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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.
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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.
Assuntos
Neoplasias da Mama , Cálcio , Neoplasias da Mama/genética , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio da Dieta , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Apoptose , Neoplasias da Mama/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Molécula 1 de Interação Estromal/genéticaRESUMO
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.
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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.
Assuntos
Sinalização do Cálcio/fisiologia , Proteína ORAI1/metabolismo , Canais de Cátion TRPC/metabolismo , Vimentina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Citocalasina D/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Tapsigargina/farmacologiaRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Ceramidas/farmacologia , Citosol/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , TransfecçãoRESUMO
AIMS: To assess levels of the calcium permeable transient receptor potential cation channel, subfamily melastatin, member 8 (TRPM8) in breast cancer molecular subtypes and to assess the consequences of TRPM8 pharmacological inhibition with AMTB (an inhibitor of TRPM8) on breast cancer cell lines. MATERIALS AND METHODS: Cell viability and migration of breast cancer cells was determined using MTS assays and wound healing assays, respectively. RNA-Seq analysis of breast tumours and qPCR in breast cancer cell lines were used to assess mRNA levels of ion channels. Membrane potential assays were employed to assess the effects of AMTB against specific voltage gated sodium channels (NaV). KEY FINDINGS: TRPM8 levels were significantly higher in breast cancers of the basal molecular subtype. AMTB decreased viable cell number in MDA-MB-231 and SK-BR-3 breast cancer cell lines (30 and 100⯵M), and also reduced the migration of MDA-MB-231 cells (30⯵M). However, these effects were independent of TRPM8, as no TRPM8 mRNA was detected in MDA-MB-231 cells. AMTB was identified as an inhibitor of NaV isoforms. NaV1.1-1.9 were expressed in a number of breast cancer cell lines, with NaV1.5 mRNA highest in MDA-MB-231 cells compared to the other breast cancer cell lines assessed. SIGNIFICANCE: TRPM8 levels may be elevated in basal breast cancers, however, TRPM8 expression appears to be lost in many breast cancer cell lines. Some of the effects of AMTB attributed to TRPM8 may be due to effects on NaV channels.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Neoplasias da Mama/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Tiofenos/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Potenciais da Membrana , Reação em Cadeia da PolimeraseRESUMO
AKT is an enzyme of the PI3K/pAKT pathway, regulating proliferation and cell survival. High basal levels of active, phosphorylated AKT (pAKT) are associated with tumor progression and therapeutic resistance in some breast cancer subtypes, including HER2 positive breast cancers. Various stimuli can increase pAKT levels and elevated basal pAKT levels are a feature of PTEN-deficient breast cancer cell lines. The aim of this study was to develop an assay able to identify modulators of pAKT levels using an automated epifluorescence microscope and high content analysis. To develop this assay, we used HCC-1569, a PTEN-deficient, HER2-overexpressing breast cancer cell line with elevated basal pAKT levels. HCC-1569 cells were treated with a selective pharmacological inhibitor of AKT (MK-2206) to reduce basal pAKT levels or EGF to increase pAKT levels. Immunofluorescence images were acquired using an automated epifluorescence microscope and integrated intensity of cytoplasmic pAKT staining was calculated using high content analysis software. Mean and median integrated cytoplasmic intensity were normalized using fold change and standard score to assess assay quality and to identify most robust data analysis. The highest z' factor was achieved for median data normalization using the standard score method (z'â¯=â¯0.45). Using our developed assay we identified the calcium homeostasis regulating proteins TPRV6, STIM1 and TRPC1 as modulators of pAKT levels in HCC-1569 cells. Calcium signaling controls a diverse array of cellular processes and some calcium homeostasis regulating proteins are involved in modulating pAKT levels in cancer cells. Thus, these identified hits present promising targets for further assessment.
Assuntos
Neoplasias da Mama/metabolismo , Sinalização do Cálcio , Microscopia de Fluorescência/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Antineoplásicos/farmacologia , Automação , Neoplasias da Mama/tratamento farmacológico , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Proteínas de Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Receptor ErbB-2/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
Regulation of Ca(2+) transport is vital in physiological processes, including lactation, proliferation and apoptosis. The plasmalemmal Ca(2+) pump isoform 2 (PMCA2) a calcium ion efflux pump, was the first protein identified to be crucial in the transport of Ca(2+) ions into milk during lactation in mice. In these studies we show that PMCA2 is also expressed in human epithelia undergoing lactational remodeling and also report strong PMCA2 staining on apical membranes of luminal epithelia in approximately 9% of human breast cancers we assessed. Membrane protein expression was not significantly associated with grade or hormone receptor status. However, PMCA2 mRNA levels were enriched in Basal breast cancers where it was positively correlated with survival. Silencing of PMCA2 reduced MDA-MB-231 breast cancer cell proliferation, whereas silencing of the related isoforms PMCA1 and PMCA4 had no effect. PMCA2 silencing also sensitized MDA-MB-231 cells to the cytotoxic agent doxorubicin. Targeting PMCA2 alone or in combination with cytotoxic therapy may be worthy of investigation as a therapeutic strategy in breast cancer. PMCA2 mRNA levels are also a potential tool in identifying poor responders to therapy in women with Basal breast cancer.
Assuntos
Neoplasias da Mama/genética , Cálcio/metabolismo , Carcinoma Basocelular/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Sinalização do Cálcio , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/mortalidade , Carcinoma Basocelular/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/patologia , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Doxorrubicina/farmacologia , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Feminino , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lactação/fisiologia , Glândulas Mamárias Humanas/enzimologia , Glândulas Mamárias Humanas/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de SobrevidaRESUMO
BACKGROUND: Understanding the cause of therapeutic resistance and identifying new biomarkers in breast cancer to predict therapeutic responses will help optimise patient care. Calcium (Ca(2+))-signalling is important in a variety of processes associated with tumour progression, including breast cancer cell migration and proliferation. Ca(2+)-signalling is also linked to the acquisition of multidrug resistance. This study aimed to assess the expression level of proteins involved in Ca(2+)-signalling in an in vitro model of trastuzumab-resistance and to assess the ability of identified targets to reverse resistance and/or act as potential biomarkers for prognosis or therapy outcome. METHODS: Expression levels of a panel of Ca(2+)-pumps, channels and channel regulators were assessed using RT-qPCR in resistant and sensitive age-matched SKBR3 breast cancer cells, established through continuous culture in the absence or presence of trastuzumab. The role of Cav3.2 in the acquisition of trastuzumab-resistance was assessed through pharmacological inhibition and induced overexpression. Levels of Cav3.2 were assessed in a panel of non-malignant and malignant breast cell lines using RT-qPCR and in patient samples representing different molecular subtypes (PAM50 cohort). Patient survival was also assessed in samples stratified by Cav3.2 expression (METABRIC and KM-Plotter cohort). RESULTS: Increased mRNA of Cav3.2 was a feature of both acquired and intrinsic trastuzumab-resistant SKBR3 cells. However, pharmacological inhibition of Cav3.2 did not restore trastuzumab-sensitivity nor did Cav3.2 overexpression induce the expression of markers associated with resistance, suggesting that Cav3.2 is not a driver of trastuzumab-resistance. Cav3.2 levels were significantly higher in luminal A, luminal B and HER2-enriched subtypes compared to the basal subtype. High levels of Cav3.2 were associated with poor outcome in patients with oestrogen receptor positive (ER+) breast cancers, whereas Cav3.2 levels were correlated positively with patient survival after chemotherapy in patients with HER2-positive breast cancers. CONCLUSION: Our study identified elevated levels of Cav3.2 in trastuzumab-resistant SKBR3 cell lines. Although not a regulator of trastuzumab-resistance in HER2-positive breast cancer cells, Cav3.2 may be a potential differential biomarker for survival and treatment response in specific breast cancer subtypes. These studies add to the complex and diverse role of Ca(2+)-signalling in breast cancer progression and treatment.
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Increased expression of specific calcium channels in some cancers and the role of calcium signaling in proliferation and invasion have led to studies assessing calcium channel inhibitors as potential therapies for some cancers. The use of channel activators to promote death of cancer cells has been suggested, but the risk of activators promoting cancer cell proliferation and the importance of the degree of channel over-expression is unclear. We developed an MCF-7 breast cancer cell line with inducible TRPV1 overexpression and assessed the role of TRPV1 levels on cell death mediated by the TRPV1 activator capsaicin and the potential for submaximal activation to promote proliferation. The TRPV1 level was a determinant of cell death induced by capsaicin. A concentration response curve with varying TRPV1 expression levels identified the minimum level of TRPV1 required for capsaicin induced cell death. At no level of TRPV1 over-expression or capsaicin concentration did TRPV1 activation enhance proliferation. Cell death induced by capsaicin was necrotic and associated with up-regulation of c-Fos and RIP3. These studies suggest that activators of specific calcium channels may be an effective way to induce necrosis and that this approach may not always be associated with enhancement of cancer cell proliferation.
Assuntos
Canais de Cátion TRPV/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Capsaicina/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/toxicidade , Feminino , Humanos , Células MCF-7 , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Canais de Cátion TRPV/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Calcium signaling is a key regulator of processes important in differentiation. In colon cancer cells differentiation is associated with altered expression of specific isoforms of calcium pumps of the endoplasmic reticulum and the plasma membrane, suggesting that differentiation of colon cancer cells is associated with a major remodeling of calcium homeostasis. Purinergic and neurotensin receptor activation are known regulators of cytosolic free Ca(2+) levels in colon cancer cells. This study aimed to assess changes in cytosolic free Ca(2+) levels in response to ATP and neurotensin with differentiation induced by sodium butyrate or culturing post-confluence. Parameters assessed included peak cytosolic free Ca(2+) level after activation; time to reach peak cytosolic free Ca(2+) and the EC50 of dose response curves. Our results demonstrate that differentiation of HT-29 colon cancer cells is associated with a remodeling of both ATP and neurotensin mediated Ca(2+) signaling. Neurotensin-mediated calcium signaling appeared more sensitive to differentiation than ATP-mediated Ca(2+) signaling.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Neurotensina/metabolismo , Receptores de Neurotensina/metabolismo , Receptores Purinérgicos/metabolismo , Trifosfato de Adenosina/metabolismo , Butiratos/farmacologia , Cálcio/metabolismo , Diferenciação Celular , Citosol/metabolismo , Relação Dose-Resposta a Droga , Células HT29 , Humanos , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
The mitochondrial calcium uniporter (MCU) transports free ionic Ca(2+) into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca(2+) levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca(2+) levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.
Assuntos
Neoplasias da Mama/patologia , Canais de Cálcio/metabolismo , Caspases/metabolismo , Inativação Gênica , Mitocôndrias/metabolismo , Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Canais de Cálcio/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Ionomicina/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Sulfonamidas/farmacologiaRESUMO
Calcium signaling is a critical regulator of cell proliferation. Elevated expression of calcium channels and pumps is a characteristic of some cancers, including breast cancer. We show that the plasma membrane calcium channel TRPV6, which is highly selective for Ca(2+), is overexpressed in some breast cancer cell lines. Silencing of TRPV6 expression in a breast cancer cell line with increased endogenous TRPV6 expression leads to a reduction in basal calcium influx and cellular proliferation associated with a reduction in DNA synthesis. TRPV6 gene amplification was identified as one mechanism of TRPV6 overexpression in a subset of breast cancer cell lines and breast tumor samples. Analysis of two independent microarray expression datasets from breast tumor samples showed that increased TRPV6 expression is a feature of estrogen receptor (ER)-negative breast tumors encompassing the basal-like molecular subtype, as well as HER2-positive tumors. Breast cancer patients with high TRPV6 levels had decreased survival compared with patients with low or intermediate TRPV6 expression. Our findings suggest that inhibitors of TRPV6 may offer a novel therapeutic strategy for the treatment of ER-negative breast cancers.
Assuntos
Neoplasias da Mama/metabolismo , Canais de Cálcio/metabolismo , Receptores de Estrogênio/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cálcio/metabolismo , Canais de Cálcio/genética , Contagem de Células , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Regulação para Baixo/genética , Feminino , Amplificação de Genes/genética , Dosagem de Genes/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Análise de Sobrevida , Canais de Cátion TRPV/genéticaRESUMO
In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca(2+) reserves, molecular components of the store-operated Ca(2+) entry pathway regulate breast cancer metastasis. A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT). In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca(2+) influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca(2+) influx. The potential roles for specific Ca(2+) channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1) channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca(2+) influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca(2+) influx in a manner dependent on Ca(2+) influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca(2+) release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (time to peak [Ca(2+)](CYT) = 188.7 ± 34.6 s (TRPC1 siRNA) versus 124.0 ± 9.5 s (non-targeting siRNA); P<0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca(2+) influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca(2+) influx in non-stimulated cells.
Assuntos
Neoplasias da Mama/patologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Agonistas Purinérgicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Canais de Cálcio/deficiência , Canais de Cálcio/genética , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína ORAI1 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Canais de Cátion TRPC/deficiência , Canais de Cátion TRPC/genéticaRESUMO
The entry of calcium into the mammary epithelial cell from the maternal plasma (i.e., calcium influx mechanisms) during lactation is poorly understood. As alterations in calcium channels and pumps are a key feature of some cancers, including breast cancer, understanding these calcium influx pathways may have significance beyond mammary biology. We show that the store-operated calcium influx protein, Orai1, is increased during lactation whereas the Orai1 activator Stim1, but not Stim2, is downregulated. Stim2 siRNA reduced basal calcium levels in a lactation model. Our results suggest that calcium influx is remodeled in mammary epithelial cells during lactation, with calcium influx increased through Orai1, activated by Stim2. Breast cancer cell lines had increased levels of ORAI1. ORAI1 siRNA in breast cancer cells reduced store-operated calcium entry and remodeled the calcium influx associated with invasive stimuli. Analysis of microarray data from 295 breast cancers showed that the transcriptional breast cancer subtype with the poorest prognosis (basal) was associated with an altered relationship between the ORAI1 regulators STIM1 and STIM2, and that women with breast cancers with STIM1(high)/STIM2(low) tumors had a significantly poorer prognosis. Our studies show that during lactation there is a remodeling in the nature of calcium influx and that alteration in the ORAI1 influx pathway may be a feature of some breast cancers, particularly those with the poorest prognosis. Our studies suggest that this pathway may be a novel therapeutic target for breast cancer treatment in these women.
Assuntos
Neoplasias da Mama/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Lactação/metabolismo , Animais , Canais de Cálcio/análise , Canais de Cálcio/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , RNA Interferente Pequeno , Molécula 1 de Interação Estromal , Molécula 2 de Interação EstromalRESUMO
ORAI1 is a protein located on the plasma membrane that acts as a calcium channel. Calcium enters via ORAI1 as a mechanism to refill the sarcoplasmic/endoplasmic reticulum calcium stores, the depletion of which can be detected by the sensor protein STIM1. Isoforms of these proteins ORAI2, ORAI3 and STIM2 also have roles in cellular calcium homeostasis but are less well characterized. This pathway of filling the calcium stores is termed store-operated calcium entry and while the pathway itself was proposed in 1986, the identity of the key molecular components was only discovered in 2005 and 2006. The characterization of the ORAI and STIM proteins has provided clearer information on some calcium-regulated pathways that are important in processes from gene transcription to immune cell function. Recent studies have also suggested the importance of the components of ORAI-mediated calcium entry in some diseases or processes significant in disease including the migration of breast cancer cells and thrombus formation. This review will provide a brief overview of ORAI-mediated calcium entry, its role in physiological and pathophysiological processes, as well as current and potential pharmacological modulators of the components of this important cellular calcium entry pathway.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Homeostase , Humanos , Proteína ORAI1 , Isoformas de Proteínas , Retículo Sarcoplasmático/metabolismo , Molécula 1 de Interação EstromalRESUMO
A remodeling of calcium homeostasis has been identified as a characterizing feature of some cancers. Possible consequences of this include alterations in many pivotal physiological responses including apoptosis, proliferation and gene transcription. An alteration in calcium homeostasis can occur via changes in the expression of proteins that transport calcium and examples of cancers where this is seen includes the prostate and breast. A specific isoform of the calcium efflux pump, plasma membrane Ca(2+)-ATPase (PMCA) 4, is significantly upregulated during differentiation of the HT-29 colon cancer cell line suggesting that it may also be altered in colon cancer. We now report that differentiated HT-29 colon cancer cells have pronounced plasma membrane PMCA4 localization, consistent with augmented calcium efflux. Assessment of PMCA4 transcription in human colon cancer samples suggests that PMCA4 is significantly (P < 0.000001) downregulated early in the progression of some colon cancers as these cells become less differentiated. Inhibition of PMCA4 using small interfering RNA did not induce cell death or augment sensitivity to the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or tumor necrosis factor-related apoptosis-inducing ligand. Reversing the colon cancer remodeling of PMCA4 by overexpression reduced cellular proliferation (P < 0.01) and downregulated transcription of the calcium sensitive early response gene FOS. Our studies suggest that the remodeling of the calcium signal in colon cancer is associated with compromised calcium efflux at a level that promotes proliferative pathways while avoiding increased sensitivity to apoptotic stimuli.
Assuntos
Cálcio/metabolismo , Neoplasias do Colo/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Genes fos , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , RNA Interferente Pequeno , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transcrição Gênica , Desacopladores/farmacologia , Regulação para CimaRESUMO
There is emerging evidence that the balance between estrogen receptor-alpha (ER(alpha)) and androgen receptor (AR) signaling is a critical determinant of growth in the normal and malignant breast. In this study, we assessed AR status in a cohort of 215 invasive ductal breast carcinomas. AR and (ER(alpha)) were coexpressed in the majority (80-90%) of breast tumor cells. Kaplan-Meier product limit analysis and multivariate Cox regression showed that AR is an independent prognostic factor in (ER(alpha))-positive disease, with a low level of AR (less than median of 75% positive cells) conferring a 4.6-fold increased risk of cancer-related death (P = 0.002). Consistent with a role for AR in breast cancer outcome, AR potently inhibited (ER(alpha))transactivation activity and 17beta-estradiol-stimulated growth of breast cancer cells. Transfection of MDA-MB-231 breast cancer cells with either functionally impaired AR variants or the DNA-binding domain of the AR indicated that the latter is both necessary and sufficient for inhibition of (ER(alpha)) signaling. Consistent with molecular modeling, electrophoretic mobility shift assays showed binding of the AR to an estrogen-responsive element (ERE). Evidence for a functional interaction of the AR with an ERE in vivo was provided by chromatin immunoprecipitation data, revealing recruitment of the AR to the progesterone receptor promoter in T-47D breast cancer cells. We conclude that, by binding to a subset of EREs, the AR can prevent activation of target genes that mediate the stimulatory effects of 17beta-estradiol on breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptores Androgênicos/metabolismo , Animais , Neoplasias da Mama/patologia , Células COS , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Chlorocebus aethiops , DNA de Neoplasias/metabolismo , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Humanos , Modelos Moleculares , Prognóstico , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Elementos de Resposta , Transdução de Sinais , Ativação TranscricionalRESUMO
The androgen receptor (AR) is an important signaling molecule in multiple tissues, yet its mode of action and cell-specific activities remain enigmatic. AR function has been best studied in the prostate, in which it is essential for growth and homeostasis of the normal organ as well as each stage of cancer development. Investigation of mechanisms responsible for continued AR action that evolve during prostate cancer progression or after hormonal management of the disease have been instructive in defining AR signaling pathways. In the current paper, we use sequence similarity and the collocation of somatic mutations in prostate cancer to define residues 501-535 of the AR amino-terminal domain as an important mediator of receptor function. Specifically, the 501-535 region is required for optimal interaction of the amino-terminal domain with both the p160 coactivator, nuclear receptor coactivator-2, and the AR-ligand binding domain in the amino/carboxyl (N/C) interaction. The N/C interaction is decreased by deletion of the 501-535 region but is distinct from deletion of the (23)FQNLF(27) peptide in that it does not affect the capacity of the AR to activate transcription from a chromatin integrated reporter or recruitment of the receptor to androgen-responsive loci in vivo. Collectively, we have been able to outline two classes of N/C-deficient AR variant that are divergent in their capacity to act in a chromatin context, thereby further defining the interplay between N/C interaction and coregulator recruitment via multiple receptor domains. These mechanisms are likely to be key determinants of the cell and promoter specific activities of the AR.