The mammalian anx7 gene codes for a Ca 2؉ -activated GTPase, which supports Ca 2؉ ͞GTP-dependent secretion events and Ca 2؉ channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca 2؉ signaling in secreting pancreatic  cells, we knocked out the anx7 gene in the mouse and tested the insulinsecretory properties of the  cells. The nullizygous anx7 (؊͞؊) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (؉͞؊) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (؉͞؊) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8-to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca 2؉ channel functions are normal. However, electrooptical recordings indicate that the (؉͞؊) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP3)-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP3 receptor expression and function in pancreatic islets. The profound increase in islets,  cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic  cells. This is a direct demonstration of a connection between glucoseactivated insulin secretion and Ca 2؉ signaling through IP3-sensitive Ca 2؉ stores.
The ANX7 gene is located on human chromosome 10q21, a site long hypothesized to harbor a tumor suppressor gene(s) (TSG) associated with prostate and other cancers. To test whether ANX7 might be a candidate TSG, we examined the ANX7-dependent suppression of human tumor cell growth, stage-specific ANX7 expression in 301 prostate specimens on a prostate tissue microarray, and loss of heterozygosity (LOH) of microsatellite markers at or near the ANX7 locus. Here we report that human tumor cell proliferation and colony formation are markedly reduced when the wild-type ANX7 gene is transfected into two prostate tumor cell lines, LNCaP and DU145. Consistently, analysis of ANX7 protein expression in human prostate tumor microarrays reveals a significantly higher rate of loss of ANX7 expression in metastatic and local recurrences of hormone refractory prostate cancer as compared with primary tumors (P ؍ 0.0001). Using four microsatellite markers at or near the ANX7 locus, and laser capture microdissected tumor cells, 35% of the 20 primary prostate tumors show LOH. The microsatellite marker closest to the ANX7 locus showed the highest rate of LOH, including one homozygous deletion. We conclude that the ANX7 gene exhibits many biological and genetic properties expected of a TSG and may play a role in prostate cancer progression.cancer genetics ͉ chromosome 10q21 ͉ loss of heterozygosity T he gene for annexin 7 (ANX7 ‡ ‡ , synexin; refs. 1-6) is located on human chromosome 10q21, where potential tumor suppressor genes (TSGs) have been hypothesized to exist for prostate and other cancers (5, 7-15). However, the specific relevance of the ANX7 gene for cancer only became apparent after we created a knockout for this gene in the mouse (16). Although the homozygous Anx7(Ϫ͞Ϫ) deletion is embryonically lethal, the phenotype of the Anx7(ϩ͞Ϫ) heterozygote includes calcium signaling deficits and growth defects such as gigantism, and selective organomegaly. As these mice aged, we also began to observe a profoundly increased frequency of disparate spontaneous tumors in both male and female Anx7(ϩ͞Ϫ) mutants (17).Because of these observations, and the chromosomal location of the gene, we hypothesized that ANX7 might be a candidate TSG associated with 10q21 locus. Commonly, TSGs can suppress growth of tumor cells, in vitro, and are frequently inactivated by mutations, deletions, or loss of expression in tumors, in vivo. In addition, loss of heterozygosity (LOH) often is observed for these genes in clinical tumor specimens. Therefore, to test this hypothesis for the ANX7 gene, we analyzed the action of the ANX7 gene on colony formation by human tumor cell lines. We also examined the expression of the ANX7 protein in hundreds of prostate cancers by using tumor tissue microarray technology. Finally, we tested a panel of primary and metastatic prostate cancers for evidence of LOH.In this paper we show that the ANX7 gene suppresses the growth of the prostate tumor cell lines DU145 and LNCaP. Consistently, in a prostate tissue microarray, we...
Digitoxin and structurally related cardiac glycoside drugs potently block activation of the TNF-␣͞NF-B signaling pathway. We have hypothesized that the mechanism might be discovered by searching systematically for selective inhibitory action through the entire pathway. We report that the common action of these drugs is to block the TNF-␣-dependent binding of TNF receptor 1 to TNF receptor-associated death domain. This drug action can be observed with native cells, such as HeLa, and reconstituted systems prepared in HEK293 cells. All other antiinflammatory effects of digitoxin on NF-B and c-Jun N-terminal kinase pathways appear to follow from the blockade of this initial upstream signaling event.digitoxin ͉ inflammation
Annexin 7 (ANX7) acts as a tumor suppressor gene in prostate cancer, where loss of heterozygosity and reduction of ANX7 protein expression is associated with aggressive metastatic tumors. To investigate the mechanism by which this gene controls tumor development, we have developed an Anx7(؉͞؊) knockout mouse. As hypothesized, the Anx7(؉͞؊) mouse has a cancer-prone phenotype. The emerging tumors express low levels of Anx7 protein. Nonetheless, the wild-type Anx7 allele is detectable in laser-capture microdissection-derived tumor tissue cells. Genome array analysis of hepatocellular carcinoma tissue indicates that the Anx7(؉͞؊) genotype is accompanied by profound reductions of expression of several other tumor suppressor genes, DNA repair genes, and apoptosis-related genes. In situ analysis by tissue imprinting from chromosomes in the primary tumor and spectral karyotyping analysis of derived cell lines identify chromosomal instability and clonal chromosomal aberrations. Furthermore, whereas 23% of the mutant mice develop spontaneous neoplasms, all mice exhibit growth anomalies, including gender-specific gigantism and organomegaly. We conclude that haploinsufficiency of Anx7 expression appears to drive disease progression to cancer because of genomic instability through a discrete signaling pathway involving other tumor suppressor genes, DNA-repair genes, and apoptosis-related genes.hepatocellular carcinoma ͉ lymphosarcoma of the thymus ͉ SKY analysis ͉ cDNA microarray A nnexin 7 (ANX7) is a newly described tumor suppressor gene (TSG) for prostate cancer in men, featuring loss of heterozygosity and reduced ANX7 § protein expression in a large fraction of archived metastatic tumors (1). ANX7 maps to chromosome 10q21, where unknown TSGs had been postulated but not yet identified (2-4). In a previous work, we established an Anx7(ϩ͞Ϫ) knockout mouse, in which Ca 2ϩ -dependent endocrine secretory defects were identified (5). We also noted substantial reductions of Anx7 expression in the pancreas, heart, and other tissues. The nullizygous Anx7(Ϫ͞Ϫ) knockout could not be studied. The embryo failed to develop beyond embryonic day 10 in utero. However, as the Anx7(ϩ͞Ϫ) colony aged, we began to observe an increasing frequency of disparate tumors (6). The mechanism of tumorigenesis in humans and mice often differ (7). Therefore, given the apparent TSG-like ANX7 parallel between tumors in mice and humans, we felt compelled to investigate the mechanisms of tumor transformation in the Anx7(ϩ͞Ϫ) mouse model.Mechanistically, we reasoned that we might be able to distinguish between a Knudson two-hit model, as might be considered possible based on the human prostate tumor data, and a haploinsufficiency model, based on low Anx7 protein levels in otherwise normal knockout-mouse tissues. We find that the data clearly support a haploinsufficiency model for tumor development in the knockout mouse. In addition, the tumors demonstrate an increased level of genetic instability, based on spectral karyotyping (SKY) analysis of primary tumors...
The ANX7 gene codes for a Ca2+-activated GTPase, which has been implicated in both exocytotic secretion in cells and control of growth. In this review, we summarize information regarding increased tumor frequency in the Anx7 knockout mice, ANX7 growth suppression of human cancer cell lines, and ANX7 expression in human tumor tissue micro-arrays. The loss of ANX7 is significant in metastatic and hormone refractory prostate cancer compared to benign prostatic hyperplasia. In addition, ANX7 expression has prognostic value for predicting survival of breast cancer patients.
Tumor suppressor function for Annexin A7 (ANXA7; 10q21) is based on cancer-prone phenotype in Anxa7(+/À) mouse and ANXA7 prognostic role in human cancers. Because ANXA7-caused liposome aggregation can be promoted by arachidonic acid (AA), we hypothesized that the phospholipid-binding tumor suppressor ANXA7 is associated with AA cascade. In a comparative study of ANXA7 versus canonical tumor suppressor p53 effects on AA lipoxygenation pathway in the p53-mutant and androgen-insensitive DU145 prostate cancer cells, both tumor suppressors altered gene expression of major 5-lipoxygenase (LOX) and 15-LOXs, including response to T helper 2 (Th2)-cytokine [interleukin-4 (IL-4)] and endogenous steroids (mimicked by dexamethasone). Wild-type and mutant ANXA7 distinctly affected expression of the dexamethasone-induced 15-LOX-2 (a prostate-specific endogenous tumor suppressor) as well as the IL-4-induced 15-LOX-1. On the other hand, wild-type p53 restored 5-LOX expression in DU145 to levels comparable to benign prostate epithelial cells. Using mass spectrometry of DNA affinity-enriched nuclear proteins, we detected different proteins that were bound to adjacent p53 and estrogen response elements in the 5-LOX promoter in DU145 cells introduced with ANXA7 versus p53. Sex hormone regulator 17-B hydroxysteroid dehydrogenase 4 was identified under p53 introduction, which induced the 5-LOX expression. Meantime, nuclear proteins bound to the same 5-LOX promoter site under introduction of ANXA7 (that was associated with the repressed 5-LOX) were identified as zinc finger proteins ZNF433 and Aiolos, pyrin domaincontaining NALP10, and the p53-regulating DNA repair enzyme APEX1. Thus, ANXA7 and p53 can distinctly regulate LOX transcription that is potentially relevant to the AAmediated cell growth control in tumor suppression.
Annexin-A7 (ANXA7) tumor suppressor role has been shown in various tumors, and ANXA7 expression has been particularly lost in androgen-resistant prostate cancers. In this study, we studied ANXA7 regulation in normal prostate versus androgen-sensitive and -resistant prostate cancer cells. Deletion mapping analysis showed lowest ANXA7-promoter activities in androgen-sensitive LNCaP prostate cancer cells. Genomatix analysis of ANXA7 promoter identified a cluster of steroid nuclear hormone receptor elements, including V$GREF (V$GRE.02/ ARE.02). Gelshift analysis clearly indicated distinct nuclear protein occupancy at this ANXA7-promoter site (À1086/À890) in prostate cancer (LNCaP, DU145, and PC3) versus normal prostate (PrEC) cells. In matrixassisted laser desorption time-of-flight mass spectrometrybased search for ANXA7 nuclear regulators, we identified several heterogeneous nuclear ribonucleoproteins (hnRNPs) (A1, A2/B1 and K) attached to the steroidassociated ANXA7-promoter site in the androgen-resistant PC3 prostate cancer cells with high ANXA7 gene copy number, but not in PrEC. The hnPNP role in ANXA7 regulation (that was validated by hnRNPA2/B1 antibody interference) resulted in multiple ANXA7 cDNA and protein products in PC3, but not in PrEC. Ingenuity pathways analysis showed plausible molecular paths between ANXA7 and the hnRNP-associated network in prostate cancer progression. Thus, a multi-hnRNP complex can be responsible for aberrant ANXA7 transcription and splicing, thereby affecting ANXA7 expression pattern and tumor suppressor function in prostate cancer.
Background: Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(ϩ/Ϫ) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(ϩ/Ϫ) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(ϩ/Ϫ) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca 2ϩ signaling pathway plays a role in  cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. Materials and Methods: To test this hypothesis, we subjected Anx7(ϩ/Ϫ) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. Results: Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA M.
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