HERG1 channels drive tumour malignancy and may serve as prognostic factor in pancreatic ductal adenocarcinoma.
ABSTRACT: hERG1 channels are aberrantly expressed in human cancers. The expression, functional role and clinical significance of hERG1 channels in pancreatic ductal adenocarcinoma (PDAC) is lacking.hERG1 expression was tested in PDAC primary samples assembled as tissue microarray by immunohistochemistry using an anti-hERG1 monoclonal antibody (?-hERG1-MoAb). The functional role of hERG1 was studied in PDAC cell lines and primary cultures. ERG1 expression during PDAC progression was studied in Pdx-1-Cre,LSL-Kras(G12D/+),LSL-Trp53(R175H/+) transgenic (KPC) mice. ERG1 expression in vivo was determined by optical imaging using Alexa-680-labelled ?-hERG1-MoAb.(i) hERG1 was expressed at high levels in 59% of primary PDAC; (ii) hERG1 blockade decreased PDAC cell growth and migration; (iii) hERG1 was physically and functionally linked to the Epidermal Growth Factor-Receptor pathway; (iv) in transgenic mice, ERG1 was expressed in PanIN lesions, reaching high expression levels in PDAC; (v) PDAC patients whose primary tumour showed high hERG1 expression had a worse prognosis; (vi) the ?-hERG1-MoAb could detect PDAC in vivo.hERG1 regulates PDAC malignancy and its expression, once validated in a larger cohort also comprising of late-stage, non-surgically resected cases, may be exploited for diagnostic and prognostic purposes in PDAC either ex vivo or in vivo.
Project description: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 (I(hERG1)) 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 I(hERG1) density in tumors, an effect that apparently regulates neurite outgrowth in neuroblastoma cells and apoptosis in leukemia cells.
Project description:Background:Breast cancer (BC) is the most frequent malignancy among females worldwide. Despite several efforts and improvements in early diagnosis and treatment, there are still tumors characterized by an aggressive behavior due to unfavorable biology, thus quite difficult to treat. In this view, searching for novel potential biomarkers is mandatory. Among them, in the recent years data have been gathered addressing ion channel as important players in oncology. Methods:A retrospective pilot study was performed on 40 BC samples by means of immunohistochemistry in order to evaluate hERG1 potassium channels expression in BC. Results:We provide evidence that hERG1 is expressed in all the BC samples analyzed. hERG1 expression was significantly associated with molecular subtype with the highest expression in Luminal A and the lowest in basal-like tumors (p?=?0.001), tumor grading (the highest hERG1 expression in well-moderate differentiated tumors, p?=?0.020), estrogen receptors (high hERG1 expression in ER-positive samples, p?=?0.008) and Ki67 proliferative index (high hERG1 scoring in samples with low proliferative index, p?=?0.038). Also, a p value close to significance was noticed for the association between hERG1 and HER2 expression (p?=?0.079). At the survival analysis, patients with high hERG1 expression turned out to have a longer progression-free survival, although statistical significance was not reached (p?=?0.195). The same trend was observed analyzing local relapse free-survival (LRFS) and metastases-free survival (MFS): patients with higher hERG1 scoring had longer LRFS and MFS (p?=?0.124 and p?=?0.071, respectively). Conclusions:The results of this pilot study provide the first evidence that the hERG1 protein is expressed in primary BC, and its expression associates with molecular subtype. hERG1 apparently behaves as a protective factor, since it contributes to identify a subset of patients with better outcome. Overall, these data suggest that hERG1 might be an additional tool for the management of BC, nevertheless further investigations are warranted to better clarify hERG1 role and clinical usefulness in BC.
Project description:The human ether-a-go-go-related gene 1 (hERG1) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel. Several hERG1 isoforms with different N- and C-terminal ends have been identified. The hERG1a, hERG1b, and hERG1-3.1 isoforms contain the full-length C terminus, whereas the hERG1(USO) isoforms, hERG1a(USO) and hERG1b(USO), lack most of the C-terminal domain and contain a unique C-terminal end. The mechanisms underlying the generation of hERG1(USO) isoforms are not understood. We show that hERG1 isoforms with different C-terminal ends are generated by alternative splicing and polyadenylation of hERG1 pre-mRNA. We identified an intrinsically weak, noncanonical poly(A) signal, AGUAAA, within intron 9 of hERG1 that modulates the expression of hERG1a and hERG1a(USO). Replacing AGUAAA with the strong, canonical poly(A) signal AAUAAA resulted in the predominant production of hERG1a(USO) and a marked decrease in hERG1 current. In contrast, eliminating the intron 9 poly(A) signal or increasing the strength of 5' splice site led to the predominant production of hERG1a and a significant increase in hERG1 current. We found significant variation in the relative abundance of hERG1 C-terminal isoforms in different human tissues. Taken together, these findings suggest that post-transcriptional regulation of hERG1 pre-mRNA may represent a novel mechanism to modulate the expression and function of hERG1 channels.
Project description:1. Ventricular arrhythmias are rare but life-threatening side effects of therapy with the second-generation H(1) receptor antagonists terfenadine and astemizole. Blockade of the K(+) channels encoded by the Human Ether-à-go-go-Related Gene 1 (HERG1) K(+) channels, which is the molecular basis of the cardiac repolarizing current I(Kr), by prolonging cardiac repolarization, has been recognized as the mechanism underlying the cardiac toxicity of these compounds. 2. In the present study, the potential blocking ability of the novel second-generation H(1) receptor antagonist mizolastine of the HERG1 K(+) channels heterologously expressed in Xenopus oocytes and in HEK 293 cells or constitutively present in SH-SY5Y human neuroblastoma cells has been examined and compared to that of astemizole. 3. Mizolastine blocked HERG1 K(+) channels expressed in Xenopus oocytes with an estimated IC(50) of 3.4 microM. Mizolastine blockade was characterized by a fast dissociation rate when compared to that of astemizole; when fitted to a monoexponential function, the time constants for drug dissociation from the K(+) channel were 72.4+/-11.9 s for 3 microM mizolastine, and 1361+/-306 s for 1 microM astemizole. 4. In human embryonic kidney 293 cells (HEK 293 cells) stably transfected with HERG1 cDNA, extracellular application of mizolastine exerted a dose-related inhibitory action on I(HERG1), with an IC(50) of 350+/-76 nM. Furthermore, mizolastine dose-dependently inhibited HERG1 K(+) channels constitutively expressed in SH-SY5Y human neuroblastoma clonal cells. 5. The results of the present study suggest that the novel second-generation H(1) receptor antagonist mizolastine, in concentrations higher than those achieved in vivo during standard therapy, is able to block in some degree both constitutively and heterologously expressed HERG1 K(+) channels, and confirm the heterogeneity of molecules belonging to this therapeutical class with respect to their HERG1-inhibitory action.
Project description:Voltage-gated K(+) channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K(+) selective conductance. In some K(+) channels, the selectivity filter can undergo a conformational change to reduce K(+) flux by a mechanism called C-type inactivation. In human ether-a-go-go-related gene 1 (hERG1) K(+) channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome.
Project description:Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K(+) channel with properties similar to the rapidly activating delayed rectifying K(+) current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1a(USO) isoforms.A double-stable, mammalian expression system was developed to characterize isoform-specific dominant-negative effects of G628S-containing channels when co-expressed at equivalent levels with wild-type hERG1a. Western blot and co-immunoprecipitation studies were performed to study the trafficking and co-assembly of wild-type and mutant hERG1 isoforms. Patch-clamp electrophysiology was performed to characterize hERG1 channel function and the isoform-specific dominant-negative effects associated with the G628S mutation.The non-functional hERG1a-G628S and hERG1b-G628S channels co-assembled with wild-type hERG1a and dominantly suppressed hERG1 current. In contrast, G628S-induced dominant-negative effects were absent in the context of the hERG1a(USO) isoform. hERG1a(USO)-G628S channels did not appreciably associate with hERG1a and did not significantly suppress hERG1 current when co-expressed at equivalent ratios or at ratios that approximate those found in cardiac tissue. These results suggest that the dominant-negative effects of LQT2 mutations may primarily occur in the context of the hERG1a and hERG1b isoforms.
Project description:Human ether à go-go related gene (hERG1) potassium channels underlie the repolarizing I(Kr) current in the heart. Since they are targets of various drugs with cardiac side effects we tested whether the amiodarone derivative 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015) blocks hERG1 channels like its parent compound. Using patch-clamp and two-electrode voltage-clamp techniques we found that KB130015 blocks native and recombinant hERG1 channels at high voltages, but it activates them at low voltages. The activating effect has an apparent EC(50) value of 12microM and is brought about by an about 4-fold acceleration of activation kinetics and a shift in voltage-dependent activation by -16mV. Channel activation was not use-dependent and was independent of inactivation gating. KB130015 presumably binds to the hERG1 pore from the cytosolic side and functionally competes with hERG1 block by amiodarone, E4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl] carbonyl] phenyl] methanesulfonamide dihydrochloride), and sertindole. Vice versa, amiodarone attenuates hERG1 activation by KB130015. Based on synergic channel activation by mallotoxin and KB130015 we conclude that the hERG1 pore contains at least two sites for activators that are functionally coupled among each other and to the cavity-blocker site. KB130015 and amiodarone may serve as lead structures for the identification of hERG1 pore-interacting drugs favoring channel activation vs. block.
Project description:HERG (human ether-à-go-go-related gene) K(+) currents fulfill important ionic functions in cardiac and other excitable cells. In addition, HERG channels influence cell growth and migration in various types of tumor cells. The mechanisms underlying these functions are still not resolved. Here, we investigated the role of HERG channels for cell growth in a cell line (SW2) derived from small cell lung cancer (SCLC), a malignant variant of lung cancer. The two HERG1 isoforms (HERG1a, HERG1b) as well as HERG2 and HERG3 are expressed in SW2 cells. Inhibition of HERG currents by acute or sustained application of E-4031, a specific ERG channel blocker, depolarized SW2 cells by 10-15 mV. This result indicated that HERG K(+) conductance contributes considerably to the maintenance of the resting potential of about -45 mV. Blockage of HERG channels by E-4031 for up to 72 h did not affect cell proliferation. In contrast, siRNA-induced inhibition of HERG1 protein expression decreased cell proliferation by about 50%. Reduction of HERG1 protein expression was confirmed by Western blots. HERG current was almost absent in SW2 cells transfected with siRNA against HERG1. Qualitatively similar results were obtained in three other SCLC cell lines (OH1, OH3, H82), suggesting that the HERG1 channel protein is involved in SCLC cell growth, whereas the ion-conducting function of HERG1 seems not to be important for cell growth.
Project description:Ginsenoside 20(S)-Rg3 (Rg3) is a steroid glycoside that induces human ether-à-go-go-related gene type 1 (hERG1, Kv11.1) channels to activate at more negative potentials and to deactivate more slowly than normal. However, it is unknown whether this action is unique to hERG1 channels. Here we compare and contrast the mechanisms of actions of Rg3 on hERG1 with three other members of the ether-à-go-go (EAG) K(+) channel gene family, including EAG1 (Kv10.1), ERG3 (Kv11.3), and ELK1 (Kv12.1). All four channel types were heterologously expressed in Xenopus laevis oocytes, and K(+) currents were measured using the two-microelectrode voltage-clamp technique. At a maximally effective concentration, Rg3 shifted the half-point of voltage-dependent activation of currents by -14 mV for ERG1 (EC50 = 414 nM), -20 mV for ERG3 (EC50 = 374 nM), -28 mV for EAG1 (EC50 = 1.18 ?M), and more than -100 mV for ELK1 (EC50 = 197 nM) channels. Rg3 also induced slowing of ERG1, ERG3, and ELK1 channel deactivation and accelerated the rate of EAG1 channel activation. A Markov model was developed to simulate gating and the effects of Rg3 on the voltage dependence of activation of hELK1 channels. Understanding the mechanism underlying the action of Rg3 may facilitate the development of more potent and selective EAG family channel activators as therapies for cardiovascular and neural disorders.
Project description:Block of human ether-à-go-go-related gene 1 (hERG1) K(+) channels by many drugs delays cardiac repolarization, prolongs QT interval, and is associated with an increased risk of cardiac arrhythmia. Preferential block of hERG1 channels in an inactivated state has been assumed because inactivation deficient mutant channels can exhibit dramatically reduced drug sensitivity. Here we reexamine the link between inactivation gating and potency of channel block using concatenated hERG1 tetramers containing a variable number (0-4) of subunits harboring a point mutation (S620T or S631A) that disrupts inactivation. Concatenated hERG1 tetramers containing four wild-type subunits exhibited high-affinity block by cisapride, dofetilide, and MK-499, similar to wild-type channels formed from hERG1 monomers. A single S620T subunit within a tetramer was sufficient to fully disrupt inactivation gating, whereas S631A suppressed inactivation as a graded function of the number of mutant subunits present in a concatenated tetramer. Drug potency was positively correlated to the number of S620T subunits contained within a tetramer but unrelated to mutation-induced disruption of channel inactivation. Introduction of a second point mutation (Y652W) into S620T hERG1 partially rescued drug sensitivity. The potency of cisapride was not altered for tetramers containing 0 to 3 S631A subunits, whereas the potency of dofetilide was a graded function of the number of S631A subunits contained within a tetramer. Together these findings indicate that S620T or S631A substitutions can allosterically disrupt drug binding by a mechanism that is independent of their effects on inactivation gating.