Thomas J. Ow

TP53 Disruptive Mutations Lead to Head and Neck Cancer Treatment Failure through Inhibition of Radiation-Induced Senescence

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 the in vitro radioresistance measured using clonogenic assays. p53 protein expression was inhibited using short hairpin RNA (shRNA) and overexpressed using a retrovirus. Radiation-induced apoptosis, mitotic cell death, senescence, and reactive oxygen species (ROS) assays were carried out. 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-β-gal staining, p21 expression, and release of 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, because of 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. Clin Cancer Res; 18(1); 290–300. ©2011 AACR.

Assembly and Initial Characterization of a Panel of 85 Genomically Validated Cell Lines from Diverse Head and Neck Tumor Sites

Purpose: Human cell lines are useful for studying cancer biology and preclinically modeling cancer therapy, but can be misidentified and cross-contamination is unfortunately common. The purpose of this study was to develop a panel of validated head and neck cell lines representing the spectrum of tissue sites and histologies that could be used for studying the molecular, genetic, and phenotypic diversity of head and neck cancer. Methods: A panel of 122 clinically and phenotypically diverse head and neck cell lines from head and neck squamous cell carcinoma, thyroid cancer, cutaneous squamous cell carcinoma, adenoid cystic carcinoma, oral leukoplakia, immortalized primary keratinocytes, and normal epithelium was assembled from the collections of several individuals and institutions. Authenticity was verified by carrying out short tandem repeat analysis. Human papillomavirus (HPV) status and cell morphology were also determined. Results: Eighty-five of the 122 cell lines had unique genetic profiles. HPV-16 DNA was detected in 2 cell lines. These 85 cell lines included cell lines from the major head and neck primary tumor sites, and close examination shows a wide range of in vitro phenotypes. Conclusions: This panel of 85 genomically validated head and neck cell lines represents a valuable resource for the head and neck cancer research community that can help advance understanding of the disease by providing a standard reference for cell lines that can be used for biological as well as preclinical studies. Clin Cancer Res; 17(23); 7248–64. ©2011 AACR.

Glucose, not Glutamine is the Dominant Energy Source Required for Proliferation and Survival of Head and Neck Squamous Carcinoma Cells

Tumor metabolism is an essential contributor to disease progression and response to treatment. An understanding of the metabolic phenotype of head and neck squamous cell carcinoma (HNSCC) will allow the development of appropriate antimetabolic strategies for this tumor type.

Disruptive TP53 mutation is associated with aggressive disease characteristics in an orthotopic murine model of oral tongue cancer

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.

Optimization of long‐term outcomes for patients with esthesioneuroblastoma

Esthesioneuroblastoma is a rare cancer of the anterior cranial base that arises in the region of the olfactory rootlets. The purpose of this study was to review the long‐term outcomes of patients diagnosed with esthesioneuroblastoma (ENB) treated at a single institution to determine factors associated with improved disease control and survival.

Evolutionary action score of TP53 identifies high-risk mutations associated with decreased survival and increased distant metastases in head and neck cancer

TP53 is the most frequently altered gene in head and neck squamous cell carcinoma, with mutations occurring in over two-thirds of cases, but the prognostic significance of these mutations remains elusive. In the current study, we evaluated a novel computational approach termed evolutionary action (EAp53) to stratify patients with tumors harboring TP53 mutations as high or low risk, and validated this system in both in vivo and in vitro models. Patients with high-risk TP53 mutations had the poorest survival outcomes and the shortest time to the development of distant metastases. Tumor cells expressing high-risk TP53 mutations were more invasive and tumorigenic and they exhibited a higher incidence of lung metastases. We also documented an association between the presence of high-risk mutations and decreased expression of TP53 target genes, highlighting key cellular pathways that are likely to be dysregulated by this subset of p53 mutations that confer particularly aggressive tumor behavior. Overall, our work validated EAp53 as a novel computational tool that may be useful in clinical prognosis of tumors harboring p53 mutations.

Glycolytic Inhibition Alters Anaplastic Thyroid Carcinoma Tumor Metabolism and Improves Response to Conventional Chemotherapy and Radiation

Anaplastic thyroid carcinoma (ATC) accounts for more than 50% of thyroid cancer mortality and is generally refractory to conventional treatment. On the basis of recent studies, we hypothesized that ATC metabolism can be targeted to improve response to chemoradiotherapy. Eight established and authenticated ATC cell lines were sequenced at 140 sites contained within 26 commonly mutated genes to identify novel potential therapeutic targets. Cellular proliferation, energy, and reducing potential stores were measured under conditions of specific nutrient deprivation. Tumor metabolism was evaluated using hyperpolarized 13C MRI in a murine orthotopic xenograft model of ATC. Sensitivity to chemotherapeutic agents and radiation (XRT) was assayed using cytotoxicity assays. We identified mutations in BRAF, NRAS, and KIT but failed to identify generalized novel targets for therapeutic intervention. ATC cell lines exhibited a mesenchymal phenotype and generalized dependence on glucose for energy, reducing potential and survival. Glycolytic inhibition using 2-deoxyglucose (2-DG) sensitized ATC cells to conventional chemotherapy and external beam radiation. In vivo, 2-DG induced a transient, but significant reduction in ATC metabolic activity. Generalized dependence of ATC cells on glucose catabolism makes them susceptible to the sensitizing effects of 2-DG for radiation therapy and chemotherapy. Under in vivo conditions, 2-DG can inhibit ATC metabolism. However, the modest magnitude and transient nature of this effect suggest the need for antimetabolic agents with more favorable pharmacodynamics to achieve therapeutic effects. Mol Cancer Ther; 11(6); 1373–80. ©2012 AACR.

Individualizing antimetabolic treatment strategies for head and neck squamous cell carcinoma based on TP53 mutational status.

Mutations in the tumor protein 53 (TP53) tumor suppressor gene are common in head and neck squamous cell carcinoma (HNSCC) and correlate with radioresistance. Currently, there are no clinically available therapeutic approaches targeting p53 in HNSCC. In this report, the authors propose a strategy that uses TP53 mutational status to individualize antimetabolic strategies for the potentiation of radiation toxicity in HNSCC cells.

Current Management of Advanced Resectable Oral Cavity Squamous Cell Carcinoma

The oral cavity is the most common site of head and neck squamous cell carcinoma, a disease which results in significant morbidity and mortality worldwide. Though the primary modality of treatment for patients with oral cavity cancer remains surgical resection, many patients present with advanced disease and are thus treated using a multi-disciplinary approach. Patients with extracapsular spread of lymphatic metastasis and surgical margins that remain positive have been found to be at high risk for local-regional recurrence and death from disease, and are most often recommended to receive both post-operative radiation as well as systemic chemotherapy. The basis for this approach, as well as scientific developments that underly future trials of novels treatments for patients with high-risk oral cavity cancer are reviewed.

MicroRNA-375 suppresses extracellular matrix degradation and invadopodial activity in head and neck squamous cell carcinoma

CONTEXTnHead and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated degradation of the extracellular matrix.nnnOBJECTIVEnTo determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity.nnnDESIGNnFor evaluation of the matrix degradation properties of the HNSCC lines, an invadopodial matrix degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of cortactin. Human protease arrays were used for the detection of the secreted proteases. Quantitative real time-polymerase chain reaction measurements were used to evaluate the messenger RNA (mRNA) expression of the commonly regulated proteases.nnnRESULTSnIncreased miR-375 expression in HNSCC cells suppresses extracellular matrix degradation and reduces the number of mature invadopodia. Higher miR-375 expression does not reduce cellular levels of selected invadopodia-associated proteins, nor is tyrosine phosphorylation of cortactin altered. However, HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression levels and secreted levels of specific proteases.nnnCONCLUSIONSnMicroRNA-375 regulates invadopodia maturation and function potentially by suppressing the expression and secretion of proteases.