Maureen Gilmore-Hebert

The Multifunctional Protein Glyceraldehyde-3-Phosphate Dehydrogenase Is Both Regulated and Controls Colony-Stimulating Factor-1 Messenger RNA Stability in Ovarian Cancer

Although glyceraldehyde-3-phosphate dehydrogenases (GAPDH) predilection for AU-rich elements has long been known, the expected connection between GAPDH and control of mRNA stability has never been made. Recently, we described GAPDH binding the AU-rich terminal 144 nt of the colony-stimulating factor-1 (CSF-1) 3′ untranslated region (UTR), which we showed to be an mRNA decay element in ovarian cancer cells. CSF-1 is strongly correlated with the poor prognosis of patients with ovarian cancer. We investigated the functional significance of GAPDHs association with CSF-1 mRNA and found that GAPDH small interfering RNA reduces both CSF-1 mRNA and protein levels by destabilizing CSF-1 mRNA. CSF-1 mRNA half-lives were decreased by 50% in the presence of GAPDH small interfering RNA. RNA footprinting analysis of the 144 nt CSF-1 sequence revealed that GAPDH associates with a large AU-rich–containing region. The effects of binding of GAPDH protein or ovarian extracts to mutations of the AU-rich regions within the footprint were consistent with this finding. In a tissue array containing 256 ovarian and fallopian tube cancer specimens, we found that GAPDH was regulated in these cancers, with almost 50% of specimens having no GAPDH staining. Furthermore, we found that low GAPDH staining was associated with a low CSF-1 score (P = 0.008). In summary, GAPDH, a multifunctional protein, now adds regulation of mRNA stability to its repertoire. We are the first to evaluate the clinical role of GAPDH protein in cancer. In ovarian cancers, we show that GAPDH expression is regulated, and we now recognize that one of the many functions of GAPDH is to promote mRNA stability of CSF-1, an important cytokine in tumor progression. (Mol Cancer Res 2008;6(8):1375–84)

Expression of α isoforms of the Na,K-ATPase in human heart

We studied expression of isoforms of Na,K-ATPase in normal and diseased human hearts. Na,K-ATPase alpha-isoform mRNA in samples from normal human left ventricle (LV) was composed of 62.5%, alpha 1, 15% alpha 2 and 22.5% alpha 3 on average. There was an increase in expression of the alpha 3 isoform in samples from failing hearts, but expression of all three isoforms decreased in pressure-overloaded right ventricle (RV).

cDNA microarray analysis of invasive and tumorigenic phenotypes in a breast cancer model

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets.

Cytoarchitectural relationships between [3H]ouabain binding and mRNA for isoforms of the sodium pump catalytic subunit in rat brain

We examined the cell type-specific expression of the alpha 1, alpha 2, and alpha 3 subunits of the sodium pump in rat brain using in situ hybridization and [3H]ouabain autoradiography. These techniques allowed us to colocalize mRNA and functional alpha 2/alpha 3 pumps on adjacent sections. The perikarya of many neurons possessed high levels of alpha 1 and/or alpha 3 transcripts, while alpha 2 mRNA appeared to be present in only a few neuronal types. [3H]Ouabain binding in general paralleled the distribution of alpha 3 mRNA-positive neurons. The regional variation of alpha 1 and alpha 3 transcripts was complex and varied. Large neurons of the olfactory bulb and piriform cortex expressed high levels of alpha 3 transcripts, but low levels of alpha 1 mRNA. In frontal cortex, neurons of layers II-III were enriched in alpha 1 mRNA, while those in layer V exhibited high levels of alpha 3 transcripts. In the hippocampus, principal neurons expressed all three alpha subunit mRNAs. CA subfield pyramidal neurons exhibited a high alpha 3/alpha 1 ratio, while dentate granule cells and hilar pyramidal neurons expressed approximately equal levels of alpha 1 and alpha 3. In the cerebellum, Purkinje and Golgi cells were rich in alpha 3 mRNA, while the granule cells appeared to express only alpha 1 transcripts. The distribution of functional sodium pump protein, as localized by [3H]ouabain binding, was highest in the neuropil of the hippocampus and cerebral cortex, and lowest over perikarya and white matter. [3H]ouabain did not bind to alpha 1 pump units, as confirmed by the complete absence of labeling over the choroid plexus, a tissue expressing only alpha 1 mRNA. In the cerebellum, regions of dense [3H]ouabain binding were localized to the granule cell layer, the inner third of the molecular layer in the basket region, and the deep cerebellar nuclei. Surprisingly, the dense neuropil in the outer 2/3 of the molecular layer lacked high [3H]ouabain binding. Thus, functional alpha 3 sodium pump units appear distributed to the axon terminals and not to apical dendrites of Purkinje, Golgi and basket cells. A similar pattern of increased [3H]ouabain binding in axonal but not dendritic fields of alpha 3-enriched neurons was present in the cerebral cortex and the hippocampus. Considering that many alpha 3-enriched neurons are of the Golgi I type with long axons, the alpha 3 isoform may be preferentially directed into axons to function in presynaptic membranes.

SGK1, a potential regulator of c-fms related breast cancer aggressiveness

The aggressive behavior of breast cancer cells can at times be modulated by hormonal mechanisms. Exposure to glucocorticoids (GC) has been shown to stimulate the invasiveness, motility and adhesiveness of breast cancer cells containing the glucocorticoid receptor. This is largely explained by GC-associated overexpression of the c-fms proto-oncogene, which encodes the receptor for the colony stimulating factor-1 (CSF-1). Our objective is to investigate additional GC-associated genetic alterations that could modulate c-fms related malignant behavior in breast cancer cells. A microarray technique using an oligonucleotide array representing 16,700 known expressed human genes was used to analyze the gene expression profile of breast cancer cells exposed to dexamethasone (Dex) or vehicle. Results were confirmed by western blot analysis. Six genes were found to be consistently differentially overexpressed in the Dex-exposed cells compared to control. We focused on serum-glucose kinase 1 (SGK1), a serine-threonine kinase known to be involved in intracellular signal transduction pathways and induced by GC and serum. An adhesion assay was performed on extracellular matrix after exposing the breast cancer cells to Dex, CSF-1 or to Dex or CSF-1 plus LY294002, a functional inhibitor of SGK1 action. Exposure to LY294002 significantly decreased both CSF-1 and Dex-induced adhesiveness to the level of control cells. SGK1 may act as a downstream intracellular regulator of c-fms, particularly of c-fms-induced adhesiveness of breast cancer cells after exposure to GC or CSF-1. This finding may have implications for potential therapeutic interventions aimed at decreasing the aggressiveness of breast cancer cells.

Na, K-ATPase isoform gene expression in normal and hypertrophied dog heart.

ObjectivesThe catalytic α subunit of the sodium-potassium ATPase, the target of digitalis glycosides, has three isoforms is tissuespecific and developmentally regulated. While the effect of pressure overload on Na, K-ATPase isoform expression has been studied in rodent heart, there are no systematic data on this question in hearts of larger animals, which differ from those of rodents both in isoform composition and in glycoside sensitivity. Thus, we investigated the expression of Na, K-ATPase isoforms in normal dog heart; we also examined the effect of experimental left ventricular hypertrophy on isoform expression.Methodshypertrophy was produced by aortic banding. Expression was assessed by quantitative Northern and Western blotting, immuno-fluorescence, and3H-ouabain binding.ResultsRNA blotting indicated that the α3 isoform represented 11% of Na, K-ATPase mRNA in normal dog LV. Normal dog LV expressed α1 and α3 protein, but no detectable α2; immunoreactive α1 and α3 protein were also present in Purkinje fibers. There was a statistically significant decrease in total expression of all α isoform mRNAs in hypertrophied dog LV, resulting in a greater proportion of α1. The expression level of the α3 isoform mRNA and protein was lower in hypertrophied hearts.ConclusionsThese results indicate a greater proportion of α1 isoform pumps in experimental canine hypertrophy. Thus, shifts in Na, K-ATPase isoforms occur in pressure-overloaded heart in large animals as well as rodents.

Regulation of non-AU-rich element containing c-fms proto-oncogene expression by HuR in breast cancer

The role of RNA-binding proteins in cancer biology is recognized increasingly. The nucleocytoplasmic shuttling and AU-rich RNA-binding protein HuR stabilizes several cancer-related target mRNAs. The proto-oncogene c-fms, whose 3′untranslated region (3′UTR) is not AU-rich, is associated with poor prognosis in breast cancer. Using a large breast-cancer tissue array (N=670), we found nuclear HuR expression to be associated with nodal metastasis and independently with poor survival (P=0.03, RR 1.45), as well as to be co-expressed with c-fms in the breast tumors (P=0.0007). We described c-fms mRNA as a direct target of HuR in vivo, and that HuR bound specifically to a 69-nt region containing ‘CUU’ motifs in 3′UTR c-fms RNA. Overexpressing or silencing HuR significantly up- or down-regulated c-fms RNA expression, respectively. We also found that known glucocorticoid stimulation of c-fms RNA and protein is largely dependent on the presence of HuR. HuR, by binding to the 69-nt wild type, but not mutant, c-fms sequence can regulate reporter gene expression post-transcriptionally. We are the first to describe that HuR can regulate gene expression by binding non-AU-rich sequences in 3′UTR c-fms RNA. Collectively, our findings suggest that HuR plays a supportive role for c-fms in breast cancer progression by binding a 69-nt element in its 3′UTR, thus regulating its expression.

Post-transcriptional regulation of c-fms proto-oncogene expression by dexamethasone and of CSF-1 in human breast carcinomas in vitro

The c-fms proto-oncogene encodes the receptor for a hematopoietic growth factor, CSF-1. Recently, the importance of c-fms and its ligand CSF-1 in malignancies of non-hematopoietic origin, such as breast, ovarian, endometrial, pulmonary, and trophoblastic cancers has been recognized. We have previously shown that glucocorticoids induce a large increase in c-fms mRNA and protein levels in breast carcinoma cell lines. In this report, we investigate the mechanism underlying such c-fms overexpression by dexamethasone. We show that dexamethasone treatment of two breast carcinoma cell lines (BT20-c-fms expressor, and SKBR3-co-expressor of both c-fms and CSF-1) does not increase the rate of c-fms gene transcription, suggesting a post-transcriptional mechanism of regulation of c-fms expression by dexamethasone. The effect of protein synthesis inhibition was studied to help determine whether there was a role for intermediary regulatory proteins in the regulation of c-fms expression. We find that several protein synthesis inhibitors interfere with dexamethasone induction of c-fms transcripts, suggesting the existence of regulatory proteins. These regulatory proteins do not appear to be constitutively expressed, as we show no effect of protein synthesis inhibition on c-fms transcript expression in resting BT20 cells. These findings suggest that the putative regulatory proteins are induced by dexamethasone. Furthermore, the addition of a protein synthesis inhibitor, pactamycin, to dexamethasone-treated BT20 cells results in a decrease in c-fms mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of ErbB4 and Kap1 Connect the Growth Factor and DNA Damage Response Pathways

ErbB4 is unusual among receptor tyrosine kinases because some isoforms can be efficiently cleaved at the plasma membrane to release a soluble intracellular domain. The cleavage product has high kinase activity and homes to the nucleus. A screen for proteins that associate with the ErbB4 intracellular domain identified candidate interactors including ITCH, WWP2, Nucleolin, and Krab-associated protein 1 (Kap1). Kap1 binds to multiple isoforms of ErbB4 but does not require ErbB4 kinase activity for binding, nor is it an ErbB4 substrate. Kap1 reduces ERBB4 transcription and either directly or indirectly modulates the expression of genes that are themselves regulated by ErbB4. Upregulation of ErbB4 and suppression of MDM2 jointly enhance and accelerate the accumulation of p21CIP1 in response to DNA damage. Overall, these findings further substantiate the role of ErbB4 in conjoint regulation of growth factor signaling and DNA damage responses. Mol Cancer Res; 8(10); 1388–98. ©2010 AACR.

Expression of multiple Na+,K+-adenosine triphosphatase isoform genes in human hematopoietic cells. Behavior of the novel A3 isoform during induced maturation of HL60 cells.

Multiple isoenzymes of the Na+,K+-ATPase (alpha, alpha+, and alpha 3) have been identified by molecular cloning (Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Biochemistry. 25:8125-8132; and Schneider, J. W., R. W. Mercer, and E. J. Benz, Jr. 1987. Clin. Res. 35:585A. [Abstr.]). At least one of these, the alpha 3 chain, represents a novel form for which protein products and enzymatic activities are just beginning to be defined in rodents. We have recently demonstrated that expression of alpha 3 is largely confined to neuromuscular tissues of fetal and adult rats (Schneider, J. W., R. W. Mercer, M. Gilmore-Hebert, M. F. Utset, C. Lai, A. Greene, and E. J. Benz, Jr. 1988. Proc. Natl. Acad. Sci. USA. 85:284-288). We now report that certain human leukemia cell lines including HL60, HEL, and Molt 4 express mRNA for both alpha and alpha 3 isoforms of Na+,K+-ATPase; mRNA was not detected in several other cell lines, including K562 and U937; no cell lines expressed alpha+ mRNA. In uninduced HL60 cells, alpha 3 mRNA comprised 20-30% of total Na+,K+-ATPase mRNA. Furthermore, in HL60 and HEL cells, both alpha and alpha 3 mRNA declined after induction of maturation by DMSO, retinoic acid, or hemin. However, the reduction in alpha 3 mRNA was far more dramatic. alpha 3 mRNA virtually disappeared, but alpha mRNA declined by only approximately 50%. In contrast, when maturation of HL60 cells along the monocyte/macrophage lineage was induced by exposure to phorbol esters, alpha 3 mRNA remained abundant. Moreover, mRNA for the beta subunit of the Na+,K+-ATPase increased dramatically. Our results demonstrate that the alpha 3 isoform, formerly thought to be confined to neuromuscular tissues, is expressed in restricted lineages of hematopoietic origin. These leukemia cell lines should provide a useful model for analyzing regulation of the alpha 3 isoform gene and characterization of alpha 3 isoform activities.