The stepwise evolution of the exome during acquisition of docetaxel resistance in breast cancer cells.
Resistance to taxane-based therapy in breast cancer patients is a major clinical problem that may be addressed through insight of the genomic alterations leading to taxane resistance in breast cancer cells. In the current study we used whole exome sequencing to discover somatic genomic alterations, evolving across evolutionary stages during the acquisition of docetaxel resistance in breast cancer cell lines.



Two human breast cancer in vitro models (MCF-7 and MDA-MB-231) of the step-wise acquisition of docetaxel resistance were developed by exposing cells to 18 gradually increasing concentrations of docetaxel. Whole exome sequencing performed at five successive stages during this process was used to identify single point mutational events, insertions/deletions and copy number alterations associated with the acquisition of docetaxel resistance. Acquired coding variation undergoing positive selection and harboring characteristics likely to be functional were further prioritized using network-based approaches. A number of genomic changes were found to be undergoing evolutionary selection, some of which were likely to be functional. Of the five stages of progression toward resistance, most resistance relevant genomic variation appeared to arise midway towards fully resistant cells corresponding to passage 31 (5 nM docetaxel) for MDA-MB-231 and passage 16 (1.2 nM docetaxel) for MCF-7, and where the cells also exhibited a period of reduced growth rate or arrest, respectively. MCF-7 cell acquired several copy number gains on chromosome 7, including ABC transporter genes, including ABCB1 and ABCB4, as well as DMTF1, CLDN12, CROT, and SRI. For MDA-MB-231 numerous copy number losses on chromosome X involving more than 30 genes was observed. Of these genes, CASK, POLA1, PRDX4, MED14 and PIGA were highly prioritized by the applied network-based gene ranking approach. At higher docetaxel concentration MCF-7 subclones exhibited a copy number loss in E2F4, and the gene encoding this important transcription factor was down-regulated in MCF-7 resistant cells.



Our study of the evolution of acquired docetaxel resistance identified several genomic changes that might explain development of docetaxel resistance. Interestingly, the most relevant resistance-associated changes appeared to originate midway through the evolution towards fully resistant cell lines. Our data suggest that no single genomic event sufficiently predicts resistance to docetaxel, but require genomic alterations affecting multiple pathways that in concert establish the final resistance stage.
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|Faculty of Health and Medical Sciences, Department of Veterinary Disease Biology, Section for Molecular Disease Biology, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark.
Stine Ninel
SN
Hansen
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Kemitorvet building 208, DK-2800, Lyngby, Denmark.
Natasja Spring
NS
Ehlers
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China.
Shida
S
Zhu
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|Department of Molecular Medicine, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus N, Denmark.
Mathilde Borg Houlberg
MB
Thomsen
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Kemitorvet building 208, DK-2800, Lyngby, Denmark.
Rikke Linnemann
RL
Nielsen
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China.
Dongbing
D
Liu
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China.
Guangbiao
G
Wang
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China.
Yong
Y
Hou
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China.
Xiuqing
X
Zhang
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China.
Xun
X
Xu
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|Department of Biomedicine, Aarhus University, Bartholins Allé 6, DK-8000, Aarhus C, Denmark.
Lars
L
Bolund
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China.
Huanming
H
Yang
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China.|Macau University of Science and Technology, Avenida Wai long, Taipa, Macau, 999078, China.|Department of Medicine and State Key Laboratory of Pharmaceutical Biotechnology, University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong.|Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J.B. Winsloews Vej 25, DK-5000, Odense, Denmark.|Department of Oncology, Odense University Hospital, Sdr. Boulevard 29, DK-5000, Odense, Denmark.
Jun
J
Wang
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|Faculty of Health and Medical Sciences, Department of Veterinary Disease Biology, Section for Molecular Disease Biology, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark.
Jose
J
Moreira
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200, Copenhagen, Denmark.|Princess Al Jawhara Albrahim Center of Excellence in the Research of Hereditary Disorders, King Abdulaziz University, Jeddah, Saudi Arabia.
Henrik J
HJ
Ditzel
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|Faculty of Health and Medical Sciences, Department of Veterinary Disease Biology, Section for Molecular Disease Biology, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark.
Nils
N
Brünner
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark.|Faculty of Health and Medical Sciences, Department of Veterinary Disease Biology, Section for Molecular Disease Biology, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark.
Anne-Sofie
AS
Schrohl
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark. stenvang@sund.ku.dk.|Faculty of Health and Medical Sciences, Department of Veterinary Disease Biology, Section for Molecular Disease Biology, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark. stenvang@sund.ku.dk.
Jan
J
Stenvang
Sino Danish Breast Cancer Research Center, Copenhagen, Denmark. ramneek@cbs.dtu.dk.|Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Kemitorvet building 208, DK-2800, Lyngby, Denmark. ramneek@cbs.dtu.dk.
Ramneek
R
Gupta
D000970:Antineoplastic Agents / Q000494:pharmacology*; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000235:genetics*; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D000077143:Docetaxel; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D019143:Evolution, Molecular; D059472:Exome; D005260:Female; D020869:Gene Expression Profiling; D014644:Genetic Variation; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D043823:Taxoids / Q000494:pharmacology*; D057188:Workflow
D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D043823:Taxoids; D000077143:Docetaxel
Breast cancer; Docetaxel resistance; Exome sequencing; Taxane