Purpose Mortality of patients with head and neck squamous cell carcinoma (HNSCC) is primarily driven by tumor cell radioresistance leading to locoregional recurrence (LRR). In this study, we use a classification of TP53 mutation (disruptive vs. nondisruptive) and examine impact on clinical outcomes and radiation sensitivity. Experimental Design Seventy-four patients with HNSCC treated with surgery and postoperative radiation and 38 HNSCC cell lines were assembled; for each, TP53 was sequenced and in vitro radioresistance measured using clonogenic assays. p53 protein expression was inhibited using shRNA and over-expressed using a retrovirus. Radiation-induced apoptosis, mitotic cell death, senescence, and ROS assays were performed. The effect of the drug metformin on overcoming mutant p53-associated radiation resistance was examined in vitro as well as in vivo, using an orthotopic xenograft model. Results Mutant TP53 alone was not predictive of LRR; however, disruptive TP53 mutation strongly predicted LRR (p=0.03). Cell lines with disruptive mutations were significantly more radioresistant (p<0.05). Expression of disruptive TP53 mutations significantly decreased radiation-induced senescence, as measured by SA-beta-gal staining, p21 expression, and release of reactive oxygen species (ROS). The mitochondrial agent metformin potentiated the effects of radiation in the presence of a disruptive TP53 mutation partially via senescence. Examination of our patient cohort showed that LRR was decreased in patients taking metformin. Conclusions Disruptive TP53 mutations in HNSCC tumors predicts for LRR, due to increased radioresistance via the inhibition of senescence. Metformin can serve as a radiosensitizer for HNSCC with disruptive TP53, presaging the possibility of personalizing HNSCC treatment.

Purpose: Histone deacetylase (HDAC) inhibitors have emerged recently as promising anticancer agents. They arrest cells in the cell cycle and induce differentiation and cell death. The antitumor activity of HDAC inhibitors has been linked to their ability to induce gene expression through acetylation of histone and nonhistone proteins. However, it has recently been suggested that HDAC inhibitors may also enhance the activity of other cancer therapeutics, including radiotherapy. The purpose of this study was to evaluate the ability of HDAC inhibitors to radiosensitize human melanoma cells in vitro. Experimental Design: A panel of HDAC inhibitors that included sodium butyrate (NaB), phenylbutyrate, tributyrin, and trichostatin A were tested for their ability to radiosensitize two human melanoma cell lines (A375 and MeWo) using clonogenic cell survival assays. Apoptosis and DNA repair were measured by standard assays. Results: NaB induced hyperacetylation of histone H4 in the two melanoma cell lines and the normal human fibroblasts. NaB radiosensitized both the A375 and MeWo melanoma cell lines, substantially reducing the surviving fraction at 2 Gy (SF2), whereas it had no effect on the normal human fibroblasts. The other HDAC inhibitors, phenylbutyrate, tributyrin, and trichostatin A had significant radiosensitizing effects on both melanoma cell lines tested. NaB modestly enhanced radiation-induced apoptosis that did not correlate with survival but did correlate with functional impairment of DNA repair as determined based on the host cell reactivation assay. Moreover, NaB significantly reduced the expression of the repair-related genes Ku70 and Ku86 and DNA-dependent protein kinase catalytic subunit in melanoma cells at the protein and mRNA levels. Normal human fibroblasts showed no change in DNA repair capacity or levels of DNA repair proteins following NaB treatment. We also examined g-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, g-H2AX foci persisted long after ionizing exposure in the NaB-treated cells. Conclusions: HDAC inhibitors radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation and that g-H2AX phosphorylation can be used as a predictive marker of radioresponse.

The clonogenic cell survival assay determines the ability of a cell to proliferate indefinitely, thereby retaining its reproductive ability to form a large colony or a clone. This cell is then said to be clonogenic. A cell survival curve is therefore defined as a relationship between the dose of the agent used to produce an insult and the fraction of cells retaining their ability to reproduce. Although clonogenic cell survival assays were initially described for studying the effects of radiation on cells and have played an essential role in radiobiology, they are now widely used to examine the effects of agents with potential applications in the clinic. These include, in addition to ionizing radiation, chemotherapy agents such as etoposide and cisplatin, antiangiogenic agents such as endostatin and angiostatin, and cytokines and their receptors, either alone or in combination therapy. Survival curves have been generated for many established cell lines growing in culture. One can use cell lines from various origins including humans and rodents; these cells can be neoplastic or normal. Because survival curves have wide application in evaluating the reproductive integrity of different cells, we provide here the steps involved in setting up a typical experiment using an established cell line in culture.