Characterization of E2F1's oncogenic, apoptotic and tumor-suppressive activities utilizing the K5 mouse model

Though E2F1 is deregulated in most human cancers by mutations of the p16-cyclin D-Rb pathway, it also exhibits tumor suppressive activity. A transgenic mouse model overexpressing E2F1 under the control of the bovine keratin 5 (K5) promoter exhibits epidermal hyperplasia and spontaneously develops tumors in the skin and other epithelial tissues after one year of age. In a p53-deficient background, aberrant apoptosis in K5 E2F1 transgenic epidermis is reduced and tumorigenesis is accelerated. In sharp contrast, K5 E2F1 transgenic mice are resistant to papilloma formation in the DMBA/TPA two-stage carcinogenesis protocol. K5 E2F4 and K5 DP1 transgenic mice were also characterized and both display epidermal hyperplasia but do not develop spontaneous tumors even in cooperation with p53 deficiency. These transgenic mice do not have increased levels of apoptosis in their skin and are more susceptible to papilloma formation in the two-stage carcinogenesis model. These studies show that deregulated proliferation does not necessarily lead to tumor formation and that the ability to suppress skin carcinogenesis is unique to E2F1. E2F1 can also suppress skin carcinogenesis when okadaic acid is used as the tumor promoter and when a pre-initiated mouse model is used, demonstrating that E2F1's tumor suppressive activity is not specific for TPA and occurs at the promotion stage. E2F1 was thought to induce p53-dependent apoptosis through upregulation of p19ARF tumor suppressor, which inhibits mdm2-mediated p53 degradation. Consistent with in vitro studies, the overexpression of E2F1 in mouse skin results in the transcriptional activation of the p19ARF and the accumulation of p53. Inactivation of either p19ARF or p53 restores the sensitivity of K5 E2F1 transgenic mice to DMBA/TPA carcinogenesis, demonstrating that an intact p19ARF-p53 pathway is necessary for E2F1 to suppress carcinogenesis. Surprisingly, while p53 is required for E2F1 to induce apoptosis in mouse skin, p19ARF is not, and inactivation of p19ARF actually enhances E2F1-induced apoptosis and proliferation in transgenic epidermis. This indicates that ARF is important for E2F1-induced tumor suppression but not apoptosis. Senescence is another potential mechanism of tumor suppression that involves p53 and p19ARF. K5 E2F1 transgenic mice initiated with DMBA and treated with TPA show an increased number of senescence cells in their epidermis. These experiments demonstrate that E2F1's unique tumor suppressive activity in two-stage skin carcinogenesis can be genetically separated from E2F1-induced apoptosis and suggest that senescence utilizing the p19ARF-p53 pathway plays a role in tumor suppression by E2F1. ^

The role of Akt activation in mouse skin tumor promotion

The importance of IGF-1/IGF-1R signaling is evident in human cancers including breast, colon, prostate, and lung which have been shown to overexpress IGF-1. Also, serum levels of IGF-1 have been identified as a risk factor for these cancers. IGF-1 has been primarily shown to mediate its mitogenic effects through signaling pathways such as MAPK and PI3K/Akt. In this regard, BK5.IGF-1 transgenic mice were generated and these mice displayed hyperplasia and hyperkeratosis in the epidermis. In addition, these mice were also found to have elevated MAPK, PI3K, and Akt activities. Furthermore, overexpression of IGF-1 in epidermis can act as a tumor promoter. BK5.IGF-1 transgenic mice developed papillomas after initiation with DMBA without further treatment with a tumor promoter such as TPA. Previous data has also shown that inhibition of the PI3K/Akt signaling pathway by the inhibitor LY294002 was able to reduce the number of tumors formed by IGF-1 mediated tumor promotion. The current studies presented demonstrate that Akt may be the critical effector molecule in IGF-1/IGF-1R mediated tumor promotion. We have found that inhibition of PI3K/Akt by LY294002 inhibits cell cycle components, particularly those associated with G1 to S phase transition including Cyclin D1, Cyclin E, E2F1, and E2F4, that are elevated in epidermis of BK5.IGF-1 transgenic mice. We have also demonstrated that Akt activation may be a central theme in early tumor promotion. In this regard, treatment with diverse tumor promoters such as TPA, okadaic acid, chrysarobin, and UVB was shown to activate epidermal Akt and its downstream signaling pathways after a single treatment. Furthermore, overexpression of Akt targeted to the basal cells of the epidermis led to hyperplasia and increased labeling index as determined by BrdU staining. These mice also had constitutively elevated levels of cell cycle components, particularly Cyclin D1, Cyclin E, E2F1, E2F4, and Mdm-2. These mice developed skin tumors following initiation with DMBA and were hypersensitive to the tumor promoting effects of TPA. Collectively, these studies provide evidence that Akt activation plays an important role in the process of mouse skin tumor promotion. ^

Effect of thiazolidinedione, a class of anti-diabetic drugs, on the mouse skin tumorigenesis

Thiazolidinediones (TZDs), a novel class of anti-diabetic drugs, have been known as ligands of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that belongs to the nuclear receptor superfamily. These synthetic compounds improve insulin sensitivity in patients with type II diabetes likely through activating PAPRγ. Interestingly, they were also shown to inhibit cell growth and proliferation in a wide variety of tumor cell lines. The aim of this study is to assess the potential use of TZDs in the prevention of carcinogenesis using mouse skin as a model. ^ We found that troglitazone, one of TZD drugs, strongly inhibited cultured mouse skin keratinocyte proliferation as demonstrated by [3H]thymidine incorporation assay. It also induced a cell cycle G1 phase arrest and inhibited expression of cell cycle proteins, including cyclin D1, cdk2 and cdk4. Further experiments showed that PPARγ expression in keratinocytes was surprisingly undetectable in vitro or in vivo. Consistent with this, no endogenous PPARγ function in keratinocytes was found, suggesting that the inhibition of troglitazone on keratinocyte proliferation and cell cycle was PPARγ-independent. We further found that troglitazone inhibited insulin/insulin growth factor I (IGF-1) mitogenic signaling, which may explains, at least partly, its inhibitory effect on keratinocyte proliferation. We showed that troglitazone rapidly inhibited IGF-1 induced phosphorylation of p70S6K by mammalian target of rapamycin (mTOR). However, troglitazone did not directly inhibit mTOR kinase activity as shown by in vitro kinase assay. The inhibition of p70S6K is likely to be the result of strong activation of AMP activated protein kinase (AMPK) by TZDs. Stable expression of a dominant negative AMPK in keratinocytes blocked the inhibitory effect of troglitazone on IGF-1 induced phosphorylation of p70S6K. ^ Finally, we found that dietary TZDs inhibited by up to 73% mouse skin tumor development promoted by elevated IGF-1 signaling in BK5-IGF-1 transgenic mice, while they had no or little effect on skin tumor development promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV). Since IGF-1 signaling is frequently found to be elevated in patients with insulin resistance and in many human tumors, our data suggest that TZDs may provide tumor preventive benefit particularly to these patients. ^

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

The role of Lmx1b in the control of dorsoventral compartmentalization in the distal mesenchyme of the developing mouse limb

Lmx1b encodes a LIM-homeodomain transcription factor required for dorso-ventral (D-V) patterning in the mesenchyme of the vertebrate limb. In the absence of Lmx1b function, limbs exhibit a bi-ventral pattern indicating that Lmx1b is required for cells to adopt a dorsal cell fate. However, how Lmx1b specifies dorsal cell fates in the mesenchyme of the distal limb is unknown. Lmx1b is initially expressed throughout the dorsal and ventral limb bud mesenchyme, then becomes dorsally restricted around E10.5. At this stage, there is a sharp boundary between Lmx1b expressing and Lmx1b non-expressing cells. How the dorso-ventral Lmx1b expression boundary is formed and maintained is currently unknown. One mechanism that may contribute to establishing and/or maintaining the Lmx1b expression boundary is if the dorsal mesenchyme is a lineage-based compartment, where different groups of non-mingling cells are separated. Compartment formation has been proposed to rely on compartment-specific selector gene activity which functions to restrict cells to a compartment and specifies the identity of cells within that compartment. Based on the evidence that the dorsal limb identity relies on the expression of Lmx1b in the dorsal half of the limb mesenchyme, we hypothesized that Lmx1b might function as a dorsal limb bud mesenchyme selector gene to set up a dorsal compartment. To test this hypothesis, we developed an inducible CreERT2/ loxP based fate mapping approach that permanently marks Lmx1b wild-type and mutant cells and examined the distribution of their descendents in the developing limb. Our data is the first to show that dorso-ventral lineage compartments exist in the limb bud mesenchyme. Furthermore, Lmx1b is required for maintenance of the dorso-ventral compartment lineage boundary. The behavior of Lmx1b mutant cells that cross into the ventral mesenchyme, as well as previous chimera analysis in which mutant cells spread evenly in the ventral limb and form patches in the dorsal side, suggest that cell affinity differences prevent intermingling of dorsal and ventral cells. ^

A physiological role of cytochromes P450 4Fs: Mouse model

Cytochromes P450 4Fs (CYP4F) are a subfamily of enzymes involved in arachidonic acid metabolism with highest catalytic activity towards leukotriene B 4 (LTB4), a potent chemoattractant involved in prompting inflammation. CYP4F-mediated metabolism of LTB4 leads to inactive ω-hydroxy products incapable of initiating chemotaxis and the inflammatory stimuli that result in the influx of inflammatory cells. Our hypothesis is based on the catalytic ability of CYP4Fs to inactivate pro-inflammatory LTB4 which assures these enzymes a pivotal role in the process of inflammation resolution. ^ To test this hypothesis and evaluate the changes in CYP4F expression under complex inflammatory conditions, we designed two mouse models, one challenged with lipopolysaccharide (LPS) as a sterile model of sepsis and the other challenged with a systemic live bacterial infection of Citrobacter rodentium, an equivalent of the human enterobacterium E. coli pathogen invasion. Based on the evidence that Peroxisome Proliferator Activated Receptors (PPARs) play an active role in inflammation regulation, we also examined PPARs as a regulation mechanism in CYP4F expression during inflammation using PPARα knockout mice under LPS challenge. Using the Citrobacter rodentium model of inflammation, we studied CYP4F levels to compare them to those in LPS challenged animals. LPS-triggered inflammation signal is mediated by Toll-like 4 (TLR4) receptors which specifically respond to LPS in association with several other proteins. Using TLR4 knockout mice challenged with Citrobacter rodentium we addressed possible mediation of CYP4F expression regulation via these receptors. ^ Our results show isoform- and tissue-specific CYP4F expression in all the tissues examined. The Citrobacter rodentium inflammation model revealed significant reduction in liver expression of CYP4F14 and CYP4F15 and an up-regulation of gene expression of CYP4F16 and CYP4F18. TLR4 knockout studies showed that the decrease in hepatic CYP4F15 expression is TLR4-dependent. CYP4F expression in kidney shows down-regulation of CYP4F14 and CYP4F15 and up-regulation of CYP4F18 expression. In the LPS inflammation model, we showed similar patterns of CYP4F changes as in Citrobacter rodentium -infected mice. The renal profile of CYP4Fs in PPARα knockout mice with LPS challenge showed CYP4F15 down-regulation to be PPARα dependent. Our study confirmed tissue- and isoform-specific regulation of CYP4F isoforms in the course of inflammation. ^

Metastatic lung cancer in a new mouse model inheriting p53 and latent K-ras mutations

Lung cancer is a devastating disease with very poor prognosis. The design of better treatments for patients would be greatly aided by mouse models that closely resemble the human disease. The most common type of human lung cancer is adenocarcinoma with frequent metastasis. Unfortunately, current models for this tumor are inadequate due to the absence of metastasis. Based on the molecular findings in human lung cancer and metastatic potential of osteosarcomas in mutant p53 mouse models, I hypothesized that mice with both K-ras and p53 missense mutations might develop metastatic lung adenocarcinomas. Therefore, I incorporated both K-rasLA1 and p53RI72HΔg alleles into mouse lung cells to establish a more faithful model for human lung adenocarcinoma and for translational and mechanistic studies. Mice with both mutations ( K-rasLA1/+ p53R172HΔg/+) developed advanced lung adenocarcinomas with similar histopathology to human tumors. These lung adenocarcinomas were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that seen in lung cancer patients. This mouse model also showed gender differences in cancer related death and developed pleural mesotheliomas in 23.2% of them. In a preclinical study, the new drug Erlotinib (Tarceva) decreased the number and size of lung lesions in this model. These data demonstrate that this mouse model most closely mimics human metastatic lung adenocarcinoma and provides an invaluable system for translational studies. ^ To screen for important genes for metastasis, gene expression profiles of primary lung adenocarcinomas and metastases were analyzed. Microarray data showed that these two groups were segregated in gene expression and had 79 highly differentially expressed genes (more than 2.5 fold changes and p<0.001). Microarray data of Bub1b, Vimentin and CCAM1 were validated in tumors by quantitative real-time PCR (QPCR). Bub1b , a mitotic checkpoint gene, was overexpressed in metastases and this correlated with more chromosomal abnormalities in metastatic cells. Vimentin, a marker of epithelial-mesenchymal transition (EMT), was also highly expressed in metastases. Interestingly, Twist, a key EMT inducer, was also highly upregulated in metastases by QPCR, and this significantly correlated with the overexpression of Vimentin in the same tumors. These data suggest EMT occurs in lung adenocarcinomas and is a key mechanism for the development of metastasis in K-ras LA1/+ p53R172HΔg/+ mice. Thus, this mouse model provides a unique system to further probe the molecular basis of metastatic lung cancer.^

Significance of signal transducer and activator of transcription 3 during multistage mouse skin carcinogenesis

Signal transducer and activator of transcription 3 (Stat3) is a signaling molecule that transduces signal from cell surface receptors, itself translocates into the nucleus, binds to consensus promoter sequences and activates gene transcription. Here, we showed that Stat3 is constitutively activated in both premalignant tumors (papillomas) and squamous cell carcinomas of mouse skin that is induced by topical treatment with an initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by a tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Additional data demonstrated that epidermal growth factor signaling contributes to the activation of Stat3 in this model. Using mice where Stat3 function is abrogated in keratinocytes via the Cre-LoxP system (K5Cre.Stat3 flox/flox), we demonstrated that Stat3 is required for de novo carcinogenesis since Stat3 deficiency leads to a complete abrogation of skin tumor development induced by DMBA and TPA. We subsequently showed that Stat3 plays a role in both the initiation and promotion stages of carcinogenesis. During initiation, Stat3 functions as an anti-apoptotic molecule for maintaining the survival of DNA-damaged keratinocyte stem cells. During promotion, Stat3 functions as a critical regulator for G1 to S phase cell cycle progression to confer selective clonal expansion of initiated cells into papillomas. On the other hand, using transgenic mice over-expressing a constitutively dimerized form of Stat3 (Stat3C) in keratinocytes (K5.Stat3C), we revealed a role for Stat3 in tumor progression. After treatment with DMBA and TPA, K5.Stat3C transgenic mice developed skin tumors with a shorter latency when 100% bypassed the premalignant stage and became carcinoma in situ. Histological and immunohistochemical analysis revealed these tumors as highly vascularized and poorly differentiated. More strikingly, these tumors exhibited invasion into surrounding mesenchymal tissue, some of which metastasized into lung. The tumor-mesenchymal front was characterized by partial loss of E-cadherin and elevation of vimentin, markers characterizing epithelial-mesenchymal transition. On the other hand, inhibition of Stat3 via a decoy oligonucleotide led to a significant reduction of tumor size in approximately 50% of all papillomas tested. In conclusion, we demonstrated that Stat3 plays a critical in all three stages (initiation, promotion and progression) of skin carcinogenesis, and it may potentially become a good target for cancer prevention and anti-cancer therapy. ^

Requirement of GCN5 histone acetyltransferase in mouse neural tube closure and skeletal patterning

Histone acetylation plays an essential role in many DNA-related processes such as transcriptional regulation via modulation of chromatin structure. Many histone acetytransferases have been discovered and studied in the past few years, but the roles of different histone acetyltransferases (HAT) during mammalian development are not well defined at present. Gcn5 histone acetyltransferase is highly expressed until E16.5 during development. Previous studies in our lab using a constitutive null allele demonstrated that Gcn5 knock out mice are embryonic lethal, precluding the study of Gcn5 functions at later developmental stages. The creation of a conditional Gcn5 null allele, Gcn5flox allele, bypasses the early lethality. Mice homozygous for this allele are viable and appear healthy. In contrast, mice homozygous for a Gcn5 Δex3-18 allele created by Cre-loxP mediated deletion display a phenotype identical to our original Gcn5 null mice. Strikingly, a Gcn5flox(neo) allele, which contain a neomycin cassette in the second intron of Gcn5 is only partially functional and gives rise to a hypomorphic phenotype. Initiation of cranial neural tube closure at forebrain/midbrain boundary fails, resulting in an exencephaly in some Gcn5flox(neo)/flox(neo) embryos. These defects were found at an even greater penetrance in Gcn5flox(neo)/Δ embryos and become completely penetrant in the 129Sv genetic background, suggesting that Gcn5 controls mouse neural tube closure in a dose dependent manner. Furthermore, both Gcn5flox(neo)/flox(neo) and Gcn5 flox(neo)/Δ embryos exhibit anterior homeotic transformations in lower thoracic and lumbar vertebrae. These defects are accompanied by decreased expression levels and a shift in anterior expression boundary of Hoxc8 and Hoxc9. This study provides the first evidence that Gcn5 regulates Hox gene expression and is required for normal axial skeletal patterning in mice. ^

Ser88 phosphorylation regulates dynein light chain 1 (DLC1) function in the mouse mammary gland

Dynein light chain 1 (DLC1) is a highly conserved and ubiquitously expressed protein which might have critical cellular function as total loss of DLC1 caused Drosophila embryonic death. Despite many proteins and RNAs interaction with it identified, DLC1's function(s) and regulation are largely unknown. Recently, DLC1 was identified as a physiological substrate of P21-activate kinase 1(Pak1) kinase from a human mammary cDNA library in a yeast-2-hybridization screening assay. Studies in primary human tumors and cell culture implicated that DLC1 could promote mammary cancerous phenotypes, and more importantly, Ser88 phosphorylation of DLC1by Pak1 kinase was found to be essential for DLC1's tumorigenic activities. Based on the above tissue culture studies, we hypothesized that Ser88 phosphorylation regulates DLC1. ^ To test this hypothesis, we generated two transgenic mouse models: MMTV-DLC1 and MMTV-DLC1-S88A mice with mammary specific expression of the DLC1 and DLC1-S88A cDNAs. Both of the transgenic mice mammary glands showed rare tumor incidence which indicated DLC1 alone may not be sufficient for tumorigenesis in vivo. However, these mice showed a significant alteration of mammary development. Mammary glands from the MMTV-DLC1 mice had hyperbranching and alveolar hyperplasia, with elevated cell proliferation. Intriguingly, these phenotypes were not seen in the mammary glands from the MMTV-S88A mice. Furthermore, while MMTV-DLC1 glands were normal during involution, MMTV-S88A mice showed accelerated mammary involution with increase apoptosis and altered expression of involution-associated genes. Further analysis of the MMTV-S88A glands showed they had increased steady state level of Bim protein which might be responsible for the early involution. Finally, our in vitro data showed that Ser88 phosphorylation abolished DLC1 dimer and consequently might disturb its interaction with Bim and destabilize Bim. ^ Collectively, our findings provided in vivo evidence that Ser88 phosphorylation of DLC1 can regulate DLC1's function. In addition, Ser88 phosphorylation might be critical for DLC1 dimer-monomer transition. ^

Identification of germ cell tumor modifier genes from the 129.MOLF-Chr19 consomic mouse strain

Naturally occurring genetic variants confer susceptibility to disease in the human population, including in testicular germ cell tumor development. Disease susceptibility loci for testicular germ cell tumors have been identified by genetic mapping in humans and mice. However, the identity of many of the susceptibility genes remains unclear. My study utilized a chromosome substitution strain, the 129.MOLF-Chr 19 (or M19 strain), to identify candidate testicular germ cell tumor susceptibility genes. Males of this strain have a high incidence of germ cell tumors in the testes. By forward genetic approaches, five susceptibility loci were fine-mapped and the genetic interactions were dissected. In addition, I identified three protein-coding genes and one micro-RNA as testicular tumor susceptibility genes by genomic screening. Using reverse genetic approaches, I verified one of the candidates, Splicing factor 1, as a modifier of testicular tumor. Deficiency of SF1 significantly reduces the incidence of testicular tumors in mice. This study highlights the advantage of the 129.MOLF-Chr 19 consomic strain in disease gene identification and validation. It also sets the stage to elucidate the molecular mechanisms of tumorigenesis in the testis. ^