Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer.
OBJECTIVE
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.
Division of Cancer, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.|Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Dhaka, 1000, Bangladesh.
Muhammad
M
Asaduzzaman
Division of Cancer, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.|MRC Cancer Unit, Hutchison/MRC Research Centre, Cambridge, CB2 0XZ, UK.
Stephanie
S
Constantinou
Division of Cancer, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
Haoxiang
H
Min
Division of Cancer, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
John
J
Gallon
Division of Cancer, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
Meng-Lay
ML
Lin
Centre for Pathology, Department of Medicine, Imperial College Faculty of Medicine, Charing Cross Hospital, Fulham Palace Rd, London, W6 8RF, UK.
Poonam
P
Singh
Division of Clinical Sciences, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
Selina
S
Raguz
Division of Cancer, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
Simak
S
Ali
Centre for Pathology, Department of Medicine, Imperial College Faculty of Medicine, Charing Cross Hospital, Fulham Palace Rd, London, W6 8RF, UK.
Sami
S
Shousha
Division of Cancer, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
R Charles
RC
Coombes
Division of Cancer, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
Eric W-F
EW
Lam
The 3rd Department of Breast Cancer, China Tianjin Breast Cancer Prevention, Treatment and Research Center, National Clinical Research Center of Cancer, Tianjin Medical University Cancer Institute and Hospital, Huan Hu Xi Road, Ti Yuan Bei, He xi District, Tianjin, 300060, People's Republic of China. 417501784@qq.com.
Yunhui
Y
Hu
Division of Cancer, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK. ernesto.yague@imperial.ac.uk.
Ernesto
http://orcid.org/0000-0003-2371-0032
E
Yagüe
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2 / Q000235:genetics; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D002154:Calpain / Q000235:genetics; D002272:Carcinoembryonic Antigen / Q000235:genetics; D000066670:Cell Plasticity / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D050881:E1A-Associated p300 Protein / Q000235:genetics*; D005260:Female; D058851:GPI-Linked Proteins / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D061986:MCF-7 Cells; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000235:genetics; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology; D017239:Paclitaxel / Q000008:administration & dosage; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D053781:Transforming Growth Factor beta2 / Q000235:genetics
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; C527875:CEACAM5 protein, human; D002272:Carcinoembryonic Antigen; D058851:GPI-Linked Proteins; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C509041:TGFB2 protein, human; D053781:Transforming Growth Factor beta2; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human; C518121:CAPN9 protein, human; D002154:Calpain; D017239:Paclitaxel
ABCG2; BCL2; Cancer stem cells; Drug resistance; EP300 signature; Metastasis