ATP2A3 Research Articles

Sarcoplasmic reticulum Ca2+ ATPases genes differential expression in breast cancer cells.

Sarcoplasmic reticulum Ca2+ ATPases (SERCA) enzymes are essential for intracellular Ca2+ homeostasis. SERCA genes (ATP2A1-3) encode for different functional isoforms of the protein, whose expression or function is altered in several types of cancer, such as gastric and oral, as well as colon, breast, lung, thyroid, liver, and prostate cancer, among others. However, the role played by SERCA pumps in carcinogenesis is unknown. Techniques such as real-time polymerase chain reaction, optical microscopy, proliferation and cell death assays, as well as bioinformatic analyses were used. To evaluate the expression levels of the ATP2A2 and ATP2A3 genes in cell lines representative of different subtypes of breast cancer. The results show that the MDA-MB-231 cell line expresses lower mRNA levels for the ATP2A3 gene in comparison with MCF-7 cells. The use of the phytoestrogen resveratrol induces ATP2A3 expression, decreases proliferation, and induces apoptosis in both cell types. SERCA expression might function as a tool to differentiate breast cancer subtypes, which have different treatment requirements.

Resveratrol up-regulates ATP2A3 gene expression in breast cancer cell lines through epigenetic mechanisms.

Resveratrol (RSV) is a phytoestrogen which has been related to chemoprevention of several types of cancer. In this work, we show up to a 6-fold increased expression of ATP2A3 gene induced by RSV that triggers apoptosis and changes of intracellular Ca2+ management in MCF-7 and MDA-MB-231 breast cancer cell lines. We explored epigenetic mechanisms for that RSV-induced ATP2A3 up-regulation. The results indicate that RSV-induced ATP2A3 up-regulation correlates with about 50% of reduced HDAC activity and reduced nuclear HDAC2 expression and occupancy on ATP2A3 promoter, increasing the global acetylation of histone H3 and the enrichment of histone mark H3K27Ac on the proximal promoter of the ATP2A3 gene in MDA-MB-231 cells. We also quantified HAT activity, finding that it can be boosted with RSV treatment; however, pharmacological inhibition of p300, one of the main HATs, did not have significant effects in RSV-mediated ATP2A3 gene expression. Additionally, DNMT activity was also reduced in cells treated with RSV, as well as the expression of Methyl-DNA binding proteins MeCP2 and MBD2. However, analysis of the methylation pattern of ATP2A3 gene promoter showed un-methylated promoter in both cell lines. Taken together, the results of this work help to explain, at the molecular level, how ATP2A3 gene is regulated in breast cancer cells, and the benefits of RSV intake observed in epidemiological data, studies with animals, and in vitro models.

Histone deacetylase inhibitors promote ATP2A3 gene expression in hepatocellular carcinoma cells: p300 as a transcriptional regulator.

Sarco(endo)plasmic reticulum Ca2+-ATPases (SERCA) expression is reduced or absent in several types of cancer and cancer cell lines; however, their expression and regulation in hepatocellular carcinoma (HCC) are unknown. Histone deacetylase inhibitors (HDACi) increase SERCA3 mRNA expression in gastric and breast cancer cell lines by increasing H3K9ac and binding of Sp1 and Sp3 transcription factors to the promoter; however, the molecular mechanism is not fully understood. Our results show that ATP2A3 (SERCA3) gene expression is decreased in human HCC samples and rat HCC AS-30D cells compared to normal liver, and HCC patients with high expression of ATP2A3 had longer overall survival than those with low expression. Sodium butyrate (NaB) and trichostatin A (TSA) increase SERCA3 mRNA expression in AS-30D cells, whereas SERCA2b mRNA expression did not change. NaB and TSA increase H3K9ac and H3K27ac in two ATP2A3 promoter regions. Besides, NaB treated cells increased Sp1 and Sp3 occupancy at ATP2A3 promoter; whereas TSA treated cells showed increased p300 levels at ATP2A3 promoter. Inhibition of p300 by C646, a specific inhibitor, mitigates SERCA3 mRNA induction by TSA, and reduces more than 70% of basal SERCA3 mRNA expression, suggesting that p300 is important for ATP2A3 gene transcription in AS-30D cells. Moreover, inhibition of p300 decreases H3K9ac in TSA treated cells. Our results provide evidence of decreased SERCA3 expression in human HCC samples and rat AS-30D cells and a correlation of SERCA3 expression with overall survival in HCC patients. Also, reveal new insights in SERCA3 transcriptional regulation mediated by HDACi.

Epigenetic regulation of the human ATP2A3 gene promoter in gastric and colon cancer cell lines.

The knowledge about the role of calcium-regulated pathways in cancer cell growth and differentiation could be useful for the development of new therapeutic approaches to diminish its mortality. The ATP2A genes encode for SERCA pumps, which modulate cytosolic Ca2+ concentration, regulating various cellular processes including cell growth. ATP2A3 gene transcriptional down-regulation has been reported in gastric and colon cancer, but there is still a lack of understanding about the epigenetic processes regulating its transcription. In this work, we report that butyrate, trichostatin A, and 5-azacytidine treatments increase SERCA3 expression, increased apoptosis, and decreased cell viability of the KATO-III gastric carcinoma cell line. We analyzed the methylation profile of the ATP2A3 gene promoter CpG island, finding clones with methylated status through -280 to -135 promoter region, harboring Sp1 and AP-2 binding sites, which could have a role in transcriptional repression. Post-translational modifications of histones show a major role in the ATP2A3 transcriptional regulation, and our results show histones marks linked to transcriptional repression associated with the -262 to -135 region, this repressive context changed to transcriptional permissive through SERCA3 re-expressing conditions. These results suggest that the nucleotide sequence from -280 to -135 position is an ATP2A3 epigenetic regulatory CpG region in KATO-III cells. Analyses of online-databases show a decreased SERCA3 expression in gastric and colon tumors, as well as overall survival results, showed that high SERCA3 expression could serve as a favorable prognostic marker for colon and gastric cancer patients.

ATP2A3 gene as an important player for resveratrol anticancer activity in breast cancer cells.

The Ca2+ -ATPases from the Sarco/endoplasmic reticulum (SERCA) are fundamental for maintaining intracellular [Ca2+ ] homeostasis by pumping Ca2+ into the endoplasmic reticulum (ER) of eukaryotic cells. SERCA enzymes are encoded by three different genes (ATP2A1-3), whose expression occurs in a tissue and development stage-specific manner. It has been reported alterations in the expression of SERCA2 and SERCA3 pumps in different types of cancer: oral, lung, colon, stomach, central nervous system, thyroid, breast, and prostate. Resveratrol (RSV), a phytoalexin produced by a wide variety of plants in response to stress situations can modulate cellular processes involved in all stages of carcinogenesis. In this work, we used breast cancer cell lines (MCF-7 and MDA-MB-231) to evaluate mRNA levels of ATP2A2 and ATP2A3 genes in response to RSV treatment. Our results demonstrate that RSV treatment induced the expression of ATP2A3 gene in both cell lines in a time and concentration-dependent manner, while the expression of ATP2A2 gene remained unaffected. The RSV-induced expression of SERCA3 in these breast cancer cell lines produced decreased cell viability, triggered apoptosis and changes in cytosolic Ca2+ levels, as well as changes in the capacity for Ca2+ release by the ER. These data suggest an important participation of SERCA3 genes in RSV-mediated anti-tumor effect in breast cancer cell lines. Nevertheless, further research is needed to elucidate the molecular mechanisms underlying this effect.

Insulin and Glucose Alter Death-Associated Protein Kinase 3 (DAPK3) DNA Methylation in Human Skeletal Muscle.

DNA methylation is altered by environmental factors. We hypothesized that DNA methylation is altered in skeletal muscle in response to either insulin or glucose exposure. We performed a genome-wide DNA methylation analysis in muscle from healthy men before and after insulin exposure. DNA methylation of selected genes was determined in muscle from healthy men and men with type 2 diabetes before and after a glucose tolerance test. Insulin altered DNA methylation in the 3' untranslated region of the calcium pump ATP2A3 gene. Insulin increased DNA methylation in the gene body of DAPK3, a gene involved in cell proliferation, apoptosis, and autophagy. DAPK3 methylation was reduced in patients with type 2 diabetes. Carbohydrate ingestion reduced DAPK3 DNA methylation in healthy men and men with type 2 diabetes, suggesting glucose may play a role. Supporting this, DAPK3 DNA methylation was inversely correlated with the 2-h glucose concentration. Whereas glucose incorporation to glycogen was unaltered by small interfering RNA against DAPK3, palmitate oxidation was increased. In conclusion, insulin and glucose exposure acutely alter the DNA methylation profile of skeletal muscle, indicating that DNA methylation constitutes a rapidly adaptive epigenetic mark. Furthermore, insulin and glucose modulate DAPK3 DNA methylation in a reciprocal manner, suggesting a feedback loop in the control of the epigenome.

Histone deacetylase inhibitors promote the expression of ATP2A3 gene in breast cancer cell lines.

Recent studies have shown that expression of Sarco(endo)plasmic Reticulum Ca(2+) -ATPase 2 (SERCA2) is decreased in oral cancer; whereas expression of SERCA3 is considerably decreased or absent in human colon, gastric, breast, and lung cancers. The ATP2A2 and ATP2A3 genes encode SERCA2 and SERCA3 isoforms, respectively. Promoter methylation on CpG islands was responsible for the repression of ATP2A2 gene in human oral cancer samples. On the other hand, histone deacetylase inhibitors (HDACi) up-regulate ATP2A3 expression in gastric, colon, and lung cancer cells in culture, however, the molecular mechanism is unknown. In this study, we investigate whether HDACi and DNA methylation regulate ATP2A2 and ATP2A3 expression in human breast cancer cell lines. Results show a marked induction of SERCA3a and pan-SERCA3 mRNA expression in human MCF-7 and MDA-MB-231 cells treated with sodium butyrate (NaB) or trichostatin A (TSA); whereas SERCA2b mRNA expression did not change significantly. ChIP assays show that NaB or TSA treatment of MDA-MB-231 cells increases H3K9 acetylation on ATP2A3 promoter. NaB also decreases H3K9 trimethylation; suggesting that these modifications stimulate ATP2A3 gene expression, through a chromatin remodeling mechanism. In contrast, NaB or TSA do not increase H3K9-acetylation of ATP2A2 proximal promoter. In addition, treatment with 5-aza-2'-deoxycytidine did not affect SERCA2b and SERCA3a expression, suggesting that promoter methylation status does not alter their expression in these cell lines. We propose that alteration of SERCA2b/SERCA3a isoform expression ratio could affect calcium management within the cell, and thus, the cellular pathways regulated by calcium could be compromised, such as cellular proliferation or apoptosis. © 2015 Wiley Periodicals, Inc.

KLF4 transcription factor positively regulates the transcription of ATP2A3 gene during the differentiation of human colon and gastric cancer cell lines

The sarco(endo)plasmic reticulum Ca2+‐ATPase, ATP2A3 gene, is highly expressed in gastrointestinal cells, but is selectively lost in cancer. Previously, was reported that SERCA3 protein is induced when human gastric and colon cancer cells undergo in vitro differentiation. In this work, we induced differentiation of gastric and colon cancer cells in culture, finding increased SERCA3 mRNA expression and increased transcriptional activity of hATP2A3‐gene‐promoter constructs (up to 50‐fold) after induced differentiation, narrowing the responsive elements within ‐ 135 to +142 bp 5’‐region of the human ATP2A3 promoter containing 8 consensus Sp‐factors binding sites, which previously have shown an important role in the transcriptional regulation of the gene. KLF4 transcription factor is decreased in gastric and colon cancer in a similar pattern to that of SERCA3, and both increase after induced differentiation. In EMSA assays, we found that KLF4 binding increases in differentiated cancer cell lines, suggesting that might function as an activator of ATP2A3 expression. In contrast, the results suggest that Sp3 functions as a repressor in tumor cells keeping low SERCA3 expression. By ChIP assays we found that KLF4 binds to the proximal ATP2A3 promoter as part of a co‐activator complex with the histone‐acetyltransferase p300, regulating positively the transcription of ATP2A3. Supported by grant PAPIIT IN213613.

ATP2A3 gene is involved in cancer susceptibility

The sarco/endoplasmatic reticulum calcium-ATPase (SERCA) translocates Ca 2+ from cytosol to the lumen of the ER and thus regulates Ca 2+ homeostasis, perturbations of which have been suggested to contribute to cancer. We have previously detected an increased number of alterations in the ATP2A2 gene in various cancer types and in the ATP2A3 gene in head and neck squamous cell carcinoma. Here, we further analyzed the ATP2A3 gene in colon, lung, and CNS cancers. We identified a statistically significant increase of alterations in each (colon cancer, p=0.0052, lung cancer, p=0.0026, CNS tumors, p=0.0045) cancer type, and all 3 types together ( p=0.0016). Epigenetic study of the ATP2A3 gene indicated an unchanged methylation status, whereas expression of the ATP2A3 gene was normal for exon 14 mutations and reduced in connection with a nucleotide change in intron VI in all studied cancer types. Identification of a significant number of alterations in cancer patients suggests that ATP2A3 is involved in increased cancer susceptibility in humans. The mostly normal expression and methylation status of the ATP2A3 gene, as well as the absence of somatic alterations, further suggest that the ATP2A3 gene may not act as a classical tumor suppressor gene, but rather haplo-insufficiency of this gene may be enough to change the cell and tissue environment in such a way to predispose to cancer development.