William E. Friedrichs

Inhibition of mTOR Activity Restores Tamoxifen Response in Breast Cancer Cells with Aberrant Akt Activity

The Akt kinase is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses. Studies show that high Akt activity in breast carcinoma is associated with a poor pathophenotype, as well as hormone and chemotherapy resistance. Additionally, high Akt activity is associated with other features of poor prognosis. Thus, a chemotherapeutic agent directed specifically toward tumors with high Akt activity could prove extremely potent in treating those breast tumors with the most aggressive phenotypes. Several studies have demonstrated that rapamycin, which inhibits mammalian target of rapamycin (mTOR), a downstream target of Akt, sensitizes certain resistant cancer cells to chemotherapeutic agents. This study evaluated the efficacy of mTOR inhibition in the treatment of tamoxifen-resistant breast carcinoma characterized by high Akt activity. We found that MCF-7 breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions and are resistant to the growth inhibitory effects of tamoxifen, both in vitro as well as in vivo in xenograft models. Cotreatment with the mTOR inhibitor rapamycin in vitro, or the ester of rapamycin, CCI-779 (Wyeth) in vivo, inhibited mTOR activity and restored sensitivity to tamoxifen, suggesting that Akt-induced tamoxifen resistance is mediated in part by signaling through the mTOR pathway. Although the mechanism underlying the synergism remains to be understood, the results were associated with rapamycin’s ability to block transcriptional activity mediated by estrogen receptor α, as assessed by reporter gene assays with estrogen-responsive element luciferase. These data corroborate prior findings indicating that Akt activation induces resistance to tamoxifen in breast cancer cells. Importantly, these data indicate a novel mechanism for tamoxifen resistance and suggest that blockage of the phosphatidylinositol 3′-kinase/Akt signaling pathway by mTOR inhibition effectively restores the susceptibility of these cells to tamoxifen. These data may have implication for future clinical studies of mTOR inhibition in breast carcinoma.

Extrahepatic expression of plasma protein genes during inflammation

The bodys protective responses to infection, wounding, trauma, and malignancy include the acute-phase reaction, which is modulated by various cytokines and their cellular receptors. During the acute-phase reaction, levels of specific proteins synthesized by the liver increase in the plasma. Little information is available about the extrahepatic synthesis of plasma proteins during the acute-phase reaction. The study described here analyzes the tissue-specific expression of genes encoding the plasma proteins albumin (ALB), α1,-antitrypsin (AAT), transferrin (TF), haptoglobin (HP), ceruloplasmin (CP), serum amyloid A (SAA), α1-acid glycoprotein (AGP) and α2-HS-glycoprotein (AHSG) during the acute-phase reaction in C57B1 mice. The acute-phase reaction was induced by intraperitoneal injections of bacterial lipopolysaccharide (LPS). During the acute-phase reaction, genes encoding CP, SAA, AGP, and HP demonstrate unique extrahepatic tissue specific patterns of expression in kidney, spleen, thymus, heart, brain, lung, testis, and epididymis. Different temporal patterns of HP gene expression also were observed in lung and thymus after induction by LPS. The function of extrahepatic synthesis of plasma proteins is not yet understood; however, a local provision of specific plasma proteins in mammalian tissues may offer the host a source of functionally important proteins during periods of stress.

Human α2-HS-glycoprotein/bovine fetuin homologue in mice: identification and developmental regulation of the gene

Human alpha 2-HS-glycoprotein (AHSG) is a plasma protein synthesized in liver and selectively concentrated in bone matrix. It has been reported to be involved in bone formation and resorption as well as immune responses. Recently, AHSG was found to be the species equivalent protein of fetuin, the major fetal serum protein in cattle and sheep. The function and regulation of AHSG/fetuin in different species are not understood. We have isolated a liver cDNA clone that encodes the human AHSG/bovine fetuin homologue in the mouse. The AHSG/fetuin gene may have a role in differentiation since it is expressed in mouse limb buds and brain only at certain stages during development. Mouse liver AHSG/fetuin mRNA was present at low level at 12 days gestation but its level increased during the late part of gestation and peaked between 1 to 3 months after birth. The regulation of mouse AHSG/fetuin synthesis during development was found to be significantly different from that of sheep and bovine fetuin. Compared to fetuin, which is reduced in adult to 1 to 2% of the fetal level, mouse AHSG synthesis subsides only 50% 4 months after birth.

Nox4 Mediates Renal Cell Carcinoma Cell Invasion through Hypoxia-Induced Interleukin 6- and 8- Production

Background Inflammatory cytokines are detected in the plasma of patients with renal cell carcinoma (RCC) and are associated with poor prognosis. However, the primary cell type involved in producing inflammatory cytokines and the biological significance in RCC remain unknown. Inflammation is associated with oxidative stress, upregulation of hypoxia inducible factor 1-alpha, and production of pro-inflammatory gene products. Solid tumors are often heterogeneous in oxygen tension together suggesting that hypoxia may play a role in inflammatory processes in RCC. Epithelial cells have been implicated in cytokine release, although the stimuli to release and molecular mechanisms by which they are released remain unclear. AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status and a role for AMPK in the regulation of cell inflammatory processes has recently been demonstrated. Methods and Principal Findings We have identified for the first time that interleukin-6 and interleukin-8 (IL-6 and IL-8) are secreted solely from RCC cells exposed to hypoxia. Furthermore, we demonstrate that the NADPH oxidase isoform, Nox4, play a key role in hypoxia-induced IL-6 and IL-8 production in RCC. Finally, we have characterized that enhanced levels of IL-6 and IL-8 result in RCC cell invasion and that activation of AMPK reduces Nox4 expression, IL-6 and IL-8 production, and RCC cell invasion. Conclusions/Significance Together, our data identify novel mechanisms by which AMPK and Nox4 may be linked to inflammation-induced RCC metastasis and that pharmacological activation of AMPK and/or antioxidants targeting Nox4 may represent a relevant therapeutic intervention to reduce IL-6- and IL-8-induced inflammation and cell invasion in RCC.

Varicella-zoster virus-specific gp140: a highly immunogenic and disulfide-linked structural glycoprotein.

A 140,000-dalton disulfide-linked glycoprotein (gp140) specified by varicella-zoster virus (VZV) in infected cultured cells was identified and precipitated by two murine monoclonal antibodies (VZ-151 and VZ-158). When analyzed under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gp140 was cleaved predominantly into a 66,000 lower-molecular-weight product (gp66). This protein was classified as a viral structural component, since it was observed in the polypeptide profile of metrizamide gradient-purified enveloped virions. By immunofluorescence analyses with a monoclonal antibody probe, gp140 expression was documented to be highly conserved both within cultured cells inoculated with homologous (VZV-Oka) and heterologous (VZV-32) strains and in infected human tissues from chicken pox and zoster patients. That the glycoprotein was highly immunogenic was confirmed by the presence of high-titer anti-gp140 antibody in the sera of both hyperimmunized laboratory animals and naturally infected humans. Temporally, the humoral response to gp140 following primary VZV infection preceded that against the other viral glycoproteins. These studies describe, therefore, an immunogenic, disulfide-linked viral structural glycoprotein, which must be included among the other five previously described VZV-specific fucosylated species--gp118, gp98, gp62, and gp45.

Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation

Hypoxia inducible factor-2α (HIF-2α) has a critical role in renal tumorigenesis. HIF-2α is stabilized in von Hippel–Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of rapamycin (mTOR) functions in two distinct complexes: Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2α protein in the absence of VHL; however, the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation is not clear. Complex assembly of the mRNA cap protein, eukaryotic translation initiation factor 4 (eIF4)E, with activators (eIF4 gamma (eIF4G)) and inhibitors (eIF4E-binding protein 1 (4E-BP1)) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22phox-based Nox oxidases, are enhanced in VHL-deficient cells and have a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2α through eIF4E-dependent mRNA translation and examined the effects of p22phox-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive, and further defined a novel role for p22phox-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22phox reduces HIF-2α-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

Omega-3 Fatty Acid Inhibition of Prostate Cancer Progression to Hormone Independence Is Associated With Suppression of mTOR Signaling and Androgen Receptor Expression

Currently, progression of prostate cancer to androgen independence remains the primary obstacle to improved survival. In order to improve overall survival, novel treatment strategies that are based upon specific molecular mechanisms that prolong the androgen-dependent state and that are useful for androgen-independent disease need to be identified. Both epidemiological as well as preclinical data suggest that omega-3 fatty acids are effective primary tumor prevention agents; however, their efficacy at preventing and treating refractory prostate cancer has not been as thoroughly investigated. We used an in vitro model of androgen ablation to determine the effect of treatment with omega-3 fatty acids on the progression to an androgen-independent state. The omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were able to prevent progression of LNCaP cells while the omega-6 fatty acid arachidonic acid (AA) actually promoted cell growth under conditions of hormone depletion. These results correlated with a decrease in the expression of the androgen receptor as well as suppression of the Akt/mTOR signaling pathway. Connecting the mechanisms by which omega-3 fatty acids affect phenotypic outcome is important for effective exploitation of these nutrient agents as a therapeutic approach. Understanding these processes is critical for the development of effective dietary intervention strategies that improve overall survival.

TISSUE SPECIFIC EXPRESSION OF MOUSE TRANSFERRIN DURING DEVELOPMENT AND AGING

Transferrin (TF) is a major plasma protein that binds ferric iron and transports it to all target tissues of the body. This study is the first step to identify the tissue specific expression of the transferrin gene in mice during development, into maturity and throughout the aging process. The transferrin gene expresses mainly in mouse liver, the cerebral hemispheres and cerebellum. In mouse, transferrin is expressed in peritoneal macrophages and in mouse macrophage cell line MO59. At 19 days of gestation, transferrin mRNA is detected in the fetal lung, heart, stomach and kidney. TF mRNA levels increase in liver throughout gestation with maximum expression occurring at 19 days. Transferrin mRNA was detected in placentas of pregnant mice, with levels progressively increasing throughout the term of pregnancy. The levels of liver TF mRNA in mouse vary in a cyclic manner during the development increasing with the aging processes. Because of the dynamic nature of tissue requirements for transferrin during homeostasis the TF gene serves as a promising system for analyzing tissue-specific regulation in vivo during development and aging. Results from this study designate periods in the life-span of the mouse where regulatory mechanisms interacting with the TF gene appear to dynamically alter its expression.

Immunoprecipitable polypeptides specified by varicella-zoster virus.

Abstract Polypeptides encoded by varicella-zoster virus (VZV) in infected cell cultures have been identified by radioimmune precipitation techniques. Detergent-solubilized extracts of VZV-infected cells were reacted with highly specific VZV antisera raised in strain-2 guinea pigs immunized with sonicates of syngeneic virus-infected cells. Fractionation of the immunoprecipitates in acrylamide slab gels demonstrated an average of 16 polypeptides, which ranged in molecular weight from 32,000 to a200,000. These included the three major immunogenic glycoproteins (gp 62, gp 98, and gp 118) and a prominent higher molecular weight nonglycosylated polypeptide at 155,000. One of the [ 35 S]methionine-labeled polypeptides comigrated with purified actin. Not all polypeptides were visible in any one particular fluorogram, but comparative analysis of polypeptide profiles derived from electrophoreses performed with different gel concentrations and different crosslinkers (methylene-bisacrylamide and N,N -diallyltartardiamide) clearly established a consistent and reproducible pattern of radioactive bands. A low background of radio-activity was nonspecifically precipitated by the antigen-antibody-protein A complexes; however, with the exception of a common band comigrating with actin, the electrophoretic profiles representing virus-specific and nonspecific immunoprecipitates were easily distinguished.

The Pro and Con of Measles Virus in Paget's Disease: Pro

Dr. Rima and colleagues have raised several questions about our recent publication by Frederichs et al. on the role of the measles virus in Paget’s disease of bone. The first point that was raised is that New Zealand and U.S. measles viruses sequences associated with Paget’s disease that we detected are all from clade A. Rima and colleagues said that it is unlikely that patients from different parts of the globe should be exposed to the same strain of measles virus at the same time. To our knowledge, there is no data on the global distribution of different measles virus genotypes in the first half of the century when these patients would have been exposed. It is of interest that all the sequences we have detected are clade A sequences. One hypothesis is that clade A sequences have unique properties that differ from other clades and may specifically lead to Paget’s disease in later life. Dr. Rima and his colleagues are also concerned that Paget’s disease–associated nucleocapsid gene mutations, which we detected, affect several conserved residues that are not mutated in patients with SSPE. We agree that this is an interesting and provocative observation, suggesting that these specific gene mutations may be highly “pagetogenic.” To the question of how these specific mutations arise, similar to SSPE mutations in the F, H, and M proteins of measles viral genome in patients succumbing to SSPE, they may arise as a consequence of virus evolution in the patient and may not be transmissible from patient to patient. Dr. Rima also suggested that the sequences that we detected may result from a contamination of our material by a control measles virus cDNA. We can reassure him and his colleagues that all appropriate controls have been done, and our data are correct as they are presented and do not represent an artifact. We have been extremely careful in assuring that these are not artifactual and have given these samples to other laboratories with our primer sets, and they have found similar results with appropriate controls. Finally, we agree that the role of the measles virus or any paramyxovirus in Paget’s disease is still controversial. However, a number of other laboratories have provided supporting evidence for the presence of measles virus sequences in Paget’s patients, using other techniques. Basle and coworkers have detected measles virus RNA in Paget’s disease bone tissue by in situ hybridization. Mills and coworkers have shown measles virus antigens in osteoclasts in patients with Paget’s disease by immunocytochemical techniques. Rebel and coworkers have also detected measles virus nucleocapsid antigen in osteoclasts from patients with Paget’s disease. These data support a potential role for the measles virus in Paget’s disease. In addition, work from our group in which we have transfected the measles virus nucleocapsid gene into normal osteoclast precursors has shown that these precursors form osteoclasts very similar to pagetic osteoclasts. Finally, we have shown that infection of murine bone marrow from transgenic mice that express the measles virus receptor CD46 form osteoclasts that are again very similar to pagetic osteoclasts.