Calcium sensing receptor stimulates breast cancer cell migration via the Gβγ-AKT-mTORC2 signaling pathway

Calcium sensing receptor, a pleiotropic G protein coupled receptor, activates secretory pathways in cancer cells and putatively exacerbates their metastatic behavior. Here, we show that various CaSR mutants, identified in breast cancer patients, differ in their ability to stimulate Rac, a small Rho GTPase linked to cytoskeletal reorganization and cell protrusion, but are similarly active on the mitogenic ERK pathway. To investigate how CaSR activates Rac and drives cell migration, we used invasive MDA-MB-231 breast cancer cells. We revealed, by pharmacological and knockdown strategies, that CaSR activates Rac and cell migration via the Gβγ-PI3K-mTORC2 pathway. These findings further support current efforts to validate CaSR as a relevant therapeutic target in metastatic cancer.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00662-y.     

Calcium signaling in osteoclasts

It has long been known that many bone diseases, including osteoporosis, involve abnormalities in osteoclastic bone resorption. As a result, there has been intense study of the mechanisms that regulate both the differentiation and bone resorbing function of osteoclast cells. Calcium (Ca²⁺) signaling appears to play a critical role in the differentiation and functions of osteoclasts. Cytoplasmic Ca²⁺ oscillations occur during RANKL-mediated osteoclastogenesis. Ca²⁺ oscillations provide a digital Ca²⁺ signal that induces osteoclasts to up-regulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), a Ca²⁺/calcineurin-dependent master regulator of osteoclastogenesis. Here we review previous studies on Ca²⁺ signaling in osteoclasts as well as recent breakthroughs in understanding the basis of RANKL-induced Ca²⁺ oscillations, and we discuss possible molecular players in this specialized Ca²⁺ response that appears pivotal for normal bone function. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Calcium signaling and epigenetics: A key point to understand carcinogenesis

Calcium (Ca²⁺) signaling controls a wide range of cellular processes, including the hallmarks of cancer. The Ca²⁺ signaling system encompasses several types of proteins, such as receptors, channels, pumps, exchangers, buffers, and sensors, of which several are mutated or with altered expression in cancer cells. Since epigenetic mechanisms are disrupted in all stages of carcinogenesis, and reversibly regulate gene expression, they have been studied by different research groups to understand their role in Ca²⁺ signaling remodeling in cancer cells and the carcinogenic process. In this review, we link Ca²⁺ signaling, cancer, and epigenetics fields to generate a comprehensive landscape of this complex group of diseases.     

Calcium signaling and cellular senescence

Cellular senescence is a stable cell proliferation arrest induced by a variety of stresses including telomere shortening, oncogene activation and oxidative stress. This process plays a crucial role in many physiopathological contexts, especially during aging when cellular senescence favors development of age-related diseases, shortening lifespan. However, the molecular and cellular mechanisms controlling senescence are still a matter of active research. In the last decade, there has been emerging literature indicating a key involvement of calcium signaling in cellular senescence. In this review we will initially give an account of the direct evidence linking calcium and the regulation of senescence. We will then review our current knowledge on the role of calcium in some senescence-associated features and physiopathological conditions, which will shed light on additional ways in which calcium signaling is implicated in cellular senescence.     

Calcium signaling in lacrimal glands

Lacrimal glands provide the important function of lubricating and protecting the ocular surface. Failure of proper lacrimal gland function results in a number of debilitating dry eye diseases. Lacrimal glands secrete lipids, mucins, proteins, salts and water and these secretions are at least partially regulated by neurotransmitter-mediated cell signaling. The predominant signaling mechanism for lacrimal secretion involves activation of phospholipase C, generation of the Ca²⁺-mobilizing messenger, IP₃, and release of Ca²⁺ stored in the endoplasmic reticulum. The loss of Ca²⁺ from the endoplasmic reticulum then triggers a process known as store-operated Ca²⁺ entry, involving a Ca²⁺ sensor in the endoplasmic reticulum, STIM1, which activates plasma membrane store-operated channels comprised of Orai subunits. Recent studies with deletions of the channel subunit, Orai1, confirm the important role of SOCE in both fluid and protein secretion in lacrimal glands, both in vivo and in vitro.     

Calcium signaling in Alzheimer's disease & therapies

Alzheimer's disease (AD) is the most common type of dementia and is characterized by the accumulation of amyloid (Aβ) plaques and neurofibrillary tangles in the brain. Much attention has been given to develop AD treatments based on the amyloid cascade hypothesis; however, none of these drugs had good efficacy at improving cognitive functions in AD patients suggesting that Aβ might not be the disease origin. Thus, there are urgent needs for the development of new therapies that target on the proximal cause of AD. Cellular calcium (Ca²⁺) signals regulate important facets of neuronal physiology. An increasing body of evidence suggests that age-related dysregulation of neuronal Ca²⁺ homeostasis may play a proximal role in the pathogenesis of AD as disrupted Ca²⁺ could induce synaptic deficits and promote the accumulation of Aβ plaques and neurofibrillary tangles. Given that Ca²⁺ disruption is ubiquitously involved in all AD pathologies, it is likely that using chemical agents or small molecules specific to Ca²⁺ channels or handling proteins on the plasma membrane and membranes of intracellular organelles to correct neuronal Ca²⁺ dysregulation could open up a new approach to AD prevention and treatment. This review summarizes current knowledge on the molecular mechanisms linking Ca²⁺ dysregulation with AD pathologies and discusses the possibility of correcting neuronal Ca²⁺ disruption as a therapeutic approach for AD.     

Calcium signaling and apoptosis

Ca(2+) is one of the key regulators of cell survival, but Ca(2+) can also induce apoptosis in response to a variety of pathological conditions. The pro-apoptotic effects of Ca(2+) are mediated by a diverse range of Ca(2+)-sensitive factors that are compartmentalized in various intracellular organelles including the ER, cytoplasm, and mitochondria. The Ca(2+) dynamics of these organelles appear to be modulated by the apoptosis-regulating Bcl-2 family proteins. In this paper, the recent progress of research on the mechanisms mediating the apoptosis-regulating effects of Ca(2+) and the interactions of Bcl-2 family proteins with the Ca(2+) storage organelles are discussed.     

Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca²⁺) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca²⁺ signaling is essential for the cellular and developmental changes that support T. gondii parasitism.The characterization of the molecular players and pathways directly activated by Ca²⁺ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca²⁺ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca²⁺ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.     

Distinctive molecular signaling in triple-negative breast cancer cell death triggered by hexadecylphosphocholine (miltefosine)

This study describes the molecular signaling involved in the different cell death modes of triple-negative breast cancer cells induced by hexadecylphosphocholine (HePC/miltefosine), a clinically relevant anticancer alkylphosphocholine. We found that the HePC treatment triggers cell-type-dependent apoptotic and non-apoptotic cell death processes. Moreover, the expression level of the apoptosis activator Fas, and Fas/Fas ligand signaling capacity are not attributing factors for the preference toward apoptosis. Using Fas siRNA and overexpression approaches we establish that Fas is not a pro-apoptotic factor but a contributor to cell protection in HePC-apoptosis-sensitive cells. The insight in the multi-modal anticancer capability of HePC in triple-negative breast cancer cells may facilitate the targeted design of therapeutic strategies against triple-negative breast cancers.     

Pro-metastatic functions of Notch signaling is mediated by CYR61 in breast cells

Metastasis is the main cause of cancer related deaths, and unfolding the molecular mechanisms underlying metastatic progression is critical for the development of novel therapeutic approaches. Notch is one of the key signaling pathways involved in breast tumorigenesis and metastasis. Notch activation induces pro-metastatic processes such as migration, invasion and epithelial to mesenchymal transition (EMT). However, molecular mediators working downstream of Notch in these processes are not fully elucidated. CYR61 is a secreted protein implicated in metastasis, and its inhibition by a monoclonal antibody suppresses metastasis in xenograft breast tumors, indicating the clinical importance of CYR61 targeting. Here, we aimed to investigate whether CYR61 works downstream of Notch in inducing pro-metastatic phenotypes in breast cells. We showed that CYR61 expression is positively regulated by Notch activity in breast cells. Notch1-induced migration, invasion and anchorage independent growth of a normal breast cell line, MCF10A, were abrogated by CYR61 silencing. Furthermore, upregulation of core EMT markers upon Notch1-activation was impaired in the absence of CYR61. However, reduced migration and invasion of highly metastatic cell line, MDA MB 231, cells upon Notch inhibition was not dependent on CYR61 downregulation. In conclusion, we showed that in normal breast cell line MCF10A, CYR61 is a mediator of Notch1-induced pro-metastatic phenotypes partly via induction of EMT. Our results imply CYR61 as a prominent therapeutic candidate for a subpopulation of breast tumors with high Notch activity.     

Calcium signaling and sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells

In this work, we show that glucose-induced activation of plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is strongly dependent on calcium metabolism and that the glucose sensor Snf3p works in a parallel way with the G protein Gpa2p in the control of the pathway. The role of Snf3p is played by the Snf3p C-terminal tail, since in a strain with the deletion of the SNF3 gene, but also expressing a chimera protein formed by Hxt1p (a glucose transporter) and the Snf3p C-terminal tail, a normal glucose-activation process can be observed. We present evidences indicating that Snf3p would be the sensor for the internal signal (phosphorylated sugars) of this pathway that would connect calcium signaling and activation of the plasma membrane ATPase. We also show that Snf3p could be involved in the control of Pmc1p activity that would regulate the calcium availability in the cytosol.     

MicroRNA-216b targets HK2 to potentiate autophagy and apoptosis of breast cancer cells via the mTOR signaling pathway

Patients suffering from breast cancer (BC) still have a poor response to treatments, even though early detection and improved therapy have contributed to a reduced mortality. Recent studies have been inspired on the association between microRNAs (miRs) and therapies of BC. The current study set out to investigate the role of miR-216b in BC, and further analyze the underlining mechanism. Firstly, hexokinase 2 (HK2) and miR-216b were characterized in BC tissues and cells by RT-qPCR and Western blot assay. In addition, the interaction between HK2 and miR-216b was analyzed using dual luciferase reporter assay. BC cells were further transfected with a series of miR-216b mimic or inhibitor, or siRNA targeting HK2, so as to analyze the regulatory mechanism of miR-216b, HK2 and mammalian target of rapamycin (mTOR) signaling pathway, and to further explore their regulation in BC cellular behaviors. The results demonstrated that HK2 was highly expressed and miR-216b was poorly expressed in BC cells and tissues. HK2 was also verified as a target of miR-216b with online databases and dual luciferase reporter assay. Functionally, miR-216b was found to be closely associated with BC progression via inactivating mTOR signaling pathway by targeting HK2. Moreover, cell viability, migration and invasion were reduced as a result of miR-216b upregulation or HK2 silencing, while autophagy, cell cycle arrest and apoptosis were induced. Taken together, our findings indicated that miR-216b down-regulates HK2 to inactivate the mTOR signaling pathway, thus inhibiting the progression of BC. Hence, this study highlighted a novel target for BC treatment.     

A neural network model of basal ganglia’s decision-making circuitry

The basal ganglia have been increasingly recognized as an important structure involved in decision making. Neurons in the basal ganglia were found to reflect the evidence accumulation process during decision making. However, it is not well understood how the direct and indirect pathways of the basal ganglia work together for decision making. Here, we create a recurrent neural network model that is composed of the direct and indirect pathways and test it with the classic random dot motion discrimination task. The direct pathway drives the outputs, which are modulated through a gating mechanism controlled by the indirect pathway. We train the network to learn the task and find that the network reproduces the accuracy and reaction time patterns of previous animal studies. Units in the model exhibit ramping activities that reflect evidence accumulation. Finally, we simulate manipulations of the direct and indirect pathways and find that the manipulations of the direct pathway mainly affect the choice while the manipulations of the indirect pathway affect the model’s reaction time. These results suggest a potential circuitry mechanism of the basal ganglia’s role in decision making with predictions that can be tested experimentally in the future.     

CCN5/WISP-2: A micromanager of breast cancer progression

The gain of plasticity by a subset of cancer cells is a unique but common sequence of cancer progression from epithelial phenotype to mesenchymal phenotype (EMT) that is followed by migration, invasion and metastasis to a distant organ, and drug resistance. Despite multiple studies, it is still unclear how cancer cells regulate plasticity. Recent studies from our laboratory and others’ proposed that CCN5/WISP-2, which is found intracellularly (in the nucleus and cytoplasm) and extracellularly, plays a negative regulator of plasticity. It prevents the EMT process in breast cancer cells as well as pancreatic cancer cells. Multiple genetic insults, including the gain of p53 mutations that accumulate over the time, may perturb CCN5 expression in non-invasive breast cancer cells, which ultimately helps cells to gain invasive phenotypes. Moreover, emerging evidence indicates that several oncogenic lesions such as miR-10b upregulation and activation of TGF-β-signaling can accumulate during CCN5 crisis in breast cancer cells. Collectively, these studies indicate that loss of CCN5 activity may promote breast cancer progression; application of CCN5 protein may represent a novel therapeutic intervention in breast cancer and possibly pancreatic cancer.     

Nuclear calcium signaling: an emerging topic in plants

The calcium ion is probably one of the most studied second messenger both in plant and animal fields. A large number of reviews have browsed the diversity of cytosolic calcium signatures and evaluated their pleiotropic roles in plant and animal cells. In the recent years, an increasing number of reviews has focused on nuclear calcium, especially on the possible roles of nuclear calcium concentration variations on nuclear activities. Experiments initially performed on animal cells gave conflicting results that brought about a controversy about the ability of the nucleus to generate its own calcium signals and to regulate its calcium level. But in plant cells, several converging scientific pieces of evidence support the hypothesis of nucleus autonomy. The present review briefly summarizes data supporting this hypothesis and tries to put forward some possible roles for these nucleus-generated calcium signals in controlling nuclear activity.     

Multilevel complexity of calcium signaling: Modeling angiogenesis

Intracellular calcium signaling is a universal,evolutionary conserved and versatile regulator of cell biochemistry.The complexity of calcium signaling and related cell machinery can be investigated by the use of experimental strategies,as well as by computational approaches.Vascular endothelium is a fascinating model to study the specific properties and roles of calcium signals at multiple biological levels.During the past 20 years,live cell imaging,patch clamp and other techniques have allowed us to detect and interfere with calcium signaling in endothelial cells(ECs),providing a huge amount of information on the regulation of vascularization(angiogenesis) in normal and tumoral tissues.These data range from the spatiotemporal dynamics of calcium within different cell microcompartments to those in entire multicellular and organized EC networks.Beside experimental strategies,in silico endothelial models,specifically designed for simulating calcium signaling,are contributing to our knowledge of vascular physiol-ogy and pathology.They help to investigate and predict the quantitative features of proangiogenic events moving through subcellular,cellular and supracellular levels.This review focuses on some recent developments of computational approaches for proangiogenic endothelial calcium signaling.In particular,we discuss the creation of hybrid simulation environments,which combine and integrate discrete Cellular Potts Models.They are able to capture the phenomenological mechanisms of cell morphological reorganization,migration,and intercellular adhesion,with single-cell spatiotemporal models,based on reaction-diffusion equations that describe the agonist-induced intracellular calcium events.     

Assessment of cytosolic free calcium changes during ceramide-induced cell death in MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m

Alterations in Ca²⁺ signaling can regulate key cancer hallmarks such as proliferation, invasiveness and resistance to cell death. Changes in the regulation of intracellular Ca²⁺ and specific components of Ca²⁺ influx are a feature of several cancers and/or cancer subtypes, including the basal-like breast cancer subtype, which has a poor prognosis. The development of genetically encoded calcium indicators, such as GCaMP6, represents an opportunity to measure changes in intracellular free Ca²⁺ during processes relevant to breast cancer progression that occur over long periods (e.g. hours), such as cell death. This study describes the development of a MDA-MB-231 breast cancer cell line stably expressing GCaMP6m. The cell line retained the key features of this aggressive basal-like breast cancer cell line. Using this model, we defined alterations in relative cytosolic free Ca²⁺ ([Ca²⁺]_CYT) when the cells were treated with C2-ceramide. Cell death was measured simultaneously via assessment of propidium iodide permeability. Treatment with ceramide produced delayed and heterogeneous sustained increases in [Ca²⁺]_CYT. Where cell death occurred, [Ca²⁺]_CYT increases preceded cell death. The sustained increases in [Ca²⁺]_CYT were not related to the rapid morphological changes induced by ceramide. Silencing of the plasma membrane Ca²⁺ ATPase isoform 1 (PMCA1) was associated with an augmentation in ceramide-induced increases in [Ca²⁺]_CYT and also cell death. This work demonstrates the utility of GCaMP6 Ca²⁺ indicators for investigating [Ca²⁺]_CYT changes in breast cancer cells during events relevant to tumor progression, which occur over hours rather than minutes.     

Calcium and secondary CPK signaling in plants in response to herbivore attack

Plant Ca²⁺ signals are involved in a sizable array of intracellular signaling pathways after pest invasion. Upon herbivore feeding there is a dramatic Ca²⁺ influx, followed by the activation of Ca²⁺-dependent signal transduction pathways that include interacting downstream networks of kinases for defense responses. Notably, Ca²⁺-binding sensory proteins such as Ca²⁺-dependent protein kinases (CPKs) have recently been documented to mediate the signaling following Ca²⁺ influx after herbivory, in phytohormone-independent manners. Here, we review the sequence of signal transductions triggered by herbivory-evoked Ca²⁺ signaling leading to CPK actions for defense responses, and discuss in a comparative way the involvement of CPKs in the signal transduction of a variety of other biotic and abiotic stresses.     

Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines

The plasma membrane Ca²⁺ ATPase (PMCA) is an important regulator of free intracellular calcium, with dynamic regulation in the rat mammary gland during lactation. Recent studies suggest that Ca²⁺ plays a role in cellular proliferation. To determine if PMCA expression is altered in tumorigenesis, we compared relative levels of PMCA1 mRNA. We found that the relative expression of PMCA1 mRNA is increased, by approximately 270% and 170%, in MCF-7 and MDA-MB-231 human breast cancer cell lines deprived of serum for 72 h, respectively, compared to the similarly treated MCF-10A human mammary gland epithelial cell line. Characterization of PMCA mRNA isoforms revealed that PMCA1b and PMCA4 mRNA are expressed in MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and BT-483 breast cancer cell lines. We also detected PMCA2 mRNA expression in all the breast cancer cell lines examined. However, PMCA3 mRNA was only detected in BT-483 cells. Our results suggest that PMCA expression may be altered in breast cancer cell lines, suggesting altered Ca²⁺ regulation in these cell lines. Our results also indicate that breast cancer cell lines can express mRNAs for a variety PMCA isoforms.