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

New Castanospermine Glycoside Analogues Inhibit Breast Cancer Cell Proliferation and Induce Apoptosis without Affecting Normal Cells

sp2-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancerMinisterio de Economía y Competitividad SAF2010-15670 y CTQ2010-1584

Functional cooperation between KCa3.1 and TRPC1 channels in human breast cancer: Role in cell proliferation and patient prognosis

Intracellular Ca2+ levels are important regulators of cell cycle and proliferation. We, and others, have previously reported the role of KCa3.1 (KCNN4) channels in regulating the membrane potential and the Ca2+ entry in association with cell proliferation. However, the relevance of KC3.1 channels in cancer prognosis as well as the molecular mechanism of Ca2+ entry triggered by their activation remain undetermined. Here, we show that RNAi-mediated knockdown of KCa3.1 and/or TRPC1 leads to a significant decrease in cell proliferation due to cell cycle arrest in the G1 phase. These results are consistent with the observed upregulation of both channels in synchronized cells at the end of G1 phase. Additionally, knockdown of TRPC1 suppressed the Ca2+ entry induced by 1-EBIO-mediated KCa3.1 activation, suggesting a functional cooperation between TRPC1 and KCa3.1 in the regulation of Ca2+ entry, possibly within lipid raft microdomains where these two channels seem to co-localize. We also show significant correlations between KCa3.1 mRNA expression and poor patient prognosis and unfavorable clinical breast cancer parameters by mining large datasets in the public domain. Together, these results highlight the importance of KCa3.1 in regulating the proliferative mechanisms in breast cancer cells as well as in providing a promising novel target in prognosis and therap

Expression of TRPC6 channels in human epithelial breast cancer cells

<p>Abstract</p> <p>Background</p> <p>TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues.</p> <p>Methods</p> <p>Molecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration).</p> <p>Results</p> <p>A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La³⁺. TRPC6, but not TRPC7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours.</p> <p>Conclusion</p> <p>Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.</p

Estrogen regulation of TRPM8 expression in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>The calcium-permeable cation channel TRPM8 (melastatin-related transient receptor potential member 8) is over-expressed in several cancers. The present study aimed at investigating the expression, function and potential regulation of TRPM8 channels by ER alpha (estrogen receptor alpha) in breast cancer.</p> <p>Methods</p> <p>RT-PCR, Western blot, immuno-histochemical, and siRNA techniques were used to investigate TRPM8 expression, its regulation by estrogen receptors, and its expression in breast tissue. To investigate the channel activity in MCF-7 cells, we used the whole cell patch clamp and the calcium imaging techniques.</p> <p>Results</p> <p>TRPM8 channels are expressed at both mRNA and protein levels in the breast cancer cell line MCF-7. Bath application of the potent TRPM8 agonist Icilin (20 μM) induced a strong outwardly rectifying current at depolarizing potentials, which is associated with an elevation of cytosolic calcium concentration, consistent with established TRPM8 channel properties. RT-PCR experiments revealed a decrease in TRPM8 mRNA expression following steroid deprivation for 48 and 72 hours. In steroid deprived medium, addition of 17-beta-estradiol (E₂, 10 nM) increased both TRPM8 mRNA expression and the number of cells which respond to Icilin, but failed to affect the Ca²⁺entry amplitude. Moreover, silencing ERα mRNA expression with small interfering RNA reduced the expression of TRPM8. Immuno-histochemical examination of the expression of TRPM8 channels in human breast tissues revealed an over-expression of TRPM8 in breast adenocarcinomas, which is correlated with estrogen receptor positive (ER⁺) status of the tumours.</p> <p>Conclusion</p> <p>Taken together, these results show that TRPM8 channels are expressed and functional in breast cancer and that their expression is regulated by ER alpha.</p

Rôle des canaux calciques de type TRP dans la cancérisation du sein (étude dans le tissu mammaire humain sain et cancéreux)

Le but de ce travail est d établir le profil d expression des canaux Transient Receptor Potential (TRP dans le cancer du sein, d étudier leurs propriétés électrophysiologiques et leurs rôles dans la prolifération, l'adhésion et la migration des cellules cancéreuses non invasives exprimant les récepteurs aux oestrogènes (ER+) MCF-7, et invasives ER- MDA-MB-231. Nos observations dans les carcinomes canalaire infiltrants ont montré une surexpression des canaux TRPV6 exclusivement dans les zones invasives ER+. Les canaux TRPC1, et TRPM8 sont surexprimés dans les tumeurs bien différenciées, de petites tailles ou avec un faible Ki67, et la surexpression des canaux TRPM8 est corrélée à l expression des ER. Par ailleurs, les canaux TRPM7 sont surexprimés dans des tumeurs indifférenciées, de grandes tailles ou avec un Ki67 élevé. De plus, cette surexpression est plus importante dans les zones non invasives ER+, et invasives ER-. Ces cinq canaux TRP sont fonctionnels dans les cellules MCF-7, et l expression des canaux TRPM8 dépend des œstrogènes. Les canaux TRPC6 et TRPM7 sont également fonctionnels dans les cellules épithéliales cancéreuses mammaires humaines issues de cultures primaires. Les canaux TRPM7 sont d une part, impliqués dans la prolifération des cellules MCF-7, via une entrée basale de Ca2+, et d autre part, régulent l'adhésion et la migration des cellules MDA-MB-231. En conclusion, nos travaux nous permettent de proposer les canaux TRPC1 et TRPM8 comme des marqueurs pronostics, les canaux TRPM7 comme des marqueurs diagnostics potentiels de la prolifération cellulaire, et les canaux TRPV6 comme marqueurs potentiels de mauvais pronostics.AMIENS-BU Santé (800212102) / SudocSudocFranceF

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

Crosstalk between Ca(2+) Signaling and Cancer Stemness: The Link to Cisplatin Resistance.

International audienceIn the fight against cancer, therapeutic strategies using cisplatin are severely limited by the appearance of a resistant phenotype. While cisplatin is usually efficient at the beginning of the treatment, several patients endure resistance to this agent and face relapse. One of the reasons for this resistant phenotype is the emergence of a cell subpopulation known as cancer stem cells (CSCs). Due to their quiescent phenotype and self-renewal abilities, these cells have recently been recognized as a crucial field of investigation in cancer and treatment resistance. Changes in intracellular calcium (Ca(2+)) through Ca(2+) channel activity are essential for many cellular processes such as proliferation, migration, differentiation, and survival in various cell types. It is now proved that altered Ca(2+) signaling is a hallmark of cancer, and several Ca(2+) channels have been linked to CSC functions and therapy resistance. Moreover, cisplatin was shown to interfere with Ca(2+) homeostasis; thus, it is considered likely that cisplatin-induced aberrant Ca(2+) signaling is linked to CSCs biology and, therefore, therapy failure. The molecular signature defining the resistant phenotype varies between tumors, and the number of resistance mechanisms activated in response to a range of pressures dictates the global degree of cisplatin resistance. However, if we can understand the molecular mechanisms linking Ca(2+) to cisplatin-induced resistance and CSC behaviors, alternative and novel therapeutic strategies could be considered. In this review, we examine how cisplatin interferes with Ca(2+) homeostasis in tumor cells. We also summarize how cisplatin induces CSC markers in cancer. Finally, we highlight the role of Ca(2+) in cancer stemness and focus on how they are involved in cisplatin-induced resistance through the increase of cancer stem cell populations and via specific pathways

Orai3, un canal calcique, surexprimé dans l'adénocarcinome primitif du poumon et associé à un mauvais pronostic

Le cancer pulmonaire est la première cause de mortalité par cancer. Les adénocarcinomes représentent 40 à 76% des cancers non à petites cellules. L homéostasie tissulaire, régulée par calcium, est basée sur une balance entre prolifération et apoptose cellulaires. Les canaux calciques Orai jouent un rôle majeur dans l entrée calcique cellulaire. Le canal Orai3 est impliqué dans prolifération des cellules cancéreuses mammaires. Afin de déterminer si ce mécanisme est spécifique ou non du cancer du sein, nous avons évalué l'expression de ce canal dans les AC primitifs du poumon inclus en paraffine (n=60) et son association potentielle aux paramètres anatomo-cliniques. Nous avons étudié son activité canalaire en imagerie calcique et son rôle dans la prolifération des cellules cancéreuses pulmonaires NCI-H23 et NCI-H460. En immunohistochimie, le canal Orai3 est surexprimé dans les tissus cancéreux par rapport tissus non-tumoraux appariés. Une association a été démontrée entre la différenciation tumorale l'expression d Orai3 (p<0,001) puis entre la surexpression d'Orai3 et la survie des patients (p=0,029). Dans les NCI-H23 et NCI-460, nous avons démontré que le canal Orai3 permet l entrée du Ca2+. A 72h post-transfection d'un siOrai3, la prolifération cellulaire a diminué drastiquement (p<0,001) avec une accumulation des cellules en phase G1 (p<0,001). Cette étude met en évidence le rôle du canal Orai3 dans la prolifération des cellules pulmonaires tumorales en contrôlant la progression du cycle cellulaire, dans la différenciation tumorale et la survie. Ces résultats indiquent que la surexpression du canal Orai3 est associée à un mauvais pronostic.AMIENS-BU Santé (800212102) / SudocSudocFranceF