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Accurate assessment of TP53 gene status in sporadic tumors and in the germline of individuals at high risk of cancer due to Li–Fraumeni Syndrome (LFS) has important clinical implications for diagnosis, surveillance, and therapy. Genomic data from more than 20,000 cancer genomes provide a wealth of information on cancer gene alterations and have confirmed TP53 as the most commonly mutated gene in human cancer. Analysis of a database of 70,000 TP53 variants reveals that the two newly discovered exons of the gene, exons 9β and 9γ, generated by alternative splicing, are the targets of inactivating mutation events in breast, liver, and head and neck tumors. Furthermore, germline rearrange-ments in intron 1 of TP53 are associated with LFS and are frequently observed in sporadic osteosarcoma. In this context of constantly growing genomic data, we discuss how screening strategies must be improved when assessing TP53 status in clinical samples. Finally, we discuss how TP53 alterations should be described by using accurate nomenclature to avoid confusion in scientific and clinical reports. Cancer Res; 77(6); 1250–60. ©2017 AACR.

Purpose:Neoadjuvant chemotherapy prior to definitive surgery has been used widely for locally advanced oral squamous cell carcinoma (OSCC). We evaluated neoadjuvant erlotinib with platinum-docetaxel versus placebo with platinum-docetaxel in patients with stage III–IVB OSCC.Patients and Methods:Patients with newly diagnosed stage III, IVA, and IVB (American Joint Committee on Cancer 7th) OSCC amenable to surgical resection were included. Patients were randomized to receive up to 3 cycles of chemotherapy with concurrent erlotinib or placebo, followed by surgery. The primary endpoint was major pathologic response (MPR) rate; secondary endpoints included safety, overall survival (OS), and progression-free survival (PFS).Results:Fifty-two patients received at least 1 cycle of treatment and 47 were evaluable with surgical resection. MPR rate was not different between erlotinib (30%, 7/23) and placebo arms (41.7%, 10/24; P = 0.55). At median follow up of 26.5 months, there was no difference on OS or PFS between groups. Patients who received erlotinib with chemotherapy and achieved MPR (n = 7) had no recurrence. The treatment-related adverse event rates were not different between the two groups (96% vs. 96%). However, rash, mostly low grade, was more common in the erlotinib arm (79% vs. 50%). Transcriptomic analysis in the pretreatment samples indicated that genes in protein glycosylation and Wnt signaling pathways were associated with benefit in those treated with erlotinib plus chemotherapy.Conclusions:The addition of erlotinib to platinum-taxane chemotherapy was well tolerated but did not induce higher rates of MPR or PFS or OS survival benefit. Patients who received chemotherapy with erlotinib and achieved major pathologic responses had excellent clinical outcome.

Purpose:In locoregionally advanced, resectable cutaneous squamous cell carcinoma of the head and neck (CSCC-HN), surgery followed by radiotherapy is standard but can be cosmetically and functionally devastating, and many patients will have recurrence.Patients and Methods:Newly diagnosed or recurrent stage III–IVA CSCC-HN patients amenable to curative-intent surgery received two cycles of neoadjuvant PD-1 inhibition. The primary endpoint was ORR per RECIST 1.1. Secondary endpoints included pathologic response [pathologic complete response (pCR) or major pathologic response (MPR; ≤10% viable tumor)], safety, DSS, DFS, and OS. Exploratory endpoints included immune biomarkers of response.Results:Of 20 patients enrolled, 7 had recurrent disease. While only 6 patients [30%; 95% confidence interval (CI), 11.9–54.3] had partial responses by RECIST, 15 patients (75%; 95% CI, 50.9–91.3) had a pCR (n = 11) or MPR (n = 4). No SAEs ocurred during or after the neoadjuvant treatment. At a median follow-up of 22.6 months (95% CI, 21.7–26.1), one patient progressed and died, one died without disease, and two developed recurrence. The 12-month DSS, DFS, and OS rates were 95% (95% CI, 85.9–100), 89.5% (95% CI, 76.7–100), and 95% (95% CI, 85.9–100), respectively. Gene expression studies revealed an inflamed tumor microenvironment in patients with pCR or MPR, and CyTOF analyses demonstrated a memory CD8+ T-cell cluster enriched in patients with pCR.Conclusions:Neoadjuvant immunotherapy in locoregionally advanced, resectable CSCC-HN is safe and induces a high pathologic response rate. Pathologic responses were associated with an inflamed tumor microenvironment.

Supplementary Data from Targeted Inhibition of Inducible Nitric Oxide Synthase Inhibits Growth of Human Melanoma In vivo and Synergizes with Chemotherapy

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Supplementary Figure and Table Legends from High-Risk TP53 Mutations Are Associated with Extranodal Extension in Oral Cavity Squamous Cell Carcinoma

Supplementary Materials and Methods and Figure Legends

Supplementary Figure from Induction Chemotherapy with or without Erlotinib in Patients with Head and Neck Squamous Cell Carcinoma Amenable for Surgical Resection

Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach.Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis.Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1–low expression group (P = 0.01, 1.9 × 10−3, and 9 × 10−4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10−4 and P = 4 × 10−4, respectively).Conclusions: Taken together, our data point to a targetable Axl–PI3 kinase–PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713–22. ©2017 AACR.

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Supplementary Data from Induction Chemotherapy with or without Erlotinib in Patients with Head and Neck Squamous Cell Carcinoma Amenable for Surgical Resection

Purpose: Although the majority of patients with HPV+ oropharyngeal cancers have a favorable prognosis, there are some patients with tumors that are resistant to aggressive chemoradiotherapy with unusual patterns of locoregional and systemic recurrences. Therefore, more effective therapies are needed. In this study, we investigated the chemosensitizing efficacy of the selective Wee-1 kinase inhibitor, AZD-1775, in HPV+ head and neck squamous cell carcinoma (HNSCC).Experimental Design: Clonogenic survival assays and an orthotopic mouse model of HPV+ oral cancer were used to examine the in vitro and in vivo sensitivity of HPV+ HNSCC cell lines to AZD-1775 in combination with cisplatin, respectively. Cell-cycle analysis, DNA damage (γH2AX), homologous recombination (HR), and apoptosis were examined to dissect molecular mechanisms.Results: We found that AZD-1775 displays single-agent activity and enhances the response of HPV+ HNSCC cells to cisplatin both in vitro and in vivo. The sensitivity of the HPV+ HNSCC cells to AZD-1775 alone or in combination with cisplatin was associated with G2 checkpoint abrogation, persistent DNA damage, and apoptosis induction. This finding of AZD-1775 increasing the sensitivity of HPV+ HNSCC cells to cisplatin through apoptosis was not seen previously in the HPV− HNSCC cancer cells and is accompanied by a decreased expression of the antiapoptotic proteins, MCl-1and XIAP, which appear to be cleaved following AZD-1775 treatment.Conclusions: AZD-1775 selectively sensitizes HPV+ HNSCC cells and orthotopic oral xenografts to cisplatin through apoptosis and support the clinical investigation of AZD-1775 in combination with cisplatin particularly in patients with advanced and recurrent metastatic HPV+ HNSCC tumors. Clin Cancer Res; 21(21); 4831–44. ©2015 AACR.

Purpose: Sinonasal undifferentiated carcinoma (SNUC) is a rare and aggressive cancer. Despite the use of multimodality treatment, the overall prognosis remains poor. To better understand the biologic features of SNUC and help develop new therapies for the disease, we established SNUC cell lines and characterized their biologic behaviors.Experimental Design: Cell lines were established from a patient with a T4N0M0 SNUC of the right maxillary sinus who was treated with surgical resection at our center. Tumor colonies were harvested and were sequentially replated onto larger plates. Two populations were developed and labeled MDA8788-6 and MDA8788-7. These cell lines were characterized with molecular, biomarker, functional, and histologic analyses.Results: Short tandem repeat genotyping revealed that the cell line is isogenic to the parental tumor, and cytogenetic analysis identified 12 chromosomal translocations. The SNUC cell lines do not form colonies in soft agar but are tumorigenic and nonmetastatic in an orthotopic mouse model of sinonasal cancer. Western blot analysis revealed that both MDA8788 cell lines express epithelial markers but do not express mesenchymal markers or the endocrine marker synaptophysin.Conclusions: This is the first report of the establishment of stable human-derived SNUC cell lines. The lines were highly tumorigenic and maintain the histologic and molecular features of the original tumor. These cell lines should serve as useful tools for the future study of SNUC biology and the development and testing of novel therapies for this deadly disease. Clin Cancer Res; 18(22); 6178–87. ©2012 AACR.

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PDF file - 157K, Expression pattern of epithelial-mesenchymal transition markers in early and late passage SNUC cells. Expression pattern of epithelial markers (pan-keratin and E-cadherin) and mesenchymal markers (α-SMA and vimentin) in both early (at passage number 17 in MDA8788-6 and at passage number 15 in MDA8788-7) and late passage (at passage number 40) in the SNUC cells was analyzed by Western blotting. Both early and late passage of SNUC cells expressed only the epithelial markers and did not expressed mesenchymal markers.

PDF file - 79K, Cytogenetic analysis of the MDA8788-7 cell line. Representative G-banding of MDA8788-7 showed the same translocation in MDA8788-6; m1 t (1:17), m2 t (1:22), m3 t (1:15), m4 t (2:12), m5 t (4:15), m6 t (3:6), m7 t (8:19), m8 t (9:10), m9 t (9:16), m10 t (13:13), m11 t (14, 14) and m12 t (18:19).

Supplemental data

Purpose: Head and neck squamous cell carcinoma (HNSCC) is commonly treated with radiotherapy, and local failure after treatment remains the major cause of disease-related mortality. To date, human papillomavirus (HPV) is the only known clinically validated, targetable biomarkers of response to radiation in HNSCC.Experimental Design: We performed proteomic and transcriptomic analysis of targetable biomarkers of radioresistance in HPV-negative HNSCC cell lines in vitro, and tested whether pharmacologic blockade of candidate biomarkers sensitized cells to radiotherapy. Candidate biomarkers were then investigated in several independent cohorts of patients with HNSCC.Results: Increased expression of several targets was associated with radioresistance, including FGFR, ERK1, EGFR, and focal adhesion kinase (FAK), also known as PTK2. Chemical inhibition of PTK2/FAK, but not FGFR, led to significant radiosensitization with increased G2–M arrest and potentiated DNA damage. PTK2/FAK overexpression was associated with gene amplification in HPV-negative HNSCC cell lines and clinical tumors. In two independent cohorts of patients with locally advanced HPV-negative HNSCC, PTK2/FAK amplification was highly associated with poorer disease-free survival (DFS; P = 0.012 and 0.034). PTK2/FAK mRNA expression was also associated with worse DFS (P = 0.03). Moreover, both PTK2/FAK mRNA (P = 0.021) and copy number (P = 0.063) were associated with DFS in the Head and Neck Cancer subgroup of The Cancer Genome Atlas.Conclusions: Proteomic analysis identified PTK2/FAK overexpression is a biomarker of radioresistance in locally advanced HNSCC, and PTK2/FAK inhibition radiosensitized HNSCC cells. Combinations of PTK2/FAK inhibition with radiotherapy merit further evaluation as a therapeutic strategy for improving local control in HPV-negative HNSCC. Clin Cancer Res; 22(18); 4643–50. ©2016 AACR.

Supplemental Figures S1-S2 and Methods. Figure S1: Mutation types and number by previous radiation treatment. Figure S2: Copy number alterations.

Supplementary Table from Pilot Phase II Trial of Neoadjuvant Immunotherapy in Locoregionally Advanced, Resectable Cutaneous Squamous Cell Carcinoma of the Head and Neck

Supplemental Table S5: mutation annotation file.

Purpose:In oropharyngeal squamous cell carcinoma (OPC), high CD8+ tumor-infiltrating lymphocyte (CD8+TIL) density confers improved prognosis. We compared neoadjuvant durvalumab (PD-L1 inhibitor) with durvalumab + tremelimumab (CTLA-4 inhibitor) in terms of impact on CD8+TIL density, safety, and efficacy in patients with OPC.Patients and Methods:Patients with newly diagnosed stage II–IVA OPC or locoregionally recurrent OPC amenable to resection were included. Patients were randomized to two cycles of durvalumab or durvalumab + tremelimumab before surgery. The primary endpoint was change between baseline and resection specimen in CD8+TIL density between arms. Secondary endpoints included safety, response rate per RECIST, major pathologic response (MPR; ≤10% viable tumor cells) rate, and patient-reported outcomes.Results:Of 28 eligible patients (14/arm), 20 (71%) had newly diagnosed OPC, and 24 (86%) were p16-positive. The posttreatment to pretreatment median CD8+TIL density ratio was 1.31 for durvalumab and 1.15 for combination treatment (P = 0.97; 95% CI: −1.07–2.28). In each group, 6 patients (43%, 95% CI: 17.66–71.14) had a response. Eight patients (29%) had a MPR at the primary tumor and/or nodal metastases. Neither baseline CD8+TIL density nor PD-L1 expression level correlated with overall response, but a trend toward greater CD8+TIL change in patients with a MPR was seen (P = 0.059; 95% CI: −0.33–3.46). Four patients (14%) had grade ≥3 adverse events. At median follow-up time of 15.79 months, all patients were alive, and one had an additional recurrence.Conclusions:Durvalumab + tremelimumab did not increase CD8+TIL density more than durvalumab alone did. The observed safety and activity support further investigation of neoadjuvant checkpoint inhibitor for OPC.

Checkpoint kinase inhibitors (CHKi) exhibit striking single-agent activity in certain tumors, but the mechanisms accounting for hypersensitivity are poorly understood. We screened a panel of 49 established human head and neck squamous cell carcinoma (HNSCC) cell lines and report that nearly 20% are hypersensitive to CHKi monotherapy. Hypersensitive cells underwent early S-phase arrest at drug doses sufficient to inhibit greater than 90% of CHK1 activity. Reduced rate of DNA replication fork progression and chromosomal shattering were also observed, suggesting replication stress as a root causative factor in CHKi hypersensitivity. To explore genomic underpinnings of CHKi hypersensitivity, comparative genomic analysis was performed between hypersensitive cells and cells categorized as least sensitive because they showed drug IC50 value greater than the cell panel median and lacked early S-phase arrest. Novel association between CDKN2A/p16 copy number loss, CDK2 activation, replication stress, and hypersensitivity of HNSCC cells to CHKi monotherapy was found. Restoring p16 in cell lines harboring CDKN2A/p16 genomic deletions alleviated CDK2 activation and replication stress, attenuating CHKi hypersensitivity. Taken together, our results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from CHKi therapy.Significance: These results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from therapy with CHK inhibitors. Cancer Res; 78(3); 781–97. ©2017 AACR.

Supplementary Table S1.

PDF file - 169K, Tumorigenicity of MDA8788-7 cells. Tumorigenicity was examined by injecting cells into the flanks of the mice. (A) The cells injected into the flank without Matrigel formed in 75% of mice (6 of 8 mice). (B) By adding Matrigel, tumors grew much larger than the ones without adding Matrigel. (C) Average size of the tumors. (D) H& E staining from the tumor of the flank injection showed very similar appearance to the surgical specimen from the SNUC patient. The original magnifications x40 (left) and x200 (right).

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Supplementary Tables

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Supplementary Figure Legends

Analysis of combined drug effects; Immunofluorescence; Caspase-Glo 3/7 activity assay; Quantitative reverse-transcriptase PCR (qRT-PCR); Orthotopic Mouse Model of Oral Cavity Cancer and Tumor Growth Delay; Generation of patient-derived HNSCC xenografts and treatment with AZD-1775; Immunohistochemistry; and In vivo TUNEL assay.

Purpose: To characterize tumor growth and metastatic potential in head and neck squamous cell carcinoma (HNSCC) cell lines in an orthotopic murine model of oral tongue cancer and to correlate TP53 mutation status with these findings.Experimental Design: Cells from each of 48 HNSCC cell lines were orthotopically injected into the oral tongues of nude mice. Tumor volume, cervical lymph node metastasis, and mouse survival were recorded. Direct sequencing of the TP53 gene and Western blot analysis for the p53 protein after induction with 5-fluorouracil was conducted. Cell lines were categorized as either mutant TP53 or wild-type TP53, and lines with TP53 mutation were further categorized on the basis of type of mutation (disruptive or nondisruptive) and level of p53 protein expression. The behavior of tumors in these different groups was compared.Results: These 48 HNSCC cell lines showed a wide range of behavior from highly aggressive and metastatic to no tumor formation. Mice injected with cells harboring disruptive TP53 mutations had faster tumor growth, greater incidence of cervical lymph node metastasis, and shorter survival than mice injected with cells lacking these mutations.Conclusions: HNSCC cell lines display a wide spectrum of behavior in an orthotopic model of oral cancer. Cell lines with disruptive TP53 mutations are more aggressive in this system, corroborating clinical reports that have linked these mutations to poor patient outcome. Clin Cancer Res; 17(21); 6658–70. ©2011 AACR.

PDF file - 133K, In vitro migration and invasion assays in MDA8788-6 cells. 4 x 104 cells were plated in insert wells or Matrigel-coated wells in the presence of 10% FBS. After 48 hours, OSC19 used as a positive control showed massive migration and invasion, whereas MDA8788-6 did not show any of them. The original magnification is x50.

Supplementary Figure S1. Synergistic effects of vorinostat and AZD1775 in vitro in established isogenic pair HNSCC cell lines with known TP53 mutational status. Supplementary Figure S2. Synergistic effects of vorinostat and AZD1775 in vitro in various established HNSCC cell lines with known TP53 mutational status. Supplementary Figure S3. Laser scanning confocal microscopy showing stress-induced membrane blebbing prior to apoptosis in selected populations of PCI13-G245D cells. Supplementary Figure S4. Combination of vorinostat and AZD1775 is associated with subsequent accumulation of cells in G2/M phase in HNSCC mutp53 cells. Supplementary Figure S5. Vorinostat interacts synergistically with AZD1775 in HNSCC to induce markers of DNA damge, replication stress and apoptosis. Supplementary Figure S6. Correlation between overall survival and expression levels of genes regulated by vorinostat alone or in combination with AZD1775 in HNSCC TCGA dataset. Supplementary Figure S7. Proteomic profiling of HNSCC mutant p53 cells treated with vorinostat and AZD1775 identifies proteins involved in cell cycle regulation. Supplementary Figure S8. Tumor volume growth curves for individual mice in each treatment group. Supplementary Figure S9. Immunostaining of p21, PCNA, RRM2 and survivin in tumor of HNSCC oral xenografts mice. Supplementary Figure S10. Combination of vorinostat and AZD1775 is well tolerated in an orthotopic mouse model of oral cancer. Supplementary Table S1. Combination index of vorinostat in combination with AZD1775 in various HNSCC cell lines.

Purpose: We investigated whether vandetanib, an inhibitor of the tyrosine kinase activities of vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET), could augment the antitumor activity of radiation with or without cisplatin in preclinical in vitro and in vivo models of human head and neck squamous cell carcinoma (HNSCC).Experimental Design: OSC-19 and HN5 HNSCC cells that were cisplatin and radioresistant were treated with vandetanib, cisplatin, and radiation alone or in combination in vitro and in vivo using an orthotopic nude mouse model. Treatment effects were assessed using clonogenic survival assay, tumor volume, bioluminescence imaging, tumor growth delay, survival, microvessel density, tumor and endothelial cell apoptosis, and EGFR and Akt phosphorylation data.Results: Vandetanib plus cisplatin radiosensitized HNSCC cells in vitro and in vivo. The combination treatment with vandetanib, cisplatin, and radiation was superior to the rest of treatments (including the double combinations) in antitumoral effects, prolonging survival, decreasing cervical lymph node metastases in vivo. It also increased both tumor and tumor-associated endothelial cell apoptosis and decreased microvessel density in vivo. An analysis of tumor growth delay data revealed that vandetanib plus cisplatin enhanced radioresponse in vivo. All vandetanib-containing treatments inhibited EGFR and Akt phosphorylation in vitro and in vivo.Conclusion: The addition of vandetanib to combination therapy with cisplatin and radiation was able to effectively overcome cisplatin and radioresistance in in vitro and in vivo models of HNSCC. Further study of this regimen in clinical trials may be warranted. Clin Cancer Res; 17(7); 1815–27. ©2011 AACR.

Supplemental methods Supplementary Figure 1. Total and phospho-PTK2/FAK is overexpressed in selected radioresistant HNSCC cells compared to radiosensitive cell lines via immunoblot. Supplementary Figure 2. A.) Inhibition of PTK2/FAK expression via stable expression of shRNA to PTK2/FAK in HN31 HNSCC cells. B.) Stable inhibition of PTK2/FAK leads to radiosensitization in vitro. C & D.) The combination of PF562271 and radiation leads to C.) increased γ-H2AX expression and D.) higher levels of G2/M arrest and sub-G1 staining in Fadu HNSCC cells. Supplementary Figure 3. A & B.) PTK2/FAK is highly expressed in a large public database of HNSCC cell lines (www.oncomine.com) (A.) and tumor specimens (TCGA) (B.) compared to other tumor types. Supplementary Table 1. HNSCC cell lines included in RPPA. Supplementary Table 2. Tumor characteristics of the institutional cohorts. Supplementary Table 3. Univariate and multivariate analysis of PTK2/FAK copy number and disease free survival (DFS) in the two institutional cohorts and the Head and Neck Cancer TCGA cohort. NA - not included in the model. HR - Hazard ratio. 95% CI- 95% Confidence Interval. Supplementary Table 4. Tumor characteristics of the HNSCC TCGA cohort.

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Supplemental Methods. Description of additional methods and procedures used in the study.

TP53 is the most frequently altered gene in head and neck squamous cell carcinoma (HNSCC), with mutations occurring in over two thirds of cases; however, the predictive response of these mutations to cisplatin-based therapy remains elusive. In the current study, we evaluate the ability of the Evolutionary Action score of TP53-coding variants (EAp53) to predict the impact of TP53 mutations on response to chemotherapy. The EAp53 approach clearly identifies a subset of high-risk TP53 mutations associated with decreased sensitivity to cisplatin both in vitro and in vivo in preclinical models of HNSCC. Furthermore, EAp53 can predict response to treatment and, more importantly, a survival benefit for a subset of head and neck cancer patients treated with platinum-based therapy. Prospective evaluation of this novel scoring system should enable more precise treatment selection for patients with HNSCC. Cancer Res; 75(7); 1205–15. ©2015 AACR.

Supplementary Table 1: TP53 mutations scored and stratified by EAp53 and Poeta classification in the platinum based induction chemotherapy cohort.

Supplementary Figure 3: Effect of cisplatin therapy on tumor growth and survival in an orthotopic nude mouse model of oral tongue cancer harboring TP53 mutations presented as area under the growth curve (AUC).

Supplementary Figure 1 from Lupeol Suppresses Cisplatin-Induced Nuclear Factor-κB Activation in Head and Neck Squamous Cell Carcinoma and Inhibits Local Invasion and Nodal Metastasis in an Orthotopic Nude Mouse Model

A poor prognosis in head and neck squamous cell carcinoma (HNSCC) patients is commonly associated with the presence of regional metastasis. Cisplatin-based chemotherapy concurrent with radiation therapy is commonly used in the treatment of locally advanced HNSCC. However, the result is dismal due to common acquisition of chemoresistance and radioresistance. Epidemiologic studies have shown the importance of dietary substances in the prevention of HNSCC. Here, we found that lupeol, a triterpene found in fruits and vegetables, selectively induced substantial HNSCC cell death but exhibited only a minimal effect on a normal tongue fibroblast cell line in vitro. Down-regulation of NF-κB was identified as the major mechanism of the anticancer properties of lupeol against HNSCC. Lupeol alone was not only found to suppress tumor growth but also to impair HNSCC cell invasion by reversal of the NF-κB–dependent epithelial-to-mesenchymal transition. Lupeol exerted a synergistic effect with cisplatin, resulting in chemosensitization of HNSCC cell lines with high NF-κB activity in vitro. In in vivo studies, using an orthotopic metastatic nude mouse model of oral tongue squamous cell carcinoma, lupeol at a dose of 2 mg/animal dramatically decreased tumor volume and suppressed local metastasis, which was more effective than cisplatin alone. Lupeol exerted a significant synergistic cytotoxic effect when combined with low-dose cisplatin without side effects. Our results suggest that lupeol may be an effective agent either alone or in combination for treatment of advanced tumors. [Cancer Res 2007;67(18):8800–9]

Supplemental Table S1. Summary of patient data and prognostic data for both the test and validation datasets.

Supplementary Figure 3 from Lupeol Suppresses Cisplatin-Induced Nuclear Factor-κB Activation in Head and Neck Squamous Cell Carcinoma and Inhibits Local Invasion and Nodal Metastasis in an Orthotopic Nude Mouse Model

Supplementary Figure 2: TP53 knockdown illustrates both loss and gain of function in vitro in HNSCC cells.

Supplementary Figure 2 from Lupeol Suppresses Cisplatin-Induced Nuclear Factor-κB Activation in Head and Neck Squamous Cell Carcinoma and Inhibits Local Invasion and Nodal Metastasis in an Orthotopic Nude Mouse Model

Supplementary Table 2: Univariate analysis of overall survival.

Supplementary Figure Legends 1-3 from Lupeol Suppresses Cisplatin-Induced Nuclear Factor-κB Activation in Head and Neck Squamous Cell Carcinoma and Inhibits Local Invasion and Nodal Metastasis in an Orthotopic Nude Mouse Model

MAPS allow differentiation of delivery and molecular interactions.

Supplemental Figure S2. Cellular proliferation is independent of TP53 status.