Thomas R. Berton

Inactivating E2f1 reverts apoptosis resistance and cancer sensitivity in Trp53-deficient mice

The E2f1 transcription factor, which regulates genes required for S-phase entry, also induces apoptosis by transcriptional and post-translational mechanisms. As E2f1 is inducible by DNA damage we investigated its importance in vivo in ultraviolet (UV)-induced apoptosis, a protective mechanism that prevents the epidermis from accumulating UV-induced mutations. Contrary to expectation, E2f1−/− mice demonstrated enhanced keratinocyte apoptosis after UVB exposure, whereas apoptosis was suppressed by epidermis-specific overexpression of human E2F1. Apoptosis induced by γ-radiation was also repressed by E2f1. E2f1−/−;Trp53−/− double knockout mice exhibited the elevated UVB-induced apoptosis of E2f1−/− alone, rather than the profound apoptosis defect seen in Trp53−/− mice, indicating that Trp53 (p53) lies functionally upstream of E2f1. Transfecting E2F1 into E2f1−/−;Trp53−/− primary fibroblasts suppressed UVB-induced apoptosis and this suppression was relieved by Trp53. The double knockout also reverted the abnormal sex ratio and early-onset tumours of Trp53−/− mice. These results imply that E2f1 functions as a suppressor of an apoptosis pathway that is initiated by DNA photoproducts and perhaps genetic abnormalities; p53 relieves this suppression.

Regulation of epidermal apoptosis and DNA repair by E2F1 in response to ultraviolet B radiation

The E2F1 transcription factor regulates the expression of genes involved in cell proliferation, apoptosis and DNA repair. Following DNA damage, E2F1 is phosphorylated and stabilized, but the physiological role of E2F1 in the response to DNA damage is unclear. We find that mice lacking E2F1 have increased levels of epidermal apoptosis compared to wild-type mice following exposure to ultraviolet B (UVB) radiation. Moreover, transgenic overexpression of E2F1 in basal layer keratinocytes suppresses apoptosis induced by UVB. Inhibition of UVB-induced apoptosis by E2F1 is unexpected given that most studies have demonstrated a proapoptotic function for E2F1. E2F1-mediated suppression of apoptosis does not involve alterations in mitogen-activated protein kinase activation or Bcl-2 downregulation in response to UVB and is independent of p53. Instead, inhibition of UVB-induced apoptosis by E2F1 correlates with a stimulation of DNA repair. Mice lacking E2F1 are impaired for the removal of DNA photoproducts, while E2F1 transgenic mice repair UVB-induced DNA damage at an accelerated rate compared to wild-type mice. These findings suggest that E2F1 participates in the response to UVB by promoting DNA repair and suppressing apoptosis.

E2F1 Localizes to Sites of UV-induced DNA Damage to Enhance Nucleotide Excision Repair

The E2F1 transcription factor is a well known regulator of cell proliferation and apoptosis, but its role in the DNA damage response is less clear. Using a local UV irradiation technique and immunofluorescence staining, E2F1 is shown to accumulate at sites of DNA damage. Localization of E2F1 to UV-damaged DNA requires the ATM and Rad3-related (ATR) kinase and serine 31 of E2F1 but not an intact DNA binding domain. E2F1 deficiency does not appear to affect the expression of nucleotide excision repair (NER) factors, such as XPC and XPA. However, E2F1 depletion does impair the recruitment of NER factors to sites of damage and reduces the efficiency of DNA repair. E2F1 mutants unable to bind DNA or activate transcription retain the ability to stimulate NER. These findings demonstrate that E2F1 has a direct, non-transcriptional role in DNA repair involving increased recruitment of NER factors to sites of damage.

Mouse Models for the p53 R72P Polymorphism Mimic Human Phenotypes

The p53 tumor suppressor gene contains a common single nucleotide polymorphism (SNP) that results in either an arginine or proline at position 72 of the p53 protein. This polymorphism affects the apoptotic activity of p53 but the mechanistic basis and physiologic relevance of this phenotypic difference remain unclear. Here, we describe the development of mouse models for the p53 R72P SNP using two different approaches. In both sets of models, the human or humanized p53 proteins are functional as evidenced by the transcriptional induction of p53 target genes in response to DNA damage and the suppression of early lymphomagenesis. Consistent with in vitro studies, mice expressing the 72R variant protein (p53R) have a greater apoptotic response to several stimuli compared with mice expressing the p53P variant. Molecular studies suggest that both transcriptional and nontranscriptional mechanisms may contribute to the differential abilities of the p53 variants to induce apoptosis. Despite a difference in the acute response to UV radiation, no difference in the tumorigenic response to chronic UV exposure was observed between the polymorphic mouse models. These findings suggest that under at least some conditions, the modulation of apoptosis by the R72P polymorphism does not affect the process of carcinogenesis.

Tumor formation in mice with conditional inactivation of Brca1 in epithelial tissues.

The BRCA1 tumor-suppressor protein has been implicated in the regulation of transcription, DNA repair, proliferation, and apoptosis. BRCA1 is expressed in many proliferative tissues and this is at least in part due to E2F-dependent transcriptional control. In this study, inactivation of a conditional murine Brca1 allele was achieved in a variety of epithelial tissues via expression of the Cre recombinase under the control of a keratin 5 (K5) promoter. The K5 Cre:Brca1 conditional knockout mice exhibited modest epidermal hyperproliferation, increased apoptosis, and were predisposed to developing tumors in the skin, the inner ear canal, and the oral epithelium after 1 year of age. Overexpression of the E2F1 transcription factor in K5 Cre:Brca1 conditional knockout mice dramatically accelerated tumor development. In addition, Brca1 heterozygous female mice that had elevated E2F1 expression developed tumors of the reproductive tract at high incidence. These findings demonstrate that in mice Brca1 functions as a tumor suppressor in other epithelial tissues in addition to the mammary gland. Moreover, inactivation of Brca1 is shown to cooperate with deregulation of the Rb-E2F1 pathway to promote tumorigenesis.

SAGE profiling of UV-induced mouse skin squamous cell carcinomas, comparison with acute UV irradiation effects

Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV‐induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH‐1 mice epidermis with that of UV‐induced squamous cell carcinomas (SCCs) from SKH‐1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase‐14 (reduced 8.5‐fold in SCCs); cathepsins D and S (reduced 3‐fold and increased 11.3‐fold, respectively, in SCCs); decorin, glutathione S‐transferase omega‐1, hypoxia‐inducible factor 1α, insulin‐like growth factor binding protein‐7, and matrix metalloproteinase‐13 (increased 18‐, 12‐, 12‐, 18.3‐, and 11‐folds, respectively, in SCCs). Chemokine (C‐C motif), ligand 27 (CCL27), which was found downregulated 12.7‐fold in SCCs by SAGE, was also observed to be strongly downregulated 6–24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV‐irradiated versus sham‐treated SKH‐1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV‐treated skin and SCCs included keratins 6 and 16, small proline‐rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.

Developmental stage determines estrogen receptor alpha expression and non-genomic mechanisms that control IGF-1 signaling and mammary proliferation in mice

Insulin like growth factor-1 (IGF-1) stimulates increased proliferation and survival of mammary epithelial cells and also promotes mammary tumorigenesis. To study the effects of IGF-1 on the mammary gland in vivo, we used BK5.IGF-1 transgenic (Tg) mice. In these mice, IGF-1 overexpression is controlled by the bovine keratin 5 promoter and recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 that is seen in women. Studies have shown that BK5.IGF-1 Tg mice are more susceptible to mammary tumorigenesis than wild-type littermates. Investigation of the mechanisms underlying increased mammary cancer risk, reported here, revealed that IGF-1 preferentially activated the PI3K/Akt pathway in glands from prepubertal Tg mice, resulting in increased cyclin D1 expression and hyperplasia. However, in glands from postpubertal Tg mice, a pathway switch occurred and activation of the Ras/Raf/MAPK pathway predominated, without increased cyclin D1 expression or proliferation. We further showed that in prepubertal Tg glands, signaling was mediated by formation of an ERα/IRS-1 complex, which activated IRS-1 and directed signaling via the PI3K/Akt pathway. Conversely, in postpubertal Tg glands, reduced ERα expression failed to stimulate formation of the ERα/IRS-1 complex, allowing signaling to proceed via the alternate Ras/Raf/MAPK pathway. These in vivo data demonstrate that changes in ERα expression at different stages of development direct IGF-1 signaling and the resulting tissue responses. As ERα levels are elevated during the prepubertal and postmenopausal stages, these may represent windows of susceptibility during which increased IGF-1 exposure maximally enhances breast cancer risk.

E2F1 suppresses skin carcinogenesis via the ARF-p53 pathway

The E2F1 transcription factor, which is deregulated in most human cancers by mutations in the p16-cyclin D-Rb pathway, has both oncogenic and tumor-suppressive properties. This is dramatically illustrated by the phenotype of an E2F1 transgenic mouse model that spontaneously develops tumors in the skin and other epithelial tissues but is resistant to papilloma formation when subjected to a two-stage carcinogenesis protocol. Here, this E2F1 transgenic model was used to further explore the tumor-suppressive property of E2F1. Transgenic expression of E2F1 was found to inhibit ras-driven skin carcinogenesis at the promotion stage independent of the type of promoting agent used. E2F1 transgenic epidermis displayed increased expression of p19ARF, p53, and p21Cip1. Inactivation of either p53 or Arf in E2F1 transgenic mice restored sensitivity to two-stage skin carcinogenesis. While Arf inactivation impaired tumor suppression and p21 induction by E2F1, it did not reduce the level of apoptosis observed in E2F1 transgenic mice. Based on these findings, we propose that E2F1 suppresses ras-driven skin carcinogenesis through a nonapoptotic mechanism involving ARF and p53.

Repression of androgen receptor transcription through the E2F1/DNMT1 axis.

Although androgen receptor (AR) function has been extensively studied, regulation of the AR gene itself has been much less characterized. In this study, we observed a dramatic reduction in the expression of androgen receptor mRNA and protein in hyperproliferative prostate epithelium of keratin 5 promoter driven E2F1 transgenic mice. To confirm an inhibitory function for E2F1 on AR transcription, we showed that E2F1 inhibited the transcription of endogenous AR mRNA, subsequent AR protein, and AR promoter activity in both human and mouse epithelial cells. E2F1 also inhibited androgen-stimulated activation of two AR target gene promoters. To elucidate the molecular mechanism of E2F-mediated inhibition of AR, we evaluated the effects of two functional E2F1 mutants on AR promoter activity and found that the transactivation domain appears to mediate E2F1 repression of the AR promoter. Because DNMT1 is a functional intermediate of E2F1 we examined DNMT1 function in AR repression. Repression of endogenous AR in normal human prostate epithelial cells was relieved by DNMT1 shRNA knock down. DNMT1 was shown to be physically associated within the AR minimal promoter located 22 bps from the transcription start site; however, methylation remained unchanged at the promoter regardless of DNMT1 expression. Taken together, our results suggest that DNMT1 operates either as a functional intermediary or in cooperation with E2F1 inhibiting AR gene expression in a methylation independent manner.

Transgenic insulin-like growth factor-1 stimulates activation of COX-2 signaling in mammary glands.

Studies show that elevated insulin‐like growth factor‐1 (IGF‐1) levels are associated with an increased risk of breast cancer; however, mechanisms through which IGF‐1 promotes mammary tumorigenesis in vivo have not been fully elucidated. To assess the possible involvement of COX‐2 signaling in the pro‐tumorigenic effects of IGF‐1 in mammary glands, we used the unique BK5.IGF‐1 mouse model in which transgenic (Tg) mice have significantly increased incidence of spontaneous and DMBA‐induced mammary cancer compared to wild type (WT) littermates. Studies revealed that COX‐2 expression was significantly increased in Tg mammary glands and tumors, compared to age‐matched WTs. Consistent with this, PGE2 levels were also increased in Tg mammary glands. Analysis of expression of the EP receptors that mediate the effects of PGE2 showed that among the four G‐protein‐coupled receptors, EP3 expression was elevated in Tg glands. Up‐regulation of the COX‐2/PGE2/EP3 pathway was accompanied by increased expression of VEGF and a striking enhancement of angiogenesis in IGF‐1 Tg mammary glands. Treatment with celecoxib, a selective COX‐2 inhibitor, caused a 45% reduction in mammary PGE2 levels, attenuated the influx of mast cells and reduced vascularization in Tg glands. These findings indicate that the COX‐2/PGE2/EP3 signaling pathway is involved in IGF‐1‐stimulated mammary tumorigenesis and that COX‐2‐selective inhibitors may be useful in the prevention or treatment of breast cancer associated with elevated IGF‐1 levels in humans.