Background: Protein lysine methyltransferases (PKMTs) are a class of histone modifiers that have recently been reported to be mutated, amplified and overexpressed in a variety of cancers and are emerging as attractive targets for drug development. Squamous cell carcinoma of the head and neck (SCCHN) is a common malignancy with a poor prognosis. Recently, we found that WHSC1, a protein methyltransferase of the NSD family of PKMTs (NSD1, WHSC1/NSD2, WHSC1L1/NSD3), is overexpressed in 75% of SCCHN patients. In addition, the TCGA project recently reported that the NSD-family of PKMTs is altered in 26% of SCCHN patients in a mutually exclusive pattern. Given the above and the fact that an upcoming WHSC1-inhibitor is under development, we decided to further investigate the role of WHSC1 as a mediator of oncogenesis and thus as a potential novel therapeutic target for SCCHN.
Methods: Cytotoxicity assays (MTTs) were performed in one HPV-positive and 3 HPV-negative SCCHN cell lines using WHSC1-specific siRNAs. Annexin V assays were performed to evaluate whether cell death was mediated through apoptosis. Cell cycle analysis by flow cytometry was also performed. Immunohistochemistry for WHSC1 and H3K36me2 was conducted in a cohort of 123 patients with SCCHN. To identify downstream targets of WHSC1, cDNA microarrays were conducted in a loss-of-function SCCHN cell line. ChIP assays were performed to assess transcriptional downstream targets directly regulated by WHSC1. Immunoprecipitation assays and mass spectrometry analysis were conducted and candidate substrates were further characterized by in vitro methyltransferase assay.
Results: Knockdown of WHSC1 with WHSC1-specific siRNAs caused significant growth suppression in 4 SCCHN cell lines. Cell death was mediated partially through induction of apoptosis, as evidence by an increase of sub-G1 and annexin V-positive cells. Cell cycle analysis also revealed a significant decrease in the S-phase cells. Survival correlations of WHSC1 in a cohort of 123 SCCHN patients is currently underway. cDNA microarray analysis revealed that knockdown of WHSC1 in a SCCHN cell line was associated with downregulation of genes involved in cellular differentiation, apoptosis and mitosis, including NEK7, MAPK8 and HIPK3, which were validated with qPCR. NEK7 was further validated to be downregulated at the protein level after WHSC1 knockdown. ChIP assay for NEK7 revealed that WHSC1 directly binds to four different gene body regions of NEK7.
Conclusions: Based on these data, it is possible that WHSC1 may function as a driver of oncogenesis in SCCHN, and could thus serve as a novel therapeutic target for this disease. With a WHSC1-inhibitor already under development, advancing our knowledge of the function of WHSC1 and the other NSD-PKMTs in SCCHN and other cancer types with NSD-aberrancies could accelerate the introduction of relevant inhibitors in clinical trials.
Citation Format: Vassiliki Saloura, Makoto Nakakido, Houda Alachkar, Tanguy Seiwert, Mark Lingen, Ezra Cohen, Yusuke Nakamura, Ryuji Hamamoto. Wolf-Hirschhorn syndrome candidate 1 as a potential novel therapeutic target for head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5153. doi:10.1158/1538-7445.AM2014-5153