Non-Canonical p16-SP1 Signaling in Head and Neck Cancer Potentially Affects Cell Plasticity

Abstract

We have previously identified a non-canonical role for p16 in head and neck cancer (HNSCC) to repress homologous recombination (HR) via activation of a ubiquitin-mediated pathway. This was potentially via interaction with the transcription factor Sp1, however this interaction, as well as its downstream effects remain to be determined. HPV- HN5 cells forced to overexpress p16 and HPV+ UM-SCC-47 cells p16 CRISPR knockout or shRNA to Sp1 were used. Following modulation, cells were evaluated via clonogenic assay and RNA Sequencing. Additionally, a nude mouse flank model of tumors derived from UM-SCC-47 cells expressing either scrambled control or Sp1 shRNA was performed to evaluate effects on tumor growth (n = 7/group). Clonogenic assay revealed that, as expected, forced expression of p16 in HPV- cells significantly increased radiosensitivity, while CRISPR KO of p16 in HPV+ cells led to radioresistance. Preliminary results indicate a similar pattern observed when Sp1 is inhibited via shRNA. RNASeq data were examined to identify genes likely regulated by p16. Specifically, genes that were significant between p16 modulated cells (either forced expression or CRISPR KO; False Discovery Rate (FDR 1%)) with differences in expression tracking p16 (at least 1.5x higher in cells with intact p16) were evaluated via Ingenuity Pathway Analysis (IPA), with the most significant pathways repressed by p16 including: Stem cell signaling (p = 5e-4), Regulation of EMT (p = 9e-4) and WNT pathways (p = 1.6e-3). Initial results from in vivo studies indicate minimal, non-significant inhibitory effects on tumor growth following inhibition of Sp1 in UM-SCC-47. Confirmatory and radiation experiments are ongoing. Our results indicate that non-canonical functions of p16 may extend beyond direct effects on HR to modulation of aspects of cell differentiation and plasticity. If accurate, p16 may be repressing EMT, contributing to its effects on radiosensitization.