Head and neck squamous cell carcinoma (HNSCC) often presents clinically past stage II and has a poor 5- year survival (52-62%). Late stage presentation indicates more systemic therapy and radiation in addition to surgery. Previous studies have demonstrated that the numbers of tumor infiltrating lymphocytes (TILs), predict patient prognosis, however, these studies fail to identify the role of natural killer (NK) cells. NK cell number positively correlates with increased survival. HNSCC patients have decreased NK cell infiltration. Despite indications of NK importance in HNSCC, mechanisms of NK suppression are not well understood. We previously reported that collagen type XI alpha I (COL11A1) is overexpressed in HNSCC tumors and contributes to tumor progression. Mutations in COL11A1 in patients with Marshall or Stickler syndrome are associated with inflammation. We hypothesize that COL11A1 is associated with NK cell suppression. The putative receptor for COL11A1 and downstream signaling was determined. Further we correlated expression of COL11A1 and its signal transducers with NK cell signatures and activity.
Gene expression and clinical data from primary tumors (n=522) was downloaded from The Cancer Genome Atlas (TCGA). Multiple genes were correlated using Spearman R correlation and statistical significance calculated using a Mann-Whitney test. CIBERSORT was used to analyze tumor immune cell infiltrates based on TCGA sequencing data. Following CIBERSORT analysis, NK cell activity was correlated with COL11A1 expression for each patient and plot as NK activity vs. mean COL11A1 expression. In vitro studies were performed using established HNSCC cell line UMSCC-1 and immortalized NK cell line NK92MI. UMSCC-1 cells were transduced with a luciferase reporter and co-cultured with NK92MI cells. NK cytotoxic activity was measured by the amount of luciferase released from UMSCC-1 cells treated with siCOL11A1 or sicontrol. Percent specific lysis was calculated using the equation [(test luc release − spontaneous luc release)/(maximum luc release − spontaneous luc release)] × 100. Protein sequences for various known collagen receptors were screened for the consensus binding sites to COL11A1. Immunoblotting was used to determine the downstream signaling mediators of COL11A1 in HNSCC.
COL11A1 expression was significantly increased by tumor stage (T1 vs. T4; p=0.03, T3 vs. T4 p=0.01), clinical stage (stage III vs. stage IV; p=0.02). Patients with low NK cell activity had significantly higher COL11A1 expression compared to patients with high NK cell activity (NKactive vs NKinactive; p=0.002). COL11A1 expression was positively correlated with Hippo/YAP pathway target genes COL11A1 vs. CTGF (R=0.67; p<0.001), COL11A1 vs. CYR61 (R=0.43; p<0.001). Specific lysis of HNSCC cells by NK92MI cells was increased by 20% in the COL11A1 knockdown (siCOL11A1) cells compared to sicontrol cells. The consensus binding sequence of ITGA2B1 was identified in the COL11A1 protein sequence. COL11A1 influenced Hippo signaling in HNSCC regulating levels of immune evasive markers.
COL11A1 expression is increased by clinical and tumor stage in HNSCC suggesting its expression influences tumor progression. Further, COL11A1 is positively associated with Hippo/Yap pathway target genes CTGF and CYR61, and correlates with decreased NK cell activity. Targeting COL11A1 in HNSCC may improve antitumor immune responses in HNSCC.