Signal and Transcription Factor Network interactions in Head and Neck Cancer

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Although constitutively activated NF-κB, attenuated TGFβ signaling and TP53 mutations frequently occur in human cancers, how these pathways interact and together contribute to malignancy remains uncertain. Here, we found an association between overexpression of NF-κB related genes, reduced expression of TGFβ receptor (TBR) subunits and downstream targets, and TP53 genotype in head and neck squamous cell carcinoma (HNSCC). In response to recombinant TGFβ1, both growth inhibition and TGFβ target gene modulation were attenuated or absent in a panel of human HNSCC lines. However, in HNSCC cells that retained residual TGFβ signaling, TGFβ1 inhibited both constitutive and TNFα-stimulated NF-κB activity. Furthermore, HNSCC lines overexpressing mutant (mt)TP53 and human tumor specimens with positive TP53 nuclear staining exhibited reduced TβRII, and knocking down mtTP53 induced TβRII, increasing TGFβ downstream gene expression while inhibiting proinflammatory NF-κB target gene expression. Transfection of ectopic TβRII directly restored TGFβ signaling while inhibiting IκBα degradation and suppressing serine 536 phosphorylation of NF-κB p65 and NF-κB transcriptional activation, linking these alterations. Finally, experiments with TβRII conditional knockout mice demonstrate that abrogation of TGFβ signaling promotes the sustained induction of NF-κB and its proinflammatory target genes during HNSCC tumorigenesis and progression. Together, these findings elucidate a regulatory framework in which attenuated TGFβ signaling promotes NF-κB activation and squamous epithelial malignancy in the setting of altered TP53 status. Inflammation and inflammatory signaling is implicated in promoting cancer development, including human head and neck squamous cell carcinomas (HNSCC). How inflammatory signals can inhibit cell growth arrest and apoptosis while promoting many genes involved in migration, angiogenesis, and metastasis is unclear. Inflammatory cytokine Tumor Necrosis Factor-alpha (TNF-alpha) induces nuclear translocation of NF-kB/REL transcription factors containing REL-A or c-REL, which mediate resistance to its cytotoxic effects and cell survival. REL-A induces pro-survival genes, but the distinctive functional role of c-REL is unclear. We recently collaborated with King, Weinberg and colleagues in the FDA, demonstrating that c-REL forms a novel nuclear complex with the p53 homologue DeltaNp63alpha in HNSCC (King K et al., Cancer Res, 2008). The TP53 family includes TP53 and TAp73, which can induce apoptosis, while deltaNp63alpha can inhibit their function and activate other genes. Examination of c-REL and TP53 family protein expression in human HNSCC reveals nuclear co-localization of c-REL and dNp63, while nuclear expression of pro-apoptotic family members p73 and TP53 is attenuated in a majority of tumors and cell lines. Interestingly, nuclear c-REL, dNp63, TAp73 and TP53 is observed in a subset of HNSCC cell lines with mutations of TP53 cultured in the absence of TNF-a. Addition of TNF-a enhanced c-REL nuclear interaction with dNp63a, displacing TAp73 from the nucleus and promoters of the key growth arrest and apoptotic genes p21CIP1/WAF1, NOXA and PUMA. Conversely, siRNA depletion of constitutive c-REL enhanced TAp73 promoter binding and expression of these genes, and inhibited cell proliferation and survival of cells containing mutant TP53. Thus, constitutive and TNF-a-induced c-REL/dNp63a represses TAp73 target pro-apoptotic genes in HNSCC. This novel cross-talk involving c-REL and the TP53 family provides a distinctive mechanism whereby inflammatory cytokines such as TNF-a can dysregulate the compensatory role of TAp73 in growth arrest and apoptosis of cancer cells with mutant TP53 (Lu et al, manuscript submitted). We further investigated the potential role of signaling and transcriptional regulation of a co-factor of p73 called Yes associated protein (YAP). We previously observed differential expression and cytoplasmic-nuclear distribution of YAP in HNSCC (Lee et al, Clin Cancer Res 2007), but the mechanisms underlying these differences as well as the functional role of YAP, as an oncogene or tumor-suppressor, remained undetermined. In this study, we found that a subset of cell lines and human tissue samples with elevated, predominately cytoplasmic YAP expression, exhibit low expression of p53 family members and wt p53 genotype, while another subset exhibits weaker, nuclear YAP expression, together with overexpression of p53 family members and mt p53 genotype. Elevated YAP was correlated with cytoplasmic sequestration related to activated AKT kinase and YAP phosphorylation. Decreased YAP levels were correlated to transcriptional repression by dNp63. Increasing nuclear YAP by transfection of a YAP-serine-127-alanine AKT phosphoacceptor-site mutant or YAP transcription by dNp63 siRNA significantly increased cell death, while knocking down YAP enhanced cell proliferation, survival, migration, and decreased chemosensitivity. Thus, YAP functions as a tumor suppressor in HNSCC, and is predominantly dysregulated by cytoplasmic sequestration via protein phosphorylation or dNp63-mediated transcriptional repression These findings suggest that differences in YAP function may be related to transcriptional or post-translational mechanisms, and that dNp63 and/or AKT may represent important targets for enhancing YAP-mediated apoptosis and chemosensitivity. AKT inhibitor was shown to enhance nuclear YAP and apoptosis (Ehsanian et al, manuscript in preparation). We hypothesized that dNp63 may have a potentially broader role in regulating NF-kB related as well as p53/p63/p73 target genes that promote cell proliferation, survival, migration, inflammation and angiogenesis in cancer. We found that deltaNp63, is often overexpressed with mutant TP53 and regulates a broad gene program implicated in HNSCC. This gene program substantially overlaps with the NF-kappaB transcriptome involved in cell growth, survival, inflammation and adhesion. This regulation includes a novel interaction where deltaNp63 complexes with NF-kappaB family member, cRel, to coordinately bind to p63 or p65/cRel sites of target gene promoters. Overexpression of deltaNp63 or TNF-alpha activates IL-8 reporter activity via a p65/cRel site in the proximal IL-8 promoter. Consistent with this diverse gene program, modulation of deltaNp63 significantly altered the HNSCC malignant phenotype. Our studies reveal a novel link between TP53 and NF-kappaB pathways, through a newly identified transcriptional mechanism via deltaNp63/cRel complex. This complex and its regulated downstream genes could serve as novel biomarkers and therapeutic targets in tumors with defective TP53 (Yang et al, manuscript in preparation).