Effects of In Utero Exposure to Ethinyl Estradiol on Tamoxifen Resistance and Breast Cancer Recurrence in a Preclinical Model.
Responses to endocrine therapies vary among patients with estrogen receptor (ER+) breast cancer. We studied whether in utero exposure to endocrine-disrupting compounds might explain these variations.



We describe a novel ER+ breast cancer model to study de novo and acquired tamoxifen (TAM) resistance. Pregnant Sprague Dawley rats were exposed to 0 or 0.1 ppm ethinyl estradiol (EE2), and the response of 9,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors to 15 mg/kg TAM, with (n = 17 tumors in the controls and n = 20 tumors in EE2 offspring) or without 1.2 g/kg valproic acid and 5 mg/kg hydralazine (n = 24 tumors in the controls and n = 32 tumors in EE2 offspring) in the female offspring, was assessed. One-sided Chi2 tests were used to calculate P values. Comparisons of differentially expressed genes between mammary tumors in in utero EE2-exposed and control rats, and between anti-estrogen-resistant LCC9 and -sensitive LCC1 human breast cancer cells, were also performed.



In our preclinical model, 54.2% of mammary tumors in the control rats exhibited a complete response to TAM, of which 23.1% acquired resistance with continued anti-estrogen treatment and recurred. Mammary tumors in the EE2 offspring were statistically significantly less likely to respond to TAM (P = .047) and recur (P = .007). In the EE2 offspring, but not in controls, adding valproic acid and hydralazine to TAM prevented recurrence (P < .001). Three downregulated and hypermethylated genes (KLF4, LGALS3, MICB) and one upregulated gene (ETV4) were identified in EE2 tumors and LCC9 breast cancer cells, and valproic acid and hydralazine normalized the altered expression of all four genes.



Resistance to TAM may be preprogrammed by in utero exposure to high estrogen levels and mediated through reversible epigenetic alterations in genes associated with epithelial-mesenchymal transition and tumor immune responses.
Department of Oncology, Georgetown University, Washington, DC.
Leena
L
Hilakivi-Clarke
Department of Oncology, Georgetown University, Washington, DC.|Institute of Biomedicine, University of Turku Medical Faculty, Turku, Finland.
Anni
A
Wärri
Department of Oncology, Georgetown University, Washington, DC.
Kerrie B
KB
Bouker
Department of Oncology, Georgetown University, Washington, DC.
Xiyuan
X
Zhang
Department of Oncology, Georgetown University, Washington, DC.|Department of Surgery, Wake Forest University, Winston-Salem, NC.
Katherine L
KL
Cook
Department of Oncology, Georgetown University, Washington, DC.
Lu
L
Jin
Department of Oncology, Georgetown University, Washington, DC.
Alan
A
Zwart
Department of Oncology, Georgetown University, Washington, DC.
Nguyen
N
Nguyen
Department of Oncology, Georgetown University, Washington, DC.
Rong
R
Hu
Department of Oncology, Georgetown University, Washington, DC.
M Idalia
MI
Cruz
Department of Oncology, Georgetown University, Washington, DC.
Sonia
S
de Assis
Department of Electrical and Computer Engineering, Virginia Tech, Arlington, VA.
Xiao
X
Wang
Department of Electrical and Computer Engineering, Virginia Tech, Arlington, VA.
Jason
J
Xuan
Department of Electrical and Computer Engineering, Virginia Tech, Arlington, VA.
Yue
Y
Wang
University of Texas Health Science Center, Houston, TX.
Bryan
B
Wehrenberg
Department of Oncology, Georgetown University, Washington, DC.
Robert
R
Clarke
D015127:9,10-Dimethyl-1,2-benzanthracene; D017500:Adenovirus E1A Proteins / Q000235:genetics; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001943:Breast Neoplasms / Q000188:drug therapy*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D004997:Ethinyl Estradiol / Q000009:adverse effects*; D005260:Female; D037502:Galectin 3 / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing; D015395:Histocompatibility Antigens Class I / Q000235:genetics; D006801:Humans; D006830:Hydralazine / Q000008:administration & dosage; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D008325:Mammary Neoplasms, Experimental / Q000139:chemically induced / Q000737:chemistry / Q000188:drug therapy*; D009364:Neoplasm Recurrence, Local / Q000235:genetics* / Q000517:prevention & control; D011247:Pregnancy; D011297:Prenatal Exposure Delayed Effects / Q000139:chemically induced*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D050783:Proto-Oncogene Proteins c-ets; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000032:analysis; D013629:Tamoxifen / Q000008:administration & dosage / Q000627:therapeutic use*; D015534:Trans-Activators / Q000235:genetics; D014635:Valproic Acid / Q000008:administration & dosage
D017500:Adenovirus E1A Proteins; C079853:ETV4 protein, human; C510951:ETV4 protein, rat; D037502:Galectin 3; D015395:Histocompatibility Antigens Class I; C000715467:KLF4 protein, human; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C098080:MICB antigen; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011960:Receptors, Estrogen; D015534:Trans-Activators; D013629:Tamoxifen; D006830:Hydralazine; D004997:Ethinyl Estradiol; D015127:9,10-Dimethyl-1,2-benzanthracene; D014635:Valproic Acid