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OBJECTIVES: To analyze charges, complications, survival, and functional outcomes for definitive surgery of mandibular osteoradionecrosis (ORN). MATERIALS AND METHODS: Retrospective analysis of 76 patients who underwent segmental mandibulectomy with reconstruction from 2000 to 2009. RESULTS: Complications occurred in 49 (65%) patients and were associated with preoperative drainage (odds ratio [OR] 4.40, 95% confidence interval [CI] 1.01–19.27). The adjusted median charge was $343 000, and higher charges were associated with double flap reconstruction (OR 8.15, 95% CI 2.19–30.29) and smoking (OR 5.91, 95% CI 1.69–20.72). Improved swallow was associated with age

Purpose:Human papillomavirus (HPV) causes >5% of cancers, but no therapies uniquely target HPV-driven cancers.Experimental Design:We tested the cytotoxic effect of 864 drugs in 16 HPV-positive and 17 HPV-negative human squamous cancer cell lines. We confirmed apoptosis in vitro and in vivo using patient-derived xenografts. Mitotic pathway components were manipulated with drugs, knockdown, and overexpression.Results:Aurora kinase inhibitors were more effective in vitro and in vivo in HPV-positive than in HPV-negative models. We hypothesized that the mechanism of sensitivity involves retinoblastoma (Rb) expression because the viral oncoprotein E7 leads to Rb protein degradation, and basal Rb protein expression correlates with Aurora inhibition–induced apoptosis. Manipulating Rb directly, or by inducing E7 expression, altered cells’ sensitivity to Aurora kinase inhibitors. Rb affects expression of the mitotic checkpoint genes MAD2L1 and BUB1B, which we found to be highly expressed in HPV-positive patient tumors. Knockdown of MAD2L1 or BUB1B reduced Aurora kinase inhibition–induced apoptosis, whereas depletion of the MAD2L1 regulator TRIP13 enhanced it. TRIP13 is a potentially druggable AAA-ATPase. Combining Aurora kinase inhibition with TRIP13 depletion led to extensive apoptosis in HPV-positive cancer cells but not in HPV-negative cancer cells.Conclusions:Our data support a model in which HPV-positive cancer cells maintain a balance of MAD2L1 and TRIP13 to allow mitotic exit and survival in the absence of Rb. Because it does not affect cells with intact Rb function, this novel combination may have a wide therapeutic window, enabling the effective treatment of Rb-deficient cancers.

Supplementary Data from Combined TRIP13 and Aurora Kinase Inhibition Induces Apoptosis in Human Papillomavirus–Driven Cancers

PDF file - 196K, Table summarizing the antibodies used for immunoblotting and IHC.

PDF file - 202K, Knockdown of TMEM16A in T24 cancer cells decreases motility and migration.

Figure S3: Sensitivity to PI3K/mTOR pathway inhibitors is not significantly associated with PI3K/mTOR pathway alterations A. The heat map depicts the association between 19 genetic mutations in the PI3K/mTOR pathway (left column) and GSK2126458 sensitivity in 59 head and neck squamous cell carcinoma (HNSCC) cell lines. P values were calculated using the Fisher exact test; no significant associations were observed. Similar results were obtained with other PI3K inhibitors using the Wilcoxon rank-sum test (data not shown). Black boxes in the heatmap indicate mutations; white (i.e., absent) boxes indicate wild type genes; blue boxes indicate non-canonical mutations in the PIK3CA gene. The black vertical line in the bar graph indicates the peak plasma concentration of GSK2126458. B. Spearman correlation analysis was used to assess associations between IC70 values and PI3K proteomic scores, including for p-AKT (T308), p-AKT (S473), p-GSK3 (S9), p-GSK3-α/β (S9/21), p-p27 (T157), p-p27 (T198), p-PRAS40 (T246), INPP4B, and PTEN; no significant correlations were detected. C. Spearman correlation analysis was used to assess associations between IC70 values and mTOR proteomic scores, including for Tp-4EBP1 (S65), p-4EBP1 (T37/46), p4EBP1 (T70), p-mTOR (S2488), p-p70S6K (T389), p-S6 (S235/236), and p-S6 (S240/244); no significant correlations were detected. The dashed blue lines indicate trend line and the shaded area represent 95% confidence interval.

PDF file - 133K, Table summarizing the data from Figure 6.

PDF file - 132K, TMEM16A expression influences cell size measured via flow cytometry.

Table S3. Spearman correlation of protein expression to drug sensitivity based on 70% inhibitory concentration (IC70) or area under the curve (AUC)

PDF file - 3718K, TMEM16A shRNA resistant DNA can rescue the expression from the stable shRNA knockdown.

PDF file - 472K, Over expression of TMEM16A in T24 cancer cells decreases motility and invasion.

PDF file - 292K, Table of values for proteomics screen.

PDF file - 78K, TMEM16A expression levels in primary SCCHN does not correlate with the presence of nodal metastases.

PDF file - 267K, Proposed model describing the role of TMEM16A in tumor progression.

Purpose: Tumor metastasis is the leading cause of death in patients with cancer. However, the mechanisms that underlie metastatic progression remain unclear. We examined TMEM16A (ANO1) expression as a key factor shifting tumors between growth and metastasis.Experimental Design: We evaluated 26 pairs of primary and metastatic lymph node (LN) tissue from patients with squamous cell carcinoma of the head and neck (SCCHN) for differential expression of TMEM16A. In addition, we identified mechanisms by which TMEM16A expression influences tumor cell motility via proteomic screens of cell lines and in vivo mouse studies of metastasis.Results: Compared with primary tumors, TMEM16A expression decreases in metastatic LNs of patients with SCCHN. Stable reduction of TMEM16A expression enhances cell motility and increases metastases while decreasing tumor proliferation in an orthotopic mouse model. Evaluation of human tumor tissues suggests an epigenetic mechanism for decreasing TMEM16A expression through promoter methylation that correlated with a transition between an epithelial and a mesenchymal phenotype. These effects of TMEM16A expression on tumor cell size and epithelial-to-mesenchymal transition (EMT) required the amino acid residue serine 970 (S970); however, mutation of S970 to alanine does not disrupt the proliferative advantages of TMEM16A overexpression. Furthermore, S970 mediates the association of TMEM16A with Radixin, an actin-scaffolding protein implicated in EMT.Conclusions: Together, our results identify TMEM16A, an eight transmembrane domain Ca2+-activated Cl− channel, as a primary driver of the “Grow” or “Go” model for cancer progression, in which TMEM16A expression acts to balance tumor proliferation and metastasis via its promoter methylation. Clin Cancer Res; 20(17); 4673–88. ©2014 AACR.

PDF file - 3396K, Overexpression of TMEM16A S970A increase anchorage dependent and independent tumor cell proliferation.

Purpose:Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function.Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression.Results:PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1MUT) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/mTOR inhibition in NOTCH1MUT but not NOTCH1WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1WT HNSCC but had little effect as single agents.Conclusions:Our findings suggest that NOTCH1MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1WT HNSCC to PI3K/mTOR pathway inhibitors.

PDF file - 87K

Purpose: Human papillomavirus (HPV) causes >5% of cancers, but no therapies uniquely target HPV-driven cancers. Experimental Design: We tested the cytotoxic effect of 864 drugs in 16 HPV-positive and 17 HPV-negative human squamous cancer cell lines. We confirmed apoptosis in vitro and in vivo using patient-derived xenografts. Mitotic pathway components were manipulated with drugs, knockdown, and overexpression. Results: Aurora kinase inhibitors were more effective in vitro and in vivo in HPV-positive than in HPV-negative models. We hypothesized that the mechanism of sensitivity involves retinoblastoma (Rb) expression because the viral oncoprotein E7 leads to Rb protein degradation, and basal Rb protein expression correlates with Aurora inhibition–induced apoptosis. Manipulating Rb directly, or by inducing E7 expression, altered cells’ sensitivity to Aurora kinase inhibitors. Rb affects expression of the mitotic checkpoint genes MAD2L1 and BUB1B, which we found to be highly expressed in HPV-positive patient tumors. Knockdown of MAD2L1 or BUB1B reduced Aurora kinase inhibition–induced apoptosis, whereas depletion of the MAD2L1 regulator TRIP13 enhanced it. TRIP13 is a potentially druggable AAA-ATPase. Combining Aurora kinase inhibition with TRIP13 depletion led to extensive apoptosis in HPV-positive cancer cells but not in HPV-negative cancer cells. Conclusions: Our data support a model in which HPV-positive cancer cells maintain a balance of MAD2L1 and TRIP13 to allow mitotic exit and survival in the absence of Rb. Because it does not affect cells with intact Rb function, this novel combination may have a wide therapeutic window, enabling the effective treatment of Rb-deficient cancers.