Publications by authors named "Luca Hegedűs"

23 Publications

  • Page 1 of 1

The Plasma Membrane Ca Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton.

Cancers (Basel) 2021 Mar 17;13(6). Epub 2021 Mar 17.

Department of Transfusiology, Semmelweis University, H-1089 Budapest, Hungary.

We demonstrated that the plasma membrane Ca ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell-cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.
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http://dx.doi.org/10.3390/cancers13061354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002435PMC
March 2021

Allosteric and ATP-Competitive MEK-Inhibition in a Novel Spitzoid Melanoma Model with a RAF- and Phosphorylation-Independent Mutation.

Cancers (Basel) 2021 Feb 16;13(4). Epub 2021 Feb 16.

Department of Thoracic Surgery, University Medicine Essen-Ruhrlandklinik, Tüschener Weg 40, 45239 Essen, Germany.

Spitzoid melanoma is a rare malignancy with histological characteristics similar to Spitz nevus. It has a diverse genetic background and in adults, a similarly grim clinical outcome as conventional malignant melanoma. We established a spitzoid melanoma cell line (PF130) from the pleural effusion sample of a 37-year-old male patient. We found that the cell line carries a rare MEK1 mutation (pGlu102_Lys104delinsGln) that belongs to the RAF- and phosphorylation-independent subgroup of MEK1 alternations supposedly insensitive to allosteric MEK inhibitors. The in vivo tumorigenicity was tested in three different models by injecting the cells subcutaneously, intravenously or into the thoracic cavity of SCID mice. In the intrapleural model, macroscopic tumors formed in the chest cavity after two months, while subcutaneously and intravenously delivered cells showed limited growth. In vitro, trametinib-but not selumentinib-and the ATP-competitive MEK inhibitor MAP855 strongly decreased the viability of the cells and induced cell death. In vivo, trametinib but not MAP855 significantly reduced tumor growth in the intrapleural model. To the best of our knowledge, this is the first patient-derived melanoma model with RAF- and phosphorylation-independent MEK mutation and we demonstrated its sensitivity to trametinib.
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http://dx.doi.org/10.3390/cancers13040829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920251PMC
February 2021

Horizontal Combination of MEK and PI3K/mTOR Inhibition in BRAF Mutant Tumor Cells with or without Concomitant PI3K Pathway Mutations.

Int J Mol Sci 2020 Oct 16;21(20). Epub 2020 Oct 16.

2nd Department of Pathology, Semmelweis University, H-1091 Budapest, Hungary.

The RAS/RAF and PI3K/Akt pathways play a key regulatory role in cancer and are often hit by oncogenic mutations. Despite molecular targeting, the long-term success of monotherapy is often hampered by de novo or acquired resistance. In the case of concurrent mutations in both pathways, horizontal combination could be a reasonable approach. In our study, we investigated the MEK inhibitor selumetinib and PI3K/mTOR dual inhibitor BEZ235 alone and in combination in BRAF-only mutant and BRAF + PI3K/PTEN double mutant cancer cells using short- and long-term 2D viability assays, spheroid assays, and immunoblots. In the 2D assays, selumetinib was more effective on BRAF-only mutant lines when compared to BRAF + PI3K/PTEN double mutants. Furthermore, combination therapy had an additive effect in most of the lines while synergism was observed in two of the double mutants. Importantly, in the SW1417 BRAF + PI3K double mutant cells, synergism was also confirmed in the spheroid and in the in vivo model. Mechanistically, p-Akt level decreased only in the SW1417 cell line after combination treatment. In conclusion, the presence of concurrent mutations alone did not predict a stronger response to combination treatment. Therefore, additional investigations are warranted to identify predictive factors that can select patients who can benefit from the horizontal combinational inhibition of these two pathways.
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http://dx.doi.org/10.3390/ijms21207649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589607PMC
October 2020

HDAC Inhibition Induces PD-L1 Expression in a Novel Anaplastic Thyroid Cancer Cell Line.

Pathol Oncol Res 2020 Oct 26;26(4):2523-2535. Epub 2020 Jun 26.

Department of Thoracic Surgery, University Medicine Essen - Ruhrlandklinik, University Duisburg-Essen, Essen, Germany.

While papillary thyroid cancer (PTC) has largely favorable prognosis, anaplastic thyroid cancer (ATC) is a rare but extremely aggressive malignancy with grim clinical outcome. Even though new therapeutic options are emerging for ATC, additional preclinical models and novel combinations are needed for specific subsets of patients. We established a novel cell line (PF49) from the malignant pleural effusion of a 68-year-old male patient with ATC that rapidly transformed from a BRAF and TERT promoter mutant PTC. PF49 cells demonstrated a robust migratory activity in vitro and strong invasive capacity in vivo in a pleural carcinosis model. Combined BRAF and MEK inhibition decreased the proliferation and migration of PF49 cells, however could not induce cell death. Importantly, HDAC inhibitor treatment with SAHA or valproic acid induced cell cycle arrest and strongly increased PD-L1 expression of the tumor cells. Induction of PD-L1 expression was also present when paclitaxel-cisplatin chemotherapeutic treatment was combined with HDAC inhibitor treatment. Increased PD-L1 expression after HDAC inhibition was recapitulated in an international ATC cell model. Our data suggest that HDAC inhibition alone or in combination with standard chemotherapy may potentiate anaplastic thyroid cancer cells for immunotherapy.
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http://dx.doi.org/10.1007/s12253-020-00834-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471186PMC
October 2020

P38 MAPK Promotes Migration and Metastatic Activity of BRAF Mutant Melanoma Cells by Inducing Degradation of PMCA4b.

Cells 2020 05 13;9(5). Epub 2020 May 13.

2nd Institute of Pathology, Semmelweis University, H-1091 Budapest, Hungary.

Metastatic melanoma is the most aggressive type of skin cancer. Previously, we identified the plasma membrane Ca pump isoform 4b (PMCA4b or ) as a putative metastasis suppressor in BRAF mutant melanoma cells. Metastasis suppressors are often downregulated in cancer, therefore, it is important to identify the pathways involved in their degradation. Here, we studied the role of p38 MAPK in PMCA4b degradation and its effect on melanoma metastasis. We found that activation of p38 MAPK induces internalization and subsequent degradation of PMCA4b through the endo/lysosomal system that contributes to the low PMCA4b steady-state protein level of BRAF mutant melanoma cells. Moreover, BRAF wild type cell models including a doxycycline-inducible HEK cell system revealed that p38 MAPK is a universal modulator of PMCA4b endocytosis. Inhibition of the p38 MAPK pathway markedly reduced migration, colony formation and metastatic activity of BRAF mutant cells in vitro partially through an increase in PMCA4b and a decrease in β4 integrin abundance. In conclusion, our data suggest that the p38 MAPK pathway plays a key role in PMCA4b degradation and inhibition of this pathway-by increasing the stability of PMCA4b-may provide a potential therapeutic target for inhibition of melanoma progression and metastasis.
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http://dx.doi.org/10.3390/cells9051209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290426PMC
May 2020

HDAC inhibition synergizes with ALK inhibitors to overcome resistance in a novel ALK mutated lung adenocarcinoma model.

Lung Cancer 2020 06 18;144:20-29. Epub 2020 Apr 18.

Department of Thoracic Surgery, West German Cancer Center, University Hospital Essen - Ruhrlandklinik, University Duisburg-Essen, Essen, Germany. Electronic address:

Objectives: Somatic chromosomal rearrangements resulting in ALK fusion oncogenes are observed in 3-7 % of lung adenocarcinomas. ALK tyrosine kinase inhibitors (ALKi) induce initially response, however, various resistance mechanisms limit their efficacy. Novel therapeutic approaches are of utmost importance to tailor these targeted therapies.

Materials And Methods: A synchronous ALK-rearranged and mutated lung cancer cell line pair was established from malignant pleural effusion (PF240-PE) and carcinosis (PF240-PC) at time of ALKi resistance. Immunohistochemistry, FISH and sequencing were performed in pre- and post-treatment tumors and in both cell lines. Differentiation markers were measured by immunoblot. Viability was tested following treatment with ALKi and/or a pan-HDAC inhibitor. Additionally, a novel treatment-naïve ALK-rearranged cell line served as control. In vivo tumorigenicity was evaluated in subcutaneous xenografts.

Results: Two distinct resistance mutations were identified in different carcinosis tissues at time of resistance, the previously described resistance mutation L1152R and the hitherto uncharacterized E1161K. Strikingly, PF240-PC cells carried E1161K and PF240-PE cells harbored L1152R. Immunohistochemistry and immunoblot identified epithelial-to-mesenchymal transition markers upregulated following ALKi resistance development both in carcinosis tissues and cell lines. While both lines grew as xenografts, they differed in morphology, migration, in vivo growth and sensitivity to ALKi in vitro. Strikingly, the combination of ALKi with SAHA yielded strong synergism.

Conclusion: Using a patient-derived ALKi resistant lung cancer model we demonstrated the synergism of HDAC and ALK inhibition. Furthermore, our findings provide strong evidence for intratumoral heterogeneity under targeted therapy and highlight the importance of site-specific mutational analysis.
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http://dx.doi.org/10.1016/j.lungcan.2020.04.002DOI Listing
June 2020

Next Generation Lipophilic Bisphosphonate Shows Antitumor Effect in Colorectal Cancer In Vitro and In Vivo.

Pathol Oncol Res 2020 Jul 4;26(3):1957-1969. Epub 2020 Jan 4.

2nd Department of Pathology, Semmelweis University, Budapest, H-1091, Hungary.

Bisphosphonates, despite proven antitumor effect in vitro in many tumor types, are currently used only for treatment of osteoporosis and bone metastasis. Colorectal cancer is the third most commonly diagnosed type of cancer and lacks targeted therapy for RAS or RAF mutation carrying cases. A new lipophilic bisphosphonate showed promising results in lung cancer models, but their effect on colorectal cancer cells was not investigated excessively. Antitumor effects and impact on RAS-related signalization of zoledronic acid (ZA) and a lipophilic bisphosphonate (BPH1222) were investigated on 7 human colorectal cancer cell lines in vitro and in vivo. Furthermore, mutant KRAS dependent effect of prenylation inhibition was investigated using isogeneic cell lines. Both bisphosphonates reduced cell viability in vitro in a dose-dependent manner. Both compounds changed cell cycle distribution similarly by increasing the proportion of cells either in the S or in the subG1 phase or both. However, BPH1222 exerted higher inhibitory effect on spheroid growth than ZA. Interestingly, we found profound alterations in phosphorylation level of Erk and S6 proteins upon ZA or BPH1222 treatment. Furthermore, investigation of a mutant KRAS isogeneic model system suggests that the drugs interfere also with the mutant KRAS proteins. In vivo experiments with KRAS mutant xenograft model also revealed growth inhibitory potential of bisphosphonate treatment. Our results show that lipophilic bisphosphonates might extend the therapeutic spectrum of bisphosphonate drugs and could be considered as additional treatment approaches in colorectal cancer.
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http://dx.doi.org/10.1007/s12253-019-00789-9DOI Listing
July 2020

Molecular Diversity of Plasma Membrane Ca Transporting ATPases: Their Function Under Normal and Pathological Conditions.

Adv Exp Med Biol 2020 ;1131:93-129

2nd Department of Pathology, Semmelweis University, Budapest, Hungary.

Plasma membrane Ca transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca from the cell in order to keep the cytosolic Ca ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca homeostasis they also change the duration and amplitude of the Ca signal and can create Ca gradients across the cell. This is accomplished by generating more than twenty PMCA variants each having the character - fast or slow response, long or short memory, distinct interaction partners and localization signals - that meets the specific needs of the particular cell-type in which they are expressed. It has become apparent that these pumps are essential to normal tissue development and their malfunctioning can be linked to different pathological conditions such as certain types of neurodegenerative and heart diseases, hearing loss and cancer. In this chapter we summarize the complexity of PMCA regulation and function under normal and pathological conditions with particular attention to recent developments of the field.
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http://dx.doi.org/10.1007/978-3-030-12457-1_5DOI Listing
October 2019

The Antitumor Effect of Lipophilic Bisphosphonate BPH1222 in Melanoma Models: The Role of the PI3K/Akt Pathway and the Small G Protein Rheb.

Int J Mol Sci 2019 Oct 3;20(19). Epub 2019 Oct 3.

Department of Pathology, Semmelweis University, H-1091 Budapest, Hungary.

Malignant melanoma is one of the most metastatic cancer types, and despite recent success with novel treatment strategies, there is still a group of patients who do not respond to any therapies. Earlier, the prenylation inhibitor hydrophilic bisphosphonate zoledronic acid (ZA) was found to inhibit melanoma growth in vitro, but only a weaker effect was observed in vivo due to its hydrophilic properties. Recently, lipophilic bisphosphonates (such as BPH1222) were developed. Accordingly, for the first time, we compared the effect of BPH1222 to ZA in eight melanoma lines using viability, cell-cycle, clonogenic and spheroid assays, videomicroscopy, immunoblot, and xenograft experiments. Based on 2D and spheroid assays, the majority of cell lines were more sensitive to BPH. The activation of Akt and S6 proteins, but not Erk, was inhibited by BPH. Additionally, BPH had a stronger apoptotic effect than ZA, and the changes of Rheb showed a correlation with apoptosis. In vitro, only M24met cells were more sensitive to ZA than to BPH; however, in vivo growth of M24met was inhibited more strongly by BPH. Here, we present that lipophilic BPH is more effective on melanoma cells than ZA and identify the PI3K pathway, particularly Rheb as an important mediator of growth inhibition.
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http://dx.doi.org/10.3390/ijms20194917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801414PMC
October 2019

Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells.

BMC Cancer 2018 Oct 23;18(1):1029. Epub 2018 Oct 23.

2nd Department of Pathology, Semmelweis University, Budapest, Hungary.

Background: Remodeling of Ca signaling is an important step in cancer progression, and altered expression of members of the Ca signaling toolkit including the plasma membrane Ca ATPases (PMCA proteins encoded by ATP2B genes) is common in tumors.

Methods: In this study PMCAs were examined in breast cancer datasets and in a variety of breast cancer cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-α) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps.

Results: Three distinct datasets displayed significantly lower ATP2B4 mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of ATP2B1 and ATP2B2 was not altered. Studying the protein expression profiles of Ca pumps in a variety of breast cancer cell lines revealed low PMCA4b expression in the ER-α positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER-α pathway in MCF-7 cells that led to enhanced Ca extrusion capacity in response to 17β-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER-α positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER-α binding site in the ATP2B4 gene in MCF-7 cells but not in other ER-α positive tumor cells. In the triple negative cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared to that of the ER-α positive cells. Although, the expression of PMCA4b was relatively high in the triple negative cells, a fraction of the protein was found in intracellular compartments that could interfere with the cellular function of the protein.

Conclusions: Our results suggest that the expression of Ca pumps is highly regulated in breast cancer cells in a subtype specific manner. Our results suggest that hormonal imbalances, epigenetic modifications and impaired protein trafficking could interfere with the expression and cellular function of PMCA4b in the course of breast cancer progression.
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http://dx.doi.org/10.1186/s12885-018-4945-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199715PMC
October 2018

Histone Deacetylase Inhibitor Treatment Increases the Expression of the Plasma Membrane Ca Pump PMCA4b and Inhibits the Migration of Melanoma Cells Independent of ERK.

Front Oncol 2017 24;7:95. Epub 2017 May 24.

2nd Institute of Pathology, Semmelweis University, Budapest, Hungary.

Several new therapeutic options emerged recently to treat metastatic melanoma; however, the high frequency of intrinsic and acquired resistance among patients shows a need for new therapeutic options. Previously, we identified the plasma membrane Ca ATPase 4b (PMCA4b) as a metastasis suppressor in BRAF-mutant melanomas and found that mutant BRAF inhibition increased the expression of the pump, which then inhibited the migratory and metastatic capability of the cells. Earlier it was also demonstrated that histone deacetylase inhibitors (HDACis) upregulated PMCA4b expression in gastric, colon, and breast cancer cells. In this study, we treated one BRAF wild-type and two BRAF-mutant melanoma cell lines with the HDACis, SAHA and valproic acid, either alone, or in combination with the BRAF inhibitor, vemurafenib. We found that HDACi treatment strongly increased the expression of PMCA4b in all cell lines irrespective of their BRAF mutational status, and this effect was independent of ERK activity. Furthermore, HDAC inhibition also enhanced the abundance of the housekeeping isoform PMCA1. Combination of HDACis with vemurafenib, however, did not have any additive effects on either PMCA isoform. We demonstrated that the HDACi-induced increase in PMCA abundance was coupled to an enhanced [Ca] clearance rate and also strongly inhibited both the random and directional movements of A375 cells. The primary role of PMCA4b in these characteristic changes was demonstrated by treatment with the PMCA4-specific inhibitor, caloxin 1c2, which was able to restore the slower Ca clearance rate and higher motility of the cells. While HDAC treatment inhibited cell motility, it decreased only modestly the ratio of proliferative cells and cell viability. Our results show that in melanoma cells the expression of both PMCA4b and PMCA1 is under epigenetic control and the elevation of PMCA4b expression either by HDACi treatment or by the decreased activation of the BRAF-MEK-ERK pathway can inhibit the migratory capacity of the highly motile A375 cells.
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http://dx.doi.org/10.3389/fonc.2017.00095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442207PMC
May 2017

The plasma membrane Ca pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells.

Int J Cancer 2017 06 17;140(12):2758-2770. Epub 2016 Nov 17.

2nd Department of Pathology, Semmelweis University, Budapest, Hungary.

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca signaling is a well-known regulator of tumor progression, the crosstalk between Ca signaling and the Ras-BRAF-MEK-ERK pathway is much less explored. Here we show that in BRAF mutant melanoma cells the abundance of the plasma membrane Ca ATPase isoform 4b (PMCA4b, ATP2B4) is low at baseline but markedly elevated by treatment with the mutant BRAF specific inhibitor vemurafenib. In line with these findings gene expression microarray data also shows decreased PMCA4b expression in cutaneous melanoma when compared to benign nevi. The MEK inhibitor selumetinib-similarly to that of the BRAF-specific inhibitor-also increases PMCA4b levels in both BRAF and NRAS mutant melanoma cells suggesting that the MAPK pathway is involved in the regulation of PMCA4b expression. The increased abundance of PMCA4b in the plasma membrane enhances [Ca ] clearance from cells after Ca entry. Moreover we show that both vemurafenib treatment and PMCA4b overexpression induce marked inhibition of migration of BRAF mutant melanoma cells. Importantly, reduced migration of PMCA4b expressing BRAF mutant cells is associated with a marked decrease in their metastatic potential in vivo. Taken together, our data reveal an important crosstalk between Ca signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor.
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http://dx.doi.org/10.1002/ijc.30503DOI Listing
June 2017

Multifaceted plasma membrane Ca(2+) pumps: From structure to intracellular Ca(2+) handling and cancer.

Biochim Biophys Acta 2016 Jun 17;1863(6 Pt B):1351-63. Epub 2015 Dec 17.

2(nd) Department of Pathology, Semmelweis University, Budapest, Hungary; Molecular Oncology Research Group of the Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary. Electronic address:

Plasma membrane Ca(2+) ATPases (PMCAs) are intimately involved in the control of intracellular Ca(2+) concentration. They reduce Ca(2+) in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca(2+) release from the endoplasmic reticulum Ca(2+) pool. In mammals four genes (PMCA1-4) are expressed, and alternative RNA splicing generates more than twenty variants. The variants differ in their regulatory characteristics. They localize into highly specialized membrane compartments and respond to the incoming Ca(2+) with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca(2+) homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca(2+) homeostasis of cancer cells. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.
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http://dx.doi.org/10.1016/j.bbamcr.2015.12.011DOI Listing
June 2016

Plasma membrane Ca²⁺-ATPases can shape the pattern of Ca²⁺ transients induced by store-operated Ca²⁺ entry.

Sci Signal 2015 Feb 17;8(364):ra19. Epub 2015 Feb 17.

Hungarian National Blood Transfusion Service, Budapest H-1113, Hungary. 2nd Department of Pathology, Semmelweis University, Budapest H-1091, Hungary. Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest H-1117, Hungary.

Calcium (Ca(2+)) is a critical cofactor and signaling mediator in cells, and the concentration of cytosolic Ca(2+) is regulated by multiple proteins, including the plasma membrane Ca(2+)-ATPases (adenosine triphosphatases) (PMCAs), which use ATP to transport Ca(2+) out of cells. PMCA isoforms exhibit different kinetic and regulatory properties; thus, the presence and relative abundance of individual isoforms may help shape Ca(2+) transients and cellular responses. We studied the effects of three PMCA isoforms (PMCA4a, PMCA4b, and PMCA2b) on Ca(2+) transients elicited by conditions that trigger store-operated Ca(2+) entry (SOCE) and that blocked Ca(2+) uptake into the endoplasmic reticulum in HeLa cells, human embryonic kidney (HEK) 293 cells, or primary endothelial cell isolated from human umbilical cord veins (HUVECs). The slowly activating PMCA4b isoform produced long-lasting Ca(2+) oscillations in response to SOCE. The fast-activating isoforms PMCA2b and PMCA4a produced different effects. PMCA2b resulted in rapid and highly PMCA abundance-sensitive clearance of SOCE-mediated Ca(2+) transients, whereas PMCA4a reduced cytosolic Ca(2+), resulting in the establishment of a higher than baseline cytosolic Ca(2+) concentration. Mathematical modeling showed that slow activation was critical to the sustained oscillation induced by the "slow" PMCA4b pump. The modeling and experimental results indicated that the distinct properties of PMCA isoforms differentially regulate the pattern of SOCE-mediated Ca(2+) transients, which would thus affect the activation of downstream signaling pathways.
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http://dx.doi.org/10.1126/scisignal.2005672DOI Listing
February 2015

Histone deacetylase inhibitor- and PMA-induced upregulation of PMCA4b enhances Ca2+ clearance from MCF-7 breast cancer cells.

Cell Calcium 2014 Feb 28;55(2):78-92. Epub 2013 Dec 28.

Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; Department of Molecular Cell Biology, Hungarian National Blood Transfusion Service, Budapest, Hungary. Electronic address:

The expression of the plasma membrane Ca2+ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca2+ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression - either by treatment or overexpression - led to enhanced Ca2+ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca2+ homeostasis. These results suggest that modulation of Ca2+ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer.
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http://dx.doi.org/10.1016/j.ceca.2013.12.003DOI Listing
February 2014

Apart from its known function, the plasma membrane Ca²⁺ATPase can regulate Ca²⁺ signaling by controlling phosphatidylinositol 4,5-bisphosphate levels.

J Cell Sci 2014 Jan 6;127(Pt 1):72-84. Epub 2013 Nov 6.

Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1025 Budapest, Hungary.

Plasma membrane Ca(2+) ATPases (PMCAs, also known as ATP2B1-ATP2B4) are known targets of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], but if and how they control the PtdIns(4,5)P₂ pool has not been considered. We demonstrate here that PMCAs protect PtdIns(4,5)P₂ in the plasma membrane from hydrolysis by phospholipase C (PLC). Comparison of active and inactive PMCAs indicates that the protection operates by two mechanisms; one requiring active PMCAs, the other not. It appears that the mechanism requiring activity is the removal of the Ca(2+) required for sustained PLC activity, whereas the mechanism not requiring activity is PtdIns(4,5)P₂ binding. We show that in PMCA overexpressing cells, PtdIns(4,5)P₂ binding can lead to less inositol 1,4,5-triphosphate (InsP₃) and diminished Ca(2+) release from intracellular Ca(2+) pools. Inspection of a homology model of PMCA suggests that PMCAs have a conserved cluster of basic residues forming a 'blue collar' at the interface between the membrane core and the cytoplasmic domains. By molecular dynamics simulation, we found that the blue collar forms four binding pockets for the phosphorylated inositol head group of PtdIns(4,5)P₂; these pockets bind PtdIns(4,5)P₂ strongly and frequently. Our studies suggest that by having the ability to bind PtdIns(4,5)P₂, PMCAs can control the accessibility of PtdIns(4,5)P₂ for PLC and other PtdIns(4,5)P₂-mediated processes.
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http://dx.doi.org/10.1242/jcs.132548DOI Listing
January 2014

A C-terminal di-leucine motif controls plasma membrane expression of PMCA4b.

Biochim Biophys Acta 2013 Dec 2;1833(12):2561-2572. Epub 2013 Jul 2.

Hungarian National Blood Transfusion Service, Budapest, Hungary. Electronic address:

Recent evidences show that the localization of different plasma membrane Ca(2+) ATPases (PMCAs) is regulated in various complex, cell type-specific ways. Here we show that in low-density epithelial and endothelial cells PMCA4b localized mostly in intracellular compartments and its plasma membrane localization was enhanced upon increasing density of cells. In good correlation with the enhanced plasma membrane localization a significantly more efficient Ca(2+) clearance was observed in confluent versus non-confluent HeLa cell cultures expressing mCherry-PMCA4b. We analyzed the subcellular localization and function of various C-terminally truncated PMCA4b variants and found that a truncated mutant PMCA4b-ct24 was mostly intracellular while another mutant, PMCA4b-ct48, localized more to the plasma membrane, indicating that a protein sequence corresponding to amino acid residues 1158-1181 contained a signal responsible for the intracellular retention of PMCA4b in non-confluent cultures. Alteration of three leucines to alanines at positions 1167-1169 resulted in enhanced cell surface expression and an appropriate Ca(2+) transport activity of both wild type and truncated pumps, suggesting that the di-leucine-like motif (1167)LLL was crucial in targeting PMCA4b. Furthermore, upon loss of cell-cell contact by extracellular Ca(2+) removal, the wild-type pump was translocated to the early endosomal compartment. Targeting PMCA4b to early endosomes was diminished by the L(1167-69)A mutation, and the mutant pump accumulated in long tubular cytosolic structures. In summary, we report a di-leucine-like internalization signal at the C-tail of PMCA4b and suggest an internalization-mediated loss of function of the pump upon low degree of cell-cell contact.
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http://dx.doi.org/10.1016/j.bbamcr.2013.06.021DOI Listing
December 2013

Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein.

Cell Signal 2013 Apr 7;25(4):752-9. Epub 2013 Jan 7.

Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.

Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca(2+) indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca(2+) oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca(2+) signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.
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http://dx.doi.org/10.1016/j.cellsig.2012.12.024DOI Listing
April 2013

Modulation of endoplasmic reticulum calcium pump expression during lung cancer cell differentiation.

FEBS J 2013 Nov 11;280(21):5408-18. Epub 2012 Dec 11.

Institut National de la Santé et de la Recherche Médicale, UMR-S 940, Paris, France; Institut Universitaire d'Hématologie, Université Paris Diderot, PRES Sorbonne Paris-Cité, France.

Cellular calcium signaling plays important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. In epithelial cells calcium signaling is initiated mainly by calcium release from endoplasmic-reticulum-associated intracellular calcium pools. Because calcium is accumulated in the endoplasmic reticulum by sarco/endoplasmic reticulum calcium ATPases (SERCA), these enzymes play a critical role in the control of calcium-dependent cell activation, growth and survival. We investigated the modulation of SERCA expression and function in human lung adenocarcinoma cells. In addition to the ubiquitous SERCA2 enzyme, the SERCA3 isoform was also expressed at variable levels. SERCA3 expression was selectively enhanced during cell differentiation in lung cancer cells, and marked SERCA3 expression was found in fully differentiated normal bronchial epithelium. As studied by using a recombinant fluorescent calcium probe, induction of the expression of SERCA3, a lower calcium affinity pump, was associated with decreased intracellular calcium storage, whereas the amplitude of capacitative calcium influx remained unchanged. Our observations indicate that the calcium homeostasis of the endoplasmic reticulum in lung adenocarcinoma cells presents a functional defect due to decreased SERCA3 expression that is corrected during pharmacologically induced differentiation. The data presented in this work show, for the first time, that endoplasmic reticulum calcium storage is anomalous in lung cancer cells, and suggest that SERCA3 may serve as a useful new phenotypic marker for the study of lung epithelial differentiation.
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http://dx.doi.org/10.1111/febs.12064DOI Listing
November 2013

Plasma membrane calcium pump (PMCA) isoform 4 is targeted to the apical membrane by the w-splice insert from PMCA2.

Cell Calcium 2012 Feb 16;51(2):171-8. Epub 2012 Jan 16.

Department of Molecular Cell Biology, National Blood Center, Diószegi u. 64, Budapest, Hungary.

Local Ca(2+) signaling requires proper targeting of the Ca(2+) signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca(2+) pump (PMCA) are responsible for Ca(2+) extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca(2+) signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.
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http://dx.doi.org/10.1016/j.ceca.2011.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279596PMC
February 2012

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells.

Biochem Biophys Res Commun 2011 Jul 6;410(2):322-7. Epub 2011 Jun 6.

Department of Molecular Cell Biology, National Blood Center, Budapest, Hungary.

The "w" splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.
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http://dx.doi.org/10.1016/j.bbrc.2011.05.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139432PMC
July 2011

Additional MDA-MB-231 breast cancer cell matrix metalloproteinases promote invasiveness.

J Cell Physiol 2008 Aug;216(2):480-5

Department of Pathology, Saint Louis University School of Medicine, St. Louis, Missouri 63104-1028, USA.

We are interested in two aspects of a given type of metastatic breast cancer: which potentially cancer-relevant genes are expressed and which factors determine invasiveness. Using reverse transcription real-time PCR, we detected gene expression of 26 matrix metalloproteinases (MMPs) in MDA-MB-231 breast cancer cells, including those of MMP-12, MMP-16 variant 2, MMP-19, MMP-20, MMP-21, MMP-23, MMP-24, MMP-25, MMP-25 variant 2, MMP-L1, MMP-26, MMP-27, and MMP-28, in contrast to the 13 MMPs detected until now in these cells. We found that MMP genes are expressed at widely different levels in these cells, over five orders of magnitude. After individual siRNA-induced depletions, we found that six additional species of cancer cell MMPs promote invasiveness in MDA-MB-231 cells: MMP-3, MMP-11, MMP-12, MMP-17, MMP-19, and MMP-23, thus raising the total to 12 endogenous MMPs which do so in these cells. The data support the conclusion that some cancer cell MMPs, although expressed at low levels, are needed for cancer trait in MDA-MB-231 cells, and that several endogenous MMPs play non-redundant roles in this process. The mRNA level of MMP-11, but not of other MMPs, rose substantially following individual siRNA-targeted depletion of cancer cell MMP-17 mRNA, while no MMP mRNA increased appreciably after degradation of other MMP mRNAs. This supports the conclusion that MMP-17 may be a member of an intracellular signaling pathway which downregulates MMP-11 mRNA.
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http://dx.doi.org/10.1002/jcp.21417DOI Listing
August 2008

Cleavage of the plasma membrane Ca+ATPase during apoptosis.

Ann N Y Acad Sci 2007 Mar;1099:440-50

Membrane Research Group of the Hungarian Academy of Sciences, Budapest H-1051, Hungary.

Maintenance of Ca2+ homeostasis is essential for normal cellular function and survival. Recent evidences suggest that Ca2+ is also an important player of apoptosis. We demonstrated that the plasma membrane Ca2+ ATPase (PMCA) isoform 4b, a key element of cellular Ca2+ homeostasis, was cleaved by caspase-3 during the course of apoptosis. This cleavage of PMCA removed the entire regulatory region from the C terminus, leaving behind a 120-kDa catalytic fragment. Since loss of PMCA activity could lead to intracellular Ca2+ overload and consequently necrotic cell death, an important question is whether the apoptotic fragment of PMCA retains full activity or it is inactivated. To address this question, we constructed a C-terminally truncated mutant that corresponded to the caspase-3 fragment of PMCA4b and showed that it was fully and constitutively active. This mutant was targeted properly to the plasma membrane when it was expressed stably or transiently in several different cell lines. We followed truncation of PMCA during apoptosis induced by mitochondrial or receptor-mediated pathways and found that a similar fragment of 120 kDa was formed and remained intact for several hours after treatment. We have also demonstrated that the caspase-3 cleavage site is an important structural element of PMCA and found that the accessibility of the caspase-3 site depended strongly on the conformational state of the protein.
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http://dx.doi.org/10.1196/annals.1387.003DOI Listing
March 2007