Combined TP53 mutation/3p loss correlates with decreased radiosensitivity and increased matrix-metalloproteinase activity in head and neck carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a source of significant morbidity and mortality worldwide1. Well-established risk factors for HNSCC include tobacco, alcohol, and human papilloma virus (HPV)1. Current staging and clinical management HNSCC patients are based on anatomical location and size2. If patients undergo surgical excision, management decisions are guided by presence or absence of bony invasion, lymph-node metastasis, or lymph-node extracapsular spread. Patients with locally advanced disease are treated with multimodality therapy including chemotherapy, radiotherapy and surgery. Many prevalent malignancies such as breast, colon, lung, or prostate cancer have diagnostic assays based on genomic profiling, HER-2 and estrogen receptor expression, or ALK, EGFR and KRAS mutation. HPV has recently been identified as being responsible for the increasing incidence of oropharyngeal SCC (OPSCC)3,4, and HPV positive (HPV+) tumor status is currently the strongest prognostic factor for these patients4,5. However, there are currently no widely adopted prognostic assays for HPV-negative (HPV−) HNSCC. We previously analyzed The Cancer Genomic Atlas (TCGA), which contains the largest collection of patient HNSCC tumor specimens6. In HPV− tumors, reduced survival outcomes associated with tumor protein p53 (TP53) mutation occur only in combination with loss of chromosome 3p6. In these patients, median survival decreased from >5 years for TP53 mutations to only 1.7 years for both mutational events (“double-hit”)6. These findings are independent of clinical stage and replicated in an independent HNSCC cohort6. In HPV+ tumors, we believe E6-mediated inactivation of TP53 also acts in synergy with 3p deletion to produce poor survival outcomes6. Our prior analysis of the TCGA demonstrated that of the genes on the 3p chromosome, FHIT, a known tumor suppressor gene involved in aerodigestive cancer, best correlates with 3p status6. The FHIT gene is located on a chromosomal fragile site, and loss of the 3p arm is most commonly mediated by chromosome breakage at this site7. Thus, 3p deletion is commonly associated with decreased FHIT protein expression or reduced copy number at the 3p.14.2 FHIT locus7. Given these findings, our analysis in this study uses FHIT copy number as representative of 3p status and thus a proxy for “single hit” (TP53 only) vs “double hit” (TP53 and FHIT deletion) in HNSCC cell lines. It has also been shown that FHIT and p53 status are linked in development of lung cancer, although little is known about the molecular nature of this interaction8. Here, we analyze data from the TCGA to examine whether TP53 and 3p status are linked in HNSCC patients. The etiology for worse clinical outcomes in TCGA patients with “double-hit” cancers is unclear. Potential explanations include greater resistance to radiation monotherapy or more aggressive tumor biology in cancers with the “double hit.” We do not anticipate a difference in cisplatin sensitivity given its lack of efficacy as monotherapy in clinical practice. To examine these hypotheses, we obtained four human tongue squamous cell carcinoma cell lines from American Type Culture Collection (ATCC). We utilized the Cancer Cell Line Encyclopedia (CCLE) to examine differences in their TP53 mutational and FHIT copy number status, corresponding with the prognostic markers identified in the TCGA. To compare radiosensitivity in-vitro, we utilized a comet-tail assay to measure each cell line’s ability to repair ionizing radiation-induced double-stranded deoxyribonucleotide (DNA) damage. FHIT knockout has been previously associated with increased activation of the ATR/CHK1 pathway and stronger cell-cycle checkpoint responses, resulting in greater cellular repair in response to ionizing radiation9. We would thus expect reduced radiation-induced DNA damage in “double-hit” lines with FHIT deletion compared to “single-hit” lines with intact FHIT. We also examined the aggressiveness of “single-hit” versus “double-hit” cell lines by comparing their respective matrix-metalloproteinase (MMP) activity levels. MMP expression has previously been associated with tumor aggressiveness and patient prognosis in head and neck cancer15–19. Absolute levels of MMP2/9 have been used to differentiate between benign papillomas and carcinoma of the larynx15. Increased MMP2/9 expression has been correlated with higher cancer grade16 and reduced survival in HNSCC17–18. In tongue SCC, increased MMP2/9 expression directly correlates with incidence of lymph node metastases19. Recently, our own work has demonstrated that MMP over-expression in HPV+ tumors predicts poorer survival14. Additionally, HPV− tumors, which have a more aggressive phenotype compared to their HPV+ counterparts, also express higher levels of MMP14. We compared MMP activity in our cell lines using previously described ratiometric activatable cell-penetrating peptides (RACPP)10–12. The RACPP relies on tumor-associated MMP2/9 to cleave an intervening linker, resulting in increased Cy5/Cy7 fluorescent signal ratio, and has previously been used for margin detection during surgery12–14.