Intrauterine exposure to bisphenol A promotes different effects in both neonatal and adult prostate of male and female gerbils (Meriones unguiculatus)

Substances that mimic endogenous hormones may alter the cell signaling that govern prostate development and predispose it to developing lesions in adult and senile life. Bisphenol A is able to mimic estrogens, and studies have demonstrated that low levels of exposure to this compound have caused alterations during prostate development. The aim of this study was to describe the prostate development in both male and female neonatal gerbils in normal conditions and under exposure to BPA during intrauterine life, and also to analyze whether the effects of intrauterine exposure to BPA remain in adulthood. Morphological, stereological, three-dimensional reconstruction, and immunohistochemical methods were employed. The results demonstrated that in 1-day-old normal gerbils, the female paraurethral glands and the male ventral lobe are morphologically similar, although its tissue components-epithelial buds (EB), periurethral mesenchyme (PeM), paraurethral mesenchyme (PaM) or ventral mesenchymal pad (VMP), and smooth muscle (SM)-have presented different immunolabeling pattern for androgen receptor (AR), and for proliferating cell nuclear antigen (PCNA). Moreover, we observed a differential response of male and female prostate to intrauterine BPA exposure. In 1-day-old males, the intrauterine exposure to BPA caused a decrease of AR-positive cells in the PeM and SM, and a decrease of the proliferative status in the EB. In contrast, no morphological alterations were observed in ventral prostate of adult males. In 1-day-old females, BPA exposure promoted an increase of estrogen receptor alpha (ERα) positive cells in PeM and PaM, a decrease of AR-positive cells in EB and PeM, besides a reduction of cell proliferation in EB. Additionally, the adult female prostate of BPA-exposed animals presented an increase of AR- and PCNA-positive cells. These results suggest that the prostate of female gerbils were more susceptible to the intrauterine BPA effects, since they became more proliferative in adult life. © 2015 Wiley Periodicals, Inc. Environ Toxicol, 2015.

Bisphenol-A promotes antiproliferative effects during neonatal prostate development in male and female gerbils

Conselho Nacional de Desenvolvimento Científico e Tecnol[ogico (CNPq)

Nitric oxide is responsible for oxidative skin injury and modulation of cell proliferation after 24 hours of UVB exposures

Nitric oxide (NO) is produced by various mammalian cells and plays a variety of regulatory roles in normal physiology and in pathological processes. This article provides evidence regarding the participation of NO in UVB-induced skin lesions and in the modulation of skin cell proliferation following UVB skin irradiation. Hairless mice were subjected to UVB irradiation for 3 hours and the skin evaluated immediately, 6 and 24 hours postirradiation. The skin lipid peroxidation, and NO levels evaluated by chemiluminescence and inducible nitric oxide synthase (iNOS) and nitrotyrosine immunolabelling increased significantly 24 hours after irradiation and decreased under the treatment with aminoguanidine (AG). On the other hand, cell proliferation markers, PCNA and VEGF showed a strong labelling index when AG was used. The data indicate that NO mediates, at least in part, the lipid peroxidation and protein nitration and also promotes the down regulation of factors involved in cell proliferation. This work shows that the NO plays an important role in the oxidative stress damage and on modulation of cell proliferation pathways in UVB irradiated skin.

Serum amyloid A is a growth factor for 3T3-L1 adipocytes, inhibits differentiation and promotes insulin resistance

BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [H-3-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-H-3]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9 +/- 0.54%) compared with the control cells (39.8 +/- 2.2%, ***P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPAR gamma 2 (peroxisome proliferator-activated receptor gamma 2), C/EBP beta (CCAAT/enhancer-binding protein beta) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-H-3]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor alpha, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.

HVEM signalling promotes colitis

Background Tumor necrosis factor super family (TNFSF) members regulate important processes involved in cell proliferation, survival and differentiation and are therefore crucial for the balance between homeostasis and inflammatory responses. Several members of the TNFSF are closely associated with inflammatory bowel disease (IBD). Thus, they represent interesting new targets for therapeutic treatment of IBD. Methodology/Principal Findings We have used mice deficient in TNFSF member HVEM in experimental models of IBD to investigate its role in the disease process. Two models of IBD were employed: i) chemical-induced colitis primarily mediated by innate immune cells; and ii) colitis initiated by CD4+CD45RBhigh T cells following their transfer into immuno-deficient RAG1-/- hosts. In both models of disease the absence of HVEM resulted in a significant reduction in colitis and inflammatory cytokine production. Conclusions These data show that HVEM stimulatory signals promote experimental colitis driven by innate or adaptive immune cells.

FOXA1 Promotes Tumor Progression in Prostate Cancer and Represents a Novel Hallmark of Castration-Resistant Prostate Cancer

Forkhead box protein A1 (FOXA1) modulates the transactivation of steroid hormone receptors and thus may influence tumor growth and hormone responsiveness in prostate cancer. We therefore investigated the correlation of FOXA1 expression with clinical parameters, prostate-specific antigen (PSA) relapse-free survival, and hormone receptor expression in a large cohort of prostate cancer patients at different disease stages. FOXA1 expression did not differ significantly between benign glands from the peripheral zone and primary peripheral zone prostate carcinomas. However, FOXA1 was overexpressed in metastases and particularly in castration-resistant cases, but was expressed at lower levels in both normal and neoplastic transitional zone tissues. FOXA1 levels correlated with higher pT stages and Gleason scores, as well as with androgen (AR) and estrogen receptor expression. Moreover, FOXA1 overexpression was associated with faster biochemical disease progression, which was pronounced in patients with low AR levels. Finally, siRNA-based knockdown of FOXA1 induced decreased cell proliferation and migration. Moreover, in vitro tumorigenicity was inducible by ARs only in the presence of FOXA1, substantiating a functional cooperation between FOXA1 and AR. In conclusion, FOXA1 expression is associated with tumor progression, dedifferentiation of prostate cancer cells, and poorer prognosis, as well as with cellular proliferation and migration and with AR signaling. These findings suggest FOXA1 overexpression as a novel mechanism inducing castration resistance in prostate cancer.

CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression

Chronic myelogenous leukemia (CML) results from a chromosomal translocation in hematopoietic stem or early progenitor cells that gives rise to the oncogenic BCR/ABL fusion protein. Clinically, CML has a chronic phase that eventually evolves into an accelerated stage and blast crisis. A CML-specific immune response is thought to contribute to the control of disease. Whether the immune system can also promote disease progression is not known. In the present study, we investigated the possibility that the TNF receptor family member CD27 is present on leukemia stem cells (LSCs) and mediates effects of the immune system on CML. In a mouse model of CML, BCR/ABL+ LSCs and leukemia progenitor cells were found to express CD27. Binding of CD27 by its ligand, CD70, increased expression of Wnt target genes in LSCs by enhancing nuclear localization of active β-catenin and TRAF2- and NCK-interacting kinase (TNIK). This resulted in increased proliferation and differentiation of LSCs. Blocking CD27 signaling in LSCs delayed disease progression and prolonged survival. Furthermore, CD27 was expressed on CML stem/progenitor cells in the bone marrow of CML patients, and CD27 signaling promoted growth of BCR/ABL+ human leukemia cells by activating the Wnt pathway. Since expression of CD70 is limited to activated lymphocytes and dendritic cells, our results reveal a mechanism by which adaptive immunity contributes to leukemia progression. In addition, targeting CD27 on LSCs may represent an attractive therapeutic approach to blocking the Wnt/β-catenin pathway in CML.

Fine-tuning of substrate architecture and surface chemistry promotes muscle tissue development

Tissue engineering has been increasingly brought to the scientific spotlight in response to the tremendous demand for regeneration, restoration or substitution of skeletal or cardiac muscle after traumatic injury, tumour ablation or myocardial infarction. In vitro generation of a highly organized and contractile muscle tissue, however, crucially depends on an appropriate design of the cell culture substrate. The present work evaluated the impact of substrate properties, in particular morphology, chemical surface composition and mechanical properties, on muscle cell fate. To this end, aligned and randomly oriented micron (3.3±0.8 μm) or nano (237±98 nm) scaled fibrous poly(ε-caprolactone) non-wovens were processed by electrospinning. A nanometer-thick oxygen functional hydrocarbon coating was deposited by a radio frequency plasma process. C2C12 muscle cells were grown on pure and as-functionalized substrates and analysed for viability, proliferation, spatial orientation, differentiation and contractility. Cell orientation has been shown to depend strongly on substrate architecture, being most pronounced on micron-scaled parallel-oriented fibres. Oxygen functional hydrocarbons, representing stable, non-immunogenic surface groups, were identified as strong triggers for myotube differentiation. Accordingly, the highest myotube density (28±15% of total substrate area), sarcomeric striation and contractility were found on plasma-coated substrates. The current study highlights the manifold material characteristics to be addressed during the substrate design process and provides insight into processes to improve bio-interfaces.

Insulin-like growth factor binding protein 2 (IGFBP2) promotes glioma development and progression

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression and poor survival in many types of human cancer (such as prostate, ovarian, adrenocortical, breast, colorectal carcinomas, leukemia, and high-grade gliomas). We therefore hypothesize that IGFBP2 is a key regulator of tumor progression. We tested our hypothesis in gliomas using the somatic gene transfer RCAS-tva mouse model system, which permits the introduction of specific genes into specific, cell lineages, in this case glial cells (RCAS: Replication competent avian sarcomavirus, tv-a: avian RCAS virus receptor). Mice are transgenic and harbor the tv-a receptor under the control of a glial-specific promoter and study genes are cloned into the RCAS vector for post-natal intracranial delivery. For these experiments, the study genes were IGFBP2, platelet-derived growth factor B (PDGFB), K-Ras, Akt, and IIp45 (invasion inhibitory protein 45 kDa; known to bind and block IGFBP2 activity), which were delivered separately and in combination. Our results show that PDGFB signaling leads exclusively to the formation of low-grade (WHO grade II) oligodendrogliomas. PDGFB delivered in combination with IGFBP2 results in the formation of anaplastic oligodendrogliomas (WHO grade III), which are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. IIp45 injected in combination with PDGFB and IGFBP2 ablates IGFBP2-induced tumor progression, which results in formation of low-grade oligodendrogliomas, and an overall reduction in tumor incidence. K-Ras expression was required to form astrocytomas with either IGFBP2 or Akt, indicating the activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells co-expressing IGFBP2 versus PDGFB expression alone. This study provides definitive evidence, for the first time, that: (1) IGFBP2 plays a role in activation of the Akt pathway, (2) IGFBP2 collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma, and (3) IGFBP2-induced tumor progression can be ablated by IIp45 or by specific inhibition of the Akt pathway. ^

EphA2 expression is regulated by EGFR and KRAS and promotes non-small cell lung cancer progression

Lung cancer is the leading cause of cancer deaths worldwide. The development of improved systemic therapy is needed for the most common form of the disease, non-small cell lung cancer (NSCLC). This will depend on the identification of valid molecular targets. Recent studies point to the receptor tyrosine kinase EphA2 as a novel therapeutic target. Overexpression of EphA2 has been demonstrated in a number of epithelial cancers, and its expression has been associated with more severe disease. Regulation of EphA2 in cancer is poorly understood. Recently, regulation of EphA2 by EGFR and KRAS has been reported in a number of in vitro models, but no examination of this relationship has been undertaken in patient tumors. Because of the established importance of EGFR and KRAS in NSCLC, we have investigated the relationship between these mutations and EphA2 in NSCLC patient tissues and cell lines. The significance of Epha2 expression was further examined by testing for correlation with survival, metastases, histology, and smoking status in patient tissues, and tumor cell proliferation and migration in vitro. EphA2 expression was analyzed in by immunohistochemistry in tissue microarray (TMA) format utilizing surgically resected lung cancer specimens. EGFR and KRAS mutation status was determined for the majority of specimens. EphA2 expression was detected in >90% of NSCLC tumors. High EphA2 expression was associated with decreased time to recurrence and metastases, and predicted poorer progression free and overall survival. Expression of EphA2 was positively correlated with activated EGFR and with KRAS mutation. Expression of EphA2 was also positively correlated with a history of smoking. There was no association between gender or histology and EphA2 expression. In H322 cells, activation of EGFR or KRAS resulted in an increase in EphA2 protein expression. Downregulation of EphA2 resulted in decreased proliferation in a clonal growth assay, and inhibited migration in a wound healing assay, in a panel of cell lines. The decrease in proliferation correlated with a transient decrease in the levels of phospho-ERK, a downstream effector of EGFR and KRAS. Based on these data, the potential of EphA2 as a therapeutic target for NSCLC should be further investigated. ^

IKKalpha mediated NOTCH1 Activation Promotes Tumorigenesis via FOXA2 Suppression

Proinflammatory cytokine TNFa plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFa-mediated tumor development remains unclear. Here, we show that IKKa, an important downstream kinase of TNFa, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKa, pFOXA2 (S107/ 111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKa and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFa/IKKa-associated FOXA2 inhibition likely contributes to inflammationmediated cancer pathogenesis. Here, we report a TNFa/IKKa/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.

Loss of ovarian function promotes angiogenesis in human ovarian carcinoma

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.

A serologically identified tumor antigen encoded by a homeobox gene promotes growth of ovarian epithelial cells

Ovarian carcinomas are thought to arise from cells of the ovarian surface epithelium by mechanisms that are poorly understood. Molecules associated with neoplasia are potentially immunogenic, but few ovarian tumor antigens have been identified. Because ovarian carcinomas can elicit humoral responses in patients, we searched for novel tumor antigens by immunoscreening a cDNA expression library with ovarian cancer patient serum. Seven clones corresponding to the homeobox gene HOXB7 were isolated. ELISAs using purified recombinant HOXB7 protein revealed significant serologic reactivity to HOXB7 in 13 of 39 ovarian cancer patients and in only one of 29 healthy women (P < 0.0001). Ovarian carcinomas were found to express HOXB7 at markedly higher levels than normal ovarian surface epithelium, suggesting that immunogenicity of HOXB7 in patients could be associated with its elevated expression in ovarian carcinomas. Overexpression of HOXB7 in immortalized normal ovarian surface epithelial cells dramatically enhanced cellular proliferation. Furthermore, HOXB7 overexpression increased intracellular accumulation and secretion of basic fibroblast growth factor, a potent angiogenic and mitogenic factor. These results reveal HOXB7 as a tumor antigen whose up-regulated expression could play a significant role in promoting growth and development of ovarian carcinomas.

Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism

The Ser/Thr kinase Raf-1 is a protooncogene product that is a central component in many signaling pathways involved in normal cell growth and oncogenic transformation. Upon activation, Raf-1 phosphorylates mitogen-activated protein kinase kinase (MEK), which in turn activates mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs), leading to the propagation of signals. Depending on specific stimuli and cellular environment, the Raf-1–MEK–ERK cascade regulates diverse cellular processes such as proliferation, differentiation, and apoptosis. Here, we describe a MEK–ERK-independent prosurvival function of Raf-1. We found that Raf-1 interacts with the proapoptotic, stress-activated protein kinase ASK1 (apoptosis signal-regulating kinase 1) in vitro and in vivo. Deletion analysis localized the Raf-1 binding site to the N-terminal regulatory fragment of ASK1. This interaction allows Raf-1 to act independently of the MEK–ERK pathway to inhibit apoptosis. Furthermore, catalytically inactive forms of Raf-1 can mimic the wild-type effect, raising the possibility of a kinase-independent function of Raf-1. Thus, Raf-1 may promote cell survival through its protein–protein interactions in addition to its established MEK kinase function.

Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis.

The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 125I-labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development.