Serena Pillozzi

HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemopoietic progenitors.

An important target in the understanding of the pathogenesis of acute myeloid leukemias (AML) relies on deciphering the molecular features of normal and leukemic hemopoietic progenitors. In particular, the analysis of the mechanisms involved in the regulation of cell proliferation is decisive for the establishment of new targeted therapies. To gain further insight into this topic we report herein a novel approach by analyzing the role of HERG K+ channels in the regulation of hemopoietic cell proliferation. These channels, encoded by the human ether-a-gò-gò-related gene (herg), belong to a family of K+ channels, whose role in oncogenesis has been recently demonstrated. We report here that herg is switched off in normal peripheral blood mononuclear cells (PBMNC) as well as in circulating CD34+ cells, however, it is rapidly turned on in the latter upon induction of the mitotic cycle. Moreover, hergappears to be constitutively activated in leukemic cell lines as well as in the majority of circulating blasts from primary AML. Evidence is also provided that HERG channel activity regulates cell proliferation in stimulated CD34+ as well as in blast cells from AML patients. These results open new perspectives on the pathogenetic role of HERG K+ channels in leukemias.

Chemotherapy resistance in acute lymphoblastic leukemia requires hERG1 channels and is overcome by hERG1 blockers

Bone marrow mesenchymal cells (MSCs) can protect leukemic cells from chemotherapy, thus increasing their survival rate. We studied the potential molecular mechanisms underlying this effect in acute lymphoblastic leukemia (ALL) cells. Coculture of ALL cells with MSCs induced on the lymphoblast plasma membrane the expression of a signaling complex formed by hERG1 (human ether-à-go-go-related gene 1) channels, the β(1)-integrin subunit, and the chemokine receptor CXC chemokine receptor-4. The assembly of such a protein complex activated both the extracellular signal-related kinase 1/2 (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/Akt prosurvival signaling pathways. At the same time, ALL cells became markedly resistant to chemotherapy-induced apoptosis. hERG1 channel function appeared to be important for both the initiation of prosurvival signals and the development of drug resistance, because specific channel blockers decreased the protective effect of MSCs. NOD/SCID mice engrafted with ALL cells and treated with channel blockers showed reduced leukemic infiltration and had higher survival rates. Moreover, hERG1 blockade enhanced the therapeutic effect produced by corticosteroids. Our findings provide a rationale for clinical testing of hERG1 blockers in the context of antileukemic therapy for patients with ALL.

Identification of a Posttranslational Mechanism for the Regulation of hERG1 K+ Channel Expression and hERG1 Current Density in Tumor Cells

ABSTRACT A common feature of tumor cells is the aberrant expression of ion channels on their plasma membrane. The molecular mechanisms regulating ion channel expression in cancer cells are still poorly known. K+ channels that belong to the human ether-a-go-go-related gene 1 (herg1) family are frequently misexpressed in cancer cells compared to their healthy counterparts. We describe here a posttranslational mechanism for the regulation of hERG1 channel surface expression in cancer cells. This mechanism is based on the activity of hERG1 isoforms containing the USO exon. These isoforms (i) are frequently overexpressed in human cancers, (ii) are retained in the endoplasmic reticulum, and (iii) form heterotetramers with different proteins of the hERG family. (iv) The USO-containing heterotetramers are retained intracellularly and undergo ubiquitin-dependent degradation. This process results in decreased hERG1 current (IhERG1) density. We detailed such a mechanism in heterologous systems and confirmed its functioning in tumor cells that endogenously express hERG1 proteins. The silencing of USO-containing hERG1 isoforms induces a higher IhERG1 density in tumors, an effect that apparently regulates neurite outgrowth in neuroblastoma cells and apoptosis in leukemia cells.

The increase of endothelial progenitor cells in the peripheral blood: a new parameter for detecting onset and severity of sepsis.

Sepsis is a clinical syndrome characterized by non-specific inflammatory response with evidence of profound changes in the function and structure of endothelium. Recent evidence suggests that vascular maintenance, repair and angiogenesis are in part mediated by recruitment from bone marrow (BM) of endothelial progenitor cells (EPCs). In this study we were interested in whether EPCs are increasingly mobilized during sepsis and if this mobilization is associated with sepsis severity. Our flow cytometry data demonstrate that in the CD34+ cell gate the number of EPCs in the blood of patients with sepsis had a four-fold increase (45 ± 4.5% p<0.001) compared to healthy controls (12 ± 3.6%) and that this increase was already evident at 6 hours from diagnosis (40.6 ± 4.2%), reaching its maximum at 72 hours. Also the percentage of cEPCs identified in the patients with sepsis (35 ± 4.6% of the CD34+ cell) was statistically different (p<0.001) compared to that found in the blood of patients with severe sepsis (75 ± 4.9%). In addition, we proved that at six hours after sepsis diagnosis, VEGF, CXCL8 and CXCL12 serum levels were significantly higher in septic patients compared to healthy volunteers 559 ± 82.14 pg/ml vs 2.9 ± 0.6 (p<0.0001), 189.8 ± 67.3 pg/ml 15 vs 11.9 ± 1.6 (p=0.014) and 780.5 ± 106.5 pg/ml; vs 190.2 ± 71.4 (p < 0.001). Our data suggest that the cEPC evaluation in peripheral blood, even at early times of diagnosis, in patients with sepsis can be envisaged as a valuable parameter to confirm diagnosis and suggest further prognosis.

Physical and Functional Interaction between Integrins and hERG1 Channels in Cancer Cells

Cancer is a complex multistep disease characterized by a profound genetic instability which leads to the aberrant and uncoordinated expression of several gene products, ultimately leading to the acquisition of a malignant phenotype. The identification of molecules and pathways that contribute to cancer establishment and progression has determined an enormous progress in oncology, providing new perspectives for the design of more specific and efficacious pharmacological approaches. In this picture, ion channels represent a relatively novel and unexpected player. In fact, the expression and activity of different channel types mark and regulate specific stages of cancer progression. The contribution of ion channels to the neoplastic phenotype ranges from the control of cell proliferation and apoptosis, to the regulation of invasiveness and metastatic spread. The role of ion channels in such processes can often be attributed to novel signaling mechanisms triggered and modulated by ion channel proteins, independently from ion fluxes. Ion channels encoded by the human ether-a-go-go-related gene 1 (herg), hERG 1 channels, are often aberrantly expressed in many primary human cancers and exert pleiotropic effects in cancer cells. Some of them are strictly related to the modulation of adhesive interactions with the extracellular matrix. The latter in turn can either regulate cell differentiation, or improve cell motility and invasiveness or stimulate the process of neo-angiogenesis. hERG1 channels can induce such diverse effects since they trigger and modulate intracellular signaling cascades. This role often depends on the formation, on the plasma membrane of tumor cells, of macromolecular complexes with membrane receptors, especially integrins. The link between hERG1 and integrins is twofold: integrins, mainly the beta1 integrin subunit, can activate hERG1. Conversely, the channels, once activated by integrins, can modulate signaling pathways downstream to integrin receptors. Both effects are mediated by the formation of a hERG1/beta1 integrin complex. Based on current evidence, we hypothesize that the activity of hERG1 channels inside the complex modulates the function of the partner protein(s) mainly through conformational coupling, instead of alterations of ion flow. Moreover, the hERG1-centered plasma membrane complexes, being specific of cancer cells, could represent novel targets for antineoplastic therapy.

New Insights into the Regulation of Ion Channels by Integrins

By controlling cell adhesion to the extracellular matrix, integrin receptors regulate processes as diverse as cell migration, proliferation, differentiation, apoptosis, and synaptic stability. Because the underlying mechanisms are generally accompanied by changes in transmembrane ion flow, a complex interplay occurs between integrins, ion channels, and other membrane transporters. This reciprocal interaction regulates bidirectional signal transduction across the cell surface and may take place at all levels of control, from transcription to direct conformational coupling. In particular, it is becoming increasingly clear that integrin receptors form macromolecular complexes with ion channels. Besides contributing to the membrane localization of the channel protein, the integrin/channel complex can regulate a variety of downstream signaling pathways, centered on regulatory proteins like tyrosine kinases and small GTPases. In turn, the channel protein usually controls integrin activation and expression. We review some recent advances in the field, with special emphasis on hematology and neuroscience. Some oncological implications are also discussed.

hERG1 channels modulate integrin signaling to trigger angiogenesis and tumor progression in colorectal cancer

Angiogenesis is a potential target for cancer therapy. We identified a novel signaling pathway that sustains angiogenesis and progression in colorectal cancer (CRC). This pathway is triggered by β1 integrin-mediated adhesion and leads to VEGF-A secretion. The effect is modulated by the human ether-à-go-go related gene 1 (hERG1) K+ channel. hERG1 recruits and activates PI3K and Akt. This in turn increases the Hypoxia Inducible Factor (HIF)-dependent transcription of VEGF-A and other tumour progression genes. This signaling pathway has novel features in that the integrin- and hERG1-dependent activation of HIF (i) is triggered in normoxia, especially after CRC cells have experienced a hypoxic stage, (ii) involves NF-kB and (iii) is counteracted by an active p53. Blocking hERG1 switches this pathway off also in vivo, by inhibiting cell growth, angiogenesis and metastatic spread. This suggests that non-cardiotoxic anti-hERG1 drugs might be a fruitful therapeutic strategy to prevent the failure of anti-VEGF therapy.

Relationships between hepatic melanogenesis and respiratory conditions in the newt, Triturus carnifex.

The Kupffer cells (melanomacrophages) in the livers of lower vertebrates contain varying quantities of melanin according to the season. Specimens of Triturus carnifex raised for 2 months at 6 degrees C and then transferred to water at 22 degrees C show a rapid increase in the hepatic accumulation of the pigment. The Kupffer cells make up more than one fourth of the liver mass in chlorbutol-anesthetized animals isolated for 6-7 hr in hypoxic water at 18 degrees C (to bring the oxygen content in a 620-mL respiratory chamber from 1.1 ppm to 0.0). Thus, hepatic melanin is synthesized when the newts oxygen supply is inadequate to meet its metabolic needs; melanogenesis, however, requires the presence of oxygen and does not occur in anesthetized specimens immersed in a totally anoxic fluid such as paraffin oil. The intraperitoneal injection prior to hypoxic treatment of 1 mg/g of body weight of kojic acid (inhibitor of the enzyme tyrosinase which catalyzes melanin synthesis) blocks melanogenesis and doubles oxygen consumption. The combination of hypoxia and tyrosinase inhibition causes permanent damage to essential functions of the nervous system, while hypoxic treatment alone has no irreversible consequences. The genic expression of tyrosinase in hypoxia appears to be a physiological response aimed at prolonging survival time in anaerobiosis by lowering the metabolic level; melanin would be an inert subproduct of this function.

Glutamine depletion by crisantaspase hinders the growth of human hepatocellular carcinoma xenografts

Background:A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO).Methods:Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1.Results:Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro.Conclusions:The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.

Developmentally regulated expression of the mouse homologues of the potassium channel encoding genes m-erg1, m-erg2 and m-erg3

Deciphering the expression pattern of K+ channel encoding genes during development can help in the understanding of the establishment of cellular excitability and unravel the molecular mechanisms of neuromuscular diseases. We focused our attention on genes belonging to the erg family, which is deeply involved in the control of neuromuscular excitability in Drosophila flies and possibly other organisms. Both in situ hybridisation and RNase Protection Assay experiments were used to study the expression pattern of mouse (m)erg1, m-erg2 and m-erg3 genes during mouse embryo development, to allow the pattern to be compared with their expression in the adult. M-erg1 is first expressed in the heart and in the central nervous system (CNS) of embryonic day 9.5 (E9.5) embryos; the gene appears in ganglia of the peripheral nervous system (PNS) (dorsal root (DRG) and sympathetic (SCG) ganglia, mioenteric plexus), in the neural layer of retina, skeletal muscles, gonads and gut at E13.5. In the adult m-erg1 is expressed in the heart, various structures of the CNS, DRG and retina. M-erg2 is first expressed at E9.5 in the CNS, thereafter (E13.5) in the neural layer of retina, DRG, SCG, and in the atrium. In the adult the gene is present in some restricted areas of the CNS, retina and DRG. M-erg3 displayed an expression pattern partially overlapping that of m-erg1, with a transitory expression in the developing heart as well. A detailed study of the mouse adult brain showed a peculiar expression pattern of the three genes, sometimes overlapping in different encephalic areas.