Tim Rupp

Overexpression of insulin-like growth factor-1 induces hyperplasia, dermal abnormalities, and spontaneous tumor formation in transgenic mice

Transgenic animals were developed to assess the role of insulin-like growth factor 1 (IGF-1) in skin growth, differentiation and organization, as well as its importance in tumor formation. Expression of a human IGF-1 cDNA was targeted to the interfollicular epidermis of transgenic mice using a human keratin 1 promoter construct (HK1). Transgenic animals (HK1.IGF-1 mice) could be identified at birth by early ear unfolding and excessive ear and skin growth compared to non-transgenic littermates. Further examination of the skin from these mice showed epidermal hyperplasia and hyperkeratosis, marked thickening of the dermis and hypodermis, and early hair follicle generation in newborns. The severity of this phenotype correlated with transgene expression both of which subsided with age. Adult HK1.IGF-1 mice developed spontaneous tumors following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) alone and exhibited an exaggerated epidermal proliferative response following treatment with the tumor promoter compared to non transgenic littermates. Additionally, HK1.IGF-1 transgenic mice developed papillomas faster and in markedly greater numbers compared to non-transgenic littermates in standard initiation-promotion experiments. The data presented suggest an important role for IGF-1 in the process of multistage carcinogenesis in mouse skin.

Altered expression of epidermal growth factor receptor ligands in tumor promoter-treated mouse epidermis and in primary mouse skin tumors induced by an initiation-promotion protocol

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor α (TGFα), heparin‐binding epidermal growth factor (HB‐EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFα mRNA and protein are upregulated in epidermis during tumor‐promoter treatment of mouse skin and in skin tumors produced by initiation‐promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or chrysarobin or a single full‐thickness wound induced the expression of HB‐EGF and AR in mRNA samples isolated from whole mouse skin. However, only full‐thickness wounding significantly elevated expression of the BTC transcript. The levels of HB‐EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation‐promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full‐thickness wounding significantly elevated KGF transcript levels in whole‐skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands. Mol. Carcinog. 22:73–83, 1998.

Severe follicular hyperplasia and spontaneous papilloma formation in transgenic mice expressing the neu oncogene under the control of the bovine keratin 5 promoter

Transgenic mice were developed to explore the role of the erbB2 during epithelial homeostasis and tumorigenesis, through targeted expression of the neu oncogene (neu*). Expression of a neu* cDNA was targeted to the basal layer of skin epidermis as well as other epithelial tissues of transgenic mice via the bovine keratin 5 promoter. Two transgenic founders were obtained that were morphologically distinguishable from non‐transgenic littermates by their visibly thickened skin and patchy hair growth by day 3 after birth. The presence of the transgene was confirmed by polymerase chain reaction analysis of tail DNA and immunofluorescence analysis of neu* protein in skin sections. Histological evaluation revealed significant hyperplasia of the follicular and interfollicular epidermis, the abnormal presence of horny material in the dermis and hypodermis, and a dramatic increase in epidermal proliferation. Many areas of the dermis involving this abnormal epithelial proliferation exhibited a squamous cell carcinoma–like appearance. In addition, there was unusual proliferation of the sebaceous glands. One founder died at day 14 and the other at day 20. The latter founder had two papillomas at the time of death. Additional phenotypic changes resulting from the expression of neu* in other tissues included hyperkeratosis in the forestomach and esophagus. In addition, there was a lack of distinction of the cortical‐medullary boundaries and an increased rate of cell death in lymphocytes in the thymus. The phenotypic changes in these other tissues correlated with transgene expression. The data suggest that erbB2 signaling has an important role in epidermal proliferation. In addition, the data provide strong support for a role for erbB2 signaling during epidermal carcinogenesis in mouse skin. Mol. Carcinog. 21:2–12, 1998.

Enhancement of susceptibility to diverse skin tumor promoters by activation of the insulin-like growth factor-1 receptor in the epidermis of transgenic mice.

Insulin‐like growth factor‐1 (IGF‐1) and its receptor are believed to play an important role in mitogenesis and neoplastic transformation. The purpose of this study was to further examine the role of IGF‐1 during tumor promotion in mouse skin. HK1.IGF1 transgenic mice, which overexpress IGF‐1 in epidermis via the human keratin 1 promoter, were previously shown to be hypersensitive to skin tumor promotion by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). We examined these mice for their sensitivity to diverse classes of tumor‐promoting agents. HK1.IGF‐1 transgenic mice initiated with 7,12‐dimethylbenz[a]anthracene were more sensitive to treatment with a wide variety of tumor promoters, including chrysarobin, okadaic acid, and benzoyl peroxide, which resulted in more rapid development of tumors and a dramatic increase in the number of tumors per mouse compared with corresponding non‐transgenic mice treated with the same compounds. Histological analyses of skin from HK1.IGF‐1 mice treated with various tumor promoters revealed that these mice were also more sensitive to the induction of epidermal hyperplasia and cell proliferation. Analysis of the IGF‐1 receptor (IGF‐1r) and epidermal growth factor (EGFr) in the epidermis of TPA‐treated HK1.IGF‐1 transgenic and non‐transgenic mice revealed that both receptors were activated (hyperphosphorylated on tyrosine residues), and the level of activation was higher in transgenic mice. The mechanism for the increased sensitivity of HK1.IGF‐1 mice to tumor promoters may involve cooperation between the IGF‐1r and EGFr signaling pathways. Our data suggest that IGF‐1r signaling may play an important role in the process of tumor promotion by diverse classes of tumor promoters. Mol. Carcinog. 25:122–131, 1999.

Altered expression of insulin-like growth factor I and its receptor during multistage carcinogenesis in mouse skin.

We examined the possible role of insulin‐like growth factor‐I (IGF‐I) and IGF‐I receptor (IGF‐Ir) during multistage carcinogenesis in mouse skin. For this purpose, the expression of both IGF‐I and IGF‐Ir was investigated in mouse skin during tumor promoter treatment and in primary papillomas and squamous cell carcinomas (SCCs) obtained from SENCAR mice treated with standard initiation‐promotion regimens. IGF‐I transcripts were not detectable or only weakly detectable in normal SENCAR mouse epidermis by northern or reverse transcription (RT)‐polymerase chain reaction (PCR) analysis, respectively, whereas IGF‐I transcripts (primarily a 7.0‐kb transcript) were readily detected in RNA preparations from the dermis by both northern blot analysis and RT‐PCR analysis. In contrast, IGF‐Ir transcripts were observed in RNA samples from both epidermis and dermis of control SENCAR mice. Single and multiple topical treatments with 3.4 nmol of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) had no effect on dermal or epidermal IGF‐I and IGF‐Ir mRNA levels. In contrast, the levels of IGF‐I transcripts were elevated (2.5‐ to 15‐fold) in a significant number of mouse skin tumors (71% of all tumors examined). Transcripts of 7.0, 2.5, and 1.3 kb were more consistently overexpressed in skin tumors compared with epidermis, whereas the two smaller transcripts were most consistently overexpressed compared with the dermis. The levels of an 11.0‐kb IGF‐Ir transcript were also elevated (2.5‐ to 8‐fold) in some papillomas (20%) and SCCs (55%), but the percentage of tumors exhibiting this property (32% of all tumors examined) was lower than the percentage overexpressing IGF‐I. These data suggest that altered expression of IGF‐I and IGF‐Ir may play a role in multistage carcinogenesis in the mouse skin model. The inability of TPA to induce elevated IGF‐I or IGF‐Ir expression suggests that these changes in skin tumors are coincident with tumor formation and not a direct result of altered epidermal proliferation per se. Altered expression of IGF‐I in a high percentage of papillomas may indicate that IGF‐I has an important role in the development of autonomous growth in these tumors. The higher percentage of SCCs with altered levels of IGF‐Ir mRNA may indicate a role for these changes in the later stages (i.e., tumor progression) of carcinogenesis in this model system.