The role of lipid peroxidation products and oxidative stress in activation of the canonical wingless-type MMTV integration site (WNT) pathway in a rat model of diabetic retinopathy

Our recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes.

Caracterización del genoma del virus like-MMTV integrado en la línea del celular de adenocarcinoma pulmonar humano INER-51y análisis de su expresión.

Tesis (Maestría en Ciencias con Acentuación en Microbiología) UANL, 2012.

Regulation of wingless-type MMTV integration site family (WNT) signalling in pancreatic islets from wild-type and obese mice

TCF7L2 is a type 2 diabetes susceptibility gene and downstream effector of canonical wingless-type MMTV integration site family (WNT) signalling. However, it is unknown whether this pathway is active in adult pancreatic islets in vivo, and whether it is regulated in obesity.

MOUSE MAMMARY TUMOR VIRUS EPITOPES EXPRESSED ON TUMOR SPECIFIC TRANSPLANTATION ANTIGENS OF CHEMICALLY INDUCED C3H/HEJ SARCOMAS (TSTA, FIBROSARCOMA, CARCINOGENESIS, MMTV, METHYLCHOLANTHRONE)

This dissertation addressed the hypothesis that the unique tumor specific transplantation antigens (TSTA) of chemically induced sarcomas express epitopes encoded by endogenous viral genes. TSTA from two 3-methylcholanthrene-induced, C3H/HeJ fibrosarcomas (MCA-F and MCA-D) were serologically assessed for viral epitopes in an enzyme-linked immunospecific assay (ELISA) and by immunoaffinity chromatography. Initial evidence with an anti-TSTA antiserum suggested that TSTA were associated with mouse mammary tumor virus (MMTV) peptides, but not peptides from murine leukemia virus (MuLV). TSTA extracted from MCA-F, was assessed with specific anti viral antibodies at three levels of purification for its association with MuLV peptides (gp 70 and p 15E) and MMTV peptides (gp52, gp36 and p27). The results demonstrate that purified preparations enriched for TSTA activity are devoid of MuLV epitopes, but enriched for a subset of MMTV epitopes. Immunoaffinity supports constructed with anti-MMTV antibodies retained TSTA from partially purified MCA-F or MCA-D extracts. Immunoaffinity chromatography with antibodies against individual MMTV peptides demonstrated that the MCA-F TSTA was specifically retained by anti-gp36 and anti-p27 supports, but not by anti-gp52 supports nor a support made with bovine serum albumin. Analysis of the affinity purified TSTA preparations by HPGPC and SDS-PAGE revealed only a few components. Application of the anti-gp36 and anti-p27 retained materials to HPGPC and subsequent in vivo analysis demonstrated that the TSTA migrated in a low and a high molecular weight region. These results suggest that TSTA specificity in C3H/HeJ mice, results from MMTV recombinant proteins. ^

MMTV-trBrca 1 mice display strain-dependent abnormalities in vaginal development

The breast cancer susceptibility gene Brca1 encodes a large multi-functional protein which is implicated as a caretaker of the genome, through its role in regulation of DNA damage response pathways, including apoptosis. Here we show that in mice expressing a dominant-negative Brca1 transgene on a BALB/c background, vaginal entrance remodeling is inhibited, and that the incidence of this phenotype is increased on a p53 +/- genotype. Given that this developmental process is mediated primarily by apoptosis, we hypothesized that disruption of BRCA1 may confer a resistance to apoptosis in normal epithelial cells. Consistent with this, we show that expression of this transgene in vitro leads to resistance to ionizing radiation induced cell killing in mammary epithelial cells. This is the first time that BRCA1 has been implicated in an apoptosis-mediated normal developmental process.

Identification d'un nouveau gène suppresseur de tumeurs candidat (EphA10) dans les tumeurs mammaires des souris transgéniques MMTV/neu

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Identification d'un nouveau gène suppresseur de tumeurs candidat (EphA10) dans les tumeurs mammaires des souris transgéniques MMTV/neu

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

A fission yeast homolog of Int-6, the mammalian oncoprotein and eIF3 subunit, induces drug resistance when overexpressed

Through a screen to identify genes that induce multi-drug resistance when overexpressed, we have identified a fission yeast homolog of Int-6, a component of the human translation initiation factor eIF3. Disruption of the murine Int-6 gene by mouse mammary tumor virus (MMTV) has been implicated previously in tumorigenesis, although the underlying mechanism is not yet understood. Fission yeast Int6 was shown to interact with other presumptive components of eIF3 in vivo, and was present in size fractions consistent with its incorporation into a 43S translation preinitiation complex. Drug resistance induced by Int6 overexpression was dependent on the AP-1 transcription factor Pap1, and was associated with increased abundance of Pap1-responsive mRNAs, but not with Pap1 relocalization. Fission yeast cells lacking the int6 gene grew slowly. This growth retardation could be corrected by the expression of full length Int6 of fission yeast or human origin, or by a C-terminal fragment of the fission yeast protein that also conferred drug resistance, but not by truncated human Int-6 proteins corresponding to the predicted products of MMTV-disrupted murine alleles. Studies in fission yeast may therefore help to explain the ways in which Int-6 function can be perturbed during MMTV-induced mammary tumorigenesis.

Influence of osteocytes in the in vitro and in vivo β-tricalcium phosphate-stimulated osteogenesis

Osteocytes, known to act as the main regulators of bone homeostasis, have become a major focus in the field of bone research. Bioactive ceramics have been widely used for bone regeneration. However, there are few studies about the interaction of osteocytes with bioceramics. The effects of osteocytes on the in vitro and in vivo osteogenesis of bioceramics are also unclear. The aim of this study was to investigate the role of osteocytes on the b-tricalcium phosphate (b-TCP) stimulated osteogenesis. It was found that osteocytes responded to the b-TCP stimulation, leading to the release of Wnt (wingless-related MMTV integration site), which enhanced osteogenic differentiation of bone marrow stromal cells via Wnt signaling pathway. Receptor activator of nuclear factor kappa B ligand, an osteoclast inducer, was also upregulated, indicating that osteocytes would also participated in activation of osteoclasts, which played a major role in the degradation process of b-TCP and new bone remodeling. In vivo studies further demonstrated that when the material was completely embedded by newly formed bone, the only cell contacting with the material was osteocyte. However, the material would eventually be degraded and replaced by the new bone, requiring the participation of osteoclasts and osteoblasts, which were demonstrated by using immunostaining in this study. As the only cell contacting with the material, osteocytes probably acted in a regulatory role to regulate the surrounding osteoclasts and osteoblasts. Osteocytes were also found to participate in the maturation of osteoblasts and the mineralization process of biomaterials, by upregulating E11 (podoplanin) and dentin matrix protein 1 expression. These findings indicated that osteocytes involved in bone biomaterial-mediated osteogenesis and biomaterial degradation, providing valuable insights into the mechanism of material-stimulated osteogenesis, and a novel strategy to optimize the evaluating system for the biological properties of biomaterials.

The ADAMTS1 protease gene is required for mammary tumor growth and metastasis

A disintegrin and metalloprotease with thrombospondin motifs protein 1 (ADAMTS1) is a protease commonly up-regulated in metastatic carcinoma. Its overexpression in cancer cells promotes experimental metastasis, but whether ADAMTS1 is essential for metastatic progression is unknown. To address this question, we investigated mammary cancer progression and spontaneous metastasis in the MMTV-PyMT mouse mammary tumor model in Adamts1 knockout mice. Adamts1−/−/PyMT mice displayed significantly reduced mammary tumor and lung metastatic tumor burden and increased survival, compared with their wild-type and heterozygous littermates. Histological examination revealed an increased proportion of tumors with ductal carcinoma in situ and a lower proportion of high-grade invasive tumors in Adamts1−/−/PyMT mice, compared with Adamts1+/+/PyMT mice. Increased apoptosis with unaltered proliferation and vascular density in the Adamts1−/−/PyMT tumors suggested that reduced cell survival accounts for the lower tumor burden in ADAMTS1-deficient mice. Furthermore, Adamts1−/− tumor stroma had significantly lesser amounts of proteolytically cleaved versican and increased numbers of CD45+ leukocytes. Characterization of immune cell gene expression indicated that cytotoxic cell activation was increased in Adamts1−/− tumors, compared with Adamts1+/+ tumors. This finding is supported by significantly elevated IL-12+ cell numbers in Adamts1−/− tumors. Thus, in vivo ADAMTS1 may promote mammary tumor growth and progression to metastasis in the PyMT model and is a potential therapeutic target to prevent metastatic breast cancer.

Ectopic expression of the Ets transcription factor ER81 in transgenic mouse mammary gland enhances both urokinase plasminogen activator and stromelysin-1 transcription

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.

An immune modulatory role for the fes proto-oncogene

The Fes protein tyrosine kinase is abundantly expressed in phagocytic immune cells, including tumor associated macrophages. Fes knockout mice (fes-/-) display enhanced sensitivity to LPS, and this was shown to be associated with increased NF-κB signaling and TNFα production from fes-/- macrophages. Interestingly, tumor onset in the mouse mammary tumor virus (MMTV-Neu) transgenic mouse model of breast cancer is significantly delayed in fes-/- mice, and this was associated with increased frequency of CD11b+ myeloid and CD3+ T cells in the premalignant mammary glands. Recent studies have also implicated Fes in cross-talk between MHC-I and the NF-κB and IRF-3 pathways in macrophages. Signal 3, the production of inflammatory cytokines and Type I interferons downstream of NF-κB and IRF-3 pathways in antigen presenting cells, is considered an important component of T-cell activation, after engagement of T cell receptor by MHC presented antigen (Signal 1) and co-receptors by their ligands (Signal 2). Using a lymphocytic choriomeningitis virus (LCMV) model of immune activation, I show that LPS stimulated fes-/- macrophages promote more robust activation of LCMV antigenspecific CD8+ T cells than wild type macrophages (fes+/+). Furthermore, LPS stimulated fes-/- macrophages showed increased phosphorylation of NF-B and IRF-3. I also showed that Fes colocalizes with MHC-I in dynamic vesicular structures within macrophages. These observations are consistent with a model where Fes regulates Signal 3 in antigen presenting cells through roles in cross-talk between MHC-I and the NF-kB and IRF-3 signaling pathways. This suggests that Fes plays an immune checkpoint role at the level of Signal 3, and that Fes inhibition could promote tumor immunity through increased Signal 3 driven T cell activation.

Wnt signaling in age-related macular degeneration: human macular tissue and mouse model

BACKGROUND: The wingless-type MMTV integration site (Wnt) signaling is a group of signal transduction pathways. In canonical Wnt pathway, Wnt ligands bind to low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6), resulting in phosphorylation and activation of the receptor. We hypothesize that canonical Wnt pathway plays a role in the retinal lesion of age-related macular degeneration (AMD), a leading cause of irreversible central visual loss in elderly.

METHODS: We examined LRP6 phosphorylation and Wnt signaling cascade in human retinal sections and plasma kallistatin, an endogenous inhibitor of the Wnt pathway in AMD patients and non-AMD subjects. We also used the Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mouse models with AMD-like retinal degeneration to further explore the involvement of Wnt signaling activation in the retinal lesions in those models and to preclinically evaluate the role of Wnt signaling suppression as a potential therapeutic option for AMD.

RESULTS: We found higher levels of LRP6 (a key Wnt signaling receptor) protein phosphorylation and transcripts of the Wnt pathway-targeted genes, as well as higher beta-catenin protein in AMD macula compared to controls. Kallistatin was decreased in the plasma of AMD patients. Retinal non-phosphorylated-β-catenin and phosphorylated-LRP6 were higher in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice than that in wild type. Intravitreal administration of an anti-LRP6 antibody slowed the progression of retinal lesions in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mice. Electroretinography of treated eyes exhibited larger amplitudes compared to controls in both mouse models. A2E, a retinoid byproduct associated with AMD was lower in the treated eyes of Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice. Anti-LRP6 also suppressed the expression of Tnf-α and Icam-1 in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 retinas.

CONCLUSIONS: Wnt signaling may be disturbed in AMD patients, which could contribute to the retinal inflammation and increased A2E levels found in AMD. Aberrant activation of canonical Wnt signaling might also contribute to the focal retinal degenerative lesions of mouse models with Ccl2 and Cx3cr1 deficiency, and intravitreal administration of anti-LRP6 antibody could be beneficial by deactivating the canonical Wnt pathway.

Preactivation of B lymphocytes does not enhance mouse mammary tumor virus infection.

We investigated whether mouse mammary tumor virus (MMTV) favors preactivated or naive B cells as targets for efficient infection. We have demonstrated previously that MMTV activates B cells upon infection. Here, we show that polyclonal activation of B cells leads instead to lower infection levels and attenuated superantigen-specific T-cell responses in vivo. This indicates that naive small resting B cells are the major targets of MMTV infection and that the activation induced by MMTV is sufficient to allow efficient infection.