Cyclooxygenase-2 induces genomic instability, BCL2 expression, doxorubicin resistance, and altered cancer-initiating cell phenotype in MCF7 breast cancer cells.
BACKGROUND
Cyclooxygenase (COX2) expression in primary breast cancer correlates with a worse prognosis. We reported previously that COX2 expression in MCF10A breast epithelial cells of basal subtype induces genomic instability. To understand the role of COX2 in estrogen receptor-positive (non-basal) breast cancer, we transfected the MCF7 cell line with COX2 and analyzed its chromosomal profile, BCL2 protein expression, and resistance to doxorubicin. We also analyzed cell cultures grown as mammospheres to determine whether COX2 expression affects the cancer-initiating ("stem") cell phenotype in MCF7 cells.


METHODS
MCF7 Tet On cells (obtained from Clontech) were stably transfected with pTRE2pur-COX2 or pTRE2pur-COX2-GFP to produce COX2 or COX2-GFP protein, respectively. BCL2 protein was detected by Western blotting. Sensitivity of cells to drug treatment was analyzed by MTT assay. Groups were compared using Chi-Square test. We analyzed the genomic instability phenotype by chromosome analysis of control and COX2 transfected metaphase-arrested MCF7 cells after Giemsa staining. We assessed the tumorigenic potential of cells grown as mammospheres with a clonogenic assay.


RESULTS
Cytogenetic analysis of early passage COX2 transfected MCF7 cells demonstrated significant genomic instability as compared to parental MCF7 cells. COX2 overexpression was associated with a significant increase in chromosomal aberrations (chromatid breaks, chromosome fusions, C anaphase). COX2 transfected MCF7 cells produced a significantly higher level of the anti-apoptotic protein BCL2 than the parental cells. In a functional assay, we found that COX2 expression correlated with increased resistance to doxorubicin. In a complimentary approach to determine tumorigenic potential of cells, we found that COX2 increased the ability of MCF7 cells to grow as mammospheres in culture, which correlated with an increase in clonogenic efficiency.


CONCLUSIONS
We found that COX2 expression in MCF7 breast cancer cells induced genomic instability, BCL2 expression, and doxorubicin resistance, thus making them significantly more tumorigenic. This data suggests that COX-2 may be an important target for breast cancer treatment.
Singh
Balraj
B
Department of Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
Cook
Kendra R
KR
Vincent
Laura
L
Hall
Carol S
CS
Berry
Jacob A
JA
Multani
Asha S
AS
Lucci
Anthony
A
D000230:Adenocarcinoma / Q000201:enzymology*; D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage; D001943:Breast Neoplasms / Q000201:enzymology*; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D003114:Colony-Forming Units Assay; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D020732:Cytogenetic Analysis; D018076:DNA, Complementary; D004317:Doxorubicin / Q000008:administration & dosage; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D005260:Female; D042822:Genomic Instability / Q000502:physiology*; D006801:Humans; D010641:Phenotype; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014162:Transfection
D000903:Antibiotics, Antineoplastic; D018076:DNA, Complementary; D019253:Proto-Oncogene Proteins c-bcl-2; D004317:Doxorubicin; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human