Cellular geometry and epithelial-mesenchymal plasticity intersect with PIEZO1 in breast cancer cells.

Differences in shape can be a distinguishing feature between different cell types, but the shape of a cell can also be dynamic. Changes in cell shape are critical when cancer cells escape from the primary tumor and undergo major morphological changes that allow them to squeeze between endothelial cells, enter the vasculature, and metastasize to other areas of the body. A shift from rounded to spindly cellular geometry is a consequence of epithelial-mesenchymal plasticity, which is also associated with changes in gene expression, increased invasiveness, and therapeutic resistance. However, the consequences and functional impacts of cell shape changes and the mechanisms through which they occur are still poorly understood. Here, we demonstrate that altering the morphology of a cell produces a remodeling of calcium influx via the ion channel PIEZO1 and identify PIEZO1 as an inducer of features of epithelial-to-mesenchymal plasticity. Combining automated epifluorescence microscopy and a genetically encoded calcium indicator, we demonstrate that activation of the PIEZO1 force channel with the PIEZO1 agonist, YODA 1, induces features of epithelial-to-mesenchymal plasticity in breast cancer cells. These findings suggest that PIEZO1 is a critical point of convergence between shape-induced changes in cellular signaling and epithelial-mesenchymal plasticity in breast cancer cells.

The intersection between cysteine proteases, Ca2+ signalling and cancer cell apoptosis.

Apoptosis is a highly complex and regulated cell death pathway that safeguards the physiological balance between life and death. Over the past decade, the role of Ca2+ signalling in apoptosis and the mechanisms involved have become clearer. The initiation and execution of apoptosis is coordinated by three distinct groups of cysteines proteases: the caspase, calpain and cathepsin families. Beyond its physiological importance, the ability to evade apoptosis is a prominent hallmark of cancer cells. In this review, we will explore the involvement of Ca2+ in the regulation of caspase, calpain and cathepsin activity, and how the actions of these cysteine proteases alter intracellular Ca2+ handling during apoptosis. We will also explore how apoptosis resistance can be achieved in cancer cells through deregulation of cysteine proteases and remodelling of the Ca2+ signalling toolkit.

Intersection between calcium signalling and epithelial-mesenchymal plasticity in the context of cancer.

Epithelial-mesenchymal transition (EMT) is a form of cellular phenotypic plasticity and is considered a crucial step in the progression of many cancers. The calcium ion (Ca2+) acts as a ubiquitous second messenger and is implicated in many cellular processes, including cell death, migration, invasion and more recently EMT. Throughout this review, the complex interplay between Ca2+ signalling and EMT will be explored. An overview of the Ca2+ pathways that are remodelled as a consequence of EMT is provided and the role of Ca2+ signalling in regulating EMT and its significance is considered. Ca2+ signalling pathways may represent a therapeutic opportunity to regulate EMT. However, as will be described in this review, the complexity of these signalling pathways represents significant challenges that must be considered if Ca2+ signalling is to be manipulated with the aim of therapeutic intervention in cancer.

AKT Regulation of ORAI1-Mediated Calcium Influx in Breast Cancer Cells.

Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.

ORAI1-Regulated Gene Expression in Breast Cancer Cells: Roles for STIM1 Binding, Calcium Influx and Transcription Factor Translocation.

A remodeling of calcium homeostasis, including calcium influx via store-operated calcium entry (SOCE), is a feature of breast cancers. SOCE is critical to maintain calcium balance in the endoplasmic reticulum calcium store and is an important mechanism for calcium signaling in a variety of cell types, including breast cancer cells. The canonical mechanism of SOCE is stromal interacting molecule 1 (STIM1)-mediated activation of ORAI. Elevated ORAI1 expression is a feature of basal breast cancer cells. However, the role of ORAI1 in the regulation of transcription in breast cancer cells of the basal molecular subtype is still unclear. Using CRISPR-Cas9 gene editing, ORAI1 protein expression was disrupted in MDA-MB-231 and MDA-MB-468 basal breast cancer cells. The ORAI1 wild-type and mutants were reintroduced into ORAI1 knockout cells to study the role of ORAI1 in gene transcriptional regulation. In the absence of calcium store depletion, ORAI1 regulated PTGS2 in MDA-MB-231 cells, and this was dependent on ORAI1 pore function and STIM1 binding. The activation of SOCE by thapsigargin resulted in ORAI1-dependent increases in IL6 transcription in MDA-MB-468 cells; this was also dependent on ORAI1 pore function and STIM1 binding and was associated with the translocation of NFAT1. Given the upregulation of ORAI1 in basal breast cancer cells, our results provide further evidence that ORAI1 may contribute to cancer progression through regulation of gene expression.

Increased matrix stiffness suppresses ATP-induced sustained Ca2+ influx in MDA-MB-231 breast cancer cells.

Both matrix stiffening and remodeling of calcium signaling occur in breast cancers, with downstream consequences linked to the progression of the disease. However, the potential intersection between calcium signaling and matrix stiffness has not been fully assessed in models of cancer. Here, we describe the assessment of calcium signaling in breast cancer cells at high and low matrix stiffness using novel gel culture models (gelatin methacryloyl and polydimethylsiloxane) and MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m. Remodeling of ATP-stimulated cytosolic calcium responses in cells on different matrices was assessed using a high throughput fluorescence imaging plate reader. Our data reveal that matrices of higher stiffness attenuate ATP-induced sustained calcium influx in MDA-MB-231 breast cancer cells. This matrix-mediated attenuation of sustained calcium influx was dependent on the store-operated calcium channel component ORAI1. These studies suggest that calcium signaling in breast cancer cells can be altered as a consequence of matrix stiffness; modulation of such pathways may represent a new mechanism to target calcium signaling to regulate tumor progression in breast cancer.

ORAI1 regulates sustained cytosolic free calcium fluctuations during breast cancer cell apoptosis and apoptotic resistance via a STIM1 independent pathway.

Excessive rapid increases in cytosolic free Ca2+ have a clear association with the induction of cancer cell death. Whereas, characterizing the Ca2+ signaling events that occur during the progression of the apoptotic cascade over a period of hours or days, has not yet been possible. Now using genetically encoded Ca2+ indicators complemented with automated epifluorescence microscopy we have shown that staurosporine-induced apoptosis in MDA-MB-231 breast cancer cells was associated with delayed development of cytosolic free Ca2+ fluctuations, which were then maintained for 24 h. These cytosolic free Ca2+ fluctuations were dependent on the Ca2+ channel ORAI1. Silencing of ORAI1, but not its canonical activators STIM1 and STIM2, promoted apoptosis in this model. The pathway for this regulation implicates a mechanism previously associated with the migration of cancer cells involving ORAI1, the chaperone protein SigmaR1, and Ca2+ -activated K+ channels.

An Emerging Role for Calcium Signaling in Cancer-Associated Fibroblasts.

Tumors exist in a complex milieu where interaction with their associated microenvironment significantly contributes to disease progression. Cancer-associated fibroblasts (CAFs) are the primary component of the tumor microenvironment and participate in complex bidirectional communication with tumor cells. CAFs support the development of various hallmarks of cancer through diverse processes, including direct cell-cell contact, paracrine signaling, and remodeling and deposition of the extracellular matrix. Calcium signaling is a key second messenger in intra- and inter-cellular signaling pathways that contributes to cancer progression; however, the links between calcium signaling and CAFs are less well-explored. In this review, we put into context the role of calcium signaling in interactions between cancer cells and CAFs, with a focus on migration, proliferation, chemoresistance, and genetic instability.

Altered Calcium Influx Pathways in Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) represent an important component of the tumour microenvironment and are implicated in disease progression. Two outstanding questions in cancer biology are how CAFs arise and how they might be targeted therapeutically. The calcium signal also has an important role in tumorigenesis. To date, the role of calcium signalling pathways in the induction of the CAF phenotype remains unexplored. A CAF model was generated through exogenous transforming growth factor beta 1 (TGFß1) stimulation of the normal human mammary fibroblast cell line, HMF3S (HMF3S-CAF), and changes in calcium signalling were investigated. Functional changes in HMF3S-CAF calcium signalling pathways were assessed using a fluorescent indicator, gene expression, gene-silencing and pharmacological approaches. HMF3S-CAF cells demonstrated functionally altered calcium influx pathways with reduced store-operated calcium entry. In support of a calcium signalling switch, two voltage-gated calcium channel (VGCC) family members, CaV1.2 and CaV3.2, were upregulated in HMF3S-CAFs and a subset of patient-derived breast CAFs. Both siRNA-mediated silencing and pharmacological inhibition of CaV1.2 or CaV3.2 significantly impaired CAF activation in HMF3S cells. Our findings show that VGCCs contribute to TGFß1-mediated induction of HMF3S-CAF cells and both transcriptional interference and pharmacological antagonism of CaV1.2 and CaV3.2 inhibit CAF induction. This suggests a potential therapeutic role for targeting calcium signalling in breast CAFs.

Activation of the Ion Channel TRPV4 Induces Epithelial to Mesenchymal Transition in Breast Cancer Cells.

Epithelial to mesenchymal transition (EMT) in cancer is important in therapeutic resistance and invasiveness. Calcium signaling is key to the induction of EMT in breast cancer cells. Although inhibition of specific calcium-permeable ion channels regulates the induction of a sub-set of EMT markers in breast cancer cells, it is still unclear if activation of a specific calcium channel can be a driver for the induction of EMT events. In this study, we exploited the availability of a selective pharmacological activator of the calcium-permeable ion channel TRPV4 to assess the direct role of calcium influx in EMT marker induction. Gene association studies revealed a link between TRPV4 and gene-ontologies associated with EMT and poorer relapse-free survival in lymph node-positive basal breast cancers. TRPV4 was an important component of the calcium influx phase induced in MDA-MB-468 breast cancer cells by the EMT inducer epidermal growth factor (EGF). Pharmacological activation of TRPV4 then drove the induction of a variety of EMT markers in breast cancer cells. These studies demonstrate that calcium influx through specific pathways appears to be sufficient to trigger EMT events.

Distinct pharmacological profiles of ORAI1, ORAI2, and ORAI3 channels.

The ubiquitous Ca2+ release-activated Ca2+ (CRAC) channel is crucial to many physiological functions. Both gain and loss of CRAC function is linked to disease. While ORAI1 is a crucial subunit of CRAC channels, recent evidence suggests that ORAI2 and ORAI3 heteromerize with ORAI1 to form native CRAC channels. Furthermore, ORAI2 and ORAI3 can form CRAC channels independently of ORAI1, suggesting diverse native CRAC stoichiometries. Yet, most available CRAC modifiers are presumed to target ORAI1 with little knowledge of their effects on ORAI2/3 or heteromers of ORAIs. Here, we used ORAI1/2/3 triple-null cells to express individual ORAI1, ORAI2, ORAI3 or ORAI1/2/3 concatemers. We reveal that GSK-7975A and BTP2 essentially abrogate ORAI1 and ORAI2 activity while causing only a partial inhibition of ORAI3. Interestingly, Synta66 abrogated ORAI1 channel function, while potentiating ORAI2 with no effect on ORAI3. CRAC channel activities mediated by concatenated ORAI1-1, ORAI1-2 and ORAI1-3 dimers were inhibited by Synta66, while ORAI2-3 dimers were unaffected. The CRAC enhancer IA65 significantly potentiated ORAI1 and ORAI1-1 activity with marginal effects on other ORAIs. Further, we characterized the profiles of individual ORAI isoforms in the presence of Gd3+ (5µM), 2-APB (5 µM and 50 µM), as well as changes in intracellular and extracellular pH. Our data reveal unique pharmacological features of ORAI isoforms expressed in an ORAI-null background and provide new insights into ORAI isoform selectivity of widely used CRAC pharmacological compounds.

A new selective pharmacological enhancer of the Orai1 Ca2+ channel reveals roles for Orai1 in smooth and skeletal muscle functions.

Store operated calcium (Ca2+) entry is an important homeostatic mechanism in cells, whereby the release of Ca2+ from intracellular endoplasmic reticulum stores triggers the activation of a Ca2+ influx pathway. Mediated by Orai1, this Ca2+ influx has specific and essential roles in biological processes as diverse as lactation to immunity. Although pharmacological inhibitors of this Ca2+ influx mechanism have helped to define the role of store operated Ca2+ entry in many cellular events, the lack of isoform specific modulators and activators of Orai1 has limited our full understanding of these processes. Here we report the identification and synthesis of an Orai1 activity enhancer that concurrently potentiated Orai1 Ca2+ -dependent inactivation (CDI). This unique enhancer of Orai1 had only a modest effect on Orai3 with weak inhibitory effects at high concentrations in intact MCF-7 breast cancer cells. The Orai1 enhancer heightened vascular smooth muscle cell migration induced by platelet-derived growth factor and the unique store operated Ca2+ entry pathway present in skeletal muscle cells. These studies show that IA65 is an exemplar for the translation and development of Orai isoform selective agents. The ability of IA65 to activate CDI demonstrates that agents can be developed that can enhance Orai1-mediated Ca2+ influx but avoid the cytotoxicity associated with sustained Orai1 activation. IA65 and/or future analogues with similar Orai1 and CDI activating properties could be fine tuners of physiological processes important in specific disease states, such as cellular migration and immune cell function.

Differential engagement of ORAI1 and TRPC1 in the induction of vimentin expression by different stimuli.

The Ca2+ signal is essential in both hypoxia- and epidermal growth factor (EGF)-mediated epithelial to mesenchymal transition (EMT) in MDA-MB-468 breast cancer cells. This finding suggests that Ca2+-permeable ion channels participate in the induction of expression of some mesenchymal markers such as vimentin. However, the ion channels involved in vimentin expression induction have not been fully characterized. This work sought to define how differential modulation of the calcium signal effects the induction of vimentin and the Ca2+ influx pathways involved. We identified that the intracellular Ca2+ chelator EGTA-AM, cytochalasin D (a modulator of cytoskeletal dynamics and cell morphology), and the sarco/endoplasmic reticulum ATPase inhibitor thapsigargin are all inducers of vimentin in MDA-MB-468 breast cancer cells. EGTA-AM- and thapsigargin-mediated induction of vimentin expression in MDA-MB-468 cells involves store-operated Ca2+ entry, as evidenced by sensitivity to silencing of the molecular components of this pathway, STIM1 and ORAI1. In stark contrast, cytochalasin D-mediated vimentin induction was insensitive to silencing of ORAI1, despite sensitivity to silencing of its canonical activator the endoplasmic reticulum Ca2+ sensor STIM1. Cytochalasin D-mediated vimentin induction was, however, sensitive to silencing of another reported STIM1 target, TRPC1. Subsequent studies identified that EGTA-AM-induced vimentin expression also partially involved a TRPC1-dependent pathway. These studies define a complex interplay between vimentin expression in this model and the specific Ca2+-permeable ion channels involved. The complexity in the engagement of different Ca2+ influx pathways that regulate vimentin induction are opportunities but also potential challenges in targeting Ca2+ signaling to block EMT in cancer cells. Our findings further highlight the need to identify potential indispensable ion channels that can regulate induction of specific mesenchymal markers via different stimuli.

Transient receptor potential cation channel subfamily V and breast cancer.

Transient receptor potential cation channel subfamily V (TRPV) channels play important roles in a variety of cellular processes. One example includes the sensory role of TRPV1 that is sensitive to elevated temperatures and acidic environments and is activated by the hot pepper component capsaicin. Another example is the importance of the highly Ca2+ selective channels TRPV5 and TRPV6 in Ca2+ absorption/reabsorption in the intestine and kidney. However, in some cases such as TRPV4 and TRPV6, breast cancer cells appear to overexpress TRPV channels. Moreover, TRPV mediated Ca2+ influx may contribute to enhanced breast cancer cell proliferation and other processes important in tumor progression such as angiogenesis. It appears that the overexpression of some TRPV channels in breast cancer and/or their involvement in breast cancer cell processes, processes important in the tumor microenvironment or pain may make some TRPV channels potential targets for breast cancer therapy. In this review, we provide an overview of TRPV expression in breast cancer subtypes, the roles of TRPV channels in various aspects of breast cancer progression and consider implications for future therapeutic approaches.

Assessment of doxorubicin-induced remodeling of Ca2+ signaling and associated Ca2+ regulating proteins in MDA-MB-231 breast cancer cells.

Triple-negative breast cancers (TNBC) are often associated with high relapse rates, despite treatment with chemotherapy agents such as doxorubicin. A better understanding of the signaling and molecular changes associated with doxorubicin may provide novel insights into strategies to enhance treatment efficacy. Calcium signaling is involved in many pathways influencing the efficacy of chemotherapy agents such as proliferation and cell death. However, there are a limited number of studies exploring the effect of doxorubicin on calcium signaling in TNBC. In this study, MDA-MB-231 triple-negative, basal breast cancer cells stably expressing the genetically-encoded calcium indicator GCaMP6m (GCaMP6m-MDA-MB-231) were used to define alterations in calcium signaling. The effects of doxorubicin in GCaMP6m-MDA-MB-231 cells were determined using live cell imaging and fluorescence microscopy. Changes in mRNA levels of specific calcium regulating proteins as a result of doxorubicin treatment were also assessed using real time qPCR. Doxorubicin (1 µM) produced alterations in intracellular calcium signaling, including enhancing the sensitivity of MDA-MB-231 cells to ATP stimulation and prolonging the recovery time after store-operated calcium entry. Upregulation in mRNA levels of ORAI1, TRPC1, SERCA1, IP3R2 and PMCA2 with doxorubicin 1 µM treatment was also observed. Doxorubicin treatment is associated with specific remodeling in calcium signaling in MDA-MB-231 cells, with associated changes in mRNA levels of specific calcium-regulating proteins.

NCS-1 expression is higher in basal breast cancers and regulates calcium influx and cytotoxic responses to doxorubicin.

Neuronal calcium sensor-1 (NCS-1) is a positive modulator of IP3 receptors and was recently associated with poorer survival in breast cancers. However, the association between NCS-1 and breast cancer molecular subtypes and the effects of NCS-1 silencing on calcium (Ca2+ ) signaling in breast cancer cells remain unexplored. Herein, we report for the first time an increased expression of NCS-1 in breast cancers of the basal molecular subtype, a subtype associated with poor prognosis. Using MDA-MB-231 basal breast cancer cells expressing the GCaMP6m Ca2+ indicator, we showed that NCS-1 silencing did not result in major changes in cytosolic free Ca2+ increases as a result of endoplasmic reticulum Ca2+ store mobilization. However, NCS-1 silencing suppressed unstimulated basal Ca2+ influx. NCS-1 silencing in MDA-MB-231 cells also promoted necrotic cell death induced by the chemotherapeutic drug doxorubicin (1 µm). The effect of NCS-1 silencing on cell death was phenocopied by silencing of ORAI1, a Ca2+ store-operated Ca2+ channel that maintains Ca2+ levels in the endoplasmic reticulum Ca2+ store and whose expression was significantly positively correlated with NCS-1 in clinical breast cancer samples. This newly identified association between NCS-1 and basal breast cancers, together with the identification of the role of NCS-1 in the regulation of the effects of doxorubicin in MDA-MB-231 breast cancer cells, suggests that NCS-1 and/or pathways regulated by NCS-1 may be important in the treatment of basal breast cancers in women.

The Calcium-Signaling Toolkit in Cancer: Remodeling and Targeting.

Processes that are important in cancer progression, such as sustained cell growth, invasion to other organs, and resistance to cell death inducers, have a clear overlap with pathways regulated by Ca2+ signaling. It is therefore not surprising that proteins important in Ca2+ signaling, sometimes referred to as the "Ca2+ signaling toolkit," can contribute to cancer cell proliferation and invasiveness, and the ability of agents to induce cancer cell death. Ca2+ signaling is also critical in other aspects of cancer progression, including events in the tumor microenvironment and processes involved in the acquisition of resistance to anticancer therapies. This review will consider the role of Ca2+ signaling in tumor progression and highlight areas in which a better understanding of the interplay between the Ca2+-signaling toolkit and tumorigenesis is still required.

ORAI1 and ORAI3 in Breast Cancer Molecular Subtypes and the Identification of ORAI3 as a Hypoxia Sensitive Gene and a Regulator of Hypoxia Responses.

The remodeling of specific calcium-permeable ion channels is a feature of some breast cancer subtypes. ORAI1 is a protein that forms a calcium-permeable ion channel responsible for store-operated calcium entry (SOCE) in a variety of cell types. ORAI3, a related isoform, is not a regulator of SOCE in most cell types. However, ORAI3 does control SOCE in many estrogen receptor-positive breast cancer cell lines, where it also controls proliferation. ORAI1 is a well-characterized regulator of the proliferation and migration of many basal breast cancer cells; however, the role of ORAI3 in these types of breast cancer cells remains unclear. Here, we sought to define ORAI1 and ORAI3 expression in breast cancer cell lines of different molecular subtypes and assess the potential role and regulation of ORAI3 in basal breast cancer cells. Our study demonstrates that elevated ORAI1 is a feature of basal-like breast cancers, while elevated ORAI3 is a feature of luminal breast cancers. Intriguingly, we found that ORAI3 is over-expressed in the mesenchymal subtype of triple-negative breast cancer. Given this, we assessed ORAI3 levels in the presence of two inducers of the mesenchymal phenotype, hypoxia and epidermal growth factor (EGF). Hypoxia induced ORAI3 levels in basal breast cancer cell lines through a pathway involving hypoxia-inducible factor-1 alpha (HIF1α. The silencing of ORAI3 attenuated hypoxia-associated phosphorylation of the EGF receptor (EGFR) and the expression of genes associated with cell migration and inflammatory/immune responses in the MDA-MB-468 model of basal breast cancer. Although elevated ORAI3 levels were not associated with survival; basal, estrogen receptor-negative and triple-negative breast cancers with high ORAI3 and low ORAI1 levels were associated with poorer clinical outcomes. This study defines ORAI3 as a potential fine-tuner for processes relevant to the progression of basal breast cancers.