Chronic stress promotes tumor growth through increased BDNF production and neo-innervation

Background: Activation of the sympathetic nervous system (SNS) in response to chronic biobehavioral stress results in high levels of catecholamines and persistent activation of adrenergic signaling, which promotes tumor growth and progression. However it is unknown how catecholamine levels within the tumor exceed systemic levels in circulation. I hypothesized that neo-innervation of tumors is required for stress-mediated effects on tumor growth. Results: First, I examined whether sympathetic nerves are present in human ovarian cancer samples as well as orthotopic ovarian cancer models. Immunohistochemical (IHC) staining for neurofilament revealed that catecholaminergic neurons are present within tumor tissue. In order to determine whether chronic stress affects the density of nerves in the tumor, I utilized an orthotopic mouse model of ovarian cancer that was exposed to daily restraint stress. IHC analysis revealed that nerve density in tumors increased by more than three-fold in stressed animals versus non-stressed controls. IHC analysis suggested that this results from both recruitment of existing neurons (axonogenesis) as well as new neuron formation (neurogenesis) within the tumor. To determine how tumors are recruiting nerve growth, I utilized a PCR array analysis of 84 nerve growth related genes and their receptors, which showed that stimulation of the SKOV3 ovarian cancer cell line with norepinephrine (NE) leads to increased expression of several neurotrophins, including brain-derived neurotrophic factor (BDNF). Neurite extension assays showed that media conditioned by ovarian cancer cell lines is capable of inducing neurite outgrowth in differentiated neuron-like PC12 cells, and NE treatment of cancer cells potentiates this effect. Norepinephrine-induced neurite extension was abolished after BDNF silencing by siRNA, suggesting that BDNF is critical to tumor cell-induced nerve growth. in vivo BDNF inhibition resulted in complete abrogation of stress-induced increases in tumor weight and nerve density, as well as downstream markers of stress. Discussion: These studies indicate that adrenergic signalling induced by chronic stress promotes neo-innervation in the tumor microenvironment. This results in a mutually beneficial relationship between the tumor cells and neurons. This work is crucial for providing a link between chronic stress and its effects on the tumor and its microenvironment. The data shown here aims to open new venues that can be used in development of therapies designed to block the stress effects on tumor growth.

The Role of K63-linked Ubiquitination Cycles in Akt Kinase Activation

Akt (also known as protein kinase B) serves a central regulator in PI3K/Akt signaling pathways to regulate numerous physiological functions including cell proliferation, survival and metabolism. Akt activation requires the binding of Akt to phospholipid PIP3 on the plasma membrane and subsequent phosphorylation of Akt by its kinases. Growth factor-mediated membrane recruitment of Akt is a crucial step for Akt activation. However, the mechanism of Akt membrane translocation is unclear. Protein ubiquitination is a significant posttranslational modification that controls many biological functions such as protein trafficking and signaling activation. Therefore, we hypothesize that ubiquitination may be involved in Akt signaling activation. We have demonstrated that Akt could be conjugated with non-proteolytic K63-linked ubiquitination by TRAF6 ubiquitin E3 ligase. This modification on Akt was required for membrane recruitment, phosphorylation and activation of Akt in response to growth factor stimulation. The human cancer-associated Akt E17K mutant exhibited an increase in K63-linked ubiquitination, which contributes to the enrichment of membrane recruitment and phosphorylation of Akt. Thus, we conclude that K63-linked ubiquitination is a critical step for oncogenic Akt activation and also involved in human cancer development. Notably, the process of protein ubiquitination can be reversed by deubiquitinating enzymes (DUBs), which play a critical role to terminate signaling activation induced by ubiquitination. To further investigate how ubiquitination cycles regulate Akt activation, we have identified that CYLD as a DUB for Akt, and CYLD inhibited growth factor-induced ubiquitination and activation of Akt. Under serum-depletion condition, CYLD interacts with Akt and keep Akt under inactive state by directly removing K63-linked ubiquitination of Akt. CYLD disassociates with Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. We also demonstrated that CYLD deficiency promoted cancer cell proliferation, survival, glucose metabolism and human prostate cancer development. Therefore, we conclude that CYLD plays a critical role for negatively regulating Akt signaling activation through deubiquitination of Akt. In summary, this study delineated the important mechanism of cycles of ubiquitination and deubiquitination of Akt in regulating membrane translocation and activation of Akt, and TRAF6 and CYLD as central switches for these processes.

Patenting genes and gene sequences: Analysis of public health implications and the law

At issue is whether or not isolated DNA is patent eligible under the U.S. Patent Law and the implications of that determination on public health. The U.S. Patent and Trademark Office has issued patents on DNA since the 1980s, and scientists and researchers have proceeded under that milieu since that time. Today, genetic research and testing related to the human breast cancer genes BRCA1 and BRCA2 is conducted within the framework of seven patents that were issued to Myriad Genetics and the University of Utah Research Foundation between 1997 and 2000. In 2009, suit was filed on behalf of multiple researchers, professional associations and others to invalidate fifteen of the claims underlying those patents. The Court of Appeals for the Federal Circuit, which hears patent cases, has invalidated claims for analyzing and comparing isolated DNA but has upheld claims to isolated DNA. The specific issue of whether isolated DNA is patent eligible is now before the Supreme Court, which is expected to decide the case by year's end. In this work, a systematic review was performed to determine the effects of DNA patents on various stakeholders and, ultimately, on public health; and to provide a legal analysis of the patent eligibility of isolated DNA and the likely outcome of the Supreme Court's decision. ^ A literature review was conducted to: first, identify principle stakeholders with an interest in patent eligibility of the isolated DNA sequences BRCA1 and BRCA2; and second, determine the effect of the case on those stakeholders. Published reports that addressed gene patents, the Myriad litigation, and implications of gene patents on stakeholders were included. Next, an in-depth legal analysis of the patent eligibility of isolated DNA and methods for analyzing it was performed pursuant to accepted methods of legal research and analysis based on legal briefs, federal law and jurisprudence, scholarly works and standard practice legal analysis. ^ Biotechnology, biomedical and clinical research, access to health care, and personalized medicine were identified as the principle stakeholders and interests herein. Many experts believe that the patent eligibility of isolated DNA will not greatly affect the biotechnology industry insofar as genetic testing is concerned; unlike for therapeutics, genetic testing does not require tremendous resources or lead time. The actual impact on biomedical researchers is uncertain, with greater impact expected for researchers whose work is intended for commercial purposes (versus basic science). The impact on access to health care has been surprisingly difficult to assess; while invalidating gene patents might be expected to decrease the cost of genetic testing and improve access to more laboratories and physicians' offices that provide the test, a 2010 study on the actual impact was inconclusive. As for personalized medicine, many experts believe that the availability of personalized medicine is ultimately a public policy issue for Congress, not the courts. ^ Based on the legal analysis performed in this work, this writer believes the Supreme Court is likely to invalidate patents on isolated DNA whose sequences are found in nature, because these gene sequences are a basic tool of scientific and technologic work and patents on isolated DNA would unduly inhibit their future use. Patents on complementary DNA (cDNA) are expected to stand, however, based on the human intervention required to craft cDNA and the product's distinction from the DNA found in nature. ^ In the end, the solution as to how to address gene patents may lie not in jurisprudence but in a fundamental change in business practices to provide expanded licenses to better address the interests of the several stakeholders. ^

Upregulation of VEGF through p70S6K in ErbB2 -mediated angiogenesis and inhibition by combined herceptin plus taxol therapy

ErbB2 overexpression in breast tumors increases metastasis, angiogenesis, and reduces survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, a spontaneous metastasis assay was performed using human breast cancer cells transfected with constitutively active ErbB2 kinase (V659E), an ErbB2 kinase-dead mutant (K753M), or vector control. Mice injected with V659E had increased metastasis and tumor microvessel density; and the increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro, which resulted from increased VEGF by increased protein synthesis. This appeared to be mediated through a PI3K, Akt, mTOR, p70S6K-signaling pathway. Furthermore, V659E xenografts had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with control. To validate the clinical relevance of these findings, human breast tumor samples were examined. Tumors overexpressing ErbB2 correlated with p70S6K phosphorylation and VEGF expression, which significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Together, ErbB2 increases VEGF expression by activating the p70S6K signaling pathway, which may serve as targets for antiangiogenic and antimetastatic therapies. ^ Herceptin is an anti-ErbB2 antibody that demonstrated anti-tumor function, especially in combination with other chemotherapies such as Taxol, in patients with ErbB2-overexpressing tumors. Since the repeated administration of low-dose chemotherapy endorsed an antiangiogenic effect in vitro, and Herceptin was shown to inhibit angiogenesis in tumor xenografts, I investigated whether combined Taxol plus Herceptin treatment inhibits ErbB2-mediated angiogenic responses more effectively. Mice with ErbB2-overexpressing xenografts were treated with control, Herceptin, Taxol, or combination Herceptin plus Taxol. Mice treated with the combination exhibited reduced tumor volumes, tumor microvessel densities, and lung metastasis; and ErbB2-overexpressing cells treated with the combination secreted less VEGF, and stimulated less endothelial cell migration. Furthermore, Akt phosphorylation contributed to VEGF upregulation and was most effectively reduced by combination treatment. ^ In summary, ErbB2 activates signaling to Akt and p70S6K leading to increased VEGF and angiogenesis. Combination Herceptin plus Taxol treatment most effectively inhibited ErbB2-mediated angiogenesis, resulting in pronounced tumoricidal effects, and may be mediated through reduction of phosphorylated Akt, a positive regulator in the p70S6K pathway. ^

Regulation of p53 expression by thymidylate synthase

Previous studies showed that thymidylate synthase (TS), as an RNA binding protein, regulates its own synthesis by impairing the translation of TS mRNA. In this report, we present evidence that p53 expression is affected in a similar manner by TS. For these studies, we used a TS-depleted human colon cancer HCT-C cell that had been transfected with either the human TS cDNA or the Escherichia coli TS gene. The level of p53 protein in transfected cells overexpressing human TS was significantly reduced when compared with its corresponding parent HCT-C cells. This suppression of p53 expression was the direct result of decreased translational efficiency of p53 mRNA. Similar results were obtained upon transfection of HCT-C cells with pcDNA 3.1 (+) containing the E. coli TS gene. These findings provide evidence that TS, from diverse species, specifically regulates p53 expression at the translational level. In addition, TS-overexpressing cells with suppressed levels of p53 are significantly impaired in their ability to arrest in G1 phase in response to exposure to a DNA-damaging agent such as γ-irradiation. These studies provide support for the in vivo biological relevance of the interaction between TS and p53 mRNA and identify a molecular pathway for controlling p53 expression.

Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin

Infection with Helicobacter pylori is associated with different human gastric diseases. Biochemical studies, in vitro adherence assays, and in vivo animal models revealed that epithelial attachment of H. pylori can be mediated by the blood-group antigen-binding adhesin (BabA) targeting human Lewisb surface epitopes. Studies with transgenic mice expressing the Lewisb epitope have shown that such attachment can alter disease outcome. In the current study, the presence of the babA2 gene encoding the adhesin was investigated in clinical isolates from a German population by using PCR and reverse transcription–PCR. A positive genotype was correlated to allelic variations in the genes encoding VacA and CagA and also to the prevalence of duodenal ulcer, distal gastric adenocarcinoma, mucosa-associated lymphoid tissue lymphoma, and antral gastritis. The presence of babA2 was significantly associated with duodenal ulcer (P = 0.0002) and adenocarcinoma (P = 0.033). In contrast, type 1 strains (vacAs1- and cagA-positive) were associated with only duodenal ulcer (P = 0.004) but not adenocarcinoma (P = 0.235). Genotype presence of babA2, vacAs1, and cagA (“triple-positive” strains) showed a highly significant correlation to the prevalence of ulcer (P = 0.000002) and adenocarcinoma (P = 0.014) and discriminated significantly better between disease outcome than did the current type 1 classification. These results indicate that the babA2 gene is of high clinical relevance and would be a useful marker to identify patients who are at higher risk for specific H. pylori-related diseases.

Induction of host signal transduction pathways by Helicobacter pylori

Adherence of Helicobacter pylori to cultured gastric epithelial cells is associated with several cellular events, including the tyrosine phosphorylation of a 145-kDa host protein; the reorganization of the host cell actin and associated cellular proteins, like vasodilator-stimulated phosphoprotein, adjacent to the attached bacterial cell; and the subsequent release of the cytokine, interleukin 8 (IL-8). H. pylori isolated from patients with ulcer disease and gastric cancer contain a DNA insertion, the cag pathogenicity island (PAI), that is not present in bacteria isolated from individuals with asymptomatic infection. Mutations in a number of PAI genes abolish tyrosine phosphorylation and IL-8 synthesis but not the cytoskeletal rearrangements. Kinase inhibition studies suggest there are two distinct pathways operative in stimulating IL-8 release from host cells and one of these H. pylori pathways is independent of the tyrosine phosphorylation step.

A model-based approach for assessing in vivo combination therapy interactions

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach compares in vivo tumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RIα subunit of protein kinase A after oral administration

Overexpression of the RIα subunit of cAMP-dependent protein kinase (PKA) has been demonstrated in various human cancers. PKA has been suggested as a potential target for cancer therapy. The goal of the present study was to evaluate an anti-PKA antisense oligonucleotide (mixed-backbone oligonucleotide) as a therapeutic approach to human cancer treatment. The identified oligonucleotide inhibited the growth of cell lines of human colon cancer (LS174T, DLD-1), leukemia (HL-60), breast cancer (MCF-7, MDA-MB-468), and lung cancer (A549) in a time-, concentration-, and sequence-dependent manner. In a dose-dependent manner, the oligonucleotide displayed in vivo antitumor activity in severe combined immunodeficient and nude mice bearing xenografts of human cancers of the colon (LS174T), breast (MDA-MB-468), and lung (A549). The routes of drug administration were intraperitoneal and oral. Synergistic effects were found when the antisense oligonucleotide was used in combination with the cancer chemotherapeutic agent cisplatin. The pharmacokinetics of the oligonucleotide after oral administration of 35S-labeled oligonucleotide into tumor-bearing mice indicated an accumulation and retention of the oligonucleotide in tumor tissue. This study further provides a basis for clinical studies of the antisense oligonucleotide targeted to the RIα subunit of PKA (GEM 231) as a cancer therapeutic agent used alone or in combination with conventional chemotherapy.

Coordinate regulation of lipogenic gene expression by androgens: Evidence for a cascade mechanism involving sterol regulatory element binding proteins

To gain more insight into the molecular mechanisms by which androgens stimulate lipogenesis and induce a marked accumulation of neutral lipids in the human prostate cancer cell line LNCaP, we studied their impact on the expression of lipogenic enzymes. Northern blot analysis of the steady-state mRNA levels of seven different lipogenic enzymes revealed that androgens coordinately stimulate the expression of enzymes belonging to the two major lipogenic pathways: fatty acid synthesis and cholesterol synthesis. In view of the important role of the recently characterized sterol regulatory element binding proteins (SREBPs) in the coordinate induction of lipogenic genes, we examined whether the observed effects of androgens on lipogenic gene expression are mediated by these transcription factors. Our findings indicate that androgens stimulate the expression of SREBP transcripts and precursor proteins and enhance the nuclear content of the mature active form of the transcription factor. Moreover, by using the fatty acid synthase gene as an experimental paradigm we demonstrate that the presence of an SREBP-binding site is essential for its regulation by androgens. These data support the hypothesis that SREBPs are involved in the coordinate regulation of lipogenic gene expression by androgens and provide evidence for the existence of a cascade mechanism of androgen-regulated gene expression.

BIM1 Encodes a Microtubule-binding Protein in Yeast

A previously uncharacterized yeast gene (YER016w) that we have named BIM1 (binding to microtubules) was obtained from a two-hybrid screen of a yeast cDNA library using as bait the entire coding sequence of TUB1 (encoding α-tubulin). Deletion of BIM1 results in a strong bilateral karyogamy defect, hypersensitivity to benomyl, and aberrant spindle behavior, all phenotypes associated with mutations affecting microtubules in yeast, and inviability at extreme temperatures (i.e., ≥37°C or ≤14°C). Overexpression of BIM1 in wild-type cells is lethal. A fusion of Bim1p with green fluorescent protein that complements the bim1Δ phenotypes allows visualization in vivo of both intranuclear spindles and extranuclear microtubules in otherwise wild-type cells. A bim1 deletion displays synthetic lethality with deletion alleles of bik1, num1, and bub3 as well as a limited subset of tub1 conditional-lethal alleles. A systematic study of 51 tub1 alleles suggests a correlation between specific failure to interact with Bim1p in the two-hybrid assay and synthetic lethality with the bim1Δ allele. The sequence of BIM1 shows substantial similarity to sequences from organisms across the evolutionary spectrum. One of the human homologues, EB1, has been reported previously as binding APC, itself a microtubule-binding protein and the product of a gene implicated in the etiology of human colon cancer.

Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution

A remarkable instability at simple repeated sequences characterizes gastrointestinal cancer of the microsatellite mutator phenotype (MMP). Mutations in the DNA mismatch repair gene family underlie the MMP, a landmark for hereditary nonpolyposis colorectal cancer. These tumors define a distinctive pathway for carcinogenesis because they display a particular spectrum of mutated cancer genes containing target repeats for mismatch repair deficiency. One such gene is BAX, a proapoptotic member of the Bcl-2 family of proteins, which plays a key role in programmed cell death. More than half of colon and gastric cancers of the MMP contain BAX frameshifts in a (G)8 mononucleotide tract. However, the functional significance of these mutations in tumor progression has not been established. Here we show that inactivation of the wild-type BAX allele by de novo frameshift mutations confers a strong advantage during tumor clonal evolution. Tumor subclones with only mutant alleles frequently appeared after inoculation into nude mice of single-cell clones of colon tumor cell lines with normal alleles. In contrast, no clones of BAX-expressing cells were found after inoculation of homozygous cell clones without wild-type BAX. These results support the interpretation that BAX inactivation contributes to tumor progression by providing a survival advantage. In this context, survival analyses show that BAX mutations are indicators of poor prognosis for both colon and gastric cancer of the MMP.

The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway

The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.

Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor

The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (≈3–10 μM) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.

Nuclear Factor-κB (NF-κB) Regulates Proliferation and Branching in Mouse Mammary Epithelium

The nuclear factor-κB (NF-κB) family of transcription factors has been shown to regulate proliferation in several cell types. Although recent studies have demonstrated aberrant expression or activity of NF-κB in human breast cancer cell lines and tumors, little is known regarding the precise role of NF-κB in normal proliferation and development of the mammary epithelium. We investigated the function of NF-κB during murine early postnatal mammary gland development by observing the consequences of increased NF-κB activity in mouse mammary epithelium lacking the gene encoding IκBα, a major inhibitor of NF-κB. Mammary tissue containing epithelium from inhibitor κBα (IκBα)-deficient female donors was transplanted into the gland-free mammary stroma of wild-type mice, resulting in an increase in lateral ductal branching and pervasive intraductal hyperplasia. A two- to threefold increase in epithelial cell number was observed in IκBα-deficient epithelium compared with controls. Epithelial cell proliferation was strikingly increased in IκBα-deficient epithelium, and no alteration in apoptosis was detected. The extracellular matrix adjacent to IκBα-deficient epithelium was reduced. Consistent with in vivo data, a fourfold increase in epithelial branching was also observed in purified IκBα-deficient primary epithelial cells in three-dimensional culture. These data demonstrate that NF-κB positively regulates mammary epithelial proliferation, branching, and functions in maintenance of normal epithelial architecture during early postnatal development.