The embryonic expression of COUP-TFII, an orphan nuclear receptor, suggests that it may participate in mesenchymal-epithelial interactions required for organogenesis. Targeted deletion of the COUP-TFII gene results in embryonic lethality with defects in angiogenesis and heart development. COUP-TFII mutants are defective in remodeling the primitive capillary plexus into large and small microcapillaries. In the COUP-TFII mutant heart, the atria and sinus venosus fail to develop past the primitive tube stage. Reciprocal interactions between the endothelium and the mesenchyme in the vascular system and heart are essential for normal development of these systems. In fact, the expression of Angiopoietin-1, a proangiogenic soluble factor thought to mediate the mesenchymal-endothelial interactions during heart development and vascular remodeling, is down-regulated in COUP-TFII mutants. This down-regulation suggests that COUP-TFII may be required for bidirectional signaling between the endothelial and mesenchymal compartments essential for proper angiogenesis and heart development.

Recent studies describing the mutational landscape of head and neck squamous cell carcinoma (HNSCC) on a genomic scale by our group and others, including The Cancer Genome Atlas, have provided unprecedented perspective for understanding the molecular pathogenesis of HNSCC progression and response to treatment. These studies confirmed that mutations of the TP53 tumor suppressor gene were the most frequent of all somatic genomic alterations in HNSCC, alluding to the importance of the TP53 gene in suppressing the development and progression of this disease. Clinically, TP53 mutations are significantly associated with short survival time and tumor resistance to radiotherapy and chemotherapy in HNSCC patients, which makes the TP53 mutation status a potentially useful molecular factor for risk stratification and predictor of clinical response in these patients. In addition to loss of wild-type p53 function and the dominant-negative effect on the remaining wild-type p53, some p53 mutants often gain oncogenic functions to promote tumorigenesis and progression. Different p53 mutants may possess different gain-of-function properties. Therefore, mutant p53 is not just one protein but actually a variety of proteins that contribute to an exceptionally vast network of tumor-promoting processes. Herein we review the most up-to-date information about TP53 mutations available via The Cancer Genome Atlas-based analysis of HNSCC and discuss our current understanding of the potential tumor-suppressive role of p53, focusing on gain-of-function activities of p53 mutations. We also summarize our knowledge regarding use of the TP53 mutation status as a potential evaluation or stratification biomarker for prognosis and a predictor of clinical response to radiotherapy and chemotherapy in HNSCC patients. Finally, we discuss possible strategies for targeting HNSCCs bearing TP53 mutations.

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE) Proteomics (1) includes the systematic cataloging of protein expression on a large scale, providing complementary information to that obtained from mRNA profiling by microarray (2, 3). Such studies could lead to the molecular characterization of cellular events associated with cancer progression, cellular signaling, and developmental stages (4 -7). Proteomics studies of clinical tumor samples have led to the identification of cancer-specific protein markers, which provide a basis for developing new methods for early diagnosis and early detection and clues to understand the molecular characterization of cancer progression (5, 8 -10).,A mainstay of conventional proteomics is high resolution 2D 1 gel electrophoresis (11, 12) followed by protein identification using mass spectrometry (13-15). The state of the art 2D gel system can be loaded with a few milligrams of protein and separates thousands of protein spots (5, 16). Although the technique has been widely used and successfully applied in a variety of biological systems, several technical limitations exist. Because of subtle changes in experimental conditions, the protein expression patterns on a single 2D gel usually cannot be fully duplicated, which makes it difficult to find the proteins changed between gels and to quantify the changes in protein expression. Although a comparison of protein expression profiles from regular 2D gel electrophoresis can be carried out with the assistance of various software programs, it typically requires some computerized justification of 2D gel images so that two images can be superimposed and compared. These difficulties limit the speed and accuracy of quantitation of protein spots in 2D gel electrophoresis.The differential in-gel electrophoresis (DIGE) technique recently introduced by Amersham Biosciences, Inc. is aimed at improving reproducibility. The concept of DIGE was originally developed by Minden and colleagues (17). To analyze the samples in DIGE, two pools of protein extracts are labeled covalently with fluorescent cyanine dyes, Cy3 and Cy5, re-

Head and neck squamous cell carcinoma (HNSCC) remains a significant public health problem, accounting for over 5% of all cancer-related deaths, and these deaths primarily result from metastatic disease. The molecular processes involved in HNSCC pathogenesis and progression are poorly understood, and here we present experimental evidence for a direct role of the cell surface receptor tyrosine kinase, TrkB, in HNSCC tumor progression. Using immunohistochemical analysis and transcriptional profiling of archival HNSCC tumor specimens, we found that TrkB and its secreted ligand, brain-derived neurotrophic factor (BDNF), are expresses in greater than 50% of human HNSCC tumors, but not in normal upper aerodigestive tract (UADT) epithelia. Studies with HNSCC cell lines reveal that in vitro stimulation with BDNF, the ligand for TrkB, upregulates the migration and invasion of HNSCC cells, and both transient and stable suppressions of TrkB result in significant abrogation of constitutive and ligand-mediated migration and invasion. Furthermore, enforced over-expression of TrkB results in altered expression of molecular mediators of epithelial-to-mesenchymal transition (EMT), including downregulation of E-cadherin and upregulation of Twist. Using an in vivo mouse model of HNSCC, we were able to show that downregulation of TrkB suppresses tumor growth. These results directly implicate TrkB in EMT and the invasive behavior of HNSCC, and correlate with the in vivo overexpression of TrkB in human HNSCC. Taken together, these data suggest that the TrkB receptor may be a critical component in the multi-step tumor progression of HNSCC, and may be an attractive target for much needed new therapies for this disease.

SUMMARY 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.