Top

Published in:

Open Access 01-06-2017 | Preclinical study

Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer

Authors: Muhammad Asaduzzaman, Stephanie Constantinou, Haoxiang Min, John Gallon, Meng-Lay Lin, Poonam Singh, Selina Raguz, Simak Ali, Sami Shousha, R. Charles Coombes, Eric W.-F. Lam, Yunhui Hu, Ernesto Yagüe

Published in: Breast Cancer Research and Treatment | Issue 3/2017

Abstract

Purpose

We have previously described a novel pathway controlling drug resistance, epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer cells. Upstream in the pathway, three miRs (miR-106b, miR-93 and miR-25) target EP300, a transcriptional activator of E-cadherin. Upregulation of these miRs leads to the downregulation of EP300 and E-cadherin with initiation of an EMT. However, miRs regulate the expression of many genes, and the contribution to EMT by miR targets other than EP300 cannot be ruled out.

Methods

We used lentiviruses expressing EP300-targeting shRNA to downregulate its expression in MCF-7 cells as well as an EP300-knocked-out colon carcinoma cell line. An EP300-expression plasmid was used to upregulate its expression in basal-like CAL51 and MDA-MB-231 breast cancer cells. Drug resistance was determined by short-term proliferation and long-term colony formation assays. Stemness was determined by tumour sphere formation in both soft agar and liquid cultures as well as by the expression of CD44/CD24/ALDH markers. Gene expression microarray analysis was performed in MCF-7 cells lacking EP300. EP300 expression was analysed by immunohistochemistry in 17 samples of metaplastic breast cancer.

Results

Cells lacking EP300 became more resistant to paclitaxel whereas EP300 overexpression increased their sensitivity to the drug. Expression of cancer stem cell markers, as well as tumour sphere formation, was also increased in EP300-depleted cells, and was diminished in EP300-overexpressing cells. The EP300-regulated gene signature highlighted genes associated with adhesion (CEACAM5), cytoskeletal remodelling (CAPN9), stemness (ABCG2), apoptosis (BCL2) and metastasis (TGFB2). Some genes in this signature were also validated in a previously generated EP300-depleted model of breast cancer using minimally transformed mammary epithelial cells. Importantly, two key genes in apoptosis and stemness, BCL2 and ABCG2, were also upregulated in EP300-knockout colon carcinoma cells and their paclitaxel-resistant derivatives. Immunohistochemical analysis demonstrated that EP300 expression was low in metaplastic breast cancer, a rare, but aggressive form of the disease with poor prognosis that is characterized by morphological and physiological features of EMT.

Conclusions

EP300 plays a major role in the reprogramming events, leading to a more malignant phenotype with the acquisition of drug resistance and cell plasticity, a characteristic of metaplastic breast cancer.

Available only for authorised users

Sledge GW, Mamounas EP, Hortobagyi GN, Burstein HJ, Goodwin PJ, Wolff AC (2014) Past, present, and future challenges in breast cancer treatment. J Clin Oncol 32(19):1979–1986. doi:10.1200/JCO.2014.55.4139 CrossRefPubMedPubMedCentral

Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133(4):704–715. doi:10.1016/j.cell.2008.03.027 CrossRefPubMedPubMedCentral

Singh A, Settleman J (2010) EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29(34):4741–4751. doi:10.1038/onc.2010.215 CrossRefPubMedPubMedCentral

Dave B, Mittal V, Tan NM, Chang JC (2012) Epithelial-mesenchymal transition, cancer stem cells and treatment resistance. Breast Cancer Res 14(1):202. doi:10.1186/bcr2938 CrossRefPubMedPubMedCentral

Liu YN, Lee WW, Wang CY, Chao TH, Chen Y, Chen JH (2005) Regulatory mechanisms controlling human E-cadherin gene expression. Oncogene 24(56):8277–8290CrossRefPubMed

Kalkhoven E (2004) CBP and p300: HATs for different occasions. Biochem Pharmacol 68(6):1145–1155. doi:10.1016/j.bcp.2004.03.045 CrossRefPubMed

Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y (1996) The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87(5):953–959CrossRefPubMed

Goodman RH, Smolik S (2000) CBP/p300 in cell growth, transformation, and development. Genes Dev 14(13):1553–1577PubMed

Bryan EJ, Jokubaitis VJ, Chamberlain NL, Baxter SW, Dawson E, Choong DY, Campbell IG (2002) Mutation analysis of EP300 in colon, breast and ovarian carcinomas. Int J Cancer 102(2):137–141. doi:10.1002/ijc.10682 CrossRefPubMed

10.

Gayther SA, Batley SJ, Linger L, Bannister A, Thorpe K, Chin SF, Daigo Y, Russell P, Wilson A, Sowter HM, Delhanty JD, Ponder BA, Kouzarides T, Caldas C (2000) Mutations truncating the EP300 acetylase in human cancers. Nat Genet 24(3):300–303. doi:10.1038/73536 CrossRefPubMed

11.

Krubasik D, Iyer NG, English WR, Ahmed AA, Vias M, Roskelley C, Brenton JD, Caldas C, Murphy G (2006) Absence of p300 induces cellular phenotypic changes characteristic of epithelial to mesenchyme transition. Br J Cancer 94(9):1326–1332. doi:10.1038/sj.bjc.6603101 CrossRefPubMedPubMedCentral

12.

Zhou Y, Hu Y, Yang M, Jat P, Li K, Lombardo Y, Xiong D, Coombes RC, Raguz S, Yague E (2014) The miR-106b ~ 25 cluster promotes bypass of doxorubicin-induced senescence and increase in motility and invasion by targeting the E-cadherin transcriptional activator EP300. Cell Death Differ 21(3):462–474. doi:10.1038/cdd.2013.167 CrossRefPubMed

13.

Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin SF, Dunning MJ, Barbosa-Morais NL, Teschendorff AE, Green AR, Ellis IO, Tavare S, Caldas C, Miska EA (2007) MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biol 8(10):R214CrossRefPubMedPubMedCentral

14.

Shiota M, Yokomizo A, Kashiwagi E, Tada Y, Inokuchi J, Tatsugami K, Kuroiwa K, Uchiumi T, Seki N, Naito S (2010) Foxo3a expression and acetylation regulate cancer cell growth and sensitivity to cisplatin. Cancer Sci 101(5):1177–1185. doi:10.1111/j.1349-7006.2010.01503.x CrossRefPubMed

15.

Takeuchi A, Shiota M, Tatsugami K, Yokomizo A, Tanaka S, Kuroiwa K, Eto M, Naito S (2012) p300 mediates cellular resistance to doxorubicin in bladder cancer. Mol Med Rep 5(1):173–176. doi:10.3892/mmr.2011.593 PubMed

16.

Zhao JJ, Gjoerup OV, Subramanian RR, Cheng Y, Chen W, Roberts TM, Hahn WC (2003) Human mammary epithelial cell transformation through the activation of phosphatidylinositol 3-kinase. Cancer Cell 3(5):483–495CrossRefPubMed

17.

Hu Y, Li K, Asaduzzaman M, Cuella R, Shi H, Raguz S, Coombes RC, Zhou Y, Yague E (2016) miR-106b ~ 25 cluster regulates multidrug resistance in an ABC transporter-independent manner via downregulation of EP300. Oncol Rep 35:1170–1178. doi:10.3892/or.2015.4412 CrossRefPubMed

18.

Raguz S, Adams C, Masrour N, Rasul S, Papoutsoglou P, Hu Y, Cazzanelli G, Zhou Y, Patel N, Coombes C, Yague E (2013) Loss of O(6)-methylguanine-DNA methyltransferase confers collateral sensitivity to carmustine in topoisomerase II-mediated doxorubicin resistant triple negative breast cancer cells. Biochem Pharmacol 85(2):186–196. doi:10.1016/j.bcp.2012.10.020 CrossRefPubMed

19.

Chen LF, Mu Y, Greene WC (2002) Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB. EMBO J 21(23):6539–6548CrossRefPubMedPubMedCentral

20.

Yague E, Armesilla AL, Harrison G, Elliott J, Sardini A, Higgins CF, Raguz S (2003) P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation. J Biol Chem 278(12):10344–10352CrossRefPubMed

21.

Vichai V, Kirtikara K (2006) Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1(3):1112–1116CrossRefPubMed

22.

Rasul S, Balasubramanian R, Filipovic A, Slade MJ, Yague E, Coombes RC (2009) Inhibition of gamma-secretase induces G2/M arrest and triggers apoptosis in breast cancer cells. Br J Cancer 100(12):1879–1888. doi:10.1038/sj.bjc.6605034 CrossRefPubMedPubMedCentral

23.

Lombardo Y, Filipovic A, Molyneux G, Periyasamy M, Giamas G, Hu Y, Trivedi PS, Wang J, Yague E, Michel L, Coombes RC (2012) Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo. Proc Natl Acad Sci USA 109(41):16558–16563. doi:10.1073/pnas.1206268109 CrossRefPubMedPubMedCentral

24.

Liu LK, Jiang XY, Zhou XX, Wang DM, Song XL, Jiang HB (2010) Upregulation of vimentin and aberrant expression of E-cadherin/beta-catenin complex in oral squamous cell carcinomas: correlation with the clinicopathological features and patient outcome. Mod Pathol 23(2):213–224. doi:10.1038/modpathol.2009.160 CrossRefPubMed

25.

Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1(5):555–567. doi:10.1016/j.stem.2007.08.014 CrossRefPubMedPubMedCentral

26.

Calcagno AM, Salcido CD, Gillet JP, Wu CP, Fostel JM, Mumau MD, Gottesman MM, Varticovski L, Ambudkar SV (2010) Prolonged drug selection of breast cancer cells and enrichment of cancer stem cell characteristics. J Natl Cancer Inst 102(21):1637–1652CrossRefPubMedPubMedCentral

27.

Kao J, Salari K, Bocanegra M, Choi YL, Girard L, Gandhi J, Kwei KA, Hernandez-Boussard T, Wang P, Gazdar AF, Minna JD, Pollack JR (2009) Molecular profiling of breast cancer cell lines defines relevant tumor models and provides a resource for cancer gene discovery. PLoS One 4(7):e6146. doi:10.1371/journal.pone.0006146 CrossRefPubMedPubMedCentral

28.

Calvet CY, Andre FM, Mir LM (2014) The culture of cancer cell lines as tumorspheres does not systematically result in cancer stem cell enrichment. PLoS One 9(2):e89644. doi:10.1371/journal.pone.0089644 CrossRefPubMedPubMedCentral

29.

Schwartz TL, Mogal H, Papageorgiou C, Veerapong J, Hsueh EC (2013) Metaplastic breast cancer: histologic characteristics, prognostic factors and systemic treatment strategies. Exp Hematol Oncol 2(1):31. doi:10.1186/2162-3619-2-31 CrossRefPubMedPubMedCentral

30.

Weigelt B, Eberle C, Cowell CF, Ng CK, Reis-Filho JS (2014) Metaplastic breast carcinoma: more than a special type. Nat Rev Cancer 14(3):147–148CrossRefPubMed

31.

Ponten F, Jirstrom K, Uhlen M (2008) The human protein atlas–a tool for pathology. J Pathol 216(4):387–393. doi:10.1002/path.2440 CrossRefPubMed

32.

Chaffer CL, Weinberg RA (2011) A perspective on cancer cell metastasis. Science 331(6024):1559–1564. doi:10.1126/science.1203543 CrossRefPubMed

33.

Snowden AW, Anderson LA, Webster GA, Perkins ND (2000) A novel transcriptional repression domain mediates p21(WAF1/CIP1) induction of p300 transactivation. Mol Cell Biol 20(8):2676–2686CrossRefPubMedPubMedCentral

34.

Girdwood D, Bumpass D, Vaughan OA, Thain A, Anderson LA, Snowden AW, Garcia-Wilson E, Perkins ND, Hay RT (2003) P300 transcriptional repression is mediated by SUMO modification. Mol Cell 11(4):1043–1054CrossRefPubMed

35.

Yuan ZM, Huang Y, Ishiko T, Nakada S, Utsugisawa T, Shioya H, Utsugisawa Y, Shi Y, Weichselbaum R, Kufe D (1999) Function for p300 and not CBP in the apoptotic response to DNA damage. Oncogene 18(41):5714–5717. doi:10.1038/sj.onc.1202930 CrossRefPubMed

36.

Fujii Y, Kumatori A, Nakamura M (2003) SATB1 makes a complex with p300 and represses gp91(phox) promoter activity. Microbiol Immunol 47(10):803–811CrossRefPubMed

37.

Gong F, Sun L, Sun Y (2010) A novel SATB1 binding site in the BCL2 promoter region possesses transcriptional regulatory function. J Biomed Res 24(6):452–459. doi:10.1016/S1674-8301(10)60060-7 CrossRefPubMedPubMedCentral

38.

Gai X, Tu K, Li C, Lu Z, Roberts LR, Zheng X (2015) Histone acetyltransferase PCAF accelerates apoptosis by repressing a GLI1/BCL2/BAX axis in hepatocellular carcinoma. Cell Death Dis 6:e1712. doi:10.1038/cddis.2015.76 CrossRefPubMedPubMedCentral

39.

Zeisberg M, Neilson EG (2009) Biomarkers for epithelial-mesenchymal transitions. J Clin Investig 119(6):1429–1437. doi:10.1172/JCI36183 CrossRefPubMedPubMedCentral

40.

Chen J, Wei D, Zhao Y, Liu X, Zhang J (2013) Overexpression of EFEMP1 correlates with tumor progression and poor prognosis in human ovarian carcinoma. PLoS One 8(11):e78783. doi:10.1371/journal.pone.0078783 CrossRefPubMedPubMedCentral

41.

Fukai J, Yokote H, Yamanaka R, Arao T, Nishio K, Itakura T (2008) EphA4 promotes cell proliferation and migration through a novel EphA4-FGFR1 signaling pathway in the human glioma U251 cell line. Mol Cancer Ther 7(9):2768–2778. doi:10.1158/1535-7163.MCT-07-2263 CrossRefPubMed

42.

Brantley-Sieders DM, Jiang A, Sarma K, Badu-Nkansah A, Walter DL, Shyr Y, Chen J (2011) Eph/ephrin profiling in human breast cancer reveals significant associations between expression level and clinical outcome. PLoS One 6(9):e24426. doi:10.1371/journal.pone.0024426 CrossRefPubMedPubMedCentral

43.

Katoh M, Katoh M (2003) Identification and characterization of human KIAA1391 and mouse Kiaa1391 genes encoding novel RhoGAP family proteins with RA domain and ANXL repeats. Int J Oncol 23(5):1471–1476PubMed

44.

Staub E, Groene J, Heinze M, Mennerich D, Roepcke S, Klaman I, Hinzmann B, Castanos-Velez E, Pilarsky C, Mann B, Brummendorf T, Weber B, Buhr HJ, Rosenthal A (2009) An expression module of WIPF1-coexpressed genes identifies patients with favorable prognosis in three tumor types. J Mol Med 87(6):633–644. doi:10.1007/s00109-009-0467-y CrossRefPubMedPubMedCentral

45.

Davis J, Martin SG, Patel PM, Green AR, Rakha EA, Ellis IO, Storr SJ (2014) Low calpain-9 is associated with adverse disease-specific survival following endocrine therapy in breast cancer. BMC Cancer 14:995. doi:10.1186/1471-2407-14-995 CrossRefPubMedPubMedCentral

46.

Akhurst RJ, Hata A (2012) Targeting the TGFbeta signalling pathway in disease. Nat Rev Drug Discov 11(10):790–811. doi:10.1038/nrd3810 CrossRefPubMedPubMedCentral

47.

Bhola NE, Balko JM, Dugger TC, Kuba MG, Sanchez V, Sanders M, Stanford J, Cook RS, Arteaga CL (2013) TGF-beta inhibition enhances chemotherapy action against triple-negative breast cancer. J Clin Investig 123(3):1348–1358. doi:10.1172/JCI65416 CrossRefPubMedPubMedCentral

48.

Shirakihara T, Horiguchi K, Miyazawa K, Ehata S, Shibata T, Morita I, Miyazono K, Saitoh M (2011) TGF-beta regulates isoform switching of FGF receptors and epithelial-mesenchymal transition. EMBO J 30(4):783–795. doi:10.1038/emboj.2010.351 CrossRefPubMedPubMedCentral

49.

Strutz F, Zeisberg M, Ziyadeh FN, Yang CQ, Kalluri R, Muller GA, Neilson EG (2002) Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Kidney Int 61(5):1714–1728. doi:10.1046/j.1523-1755.2002.00333.x CrossRefPubMed

50.

Zhang Y, Toy KA, Kleer CG (2012) Metaplastic breast carcinomas are enriched in markers of tumor-initiating cells and epithelial to mesenchymal transition. Mod Pathol 25(2):178–184. doi:10.1038/modpathol.2011.167 CrossRefPubMed

51.

Lien HC, Hsiao YH, Lin YS, Yao YT, Juan HF, Kuo WH, Hung MC, Chang KJ, Hsieh FJ (2007) Molecular signatures of metaplastic carcinoma of the breast by large-scale transcriptional profiling: identification of genes potentially related to epithelial-mesenchymal transition. Oncogene 26(57):7859–7871. doi:10.1038/sj.onc.1210593 CrossRefPubMed

52.

Weigelt B, Kreike B, Reis-Filho JS (2009) Metaplastic breast carcinomas are basal-like breast cancers: a genomic profiling analysis. Breast Cancer Res Treat 117(2):273–280. doi:10.1007/s10549-008-0197-9 CrossRefPubMed

53.

Goswami S, Sharma-Walia N (2015) Osteoprotegerin secreted by inflammatory and invasive breast cancer cells induces aneuploidy, cell proliferation and angiogenesis. BMC Cancer 15:935. doi:10.1186/s12885-015-1837-1 CrossRefPubMedPubMedCentral

Title: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer
Authors: Muhammad Asaduzzaman
Stephanie Constantinou
Haoxiang Min
John Gallon
Meng-Lay Lin
Poonam Singh
Selina Raguz
Simak Ali
Sami Shousha
R. Charles Coombes
Eric W.-F. Lam
Yunhui Hu
Ernesto Yagüe
Publication date: 01-06-2017
Publisher: Springer US
Published in: Breast Cancer Research and Treatment / Issue 3/2017
Print ISSN: 0167-6806
Electronic ISSN: 1573-7217
DOI: https://doi.org/10.1007/s10549-017-4202-z

Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras

Prof. Fabrice Barlesi

Developed by: Springer Medicine

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 3/2017

ΔNp63 activates EGFR signaling to induce loss of adhesion in triple-negative basal-like breast cancer cells

Failure patterns according to molecular subtype in patients with invasive breast cancer following postoperative adjuvant radiotherapy: long-term outcomes in contemporary clinical practice

A phase II study of combined ridaforolimus and dalotuzumab compared with exemestane in patients with estrogen receptor-positive breast cancer

FOXM1 transcriptionally regulates expression of integrin β1 in triple-negative breast cancer

Relationship of breast MRI to recurrence rates in patients undergoing breast-conservation treatment

A83-01 inhibits TGF-β-induced upregulation of Wnt3 and epithelial to mesenchymal transition in HER2-overexpressing breast cancer cells

Keynote webinar | Spotlight on antibody–drug conjugates in cancer