Profiling gene promoter occupancy of Sox2 in two phenotypically distinct breast cancer cell subsets using chromatin immunoprecipitation and genome-wide promoter microarrays.
BACKGROUND
Aberrant expression of the embryonic stem cell marker Sox2 has been reported in breast cancer (BC). We previously identified two phenotypically distinct BC cell subsets separated based on their differential response to a Sox2 transcription activity reporter, namely the reporter-unresponsive (RU) and the more tumorigenic reporter-responsive (RR) cells. We hypothesized that Sox2, as a transcription factor, contributes to their phenotypic differences by mediating differential gene expression in these two cell subsets.


METHODS
We used chromatin immunoprecipitation and a human genome-wide promoter microarray (ChIP-chip) to determine the promoter occupancies of Sox2 in the MCF7 RU and RR breast cancer cell populations. We validated our findings with conventional chromatin immunoprecipitation, quantitative reverse transcription polymerase chain reaction (qPCR), and western blotting using cell lines, and also performed qPCR using patient RU and RR samples.


RESULTS
We found a largely mutually exclusive profile of gene promoters bound by Sox2 between RU and RR cells derived from MCF7 (1830 and 456 genes, respectively, with only 62 overlapping genes). Sox2 was bound to stem cell- and cancer-associated genes in RR cells. Using quantitative RT-PCR, we confirmed that 15 such genes, including PROM1 (CD133), BMI1, GPR49 (LGR5), and MUC15, were expressed significantly higher in RR cells. Using siRNA knockdown or enforced expression of Sox2, we found that Sox2 directly contributes to the higher expression of these genes in RR cells. Mucin-15, a novel Sox2 downstream target in BC, contributes to the mammosphere formation of BC cells. Parallel findings were observed in the RU and RR cells derived from patient samples.


CONCLUSIONS
In conclusion, our data supports the model that the Sox2 induces differential gene expression in the two distinct cell subsets in BC, and contributes to their phenotypic differences.
Department of Oncology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. karenktjung@ualberta.ca.
Karen
K
Jung
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. pw2@ualberta.ca.
Peng
P
Wang
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. nidhi2@ualberta.ca.
Nidhi
N
Gupta
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. gopal@ualberta.ca.
Keshav
K
Gopal
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. fw2@ualberta.ca.
Fang
F
Wu
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. xiaoxia@ualberta.ca.
Xiaoxia
X
Ye
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. al15@ualberta.ca.
Abdulraheem
A
Alshareef
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. gilbert.bigras@albertahealthservices.ca.
Gilbert
G
Bigras
Department of Oncology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. todd.mcmullen@ualberta.ca.|Department of Surgery, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. todd.mcmullen@ualberta.ca.
Todd P
TP
McMullen
Department of Oncology, McGill University, Montreal, Canada. bassam.abdulkarim@mcgill.ca.
Bassam S
BS
Abdulkarim
Department of Oncology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. rlai@ualberta.ca.|Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. rlai@ualberta.ca.|DynaLIFEDx Medical Laboratories, Edmonton, Canada. rlai@ualberta.ca.
Raymond
R
Lai
D000071916:AC133 Antigen; D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D005260:Female; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D061986:MCF-7 Cells; D009077:Mucins / Q000235:genetics; D010455:Peptides / Q000235:genetics; D010641:Phenotype; D063150:Polycomb Repressive Complex 1 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics* / Q000378:metabolism
D000071916:AC133 Antigen; D015703:Antigens, CD; C069077:BMI1 protein, human; D006023:Glycoproteins; C116884:LGR5 protein, human; C460499:MUC15 protein, human; D009077:Mucins; C000606023:PROM1 protein, human; D010455:Peptides; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D063150:Polycomb Repressive Complex 1