P16 Regulates Transcription Factor Sp1 Function to Affect Outcome in Oropharyngeal Cancer

Abstract

<h3>Purpose/Objective(s)</h3> Our group has previously demonstrated that p16 activates a ubiquitin mediated pathway to repress homologous recombination via activation of HUWE1, potentially via the transcription factor Sp1. However, the specific mechanism of this interaction and its clinical significance remain to be determined. We performed <i>in vitro</i> analysis of p16 and Sp1 interaction in multiple contexts and performed correlative analysis using the head and neck TCGA clinical data, as well as analysis of HPV+ and HPV- oropharyngeal cancer (OPC) treated with chemoradiation (CRT) to evaluate the combined importance of p16 and Sp1 on outcome. We hypothesized that p16 increases the Sp1-HUWE1 interaction. <h3>Materials/Methods</h3> For clinical outcome, 100 patients with oropharyngeal cancer evenly divided between p16+ and p16- and treated with chemoradiation were evaluated for gene expression using targeted multiplex analysis platform. Additional clinical correlative analyses between HPV, p16, HUWE1 and Sp1 were performed using the TCGA Head and Neck cohort (n=530). UM-SCC-47 p16 CRISPR KO and HN5 cells harboring doxycycline-inducible p16 were used to perform <i>in vitro</i> studies. Immunoprecipitation (IP) was used to confirm the protein-protein interaction between protein p16 and transcription factor Sp1. Chromatin immunoprecipitation (ChIP) assay was used to determine the existence of the Sp1 protein-HUWE1 DNA promoter interaction. <h3>Results</h3> Sp1 (p=7.9e⁻¹⁶) and HUWE1 (p=7.9e⁻⁴) were expressed at significantly higher levels in HPV+ tumors and Sp1 and HUWE1 gene expression were highly correlated (p=5.3e10⁻⁴⁵) in the TCGA Head and Neck cohort. In addition, Sp1 expression was significantly higher in p16 positive OPC tumors in a separate patient cohort (p=3e⁻³). In OPC patents treated uniformly with CRT, the combination of p16 and Sp1 expression were associated with reduced locoregional recurrence following CRT (p=7e⁻⁶), effectively delineating good prognosis p16- patients and poor prognosis p16+ patients. IP of p16 in HN5 cells showed increased interaction with Sp1 after doxycycline-induced p16INK4a expression, and in combination with IR. ChIP assay demonstrated SP1 dependent enrichment in 2 of 5 GC box regions of the human HUWE1 promoter. More importantly, these SP1 protein-HUWE1 promoter interactions were significantly enhanced after p16 overexpression. <h3>Conclusion</h3> We show Sp1 directly interacts with HUWE1 promoter resulting in an increase of HUWE1 expression, and this interaction is mediated by p16. This increase of HUWE1 expression then leads to down-regulation of homologous recombination, sensitizing cells to DNA damage. Clinical data shows the combined expression of p16, Sp1 and HUWE1 are highly correlated and that the combination of p16 and Sp1 may be an effective biomarker in OPC.