Abstract
Breast cancer-associated deaths are related mainly to specific molecular subtypes and the presence of metastasis. The Epithelial-to-Mesenchymal Transition (EMT) and Ca2+ signaling pathways are involved in breast cancer metastasis, and they are regulated in part by epigenetic mechanisms. Moreover, activation of EMT modulates Ca2+ concentration and in turn, Ca2+ signaling regulates the expression of EMT markers. Also, activation of Ca2+ signaling genes with epigenetic inhibitors reverts the EMT. Thus, Ca2+ signaling might have an important role in breast cancer metastasis and EMT, particularly through the epigenetic regulation of genes involved in its signaling. However, little is known due to that an estimate of 1670 genes participate in the Ca2+ signaling and only a few genes have been studied. Here, we aimed to explore the expression of all genes involved in Ca2+ signaling in all breast cancer subtypes and EMT, and whether modulation of epigenetic mechanisms is related to their expression. Several genes of the Ca2+ signaling are altered in all breast cancer subtypes, being the cadherins and voltage channels the most frequent altered genes. Also, DNA methylation and histone posttranslational modifications showed a good correlation with their altered expression. The expression of the cadherins and voltage channels is also modulated during breast EMT, and ATAC-seq results suggest that chromatin rearrangement at their promoter is involved. In conclusion, the expression of the genes involved in Ca2+ signaling is altered in all breast cancer subtypes and during EMT, and epigenetic mechanisms are an attractive target to regulate their expression.
This is a preview of subscription content, log in via an institution to check access.
References
Adhikary S, Chakravarti D, Terranova C, Sengupta I, Maitituoheti M, Dasgupta A, Srivastava DK, Ma J, Raman AT, Tarco E, Sahin AA, Bassett R, Yang F, Tapia C, Roy S, Rai K, Das C (2019) Atypical plant homeodomain of UBR7 functions as an H2BK120Ub ligase and breast tumor suppressor. Nat Commun 10(1):1398. https://doi.org/10.1038/s41467-019-08986-5
Adiga D, Radhakrishnan R, Chakrabarty S, Kumar P, Kabekkodu SP (2020) The role of calcium signaling in regulation of epithelial-mesenchymal transition. Cells Tissues Organs. https://doi.org/10.1159/000512277
Agiostratidou G, Li M, Suyama K, Badano I, Keren R, Chung S, Anzovino A, Hulit J, Qian B, Bouzahzah B, Eugenin E, Loudig O, Phillips GR, Locker J, Hazan RB (2009) Loss of retinal cadherin facilitates mammary tumor progression and metastasis. Can Res 69(12):5030–5038. https://doi.org/10.1158/0008-5472.CAN-08-4007
Bhargava A, Saha S (2019) T-type voltage gated calcium channels: a target in breast cancer? Breast Cancer Res Treat 173(1):11–21. https://doi.org/10.1007/s10549-018-4970-0
Bolanz KA, Hediger MA, Landowski CP (2008) The role of TRPV6 in breast carcinogenesis. Mol Cancer Ther 7(2):271–279. https://doi.org/10.1158/1535-7163.MCT-07-0478
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68(6):394–424. https://doi.org/10.3322/caac.21492
Cardoso F, Spence D, Mertz S, Corneliussen-James D, Sabelko K, Gralow J, Cardoso M-J, Peccatori F, Paonessa D, Benares A, Sakurai N, Beishon M, Barker S-J, Mayer M (2018) Global analysis of advanced/metastatic breast cancer: decade report (2005–2015). Breast 39:131–138. https://doi.org/10.1016/j.breast.2018.03.002
Carlsson H, Petersson S, Enerbäck C (2005) Cluster analysis of S100 gene expression and genes correlating to psoriasin (S100A7) expression at different stages of breast cancer development. Int J Oncol 27(6):1473–1481. https://doi.org/10.3892/ijo.27.6.1473
Chang P-H, Chen M-C, Tsai Y-P, Tan GYT, Hsu P-H, Jeng Y-M, Tsai Y-F, Yang M-H, Hwang-Verslues WW (2021) Interplay between desmoglein2 and hypoxia controls metastasis in breast cancer. Proc Natl Acad Sci 118(3):e2014408118. https://doi.org/10.1073/pnas.2014408118
Chikarmane SA, Tirumani SH, Howard SA, Jagannathan JP, DiPiro PJ (2015) Metastatic patterns of breast cancer subtypes: what radiologists should know in the era of personalized cancer medicine. Clin Radiol 70(1):1–10. https://doi.org/10.1016/j.crad.2014.08.015
Connolly RM, Li H, Jankowitz RC, Zhang Z, Rudek MA, Jeter SC, Slater SA, Powers P, Wolff AC, Fetting JH, Brufsky A, Piekarz R, Ahuja N, Laird PW, Shen H, Weisenberger DJ, Cope L, Herman JG, Somlo G, Stearns V (2017) Combination epigenetic therapy in advanced breast cancer with 5-azacitidine and entinostat: a phase II national cancer institute/stand up to cancer study. Clin Cancer Res 23(11):2691–2701. https://doi.org/10.1158/1078-0432.CCR-16-1729
Cope LM, Fackler MJ, Lopez-Bujanda Z, Wolff AC, Visvanathan K, Gray JW, Sukumar S, Umbricht CB (2014) Do breast cancer cell lines provide a relevant model of the patient tumor methylome? PLoS One 9(8):e105545. https://doi.org/10.1371/journal.pone.0105545
da Costa Prando É, Cavalli LR, Rainho C (2011) Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines. Epigenetics 6(12):1413–1424. https://doi.org/10.4161/epi.6.12.18271
Dang DK, Makena MR, Llongueras JP, Prasad H, Ko M, Bandral M, Rao R (2019) A Ca2þ-ATPase regulates E-cadherin biogenesis and epithelial–mesenchymal transition in breast cancer cells. Mol Cancer Res 17(8):1735–1747. https://doi.org/10.1158/1541-7786.MCR-19-0070
Davis FM, Azimi I, Faville RA, Peters AA, Jalink K, Putney JW, Goodhill GJ, Thompson EW, Roberts-Thomson SJ, Monteith GR (2014) Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent. Oncogene 33(18):2307–2316. https://doi.org/10.1038/onc.2013.187
Dongre A, Weinberg RA (2019) New insights into the mechanisms of epithelial–mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol 20(2):69–84. https://doi.org/10.1038/s41580-018-0080-4
Faouzi M, Hague F, Potier M, Ahidouch A, Sevestre H, Ouadid-Ahidouch H (2011) Down-regulation of Orai3 arrests cell-cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells. J Cell Physiol 226(2):542–551. https://doi.org/10.1002/jcp.22363
Fares J, Fares MY, Khachfe HH, Salhab HA, Fares Y (2020) Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduct Target Ther. https://doi.org/10.1038/s41392-020-0134-x
Felipe Lima J, Nofech-Mozes S, Bayani J, Bartlett J (2016) EMT in breast carcinoma—a review. J Clin Med 5(7):65. https://doi.org/10.3390/jcm5070065
Fiegl H, Millinger S, Goebel G, Müller-Holzner E, Marth C, Laird PW, Widschwendter M (2006) Breast cancer DNA methylation profiles in cancer cells and tumor stroma: association with HER-2/neu status in primary breast cancer. Can Res 66(1):29–33. https://doi.org/10.1158/0008-5472.CAN-05-2508
Franco HL, Nagari A, Malladi VS, Li W, Xi Y, Richardson D, Allton KL, Tanaka K, Li J, Murakami S, Keyomarsi K, Bedford MT, Shi X, Li W, Barton MC, Dent SYR, Kraus WL (2018) Enhancer transcription reveals subtype-specific gene expression programs controlling breast cancer pathogenesis. Genome Res 28(2):159–170. https://doi.org/10.1101/gr.226019.117
Hao Y, Hao S, Andersen-Nissen E, Mauck WM, Zheng S, Butler A, Lee MJ, Wilk AJ, Darby C, Zager M, Hoffman P, Stoeckius M, Papalexi E, Mimitou EP, Jain J, Srivastava A, Stuart T, Fleming LM, Yeung B, Satija R (2021) Integrated analysis of multimodal single-cell data. Cell 184(13):3573-3587.e29. https://doi.org/10.1016/j.cell.2021.04.048
Harbeck N, Penault-Llorca F, Cortes J, Gnant M, Houssami N, Poortmans P, Ruddy K, Tsang J, Cardoso F (2019) Breast cancer. Nat Rev Dis Primers. https://doi.org/10.1038/s41572-019-0111-2
Heldin C-H, Vanlandewijck M, Moustakas A (2012) Regulation of EMT by TGFβ in cancer. FEBS Lett 586(14):1959–1970. https://doi.org/10.1016/j.febslet.2012.02.037
Hernández-Oliveras A, Izquierdo-Torres E, Hernández-Martínez G, Zarain-Herzberg Á, Santiago-García J (2021) Transcriptional and epigenetic landscape of Ca2+-signaling genes in hepatocellular carcinoma. J Cell Commun Signal 15(3):433–445. https://doi.org/10.1007/s12079-020-00597-w
Hörtenhuber M, Toledo EM, Smedler E, Arenas E, Malmersjö S, Louhivuori L, Uhlén P (2017) Mapping genes for calcium signaling and their associated human genetic disorders. Bioinformatics (Oxford, England) 33(16):2547–2554. https://doi.org/10.1093/bioinformatics/btx225
Iamshanova O, Fiorio Pla A, Prevarskaya N (2017) Molecular mechanisms of tumour invasion: regulation by calcium signals. J Physiol 595(10):3063–3075. https://doi.org/10.1113/JP272844
Izquierdo-Torres E, Hernández-Oliveras A, Meneses-Morales I, Rodríguez G, Fuentes-García G, Zarain-Herzberg Á (2019) Resveratrol up-regulates ATP2A3 gene expression in breast cancer cell lines through epigenetic mechanisms. Int J Biochem Cell Biol. https://doi.org/10.1016/j.biocel.2019.05.020
Izquierdo-Torres E, Hernández-Oliveras A, Fuentes-García G, Zarain-Herzberg Á (2020) Calcium signaling and epigenetics: a key point to understand carcinogenesis. Cell Calcium 91:102285. https://doi.org/10.1016/j.ceca.2020.102285
Kanwar N, Carmine-Simmen K, Nair R, Wang C, Moghadas-Jafari S, Blaser H, Tran-Thanh D, Wang D, Wang P, Wang J, Pasculescu A, Datti A, Mak T, Lewis JD, Done SJ (2020) Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis. EBioMedicine 52:102646. https://doi.org/10.1016/j.ebiom.2020.102646
Kaszak I, Witkowska-Piłaszewicz O, Niewiadomska Z, Dworecka-Kaszak B, Ngosa Toka F, Jurka P (2020) Role of cadherins in cancer—a review. Int J Mol Sci 21(20):7624. https://doi.org/10.3390/ijms21207624
Khaled N, Bidet Y (2019) New insights into the implication of epigenetic alterations in the EMT of triple negative breast cancer. Cancers 11(4):1–21. https://doi.org/10.3390/cancers11040559
Koboldt DC, Fulton RS, McLellan MD, Schmidt H, Kalicki-Veizer J, McMichael JF, Fulton LL, Dooling DJ, Ding L, Mardis ER, Wilson RK, Ally A, Balasundaram M, Butterfield YSN, Carlsen R, Carter C, Chu A, Chuah E, Chun HJE, Palchik JD (2012) Comprehensive molecular portraits of human breast tumours. Nature 490(7418):61–70. https://doi.org/10.1038/nature11412
Kwaepila N, Burns G, Leong AS-Y (2006) Immunohistological localisation of human FAT1 (hFAT) protein in 326 breast cancers. Does this adhesion molecule have a role in pathogenesis? Pathology 38(2):125–131. https://doi.org/10.1080/00313020600559975
Li D, Hsu S, Purushotham D, Sears RL, Wang T (2019) WashU epigenome browser update 2019. Nucleic Acids Res 47(W1):W158–W165. https://doi.org/10.1093/nar/gkz348
Luu T, Kim K, Blanchard S, Anyang B, Hurria A, Yang L, Beumer JH, Somlo G, Yen Y (2018) Phase IB trial of ixabepilone and vorinostat in metastatic breast cancer. Breast Cancer Res Treat 167(2):469–478. https://doi.org/10.1007/s10549-017-4516-x
Makena MR, Ko M, Dang DK, Rao R (2021) Epigenetic modulation of spca2 reverses epithelial to mesenchymal transition in breast cancer cells. Cancers 13(2):1–18. https://doi.org/10.3390/cancers13020259
McAndrew D, Grice DM, Peters AA, Davis FM, Stewart T, Rice M, Smart CE, Brown MA, Kenny PA, Roberts-Thomson SJ, Monteith GR (2011) ORAI1-mediated calcium influx in lactation and in breast cancer. Mol Cancer Ther 10(3):448–460. https://doi.org/10.1158/1535-7163.MCT-10-0923
McFaline-Figueroa JL, Hill AJ, Qiu X, Jackson D, Shendure J, Trapnell C (2019) A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transition. Nat Genet 51(9):1389–1398. https://doi.org/10.1038/s41588-019-0489-5
Mi H, Muruganujan A, Thomas PD (2012) PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res 41(D1):D377–D386. https://doi.org/10.1093/nar/gks1118
Novak P, Jensen T, Oshiro MM, Watts GS, Kim CJ, Futscher BW (2008) Agglomerative epigenetic aberrations are a common event in human breast cancer. Can Res 68(20):8616–8625. https://doi.org/10.1158/0008-5472.CAN-08-1419
Oshiro MM, Watts GS, Wozniak RJ, Junk DJ, Munoz-Rodriguez JL, Domann FE, Futscher BW (2003) Mutant p53 and aberrant cytosine methylation cooperate to silence gene expression. Oncogene 22(23):3624–3634. https://doi.org/10.1038/sj.onc.1206545
Oshiro MM, Kim CJ, Wozniak RJ, Junk DJ, Muñoz-Rodríguez JL, Burr JA, Fitzgerald M, Pawar SC, Cress AE, Domann FE, Futscher BW (2005) Epigenetic silencing of DSC3 is a common event in human breast cancer. Breast Cancer Res 7(5):R669. https://doi.org/10.1186/bcr1273
Pastushenko I, Mauri F, Song Y, de Cock F, Meeusen B, Swedlund B, Impens F, Van Haver D, Opitz M, Thery M, Bareche Y, Lapouge G, Vermeersch M, Van Eycke Y-R, Balsat C, Decaestecker C, Sokolow Y, Hassid S, Perez-Bustillo A, Blanpain C (2021) Fat1 deletion promotes hybrid EMT state, tumour stemness and metastasis. Nature 589(7842):448–455. https://doi.org/10.1038/s41586-020-03046-1
Pera E, Kaemmerer E, Milevskiy MJG, Yapa KTDS, O’Donnell JS, Brown MA, Simpson F, Peters AA, Roberts-Thomson SJ, Monteith GR (2016) The voltage gated Ca2+-channel Cav3.2 and therapeutic responses in breast cancer. Cancer Cell Int 16(1):24. https://doi.org/10.1186/s12935-016-0299-0
Qi C, Zhu YT, Hu L, Zhu Y-J (2009) Identification of Fat4 as a candidate tumor suppressor gene in breast cancers. Int J Cancer 124(4):793–798. https://doi.org/10.1002/ijc.23775
Redig AJ, Mcallister SS (2013) Breast cancer as a systemic disease: a view of metastasis. J Intern Med 274(2):113–126. https://doi.org/10.1111/joim.12084
Ribatti D, Tamma R, Annese T (2020) Epithelial-mesenchymal transition in cancer: a historical overview. Translational Oncol 13(6):100773. https://doi.org/10.1016/j.tranon.2020.100773
Schröder R, Illert A-L, Erbes T, Flotho C, Lübbert M, Duque-Afonso J (2021) The epigenetics of breast cancer–opportunities for diagnostics, risk stratification and therapy. Epigenetics. https://doi.org/10.1080/15592294.2021.1940644
So CL, Saunus JM, Roberts-Thomson SJ, Monteith GR (2019) Calcium signalling and breast cancer. Semin Cell Dev Biol 94:74–83. https://doi.org/10.1016/j.semcdb.2018.11.001
Su Y, Hopfinger NR, Nguyen TD, Pogash TJ, Santucci-Pereira J, Russo J (2018) Epigenetic reprogramming of epithelial mesenchymal transition in triple negative breast cancer cells with DNA methyltransferase and histone deacetylase inhibitors. J Exp Clin Cancer Res 37(1):1–18. https://doi.org/10.1186/s13046-018-0988-8
Sun S, Lee YR, Enfield B (2019) Hemimethylation patterns in breast cancer cell lines. Cancer Inform 18:117693511987295. https://doi.org/10.1177/1176935119872959
Tu Y, Hershman DL, Bhalla K, Fiskus W, Pellegrino CM, Andreopoulou E, Makower D, Kalinsky K, Fehn K, Fineberg S, Negassa A, Montgomery LL, Wiechmann LS, Alpaugh RK, Huang M, Sparano JA (2014) A phase I-II study of the histone deacetylase inhibitor vorinostat plus sequential weekly paclitaxel and doxorubicin-cyclophosphamide in locally advanced breast cancer. Breast Cancer Res Treat 146(1):145–152. https://doi.org/10.1007/s10549-014-3008-5
Vagia E, Mahalingam D, Cristofanilli M (2020) The Landscape of targeted therapies in TNBC. Cancers 12(4):916. https://doi.org/10.3390/cancers12040916
Varga K, Hollósi A, Pászty K, Hegedűs L, Szakács G, Tímár J, Papp B, Enyedi Á, Padányi R (2018) Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells. BMC Cancer 18(1):1029. https://doi.org/10.1186/s12885-018-4945-x
Vega-Benedetti AF, Loi E, Moi L, Blois S, Fadda A, Antonelli M, Arcella A, Badiali M, Giangaspero F, Morra I, Columbano A, Restivo A, Zorcolo L, Gismondi V, Varesco L, Bellomo SE, Giordano S, Canale M, Casadei-Gardini A, Zavattari P (2019) Clustered protocadherins methylation alterations in cancer. Clin Epigenetics 11(1):100. https://doi.org/10.1186/s13148-019-0695-0
Vodnala SK, Eil R, Kishton RJ, Sukumar M, Yamamoto TN, Ha NH, Lee PH, Shin MH, Patel SJ, Yu Z, Palmer DC, Kruhlak MJ, Liu X, Locasale JW, Huang J, Roychoudhuri R, Finkel T, Klebanoff CA, Restifo NP (2019) T cell stemness and dysfunction in tumors are triggered by a common mechanism. Science. https://doi.org/10.1126/science.aau0135
Waks AG, Winer EP (2019) Breast cancer treatment: a review. JAMA J Am Med Assoc 321(3):288–300. https://doi.org/10.1001/jama.2018.19323
Wang C-Y, Lai M-D, Phan NN, Sun Z, Lin Y-C (2015) Meta-analysis of public microarray datasets reveals voltage-gated calcium gene signatures in clinical cancer patients. PLoS One 10(7):e0125766. https://doi.org/10.1371/journal.pone.0125766
Weng J, Xiao J, Mi Y, Fang X, Sun Y, Li S, Qin Z, Li X, Liu T, Zhao S, Zhou L, Wen Y (2018) PCDHGA9 acts as a tumor suppressor to induce tumor cell apoptosis and autophagy and inhibit the EMT process in human gastric cancer. Cell Death Dis 9(2):27. https://doi.org/10.1038/s41419-017-0189-y
Wozniak RJ, Klimecki WT, Lau SS, Feinstein Y, Futscher BW (2007) 5-Aza-2′-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation. Oncogene 26(1):77–90. https://doi.org/10.1038/sj.onc.1209763
Wu Y, Sarkissyan M, Vadgama J (2016) Epithelial-mesenchymal transition and breast cancer. J Clin Med 5(2):13. https://doi.org/10.3390/jcm5020013
Xiong G, Chen J, Zhang G, Wang S, Kawasaki K, Zhu J, Zhang Y, Nagata K, Li Z, Zhou BP, Xu R (2020) Hsp47 promotes cancer metastasis by enhancing collagen-dependent cancer cell-platelet interaction. Proc Natl Acad Sci 117(7):3748–3758. https://doi.org/10.1073/pnas.1911951117
Xu D, Yuan H, Meng Z, Yang C, Li Z, Li M, Zhang Z, Gan Y, Tu H (2020) Cadherin 13 inhibits pancreatic cancer progression and epithelial-mesenchymal transition by Wnt/β-catenin signaling. J Cancer 11(8):2101–2112. https://doi.org/10.7150/jca.37762
Xu M, Liu C, Pu L, Lai J, Li J, Ning Q, Liu X, Deng S (2021) Systemic analysis of the expression levels and prognosis of breast cancer-related cadherins. Exp Biol Med 246(15):1706–1720. https://doi.org/10.1177/15353702211010417
Yang J, Niu H, Huang Y, Yang K (2016) A systematic analysis of the relationship of CDH13 promoter methylation and breast cancer risk and prognosis. PLoS One 11(5):e0149185. https://doi.org/10.1371/journal.pone.0149185
Zenkova D, Kamenev V, Sablina R, Artyomov M, Sergushichev A (2018) Phantasus: visual and interactive gene expression analysis. https://doi.org/10.18129/B9.bioc.phantasus, https://genome.ifmo.ru/phantasus
Zhang Q, Liu J, Ao N, Yu H, Peng Y, Ou L, Zhang S (2020) Secondary cancer risk after radiation therapy for breast cancer with different radiotherapy techniques. Sci Rep 10(1):1–12. https://doi.org/10.1038/s41598-020-58134-z
Zhang N, Li L, Long Z, Du J, Li S, Yin H, Xie K, Wu Z, Chen Y, Volontovich D, Cheng H, Wang F (2021) Are dietary factors involved in the association of CDH4 methylation and breast cancer risk? Br J Nutr. https://doi.org/10.1017/S0007114521002804
Zhu P, Lv J, Yang Z, Guo L, Zhang L, Li M, Han W, Chen X, Zhuang H, Lu F (2014) Protocadherin 9 inhibits epithelial–mesenchymal transition and cell migration through activating GSK-3β in hepatocellular carcinoma. Biochem Biophys Res Commun 452(3):567–574. https://doi.org/10.1016/j.bbrc.2014.08.101
Zolota V, Tzelepi V, Piperigkou Z, Kourea H, Papakonstantinou E, Argentou M-I, Karamanos NK (2021) Epigenetic alterations in triple-negative breast cancer—the critical role of extracellular matrix. Cancers 13(4):713. https://doi.org/10.3390/cancers13040713
Acknowledgements
This work was supported by grants from Dirección General de Asuntos del Personal Académico-PAPIIT-UNAM IN219219 and IN208422 to A. Z-H. A DGAPA-UNAM postdoctoral fellowship supported A. H-O. We thank Dr. Eduardo Izquierdo-Torres for his comments to improve this manuscript.
Funding
All authors declare no financial or non-financial interests that are directly or indirectly related to the work submitted for publication.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Hernández-Oliveras, A., Zarain-Herzberg, Á. Expression and associated epigenetic mechanisms of the Ca2+-signaling genes in breast cancer subtypes and epithelial-to-mesenchymal transition. J. Cell Commun. Signal. 16, 461–474 (2022). https://doi.org/10.1007/s12079-021-00655-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12079-021-00655-x