Abstract
Background
Breast cancer patients undergoing chemotherapy encounter a significant challenge of chemoresistance because of drug efflux by ATP-binding cassette (ABC) transporters. Breast cancer cell density alters considerably throughout the early stages of primary and secondary tumor development. Although cell density in culture influences kinetics, the effects of varying cell densities on the chemoresistance of breast cancer cells remains largely unexplored.
Methods and results
We observed chemotherapeutics-induced differential gene and protein expression of ABC transporters in luminal and basal breast cancer cells cultured at low and high seeding densities. Low-density cultures depicted a significant increase in the mRNA expression of ABC transporters—ABCG2, ABCG1, ABCC4, ABCA2, ABCA3, ABCC2, ABCC3, ABCC6, ABCC7, and ABCC9 as compared with high-density cultures. Next, cells at both low and high seeding densities when pre-treated with cyclosporine A (CsA), a pan-inhibitor of ABC transporters, resulted in increased sensitization to chemotherapeutics—doxorubicin and tamoxifen at markedly low IC50 concentrations suggesting the role of ABC transporters. Finally, markedly high doxorubicin-drug accumulation, significantly increased expression of N-cadherin, and a significant decrease in chemotherapeutics-induced in vitro tumorigenesis was observed in low-density seeded breast cancer cells when pre-treated with CsA suggesting ABC transporters inhibition-mediated increased efficacy of chemotherapeutics.
Conclusion
These findings suggest that breast cancer cells grown at low seeding density imparts chemoresistance towards doxorubicin or tamoxifen by a differential increase in the expression of ABC transporters. Thus, a combinatorial treatment strategy including ABC transporter inhibitors and chemotherapeutics can be a way forward for overcoming the breast cancer chemoresistance.
Similar content being viewed by others
References
Siegel RL, Miller KD, Fuchs HE, Jemal A (2022) Cancer statistics, 2022. CA Cancer J Clin 72(1):7–33. https://doi.org/10.3322/caac.21708
Waks AG, Winer EP (2019) Breast cancer treatment: a review. JAMA 321(3):288–300. https://doi.org/10.1001/jama.2018.19323
Zhou HM, Zhang JG, Zhang X, Li Q (2021) Targeting cancer stem cells for reversing therapy resistance: mechanism, signaling, and prospective agents. Sig Transduct Target Ther 6(1):62. https://doi.org/10.1038/s41392-020-00430-1
Skarping I, Förnvik D, Heide-Jørgensen U, Sartor H, Hall P, Zackrisson S, Borgquist S (2021) Mammographic density as an image-based biomarker of therapy response in neoadjuvant-treated breast cancer patients. Cancer Causes Control: CCC 32(3):251–260. https://doi.org/10.1007/s10552-020-01379-w
Pizzato M, Carioli G, Rosso S, Zanetti R, La Vecchia C (2021) Mammographic breast density and characteristics of invasive breast cancer. Cancer Epidemiol 70:101879. https://doi.org/10.1016/j.canep.2020.101879
Ramamoorthi G, Kodumudi K, Gallen C, Zachariah NN, Basu A, Albert G, Czerniecki BJ (2022) Disseminated cancer cells in breast cancer: Mechanism of dissemination and dormancy and emerging insights on therapeutic opportunities. Semin Cancer Biol 78:78–89. https://doi.org/10.1016/j.semcancer.2021.02.004
Gupta SK, Singh P, Ali V, Verma M (2020) Role of membrane-embedded drug efflux ABC transporters in the cancer chemotherapy. Oncol Rev. https://doi.org/10.4081/oncol.2020.448
Chen J, Wang Z, Gao S, Wu K, Bai F, Zhang Q, Wang H, Ye Q, Xu F, Sun H, Lu Y, Liu Y (2021) Human drug efflux transporter ABCC5 confers acquired resistance to pemetrexed in breast cancer. Cancer Cell Int 21(1):136. https://doi.org/10.1186/s12935-021-01842-x
Giddings EL, Champagne DP, Wu MH, Laffin JM, Thornton TM, Valenca-Pereira F, Rincon M (2021) Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance. Nat Commun 12(1):1–19. https://doi.org/10.1038/s41467-021-23071-6
Dean M, Hamon Y, Chimini G (2001) The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res 42(7):1007–1017. https://doi.org/10.1016/S0022-2275(20)31588-1
Saxena M, Stephens MA, Pathak H, Rangarajan A (2011) Transcription factors that mediate epithelial–mesenchymal transition lead to multidrug resistance by upregulating ABC transporters. Cell Death Dis 2(7):e179–e179. https://doi.org/10.1038/cddis.2011.61
Leonard GD, Fojo T, Bates SE (2003) The role of ABC transporters in clinical practice. Oncologist 8(5):411–424. https://doi.org/10.1634/theoncologist.8-5-411
Xiao H, Zheng Y, Ma L, Tian L, Sun Q (2021) Clinically-relevant ABC transporter for anti-cancer drug resistance. Front Pharmacol 12:648407. https://doi.org/10.3389/fphar.2021.648407
Manupati K, Dhoke NR, Debnath T, Yeeravalli R, Guguloth K, Saeidpour S, Das A (2017) Inhibiting epidermal growth factor receptor signalling potentiates mesenchymal–epithelial transition of breast cancer stem cells and their responsiveness to anticancer drugs. FEBS J 284(12):1830–1854. https://doi.org/10.1111/febs.14084
Yeeravalli R, Kaushik K, Das A (2021) TWIST1-mediated transcriptional activation of PDGFRβ in breast cancer stem cells promotes tumorigenesis and metastasis. Biochimica et Biophysica Acta (BBA)-Mol Basis Dis 1867(7):166141. https://doi.org/10.1016/j.bbadis.2021.166141
Reddy L, Dharmabalan ST, Manupati K, Yeeravalli R, Vijay LD, Donthiboina K, Das A (2020) Concise synthesis of 1, 1-diarylvinyl sulfones and investigations on their anti-proliferative activity via tubulin inhibition. Anticancer Agents Med Chem (Formerly Current Medicinal Chemistry-Anti-Cancer Agents) 20(12):1469–1474. https://doi.org/10.2174/1871520620666200423075630
Sampson A, Peterson BG, Tan KW, Iram SH (2019) Doxorubicin as a fluorescent reporter identifies novel MRP1 (ABCC1) inhibitors missed by calcein-based high content screening of anticancer agents. Biomed Pharmacother 118:109289. https://doi.org/10.1016/j.biopha.2019.109289
Manupati K, Debnath S, Goswami K, Bhoj PS, Chandak HS, Bahekar SP, Das A (2019) Glutathione S-transferase omega 1 inhibition activates JNK‐mediated apoptotic response in breast cancer stem cells. FEBS J 286(11):2167–2192. https://doi.org/10.1111/febs.14813
Singh D, Deshmukh RK, Das A (2021) SNAI1-mediated transcriptional regulation of epithelial-to-mesenchymal transition genes in breast cancer stem cells. Cell Signal 87:110151. https://doi.org/10.1016/j.cellsig.2021.110151
Lee HH, Bellat V, Law B (2017) Chemotherapy induces adaptive drug resistance and metastatic potentials via phenotypic CXCR4-expressing cell state transition in ovarian cancer. PLoS ONE 12(2):e0171044. https://doi.org/10.1371/journal.pone.0171044
Madden EC, Gorman AM, Logue SE, Samali A (2020) Tumour cell secretome in chemoresistance and tumour recurrence. Trends Cancer 6(6):489–505. https://doi.org/10.1016/j.trecan.2020.02.020
Muriithi W, Macharia LW, Heming CP, Echevarria JL, Nyachieo A, Niemeyer Filho P, Neto VM (2020) ABC transporters and the hallmarks of cancer: roles in cancer aggressiveness beyond multidrug resistance. Cancer Biol Med 17(2):253. https://doi.org/10.20892%2Fj.issn.2095-3941.2019.0284
Citron ML (2008) Dose-dense chemotherapy: principles, clinical results and future perspectives. Breast Care 3(4):251–255. https://doi.org/10.1159/000148914
Fan D, Beltran P, Wang Y, Bucana C, Yoon S, Deguzman A, Fidler I (1996) Cell density-dependent regulation of mdr-1 gene expression in murine colon cancer cells. Int J Oncol 9(5):865–878. https://doi.org/10.3892/ijo.9.5.865
Furukawa T, Wakabayashi K, Tamura A, Nakagawa H, Morishima Y, Osawa Y, Ishikawa T (2009) Major SNP (Q141K) variant of human ABC transporter ABCG2 undergoes lysosomal and proteasomal degradations. Pharm Res 26(2):469–479. https://doi.org/10.1007/s11095-008-9752-7
Hembruff SL, Laberge ML, Villeneuve DJ, Guo B, Veitch Z, Cecchetto M, Parissenti AM (2008) Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance. BMC Cancer 8(1):1–16. https://doi.org/10.1186/1471-2407-8-318
Maeda M, Johnson KR, Wheelock MJ (2005) Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition. J Cell Sci 118(5):873–887. https://doi.org/10.1242/jcs.01634
Manupati K, Yeeravalli R, Kaushik K, Singh D, Mehra B, Gangane N, Das A (2021) Activation of CD44-Lipoprotein lipase axis in breast cancer stem cells promotes tumorigenesis. Biochim et Biophys Acta (BBA)-Mol Basis Dis 1867(11):166228. https://doi.org/10.1016/j.bbadis.2021.166228
Bai X, Ni J, Beretov J, Graham P, Li Y (2018) Cancer stem cell in breast cancer therapeutic resistance. Cancer Treat Rev 69:152–163. https://doi.org/10.1016/j.ctrv.2018.07.004
Sharif GM, Wellstein A (2015) Cell density regulates cancer metastasis via the Hippo pathway. Future Oncol 11(24):3253–3260. https://doi.org/10.2217/fon.15.268
Winkler J, Abisoye-Ogunniyan A, Metcalf KJ, Werb Z (2020) Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nat Commun 11(1):1–19. https://doi.org/10.1038/s41467-020-18794
Zboralski D et al (2022) Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy. Eur J Nucl Med Mol Imaging 49:3651–3667. https://doi.org/10.1007/s00259-022-05842-5
D’Andrea MR, Cereda V, Coppola L, Giordano G, Remo A, De Santis E (2021) Propensity for early metastatic spread in breast cancer: role of tumor vascularization features and tumor immune infiltrate. Cancers 13(23):5917. https://doi.org/10.3390/cancers13235917
Spitzwieser M, Pirker C, Koblmüller B, Pfeiler G, Hacker S, Berger W, Cichna-Markl M (2016) Promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in human cancer cell lines, multidrug-resistant cell models and tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients. Oncotarget 7(45):73347. https://doi.org/10.18632%2Foncotarget.12332
Balaji SA, Udupa N, Chamallamudi MR, Gupta V, Rangarajan A (2016) Role of the drug transporter ABCC3 in breast cancer chemoresistance. PLoS ONE 11(5):e0155013. https://doi.org/10.1371/journal.pone.0155013
Tsou SH, Chen TM, Hsiao HT, Chen YH (2015) A critical dose of doxorubicin is required to alter the gene expression profiles in MCF-7 cells acquiring multidrug resistance. PLoS ONE 10(1):e0116747. https://doi.org/10.1371/journal.pone.0116747
Zhao Y, Lu H, Yan A, Yang Y, Meng Q, Sun L, Cai L (2013) ABCC3 as a marker for multidrug resistance in non-small cell lung cancer. Sci Rep 3(1):1–6. https://doi.org/10.1038/srep03120
Sims JT, Ganguly SS, Bennett H, Friend JW, Tepe J, Plattner R (2013) Imatinib reverses doxorubicin resistance by affecting activation of STAT3-dependent NF-κB and HSP27/p38/AKT pathways and by inhibiting ABCB1. PLoS ONE 8(1):e55509. https://doi.org/10.1371/journal.pone.0055509
Gujral TS, Kirschner MW (2017) Hippo pathway mediates resistance to cytotoxic drugs. Proc Natl Acad Sci 114(18):E3729–E3738. https://doi.org/10.1073/pnas.1703096114
Acknowledgements
AD acknowledges institutional funding by CSIR-IICT. The fellowship provided by DST INSPIRE to SS is gratefully acknowledged. (Manuscript Communication number: IICT/Pubs./2022/011).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Shivhare, S., Das, A. Cell density modulates chemoresistance in breast cancer cells through differential expression of ABC transporters. Mol Biol Rep 50, 215–225 (2023). https://doi.org/10.1007/s11033-022-08028-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-022-08028-2