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Cancer Research 67, 9158-9168, October 1, 2007. doi: 10.1158/0008-5472.CAN-07-0590
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Reduction in I{kappa}B Kinase {alpha} Expression Promotes the Development of Skin Papillomas and Carcinomas

Eunmi Park, Feng Zhu, Bigang Liu, Xiaojun Xia, Jianjun Shen, Tracie Bustos, Susan M. Fischer and Yinling Hu

Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas

Requests for reprints: Yinling Hu, Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, 1808 Park Road 1-C, Smithville, TX 78957. Phone: 512-237-9338; Fax: 512-237-4375; E-mail: yhu{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We reported recently a marked reduction in I{kappa}B kinase {alpha} (IKK{alpha}) expression in a large proportion of human poorly differentiated squamous cell carcinomas (SCC) and the occurrence of Ikk{alpha} mutations in human SCCs. In addition, overexpression of IKK{alpha} in the epidermis inhibited the development of skin carcinomas and metastases in mice. However, whether a reduction in IKK{alpha} expression promotes skin tumor development is currently unknown. Here, we assessed the susceptibility of Ikk{alpha} hemizygotes to chemical carcinogen-induced skin carcinogenesis. Ikk{alpha}+/– mice developed 2 times more papillomas and 11 times more carcinomas than did Ikk{alpha}+/+ mice. The tumors were larger in Ikk{alpha}+/– than in Ikk{alpha}+/+ mice, but tumor latency was shorter in Ikk{alpha}+/– than in Ikk{alpha}+/+ mice. Some of the Ikk{alpha}+/– papillomas and most Ikk{alpha}+/– carcinomas lost the remaining Ikk{alpha} wild-type allele. Somatic Ikk{alpha} mutations were detected in carcinomas and papillomas. The chemical carcinogen-induced H-Ras mutations were detected in all the tumors. The phorbol ester tumor promoter induced higher mitogenic and angiogenic activities in Ikk{alpha}+/– than in Ikk{alpha}+/+ skin. These elevated activities were intrinsic to keratinocytes, suggesting that a reduction in IKK{alpha} expression provided a selective growth advantage, which cooperated with H-Ras mutations to promote papilloma formation. Furthermore, excessive extracellular signal-regulated kinase and IKK kinase activities were observed in carcinomas compared with those in papillomas. Thus, the combined mitogenic, angiogenic, and IKK activities might contribute to malignant conversion. Our findings provide evidence that a reduction in IKK{alpha} expression promotes the development of papillomas and carcinomas and that the integrity of the Ikk{alpha} gene is required for suppressing skin carcinogenesis. [Cancer Res 2007;67(19):9158–68]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
We recently reported somatic I{kappa}B kinase {alpha} (Ikk{alpha}) mutations in human squamous cell carcinomas (SCC) and a marked reduction in IKK{alpha} expression in poorly differentiated human and mouse cutaneous SCCs (1), which highlights the importance of IKK{alpha} in human skin cancer. However, the natural role for IKK{alpha} in skin tumor development is unclear. The animal model provides an appropriate tool to address these questions.

IKK{alpha} is one subunit of the IKK complex, which is central for nuclear factor-{kappa}B (NF-{kappa}B) activation (2). Its involvement in the development of lymphoid organs and innate immunity requires IKK/NF-{kappa}B activity (3, 4). IKK{alpha} also plays an essential role in the formation of the epidermis during embryonic development in mice (5–7). Ikk{alpha}–/– mice develop a striking hyperplastic epidermis that lacks terminal differentiation and these mice die at birth (5–7). Ikk{alpha}–/– keratinocytes and skin preserve IKK/NF-{kappa}B activity (5, 8). Reintroduction of IKK{alpha} or kinase-inactive IKK{alpha} induced terminal differentiation in Ikk{alpha}–/– keratinocytes, but reintroduction of IKKß, RelA p65, or I{kappa}B{alpha} (an inhibitor for NF-{kappa}B) failed to do so (8). Furthermore, Sil et al. (9) reported that IKK{alpha} or kinase-inactive IKK{alpha} transgene driven by the keratin 14 promoter rescued the skin phenotype of Ikk{alpha}–/– mice. These results suggest that the function of IKK{alpha} in determining the epidermal formation is IKK/NF-{kappa}B independent.

We reported recently that Lori.IKK{alpha} transgenic mice developed normal skin and that the elevated IKK{alpha} in the suprabasal epidermis enhanced terminal differentiation markers (1). These transgenic mice developed significantly fewer malignant carcinomas and metastases than did wild-type (WT) mice when they were challenged with the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The mitogenic and angiogenic activities were reduced in the DMBA/TPA–treated skin of Lori.IKK{alpha} mice relative to those in the DMBA/TPA–treated skin of WT mice. These results suggest that elevated IKK{alpha} expression antagonizes chemical carcinogen-induced mitogenesis and angiogenesis, thereby repressing the development of malignant carcinomas and metastases. However, the effect of reduced IKK{alpha} on skin tumor development is unknown.

Ras plays a prominent role in the development of human SCCs and H-Ras mutations are frequently found in human SCCs (10, 11). Ras activation is required for chemical carcinogen-induced skin carcinogenesis in mice. The carcinogen DMBA causes activating H-Ras mutations, and the tumor promoter TPA expands the population of Ras-initiated cells (12, 13). Most papillomas eventually regress, and only a few become carcinomas in mice with a C57BL6 or a C56BL/129/Sv background (14, 15). Mice lacking H-Ras developed significantly fewer skin tumors than did WT mice induced by DMBA/TPA (16). In the present study, we tested the susceptibility of Ikk{alpha} hemizygotes to DMBA/TPA–induced skin carcinogenesis. Ikk{alpha}+/– mice developed 2 times more benign tumors (papillomas) and 11 times more malignant carcinomas than did WT mice. Furthermore, we found that most Ikk{alpha}+/– carcinomas and some Ikk{alpha}+/– papillomas lost the remaining WT Ikk{alpha} allele. These findings show that reduction in IKK{alpha} expression promotes papilloma formation and malignant conversion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and skin carcinogenesis. All the mice used in this study were cared for in accordance with the guidelines of our institution's Animal Care and Use Committee (animal protocol 04-01-05732). DMBA (Sigma-Aldrich, Inc.) and TPA (LC Laboratories) were used to treat mice. Six- to 8-week-old female Ikk{alpha}+/+ and Ikk{alpha}+/– mice with a C57BL6 background (C57BL6/129/Sv mice were backcrossed with C57BL6 mice thrice) were topically treated with 100 µg DMBA in 200 µL acetone; 2 weeks later, the mice were treated with 2.5 µg TPA in 200 µL acetone five times weekly for 28 weeks. For controls, 15 Ikk{alpha}+/+ and 15 Ikk{alpha}+/– mice were treated with 100 µg DMBA in acetone (no TPA) and 15 Ikk{alpha}+/+ and 15 Ikk{alpha}+/– mice were treated with 2.5 µg TPA in acetone (no DMBA) five times weekly for 28 weeks. For bromodeoxyuridine (BrdUrd; Sigma-Aldrich) incorporation experiment, mice were treated with 2.5 µg TPA in 200 µL acetone or 200 µL acetone alone as a control five times weekly for 4 weeks or with 100 µg DMBA in 200 µL acetone once. BrdUrd (0.05 mg/g body weight) was administered i.p. to mice 1 h before they were killed. Skin specimens were embedded in paraffin and then sectioned, and BrdUrd labeling was counted in 1,000 cells in the basal epidermis. Immunohistochemical BrdUrd detection was done in our Histology and Tissue Core.1 Paraffin-embedded sections of tumor tissue and skin were prepared, and routine H&E and immunohistochemical staining of these sections with antibodies against proliferating cell nuclear antigen (PCNA) and IKK{alpha} were done in our Histology and Tissue Core.

Detection of H-Ras (V61 and V12) mutations. Analyses for H-Ras mutations were done in our Molecular Biology Core.2 PCR primers for H-Ras codon 61 mutation detection were 5'-ggtgtaggctggttctgtggattctc-3' and 5'-gcacacggaaccttcctcac-3', which generated a 329-bp band that was gel purified and used for a second PCR round. PCR primers for the second round were 5'-tgtggattctctggtctgaggagag-3' and 5'-cataggtggctcacctgtactgatg-3', which produced a 269-bp fragment. The PCR products were digested with XhaI. PCR primers for H-Ras codon 12 mutation detection were 5'-cctggattggcagccgctgt-3' and 5'-tcatactcgtccacaaagtg-3', which generated a 125-bp fragment. The fragments were digested with MnII.

Real-time PCR. Total RNA was isolated from skin, tumors, and keratinocytes by TRI reagent (Molecular Research Center, Inc.), and cDNA was synthesized by a RETROscript kit (Ambion, Inc.). The PCR primers and probes were purchased from Applied Biosystems [tumor necrosis factor {alpha} (TNF{alpha}), Mm00443258_m1; interleukin-1{alpha} (IL-1{alpha}), Mm00439620_m1; transforming growth factor {alpha} (TGF{alpha}), Mm00446231_m1; vascular endothelial growth factor-A (VEGF-A), Mm00437304_m1; epidermal growth factor (EGF), Mm00438696_m1; amphiregulin, Mm00437583_m1; heparin-binding EGF (HB-EGF), Mm00439307_m1; fibroblast growth factor 2 (FGF2), Mm00433287_m1; and FGF13, Mm00438910_m1]. The reactions were done according to the manufacturer's instructions. Each cDNA sample was analyzed in triplicate by an ABI 7900 sequence detector (Applied Biosystems). Thermal cycling was done as follows: 1 cycle at 50°C for 2 min and 95°C for 10 min and 40 cycles at 95°C for 15 s and 60°C for 1 min. Data were analyzed by the Prism Dissociation Curve software program (SDS 2. 2. 2, Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Southern blot analysis. DNA was isolated from tumors and normal skin by an extraction kit (Promega). DNA (10 µg) was digested with BamHI overnight, applied to a 0.9% agarose gel, transferred to blotting membranes (Zeta-Probe GT, Bio-Rad), and fixed by UV light. The DNA blotting membranes were hybridized with an NH2-terminal IKK{alpha} cDNA (nucleotides 1–575) probe and a 400-bp NH2-terminal GAPDH cDNA probe as a control. Southern blotting was done according to the manufacturer's instructions (Bio-Rad).

Western blotting. A cell lysate (40 µg) was applied to SDS gel, and specific protein levels were measured by Western blotting as described previously (8) with the following antibodies against IKK{alpha} (Imgenex), phosphorylated extracellular signal-regulated kinase (p-ERK; Cell Signaling Technology), ERK1 (Santa Cruz Biotechnology), ERK2 (Santa Cruz Biotechnology), and ß-actin (Sigma).

Keratinocyte culture. Mouse primary keratinocytes were isolated and cultured as described previously (8). Briefly, skin specimens isolated from newborn mice or E19 embryos were treated with 0.25% trypsin (Life Technologies) for 8 to 10 h at 4°C; the epidermis was separated from the dermis. Isolated keratinocytes were plated on 60-cm cell dishes containing keratinocyte serum-free medium (Life Technologies).

Ikk{alpha} mutation analysis. Total RNA was isolated from skin or tumors with TRI reagent, and cDNA from these samples was synthesized by a RETROscript kit. The PCR fragments were generated by an expanded high-fidelityplus PCR system (Roche Diagnostics GmbH) with primers (IKK{alpha}, 5'-ccattcactattctgaggttggtgtc-3' and 5'-tactggaggggttactgtgccttc-3'). The PCR products were subcloned into pGEM-T vectors (Promega) and sequenced (1). The sequences were compared with the National Center for Biotechnology Information, gi 6680941.

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (ChIP) assay was done following the manufacturer's instructions (Upstate). Keratinocytes were starved for 24 h (no growth factors) and cells were treated with EGF (10 ng/mL) for 24 h or with TPA (48 nmol/L) for 2 h. After cells were fixed, washed, and collected, their DNA was sheared by sonication in 200 µL lysis buffer. Then, 20 µL sonicated DNA from each sample was purified and used as an input DNA control. The rest of the DNA was precleared. The chromatin DNA was precipitated with an anti-IKK{alpha} antibody and protein A/G beads, and the DNA was eluted from the beads. The precipitated DNA was analyzed by PCR for 1 cycle at 95°C for 5 min; 35 cycles at 95°C for 20 s, 55°C for 20 s, and 72°C for 30 s; and 1 cycle at 72°C for 10 min. The PCR primers for proximal VEGF-A promoters (–903 to –1,233 bp) were 5'-gtgttcctgagcccagtttgaag-3' and 5'-agtccgctgaatagtctgccttg-3' and the primers for distal VEGF-A promoter (–2,414 to –2,065 bp) were 5'-tgggttagaggtgggggttttg-3' and 5'-aactgaagccagggtgccaatg-3'. PCR primers for analyzing the mRNA level of VEGF-A (491 bp) were 5'-gcacccacgacagaaggagagcaga-3' and 5'-cgccttggcttgtcacatctgcaa-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ikk{alpha}+/– mice develop many more and larger skin tumors earlier than do Ikk{alpha}+/+ mice. To determine the relationship between IKK{alpha} expression and skin carcinogenesis, we topically treated Ikk{alpha}+/+ and Ikk{alpha}+/– mice with a single dose of DMBA followed by repeated treatment with TPA. At week 28 of TPA treatment, we collected and weighed the tumors. Ikk{alpha}+/– mice developed significantly more tumors than did Ikk{alpha}+/+ mice (P < 0.0005, linear regression model; Fig. 1A ; Supplementary Table S1A). Incidence of tumors in the Ikk{alpha}+/+ mice reached 100% by 19 weeks and in the Ikk{alpha}+/– mice by 14 weeks (P = 0.0014, log-rank test; Fig. 1B). Papillomas and carcinomas in Ikk{alpha}+/– mice were significantly larger than those in Ikk{alpha}+/+ mice (P < 0.0005, two-sided Fisher's exact test; Fig. 1C and D). Control mice treated with DMBA once or TPA (five times weekly) alone did not develop any tumors at week 28. The repeated experiment 2 showed similar results (Supplementary Fig. S1A and B; Supplementary Table S1B). To determine the rate of carcinoma conversion, we histologically examined all the tumors that weighed >0.03 g. Four of the 125 (3.2%) tumors that developed in the 15 Ikk{alpha}+/+ mice and 47 of the 285 (16.5%) tumors that developed in the 15 Ikk{alpha}+/– mice were carcinomas (P < 0.005, Fisher's exact test; Fig. 1C). These results suggested that Ikk{alpha}+/– mice were far more susceptible to skin carcinogenesis than Ikk{alpha}+/+ mice. Genetic background affects the susceptibility of mice to chemical carcinogen-induced skin carcinogenesis (15). Although the mice used for this study were not 100% C57BL6, Ikk{alpha}+/– and Ikk{alpha}+/+ mice were sisters. Mice with a mixed C57BL6 genetic background have been used for the studies of chemical carcinogen-induced skin carcinogenesis (14, 17). In addition, the differences in the tumor incidence and the tumor multiplicity between Ikk{alpha}+/+ group and Ikk{alpha}+/– group were dramatically significant. Thus, it was likely that the genetic background was not a major factor that caused these differences. We also examined apoptotic cells in tumors from Ikk{alpha}+/+ and Ikk{alpha}+/– mice by immunostaining analysis and did not observe a significant difference for this end point between Ikk{alpha}+/+ and Ikk{alpha}+/– tumors (data not shown).


Figure 1
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  		Figure 1. Ikk{alpha}+/– mice are more susceptible to DMBA/TPA–induced skin carcinogenesis than Ikk{alpha}+/+ mice. A, tumor multiplicity in Ikk{alpha}+/+ and Ikk{alpha}+/– mice (P < 0.0005, linear regression model). B, tumor incidence in Ikk{alpha}+/+ and Ikk{alpha}+/– mice (P = 0.0014, log-rank test). C, tumor weights in Ikk{alpha}+/+ and Ikk{alpha}+/– mice at week 28 (P < 0.0005, Fisher's exact test). D, Ikk{alpha}+/+ and Ikk{alpha}+/– mice with carcinomas and papillomas at week 28.

 
H-Ras mutations are detected in papillomas and carcinomas. The papillomas in the Ikk{alpha}+/+ and Ikk{alpha}+/– mice displayed well-differentiated patterns with a thick surface layer of keratin. The carcinomas were less differentiated or undifferentiated in the Ikk{alpha}+/+ and Ikk{alpha}+/– mice (Fig. 2A ). The carcinomas expressed PCNA in the entire tumor, but the papillomas expressed PCNA only in the hyperproliferating basal cells (Fig. 2A).


Figure 2
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  		Figure 2. H-Ras mutations are detected in papillomas and carcinomas. A, histology of Ikk{alpha}+/+ and Ikk{alpha}+/– papillomas and carcinomas by staining with H&E. Original magnification (left). Carcinomas expressed PCNA across the entire tumor, but papillomas expressed PCNA only in the hyperproliferating basal cells. #, sample numbers; dark brown nuclear staining, positive immunohistochemical staining; blue staining, hematoxylin counterstaining. B, PCR fragments containing V61 mutations (CAA to CTA) of H-Ras were digested with XhaI to generate two bands. C, PCR fragments containing V12 mutations of H-Ras (GGA to GGC) were not digested with MnII.

 
To determine whether these skin tumors were initiated by DMBA-mediated H-Ras mutations, we examined V61 mutations (CAA->CTA) of H-Ras in randomly selected papillomas and carcinomas from Ikk{alpha}+/+ and Ikk{alpha}+/– mice by using PCR. The H-Ras (V61) mutations were detected in 19 of 23 (82.6%) Ikk{alpha}+/+ tumors and in 24 of 28 (85.7%) Ikk{alpha}+/– tumors (Fig. 2B). Moreover, we found V12 mutations (GGA->GGC) in the tumors that were negative for V61 mutations of H-Ras (Fig. 2C). We did not detect any H-Ras mutations in seven untreated skin specimens of Ikk{alpha}+/+ and Ikk{alpha}+/– mice, respectively, and in eight DMBA/TPA–treated skin specimens of Ikk{alpha}+/+ and Ikk{alpha}+/– mice, respectively. These results suggested that all the skin tumors were initiated by DMBA-mediated H-Ras mutations.

Levels of IKK{alpha} proteins are markedly reduced in most Ikk{alpha}+/– carcinomas and some Ikk{alpha}+/– papillomas. We further evaluated the IKK{alpha} level in papillomas and carcinomas. Western blotting analysis showed higher levels of IKK{alpha} in 10 papillomas than in skin specimens from Ikk{alpha}+/+ mice (Fig. 3A ). The increased IKK{alpha} could be induced by long-term treatment with TPA, as suggested by Saleem et al. (18). We reported previously that poorly differentiated SCCs expressed markedly reduced IKK{alpha} (1). Reduction in IKK{alpha} was also detected in the 8 poorly differentiated carcinomas of the 15 Ikk{alpha}+/+ carcinomas (Fig. 3A; Supplementary S2A and B). Furthermore, some of the 17 Ikk{alpha}+/– papillomas expressed elevated IKK{alpha}, but the remainder expressed reduced IKK{alpha} (Fig. 3A). All 18 Ikk{alpha}+/– carcinomas expressed markedly reduced IKK{alpha}. Comparison of IKK{alpha} relative levels suggested that the IKK{alpha} levels were inversely correlated with the increased numbers of carcinomas (Fig. 3B). Immunohistochemical staining showed a reduction in IKK{alpha} expression only in carcinomas, not in the skin that surrounded the tumors (Fig. 3C). Collectively, these results suggested that a reduction in IKK{alpha} expression contributed to the formation of papillomas; a further reduction greatly enhanced the development of carcinomas, although the mechanism by which IKK{alpha} was reduced might be different in Ikk{alpha}+/+ and Ikk{alpha}+/– tumors.


Figure 3
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  		Figure 3. Reduction in IKK{alpha} proteins in most Ikk{alpha}+/– carcinomas, some of Ikk{alpha}+/– papillomas, and poorly differentiated Ikk{alpha}+/+ carcinomas. A, IKK{alpha} levels in papillomas and carcinomas, detected by Western blotting. ß-Actin, a protein loading control; control, normal skin. Ratio, densities of the IKK{alpha} signal normalized to those of the ß-actin signal (ratio for WT skin was set as 1). Signals were scanned by a Kodak Image Station 440 with the ID3.6 software program (Kodak) and analyzed by the ImageQuant TL software program (version 2003.02). B, comparison of relative IKK{alpha} expression levels in papillomas and carcinomas. Ikk{alpha}+/+ carcinomas were obtained from experiments 1 and 2. C, IKK{alpha} expression in Ikk{alpha}+/+ and Ikk{alpha}+/– skin, papillomas, and carcinomas, detected by immunohistochemical staining. +/+, Ikk{alpha}+/+; +/–, Ikk{alpha}+/–; –/–, Ikk{alpha}–/–; #, sample numbers; dark brown staining, IKK{alpha} staining; blue staining, hematoxylin counterstaining. Original magnifications, x100 (papillomas and carcinomas) and x200 (skin).

 
Genetic alterations of Ikk{alpha} occur in skin tumors. We reported previously that the poorly differentiated carcinomas expressed reduced IKK{alpha} proteins; however, IKK{alpha} mRNA levels were not reduced in these carcinomas obtained from WT mice (1), and we observed similar results in this study (Supplementary Fig. S2B). In addition, we observed that papillomas and carcinomas from Ikk{alpha}+/– mice tended to lose IKK{alpha} (Fig. 3A), suggesting that loss of heterozygosity (LOH) of Ikk{alpha} might be responsible for loss of IKK{alpha} protein in Ikk{alpha}+/– tumors. To test this hypothesis, we examined the BamHI-digested Ikk{alpha} gene in papillomas and carcinomas by Southern blotting with an NH2-terminal IKK{alpha} cDNA probe covering exons 1 to 6 of Ikk{alpha} (Fig. 4A ). This probe hybridized with three fragments, including a 9.3-kb fragment from exon 2 to upstream of the Ikk{alpha} gene, a 3.6-kb fragment from exons 3 to 4, and a 5.2-kb fragment from exons 5 to 9 in Ikk{alpha}+/+ cells (Fig. 4B). In the Ikk{alpha}–/– cells, the 5.2-kb fragment disappeared because exon 7 in the Ikk{alpha} allele included a neo gene that contained two additional BamHI sites at its 5' and 3' ends (Fig. 4A and B; ref. 5). After BamHI digestion, this probe only recognized a 1.3-kb fragment from exons 5 to 7 but not the fragment from exons 7 to 9. Thus, three DNA fragments (9.2, 3.6, and 1.3 kb) in Ikk{alpha}–/– cells and four DNA fragments (9.3, 3.6, 5.2, and 1.3 kb) in Ikk{alpha}+/– cells were detected. When Ikk{alpha} LOH occurred in Ikk{alpha}+/– cells, only 9.3, 3.6, and 1.3 kb were detected. We found LOH in 4 of the 9 (44%) papillomas and 22 of the 23 (95%) carcinomas (Fig. 4B; Supplementary Table S2). The faint WT bands (5.2 kb) in Ikk{alpha}+/– carcinomas were presumably caused by the presence of contaminating non-carcinoma cells in the tumor samples. We confirmed LOH in the Ikk{alpha}+/– carcinomas by PCR (Supplementary Fig. S3A; ref. 5). Reverse transcription-PCR (RT-PCR) confirmed that Ikk{alpha}+/– carcinomas retained low levels of IKK{alpha} mRNA (Supplementary Fig. S3B). Collectively, these results indicated that LOH of Ikk{alpha} might be largely responsible for loss of IKK{alpha} protein in Ikk{alpha}+/– carcinomas and papillomas.


Figure 4
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  		Figure 4. Genetic alterations in skin tumors. A, a map for BamHI-digested Ikk{alpha} genomic DNA. Numbered boxes represent exons. Lines between the exons represent introns. Numbers for the IKK{alpha} cDNA probes (bp) indicate nucleotide positions. Numbers for the Ikk{alpha} gene (kb) indicate the sizes of the BamHI-digested genomic DNA fragments. B, BamHI; Neo, neomycin gene inserted into exon 7 in the Ikk{alpha} knockout allele. B and C, BamHI-digested DNA samples of skin, papillomas, and carcinomas were analyzed by Southern blotting that was probed with an NH2-terminal IKK{alpha} cDNA. Control, skin; C, carcinoma; P, papilloma; +/+, Ikk{alpha}+/+; +/–, Ikk{alpha}+/–; –/–, Ikk{alpha}–/–. D, detection of Ikk{alpha} mutations including mutations, deletions, and insertions in 45 clones from four Ikk{alpha}+/+ carcinomas. *, mutations; {downarrow}, insertion; }, deletion. Thirty-three clones from two acetone-treated skin specimens were used as background controls.

 
We then examined the Ikk{alpha} gene in 11 papillomas and 7 carcinomas from Ikk{alpha}+/+ mice by Southern blotting with the IKK{alpha} cDNA probe and found no alterations in Ikk{alpha} (Fig. 4C; Supplementary Table S3). Ikk{alpha} mutations in human SCCs were reported previously (1). To identify mutations in the Ikk{alpha} murine gene, we used RT-PCR to amplify an IKK{alpha} transcript (nucleotides 1606–2090; amino acids 518–679 of IKK{alpha}; NM_007700) from either acetone-treated control skins or Ikk{alpha}+/+ carcinomas. Amplified PCR products were subcloned into the pGEM vectors and sequenced. We sequenced 33 clones from two acetone-treated skin samples, which were used as PCR background controls (5 and 6 in Supplementary Table S4). Many Ikk{alpha} mutations, including insertions and deletions, were detected in 45 clones from four Ikk{alpha}+/+ carcinomas (Fig. 4D; Supplementary Table S4). Ikk{alpha} mutations were also detected in Ikk{alpha}+/– papillomas and Ikk{alpha}+/– carcinomas (Supplementary Table S5). Interestingly, we detected many Ikk{alpha} mutations in the IKK{alpha} transcript of one Ikk{alpha}+/– carcinoma (Supplementary Table S5). Ikk{alpha} LOH was not detected in all the Ikk{alpha}+/– carcinomas (Supplementary Table S2). Thus, Ikk{alpha} mutations might also contribute to loss of IKK{alpha} proteins in carcinomas if some cells in carcinomas retained the remaining WT Ikk{alpha} allele. Apparently, there were more Ikk{alpha} mutations in carcinomas than in papillomas. These transition and transversion nucleotide substitutions caused missense and nonsense mutations (Fig. 4D). Although we detected some repeated mutations in the same tumors, the frequency of the repeated mutations was low, suggesting that these somatic mutations were not amplified, which was consistent with our previous results (1). In addition, sequencing results confirmed that the mutations were not homologous (Supplementary Fig. S3C). Collectively, Ikk{alpha} mutations were found in both human and mouse SCCs.

Reduction in IKK{alpha} expression enhances mitogenic activity in skin and carcinomas. A previous study suggested that ERK activity was a signature for Ras-initiated skin tumors in two-stage chemical carcinogenesis settings (17). We found H-Ras mutations in the tested papillomas and carcinomas. Because control mice treated with DMBA alone did not produce any tumors at 28 weeks, we investigated the effect of reduced IKK{alpha} on the promotion of TPA-induced tumors. Immunohistochemical staining showed a higher fraction of BrdUrd-positive cells in the epidermis of Ikk{alpha}+/– mice than in the epidermis of Ikk{alpha}+/+ mice treated with TPA (P = 0.009, Student's t test; Fig. 5A ), suggesting that TPA enhanced cell proliferation in Ikk{alpha}+/– skin. We next examined phosphorylation of ERK, a downstream target of Ras. ERK activity was higher in Ikk{alpha}+/– than in Ikk{alpha}+/+ cultured keratinocytes under routine culture conditions (Supplementary Fig. S4). ERK activity was much higher in TPA-treated Ikk{alpha}+/– than in TPA-treated Ikk{alpha}+/+ skin (Fig. 5B). Furthermore, examination of several growth factors that are downstream targets of TPA (12) by real-time PCR showed excessive expression of TGF{alpha}, EGF, amphiregulin, FGF2, FGF13, VEGF-A, and TNF{alpha} in TPA-treated Ikk{alpha}+/– skin specimens relative to that in TPA-treated Ikk{alpha}+/+ skin specimens (Fig. 5C). TPA moderately elevated expression of HB-EGF and IL-1 in Ikk{alpha}+/– compared with that in Ikk{alpha}+/+ skin. The basal levels of ERK activity and expression of these growth factors were slightly higher in Ikk{alpha}+/– than in Ikk{alpha}+/+ skin (Fig. 5B and C). Moreover, we isolated keratinocytes from Ikk{alpha}+/+ and Ikk{alpha}+/– newborn mice and found that TPA treatment induced significant higher levels of ERK activities and higher expression levels of the growth factors in Ikk{alpha}+/– keratinocytes than in Ikk{alpha}+/+ keratinocytes (Fig. 5D and E), suggesting that the increased mitogenic activity was intrinsic to keratinocytes. Western blotting showed considerably higher ERK activity in carcinomas than in papillomas (Fig. 5F). Collectively, these results indicated that reduced IKK{alpha} markedly elevated TPA-induced ERK activity and expression of these growth factors, which provided a molecular basis for promoting keratinocyte proliferation, papilloma formation, and malignant conversion in Ikk{alpha}+/– mice.


Figure 5
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  		Figure 5. Reduced IKK{alpha} expression elevates ERK activity and expression of growth factors and cytokines in TPA-treated skin and carcinomas. A, percentage of cells with BrdUrd incorporation in skin specimens treated with acetone, TPA, or DMBA for 4 wks. +/+, Ikk{alpha}+/+; +/–, Ikk{alpha}+/–. B, levels of p-ERK and ERK1/2 in skin specimens treated with TPA for 4 wks, detected by Western blotting. ß-Actin, loading control. C, relative levels of indicated growth factor and cytokine mRNA in the skin specimens, detected by real-time PCR. D, ERK activities in Ikk{alpha}+/– (+/–) and Ikk{alpha}+/+ (+/+) primary cultured keratinocytes, which were starved overnight before TPA treatment, detected by Western blotting. ß-Actin, protein loading control. E, relative levels of indicated growth factor mRNA in primary cultured keratinocytes as detected by real-time PCR. Control (C); TPA treatment for 2 h (T). F, levels of p-ERK and ERK in Ikk{alpha}+/+ and Ikk{alpha}+/– skin specimens, papillomas, and carcinomas, detected by Western blotting (WB). G, IKK kinase activity and IKKß levels in papillomas, carcinomas, and skin specimens, detected by immunocomplex kinase assay (KA) with an anti-IKK{gamma} antibody. IKK{gamma} recovery was determined by Western blotting. GST-I{kappa}B{alpha} (1–54 amino acids), IKK kinase substrate; IP, immunoprecipitation; Su-KA, kinase substrate; +/+, Ikk{alpha}+/+; +/–, Ikk{alpha}+/–.

 
Because IKK{alpha} is one subunit of the IKK complex, we then evaluated IKK activity. Kinase assay showed that IKK activity was only slightly increased in TPA-treated Ikk{alpha}+/– compared with that in TPA-treated Ikk{alpha}+/+ skin specimens; the levels of IKKß were similar in these skin specimens (Supplementary Fig. S5). However, the IKK activity was higher in carcinomas than in papillomas, although the IKKß level was not increased in carcinomas (Fig. 5G). The levels of I{kappa}B{alpha} were found to be lower in some carcinomas than in papillomas (Supplementary Fig. S6A). To determine whether IKK{alpha} loss deregulated expression of IKKß and I{kappa}B{alpha}, we examined their levels in the epidermis. Western blotting showed that Ikk{alpha}+/+ and Ikk{alpha}–/– epidermis expressed similar levels of IKKß, I{kappa}B{alpha}, and p65 (Supplementary Fig. S6B). Presumably, the replacement of IKK{alpha} by IKKß in the IKK complex might contribute to the increased IKK kinase activity in IKK{alpha}-deficient cells as suggested by three previous reports (8, 19–21).

Loss of IKK{alpha} promotes VEGF-A expression and blood vessel formation. Our skin carcinogenesis experiments showed that Ikk{alpha}+/– mice developed 11 times more carcinomas than did Ikk{alpha}+/+ mice (Fig. 1C). VEGF-A, an important angiogenesis factor, promotes neovascularization and the onset of tumor invasion (22). We reported previously that overexpression of IKK{alpha} repressed Ras-induced VEGF-A expression; binding of IKK{alpha} to a distal VEGF-A promoter (–2,414 to –2,065 bp) was correlated to a decrease in VEGF-A expression (1). In the present study, we observed elevated VEGF-A expression in Ikk{alpha}+/– compared with that in Ikk{alpha}+/+ skin specimens and keratinocytes (Fig. 5C and E). It has been reported that EGF enhances VEGF-A expression (23). We thus examined whether reduced IKK{alpha} elevated VEGF-A expression and blood vessel formation. ChIP assay showed that EGF treatment reduced binding of IKK{alpha} to the distal VEGF-A promoter, which was associated with elevated EGF-A expression in Ikk{alpha}+/+ keratinocytes (Fig. 6A–C ). Reintroduced IKK{alpha} bound to the distal VEGF-A promoter and repressed VEGF-A expression in Ikk{alpha}–/– keratinocytes (Fig. 6A–C). IKK{alpha} did not bind to the proximal VEGF-A promoter (Fig. 6A), which was consistent with our previous finding (1). Furthermore, we found that TPA and DMBA dramatically enhanced the formation of blood vessels in the skin of Ikk{alpha}+/– mice relative to that in the skin of Ikk{alpha}+/+ mice (Fig. 6D). Significantly more blood microvessels in the skin stroma of Ikk{alpha}+/– mice than in the skin stroma of Ikk{alpha}+/+ mice were also observed after treatment with DMBA/TPA for 14 weeks (Supplementary Fig. S7A and B). Thus, IKK{alpha} loss-enhanced VEGF-A expression might foster the development of carcinomas in Ikk{alpha}+/– mice.


Figure 6
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  		Figure 6. Reduction in IKK{alpha} expression promotes the expression of VEGF-A and the formation of blood vessels. A, binding of IKK{alpha} to the distal VEGF-A promoter (dist-P), not proximal VEGF-A promoter (pro-P), detected by ChIP with an anti-IKK{alpha} antibody. Ikk{alpha}+/+ (+/+) keratinocytes were starved with medium without EGF overnight and treated with EGF (10 ng/mL). –/–, Ikk{alpha}–/–; Input, PCR control. B, levels of VEGF-A mRNA in Ikk{alpha}+/+ primary cultured keratinocytes that were starved (–) or treated with 10 ng/mL EGF (+) and in Ikk{alpha}–/– primary cultured keratinocytes infected with adenovirus expressing green fluorescent protein (–) or IKK{alpha} (+), detected by real-time PCR. NS, nonspecific PCR bands; GAPDH, loading control. C, comparison of levels of VEGF-A expression in B. *, P < 0.05, t test. +/+, Ikk{alpha}+/+; –/–, Ikk{alpha}–/–. D, comparison of the formation of blood vessels in the skin specimens of Ikk{alpha}+/– and Ikk{alpha}+/– mice treated with acetone, TPA, or DMBA. Six-week-old female Ikk{alpha}+/+ and Ikk{alpha}+/– mice were treated with acetone (200 µL), TPA (2.5 µg in 200 µL acetone), or DMBA (100 µg in 200 µL acetone) once. The skin specimens of the mice were photographed 72 h after treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we found that Ikk{alpha}+/– mice developed many more and larger tumors than did Ikk{alpha}+/+ mice; the latency period for tumor appearance was shorter in Ikk{alpha}+/– mice than in Ikk{alpha}+/+ mice (Fig. 1A–D), indicating that a reduction in IKK{alpha} expression provided a selective growth advantage, which promoted the formation of skin tumors. Elevated ERK activity has been suggested as a signature event for Ras-initiated skin tumors (17). For example, ERK activity and H-Ras mutations were not detected in skin carcinomas that lost the remaining WT Pten allele in Pten+/– mice (17). H-Ras mutations were detected in all the tumors in our mice and elevated ERK activity was observed in carcinomas compared with those in papillomas, which suggested that reduced IKK{alpha} provided a selective growth advantage that cooperated with DMBA-induced H-Ras mutations to promote skin carcinogenesis. Our results further showed that TPA treatment induced excessive ERK activity and excessive expression of EGF, TGF{alpha}, amphiregulin, FGF2, FGF13, and VEGF-A in Ikk{alpha}+/– skin specimens. The elevated mitogenic activity was intrinsic to keratinocytes, which was consistent with a previous study, in which the expression levels of a group of FGFs and ERK activity were dramatically higher in Ikk{alpha}–/– skin and keratinocytes than in Ikk{alpha}+/+ skin and keratinocytes and that reintroduction of IKK{alpha} or kinase-inactive IKK{alpha} repressed ERK activity and expression of FGFs (9). The mitogenic activity was associated with the nuclear function of IKK{alpha} (9). Thus, a reduction in IKK{alpha} expression induced excessive mitogenic activities following TPA treatment, which provided a molecular basis for promoting keratinocyte proliferation and papilloma formation. Elevated ERK activity and excessive expression of growth factors were reported previously to contribute to the malignant conversion during skin carcinogenesis (17, 24). In addition, IKK{alpha} loss enhanced expression of VEGF-A that was likely to be important for tumor invasion (Fig. 6A–D; Supplementary S7; ref. 25). Thus, IKK{alpha} loss-mediated mitogenic and angiogenic activities might facilitate malignant conversion as well.

We showed previously that elevated IKK{alpha} expression repressed chemical carcinogen-induced mitogenic and angiogenic activities in the epidermis and the dermis of Lori.IKK{alpha} transgenic mice (1). The IKK{alpha} transgenic mice developed significantly fewer malignant carcinomas and metastases than did WT mice. Here, we report that reduced IKK{alpha} expression promoted chemical carcinogen- or growth factor–induced mitogenic and angiogenic activities and that Ikk{alpha}+/– mice were far more prone to skin carcinogenesis than were Ikk{alpha}+/+ mice. These findings suggest that IKK{alpha}-associated mitogenic and angiogenic activities are important mechanism in repressing or promoting skin carcinogenesis.

Moreover, we observed elevated IKK kinase activity in carcinomas compared with that in papillomas, although the levels of IKKß were not elevated in the carcinomas. In addition, the levels of I{kappa}B{alpha} were reduced in some carcinomas (Fig. 5G; Supplementary S6A). Loss of IKK{alpha} was not found to elevate the levels of IKKß or down-regulate the levels of I{kappa}B{alpha} in the epidermis (Supplementary Fig. S6B). Previous findings suggested that IKKß replacement for IKK{alpha} in the IKK complex elevated the IKK kinase activity in IKK{alpha}-deficient cells because IKKß showed a stronger kinase activity for I{kappa}Bs than did IKK{alpha} (8, 19–21). Thus, IKK{alpha} loss or other indirect causes might be involved in the IKK kinase activation in the carcinomas. Increased IKK kinase activity has been implied to promote tumor progression through a variety of avenues (26). Increased IKKß-dependent IKK activity was reported to promote human cancer development and colitis-associated cancer in mice (27, 28). Reduced I{kappa}B{alpha} levels and increased NF-{kappa}B activities were observed previously in chemical carcinogen-induced carcinomas compared with those in papillomas (29). In addition, IKK{alpha} loss was reported to promote the expression of cytokines, such as TNF{alpha} (19), and we also detected elevated expression of TNF{alpha} in IKK{alpha}-deficient keratinocytes (data not shown). Thus, elevated IKK/NF-{kappa}B activity might promote skin carcinogenesis in Ikk{alpha}+/– mice, although the detailed mechanisms remain to be elucidated.

We previously reported somatic Ikk{alpha} mutations in human SCCs (1). In this study, we also observed Ikk{alpha} mutations in carcinomas and papillomas, suggesting that the Ikk{alpha} gene might be a susceptible target for mutagenesis during skin carcinogenesis. Recently, Greenman et al. (30) reported that Ikk{alpha} (Chuk) mutations were frequently detected in human cancers after examining mutations in 518 genes. We found more transition mutations (T->C and A->G) than transversion mutations (Supplementary Tables S4 and S5), which was consistent with our previous report (1). Some of the mutations resulted in changed amino acids, which might destabilize IKK{alpha} or alter IKK{alpha} activity. Some of the mutations created the stop codon, which presumably generated a truncated IKK{alpha} protein. We also detected deletions and insertions in the IKK{alpha} transcripts, which caused frameshift mutations for IKK{alpha}. Seemingly, there were more Ikk{alpha} mutations in carcinomas than in papillomas, suggesting that the increased numbers of Ikk{alpha} mutations might contribute to destabilization of IKK{alpha} in skin carcinomas. Furthermore, we found some repeated mutations in different tumors, which might be "hotspots." We also noticed more Ikk{alpha} mutations in DMBA/TPA–induced mouse SCCs than those in human SCCs (1), which might be due to repeated treatments with a high dose of TPA for inducing skin tumors in mice. It is known that TPA can increase the amount of intracellular oxidative damage that influences the potential for DNA damage in cells (31). However, we previously observed markedly reduced IKK{alpha} expression in poorly differentiated human SCCs (1). Possibly, in addition to Ikk{alpha} mutations, IKK{alpha} in human SCCs can be down-regulated by alternative pathways. For example, p63 was reported to regulate IKK{alpha} expression in the formation of the epidermis, and deregulated p63 expression was involved in skin tumor development (32–34). Oncogenic stress, mutations, or unbalanced alleles can cause LOH of genes in tumors (14, 35, 36). We observed that 44% of the Ikk{alpha}+/– papillomas and 95% of the Ikk{alpha}+/– carcinomas lost the remaining WT Ikk{alpha} allele. Ikk{alpha}+/– mice developed 11 times more carcinomas than did Ikk{alpha}+/+ mice. These results suggested that the integrity of the Ikk{alpha} gene was important for suppressing malignant conversion. The development of malignancies is complex; more mechanisms remain to be revealed.


   Acknowledgments
 
Grant support: National Cancer Institute grants CA102510 and CA117314 (Y. Hu), CA105345 (S.M. Fischer), and CA16672 (comprehensive center grant) and National Institute of Environmental Health Science grant ES07784.

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.

We thank L. Schroeder and S. Hensley for analyzing Ikk{alpha} mutations; I.B. Gimenez-Conti and N.W. Abbey for doing immunohistochemical and histological examinations; H. Thames for conducting the statistical analyses; K. Bouic (University of California, San Diego, La Jolla, CA) for generating adenoviruses; M. Aldaz and M. MacLeod for helpful discussions; and V. Edwards for editorial assistance.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

E. Park and F. Zhu contributed equally to this work.

1 http://sciencepark.mdanderson.org/histology/ihc.html Back

2 http://sciencepark.mdanderson.org/mbcore/ihc.html Back

Received 2/12/07. Revised 5/11/07. Accepted 6/ 4/07.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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