David K. Bol

Tumor Development by Transgenic Expression of a Constitutively Active Insulin-Like Growth Factor I Receptor

The insulin-like growth factor I receptor (IGF-IR) is a transmembrane tyrosine kinase that is essential to growth and development and also thought to provide a survival signal for the maintenance of the transformed phenotype. There has been increasing interest in further understanding the role of IGF-I signaling in cancer and in developing receptor antagonists for therapeutic application. We describe herein a novel animal model that involves transgenic expression of a fusion receptor that is constitutively activated by homodimerization. Transgenic mice that expressed the activated receptor showed aberrant development of the mammary glands and developed salivary and mammary adenocarcinomas as early as 8 weeks of age. Xenograft tumors and a cell line were derived from the transgenic animals and are sensitive to inhibition by a novel small-molecule inhibitor of the IGF-IR kinase. This new model should provide new opportunities for further understanding how aberrant IGF-IR signaling leads to tumorigenesis and for optimizing novel antagonists of the receptor kinase.

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.

Constitutive expression of erbB2 in epidermis of transgenic mice results in epidermal hyperproliferation and spontaneous skin tumor development.

The erbB family of receptor tyrosine kinases, which consists of the epidermal growth factor receptor (EGFr/erbB1), erbB2 (neu), erbB3 and erbB4, has been shown to be important for both normal development as well as neoplasia. The expression of rat erbB2 was targeted to the basal layer of mouse epidermis with the bovine keratin 5 promoter. Overexpression of wild type rat erbB2 in the basal layer of epidermis led to alopecia, follicular hyperplasia and sebaceous gland enlargement as well as hyperplasia of the interfollicular epidermis. Spontaneous papillomas, some of which converted to squamous cell carcinomas, arose in homozygous erbB2 transgenic mice as early as 6 weeks of age with >90% incidence by 6 months. Analysis of several proliferation/differentiation markers indicated that erbB2 overexpression led to epidermal hyperproliferation and a possible delay in epidermal differentiation. Transgenic mice were also hypersensitive to the proliferative effects of the skin tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA) and were more sensitive to two-stage carcinogenesis. Elevations in EGFr and erbB2 protein as well as erbB2:EGFr and erbB2:erbB3 heterodimers were observed in skin of the erbB2 transgenic mice. Phosphotyrosine levels of the EGFr, erbB2 and erbB3 proteins were also elevated. These results indicate an important role for erbB2 signaling in epidermal growth, development and neoplasia.

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.

Changes in protein expression during multistage mouse skin carcinogenesis

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12‐dimethylbenz[a]anthracene–initiated, 12‐O‐tetradecanoylphorbol‐13‐acetate–promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor‐β1 (TGFβ1) were strongly expressed in the suprabasal layers, whereas integrin α6β4 was expressed only in basal cells and only moderate staining for transforming growth factor‐α (TGFα) was seen. In hyperplastic skin, TGFα expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin‐binding EGF‐like growth factor (HB‐EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap‐junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of α6β4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20–30 wk), focal areas of γ‐glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFβ1 expression began to decrease. Cx26 and TGFα staining became patchier in some late‐stage papillomas (30–40 wk), whereas suprabasal α6β4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFα, HB‐EGF, TGFβ1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for α6β4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity. Mol. Carcinog. 20:125–136, 1997.

Apoptotic and cytostatic farnesyltransferase inhibitors have distinct pharmacology and efficacy profiles in tumor models

BMS-214662 and BMS-225975 are tetrahydrobenzodiazepine-based farnesyltransferase inhibitors (FTIs) that have nearly identical structures and very similar pharmacological profiles associated with farnesyltransferase (FT) inhibition. Despite their similar activity against FT in vitro and in cells, these compounds differ dramatically in their apoptotic potency and tumor-regressing activity in vivo. BMS-214662 is the most potent apoptotic FTI known and exhibits curative responses in mice bearing a variety of staged human tumor xenografts such as HCT-116 human colon tumor. By contrast, BMS-225975 does not cause tumor regression and at best causes partial tumor growth inhibition in staged HCT-116 human colon tumor xenografts. Lack of tumor regression activity in BMS-225975 was attributable to its relatively weak apoptotic potency, not to poor cell permeability or pharmacokinetics. Both compounds were equally effective in inhibiting Ras processing and causing accumulation of a variety of nonfarnesylated substrates of FT in HCT-116 cells. Because BMS-225975 has poor apoptotic activity compared with BMS-214662 but inhibits FT to the same extent as BMS-214662, it is very unlikely that FT inhibition alone can account for the apoptotic potency of BMS-214662. Clearly distinct patterns of sensitivities in a cell line panel were obtained for the apoptotic FTI BMS-214662 and the cytostatic FTI BMS-225975. Activation of the c-Jun-NH2-terminal kinase pathway was readily observed with BMS-214662 but not with BMS-225975. We developed a highly sensitive San-1 murine xenograft tumor model that is particularly useful for evaluating the in vivo activity of cytostatic FTIs such as BMS-225975.

A Phase I Dose‐Ranging Study of the Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of AVN944, an IMPDH Inhibitor, in Healthy Male Volunteers

A phase I study of AVN944, an inosine monophosphate dehydrogenase (IMPDH) inhibitor, was carried out to assess its safety, tolerability, pharmacokinetics, and pharmacodynamics. Healthy male volunteers (N = 25) participated in this double‐blind, randomized, placebo‐controlled trial. Sixteen received oral doses ranging from 25 to 250 mg on 3 separate occasions at intervals of 3 or 6 days after overnight fasting. Six participants received two 100‐mg doses, and 2 participants received 2 placebo doses, one with food and the other after fasting overnight. Clinical and laboratory parameters, including excretion of AVN944 and thiocyanate and IMPDH inhibition, were made at intervals for 48 hours postdosing. There were 13 mild and 2 moderate but no serious adverse events. One mild and 1 moderate event could be treatment related. AVN944 disappeared rapidly from plasma, but clearance decreased at doses >50 mg. Food reduced absorption, with a geometric mean Cmax ratio of 33% and a geometric mean AUC0‐∞ ratio of 44%. Urinary excretion was negligible. AVN944 doses >100 mg showed definite IMPDH inhibition lasting at least 4 to 6 hours. AVN944, when administered orally to healthy volunteers, is well tolerated, absorbs better with fasting, and exhibits a pharmacodynamic profile that suggests potential for significant anticancer activity.

Constitutively active receptor tyrosine kinases as oncogenes in preclinical models for cancer therapeutics

Receptor tyrosine kinases (RTK) remain an area of therapeutic interest because of their role in epithelial tumors, and experimental models specific to these targets are highly desirable. Chimeric receptors were prepared by in-frame fusion of the CD8 extracellular sequence with the cytoplasmic sequences of RTKs. A CD8HER2 fusion protein was shown to form disulfide-mediated homodimers and to transform fibroblasts and epithelial cells. CD8RTK fusion proteins transform rat kidney epithelial cells and impart phenotypes that may reflect signaling specificity inherent in the native receptors. Transgenic expression of CD8HER2 and CD8Met in mice resulted in the formation of salivary and mammary gland tumors. The transgenic tumors allow the derivation of allograft tumors and cell lines that are sensitive to inhibition by small molecule kinase inhibitors. This approach provides excellent cell and tumor models for the characterization of signaling properties of diverse RTKs and for the evaluation of rationally designed antagonists targeting these kinases. [Mol Cancer Ther 2006;5(6):1571–6]