Regulation of IGF-1-dependent cyclin D1 and E expression by hEag1 channels in MCF-7 cells: The critical role of hEag1 channels in G1 phase progression

AbstractInsulin-like Growth Factor-1 (IGF-1) plays a key role in breast cancer development and cell cycle regulation. It has been demonstrated that IGF-1 stimulates cyclin expression, thus regulating the G1 to S phase transition of the cell cycle. Potassium (K+) channels are involved in the G1 phase progression of the cell cycle induced by growth factors. However, mechanisms that allow growth factors to cooperate with K+ channels in order to modulate the G1 phase progression and cyclin expression remain unknown. Here, we focused on hEag1 K+ channels which are over-expressed in breast cancer and are involved in the G1 phase progression of breast cancer cells (MCF-7). As expected, IGF-1 increased cyclin D1 and E expression of MCF-7 cells in a cyclic manner, whereas the increase of CDK4 and 2 levels was sustained. IGF-1 stimulated p21WAF1/Cip1 expression with a kinetic similar to that of cyclin D1, however p27Kip1 expression was insensitive to IGF-1. Interestingly, astemizole, a blocker of hEag1 channels, but not E4031, a blocker of HERG channels, inhibited the expression of both cyclins after 6–8h of co-stimulation with IGF-1. However, astemizole failed to modulate CDK4, CDK2, p21WAF1/Cip1 and p27Kip1 expression. The down-regulation of hEag1 by siRNA provoked a decrease in cyclin expression. This study is the first to demonstrate that K+ channels such as hEag1 are directly involved in the IGF-1-induced up-regulation of cyclin D1 and E expression in MCF-7 cells. By identifying more specifically the temporal position of the arrest site induced by the inhibition of hEag1 channels, we confirmed that hEag1 activity is predominantly upstream of the arrest site induced by serum-deprivation, prior to the up-regulation of both cyclins D1 and E

Are Orai1 and Orai3 channels more important than calcium influx for cell proliferation?

AbstractTransformed and tumoral cells share the characteristic of being able to proliferate even when external calcium concentration is very low. We have investigated whether Human Embryonic Kidney 293 cells, human hepatoma cell Huh-7 and HeLa cells were able to proliferate when kept 72h in complete culture medium without external calcium. Our data showed that cell proliferation rate was similar over a range of external calcium concentration (2μM to 1.8mM). Incubation in the absence of external calcium for 72h had no significant effect on endoplasmic reticulum (ER) Ca2+ contents but resulted in a significant decrease in cytosolic free calcium concentration in all 3 cell types. Cell proliferation rates were dependent on Orai1 and Orai3 expression levels in HEK293 and HeLa cells. Silencing Orai1 or Orai3 resulted in a 50% reduction in cell proliferation rate. Flow cytometry analysis showed that Orai3 induced a small but significant increase in cell number in G2/M phase. RO-3306, a cdk-1 inhibitor, induced a 90% arrest in G2/M reversible in less than 15min. Our data showed that progression through G2/M phase after release from RO-3306-induced cell cycle arrest was slower in both Orai1 and Orai3 knock-downs. Overexpressing Orai1, Orai3 and the dominant negative non-permeant mutants E106Q-Orai1 and E81Q-Orai3 induced a 50% increase in cell proliferation rate in HEK293 cells. Our data clearly demonstrated that Orai1 and Orai3 proteins are more important than calcium influx to control cell proliferation in some cell lines and that this process is probably independent of ICRAC and Iarc

Remodeling of Channel-Forming ORAI Proteins Determines an Oncogenic Switch in Prostate Cancer

SummaryORAI family channels have emerged as important players in malignant transformation, yet the way in which they reprogram cancer cells remains elusive. Here we show that the relative expression levels of ORAI proteins in prostate cancer are different from that in noncancerous tissue. By mimicking ORAI protein remodeling observed in primary tumors, we demonstrate in in vitro models that enhanced ORAI3 expression favors heteromerization with ORAI1 to form a novel channel. These channels support store-independent Ca2+ entry, thereby promoting cell proliferation and a smaller number of functional homomeric ORAI1-based store-operated channels, which are important in supporting susceptibility to apoptosis. Thus, our findings highlight disrupted dynamic equilibrium of channel-forming proteins as an oncogenic mechanism

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Rôle déterminant des canaux potassiques hEag1 dans le cycle cellulaire induit par l'IGF-1 (implication dans le cancer du sein)

Le cancer du sein est une préoccupation majeure de santé publique. Il est établi que les oestrogènes favorisent la croissance des cellules cancéreuses mammaires. C'est pourquoi, les principales stratégies thérapeutiques utilisées reposent sur l'hormonothérapie anti-oestrogéniques. Cependant, ces traitements ne possèdent pas une efficacité absolue, qui peut être expliquée par l'action mitogène des facteurs de croissance, tels que l'IGF-1. Ce facteur est défini comme un facteur de risque dans le cancer du sein et un puissant mitogène des cellules cancéreuses. Par ailleurs, il apparait que les canaux potassiques jouent un rôle prépondérant dans la croissance des cellules, et notamment les canaux potassiques de type hEag1 dont le potentiel oncogénique est reconnu. Le but de ce mémoire a été de comprendre le rôle de ces canaux dans l'effet mitogène de l'IGF-1 sur les cellules MCF-7. Nos travaux montrent que l' IGF-1 est capable de moduler l'activité et l'expression des canaux hEag1 impliquant la voie PI3K. Cet effet apparait essentiel pour la progression des cellules cancéreuses mammaires dans le cycle cellulaire favorisant ainsi leur croissance. De plus, nous démontrons que l'activité des canaus hEag1 est nécessaire à la mise en place des évènements précédents la synthèse d'ADN, à savoir l'augmentation de l'expression des cyclines D1 et E induite par l'IGF-1. Ainsi, nous démontrons l'importance des canaux hEag1 dans la prolifération des cellules cancéreuses mammaires induite par l'IGF-1. En conclusion, nos travaux permettent de proposer les canaux hEag1 comme cible potentielle dans le cadre des traitements du cancer du sein, plus particulièrement ceux présentant une forte activité du système IGF.Breast cancer has the highest incidence rate for cancer in women in industrialized countries including France. Statistically, it is estimated that one woman out of nine will develop breast cancer at some point in her life. The main treatment for breast cancer consists of cell growth inhibition by using anti-estrogenic treatments. However, this results only in a partial regression of the disease. Therefore, other growth factors, like IGF-1 is considered to be important in the breast cancer development. Indeed, elevated levels of circulating IGF-1 are associated with increased risk of breast cancer. Furthermore, ether à go-go (hEag1) K+ channels have been reported to be crucial for breast cancer cell proliferation, cell cycle progression and considered as a tumour marker. The goal of this study was to investigate the regulation of hEag1 K+ channel by IGF-1 and to determine whether this regulation is involved in the IGF-1 mitogenic effects on human breast cancer cells MCF-7. Our results show that IGF-1 modulates both the activity and the expression of hEag1 K+ channels through the PI3K pathway. By these two mechanisms, we suggest that IGF-1 stimulates the proliferation of MCF-7 cells. We demonstrated, for the first time, the involvement of hEag1 channels in the regulation of cyclin D1 and E expression induced by IGF-1 in MCF-7 cells leading to G1 phase progression and G1/S transition and therefore to cell proliferation. In conclusion, our study brings very new elements in the understanding of function of hEag1 channels and may lead to the development of ion channel targeted cancer therapy through IGF-1 modulation.AMIENS-BU Santé (800212102) / SudocSudocFranceF

Fine-tuning of eTRPM8 expression and activity conditions keratinocyte fate

International audienceRecently, we reported the cloning and characterization of short isoform of the icilin-activated cold receptor TRPM8 channel in keratinocytes, dubbed eTRPM8. We demonstrated that eTRPM8 via fine tuning of the endoplasmic reticulum (ER) – mitochondria Ca2+ shuttling regulates mitochondrial ATP and superoxide (O2•-) production and, thereby, mediates control of epidermal homeostasis by mild cold. Here, we provide additional information explaining why eTRPM8 suppression and TRPM8 stimulation both inhibit keratinocyte growth. We also demonstrate that stimulation of eTRPM8 with icilin may give rise to sustained oscillatory responses. Furthermore, we show that ATP-induced cytosolic and mitochondrial Ca2+ responses are attenuated by eTRPM8 suppression. This suggests positive interplay between eTRPM8 and purinergic signaling pathways, what may serve to facilitate the ER-mitochondria Ca2+ shuttling. Finally, we demonstrate that cold (25°C) induces eTRPM8-dependent superoxide-mediated necrosis of keratinocytes. Altogether, these results are in line with our model of eTRPM8-mediated cold-dependent balance between keratinocyte proliferation and differentiatio

Optimal differentiation of in vitro keratinocytes requires multifactorial external control.

For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32 °C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic stimulation by calcium, serum, confluence and lower incubation temperature amplified the differentiation rate

Calcium channels, external calcium concentration and cell proliferation

International audienceEvidence for a role for calcium channel proteins in cell proliferation is numerous suggesting that calcium influx is essential in this physiological process. Several studies in the past thirty years have demonstrated that calcium channel expression levels are determinant in cell proliferation. Voltage-gated, storeoperated, second messengers and receptor-operated calcium channels have been associated to cell proliferation. However, the relationship between calcium influx and cell proliferation can be uncoupled in transformed and cancer cells, resulting in an external calcium-independent proliferation. Thus, protein expression could be more important than channel function to trigger cell proliferation suggesting that additional channel functions may be responsible to reconcile calcium channel expression and cell proliferation. When needed, external calcium concentration is obviously important for calcium channel function but it also regulates calcium sensing receptor (CaSR) activity. CaSR can up-or down-regulate cell proliferation depending on physiological conditions. CaSR sensitivity to external calcium is within the 0.5 to 5 mM range and therefore, the role of these receptors in cell proliferation must be taken into account. We therefore suggest here that cell proliferation rates could depend on the relative balance between calcium influx and CaSR activation