Suzana Kovac

The role of hypoxia-inducible factor 1α in determining the properties of castrate-resistant prostate cancers.

Background Castrate-resistant prostate cancer (CRPC) is a lethal condition in patients receiving androgen deprivation therapy for prostate cancer (PC). Despite numerous studies showing the expression of HIF1α protein under normoxia in PC cell lines, the role of this normoxic HIF1α expression in chemo-resistance and migration has not been investigated previously. As no method is currently available to determine which tumors will progress to CRPC, the role of HIF1α in PC and its potential for predicting the development of CRPC was also investigated. Methods The effect of HIF1α protein knockdown on chemo-resistance and migration of PC3 cells was assessed by cell counting and Transwell assays, respectively. Translation efficiency of HIF1α mRNA was determined in PC cells using a HIF1α 5′UTR-luciferase construct. Clinical outcomes were correlated following the staining of 100 prostate tumors for HIF1α expression. Results The CRPC-like cell lines (PC3 and DU145) expressed more HIF1α protein than an androgen sensitive cell line (LNCaP). Migration rate and chemo-resistance were higher in the PC3 cells and both were decreased when HIF1α expression was reduced. Increased translation of HIF1α mRNA may be responsible for HIF1α overexpression in PC3 cells. Patients whose tumors expressed HIF1α had significantly decreased metastasis-free survival and the patients who were on androgen-deprivation therapy had decreased CRPC-free survival on Kaplan-Meier analysis. On multivariate analysis HIF1α was an independent risk factor for progression to metastatic PC (Hazard ratio (HR) 9.8, p = 0.017) and development of CRPC (HR 10.0, p = 0.021) in patients on androgen-deprivation therapy. Notably the tumors which did not express HIF1α did not metastasize or develop CRPC. Conclusions HIF1α is likely to contribute to metastasis and chemo-resistance of CRPC and targeted reduction of HIF1α may increase the responsiveness of CRPCs to chemotherapy. Expression of HIF1α may be a useful screening tool for development of CRPC.

Gastrin increases its own synthesis in gastrointestinal cancer cells via the CCK2 receptor.

The involvement of the gastrointestinal hormone gastrin in the development of gastrointestinal cancer is highly controversial. Here we demonstrate a positive‐feedback loop whereby gastrin, acting via the CCK2 receptor, increases its own expression. Such an autocrine loop has not previously been reported for any other gastrointestinal hormone. Gastrin promoter activation was dependent on the MAP kinase pathway and did not involve Sp1 binding sites or epidermal growth factor receptor transactivation. As the treatment of gastrointestinal cancer cells with amidated gastrin led to increased expression of non‐amidated gastrins, the positive‐feedback loop may contribute to the sustained increase in circulating gastrins observed in colorectal cancer patients.

A Novel Effect of Bismuth Ions SELECTIVE INHIBITION OF THE BIOLOGICAL ACTIVITY OF GLYCINE-EXTENDED GASTRIN

Although bismuth salts have been used for over two centuries for the treatment of various gastrointestinal disorders, the mechanism of their therapeutic action remains controversial. Because gastrins bind two trivalent ferric ions with high affinity, and because ferric ions are essential for the biological activity of glycine-extended gastrin 17, we have investigated the hypothesis that trivalent bismuth ions influence the biological activity of gastrins. Binding of bismuth ions to gastrins was measured by fluorescence quenching and NMR spectroscopy. The effects of bismuth ions on gastrin-stimulated biological activities were measured in inositol phosphate, cell proliferation, and cell migration assays. Fluorescence quenching experiments indicated that both glycine-extended and amidated gastrin 17 bound two bismuth ions. The NMR spectral changes observed on addition of bismuth ions revealed that Glu-7 acted as a ligand at the first bismuth ion binding site. In the presence of bismuth ions the ability of glycine-extended gastrin 17 to stimulate inositol phosphate production, cell proliferation, and cell migration was markedly reduced. In contrast, bismuth ions had little effect on the affinity of the CCK-2 receptor for amidated gastrin 17, or on the stimulation of inositol phosphate production by amidated gastrin 17. We conclude that bismuth ions may act, at least in part, by blocking the effects of glycine-extended gastrin 17 on cell proliferation and cell migration in the gastrointestinal tract. This is the first report of a specific inhibitory effect of bismuth ions on the action of a gastrointestinal hormone.

Gastrins, iron homeostasis and colorectal cancer.

The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron deficiency anemia was recognized as a long-term consequence of partial gastrectomy. This review summarizes the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyze the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Stimulation of proliferation in the colorectal mucosa by gastrin precursors is blocked by desferrioxamine

Precursors of the peptide hormone gastrin stimulate proliferation in the colorectal mucosa and promote the development of colorectal carcinoma. Gastrins bind two ferric ions selectively and with high affinity, and the biological activity of glycine-extended gastrin (Ggly) in vitro is dependent on the presence of ferric ions. The aim of the present study was to determine whether or not iron is required for biological activity of progastrin and Ggly in vivo. Rats that had undergone a colostomy were infused with Ggly, and proliferation was measured in the defunctioned rectal mucosa. Proliferation was also measured in the colonic mucosa of hGAS and MTI-Ggly mice, which, by definition, overexpress progastrin and Ggly, respectively. The requirement for iron was assessed by thrice-weekly injection of the chelating agent desferrioxamine (DFO). The proliferation index in the defunctioned rectal mucosa was significantly increased in the Ggly-infused rats, and the increase was significantly reduced after treatment with DFO. Treatment with DFO significantly reduced the crypt height and proliferation index in the colonic mucosa of hGAS and MTI-Ggly mice but had no effect on the same variables in wild-type mice. These observations are consistent with the hypothesis that the biological activity of progastrin and Ggly in vivo is dependent on the presence of ferric ions and further suggest that chelating agents may block the stimulatory effects of gastrin precursors in the development of colorectal carcinoma.

Induction of Gastrin Expression in Gastrointestinal Cells by Hypoxia or Cobalt Is Independent of Hypoxia-Inducible Factor (HIF)

Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P < 0.05), respectively. The hypoxia mimetic, cobalt chloride (300 μM), increased gastrin promoter activity in AGS cells by 2.4 ± 0.3-fold (P < 0.05), and in AGS-cholecystokinin receptor 2 cells by 4.0 ± 0.3-fold (P < 0.05), respectively. The observations that either deletion from the gastrin promoter of the putative binding sites for the transcription factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

Peptide processing and biology in human disease.

Purpose of reviewTo describe recent advances in the processing of gastrointestinal hormones, and the consequences for human disease of mutations in the enzymes involved. Recent findingsAlthough gastrointestinal prohormones were long regarded as devoid of biological activity, recent data indicate that the prohormones for both gastrin and gastrin-releasing peptide are bioactive, through different receptors from the mature hormones. Mutations in the family of prohormone convertases responsible for the initial steps in the processing of gastrointestinal hormones are associated with several different pathophysiological conditions in humans. SummaryHuman mutational studies, when taken together with the phenotypes observed in mice deficient in the prohormone convertases, emphasize the crucial importance of the processing enzymes in mammalian biology. Although the phenotypes may often be ascribed to defective production of a mature hormone or growth factor, the recognition that the precursors are independently bioactive suggests that the increased precursor concentrations may also contribute to the symptoms. The observation that the precursors often act through different receptors from the mature hormones may permit the development of precursor-selective antagonists for therapeutic use.

Circulating gastrin is increased in hemochromatosis.

Gastric acid production is important in intestinal iron absorption. The peptide hormone gastrin exists in both amidated and non‐amidated forms, which stimulate and potentiate gastric acid secretion, respectively. Since non‐amidated gastrins require ferric ions for biological activity in vitro, this study investigated the connection between iron status and gastrin by measurement of circulating gastrin concentrations in mice and humans with hemochromatosis. Gastrin concentrations are increased in the plasma and gastric mucosa of Hfe −/− mice, and in the sera of humans with HFE‐related hemochromatosis. The discovery of a relationship between iron status and circulating gastrin concentrations opens a new perspective on the mechanisms of iron homeostasis.

Interrelationships between circulating gastrin and iron status in mice and humans

The observations that the peptide hormone gastrin interacts with transferrin in vitro and that circulating gastrin concentrations are increased in the iron-loading disorder hemochromatosis suggest a possible link between gastrin and iron homeostasis. This study tested the hypothesis that gastrin and iron status are interrelated by measurement of iron homeostasis in mice and humans with abnormal circulating gastrin concentrations. Intestinal iron absorption was determined by (59)Fe uptake following oral gavage, and concentrations of duodenal divalent metal transporter-1 (DMT-1) and hepatic hepcidin mRNAs were determined by quantitative real-time PCR in agastrinemic (GasKO), hypergastrinemic cholecystokinin 2 receptor-deficient (CCK2RKO), or wild-type mice. Iron status was measured by standard methods in the same mice and in hypergastrinemic humans with multiple endocrine neoplasia type 1 (MEN-1). Iron absorption was increased sixfold and DMT-1 mRNA concentration fourfold, and transferrin saturation was reduced 0.8-fold and hepcidin mRNA expression 0.5-fold in juvenile GasKO mice compared with age-matched wild-type mice. In mature mice, few differences were observed between the strains. Juvenile CCK2RKO mice were hypergastrinemic and had a 5.4-fold higher DMT-1 mRNA concentration than wild-type mice without any increase in iron absorption. In contrast to juvenile GasKO mice, juvenile CCK2RKO mice had a 1.5-fold greater transferrin saturation, which was reflected in a twofold increase in liver iron deposition at maturity compared with wild-type mice. The correlation between transferrin saturation and circulating gastrin concentration observed in mutant mice was also observed in human patients with MEN, in whom hypergastrinemia correlated positively (P = 0.004) with an increased transferrin saturation. Our data indicate that, in juvenile animals when iron demand is high, circulating gastrin concentrations may alter iron status by a CCK2R-independent mechanism.

Zinc ions upregulate the hormone gastrin via an E-box motif in the proximal gastrin promoter

Gastrin and its precursors act as growth factors for the normal and neoplastic gastrointestinal mucosa. As the hypoxia mimetic cobalt chloride upregulates the gastrin gene, the effect of other metal ions on gastrin promoter activity was investigated. Gastrin mRNA was measured by real-time PCR, gastrin peptides by RIA, and gastrin promoter activity by dual-luciferase reporter assay. Exposure to Zn(2)(+) ions increased gastrin mRNA concentrations in the human gastric adenocarcinoma cell line AGS in a dose-dependent manner, with a maximum stimulation of 55 ± 14-fold at 100 μM (P<0.05). Significant stimulation was also observed with Cd(2)(+) and Cu(2)(+), but not with Ca(2)(+), Mg(2)(+), Ni(2)(+), or Fe(3)(+) ions. Activation of MAPK and phosphatidylinositol 3-kinase pathways is necessary but not sufficient for gastrin induction by Zn(2)(+). Deletional mutation of the gastrin promoter identified an 11 bp DNA sequence, which contained an E-box motif, as necessary for Zn(2)(+)-dependent gastrin induction. The fact that E-box binding transcription factors play a crucial role in the epithelial-mesenchymal transition (EMT), together with our observation that Zn(2)(+) ions upregulate the gastrin gene in AGS cells by an E-box-dependent mechanism, suggests that Zn(2)(+) ions may induce an EMT, and that gastrin may be involved in the transition.