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Background Remodeling of Ca2+ signaling is an important step in cancer progression, and altered expression of members of the Ca2+ signaling toolkit including the plasma membrane Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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. Electronic supplementary material The online version of this article (10.1186/s12885-018-4945-x) contains supplementary material, which is available to authorized users.

Plasma membrane Ca2+ transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca2+ from the cell in order to keep the cytosolic Ca2+ ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca2+ homeostasis they also change the duration and amplitude of the Ca2+ signal and can create Ca2+ 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.

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca2+ 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 Ca2+ 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 [Ca2+ ]i clearance from cells after Ca2+ 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 Ca2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor.

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.

Several new therapeutic options emerged recently to treat metastatic melanoma, however, the high frequency of intrinsic and acquired resistance among patients show a need for new therapeutic options. Previously, we identified the plasma membrane Ca2+ 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 HDAC inhibitors up-regulated 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 histone deacetylase (HDAC) inhibitors SAHA and valproic acid either alone, or in combination with the BRAF inhibitor vemurafenib. We found that HDAC inhibitor 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 more intensely in the BRAF mutant cells. Combination of the HDAC inhibitors with vemurafenib, however, did not have any additive effects on either PMCA isoform. We demonstrated that the HDAC inhibitor-induced increase in PMCA abundance was coupled to an enhanced [Ca2+]i 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 Ca2+ 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 elevation of PMCA4b expression either by HDAC inhibitor treatment or by the decreased activation of the BRAF-MEK-ERK pathway can inhibit the migratory capacity of the highly motile A375 cells.

Preimplantation genetic testing for aneuploidy (PGT-A) is a suitable technique to identify euploid embryos, which have the highest potential to implant, thus increase the chance of a healthy live birth. The main indications of PGT-A are advanced maternal age, repeated implantation failure, repeated miscarriages and severe male infertility. Several studies have already proven that testing embryos for genetic abnormalities in the above cases results in higher implantation rate and reduced number of pregnancy loss. In spite of these - due to a legislative change in Hungary in 2015 - PGT-A was reclassified as an experimental procedure and its use became banned throughout the country. For this reason, after 4 years of successful practice, Hungarian patients were not able to participate in IVF procedure combined with PGT-A anymore. In this retrospective analysis, efficacy of PGT-A-based embryo selection was evaluated and was compared to the conventional morphology-based selection (MBS) in patients with advanced maternal age, between 2013 and 2017 at our private fertility clinic. PGT-A was performed with array comparative genomic hybridization. We found that implantation rate was significantly higher (43.62% vs. 27.88%; p = 0.0208) and miscarriage rate was significantly lower (17.07% vs. 37.93%; p = 0.0492) in the PGT-A group compared to the MBS group from 2013 to 2015. These outcomes were achieved with a significantly lower number of transferred embryos in the PGT-A group (1.25 vs. 1.58; p = 0.0003). In 2016-2017, the number of transferred embryos were significantly reduced in the MBS group as well (1.14 vs. 1.58; p < 0.0001). However, outcomes of the IVF treatments did not change significantly compared to the previous two years (2013-2015). Our results imply that PGT-A-based embryo selection is more efficient than morphology-based selection in patients with advanced maternal age. Therefore, prohibition of the use of PGT-A had significant consequences on the efficiency and safety of IVF treatment in the country.

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.

The aim of our study was to test the effects of histone deacetylase (HDAC) and BRAF inhibitors on the expression and activity of the plasma membrane Ca2+ pump isoform 4b (PMCA4b) in BRAF mutant melanoma cell lines. Remodeling of Ca2+ homeostasis during malignancy is caused by the rearrangement of the Ca2+ signaling machinery, including Ca2+ pumps, Na+/Ca2+ exchangers and channels. Plasma membrane calcium ATPases (ATP2B - or PMCA) maintain the resting low intracellular calcium concentration by pumping out excess calcium from the cytosol. Changes in PMCA expression during malignant transformation have been described previously in colorectal and breast cancer cells, and most recently by our group in melanomas. We found that in BRAF mutant melanoma cells the PMCA 4b protein level was markedly elevated by BRAF inhibitor treatment. Overexpression of PMCA4b suppressed motility and metastatic potential. Previously, it was shown that HDAC inhibitors-induced differentiation up-regulated PMCA4b expression in gastric, colon and breast cancer cells. In the present study we treated BRAF mutant and BRAF wild type melanoma cells with HDAC inhibitors (SAHA (suberanilohydroxamic acid) or valproic acid) and tested the changes in abundance, localization and function of PMCA4b. Expression levels of the PMCA proteins were analyzed by qRT-PCR and Western Blotting. Subcellular localization of the PMCAs and the effects of treatments on cytosolic Ca2+ signaling were analyzed by confocal microscopy. We found that treatment with the HDAC inhibitors increased the level of PMCA4b expression at both mRNA and protein levels in both BRAF wild type and BRAF mutant cell types. The increased PMCA4b level was coupled with enhanced plasma membrane localization and with a faster Ca2+ clearance after stimulation. Our results show that in melanoma cells the expression of PMCA4b is under epigenetic control and HDAC inhibitors efficiently enhanced PMCA4b expression independent of the BRAF status of cells. Citation Format: Luca Hegedus, Tamas Garay, Eszter Molnar, Karolina Varga, Rita Padanyi, Katalin Paszty, Balazs Hegedus, Mathias Wolf, Michael Grusch, Eniko Kallay, Agnes Enyedi. Treatment with histone deacetylase (HDAC) and BRAF inhibitors upregulates the expression of the plasma membrane Ca2+ pump, PMCA4b and alters intracellular Ca2+ handling in BRAF mutant melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1883.