Decreased STAT1 expression by promoter methylation in squamous cell carcinogenesis.
BACKGROUND
Dysregulation of signal transducers and activators of transcription (STATs) is associated with many cancers, but no role of STAT1 in human tumor progression has been demonstrated.


METHODS
We compared STAT1 protein expression in squamous cell carcinoma of the head and neck (SCCHN) tumors (n = 28) and normal oropharyngeal mucosa samples (n = 10) from patients without cancer as assessed by immunoblotting. Stable clones were established from SCCHN 1483 cells that were transfected with a STAT1 expression construct; cell growth and cisplatin-induced apoptosis of the clones and vector control-transfected 1483 cells were compared using trypan blue exclusion and Annexin V staining and expression of the cyclin-dependent kinase inhibitor p21 was assayed by immunoblotting. The growth of STAT1-overexpressing SCCHN 1483 xenograft tumors was compared with that of xenograft tumors derived from cells transfected with vector control DNA. DNA from SCCHN tumors (n = 16) and paired peripheral blood lymphocytes were analyzed for STAT1 mutations and promoter methylation using methylation-specific polymerase chain reaction and bisulfite sequencing. SCCHN cell lines (PCI-15b, 1483, and UM-22B) were treated with the demethylating agent azacytidine alone or in combination with the cytotoxic drug cisplatin, and expression of STAT1 and p21 were monitored by immunoblotting. All statistical tests were two-sided.


RESULTS
STAT1 levels were statistically significantly lower in the SCCHN tumors than normal mucosa (median = 0.8 relative units versus 2.4, difference = 1.6, 95% confidence interval [CI] = 1.3 to 2.0, P < .001). Overexpression of STAT1 abrogated the growth of SCCHN cells and xenograft tumors and increased p21 expression. STAT1 expression levels of the tumors with STAT1 promoter methylation (n = 12) were lower than those of tumors (n = 4) without promoter methylation of STAT1 (P = .008). Azacytidine treatment increased expression of STAT1 and p21 in SCCHN cell lines and increased apoptosis in cisplatin-treated 1483 cells compared with cisplatin treatment alone (mean = 61.3% versus 25.8%, difference = 35.5%, 95% CI = 24.5% to 43.4%; P = .028).


CONCLUSIONS
STAT1 can function as a tumor suppressor in SCCHN cells. Silencing of the STAT1 gene via promoter methylation may contribute to SCCHN tumor cell growth.
Xi
Sichuan
S
Department of Otolaryngology, University of Pittsburgh School of Medicine, Cancer Institute, Pittsburgh, PA, USA.
Dyer
Kevin F
KF
Kimak
Mark
M
Zhang
Qing
Q
Gooding
William E
WE
Chaillet
J Richard
JR
Chai
Raymond Liu
RL
Ferrell
Robert E
RE
Zamboni
Beth
B
Hunt
Jennifer
J
Grandis
Jennifer Rubin
JR
D000970:Antineoplastic Agents / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000627:therapeutic use; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D019175:DNA Methylation; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D020545:Respiratory Mucosa / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000378:metabolism*; D014162:Transfection; D014183:Transplantation, Heterologous; D015854:Up-Regulation
D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D008156:Luciferases; D002945:Cisplatin