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Supplementary Figure S1. Association between age, number of mutations and smoking status.

Purpose: Epidemiologic studies have identified an increasing incidence of squamous cell carcinoma of the oral tongue (SCCOT) in younger patients.Experimental Design: DNA isolated from tongue tumors of young (45 years) patients at was subjected to whole-exome sequencing and copy-number analysis. These data were compared with data from similar patients in the TCGA (The Cancer Genome Atlas) project.Results: In this study, we found that gene-specific mutation and copy-number alteration frequencies were similar between young and old patients with SCCOT in two independent cohorts. Likewise, the types of base changes observed in the young cohort were similar to those in the old cohort even though they differed in smoking history. TCGA data also demonstrate that the genomic effects of smoking are tumor site–specific, and we find that smoking has only a minor impact on the types of mutations observed in SCCOT.Conclusions: Overall, tumors from young patients with SCCOT appear genomically similar to those of older patients with SCCOT, and the cause for the increasing incidence of young SCCOT remains unknown. These data indicate that the functional impact of smoking on carcinogenesis in SCCOT is still poorly understood. Clin Cancer Res; 20(14); 3842–8. ©2014 AACR.

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Purpose: Anaplastic thyroid carcinoma (ATC) is one of the most lethal human cancers with a median survival of 6 months. The inhibition of epidermal growth factor receptor (EGFR) alone, or with VEGF receptor 2 (VEGFR2), represents an attractive approach for treatment of ATC. Several reports have examined agents that target these receptors. However, with the misidentification of as many as 60% of all commonly used ATC cell lines, the significance of these past findings is unclear.Experimental Design: Cell lines authenticated by short tandem repeat profiling were selected to establish xenograft tumors in an orthotopic murine model of ATC. These mice were then treated with vandetanib to evaluate its effects on ATC tumor growth. Dynamic contrast-enhanced (DCE) MRI was utilized to measure the impact of vandetanib on tumor vasculature.Results: Vandetanib inhibited tumor growth of the ATC cell lines Hth83 and 8505C in vivo by 69.3% (P < 0.001) and 66.6% (P < 0.05), respectively, when compared with control. Significant decreases in vascular permeability (P < 0.01) and vascular volume fraction (P < 0.05) were detected by DCE-MRI in the orthotopic xenograft tumors after 1 week of treatment with vandetanib as compared with control.Conclusion: The inhibition of EGFR and VEGFR2 by vandetanib and its tremendous in vivo antitumor activity against ATC make it an attractive candidate for further preclinical and clinical development for the treatment of this particularly virulent cancer, which remains effectively untreatable. Vandetanib disrupts angiogenesis and DCE-MRI is an effective method to quantify changes in vascular function in vivo. Clin Cancer Res; 17(8); 2281–91. ©2011 AACR.

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.

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Purpose: Development of extranodal extension (ENE) has been associated with poor survival in patients with oral cavity squamous cell carcinoma (OSCC). Here, we sought to confirm the role of ENE as a poor prognostic factor, and identify genomic and epigenetic markers of ENE in order to develop a predictive model and improve treatment selection. Experimental Design: An institutional cohort (The University of Texas MD Anderson Cancer Center) was utilized to confirm the impact of ENE on clinical outcomes and evaluate the genomic signature of primary and ENE containing tissue. OSCC data from The Cancer Genome Atlas (TCGA) were analyzed for the presence of molecular events associated with nodal and ENE status. Results: ENE was associated with decreased overall and disease-free survival. Mutation of the TP53 gene was the most common event in ENE+ OSCC. The frequency of TP53 mutation in ENE+ tumors was higher compared with ENE− tumors and wild-type (WT) TP53 was highly represented in pN0 tumors. pN+ENE+ patients had the highest proportion of high-risk TP53 mutations. Both primary tumors (PT) and lymph nodes with ENE (LN) exhibited a high rate of TP53 mutations (58.8% and 58.8%, respectively) with no significant change in allele frequency between the two tissue sites. Conclusions: ENE is one of the most significant markers of OSCC OS and DFS. There is a shift toward a more aggressive biological phenotype associated with high-risk mutations of the TP53 gene. Prospective clinical trials are required to determine whether TP53 mutational status can be used for personalized treatment decisions. Clin Cancer Res; 24(7); 1727–33. ©2018 AACR.

BACKGROUND Human immunodeficiency virus–infected individuals (HIVIIs) have a higher incidence of head and neck squamous cell carcinoma (HNSCC), and clinical and histopathological differences have been observed in their tumors in comparison with those of HNSCC patients without a human immunodeficiency virus (HIV) infection. The reasons for these differences are not clear, and molecular differences between HIV-related HNSCC and non–HIV-related HNSCC may exist. This study compared the mutational patterns of HIV-related HNSCC and non–HIV-related HNSCC. METHODS The DNA of 20 samples of HIV-related HNSCCs and 32 samples of non–HIV-related HNSCCs was sequenced. DNA libraries covering exons of 18 genes frequently mutated in HNSCC (AJUBA, CASP8, CCND1, CDKN2A, EGFR, FAT1, FBXW7, HLA-A, HRAS, KEAP1, NFE2L2, NOTCH1, NOTCH2, NSD1, PIK3CA, TGFBR2, TP53, and TP63) were prepared and sequenced on an Ion Personal Genome Machine sequencer. DNA sequencing data were analyzed with Ion Reporter software. The human papillomavirus (HPV) status of the tumor samples was assessed with in situ hybridization, the MassARRAY HPV multiplex polymerase chain reaction assay, and p16 immunostaining. Mutation calls were compared among the studied groups. RESULTS HIV-related HNSCC revealed a distinct pattern of mutations in comparison with non–HIV-related HNSCC. TP53 mutation frequencies were significantly lower in HIV-related HNSCC. Mutations in HIV+ patients tended to be TpC>T nucleotide changes for all mutated genes but especially for TP53. CONCLUSIONS HNSCC in HIVIIs presents a distinct pattern of genetic mutations, particularly in the TP53 gene. HIV-related HNSCC may have a distinct biology, and an effect of the HIV virus on the pathogenesis of these tumors should not be ruled out. Cancer 2017. © 2017 American Cancer Society.

The phosphatidylinositol 3′ kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway, which is a critical regulator of cell proliferation and survival, is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens, and hypothesised that KP372-1, an Akt inhibitor, would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells, leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling, further preclinical evaluation of this compound for treatment of thyroid cancer is warranted.

Purpose: Epidemiologic studies have identified an increasing incidence of squamous cell carcinoma of the oral tongue (SCCOT) in younger patients. Experimental Design: DNA isolated from tongue tumors of young (45 years) patients at was subjected to whole-exome sequencing and copy-number analysis. These data were compared with data from similar patients in the TCGA (The Cancer Genome Atlas) project. Results: In this study, we found that gene-specific mutation and copy-number alteration frequencies were similar between young and old patients with SCCOT in two independent cohorts. Likewise, the types of base changes observed in the young cohort were similar to those in the old cohort even though they differed in smoking history. TCGA data also demonstrate that the genomic effects of smoking are tumor site–specific, and we find that smoking has only a minor impact on the types of mutations observed in SCCOT. Conclusions: Overall, tumors from young patients with SCCOT appear genomically similar to those of older patients with SCCOT, and the cause for the increasing incidence of young SCCOT remains unknown. These data indicate that the functional impact of smoking on carcinogenesis in SCCOT is still poorly understood. Clin Cancer Res; 20(14); 3842–8. ©2014 AACR.

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined. We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells. Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth. Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation. Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function.

Background The purpose of this study was to identify mechanisms of innate resistance to an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, in a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. Specifically, we analyzed the role of HRAS mutations in erlotinib resistance. Methods Erlotinib sensitivity was determined by methyl thiazolyl-tetrazolium (MTT) assays. Molecular signaling pathways and somatic mutations were examined. Changes in sensitivity after modulation of HRAS expression were evaluated. Results All 7 cell lines were wild-type for EGFR and KRAS regardless of erlotinib sensitivity; however, 1 erlotinib-resistant cell line (HN31) harbored an HRAS G12D mutation. Downregulation of HRAS expression by small interfering RNA (siRNA) or short hairpin RNA (shRNA) in HN31 led to increased erlotinib sensitivity in vitro and in vivo. Transfection of activating HRAS-mutant (G12D and G12V) constructs into erlotinib-sensitive cell lines made them more resistant to erlotinib. Conclusion Activating HRAS mutations can confer erlotinib resistance in an HRAS mutant HNSCC cell line. © 2014 Wiley Periodicals, Inc. Head Neck 36: 1547–1554, 2014

Mutation of the TP53 gene occurs in more than half of cases of head and neck squamous cell carcinoma (HNSCC). However, little is known about how specific TP53 mutations affect tumor progression. The objective of this study is to determine the gain of function of mutant p53 with a proline-to-serine substitution at codon 151.Laboratory-based study.A panel of HNSCC cell lines was determined with anoikis assays, and orthotopic mouse experiments were performed. TP53 was sequenced. The shRNA knockdown and overexpression approaches were used for testing mutant p53 functions. The crystal structure of the p53 protein was analyzed using an in silico approach.An anoikis-resistant cell line, Tu138, was found to have a proline-to-serine substitution at codon 151 of TP53, which results in loss of wild-type p53 transcriptional activity. Moreover, the mutant p53 was shown to promote anoikis resistance and soft agar growth. Using an in silico approach based on the crystal structure of wild-type p53 protein, substitution of proline by serine at position 151 would create a cavity in a hydrophobic pocket, the loss of van der Waals contacts, and the thermodynamically unfavorable placement of a polar group, the hydroxyl oxygen atom of the serine, within a hydrophobic region, all of which likely cause a locally altered structure.Our data suggest that mutation at position 151 leads to a structural alteration, which results in significant functional changes in the p53 protein that impact tumor progression.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole-exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papillomavirus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA, and HRAS, we identified mutations in FBXW7 and NOTCH1. Nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.

Overexpression of insulin-like growth factor-I receptor (IGF-IR) is seen in a multitude of human thyroid cancers and correlates with poor prognosis. However, recent studies suggest that low phospho-IGF-IR (pIGF-IR) expression rather than its overexpression may be an indicator of poorly differentiated disease. No previous study has evaluated the expression of pIGF-IR to determine if activation or loss of expression of this receptor is associated with thyroid tumor progression. Accordingly, a quantitative immunohistochemical (IHC) method was used to evaluate the clinico-pathological significance of pIGF-IR expression in archival samples of human thyroid carcinomas. Quantitative analysis of pIGF-IR levels revealed a significant difference in the median index of pIGF-IR between different histological subtypes of thyroid cancer ( P < 0.001). Specifically, the median pIGF-IR index of differentiated thyroid cancers was significantly higher than the median index of other poorly differentiated thyroid cancer ( P < 0.001). This was further confirmed in individual tumor sections of thyroid carcinoma where anaplastic and differentiated components co-existed. No significant difference was noted in the pIGF-IR index of tumors grouped by size or stage but a trend towards lower mean pIGF-IR index was noted in older patients. Our data indicates that pIGF-IR is upregulated in a majority of follicular thyroid carcinomas, suggesting it may be a potential target for therapy for patients with this disease. In addition, since low pIGF-IR expression was found to correlate with aggressive human thyroid carcinoma, it also suggests that IGF-IR may not be needed for progression of anaplastic thyroid carcinoma possibly because other cell signaling pathways are activated, obviating the need for IGF-IR signaling. However, more mechanistic studies would be necessary to substantiate the possibility that pIGF-IR may be important for differentiation of thyroid tissues and is lost with disease progression.

Purpose: Adenoid cystic carcinoma (ACC) can often be controlled with surgery and postoperative adjuvant radiotherapy but is also characterized by late local recurrence and distant metastasis. No effective systemic therapeutic agents have been found to alter the natural history of ACC. Therefore, new therapeutic approaches are needed. In this study, we evaluated whether vandetanib (Zactima), a potent inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) tyrosine kinases, had antitumor efficacy in vitro and in an orthotopic nude mouse model of human ACC.Experimental Design: The in vitro effects of vandetanib were assessed in three ACC cell lines on cell growth, apoptosis, and VEGFR-2 and EGFR phosphorylation levels. The in vivo antitumor activity of vandetanib was examined in nude mice bearing parotid gland ACC tumors. The mice were treated for 4 weeks with vandetanib (50 mg/kg/d) or placebo (control). Tumors were resected at necropsy, and immunohistochemical and immunofluorescence staining were done.Results: In vitro, vandetanib caused dose-dependent inhibition of VEGFR-2 and EGFR phosphorylation in ACC cells. Vandetanib also inhibited the cell proliferation and induced their dose-dependent apoptosis. In vivo, mice in the vandetanib group had tumor volumes significantly lower than those in the control group (P < 0.01). In addition, immunohistochemical staining showed a decrease in microvessel density and an increase in apoptosis of both tumor cells and endothelial cells within the tumor xenografts.Conclusion: These results suggest that vandetanib inhibits the growth of ACC in vitro and in vivo, making it a promising novel agent for the treatment of ACC.

Overexpression of S100A7 (psoriasin), a small calcium-binding protein, has been associated with the development of psoriasis and carcinomas in different types of epithelia, but its precise functions are still unknown. Using human tissue specimens, cultured cell lines, and a mouse model, we found that S100A7 is highly expressed in preinvasive, well-differentiated and early staged human squamous cell carcinoma of the oral cavity (SCCOC), but little or no expression was found in poorly differentiated, later-staged invasive tumors. Interestingly, our results showed that S100A7 inhibits both SCCOC cell proliferation in vitro and tumor growth/invasion in vivo. Furthermore, we demonstrated that S100A7 is associated with the beta-catenin complex, and inhibits beta-catenin signaling by targeting beta-catenin degradation via a noncanonical mechanism that is independent of GSK3beta-mediated phosphorylation. More importantly, our results also indicated that beta-catenin signaling negatively regulates S100A7 expression. Thus, this reciprocal negative regulation between S100A7 and beta-catenin signaling implies their important roles in tumor development and progression. Despite its high levels of expression in early stage SCCOC tumorigenesis, S100A7 actually inhibits SCCOC tumor growth/invasion as well as tumor progression. Downregulation of S100A7 in later stages of tumorigenesis increases beta-catenin signaling, leading to promotion of tumor growth and tumor progression.

Objective. The Department of Veterans Affairs and Veterans' Administration hospitals began using the electronic medical record in 1994, streamlining provider-to-provider communication of vital clinical information, and simultaneously providing investigators access to vast amounts of clinical data for research purposes. Administrative and coded data, including symptoms and diagnoses as derived from chart content, are one of the most ready substrates for analysis of these massive databases to answer various research-related inquiries. We evaluated the capability of this type of coded data to accurately identify patients with schizophrenia from a group of 819 patients taking opioids for chronic pain in the primary care clinic of the Philadelphia Veterans' Administration Medical Center. Methods. Patients were identified for inclusion in this analysis by their sequential enrollment in an Opioid Renewal Clinic as referred by their primary care providers, as well as a nonreferred cohort of chronic pain patients maintained on opioids by their primary care providers (N = 819). The prevalence of schizophrenia obtained by coded diagnostic labeling by computerized chart review was compared with the prevalence of schizophrenia obtained by systematic independent manual chart review of all cases by a research psychiatrist. Results. Based purely on coded diagnostic labeling, the prevalence of schizophrenia in this population was 14%, nearly 14 times the estimated general population prevalence. However, manual independent chart review of the identified cases by a research psychiatrist (N = 119) resulted in the actual prevalence in this group to be estimated at 1.8%. None of the patients with three or less incidents of diagnostic labeling by code had schizophrenia. Of those with four or more incidents, 74% had schizophrenia. Conclusions. Better accuracy is obtained when cases of schizophrenia are identified by multiple coding incidents derived from longitudinal clinical encounters. These findings suggest that the extraction of up to three coding incidents for schizophrenia alone is not an accurate means of identifying this clinical population for research and epidemiological purposes. Further, when designing automated, information technology-based approaches to the identification of subjects for clinical research and extracting their data from electronic medical records using International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM) codes, mental health diagnoses must be uniquely considered on a disease-by-disease basis.

Purpose: The insulin-like growth factor-I receptor (IGF-IR) and its ligands have been implicated in the pathogenesis and progression of various cancers, including those arising in the thyroid gland. We therefore evaluated whether the IGF-IR could serve as a potential target for therapy of anaplastic thyroid carcinoma (ATC). Experimental Design: The expression and activation of the IGF-IR and some of its downstream signaling pathway components were evaluated in both human thyroid cancer specimens and thyroid cancer cell lines. The therapeutic potential of a humanized monoclonal antibody (A12) directed against IGF-IR was assessed in vitro and in vivo in an orthotopic model of ATC. Tumor volume and overall survival time were analyzed to evaluate the efficacy of A12 in vivo. Results: IGF-IR was overexpressed in 94% of the thyroid cancers. Blockade of IGF-IR with A12 was effective in attenuating IGF-IR signaling both in vitro and in vivo. However, the inhibitory effects of A12 on cell proliferation were cell line dependent, as those ATC cell lines that had detectable levels of pIGF-IR were more sensitive to A12 treatment. A12 was equally effective in vivo, where it brought ∼57% (P = 0.041) inhibition in tumor volume. The concomitant use of A12 and irinotecan produced additive effects and resulted in a 93% (P < 0.001) reduction in tumor volume. Blocking IGF-IR blocked Akt phosphorylation and decreased proliferation and microvessel density but increased apoptosis within the tumor xenografts. Our results also highlighted a previously undefined IGF-IR-mediated antiangiogenic effect on tumor-associated endothelium in thyroid cancers. Conclusion: Blocking the IGF-IR with A12 seems to be a potential avenue for treating patients with ATC by its direct antitumor effects and its effects on the tumor vasculature.

Purpose: Patients with radioiodine-resistant follicular thyroid cancer (FTC) have a poor prognosis, if metastasized, with currently available treatment modalities. Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) and their receptors (EGFR and VEGFR) have been reported to be overexpressed in FTC and have been implicated in FTC development. We hypothesized that inhibiting the phosphorylation of EGFR and VEGFR by treatment with NVP-AEE788 (AEE788), a novel dual specific EGFR and VEGFR inhibitor, either alone or in combination with paclitaxel, would inhibit the growth of FTC xenografts in an orthotopic nude mouse model. Experimental Design: To confirm previous reports, EGF and EGFR expression and vascularity were analyzed in human samples of FTC, Hürthle cell carcinoma, and normal thyroid tissues. EGFR expression in four FTC cell lines was measured using Western blotting. The antitumor effect of AEE788 on FTC cells in vitro was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and Western blotting. The effect of AEE788, alone and in combination with paclitaxel, on FTC tumor growth in an orthotopic nude mouse model was also investigated. Immunohistochemical analysis of EGFR and VEGFR signaling status, cell proliferation, apoptosis, and microvessel density was done. Results: EGF, EGFR, and vascularity were increased in human thyroid tumor samples and EGFR was increased in FTC cells. AEE788 inhibited FTC cell growth in vitro and reduced the phosphorylation status of EGFR, VEGFR, and two downstream targets, AKT and mitogen-activated protein kinase, in FTC cells. AEE788 alone and, to a greater extent, AEE788 plus paclitaxel suppressed FTC tumor growth in the thyroids of nude mice. Conclusion: Dual inhibition of EGFR and VEGFR by AEE788 could represent a novel approach to the treatment of radioiodine-resistant FTC.

The relationship between circadian phototransduction and circadian-regulated processes is poorly understood. Melatonin, commonly a circadian phase marker, may play a direct role in a myriad of physiologic processes. The circadian rhythm for pineal melatonin secretion is regulated by the hypothalamic suprachiasmatic nucleus (SCN). Its neural source of light input is a unique subset of intrinsically photosensitive retinal ganglion cells expressing melanopsin, the primary circadian photopigment in rodents and primates. Action spectra of melatonin suppression by light have shown that light in the 446-477 nm range, distinct from the visual system's peak sensitivity, is optimal for stimulating the human circadian system. Breast cancer is the oncological disease entity whose relationship to circadian rhythm fluctuations has perhaps been most extensively studied. Empirical data has increasingly supported the hypothesis that higher risk of breast cancer in industrialized countries is partly due to increased exposure to light at night. Studies of tumor biology implicate melatonin as a potential mediator of this effect. Yet, causality between lifestyle factors and circadian tumor biology remains elusive and likely reflects significant variability with physiologic context. Continued rigorous empirical inquiry into the physiology and clinical implications of these habitual, integrated aspects of life is highly warranted at this time.

Therapies that target signaling pathways critical to the pathogenesis and progression of squamous cell carcinoma of the head and neck (HNSCC) are needed. One such target, phosphatidylinositol 3-kinase, and its downstream target serine/threonine kinase, Akt, are up-regulated in HNSCC. Targeted therapy could consist of inhibitors of these kinases or, alternatively, of inhibitors of the pathways that they regulate. To explore the effect of Akt inhibition on the growth and survival of HNSCC tumors, we evaluated the effect of a novel Akt inhibitor, KP372-1, on the growth, survival, and sensitivity to anoikis of HNSCC cell lines in culture. Using Western blotting of head and neck cancer cell lines and squamous mucosa and carcinoma specimens, we found that Akt was highly phosphorylated in head and neck cancer cell lines and human head and neck squamous carcinoma specimens. Treatment of HNSCC cell lines with KP372-1 blocked the activation of Akt, inhibited head and neck cancer cell proliferation, and induced apoptosis and anoikis in several HNSCC cell lines. Furthermore, KP372-1 decreased the phosphorylation of the S6 ribosomal (Ser240/244) protein, which is a downstream target of Akt. Taken together, these findings indicate that KP372-1 may be a useful therapeutic agent for HNSCC and should be further evaluated in preclinical models of HNSCC.

Objectives/Hypothesis: Vascular endothelial growth factor (VEGF) is an important mediator in tumor vascularization, growth, and metastasis. We investigated whether blockade of the VEGF receptor (VEGF-R) signaling pathway by the tyrosine kinase inhibitor PTK787 combined with CPT-11, a semisynthetic camptothecin analogue, can inhibit the tumor growth and angiogenesis of squamous cell carcinoma of the oral tongue in an orthotopic nude mouse model. Methods: JMAR human oral squamous cell carcinoma cells were injected into the tongues of nude mice. Seven days later, the mice were randomized to receive a placebo, daily oral PTK787, weekly CPT-11 injection, or PTK787 plus CPT-11. After 4 weeks of treatment, the mice underwent necropsy, and the tongue tumors, cervical lymph nodes, and lungs were removed for immunohistochemical analyses. Results: CPT-11, PTK787, and PTK787 plus CPT-11 significantly decreased tumor volumes and prolonged survival. The combination treatment group had the most significant decrease in volume and increase in survival. PTK787 alone or in combination with CPT-11 reduced the phosphorylation of VEGF-R in tumor cells and tumor-associated endothelial cells, was associated with decreased microvessel density, a decreased proliferative index, and an increased apoptotic index. PTK787 alone or the combination therapy resulted in apoptosis of both tumor cells and tumor-associated endothelial cells. Conclusions: These results suggest that targeting VEGF-R tyrosine kinase activity can be an effective therapeutic approach in squamous cell carcinoma of the oral tongue.

We investigated integrin-linked kinase (ILK), a focal adhesion serine-threonine protein kinase, as a new molecular target for treating anaplastic thyroid cancer. ILK mediates cell growth and survival signals and is overexpressed in a number of cancers. Therefore, we hypothesized that inhibition of ILK leads to growth arrest and apoptosis of thyroid cancer cells. According to Western blotting, the level of ILK protein was highly expressed in one papillary (NPA187) and four of five (Hth74, DRO, ARO, KAT4, and K4) anaplastic thyroid cancer cell lines. Immunohistochemical analysis of a human tissue microarray revealed that ILK was highly expressed in anaplastic thyroid cancer but not in normal human thyroid tissue. Treating thyroid cancer cell lines with a new ILK inhibitor, QLT0267, inhibited epidermal growth factor–induced phosphorylation of AKT, inhibited cell growth, and induced apoptosis in the NPA187, DRO, and K4 cell lines. QLT0267 also inhibited the kinase activity of immunoprecipitated ILK in four of five cell lines. Tumor volumes in mice treated with QLT0267 were significantly reduced compared with those in untreated mice. In immunohistochemical studies, QLT0267 suppressed phosphorylated p-AKT and angiogenesis (i.e., reduced mean vascular density) and induced apoptosis in both tumor cells and tumor-associated endothelial cells of the thyroid DRO xenografts. In summary, we found that ILK expression and activity were elevated in human anaplastic thyroid cancer and ILK inhibition led to growth arrest and apoptosis in vitro and in vivo. Our results provide preliminary evidence that ILK is a potential therapeutic target for treating anaplastic thyroid cancer.

Patients suffering from bone metastases of follicular thyroid carcinoma (FTC) have a poor prognosis because of the lack of effective treatment strategies. The overexpression of epidermal growth factor receptor (EGFR) associated with increased vascularity has been implicated in the pathogenesis of FTC and subsequent bone metastases. We hypothesized that inhibiting the phosphorylation of the EGFR and vascular endothelial growth factor receptor (VEGFR) by AEE788, a dual tyrosine kinase inhibitor of EGFR and VEGFR, in combination with paclitaxel would inhibit experimental FTC bone lesions and preserve bone structure. We tested this hypothesis using the human WRO FTC cell line. In culture, AEE788 inhibited the EGF-mediated phosphorylation of EGFR, VEGFR2, mitogen-activated protein kinase, and Akt in culture. AEE788, alone and in combination with paclitaxel, inhibited cell growth and induced apoptosis. When WRO cells were injected into the tibia of nude mice, tumor and endothelial cells within the lesions expressed phosphorylated EGFR, VEGFR, Akt, and mitogen-activated protein kinase that were inhibited by the oral administration of AEE788. Therapy consisting of orally given AEE788 and i.p. injected paclitaxel induced a high level of apoptosis in tumor-associated endothelial cells and tumor cells with the inhibition of tumor growth in the bone and the preservation of bone structure. Collectively, these data show that blocking the phosphorylation of EGFR and VEGFR with AEE788 combined with paclitaxel can significantly inhibit experimental human FTC in the bone of nude mice.

The phosphatidylinositol 3′ kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway, which is a critical regulator of cell proliferation and survival, is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens, and hypothesised that KP372-1, an Akt inhibitor, would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells, leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling, further preclinical evaluation of this compound for treatment of thyroid cancer is warranted.

Light is being used as a pre-launch countermeasure to circadian and sleep disruption in astronauts. The effect of light on the circadian system is readily monitored by measurement of plasma melatonin. Our group has established an action spectrum for human melatonin regulation and determined the region of 446–477 nm to be the most potent for suppressing plasma melatonin. The aim of this study was to compare the efficacy of 460 and 555 nm for suppressing melatonin using a within-subjects design. Subjects ( N = 12 ) were exposed to equal photon densities ( 7.18 × 10 12 photons / cm 2 / s ) at 460 and 555 nm. Melatonin suppression was significantly stronger at 460 nm ( p 0.02 ). An extension to the action spectrum showed that 420 nm light at 16 and 32 μ W / cm 2 significantly suppressed melatonin ( p 0.04 and p 0.002 ). These studies will help optimize lighting countermeasures to circadian and sleep disruption during spaceflight.

Purpose: We investigated whether concomitant blockade of the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways by AEE788, a dual inhibitor of EGFR and VEGFR tyrosine kinases, would inhibit the growth of cutaneous squamous cell carcinoma (SCC) cells and human cutaneous cancer xenografts in nude mice. Experimental Design: We examined the effects of AEE788 on the phosphorylation of EGFR and VEGFR-2 in cutaneous SCC cells expressing EGFR and VEGFR-2 and cutaneous SCC cell growth and apoptosis. We assessed the in vivo antitumor effects of AEE788 in a xenograft model in nude mice. AEE788 (50 mg/kg) was given orally thrice weekly to mice that had been s.c. injected with Colo16 tumor cells. Mechanisms of in vivo AEE788 activity were determined by immunohistochemical analysis. Results: Treatment of cutaneous SCC cells with AEE788 led to dose-dependent inhibition of EGFR and VEGFR-2 phosphorylation, growth inhibition, and induction of apoptosis. In mice treated with AEE788, tumor growth was inhibited by 54% at 21 days after the start of treatment compared with control mice (P < 0.01). Immunohistochemical analysis revealed that AEE788 inhibited phosphorylation of EGFR and VEGFR and induced apoptosis of tumor cells and tumor-associated endothelial cells. Conclusions: In addition to inhibiting cutaneous cancer cell growth by blocking EGFR and VEGFR signaling pathways in vitro, AEE788 inhibited in vivo tumor growth by inducing tumor and endothelial cell apoptosis.

Extensive genomic deletions involving chromosome 10 are the most common genetic alteration in glioblastoma multiforme (GBM). To localize and examine the potential roles of two chromosome arm 10q tumor suppressor regions, we used two independent strategies: mapping of allelic deletions, and functional analysis of phenotypic suppression after transfer of chromosome 10 fragments. By allelic deletion analysis, the region of 10q surrounding the MMAC/PTEN locus was shown to be frequently lost in GBMs but maintained in most low-grade astrocytic tumors. An additional region at 10q25 containing the DMBT1 locus was lost in all grades of gliomas examined. The potential biological significance of these two regions was further assessed by examining microcell hybrids that contained various fragments of 10q. Somatic cell hybrid clones that retained the MMAC/PTEN locus have a less transformed phenotype with clones exhibiting an inability to grow in soft agarose. However, presence or absence of DMBT1 did not correlate with any in vitro phenotype assessed in our model system. These results support a model of molecular progression in gliomas in which the frequent deletion of 10q25-26 is an early event and is followed by the deletion of the MMAC/PTEN during the progression to high-grade GBMs.

The effects of transforming growth factor-α (TGF-α) on cell growth were studied in human glioma U251 cells transfected with antisense TGF-α vectors (pcDNAI.neo). Several antisense clones showed a marked decrease in growth rate in serum-free medium but not in medium containing 10% FBS, compared with those of parental cells and clones from sense or vector transfectants. Antisense clones also produced fewer and smaller colonies in anchorage-independent growth assays. Moreover, there was a reduction in TGF-α expression in these antisense clones at both the protein and mRNA levels, as determined by enzyme linked immuno-sorbent assay and reverse transcriptase polymerase chain reaction analysis. A U251 clone transfected by TGF-α antisense in a different vector (pMT/Ep) also showed a marked suppression in cell growth and TGF-α mRNA level. Finally, transfected clones with either vector system, showed decreased tumorigenicity in nude mice. In summary, a strong correlation between the inhibition of glioma cell growth and TGF-α expression was obtained from two different plasmid vectors, indicating that the expression of TGF-α could be specifically and effectively down-regulated by TGF-α antisense vector, which in turn led to growth inhibition. These studies suggests that TGF-α plays an essential role in controlling human glioma cell proliferation and may serve as a potential target for treatment of malignant glioma.

Deletions involving regions of chromosome 10 occur in the vast majority (> 90%) of human glioblastoma multiformes. A region at chromosome 10q23-24 was implicated to contain a tumour suppressor gene and the identification of homozygous deletions in four glioma cell lines further refined the location. We have identified a gene, designated MMAC1, that spans these deletions and encodes a widely expressed 5.5-kb mRNA. The predicted MMAC1 protein contains sequence motifs with significant homology to the catalytic domain of protein phosphatases and to the cytoskeletal proteins, tensin and auxilin. MMAC1 coding-region mutations were observed in a number of glioma, prostate, kidney and breast carcinoma cell lines or tumour specimens. Our results identify a strong candidate tumour suppressor gene at chromosome 10q23.3, whose loss of function appears to be associated with the oncogenesis of multiple human cancers.

Genetic deletions to chromosome 10 have been extensively documented for human glioblastomas (GBMs). To identify gene products that may be involved in malignant progression, a subtractive hybridization was performed between GBM cells and hybrid cells suppressed for tumorigenicity following microcell transfer of chromosome 10. One novel cDNA isolated from this subtraction showed consistent upregulation (approximately 4 to 10-fold) that correlated with the nontumorigenic phenotype of the hybrid cells. Subsequent analysis resulted in the identification of a full length cDNA (2,569 bp) termed RIG (regulated in glioma). RIG expression was either not detected or detected only at low levels in cultured glioma cells and primary glioblastoma specimens compared to normal brain cells. The 2.6 kb RIG mRNA was expressed predominantly in normal brain with lower levels in heart and lung. Sequence analysis showed no significant homology to known gene products. Genomic alterations of RIG were present in approximately 25% of glioma cell lines examined. Also, RIG mapped to chromosome 11p15.1, a region that is known to be altered in malignant astrocytomas. The differential expression pattern, tissue distribution and chromosomal location of RIG suggests it serves as a molecular marker for or may play a role in the malignant progression of GBMs.

The purpose of this study was to identify mechanisms of innate resistance to an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, in a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. Specifically, we analyzed the role of HRAS mutations in erlotinib resistance.Erlotinib sensitivity was determined by methyl thiazolyl-tetrazolium (MTT) assays. Molecular signaling pathways and somatic mutations were examined. Changes in sensitivity after modulation of HRAS expression were evaluated.All 7 cell lines were wild-type for EGFR and KRAS regardless of erlotinib sensitivity; however, 1 erlotinib-resistant cell line (HN31) harbored an HRAS G12D mutation. Downregulation of HRAS expression by small interfering RNA (siRNA) or short hairpin RNA (shRNA) in HN31 led to increased erlotinib sensitivity in vitro and in vivo. Transfection of activating HRAS-mutant (G12D and G12V) constructs into erlotinib-sensitive cell lines made them more resistant to erlotinib.Activating HRAS mutations can confer erlotinib resistance in an HRAS mutant HNSCC cell line.

Purpose: Anaplastic thyroid carcinoma (ATC) is one of the most lethal human cancers with a median survival of 6 months. The inhibition of epidermal growth factor receptor (EGFR) alone, or with VEGF receptor 2 (VEGFR2), represents an attractive approach for treatment of ATC. Several reports have examined agents that target these receptors. However, with the misidentification of as many as 60% of all commonly used ATC cell lines, the significance of these past findings is unclear. Experimental Design: Cell lines authenticated by short tandem repeat profiling were selected to establish xenograft tumors in an orthotopic murine model of ATC. These mice were then treated with vandetanib to evaluate its effects on ATC tumor growth. Dynamic contrast-enhanced (DCE) MRI was utilized to measure the impact of vandetanib on tumor vasculature. Results: Vandetanib inhibited tumor growth of the ATC cell lines Hth83 and 8505C in vivo by 69.3% (P < 0.001) and 66.6% (P < 0.05), respectively, when compared with control. Significant decreases in vascular permeability (P < 0.01) and vascular volume fraction (P < 0.05) were detected by DCE-MRI in the orthotopic xenograft tumors after 1 week of treatment with vandetanib as compared with control. Conclusion: The inhibition of EGFR and VEGFR2 by vandetanib and its tremendous in vivo antitumor activity against ATC make it an attractive candidate for further preclinical and clinical development for the treatment of this particularly virulent cancer, which remains effectively untreatable. Vandetanib disrupts angiogenesis and DCE-MRI is an effective method to quantify changes in vascular function in vivo. Clin Cancer Res; 17(8); 2281–91. ©2011 AACR.

Background Cutaneous squamous cell carcinoma (CSCC) is the second most common nonmelanoma skin cancer. Most of the approximately 250,000 cases occurring annually in the United States are small, nonaggressive, and cured by excision alone. However, a subset of these tumors which are defined by poorly differentiated histology, large tumor size, invasion of adjacent structures, and/or regional metastases can prove resistant to treatment despite adjuvant radiotherapy and can have an increased risk of recurrence and nodal metastasis. Novel therapeutic approaches are necessary to improve the outcomes for patients with aggressive CSCC. Methods We analyzed the effect of targeted therapy on the growth and survival of CSCC cell lines using an anti-insulin-like growth factor-I receptor (IGF-IR) antibody, A12, alone or in combination with an anti-epidermal growth factor receptor (EGFR) antibody, cetuximab, both in vitro and in vivo in an athymic nude mouse model of CSCC. Results Treatment with A12 and cetuximab inhibited the signaling pathways of IGF-IR and EGFR and inhibited proliferation and induced apoptosis of squamous cell carcinoma (SCC) cell lines in vitro. Immunohistochemical staining revealed decreased proliferating cell nuclear antigen (PCNA), microvessel density, and increased apoptosis within the treated tumor xenografts. In addition, the administration of A12, alone or in combination with cetuximab inhibited the growth of tumors by 51% and 92%, respectively, and significantly enhanced survival in the nude mouse model of CSCC (p = .044 and p < .001, respectively). Conclusion These data suggest that dual treatment with monoclonal antibodies to the EGFR and IGF-IR may be therapeutically useful in the treatment of CSCC. © 2010 Wiley Periodicals, Inc. Head Neck, 2011

Purpose: Malignant sinonasal tumors are clinically challenging due to their proximity to vital structures and their diverse histogenesis and biological behavior. To date, no animal models accurately reflect the clinical behavior of these malignancies. We developed an orthotopic murine model of sinonasal malignancy that reproduces the intracranial extension, bony destruction, and spread along neural fascial planes seen in patients with aggressive sinonasal malignancies of various histologies. Experimental Design: Human squamous cell carcinoma line (DM14) and adenoid cystic carcinoma line (ACC-3) were implanted in the right maxillary sinus or soft palate in male nude mice. Animals were monitored for tumor growth and survival. Tumor specimens were removed for histopathologic evaluation to assess for intracranial extension, orbital invasion, bony invasion, perineural invasion, and distant metastasis. Statistical analysis was done to calculate P values with the Student's t test for individual tumor volumes. Differences in survival times were assessed using the log-rank test. Results: Mice with DM14 or ACC-3 implanted in either the maxillary sinus or the soft palate developed large primary tumors. A statistically significant inverse correlation between survival and the number of tumor cells implanted was found. Histopathologic evaluation revealed orbital invasion, intracranial extension, pulmonary metastasis, lymph node metastasis, and perineural invasion. Conclusions: We describe the first orthotopic model for sinonasal malignancy. Our model faithfully recapitulates the phenotype and malignant behavior of the aggressive tumor types seen in patients. This model offers an opportunity to identify and specifically target the aberrant molecular mechanisms underlying this heterogeneous group of malignancies.

Objective: This study compared changes in bodily pain, healthrelated quality of life (HRQoL), and psychological symptoms during an 8-week mindfulness-based stress reduction (MBSR) program among groups of participants with different chronic pain conditions. Methods: From 1997-2003, a longitudinal investigation of chronic pain patients (n=133) was nested within a larger prospective cohort study of heterogeneous patients participating in MBSR at a university-based Integrative Medicine center. Measures included the Short-Form 36 Health Survey and Symptom Checklist-90-Revised. Paired t tests were used to compare pre–post changes on outcome measures. Differences in treatment effect sizes were compared as a function of chronic pain condition. Correlations were examined between outcome parameters and home meditation practice. Results: Outcomes differed in significance and magnitude across common chronic pain conditions. Diagnostic subgroups of patients with arthritis, back/neck pain, or two or more comorbid pain conditions demonstrated a significant change in pain intensity and functional limitations due to pain following MBSR. Participants with arthritis showed the largest treatment effects for HRQoL and psychological distress. Patients with chronic headache/migraine experienced the smallest improvement in pain and HRQoL. Patients with fibromyalgia had the smallest improvement in psychological distress. Greater home meditation practice was associated with improvement on several outcome measures, including overall psychological distress, somatization symptoms, and self-rated health, but not pain and other quality of life scales. Conclusion: MBSR treatment effects on pain, HRQoL and psychological well-being vary as a function of chronic pain condition and compliance with home meditation practice.

Psychological distress is linked with impaired glycemic control among diabetics.Estimate changes in glycemic control, weight, blood pressure, and stress-related psychological symptoms in patients with type 2 diabetes participating in a standard Mindfulness Based Stress Reduction (MBSR) program.Prospective, observational study.Academic health center.Adult patients with type 2 diabetes mellitus.Participation in MBSR program for heterogeneous patient population. Diet and exercise regimens held constant.Glycosylated hemoglobin A1c (HA1c), blood pressure, body weight, and Symptom Checklist 90-Revised (anxiety, depression, somatization, and general psychological distress scores).Eleven of 14 patients completed the intervention. At 1 month follow-up, HA1c was reduced by 0.48% (P = .03), and mean arterial pressure was reduced by 6 mmHg (P = .009). Body weight did not change. A decrease in measures of depression, anxiety, and general psychological distress was observed.

Purpose: Anaplastic thyroid cancer (ATC) is a locally aggressive type of thyroid tumor with high rate of distant metastases. With conventional treatment, the median survival ranges from 4 to 12 months; therefore, new treatment options are needed. AZD2171 is a tyrosine kinase inhibitor of the vascular endothelial growth factor receptors (VEGFR) VEGFR-1, VEGFR-2, and VEGFR-3. The objective of the study is to determine whether AZD2171 can inhibit VEGFR-2 signaling and decrease tumor growth and prolong survival of ATC in an orthotopic nude mouse model. Experimental Design: We examined the effects of AZD2171 on phosphorylation of VEGFR-2, mitogen-activated protein kinase, and AKT in human umbilical vascular endothelial cells. To determine the antiproliferative and antiapoptotic effects of AZD2171, we did 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry assays, respectively. We assessed the antitumor effects of AZD2171 in a xenograft model of ATC using control, AZD2171, paclitaxel, and combination groups by measuring tumor size and survival. Results: Treatment with AZD2171 led to dose-dependent inhibition of VEGFR-2 phosphorylation and its downstream signaling in human umbilical vascular endothelial cells (IC50 for cell proliferation, 500 nmol/L). In the ATC cell lines DRO and ARO, IC50 was 7.5 μmol/L. AZD2171 induced apoptosis in 50% of endothelial and ATC cells at 3 and 10 μmol/L concentrations, respectively. In vivo, AZD2171 led to a significant reduction in tumor size between control and AZD2171 (P = 0.002) or AZD2171 + paclitaxel group (P = 0.002) but not the paclitaxel alone group (P = 0.11). Survival was significantly higher among AZD2171 (P < 0.001) and combination groups (P < 0.001) compared with control. Conclusions: AZD2171 effectively inhibits tumor growth and prolongs survival of ATC-bearing mice. The main effect of AZD2171 is mediated through angiogenesis inhibition.

Objective: To evaluate the effectiveness of a dual magnetic resonance-near infrared fluorescence optical imaging agent, poly(l-glutamic acid)-DTPA-Gd-NIR813, for both preoperative and intraoperative visualization and characterization of sentinel lymph nodes (SLN) in mice. Materials and Methods: Poly(l-glutamic acid) was conjugated with DTPA-Gd and NIR813 dye to obtain PG-DTPA-Gd-NIR813. To confirm drainage into the SLNs, this agent was injected subcutaneously into the front paw of nude mice followed by isosulfan blue (n = 6). Furthermore, PG-DTPA-Gd-NIR813 was injected subcutaneously at doses of 0.002 mmol Gd/kg (4.8 nmol eq. NIR813) and 0.02 mmol Gd/kg (48 nmol eq. NIR813) (n = 3/dose). To differentiate metastatic from nonmetastatic lymph nodes, nude mice bearing human oral squamous cell carcinoma (DM14) were injected intralingually with 0.02 mmol Gd/kg PG-DTPA-Gd-NIR813 (n = 3). Pre- and postcontrast images were taken using 4.7 T Bruker MRI scanner and Xenogen optical imaging system. The status of lymph nodes resected under the guidance of optical imaging was confirmed by histologic examinations. Results: PG-DTPA-Gd-NIR813 colocalized with isosulfan blue, indicating drainage to the SLN. After subcutaneous injection, axiliary and branchial lymph nodes were clearly visualized with both T1-weighted MR and optical imaging within 3 minutes of contrast injection, even at the lowest dose tested (0.002 mmol Gd/kg). After intralingual injection in tumor-bearing mice, MR imaging identified 4 of the 6 superficial cervical lymph nodes, whereas near-infrared fluorescence (NIRF) optical imaging identified all 6 cervical nodes. The pattern of contrast enhancement of SLN visualized in MR images showed a characteristic ring-shaped appearance with a central filling defect, possibly resulting from nodal infiltration of metastatic lesions. Histopathologic examination of the SLNs resected under NIRF imaging guidance revealed micrometastases in all 6 SLNs identified by NIRF imaging. Conclusions: The dual modality imaging method demonstrated in this study represents an effective technique for localization and characterization of SLN.

Anaplastic thyroid carcinoma (ATC) remains one of the most lethal human cancers. We hypothesized that sorafenib, a multikinase inhibitor of the BRaf, vascular endothelial growth factor receptor-2, and platelet-derived growth factor receptor-β kinase, would decrease tumor growth and angiogenesis in an orthotopic model of ATC. The in vitro antiproliferative and proapoptotic effects of sorafenib on ATC cell lines were examined. To study the in vivo effects of sorafenib on orthotopic ATC tumors in nude mice, sorafenib was given p.o. at 40 or 80 mg/kg daily. Intratumoral effects were studied using immunohistochemical analysis. The effect of sorafenib on survival of the mice was also studied. Sorafenib inhibited the in vitro proliferation of ATC cell lines. Sorafenib also significantly inhibited tumor angiogenesis via the induction of endothelial apoptosis in an orthotopic model of thyroid cancer. As result, the growth of orthotopic ATC xenografts was reduced and the survival of the test animals was improved. Sorafenib exerts significant antitumor activity in an orthotopic xenograft model of ATC via a potent antiangiogenic effect. The antiangiogenic effects of sorafenib suggest that its use in clinical setting may not depend on the BRAF mutational status of thyroid tumors. Given the lack of curative options for patients with ATC, sorafenib warrants further study as a therapeutic agent against ATC. [Mol Cancer Ther 2007;6(6):1785–92]

We evaluated the accuracy of diagnostic codes for schizophrenia in identifying actual cases in the historical charts of 801 primary care patients. The rate of schizophrenia by diagnostic code was 14%, whereas the estimated rate based on independent clinical chart review by a trained psychiatrist, using DSM-IV criteria, was 1.8%. The findings suggest that coded data alone should not be used to determine which patients have schizophrenia for research studies.

Objective To study the expression of integrin-linked kinase (ILK) in human squamous cell carcinoma of the head and neck (SCCHN) tumor specimens and cell lines and the efficacy of the novel small molecule QLT0267. Design Immunohistochemical analysis of 17 SCCHN tumor tissue specimens and 3 normal tongue tissue specimens for ILK expression and in vitro analysis of the effectiveness of QLT0267 on SCCHN cells. Setting Academic medical center. Main Outcome Measures Expression levels of ILK in SCCHN tumor specimens and cell lines and the efficacy of QLT0267 in inhibiting cell growth and inducing apoptosis in SCCHN cell lines. Results Most SCCHN tumor specimens stained for ILK, whereas none of the 3 normal tongue tissue specimens stained for ILK. Integrin-linked kinase was expressed in all 6 SCCHN cell lines tested. In 4 pairs of normal and SCCHN tumor specimens, ILK expression and activity were higher in most tumor samples tested. A kinase assay showed that QLT0267 inhibited the ILK activity of 2 SCCHN cell lines (TU167 and MDA1986). Modified tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA fragmentation ladder, and TUNEL (terminal deoxynucleotidyl transferase–mediated biotin–deoxyuridine triphosphate nick-end labeling) assays showed that QLT0267 inhibited cell growth and induced apoptosis in these 2 cell lines. A dose-dependent decrease in Akt phosphorylation was observed for these 2 cell lines on treatment with QLT0267. Conclusions Integrin-linked kinase is overexpressed in SCCHN tumor specimens. Targeting ILK with the small-molecule ILK inhibitor QLT0267 inhibits cell growth and induces apoptosis in SCCHN cell lines by reducing ILK activity and Akt phosphorylation. Integrin-linked kinase may be an attractive target for molecular therapy with which to enhance treatment of SCCHN.

We hypothesized that epidermal growth factor (EGF) receptor (EGFR) activation and vascular endothelial growth factor (VEGF)–induced angiogenic signals are important for the progression and metastasis of human salivary adenoid cystic carcinoma (ACC). To test this hypothesis, we evaluated the therapeutic effect of AEE788, a dual inhibitor of EGF and VEGF receptor (VEGFR) tyrosine kinases, on human salivary ACC. In clinical specimens of salivary ACC, EGF and VEGF signaling proteins were expressed at markedly higher levels than in adjacent normal glandular tissues. We examined the effects of AEE788 on salivary ACC cell growth and apoptosis and on the phosphorylation of EGFR and VEGFR-2 in salivary ACC cells. Treatment of salivary ACC cells with AEE788, alone or in combination with chemotherapy, led to growth inhibition, induction of apoptosis, and dose-dependent inhibition of EGFR and VEGFR-2 phosphorylation. To determine the in vivo antitumor effects of AEE788, nude mice with orthotopic parotid tumors were randomized to receive oral AEE788 alone, paclitaxel alone, cisplatin alone, a combination of AEE788 plus paclitaxel, a combination of AEE788 plus cisplatin, or a placebo. AEE788 inhibited tumor growth and prevented lung metastasis in nude mice. To study the mechanism of interaction between AEE788 and chemotherapy, AEE788 was found to potentiate growth inhibition and apoptosis of ACC tumor cells mediated by chemotherapy. Tumors of mice treated with AEE788 and AEE788 plus chemotherapy exhibited down-regulation of activated EGFR and VEGFR-2, increased tumor and endothelial cell apoptosis, and decreased microvessel density, which correlated with a decrease in the level of matrix metalloproteinase-9 and matrix metalloproteinase-2 expression and a decrease in the incidence of vascular metastasis. These data show that EGFR and VEGFR can be molecular targets for therapy of salivary ACC. [Mol Cancer Ther 2006;5(11):2696–705]

Abstract Studies in rodents with retinal degeneration indicated that neither the rod nor the cone photoreceptors obligatorily participate in circadian responses to light, including melatonin suppression and photoperiodic response. Yet there is a residual phase-shifting response in melanopsin knockout mice, which suggests an alternate or redundant means for light input to the SCN of the hypothalamus. The findings of Aggelopoulos and Meissl suggest a complex, dynamic interrelationship between the classic visual photoreceptors and SCN cell sensitivity to light stimuli, relative to various adaptive lighting conditions. These studies raised the possibility that the phototransductive physiology of the retinohypothalamic tract in humans might be modulated by the visual rod and cone photoreceptors. The aim of the following two-part study was to test the hypothesis that dim light adaptation will dampen the subsequent suppression of melatonin by monochromatic light in healthy human subjects. Each experiment included 5 female and 3 male human subjects between the ages of 18 and 30 years, with normal color vision. Dim white light and darkness adaptation exposures occurred between midnight and 0200 h, and a full-field 460-nm light exposure subsequently occurred between 0200 and 0330-h for each adaptation condition, at 2 different intensities. Plasma samples were drawn following the 2-h adaptation, as well as after the 460-nm monochromatic light exposure, and melatonin was measured by radioimmunoassay. Comparison of melatonin suppression responses to monochromatic light in both studies revealed a loss of significant suppression after dim white light adaptation compared with dark adaptation ( p < 0.04 and p < 0.01). These findings indicate that the activity of the novel circadian photoreceptive system in humans is subject to subthreshold modulation of its sensitivity to subsequent monochromatic light exposure, varying with the conditions of light adaptation prior to exposure.

Oral cavity squamous cell carcinoma (OSCC) is one of the leading causes of cancer deaths worldwide and most of these deaths result from local-regional recurrence and metastases. Evasion of apoptosis is an important hallmark of cancer development and progression, and previous studies have shown that evasion of anoikis, or detachment-induced apoptosis, correlates with a more aggressive phenotype of carcinoma cells in OSCC. To elucidate the cytogenetic and molecular characteristics of anoikis resistance, we generated several cell lines and clones that displayed this cellular phenotype. To test the hypothesis that chromosomal alterations may underlie this phenotypic transformation, we used karyotype analysis to observe changes in the chromosomal structure of anoikis-sensitive and anoikis-resistant cell lines. We further hypothesized that a unique pattern of gene expression was induced by cell-detachment of anoikis-resistant cell lines, and cDNA microarray analysis was performed using a panel of anoikis-resistant oral cancer cell lines grown under attached and detached growth conditions. We identified S100P, KLK6 and CTNNAL1 as genes whose expression levels were differentially regulated in the anoikis-resistant cell lines compared to the anoikis-sensitive cells under detached conditions. These results were verified using real-time RT-PCR. The anoikis-resistant phenotype of squamous cell carcinoma has a distinct genetic expression pattern that is marked by chromosomal alterations that may contribute to differential expression of genes involved in diverse cellular functions. Therapies targeting these potential mediators of anoikis resistance may prove to be beneficial in the treatment of metastatic squamous cell carcinoma.

Purpose: Anaplastic thyroid carcinoma (ATC) remains one of the most lethal known human cancers. Targeted molecular therapy with cetuximab, a monoclonal antibody against epidermal growth factor receptor, offers new treatment potentials for patient with ATC. Cetuximab has also been reported to have synergistic effects when combined with irinotecan, a topoisomerase inhibitor. Therefore, we hypothesized that cetuximab and irinotecan would be effective in inhibiting the growth and progression of ATC in a murine orthotopic model. Experimental Design: The in vitro antiproliferative effects of cetuximab and irinotecan on ATC cell line ARO were examined. We also studied the in vivo effects of cetuximab and irinotecan on the growth, invasion, and metastasis of orthotopic ATC tumors in nude mice. The in vivo antitumor efficacy of cetuximab/irinotecan combination was also compared with that of doxorubicin. Results: Cetuximab alone did not show any antiproliferative or proapoptotic effect on this cell line. However, when combined with irinotecan, cetuximab potentiated the in vitro antiproliferative and proapoptotic effect of irinotecan. Cetuximab, irinotecan, and cetuximab/irinotecan combination resulted in 77%, 79%, and 93% in vivo inhibition of tumor growth, respectively. Incidences of lymph node metastasis, laryngeal invasion, and tumor microvessel density were also significantly decreased in these treatment groups. Furthermore, the cetuximab/irinotecan combination was significantly more effective than doxorubicin in inhibiting the growth of orthotopic ATC xenografts. Conclusions: Combination therapy with cetuximab/irinotecan inhibits the growth and progression of orthotopic ATC xenografts in nude mice. Given the lack of curative options for patients with ATC, combination therapy with cetuximab and irinotecan treatment warrants further study.

Background. A preclinical evaluation of CPT-1 (Camptosar, irinotecan) and PTK787/ZK222584, a vascular endothelial growth factor receptor (VEGFR-2) tyrosine kinase inhibitor, as therapeutic agents against anaplastic thyroid carcinoma (ATC) was performed in vitro and in an orthotopic model of ATC in nude mice. Methods. The cytotoxic and cytostatic effects of CPT-11 on ATC cell lines were evaluated. The antitumor effects of CPT-11 in combination with PTK787/ZK222584 on orthotopic ATC xenografts in nude mice were also studied. Results. CPT-11 demonstrated significant antiproliferative effects on ATC cell lines. In vivo, PTK787/ZK222584, CPT-11, and the two agents together produced 61%, 82%, and 89% decrease in tumor growth, respectively. The differences in tumor volume between CPT-11 and CPT-11 + PTK787/ZK222584 groups were not statistically significant. PTK787/ZK222584 inhibited the phosphorylation of VEGFR-2 on tumor endothelium and decrease the tumor microvessel density. Conclusions. The camptothecin class of chemotherapeutic agents and antiangiogenic agents such as PTK787/ZK222584 warrant further study as novel therapeutic agents against ATC. © 2005 Wiley Periodicals, Inc. Head Neck27: 389–399, 2006

The increased breast cancer risk in female night shift workers has been postulated to result from the suppression of pineal melatonin production by exposure to light at night. Exposure of rats bearing rat hepatomas or human breast cancer xenografts to increasing intensities of white fluorescent light during each 12-hour dark phase (0-345 μW/cm2) resulted in a dose-dependent suppression of nocturnal melatonin blood levels and a stimulation of tumor growth and linoleic acid uptake/metabolism to the mitogenic molecule 13-hydroxyoctadecadienoic acid. Venous blood samples were collected from healthy, premenopausal female volunteers during either the daytime, nighttime, or nighttime following 90 minutes of ocular bright, white fluorescent light exposure at 580 μW/cm2 (i.e., 2,800 lx). Compared with tumors perfused with daytime-collected melatonin-deficient blood, human breast cancer xenografts and rat hepatomas perfused in situ, with nocturnal, physiologically melatonin-rich blood collected during the night, exhibited markedly suppressed proliferative activity and linoleic acid uptake/metabolism. Tumors perfused with melatonin-deficient blood collected following ocular exposure to light at night exhibited the daytime pattern of high tumor proliferative activity. These results are the first to show that the tumor growth response to exposure to light during darkness is intensity dependent and that the human nocturnal, circadian melatonin signal not only inhibits human breast cancer growth but that this effect is extinguished by short-term ocular exposure to bright, white light at night. These mechanistic studies are the first to provide a rational biological explanation for the increased breast cancer risk in female night shift workers.