SUMMARY Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. The DNA binding protein CTCF regulates a diverse array of epigenetic processes and is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation, and chemically induced cancer in a broad range of tissues. Ctcf+/− tumors are characterized by increased aggressiveness including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf+/− tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome-wide. These findings establish CTCF as a prominent tumor suppressor gene and point to CTCF mediated epigenetic stability as a major barrier to neoplastic progression.
Purpose To identify novel therapeutic drug targets for p53 mutant head and neck squamous cell carcinoma (HNSCC). Experimental Design RNAi kinome viability screens were performed on HNSCC cells including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was utilized to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets utilizing multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition utilizing a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Results Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2/M cell cycle checkpoint, SFK, PI3K and FAK pathways. RNAi mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53 mutant HNSCC xenograft model. Conclusions WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer.
p27kip1 is a cyclin-dependent kinase inhibitor and a tumor suppressor. In some tumors, p27 suppresses tumor growth by inhibition of cell proliferation. However, this is not universally observed, implying additional mechanisms of tumor suppression by p27. p27-deficient mice are particularly susceptibility to genotoxininduced tumors, suggesting a role for p27 in the DNA damage response. To test this hypothesis, we measured genotoxin-induced mutations and chromosome damage in p27-deficient mice. Both p27 ؉/؊ and p27 ؊/؊ mice displayed a higher N-ethyl-N-nitrosourea-induced mutation frequency in the colon than p27 ؉/؉ littermates. Furthermore, cells from irradiated p27-deficient mice exhibited a higher number of chromatid breaks and showed modestly increased micronucleus formation compared to cells from wild-type littermates. To determine if this mutator phenotype was related to the cell cycle-inhibitory function of p27, we measured cell cycle arrest in response to DNA damage. Both normal and tumor cells from p27-deficient mice showed impaired G 2 /M arrest following low doses of ionizing radiation. Thus, p27 may inhibit tumor development through two mechanisms. The first is by reducing the proliferation of cells that have already sustained an oncogenic lesion. The second is by transient inhibition of cell cycle progression following genotoxic insult, thereby minimizing chromosome damage and fixation of mutations.
MYC-induced DNA damage is exacerbated in WRN deficient cells, leading to replication stress and accelerated cellular senescence. To determine if WRN deficiency impairs MYC driven tumor development, we utilized both xenograft and autochthonous tumor models. Conditional silencing of WRN expression in c-MYC overexpressing non-small cell lung cancer xenografts impaired both tumor establishment and tumor growth. This inhibitory effect of WRN knock-down was accompanied by increased DNA damage, decreased proliferation, and tumor necrosis. In the Eμ-Myc mouse model of B-cell lymphoma, a germline mutation in the helicase domain of Wrn (WrnΔhel/Δhel) resulted in a significant delay in emergence of lethal lymphomas, extending tumor free survival by >30%. Analysis of pre-neoplastic B cells from Eμ-Myc Wrn mutant mice revealed increased DNA damage, elevation of senescence markers, and decreased proliferation in comparison with cells from age-matched Eμ-Myc mice. Immunohistochemical and global gene expression analysis of overt Eμ-Myc WrnΔhel/Δhel lymphomas demonstrated a marked increase in expression of the CDK inhibitor, p16Ink4a, as well as elevation of TAp63, a known mediator of senescence. Collectively, these studies demonstrate that in the context of Myc-associated tumorigenesis, loss of Wrn amplifies the DNA damage response, both in pre-neoplastic and neoplastic tissue, engaging activation of tumor suppressor pathways. This leads to inhibition of tumor growth and prolonged tumor free survival. Targeting WRN or its enzymatic function could prove to be an effective strategy in the treatment of MYC-associated cancers.
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