Wilms' tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck.
BACKGROUND
Wilms' tumor gene 1 (WT1) can act as a suppressor or activator of tumourigenesis in different types of human malignancies. The role of WT1 in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Overexpression of WT1 has been reported in SCCHN, suggesting a possible oncogenic role for WT1. In the present study we aimed at investigating the function of WT1 and its previously identified protein partners p63 and p53 in the SCCHN cell line FaDu.


METHODS
Silencing RNA (siRNA) technology was applied to knockdown of WT1, p63 and p53 in FaDu cells. Cell proliferation was detected using MTT assay. Chromatin immunoprecipitation (ChIP)/PCR analysis was performed to confirm the effect of WT1 on the p63 promoter. Protein co-immunoprecipitation (co-IP) was used to find protein interaction between WT1 and p53/p63. Microarray analysis was used to identify changes of gene expression in response to knockdown of either WT1 or p63. WT1 RNA level was detected using real-time quantitative PCR (RT-qPCR) in patients with SCCHN.


RESULTS
We found that WT1 and p63 promoted cell proliferation, while mutant p53 (R248L) possessed the ability to suppress cell proliferation. We reported a novel positive correlation between WT1 and p63 expression. Subsequently, p63 was identified as a WT1 target gene. Furthermore, expression of 18 genes involved in cell proliferation, cell cycle regulation and DNA replication was significantly altered by downregulation of WT1 and p63 expression. Several known WT1 and p63 target genes were affected by WT1 knockdown. Protein interaction was demonstrated between WT1 and p53 but not between WT1 and p63. Additionally, high WT1 mRNA levels were detected in SCCHN patient samples.


CONCLUSIONS
Our findings suggest that WT1 and p63 act as oncogenes in SCCHN, affecting multiple genes involved in cancer cell growth.
Department of Medical Biosciences, Clinical Chemistry, Umeå University, By 6 M, 2nd floor, Umeå, 90185, Sweden. xingru.li@medbio.umu.se.
Xingru
X
Li
Department of Medical Biosciences, Clinical Chemistry, Umeå University, By 6 M, 2nd floor, Umeå, 90185, Sweden. soot0002@student.umu.se.
Sofia
S
Ottosson
Department of Medical Biosciences, Clinical Chemistry, Umeå University, By 6 M, 2nd floor, Umeå, 90185, Sweden. sihan.wang@medbio.umu.se.
Sihan
S
Wang
Department of Medical Biosciences, Pathology, Umeå University, By 6 M, 2nd floor, Umeå, 90185, Sweden. emma.jernberg@medbio.umu.se.
Emma
E
Jernberg
Department of Medical Biosciences, Pathology, Umeå University, By 6 M, 2nd floor, Umeå, 90185, Sweden. linda.boldrup@medbio.umu.se.
Linda
L
Boldrup
Department of Medical Biosciences, Pathology, Umeå University, By 6 M, 2nd floor, Umeå, 90185, Sweden. xiaolian.gu@medbio.umu.se.
Xiaolian
X
Gu
Department of Medical Biosciences, Pathology, Umeå University, By 6 M, 2nd floor, Umeå, 90185, Sweden. karin.nylander@medbio.umu.se.
Karin
K
Nylander
Department of Medical Biosciences, Clinical Chemistry, Umeå University, By 6 M, 2nd floor, Umeå, 90185, Sweden. aihong.li@medbio.umu.se.
Aihong
A
Li
D002294:Carcinoma, Squamous Cell / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000502:physiology*; D016162:Genes, Wilms Tumor / Q000502:physiology*; D006258:Head and Neck Neoplasms / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D008565:Membrane Proteins / Q000502:physiology*; D000077195:Squamous Cell Carcinoma of Head and Neck
C081876:CKAP4 protein, human; D008565:Membrane Proteins