Overexpression of human Cripto-1 in transgenic mice delays mammary gland development and differentiation and induces mammary tumorigenesis

Overexpression of Cripto-1 has been reported in several types of human cancers including breast cancer. To investigate the role of human Cripto-1 (CR-1) in mammary gland development and tumorigenesis, we developed transgenic mice that express the human CR-1 transgene under the regulation of the whey acidic protein (WAP) promoter in the FVB/N mouse background. The CR-1 transgene was detected in the mammary gland of 15-week-old virgin WAP-CR-1 female mice that eventually developed hyperplastic lesions. From mid-pregnancy to early lactation, mammary lobulo-alveolar structures in WAP-CR-1 mice were less differentiated and delayed in their development due to decreased cell proliferation as compared to FVB/N mice. Early involution, due to increased apoptosis, was observed in the mammary glands of WAP-CR-1 mice. Higher levels of phosphorylated AKT and MAPK were detected in mammary glands of multiparous WAP-CR-1 mice as compared to multiparous FVB/N mice suggesting increased cell proliferation and survival of the transgenic mammary gland. In addition, more than half (15 of 29) of the WAP-CR-1 multiparous female mice developed multifocal mammary tumors of mixed histological subtypes. These results demonstrate that overexpression of CR-1 during pregnancy and lactation can lead to alterations in mammary gland development and to production of mammary tumors in multiparous mice.     

Regulation of Cripto-1 signaling and biological activity by caveolin-1 in mammary epithelial cells

Human and mouse Cripto-1 (CR-1/Cr-1) proteins play an important role in mammary gland development and tumorigenesis. In this study, we examined the relationship between Cripto-1 and caveolin-1 (Cav-1), a membrane protein that acts as a tumor suppressor in the mammary gland. Cripto-1 was found to interact with Cav-1 in COS7 cells and mammary epithelial cells. Using EpH4 mouse mammary epithelial cells expressing Cr-1 (EpH4 Cr-1) or Cr-1 and Cav-1 (EpH4 Cr-1/Cav-1), we demonstrate that Cav-1 expression markedly reduced the ability of Cr-1 to enhance migration, invasion, and formation of branching structures in EpH4 Cr-1/Cav-1 cells as compared to EpH4 Cr-1 cells. Furthermore, coexpression of Cav-1 together with Cr-1 in EpH4 Cr-1/Cav-1 cells inhibited Cr-1-mediated activation of c-src and mitogen-activated protein kinase signaling pathways. Conversely, primary mammary epithelial cells isolated from Cav-1 null(-/-)/mouse mammary tumor virus-CR-1 transgenic animals showed enhanced motility and activation of mitogen-activated protein kinase and c-src as compared to Cav-1(+/-)/CR-1 mammary cells. Finally, mammary tumors derived from mouse mammary tumor virus-CR-1 mice showed a dramatic reduction of Cav-1 expression as compared to mammary tissue from normal FVB/N mice, suggesting that in vivo Cav-1 is down-regulated during the process of CR-1-mediated mammary tumorigenesis.     

Transforming growth factor-alpha, epidermal growth factor receptor, and PCNA immunoexpression in uterine leiomyosarcomas and leiomyomas in B6C3F1 mice.

The role of growth factors in the development of murine uterine mesenchymal tumors is unknown. In this study, immunohistochemical expression of transforming growth factor alpha (TGF-alpha) and its receptor epidermal growth factor (EGF-R) was assessed in spontaneous uterine leiomyomas and leiomyosarcomas in B6C3F1 mice. Cell proliferation, which has been induced by some growth factors, was evaluated by immunohistochemical detection of an endogenous marker of cell proliferation, proliferating cell nuclear antigen (PCNA). PCNA labeling indices were determined and compared to the intensity and distribution of TGF-alpha staining in sequential sections of control myometrium or tumor tissue. Results showed uterine leiomyosarcomas had positive cytoplasmic staining for TGF-alpha; however, all uterine leiomyomas evaluated were negative. Positive EGF-R staining was also observed in the uterine leiomyosarcomas, but not in the leiomyomas. EGF-R immunoexpression was detected primarily within the cytoplasm of the leiomyosarcoma cells, with occasional nuclear immunoreactivity. Immunohistochemical staining for PCNA was more intense and there were increased numbers of positively staining nuclei in the leiomyosarcomas compared to samples of control myometrium or leiomyomas. The mean labeling index for the uterine leiomyosarcomas (7.40%) was significantly (p < 0.01) higher than that of leiomyomas (0.29%) and control uterine myometrium (0.13%). We conclude, that TGF-alpha and its receptor, EGF-R, are expressed more intensely in uterine leiomyosarcomas, compared to leiomyomas in B6C3F1 mice. Immunoexpression of TGF-alpha may be an important biomarker of malignancy in uterine smooth muscle tumors in mice. Futhermore, TGF-alpha may play a critical role in increased proliferation of uterine smooth muscle tumor cells as suggested by increased immunolocalization of PCNA in rodent leiomyosarcomas expressing TGF-alpha, although other factors regulating cell replication can not be ruled out.     

PECAM-1 affects GSK-3beta-mediated beta-catenin phosphorylation and degradation

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) regulates a variety of endothelial and immune cell biological responses. PECAM-1-null mice exhibit prolonged and increased permeability after inflammatory insults. We observed that in PECAM-1-null endothelial cells (ECs), beta-catenin remained tyrosine phosphorylated, coinciding with a sustained increase in permeability. Src homology 2 domain containing phosphatase 2 (SHP-2) association with beta-catenin was diminished in PECAM-1-null ECs, suggesting that lack of PECAM-1 inhibits the ability of this adherens junction component to become dephosphorylated, promoting a sustained increase in permeability. beta-Catenin/Glycogen synthase kinase 3 (GSK-3beta) association and beta-catenin serine phosphorylation levels were increased and beta-catenin expression levels were reduced in PECAM-1-null ECs. Glycogen synthase kinase 3 (GSK-3beta) serine phosphorylation (inactivation) was blunted in PECAM-1-null ECs after histamine treatment or shear stress. Our data suggest that PECAM-1 serves as a critical dynamic regulator of endothelial barrier permeability. On stimulation by a vasoactive substance or shear stress, PECAM-1 became tyrosine phosphorylated, enabling recruitment of SHP-2 and tyrosine-phosphorylated beta-catenin to its cytoplasmic domain, facilitating dephosphorylation of beta-catenin, and allowing reconstitution of adherens junctions. In addition, PECAM-1 modulated the levels of beta-catenin by regulating the activity of GSK-3beta, which in turn affected the serine phosphorylation of beta-catenin and its proteosomal degradation, affecting the ability of the cell to reform adherens junctions in a timely fashion.     

Integrin-linked kinase cytoplasmic and nuclear expression in laryngeal carcinomas

Integrin-linked kinase (ILK) has been implicated in the development and progression of several human malignancies. Previous in vitro studies also implicate ILK in the activation of Akt and beta-catenin as well as in the regulation of E-cadherin expression. However, the role of ILK in human laryngeal cancer and its possible in vivo downstream effectors in the disease are currently unknown. We examined by immunohistochemistry the protein expression of ILK, phosphorylated-Akt (p-Akt), E-cadherin, and beta-catenin in 97 invasive squamous laryngeal carcinomas. Increased cytoplasmic and nuclear expression of ILK and p-Akt decreased membranous expression of E-cadherin and nuclear accumulation of beta-catenin was found in 87.6%, 85.6%, 71.1%, and 43.3% of cases, respectively. Our results suggest that ILK expression may be implicated in human laryngeal carcinoma and its localization in the nucleus possibly proposes novel nuclear functions of this molecule. In addition, enhanced ILK expression correlates with activation of Akt but not with downregulation of E-cadherin and activation of beta-catenin. Finally, in our material while activated Akt seems to characterize well-differentiated tumors, loss of E-cadherin and activation of beta-catenin correlated with high grade carcinomas.     

TGF-beta1 is increased in a transgenic mouse model of familial Alzheimer's disease and causes neuronal apoptosis

Alzheimer's disease (AD) is a neurodegenerative disorder, due to excess amyloid-beta peptide (Abeta). TGF-beta1 and beta-catenin signaling pathways have been separately implicated in modulating Abeta-neurotoxicity. However, the underlying mechanisms remain unclear. Here, we report that TGF-beta1 and nuclear Smad7 and beta-catenin levels were markedly upregulated in cortical brain regions of the TgCRND8 mice, a mouse model of familial Alzheimer's disease. Coimmunoprecipitation of cortical brain tissue lysates revealed an interaction between Smad7 and beta-catenin. This interaction which was significantly enhanced in the TgCRND8 mice was also associated with an increase in TCF/LEF DNA-shift binding activity. TCF/LEF reporter gene activity was significantly increased in mouse primary cortical neuronal cultures (MCN) from the TgCRND8 mice, compared to controls. Interestingly, exposure of MCN to Abeta(1-42) led to an increase in TGF-beta1 and nuclear levels of both beta-catenin and Smad7. Furthermore, addition of TGF-beta1 to the MCN caused an increase in apoptosis and Smad7 levels. When Smad7 or beta-catenin levels were reduced by siRNA, TGF-beta1-induced apoptosis was suppressed, indicating that both Smad7 and beta-catenin are required for TGF-beta1-induced neurotoxicity. Since Abeta(1-42)-induced TGF-beta1, we suggest that TGF-beta1 may amplify Abeta(1-42)-mediated neurodegeneration in AD via Smad7 and beta-catenin interaction and nuclear localization.     

Mutations and nuclear accumulation of beta-catenin correlate with intestinal phenotypic expression in human gastric cancer

AIMS: Abnormal localization of beta-catenin is frequently observed in human gastric cancers. The aim of the present study was to evaluate relationships among gastrointestinal differentiation phenotypes, beta-catenin localization and mutations of Wnt signalling genes. METHODS AND RESULTS: Seventy-seven regions in 39 gastric adenocarcinomas were classified according to beta-catenin localization and gastric and intestinal phenotypes. Cases with membranous beta-catenin localization showed a gradual decrease from gastric (G) (55% = 6/11) and gastric-and-intestinal-mixed (GI) (17% = 5/29) to intestinal (I) (0% = 0/21) phenotypes, while those with nuclear localization showed a concomitant increase: 18% (2/11), 41% (12/29), 95% (20/21) and 63% (10/16) for G, GI, I and null type (N), respectively (P < 0.001, membranous versus nuclear localization in G, GI through I). Mutations in exon 3 of the beta-catenin gene were found in G (50% = 1/2), GI (67% = 8/12), I (45% = 9/20) and N (0% = 0/10) regions with nuclear beta-catenin localization (GI versus N, P < 0.01; I versus N, P < 0.05). Adenomatous polyposis coli (APC) gene mutations were demonstrated only in GI, I and N types, irrespective of beta-catenin localization. Molecular analysis of these genes revealed 10 tumours to be heterogeneous out of 16 informative cases (62.5%). CONCLUSION: Intestinal phenotypic expression is accompanied by a shift from membranous to cytoplasmic/nuclear accumulation of beta-catenin. In contrast, N-type regions may progress along a different pathway.     

Tau overexpression in transgenic mice induces glycogen synthase kinase 3beta and beta-catenin phosphorylation

The abnormal phosphorylations of tau, GSK3beta, and beta-catenin have been shown to perform a crucial function in the neuropathology of Alzheimer's disease (AD). The primary objective of the current study was to determine the manner in which overexpressed htau23 interacts and regulates the behavior and phosphorylation characteristics of tau, GSK3beta, and beta-catenin. In order to accomplish this, transgenic mice expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE/htau23) were created. Transgenic mice evidenced the following: (i) tendency toward memory impairments at later stages, (ii) dramatic overexpression of the tau transgene, coupled with increased tau phosphorylation and paired helical filaments (PHFs), (iii) high levels of GSK3beta phosphorylation with advanced age, resulting in increases in the phosphorylations of tau and beta-catenin, (iv) an inhibitory effect of lithium on the phosphorylations of tau, GSK3beta, and beta-catenin, but not in the non-transgenic littermate group. Therefore, the overexpression of NSE/htau23 in the brains of transgenic mice induces abnormal phosphorylations of tau, GSK3beta, and beta-catenin, which are ultimately linked to neuronal degeneration in cases of AD. These transgenic mice are expected to prove useful for the development of new drugs for the treatment of AD.     

Cyclin D1 overexpression in N-methyl-N′-nitro-N-nitrosoguanidine-induced rat gastric adenocarcinomas

Changes in cell cycle regulation are involved in many human cancers, including gastric cancer. In the present study, cyclin D1 expression and localization were immunohistochemically analyzed in 23 N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric adenocarcinomas and compared with findings for beta-catenin. Cyclin D1 nuclear overexpression was more frequently observed in tumors displaying nuclear (4/4=100%) and cytoplasmic (3/4=75%) beta-catenin accumulation than those with membranous (3/15=20%) localization (nuclear vs. membranous, P<0.02). In the former cases it was considered that cyclin D1 was induced with beta-catenin activation; in the latter, a direct or indirect pathway for cyclin D1 accumulation bypassing Wnt pathway might be involved. Cyclin D1 was also found to be accumulated in gastric glands within normal-looking mucosa, these perhaps representing preneoplastic lesions for cancers with membranous beta-catenin accumulation.     

Vascular endothelial growth factor expression,beta-catenin tyrosine phosphorylation,and endothelial proliferative behavior: a pathway for transformation?

The hypothesis that tumor growth is angiogenesis dependent has been documented by a considerable body of direct and indirect experimental data and has generated intense basic and pharmaceutical-related interest. In contrast, the study of endothelial cell tumors has been modest by comparison. Hemangioma is the most common tumor of any kind seen in infancy and also, perhaps, the least understood. We compared a mouse hemangioma-derived cell line (EOMA) and primary human endothelial cells (HUVEC) for their proliferative behavior and molecular alterations. EOMA cells intrinsically expressed vascular endothelial growth factor (VEGF), which acts in an autocrine manner, resulting in an increase in CD1 expression and cell proliferation, both of which were inhibited by anti-VEGF neutralizing antibodies. Such an autocrine loop is supported by constitutive VEGF receptor (Flk-1) tyrosine phosphorylation, Flk-1 and Flt-1 nuclear localization, and mitogen-activated protein kinase activation. beta-catenin was also found to exhibit significant nuclear localization and constitutively associate with Flk-1 and Flt-1 in EOMA cells but much less so in HUVEC, and immunoprecipitated Flk-1 was able to phosphorylate purified beta-catenin in an immune complex kinase assay. EOMA cells were also noted to express reduced levels of N-cadherin and gamma-catenin compared with HUVEC. Interestingly, sequestration of endogenous VEGF in EOMA cultures resulted in a dramatic decrease in nuclear beta-catenin and a reduction in CD1 levels, whereas addition of exogenous VEGF elicited increased nuclear beta-catenin localization and increased CD1 levels in HUVEC. The possible contributions of VEGF signaling pathways, cell junction component expression levels, and phosphorylation states to endothelial cell transformation and proliferation are discussed.     

Chronic oxidative stress causes amplification and overexpression of ptprz1 protein tyrosine phosphatase to activate beta-catenin pathway

Ferric nitrilotriacetate induces oxidative renal tubular damage via Fenton-reaction, which subsequently leads to renal cell carcinoma (RCC) in rodents. Here, we used gene expression microarray and array-based comparative genomic hybridization analyses to find target oncogenes in this model. At the common chromosomal region of amplification (4q22) in rat RCCs, we found ptprz1, a tyrosine phosphatase (also known as protein tyrosine phosphatase zeta or receptor tyrosine phosphatase beta) highly expressed in the RCCs. Analyses revealed genomic amplification up to eightfold. Despite scarcity in the control kidney, the amounts of PTPRZ1 were increased in the kidney after 3 weeks of oxidative stress, and mRNA levels were increased 16 approximately 552-fold in the RCCs. Network analysis of the expression revealed the involvement of the beta-catenin pathway in the RCCs. In the RCCs, dephosphorylated beta-catenin was translocated to nuclei, resulting in the expression of its target genes cyclin D1, c-myc, c-jun, fra-1, and CD44. Furthermore, knockdown of ptprz1 with small interfering RNA (siRNA), in FRCC-001 and FRCC-562 cell lines established from the induced RCCs, decreased the amounts of nuclear beta-catenin and suppressed cellular proliferation concomitant with a decrease in the expression of target genes. These results demonstrate that chronic oxidative stress can induce genomic amplification of ptprz1, activating beta-catenin pathways without the involvement of Wnt signaling for carcinogenesis. Thus, iron-mediated persistent oxidative stress confers an environment for gene amplification.     

Deregulation of the phosphatidylinositol-3 kinase signaling cascade is associated with neurodegeneration in Npc1-/- mouse brain

Niemann-Pick type C (NPC) disease is caused by mutations to genes that encode proteins critical to intracellular lipid homeostasis. The events underlying NPC progressive neurodegeneration are poorly understood but include neurofibrillary tangles of the type found in Alzheimer's disease. Here we investigated possible contributions of a phosphatidylinositol-3 kinase cascade [PI3K, Akt, glycogen synthase kinase-3beta (GSK-3beta)] that is linked to apoptosis and various degenerative conditions. Brain concentrations of phosphorylated Akt, which phosphorylates and inactivates GSK-3beta, were significantly elevated in Npc1-/- mice relative to Npc1+/+ mice. Accordingly, levels of inactive GSK-3beta were 50 to 100% higher in mutant brains than in controls. Increases in inactive GSK-3beta occurred early in postnatal development, well before neuronal loss, and were most prominent in structures with intracellular cholesterol accumulation, suggesting a contribution to subsequent degeneration. Perturbations of nuclear factor (NF)-kappaB, which is regulated by GSK-3beta, occurred in Npc1-/- mouse brains. Nuclear concentrations and DNA binding activity of NF-kappaB's transactivation subunit, p65, were significantly reduced in Npc1-/- mice compared to Npc1+/+ mice. Cytoplasmic levels of the p50 subunit and its precursor, p105, were higher in Npc1-/- mice. These results suggest that excessive activity in the PI3K-Akt pathway depresses GSK-3beta, thereby disrupting the formation and/or nuclear import of p50/p65 NF-kappaB dimers and contributing to neuronal degeneration.     

Tumor spectrum in the p53 heterozygous zeta globin-promoted Tg.AC (v-Ha-ras) bitransgenic mouse model

The use of a bitransgenic mouse model for cancer is an effective approach for studying the impact of specific carcinogens and the occurrence of tissue-specific lesions. We studied the novel p53 heterozygous zeta globin-promoted Tg.AC (v-Ha-ras) mouse model because these mice contain a carcinogen-inducible ras oncogene and one functional p53 tumor suppressor allele, both of which occur frequently in human cancers. Our aim was to characterize the short-term control and chemically induced tumor spectrum in this novel model. Mice were placed on basal semipurified diet containing 20% soy protein for 2 weeks prior to random allocation to groups. Subsequently, 15 male and 15 female mice were administered corn oil vehicle alone or containing benzo(a)pyrene (20 mg/kg body weight) via oral gavage 2 times per week for 10 weeks with subsequent observation for 18 weeks. Mice exhibited lesions characteristic of FVB/N, p53 heterozygous and Tg.AC mouse models. However, an array of unique, novel lesions were observed including uterine leiomyosarcomas, mammary gland carcinomas, mammary squamous cell carcinomas, and parotid salivary gland carcinomas suggesting tissue-specific interactions of the 2 genotypes. Thus, this bitransgenic model may provide further insight into the mechanistic interaction of 2 genes commonly mutated in neoplasia.     

Beta-catenin accumulation in the progression of human hepatocarcinogenesis correlates with loss of E-cadherin and accumulation of p53, but not with expression of conventional WNT-1 target genes

Beta-catenin integrates intracellular WNT signalling and the intercellular E-cadherin-catenin adhesion system. To date, little is known about the role of beta-catenin activation and nuclear accumulation in hepatocarcinogenesis. This study has analysed beta-catenin expression patterns in human dysplastic nodules (DNs), as well as in hepatocellular carcinomas (HCCs) in comparison with proliferation, expression of WNT-1 target genes, E-cadherin, and p53. One hundred and seventy HCCs and 25 DNs were categorized according to established criteria and analysed for the expression pattern of beta-catenin. Analysis of the proliferative activity and expression of E-cadherin, cyclin D1, MMP-7, c-myc, and p53 was performed on a representative subgroup of cases. All DNs lacked nuclear beta-catenin, while 36% of all HCCs were positive, with the number of nuclear stained cells ranging from less than 1% to more than 90%. Increasing nuclear accumulation of beta-catenin correlated with reduced membranous E-cadherin expression and nuclear p53 but not with proliferation. Cyclin D1, MMP-7, and c-myc expression was detected in 54%, 26%, and 65% of HCCs, respectively, but did not correlate with nuclear beta-catenin, proliferation, or grading. Sequence analysis of the beta-catenin gene revealed no detectable mutations in DNs, but mutations in the GSK-3beta binding site were present in 14.3% of the HCCs. In conclusion, this study has demonstrated that nuclear accumulation of beta-catenin is a frequent progression event in human hepatocarcinogenesis which correlates with nuclear p53 accumulation and loss of membranous E-cadherin, but not with the expression pattern of established WNT-1 target genes. It is hypothesized that the role of beta-catenin in human HCC differs significantly from its established function in colon carcinogenesis.