In this study, we determined the in vivo growth and metastatic characteristics of 48 HNSCC cell lines using an orthotopic xenograft nude mouse model of oral tongue carcinoma. A wide range of behavior was observed, from cell lines that formed no tumors to those that formed tumors that grew rapidly and consistently metastasized. We examined the TP53 mutation status and tumor behavior, and showed that cell lines with disruptive TP53 mutations were associated with shorter survival, faster tumor growth, and increased incidence of cervical lymph node metastasis in the orthotopic model of HNSCC.
Our study of 48 HNSCC cell lines in this in vivo model is the most comprehensive to date. We have shown that both tumor growth rate and incidence of cervical lymph node metastasis vary considerably between cell lines, consistent with the range of behavior observed clinically between tumors in individual HNSCC patients. Although we found some correlation between tumor volume and incidence of cervical lymph node metastasis, some of the cell lines with the highest rates of cervical lymph node metastasis showed only a moderate tumor volume, whereas some cell lines with fast tumor growth did not form metastases. These findings suggest that cell characteristics other than proliferation rate contribute to metastasis and that these cell lines can be differentiated by using this orthotopic model. The data presented here provide a valuable resource for future studies of HNSCC tumor progression and metastasis using the orthotopic xenograft model and HNSCC cells.
We chose to study TP53
mutation because of the fundamental role p53 appears to play in HNSCC (20
). After analyzing the TP53
sequence of 48 HNSCC cell lines, we found that only 5 (10.4%) of the cell lines retained the wild-type TP53
sequence; this is a substantially lower proportion than that reported in clinical specimens, which is typically 40–60% (7
). This observation may suggest that there is a selection for TP53
mutations when immortalized cell lines are established, or perhaps the incidence of TP53
mutation in the clinical setting is underreported, for example as a result of the inherent contamination of tumor specimens with normal tissue containing wild-type TP53
. This latter possibility is consistent with findings of a large scale exomic sequencing of oral cavity cancers that we are completing (our unpublished observations).
The strongest evidence to date linking TP53
mutation with outcome in HNSCC has been presented by Poeta et al., who found that disruptive mutations were associated with decreased overall survival (7
). In our study, we consistently observed that disruptive TP53
mutations were significantly associated with shorter survival, greater tumor volume, and increased incidence of cervical lymph node metastasis in the orthotopic xenograft model. These results indicate that disruptive TP53
mutations may play a critical role in the progression and metastasis of HNSCC and support previous clinical reports concluding that disruptive TP53
mutations are a prognostic biomarker for patients with HNSCC.
Despite our evidence supporting the disruptive/non-disruptive classification system, it is recognized that this categorization has a theoretic structural basis which predicts p53 function based on the substituted amino acid’s location and type. We have isolated the p151s mutation, classified as ‘non-disruptive’, and demonstrated that it has significant gain-of-function activity in our model. It is possible that the disruptive categorization merely selects for mutations that are deleterious, and it seems that a more concrete delineation of p53 mutations based on alteration of function or demonstration of one or several consistent gain-of-function mechanisms is necessary. The fact that TP53-null mutations were relatively benign in our study suggests that gain-of-function activity may contribute significantly to the deleterious effect of TP53 mutations associated with the poorest outcome. A large-scale analysis of TP53 mutations is required to identify and characterize the most deleterious mutations from a mechanistic standpoint.
In our study, we found that most of the insertion, deletion, and splice mutations resulted in loss of p53 expression. Therefore, we used the combination of mutation and lack of protein expression to further define a separate, p53 null group. Although previous clinical studies of ovarian cancer have suggested that patients with tumors containing p53 null mutations are at risk for distant metastases and have a poor prognosis (22
), we found that cell lines with null mutations were significantly less aggressive than those in the disruptive TP53
category, suggesting that p53 null mutations are less deleterious than disruptive mutations in HNSCC cells.
A recent publication has reported that truncating TP
53 mutations, defined as nonsense, splice, and frameshift mutations, and largely represented by the “null’ group in our study, are associated with poor prognosis among patients with HNSCC (24
). The discrepancy of our data with this literature may be due to several considerations. The most obvious is that mice in our study are not treated for their disease, whereas patients in the study by Lindenbergher et al. were treated with either surgery or surgery and post-operative radiation. It may be that specific TP53
mutations contribute greatly to tumor formation and progression, whereas other mutations are more deleterious the setting of radiation therapy. A second consideration is that a significant number of patients in the study had oropharyngeal tumors, and though HPV was evaluated in this study, microenvironmental factors specific to the lymphoid-rich oropharynx may contribute to tumor phenotype and thus alter the role of TP53
mutation. Further mechanistic evaluation is required, and our work represents a model in which these questions can be isolated and examined in future studies.
The categorization of mutations into disruptive and non-disruptive is not the only system that has been correlated with outcome in HNSCC. Another classification scheme has been described that is based on p53 transactivation activity in a yeast-based functional assay (6
). The transactivation activity of p53 plays a key role in its tumor suppressor function, and it has been shown that a genetically engineered mouse model carrying a transactivation-deficient TP53
mutation is predisposed to cancer development (26
). Perrone et al. recently reported that oral cavity tumors with wild-type TP53
or mutations that retain partial transactivation ability showed a higher response rate to induction chemotherapy (8
). In the present study, 28 of the cell lines could be categorized by functional status on the basis of transactivation ability. Splice site, deletion, and mutations resulting in a premature stop codon are not classifiable by this system, although the majority would be considered non-functional. We classified these 28 cell lines into fully functional, partially functional, and nonfunctional groups. However, only 2 of the 28 cell lines carried mutations that retained partial transactivation ability, whereas all others were classified as nonfunctional. As expected, in the orthotopic nude mouse model the cell lines with nonfunctional p53 mutations were associated with shorter survival, greater tumor volume, and increased incidence of cervical lymph node metastasis than the cell lines with wild-type or partially functional p53 mutations (data not shown). Because only a few of our panel of cell lines carried mutations that retained partial transactivation ability, it is difficult to draw conclusions about the role of partially functional mutations in our system from these results. The true impact of partially functional mutations in HNSCC remains to be elucidated, and this could perhaps be further studied in the preclinical model we describe here if more cell lines with partially functional p53 mutations are identified or created.
There are some discrepancies between TP53
mutations identified in the present study and those described in previous reports evaluating the same HNSCC cell lines (Supplementary Table S3
). In the current study, all of our cell lines were received from the American Type Culture Collection or requested directly from the laboratory from which they originated, and each line was validated using STR genotyping confirmed against that obtained by the originators (19
). We also sequenced TP53
twice in independent laboratories for each line, and confirmed p53 protein expression using western blotting. The inconsistencies between our report and those previously reported may reflect the differences of the procedures to detect TP53
status in HNSCC cells. Alternatively, these discrepancies may be the result of cell line contamination or mis-identification in the previous reports. Discrepancies among published works reporting TP53
status in cell lines have been reviewed, and this unfortunately occurs frequently (17
). Although we cannot resolve discrepancies between existing literature and our report, we attest to undertaking rigorous cell line authentification (19
) and TP53
evaluation in our study.
In summary, we have demonstrated the utility of the orthotopic xenograft model by characterizing the largest panel of HNSCC cell lines to date. Our results have corroborated clinical reports showing that disruptive TP53 mutations correlate with aggressive tumor characteristics. Furthermore, we have proposed a new approach to classify TP53 mutations by combining TP53 sequence information with p53 protein expression levels. These results suggest that disruptive TP53 mutations may play a key role in the tumor progression and cervical lymph node metastatic potential of HNSCC. Our work should help facilitate further studies examining the cellular and molecular mechanisms of TP53 mutations in HNSCC tumor progression, metastasis, and response to therapy.