Signal and Transcription Factor Network interactions in Head and Neck Cancer

尽管组成性激活的NF-kB、减弱的TGF β信号传导和TP 53突变经常在人类癌症中共同发生，但这些途径如何相互作用并共同促成恶性肿瘤及其作为治疗和预防靶点的潜力是一个重要问题。 我们先前发现头颈部鳞状细胞癌（HNSCC）中NF-κ B相关基因的过表达、TGF β受体（TBR）亚单位的表达改变和下游靶点之间存在关联。在过去的一年中，我们报道了TGF β受体I/磷酸酶张力蛋白（Tgfbr 1/Pten）在HNSCC中经常降低，并且这些基因的条件性双敲除小鼠发展出表现出PI 3 K-Akt途径和NF-κ B/REL活化增加的HNSCC（Bian，Oncogene，2012）。随后的研究证明了PI 3 K-mTOR抑制剂用于预防这些Tgfbr 1/Pten dko小鼠中的肿瘤发展和治疗携带人异种移植物的小鼠的潜力（Herzog，Bian等人，提交）。我们发现PI 3 K-mTOR拮抗剂抑制下游促生长信号传导，重新激活肿瘤抑制因子TP 5，并使肿瘤对细胞毒性化疗和放疗敏感。作为与NIDCR研究人员合作的NIH主任Bench to Bedside奖的共同获得者，我们已经开始了一项新辅助mTOR抑制剂雷帕霉素在晚期HNSCC患者中的试点临床研究（NIH方案10-D-180）。 热休克蛋白90抑制剂靶向许多癌蛋白的信号网络，我们已经表明，在HNSCC共激活。在合作研究中，我们报告了HSP 90抑制剂针对过表达的表皮生长因子受体、下游信号传导，并强烈抑制人HNSCC异种移植物（Ahsan等人，Neoplasia，2012）。在NIH的I期研究中，另一种HSP 90抑制剂证明了在人肿瘤异种移植物中抑制致癌信号和转录因子以及野生型p53的再活化（Friedman等人，提交）。 炎症和炎性信号传导涉及促进癌症发展，包括人头颈部鳞状细胞癌（HNSCC）。炎症信号如何抑制细胞生长停滞和凋亡，同时促进参与迁移、血管生成和转移的许多基因尚不清楚。 炎性细胞因子肿瘤坏死因子-α（TNF-α）诱导含有TNF-α或c-REL的NF-κ B/REL转录因子的核转位，其介导对其细胞毒性作用和细胞存活的抗性。c-REL-A诱导促存活基因，但c-REL的独特功能作用尚不清楚。我们最近与King、温伯格和FDA的同事合作，证明c-REL与HNSCC中的p53同源物DeltaNp 63 alpha形成了一种新型核复合物（King K等人，Cancer Res，2008）。TP 53家族包括TP 53和TAp 73，它们可以诱导细胞凋亡，而deltaNp 63 α可以抑制它们的功能并激活其他基因。对人HNSCC中c-REL和TP 53家族蛋白表达的检查揭示了c-REL和dNp 63的核共定位，而促凋亡家族成员p73和TP 53的核表达在大多数肿瘤和细胞系中减弱。有趣的是，在不存在TNF-α的情况下培养的具有TP 53突变的HNSCC细胞系的亚组中观察到核c-REL、dNp 63、TAp 73和TP 53。加入TNF-α增强了c-REL与dNp 63 a的核相互作用，从核中置换TAp 73以及关键生长停滞和凋亡基因p21 CIP 1/WAF 1、NOXA和TAP 73 A的启动子。相反，组成型c-REL的siRNA耗竭增强了TAp 73启动子结合和这些基因的表达，并抑制了含有突变型TP 53的细胞的细胞增殖和存活。因此，组成型和TNF-α诱导的c-REL/dNp 63 a抑制HNSCC中的TAp 73靶向促凋亡基因。这种涉及c-REL和TP 53家族的新的串扰提供了一种独特的机制，其中炎性细胞因子如TNF-α和NF-κ B家族成员cREL可以在具有突变型TP 53的癌细胞的生长停滞和凋亡中失调TAp 73的补偿作用（Lu等人，Cancer Res，2011）。 我们假设dNp 63可能在调节NF-κ B相关以及p53/p63/p73靶基因中具有潜在的更广泛的作用，这些靶基因促进癌症中的细胞增殖、存活、迁移、炎症和血管生成。我们发现，deltaNp 63，经常过表达突变型TP 53和调节一个广泛的基因程序牵连HNSCC。该基因程序与参与细胞生长、存活、炎症和粘附的NF-κ B转录组基本重叠。这种调节包括一种新的相互作用，其中deltaNp 63与NF-κ B家族成员cRel复合，以协同结合靶基因启动子的p63或p65/cRel位点。deltaNp 63或TNF-α的过表达通过近端IL-8启动子中的p65/cRel位点激活IL-8报道活性。与这种多样化的基因程序一致，deltaNp 63的调节显著改变了HNSCC恶性表型。我们的研究揭示了TP 53和NF-κ B通路之间的新联系，通过新鉴定的转录机制，通过deltaNp 63/cRel复合物。这种复合物及其调节的下游基因可以作为具有缺陷性TP 53的肿瘤中的新的生物标志物和治疗靶标（Yang等人，Cancer Res，2011）。总之，这些发现表明协调NF-kB、p63和p73相互作用和功能的信号调节在发病机制中以及作为预防和治疗的靶标可能是重要的。

Although constitutively activated NF-kB, attenuated TGFb signaling, and TP53 mutations frequently co-occur in human cancers, how these pathways interact and together contribute to malignancy and their potential as targets for therapy and prevention are an important problem. We previously found an association between overexpression of NF-kB related genes, altered expression of TGFb receptor (TBR) subunits and downstream targets in head and neck squamous cell carcinoma (HNSCC). During the past year we reported that TGFbeta receptor I/ phosphastase tensin (Tgfbr1/Pten) are frequently decreased in HNSCC, and conditional double knockout mice for these genes develop HNSCC that exhibit increased PI3K-Akt pathway and NF-kappaB/REL activation (Bian, Oncogene, 2012). Subsequent studies demonstrate the potential of PI3K-mTOR inhibitor for prevention of tumor development in these Tgfbr1/Pten dko mice and therapy of human xenograft bearing mice (Herzog, Bian et al., submitted). We found that PI3K-mTOR antagonist inhibited downstream progrowth signaling, reactivated tumor suppressor TP5, and sensitized tumors to cytotoxic chemotherapy and radiation. As co-recipients of an NIH Director's Bench to Bedside Award in collaboration with NIDCR investigators, we have initiated a pilot clinical study of neoadjuvant mTOR inhibitor rapamycin in patients with advanced HNSCC (NIH protocol 10-D-180). Heat Shock Protein 90 inhibitors target many oncoproteins in the signal network we have shown is co-activated in HNSCC. In collaborative studies, we reported that HSP90 inhibitors target overexpressed Epidermal growth factor receptor, downstream signaling, and strongly inhibits human HNSCC xenografts (Ahsan et al., Neoplasia, 2012). Another HSP90 inhibitor in phase I studies at NIH demonstrated inhibition of oncogenic signal and transcription factors and reactivation of wild type p53 in human tumor xenografts (Friedman et al., submitted). 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 and NF-kB family member cREL can dysregulate the compensatory role of TAp73 in growth arrest and apoptosis of cancer cells with mutant TP53 (Lu et al, Cancer Res, 2011). 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, Cancer Res, 2011).Together, these findings suggest signal regulation coordinating NF-kB, p63 and p73 interactions and function may be important in pathogenesis and as targets for prevention and therapy.