Genomic Analysis of Head and Neck Squamous Cell Carcinoma Cell Lines and Human Tumors: A Rational Approach to Preclinical Model Selection

This article is featured in Highlights of This Issue, p. 477

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, arising in the upper aerodigestive tract, including the oral cavity, pharynx, and larynx. HNSCC, with more than 600,000 new cases diagnosed annually, is often lethal with only 40% to 50% of HNSCC patients surviving 5 years after their diagnosis (1). Risk factors for HNSCC include tobacco and alcohol use. Infection with the human papilloma virus (HPV) is increasingly identified as a strong contributing factor, especially in oropharyngeal cancer (1, 2). Inherited disorders such as Fanconi anemia are also associated with increased susceptibility to HNSCC (3).

HNSCC cell lines have been developed in many laboratories worldwide (4). Improved understanding of the biologic underpinnings of HNSCC and a more rational approach to therapy can best be accomplished through the judicious use of models that reproduce key features of human tumors. The use of cell lines derived from human tumors dates back to derivation of HeLa cells, a robust cancer cell line developed from a cervical cancer afflicting a patient named Henrietta Lacks in 1951 (5). In 1990, cancer cell lines encompassing nine tumor types were used as preclinical models for drug-screening efforts known as the NCI60 collection. To date, more than 10,000 different potential therapeutic agents have been tested using these models (6). Although the NCI60 contains a wide variety of cancer types, the collection lacks HNSCC cell lines. In 2010, the genomic profiling and pharmacologic data for 311 cancer cell lines was released by GlaxoSmithKline via the National Cancer Institute's cancer Bioinformatics Grid (caBIG), including 10 HNSCC cell lines (7). However, none of these HNSCC cell lines contained mutations in the six genes analyzed (HRAS, KRAS, NRAS, BRAF, PIK3CA, and PTEN). Although larger cell line panels have been used for compound screening, no study to date has focused on genetically characterized HNSCC cell line panels.

The Cancer Cell Line Encyclopedia (CCLE), a comprehensive database including the mutational status of more than 1,600 genes, gene expression, and chromosome copy-number data across a panel of 947 human cancer cell lines from 36 different tissue types was recently reported by Barretina and colleagues (8). Similar to the NCI60 collection, pharmacologic profiling of 24 different anticancer drugs and compounds was carried out in 479 of these cell lines. The profiling focused on identifying preclinical, genetic indicators of sensitivity to specific compounds (8). Thirty-two HNSCC cell lines were included in the CCLE and sequencing data were reported for 30 of these models. Drug-sensitivity profiling was carried out in six of the HNSCC cell lines. In addition to the CCLE, Garnett and colleagues presented their systematic identification of genomic markers of drug sensitivity in cancer cells, which is a repository of mutation profiles of 639 cell lines, including 20 HNSCC cell lines and 130 drugs for screening across the majority of these cell lines. Eleven of these HNSCC cell lines and 25 of the 130 drugs were also studied in the CCLE. In contrast with the high-throughput sequencing approach used in the CCLE, the Garnett and colleagues database only resequenced the full coding exons for 77 genes identified in their Cancer Gene Census (9).

Cancer cell lines are typically used as preclinical models for mechanistic studies. However, the potential for established HNSCC cell lines to reflect the genetic alterations found in human HNSCC tumors has not been thoroughly investigated. Two recent studies have profiled the mutations, using whole-exome sequencing, in 106 unique HNSCC tumor samples (10, 11). These efforts revealed a number of oncogenes implicated in the pathogenesis of HNSCC (10–12). The present study was undertaken to compare the gene mutation frequencies between HNSCC human tumors and cell lines to facilitate the rational selection of cell line models for translational HNSCC research.

Databases used in this article are publicly available. The five cohorts are included:

1. HNSCC cell lines genomic and pharmacologic profiling from Barretina and colleagues database (http://www.nature.com/nature/journal/v483/n7391/full/nature11003.html and http://www.broadinstitute.org/ccle/home).

2. HNSCC cell lines genomic and pharmacologic profiling from Garnett and colleagues database (http://www.nature.com/nature/journal/v483/n7391/full/nature11005.html).

3. HNSCC tumors genomic profiling from Stransky and colleagues database (http://www.sciencemag.org/content/333/6046/1157).

4. HNSCC tumors genomic profiling from Agrawal and colleagues database (http://www.sciencemag.org/content/333/6046/1154.full).

5. The Cancer Genome Atlas (TCGA) database (http://www.cbioportal.org/public-portal/).

We used logistic regression to estimate the effects of source (cell line or tumor) and site of disease (oral cavity, pharynx, and larynx) on mutation frequency. A test of interaction between source and site was conducted. No interaction was detected for any gene, thereby prompting tests of main effects. Significant effects were reported for any genes satisfying a maximum 10% expected false discovery rate. To tabulate mutations in common, we used a comparison program, created using Microsoft Excel. The mutations (nonsynonymous mutation) and gene copy numbers in HNSCC cell lines were compared with those in HNSCC tumors side by side by this comparison program. The use of this comparison program was reported previously (13). The gene copy-number analysis used log₂ ratio as described in ref. (8). The log₂ ratio of normal (copy-neutral) clones is log₂(2/2) = 0, single copy losses is log₂(1/2) = −1, and single copy gains is log₂(3/2) = 0.58. The effects of drug treatment versus dimethyl sulfoxide (DMSO) control was repeated three times and compared using an unpaired t test with Welch's correction (P < 0.05 as statistically significant).

The HNSCC cell lines, CAL-27, and SCC-9 cells were obtained from the American Type Culture Collection (ATCC). All cell lines were genotypically verified. HNSCC cell lines were cultured in the respective culture medium containing 10% fetal calf serum, 1× penicillin/streptomycin solution (Invitrogen): CAL-27 in Dulbecco's Modified Eagle Medium (DMEM), SCC-9 in DMEM/F12 with 0.4 μg/mL hydrocortisone (Mediatech, Inc). All cell lines were maintained in a humidified cell incubator at 37°C, 5% CO₂.

Cloning and mutagenesis were performed as previously described (13).

HNSCC cells were plated at 20% confluency in T25 flasks 24 hours before transfection. Then, cells were transfected with either pMXspuro-PIK3CA wild-type (WT) or pMXs-puro-PIK3CA mutants (H1047R) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Twenty-four hours after transfection, 1 × 10⁴ cells were plated in 48-well culture plates 24 hours before drug treatment. Cells were treated with either 2.0 μmol/L or 8.0 μ mol/L erlotinib for 48 hours. MTT assay was performed.

Thirty-nine HNSCC cell lines were included in the reports by Barretina and colleagues and Garnett and colleagues. Eleven cell lines are common to both data sets while Barretina and colleagues included 30 unique HNSCC cell lines and Garnett and colleagues reported on 20 additional HNSCC cell lines (Fig. 1). Clinical and pathologic information from these 39 HNSCC cell lines is summarized in Table 1 and Supplementary Table S1. The mean age of the 28 patients with known age from whom the cell lines were derived was 58.2 years. Thirty (83% of known gender) patients were male, 6 (17% of known gender) were female, and 3 patients have no reported information for sex. The oral cavity is the most highly represented anatomic tumor site, accounting for 68% of the cell lines with known primary site. Pharyngeal tumors account for four (12%) of the cell lines, six (18%) of the cell lines were derived from the larynx, one (3%) cell line was from the nasal septum, and the anatomic site of the remaining five are unknown. Only three cell lines are reported to be HPV-negative and the remaining cell lines have no reported HPV status. Given the paucity of confirmed HPV-positive HNSCC cell lines, it is likely that none of the sequenced HNSCC cell lines are derived from HPV-positive HNSCC (14). Although the clinical and pathologic information available for the sequenced HNSCC cell lines is incomplete, the age and male predominance reflects most HNSCC clinical cohorts. Cell lines are generally derived from resected tumor specimens. Given the increasing tendency over the past few decades to treat tumors of the pharynx and larynx primarily with nonoperative therapy, it is not surprising that the majority of cell lines are derived from oral cavity tumors where the primary treatment strategy is surgical resection. Similarly, because the HPV-HNSCC tumors are largely pharyngeal (especially tonsil) cancers (14), the small number of cell lines derived from tumors arising in the pharynx likely contributes to the limited availability of HPV-HNSCC cell line models.

Cancer is thought to arise, at least in part, by the accumulation of genetic mutations, including activating oncogenes and inactivating tumor-suppressor genes (15–17). However, the dynamic evolution of mutation calling algorithms and varying approaches between institutions can limit the ability to compare sequencing results. We first compared the methods to generate the genetic data including HNSCC cell line data in these two cancer cell line databases and HNSCC primary tumor data in the two reported human cohorts (8–11). All of the studies used Affymetrix SNP6.0 arrays to elucidate genotypes. For sequencing, the cell line samples in the CCLE and the 74 tumor samples from our prior study were both performed at the Broad Institute on Illumina instruments and generated gene profiles using similar strategies (7, 9). Seventeen tumor samples in the other human HNSCC cohort were also sequenced using Illumina, whereas 15 tumor samples were performed by SOLiD (Table 2; ref. 10). Barretina and colleagues provide genomic data for 947 cell lines, including gene mutation status of 1,651 cancer-related genes for 30 HNSCC cell lines (Table 2). These cell lines harbored 1,637 mutations in 654 genes. Garnett and colleagues reported markedly fewer mutations for each cell line with 49 mutations in seven genes as only selected 77 genes were sequenced in their project. A total of 13 genes were mutated in more than 50% of the HNSCC cell lines evaluated (Supplementary Table S2). Human HNSCC tumors from the Stranksy and colleagues cohort harbored 7,165 mutations in 4,897 genes, whereas the human HNSCC tumors in the Agrawal and colleagues cohort contained 609 mutations in 561 genes.

In an attempt to further determine the potential of immortalized HNSCC cell lines to reflect the underlying biology of human HNSCC tumors by gene mutation status, the mutation rates of specific genes in HNSCC tumors and cell lines were compared. Here, we analyzed the HNSCC cell line mutation data from Barretina and colleagues and Garnett and colleagues databases as a cell line panel and the two human HNSCC tumor cohorts as a human tumor panel. Of note, 334 genes were found in both HNSCC cell lines and human tumors, representing 51% of the mutated genes in HNSCC cell lines (Fig. 2A). The 15 most commonly mutated genes in HNSCC cell lines are depicted in Table 3. TP53 is the most commonly mutated gene both in HNSCC tumors and cell lines [64.2% (68 of 106) of human tumors and 84.6% (33 of 39) of cell lines; Supplementary Table S3]. We noted that almost all patient tumors (103 of 106) and most cell lines (33 of 39) are from oral cavity, pharynx, or larynx (Supplementary Table S4). We analyzed the mutant genes with frequencies more than 5% in both human HNSCC cell lines and human tumors from these anatomic sites. The mutation frequencies were computed and tested for differences with logistic regression adjusting for disease site. The mutation rates of 19 genes were not different in HNSCC cell lines and primary tumors, whereas four genes had greater frequency of mutation among the tested cell lines compared with tumor specimens with an expected false discovery to 10% or less, including CDKN2A (42% in cell lines vs. 11% in tumors), FMN2 (30% in cell lines vs. 6% in tumors), MLL3 (30% in cell lines vs. 6% in tumors), and TTN (61% in cell lines vs. 28% in tumors; Fig. 2B and Supplementary Table S5). We further compared the gene mutation status between the HNSCC cell lines and tumors according to the primary anatomic tumor site. There were seven genes with different mutation rates by site, including CSMD3, CUBN, NAV3, NSD1, PKHD1, TP53, and TTN. TTN was the only gene with a mutation rate differing by both source cell line versus human tumor and anatomic site (Supplementary Fig. S1). There was no interaction between source and site, indicating that differences in mutation frequency by site are consistent across source and vice versa.

Some genes, however, were only mutated in HNSCC cell lines, indicating a discordance between HNSCC human tumors and cell line models. Seventeen genes mutated in more than 10% of HNSCC cell lines were never mutated in the human tumors (Table 4), whereas 11 genes were only mutated in human HNSCC tumors with frequencies more than 10% but never reported in cell lines (Supplementary Table S6). It is noteworthy that six genes were mutated in more than 50% of cell lines but never mutated in tumors including VEGFC, PRKDC, GRIA3, MAML3, GPR112, and NEK3.

Analysis of DNA copy numbers in HNSCC cell lines in the two cell lines database revealed 194 amplified genes (log₂ > 0.58), including 40 genes amplified in more than 50% of HNSCC cell lines, whereas 60 genes were deleted (log₂ < −1) in these cell lines. Because the human HNSCC tumor studies only reported gene copy alterations of 14 genes including 13 amplified genes and one deleted gene, we also compared the gene copy-number alterations in HNSCC cell lines and in tumor samples in TCGA (The Cancer Genome Atlas) cohort. Only eight genes were amplified (PIK3CA, EGFR, CCND2, KDM5A, ERBB2, PMS1, FGFR1, and WHSCIL1) and five genes were deleted (CDKN2A, SMAD4, NOTCH2, NRAS, and TRIM33) in both cell lines and tumors (Fig. 2C). All of these genes had higher alteration frequencies in HNSCC cell lines compared with primary tumors. These cumulative findings suggest that mutations and gene copy-number alterations exist in HNSCC cell lines, but not in human tumors, and may result from selection and/or propagation in tissue culture that does not reflect critical biologic properties of this cancer. In addition, tumor cells containing selected HNSCC mutations may not survive the process of cell line selection and can only be modeled by cell line engineering.

The EGF receptor (EGFR) is a member of the type 1 tyrosine kinase family and is recognized as a key regulator of cellular differentiation and proliferation by activating downstream signal transduction pathways, including the Ras–Raf–MAPK pathway and phosphatidylinositol-3 kinases (PI3K). Increased EGFR expression, by gene amplification and transcriptional activation, is one of the most frequent alterations found in HNSCC in which EGFR has been identified as a prognostic biomarker and molecular therapeutic target (18). Although EGFR overexpression is one of the most common alterations found in HNSCC, the incidence of EGFR mutations in HNSCC is negligible (19, 20). We defined the EGFR pathway and found 10 genes in the pathway mutated in both HNSCC cell lines and human tumors (Fig. 3). Genes in the Ras–Raf–MAPK pathway, including HRAS, KRAS, RAF1, and MAPK1 (ERK1) were mutated both in HNSCC cell lines and tumors, whereas N-RAS and MAP2K1 (MEK1) were only mutated in HNSCC cell lines and B-RAF was only mutated in HNSCC tumors. In the PI3K pathway, GAB1, PI3K p110 subunits (PIK3CA, PIK3CD, and PIK3CG), three MAP3Ks (MAP3K3, MAP3K4, and MAP3K6), and MAP2K4 (MEK4) were mutated in both cell lines and tumors, whereas MAPK8 (JNK1) was only mutated in HNSCC cell lines and MAPK9 (JNK2) mutations were restricted to HNSCC tumors. STAT1 was mutated both in HNSCC tumors and cell lines, whereas STAT3 mutation was only found in HNSCC tumors (Supplementary Table S7). These results suggest that mutations of genes in the EGFR signaling pathway may contribute to HNSCC either in tumorigenesis or tumor growth despite the paucity of EGFR mutations.

Six agents have been U.S. Food and Drug Administration (FDA)–approved for the treatment of HNSCC, including methotrexate (approved in 1956), 5-FU (5-fluorouracil; approved in 1957), bleomycin (approved in 1973 and rarely used in modern oncology), cisplatin (approved in 1978), and docetaxel and cetuximab (both approved in 2006). Six HNSCC cell lines were included in the pharmacologic profiling in the Barretina and colleagues database, including Cal 27, Detroit 562, FaDu, HSC-2, SCC9, and SCC25 using 24 chemotherapy and molecular-targeting agents (8). We analyzed the responses of these HNSCC cell lines to the 24 compounds by the activity area (defined as the area between the pharmacologic dose–response curve and a fixed reference A_ref = 0. Using the fixed reference, activity area = 0 corresponds to an inactive compound; ref. 8). Paclitaxel (targeting TUBB1), panobinostat (targeting HDAC), 17-AAG (targeting HSP90), and irinotecan (targeting TOP2) most potently inhibited the growth of HNSCC cell lines. However, HNSCC cell lines were resistant to PLX4720 (targeting BRAF), PF2341066 (targeting c-MET), PD-0332991 (targeting CDK4), sorafenib (a multikinase inhibitor), nultin-3 (targeting MDM2), and L-685458 (targeting Gamma Secretase; Fig. 4A). Compared with cell lines derived from other organ sites, HNSCC cell lines were significantly more sensitive to the EGFR tyrosine kinase inhibitor (TKI) erlotinib (P = 0.002). The pharmacologic data in Garnett and colleagues cohort also confirmed this finding. We then further analyzed the drug responses of these six HNSCC cell lines to EGFR inhibitors. Among the six HNSCC cell lines tested, HSC-2 and Detroit 562, derived from tumors of the oral cavity and pharynx, respectively, were resistant to the five compounds including AZD0530 (targeting Src family kinases), erlotinib (targeting EGFR), lapatinib (targeting HER2 and EGFR), TKI 258 (targeting EGFR), and ZD-6474 [targeting EGFR and VEGFR (VEGF receptor); Fig. 4B]. To determine the correlation between gene mutations and drug sensitivity, we compared the genomic profile of HSC-2 and Detroit 562 with the other four HNSCC cell lines profiled. Interestingly, PIK3CA was only mutated in HSC-2 and Detroit 562 cell lines, whereas the other four cell lines harbored WT PIK3CA. To determine if PIK3CA mutation contributes to sensitivity to EGFR-targeting agents, we introduced a PIK3CA mutation (H1047R) or WT PIK3CA into Cal 27 and SCC9 cells that harbor WT PIK3CA and assessed the impact of PIK3CA mutation on drug sensitivity. Cal 27 and SCC9-expressing mutant (H1047R) or WT PIK3CA were treated with erlotinib. Both Cal 27 and SCC9-expressing mutant PIK3CA were significantly more resistant to erlotinib treatment at both concentrations tested, comparing with isogenic cells expressing WT PIK3CA (Fig. 4C). These results indicate that PIK3CA mutation might serve as a negative predictive biomarker for erlotinib in HNSCC, indicating that the mutational status of PIK3CA may be used to select preclinical models for response to EGFR TKI.

Human tumor-derived cell lines serve as important preclinical models to identify therapeutic targets and mechanisms of anticancer agents for translational studies. The first high-throughput cancer cell line screening program was the NCI60 platform, which led to the development of many new technologies for drug screening from 1984 to 2005 (18). However, this platform is limited by the lack of representation of cell lines derived from a number of human tumors, including HNSCC. The reduced cost of gene sequencing in conjunction with the efforts of TCGA and other groups has led to the increased availability of mutation data linked to human cancer cell lines and patient tumors. In addition, the large number of molecular-targeting agents in clinical development underscores the need to link baseline tumor cell characteristics to drug responses to improve treatment selection. The Cancer Cell line Project at the Wellcome Trust's Sanger Institute is well known for resequencing the most common cancer-associated genes in human tumor-derived cell lines, and 77 genes in 770 cancer cell lines have been resequenced in this project to date. The CCLE project at the Broad Institute generated genomic profiles including a compilation of gene expression, chromosomal copy number, and massively parallel sequencing data from 947 human cancer cell lines with pharmacologic profiles for 24 anticancer drugs across 479 of the lines (8). Matching normal genomic DNA is rarely, if ever, available for cell lines as most established cell lines have been in culture for many years and the paired normal material is not available. None of the sequencing results to date on HNSCC cell lines have used matched normal DNA. In the CCLE project, gene mutations were evaluated in conjunction with dbSNP134 or allele frequency in the National Heart, Lung, and Blood Institute Exome Sequencing Project or 1000 Genomes Project to exclude common germline variants (8). There were 1,147 gene mutation sites found in HNSCC cell lines in CCLE, only 72 of which were reported single-nucleotide polymorphisms (SNP) according to dbSNP134 and the 1000 Genomes Project. Although not all of the mutations found in HNSCC cells represent somatic changes, such large annotated cell line collections can still be used to facilitate preclinical stratification for anticancer agent testing. To date, the genomic data from HNSCC cell lines and human tumors have not been linked to allow investigators to rationally select preclinical models for translational studies. The present study was undertaken in an attempt to address this gap in knowledge.

Genetic profiling of large cell line panels has been used to determine correlations between tumor and cell lines in other cancers. Neve and colleagues showed that 51 breast cancer cell lines display the same heterogeneity in copy number and gene expression as 145 primary subtype tumors. These breast cancer cell lines harbored most of the recurrent genomic abnormalities associated with clinical outcome in primary tumors (21). Lin and colleagues reported that cultured melanoma cells encompass the spectrum of significant genomic alterations present in primary tumors (22). Although both of these prior reports suggested that cell lines could mirror primary tumors from the corresponding tumor type by genomic copy number and transcriptional profiles for the cell lines with primary tumors, gene mutations were not compared. In the present study, we analyzed the correlation of mutations in 39 HNSCC cell lines and 106 human tumors. Our results suggest that the mutated genes in HNSCC cell lines can reflect many, but not all, of the mutated genes in HNSCC primary tumors. Of note, 51% (334) of mutated genes were detected in both HNSCC cell lines and primary human HNSCC tumors, and 83% (19 of the 23 genes) of genes with mutation frequencies more than 5% in cell lines have similar mutation frequencies in primary tumors. Even in a specific signaling pathway such as the EGFR pathway, the mutations in cell lines mirror most of the mutations in human tumors. These findings suggest that genes commonly mutated in HNSCC tumors are generally reflected in cell line models.

Our results indicate some discordance between mutations in HNSCC cell lines and tumors including genes, which had higher mutation frequencies in cell lines and genes that were mutated only in cell lines, but not in tumors. In breast cancer, cell lines were reported to harbor more genetic aberrations and high-level amplifications than primary tumors (23). Ross and colleagues analyzed the variation in expression of approximately 8,000 unique genes among the NCI60 cell lines and found that genes involved in proliferation were generally upregulated in cell lines (24). Others have reported that expression of p16^INK4a steadily increases in culture epithelial cells immortalized by telomerase, whereas loss of p16^INK4a expression is common in human cancers that are derived from epithelia (25, 26). It has been postulated that high-level gene amplification may provide a selective advantage for growth in vitro (22). Our analysis showed that many genes have higher mutation frequencies in HNSCC cell lines and 17 genes that are mutated only in HNSCC cell lines by more than 10%. We further extended our analysis to check these genes' mutation status in TCGA. There are 308 HNSCC tumor samples contained in the recently published TCGA, much more than the Stransky and colleagues and Agrawal and colleagues cohorts. Overall, the comparison yielded results consistent with our previous comparisons. Although the genes that had previously believed to be mutated exclusively in cell lines were also found to be mutated in TCGA tumor samples, they were found at frequencies far below their incidence in cell lines. For example, VEGFC is mutated in the majority of HNSCC cell lines (79.5%) but never in human HNSCC tumors in the Stransky and colleagues and Agrawal and colleagues cohorts. Although VEGFC is found to be mutated in TCGA, the mutation frequency is still less than 1% of the 308 tumors contained in this database. VEGFC is a member of the VEGF family and it plays an important role in lymphangiogenesis and angiogenesis in embryos and tumors. It enhances cancer cell mobility and invasiveness and contributes to the promotion of cancer cell metastasis by activating Fit-4. VEGFC overexpression in the lung cancer cell line H928 induces cell migration and invasion through the p38 mitogen—activated protein kinase (MAPK) pathway (27). It was reported by Benke and colleagues that forced expression of VEGFC in HNSCC HN4 cells with low endogenous CXCL5 levels increased cell growth and suppression of VEGFC-inhibited migration of HNSCC HN12 cells (28). Although the functional consequences of VEGFC mutations are incompletely understood, the mutations of this gene, found uniquely in HNSCC cell lines, may represent an artifact of the culture process. It suggested that these genes, which just found mutated or have much higher mutation frequencies in HNSCC cell lines might be acquired through the immortalization process.

EGFR is a type 1 receptor tyrosine kinase and contributes to cell growth, development, and differentiation. EGFR is expressed in most epithelial tissues but is upregulated in many epithelial malignancies including HNSCC in which overexpression compared with corresponding normal tissues has been reported in 80% to 90% of cases (29). Gandhi and colleagues analyzed the alterations of genes in the EGFR signaling pathway in 77 non–small cell lung cancer cell lines and copy-number gains were frequent (>10%) for EGFR, HER2, HER3, and KRAS (30). We found four genes in the EGFR pathway in HNSCC cell lines, which were amplified with the frequencies >10%, including PIK3CA, EGFR, ERBB2, and AKT 1. PIK3CA and EGFR were also found to be amplified in HNSCC tumors in primary HNSCC tumors. We and others have reported that EGFR gene amplification contributes to EGFR overexpression in HNSCC (31). PIK3CA amplification has also been found more frequently in HPV-positive HNSCC compared with HPV-negative tumors (13). In addition to gene amplification, 12 genes in the EGFR signaling pathway were mutated in both HNSCC cell lines and human tumors, including PIK3CA, RAS, RAF, STAT1, MAP3Ks, and MAP2K4. This finding suggests that genetic alteration of the components of the EGFR signaling pathway in HNSCC cell lines may reflect genetic alterations in primary HNSCC tumors.

A new generation of anticancer drugs with molecular targets has emerged as potent therapeutic agents. The EGFR signaling pathway has been implicated in HNSCC biology. Many agents inhibiting EGFR or components of the EGFR pathway have been FDA-approved for cancer treatment. In the CCLE pharmacologic profiling, six HNSCC cell lines were treated by seven agents targeting EGFR. HSC-2 and Detroit 562 cell lines, both of which harbor mutant PIK3CA (H1047R), were relatively resistant to seven EGFR pathway inhibitors compared with the other four HNSCC cell lines with WT PIK3CA. PIK3CA is the p110α catalytic subunit of the class IA PI3K and can be activated by growth factor receptor tyrosine kinases, including EGFR (32). We recently reported that genes in the PI3K pathway represent the most common alterations in HNSCC (13). In the present study, we analyzed PI3K pathway mutations in HNSCC cell lines and tumors. Eight genes in the PI3K pathway were found to be mutated both in cell lines and tumors, whereas four genes were only mutated in cell lines and four other genes were only reportedly mutated in tumors (Table 5). We also found that PIK3CA is the most commonly mutated gene in this pathway in HNSCC tumors (13). All of the PIK3CA mutants tested enhanced cell growth, and cell lines harboring endogenous PIK3CA (H1047R) mutations demonstrated increased sensitivity to PI3K pathway inhibition using a mTOR/PI3K–targeted agent (BEZ-235) in vitro and in vivo. Both HPV-positive and HPV-negative PIK3CA-mutated patient tumorgrafts were significantly more sensitive to BEZ-235 in vivo compared with tumors containing WT PIK3CA. Inhibition of tumor growth was accompanied by decreased PI3K signaling, as demonstrated by downregulation of p-AKT (S473) and p-S6(S235/236) in the BEZ-235–treated tumors. PIK3CA mutants have been reported to contribute to drug resistance in other cancers but have not been studied to date in HNSCC cell line models. Berns and colleagues reported that the oncogenic PIK3CA mutant (H1047R) contributed to trastuzumab resistance in breast cancer cell lines. The presence of PIK3CA mutations or low PTEN expression was associated with poor prognosis after trastuzumab therapy in patients with breast cancer (33). The PIK3CA mutant colorectal cancer cell line HCT116 was more resistant to EGFR-targeted monoclonal antibodies compared with PIK3CA WT controls (34, 35). In RAS mutant cancer cells, activating PIK3CA mutations could reduce the sensitivity to MEK inhibition (36). Two recent studies found that EGFR TKIs were relatively inactive in unselected HNSCC populations (37). HSC-2 and Detroit 562 cell lines were less sensitive to BRAF inhibitor RAF265 and MEK inhibitors AZD6244 and PD-0325901 in the CCLE report (8). These cumulative findings suggest that identification of individuals whose tumors harbor mutant PIK3CA may identify a subgroup of patients who may not be responsive to EGFR targeting and underscore the value of rational cell line model selection to guide the choice of therapies in the clinic.

L.A. Garraway received commercial research grant from Novartis, has ownership interest (including patents) in Foundation Medicine, and is consultant/advisory board member of Foundation Medicine, Novartis, Boehringer Ingelheim, Millennium Pharmaceuticals, and Onyx Pharmaceuticals. V.W.Y. Lui received other commercial research support from Novartis. No potential conflicts of interest were disclosed by the other authors.

Conception and design: H. Li, J.R. Grandis

Development of methodology: H. Li

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Li, J.R. Grandis

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Li, J.S. Wawrose, W.E. Gooding, L.A. Garraway, N.D. Peyser, J.R. Grandis

Writing, review, and/or revision of the manuscript: H. Li, J.S. Wawrose, W.E. Gooding, V.W.Y. Lui, J.R. Grandis

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Li, J.S. Wawrose

Study supervision: J.R. Grandis

This study was funded by grants NIH P50CA097190, R01CA77308, and R01CA098372 and the American Cancer Society (to J.R. Grandis).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.