher dose of DMBA—for a full carcinogenic effect.

With respect to the role of loss of TGF-ßRII signaling in malignant progression, ML.{Delta}ßRII tumors showed a correlation between DMBA-elicited c-rasHa mutations and malignant conversion. Both the DMBA/TPA two-stage protocol and the higher dose of DMBA treatment induced high rates of papilloma conversion to carcinomas with similar rates of c-rasHa mutations (Table 1)Citation . However, none of the TPA-elicited papillomas progressed to carcinomas, and the rate of c-rasHa mutation was also very low in this group (one of seven, Table 1Citation ). These data suggest that, although {Delta}ßRII plays important roles in both the earlier and later stages of carcinogenesis, c-rasHa activation seems to be necessary to cooperate with the loss of TGF-ß signaling for malignant conversion. Supporting this hypothesis, Glick et al. (9) reported that v-rasHa transduction of TGF-ß1 null keratinocytes accelerated malignant conversion. However, because a subcarcinogenic dose of DMBA (despite the induction of c-rasHa mutations at this dosage) did not induce tumor formation in ML.{Delta}ßRII mice, other genetic insults (induced by the higher dose of DMBA) or tumor promotion events (induced by TPA) are still required for tumorigenesis in ML.{Delta}ßRII mice.

The Rapid Metastasis in ML.{Delta}ßRII Tumors Is Associated with Increased Angiogenesis.
Metastasis is usually associated with late-stage, poorly differentiated SCC (37) . However, a unique feature of ML.{Delta}ßRII tumors is that metastatic lesions developed from well-differentiated SCCs, as identified by both histopathology and keratin staining (Fig. 2)Citation . This rapid progression to metastasis was correlated with increased vascularization in ML.{Delta}ßRII tumors (Fig. 5)Citation , which suggests that angiogenesis may play a pivotal role in ML.{Delta}ßRII tumor progression and invasion. Because TGF-ß is believed to induce angiogenesis (2 , 50) and has been reported to induce expression of VEGFs in cultured keratinocytes (51) , epithelial tumor cells, and their stromal fibroblasts (52) , we were somewhat surprised to find that ML.{Delta}ßRII tumors exhibited increased angiogenesis (Fig. 5)Citation that was correlated with increased VEGF expression and decreased TSP-1 expression (Fig. 6)Citation . This may have resulted from increased expression of endogenous TGF-ß1 in ML.{Delta}ßRII tumors (Fig. 7)Citation . It is possible that {Delta}ßRII mainly blocks TGF-ß-induced growth inhibition in the epidermis, whereas other TGF-ß functions, such as TGF-ß-induced VEGF expression, are not blocked by {Delta}ßRII in this transgenic model. Although it has been debated whether TGF-ßRI and TGF-ßRII can signal independently (19 , 20) , recent studies suggest differential suppression of TGF-ß functions may depend on the expression level of the {Delta}ßRII (18) . In agreement with this postulation, Sankar et al. (53) reported that the truncated TGF-ßRII blocked only TGF-ß1-induced growth inhibition without affecting angiogenesis, whereas the truncated TGF-ßRI blocked TGF-ß1-induced angiogenesis. Alternatively, if the intact type II receptor is still required for mediating TGF-ß-induced angiogenesis, increased angiogenesis in ML.{Delta}ßRII tumors may be a result of a paracrine effect of the increased TGF-ß on stromal cells that do not express {Delta}ßRII and are fully capable of TGF-ß signaling. This potential paracrine effect could be further enhanced in ML.{Delta}ßRII tumors inasmuch as TGF-ß1 was expressed at higher levels in ML.{Delta}ßRII tumors than nontransgenic tumors, particularly in proliferative cells (Fig. 7)Citation that are closely associated with the tumor stroma.

Because TSP-1 has been reported to be transcriptionally activated by the tumor suppressor p53 (43) and VEGF expression can also be elevated by mutant p53 (54) , changes in TSP-1 and VEGF expression in ML.{Delta}ßRII tumors may occur via the inactivation of p53. However, we did not detect p53 mutations in {Delta}ßRII tumors. Furthermore, we have shown that chemically induced tumors in either p53 knockout mice (p53-/-) or transgenic mice expressing a dominant negative p53 mutant (p53m) in the epidermis did not exhibit altered expression levels of either TSP-1 or VEGF (55) . Therefore, increased angiogenesis resulting from {Delta}ßRII expression seems to be independent of p53 status. Our present study suggests that tumors that possess both elevated TGF-ß and loss of functional TGF-ßRII may have a poor prognosis. The mutant TGF-ßRII not only allows tumor cells to escape from TGF-ß-induced growth arrest, but the subsequent elevation in TGF-ß expression levels increases angiogenesis, which facilitates tumor growth and invasion.


   ACKNOWLEDGMENTS
 
We thank Dr. Dennis R. Roop for his support of this work. We also thank Dr. Peter Polverini for providing the TSP-1 cDNA clone, Dr. Georg Breier for providing the VEGF cDNA clone, Dr. Kathleen C. Flanders for providing the TGF-ß1 antibody, and Dr. Dennis R. Roop and Kristin Liefer for critical comments on the article. Mattie Brooks provided excellent technical assistance.


   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 work was supported by a Career Development Award from the Dermatology Foundation and NIH Grant CA79998-01 (to X-J. W.) and NIH Grant CA52607 (to Dennis R. Roop). Back

2 To whom requests for reprints should be addressed, at Departments of Cell Biology and Dermatology, Baylor College of Medicine, Houston, TX 77030. Phone: (713)798-3306; Fax: (713)798-3800; E-mail: xwang{at}bcm.tmc.edu Back

3 The abbreviations used are: TGF, transforming growth factor; TGF-ßRI, TGF-ß type I receptor; TGF-ßRII, TGF-ß type II receptor; {Delta}ßRII, a dominant negative TGF-ßRII; ML, mouse loricrin promoter; DMBA, dimethylbenz[a]anthracene; TPA, 12-O-tetradecanoylphorbol-13-acetate; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SCC, squamous cell carcinoma; RPA, RNase protection assay. Back

Received 10/23/98. Accepted 4/14/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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