Overexpression of PIK3CA in murine head and neck epithelium drives tumor invasion and metastasis through PDK1 and enhanced TGFß signaling.

Head and neck squamous cell carcinoma (HNSCC) patients have a poor prognosis, with invasion and metastasis as major causes of mortality. The phosphatidylinositol 3-kinase (PI3K) pathway regulates a wide range of cellular processes crucial for tumorigenesis, and PIK3CA amplification and mutation are among the most common genetic alterations in human HNSCC. Compared with the well-documented roles of the PI3K pathway in cell growth and survival, the roles of the PI3K pathway in tumor invasion and metastasis have not been well delineated. We generated a PIK3CA genetically engineered mouse model (PIK3CA-GEMM) in which wild-type PIK3CA is overexpressed in head and neck epithelium. Although PIK3CA overexpression alone was not sufficient to initiate HNSCC formation, it significantly increased tumor susceptibility in an oral carcinogenesis mouse model. PIK3CA overexpression in mouse oral epithelium increased tumor invasiveness and metastasis by increasing epithelial-mesenchymal transition and by enriching a cancer stem cell phenotype in tumor epithelial cells. In addition to these epithelial alterations, we also observed marked inflammation in tumor stroma. AKT is a central signaling mediator of the PI3K pathway. However, molecular analysis suggested that progression of PIK3CA-driven HNSCC is facilitated by 3-phosphoinositide-dependent protein kinase (PDK1) and enhanced transforming growth factor ß (TGFß) signaling rather than by AKT. Examination of human HNSCC clinical samples revealed that both PIK3CA and PDK1 protein levels correlated with tumor progression, highlighting the significance of this pathway. In summary, our results offer significant insight into how PIK3CA overexpression drives HNSCC invasion and metastasis, providing a rationale for targeting PI3K/PDK1 and TGFß signaling in advanced HNSCC patients with PIK3CA amplification.

Redox-dependent Brca1 transcriptional regulation by an NADH-sensor CtBP1.

C-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor and metabolic sensory protein, which often represses tumor suppressor genes. Hence, we sought to determine if CtBP1 affects expression of the tumor suppressor Brca1 in head and neck tissue, as downregulation of Brca1 begins at the early stages of head and neck squamous cell carcinomas (HNSCCs). We found that CtBP1 represses Brca1 transcription by binding to the E2F4 site of the Brca1 promoter. Additionally, the recruitment of CtBP1 to the Brca1 promoter is redox-dependent, that is, increased at high NADH levels in hypoxic conditions. Further, immunostaining using a human HNSCC tissue array revealed that nuclear CtBP1 staining began to accumulate in hyperplasic lesions and HNSCCs, this staining correlated with Brca1 downregulation in these lesions. Pharmacological disruption of CtBP1 binding to Brca1 promoter by the antioxidant Tempol, which reduces NADH levels, relieved CtBP1-mediated repression of Brca1, leading to increased DNA repair in HNSCC cells. As tumor cells are generally hypoxic with increased NADH levels, the dynamic control of Brca1 by a 'metabolic switch' found in this study not only provides an important link between tumor metabolism and tumor suppressor expression but also suggests a potential chemo preventative or therapeutic strategy for HNSCC by blocking NADH-dependent CtBP1 activity at early stages of HNSCC carcinogenesis.

TGFß signaling in head and neck squamous cell carcinoma.

Transforming growth factor beta (TGFß) is a key regulator of epithelial cell proliferation, immune function and angiogenesis. Because TGFß signaling maintains epithelial homeostasis, dysregulated TGFß signaling is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Defective TGFß signaling in epithelial cells causes hyperproliferation, reduced apoptosis and increased genomic instability, and the compensatory increase in TGFß production by tumor epithelial cells with TGFß signaling defects further promotes tumor growth and metastases by increasing angiogenesis and inflammation in tumor stromal cells. Here, we review the mouse models that we used to study TGFß signaling in HNSCC.

Composite glucocorticoid regulation at a functionally defined negative glucocorticoid response element of the human corticotropin-releasing hormone gene.

Glucocorticoid-dependent negative feedback of the hypothalamic-pituitary-adrenal axis is mediated in part through direct inhibition of hypothalamic CRH gene transcription. In the present study, we sought to further localize and characterize glucocorticoid receptor (GR) and AP-1 interactions at a functionally defined negative glucocorticoid response element (nGRE) of the CRH promoter. Transient transfection studies in mouse corticotroph AtT-20 cells demonstrated that internal deletion of the nGRE (-278 to -249 nucleotides) within the context of 1 kb of the intact CRH promoter resulted in decreased 8-BrcAMP stimulation and glucocorticoid-dependent repression of CRH promoter activity. The nGRE conferred transcriptional activation by both cAMP and overexpressed c-jun or c-fos AP-1 nucleoproteins as well as specific glucocorticoid-dependent repression to a heterologous promoter. A similar profile of regulation was observed for the composite GRE derived from mouse proliferin promoter. The CRH nGRE was clearly distinct from the consensus cAMP response element (CRE) at -224 nucleotides, which increased basal activity and cAMP responsiveness of a heterologous promoter but did not confer glucocorticoid-dependent repression. High-affinity binding sites for both GR and AP-1 nucleoproteins were identified at adjacent elements within the nGRE. Mutations that disrupted either GR or AP-1 binding activity were associated with loss of glucocorticoid-dependent repression. These results are consistent with a composite mechanism of glucocorticoid-dependent repression involving direct DNA binding of GR and AP-1 nucleoproteins at discrete adjacent sites within the CRH promoter.

Localization of a negative glucocorticoid response element of the human corticotropin releasing hormone gene.

Corticotropin releasing hormone (CRH) plays a primary role in mediating suprapituitary activation of the hypothalamic-pituitary-adrenal axis and is an important physiologic target of negative feedback regulation by glucocorticoids. We sought to define cis-acting regions of the CRH promoter responsible for cAMP-dependent activation and glucocorticoid-dependent repression of CRH promoter activity. In transiently transfected AtT-20 cells, cAMP-dependent transcriptional activation was mediated largely through a classical, consensus, cAMP-response element (CRE) at - 224 bp. Dexamethasone (DEX) produced a specific 2-3-fold repression of cAMP-stimulated, but not basal, CRH promoter activity. Using a series of 5' nested deletions, dexamethasone-dependent repression of cAMP-stimulated CRH promoter activity was localized to promoter sequences between -278 and -249 bp. Specific, high-affinity binding of glucocorticoid receptor (GR) DNA-binding domain to this promoter region was observed using an eletrophoretic mobility shift assay (EMSA). We conclude that (i) cAMP dependent activation of the CRH promoter is mediated primarily by the CRE at -224 bp, (ii) glucocorticoid-dependent repression is specific for the CRH promoter, and not a generalized effect of glucocorticoid signaling or interference with the protein kinase A (PKA) signaling pathway, (iii) a highly conserved region between -278 and -249 bp is critical for glucocorticoid dependent repression, and (iv) GR is capable of interacting directly with this functionally defined negative glucocorticoid response element of the CRH promoter.