Quercetin ameliorates XIAP deficiency-associated hyperinflammation.

XIAP (X-linked inhibitor of apoptosis) deficiency is a rare inborn error of immunity. XIAP deficiency causes hyperinflammatory disease manifestations due to dysregulated TNF (tumor necrosis factor)-receptor signaling and NLRP3 (NOD- [nucleotide-binding oligomerization domain], LRR- [leucine-rich repeat] and pyrin domain-containing protein 3) inflammasome function. Safe and effective long-term treatments are needed and are especially important to help prevent the need for high-risk allogeneic hematopoietic cell transplantation. Here we evaluated inflammasome inhibitors as potential therapeutics with a focus on the natural flavonoid antioxidant quercetin. Bone marrow (BM)-derived macrophages were derived from XIAP-deficient or wild-type (WT) mice. Human monocytes were obtained from control or XIAP-deficient patients. Cells were stimulated with TLR (Toll-like receptor) agonists or TNF-α ± inhibitors or quercetin. For in vivo lipopolysaccharide (LPS) challenge experiments, XIAP-deficient or WT mice were fed mouse chow ± supplemental quercetin (50 mg/kg per day exposure) for 7 days followed by a challenge with 10 ng/kg LPS. IL-1ß (interleukin-1ß) and IL-18 were measured by ELISA (enzyme-linked immunosorbent assay). In murine studies, quercetin prevented IL-1ß secretion from XIAP knockout cells following TLR agonists or TNF-α stimulation (P < .05) and strongly reduced constitutive production of IL-18 by both WT and XIAP-deficient cells (P < .05). At 4 hours after in vivo LPS challenge, blood levels of IL-1ß and IL-18 were significantly decreased in mice that had received quercetin-supplemented chow (P < .05). In experiments using human cells, quercetin greatly reduced IL-1ß secretion by monocytes following TNF-α stimulation (P < .05). Our data suggest that quercetin may be an effective natural therapeutic for the prevention of XIAP deficiency-associated hyperinflammation. Clinical trials, including careful pharmacokinetic and pharmacodynamic studies to ensure that effective levels of quercetin can be obtained, are warranted.

Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity.

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAPon or YAPoff cancer classes that express or silence YAP, respectively. YAPoff solid cancers are neural/neuroendocrine and frequently RB1-/-, such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAPoff or YAPon cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications.

TRP53 Mutants Drive Neuroendocrine Lung Cancer Through Loss-of-Function Mechanisms with Gain-of-Function Effects on Chemotherapy Response.

Lung cancer is the leading cause of cancer-related deaths with small-cell lung cancer (SCLC) as the most aggressive subtype. Preferential occurrence of TP53 missense mutations rather than loss implicates a selective advantage for TP53-mutant expression in SCLC pathogenesis. We show that lung epithelial expression of R270H and R172H (R273H and R175H in humans), common TRP53 mutants in lung cancer, combined with RB1 loss selectively results in two subtypes of neuroendocrine carcinoma, SCLC and large cell neuroendocrine carcinoma (LCNEC). Tumor initiation and progression occur in a remarkably consistent time frame with short latency and uniform progression to lethal metastatic disease by 7 months. R270H or R172H expression and TRP53 loss result in similar phenotypes demonstrating that TRP53 mutants promote lung carcinogenesis through loss-of-function and not gain-of-function mechanisms. Tumor responses to targeted and cytotoxic therapeutics were discordant in mice and corresponding tumor cell cultures demonstrating need to assess therapeutic response at the organismal level. Rapamycin did not have therapeutic efficacy in the mouse model despite inhibiting mTOR signaling and markedly suppressing tumor cell growth in culture. In contrast, cisplatin/etoposide treatment using a patient regimen prolonged survival with development of chemoresistance recapitulating human responses. R270H, but not R172H, expression conferred gain-of-function activity in attenuating chemotherapeutic efficacy. These data demonstrate a causative role for TRP53 mutants in development of chemoresistant lung cancer, and provide tractable preclinical models to test novel therapeutics for refractory disease. Mol Cancer Ther; 16(12); 2913-26. ©2017 AACR.

IFN-α mediates the development of autoimmunity both by direct tissue toxicity and through immune cell recruitment mechanisms.

IFN-α is known to play a key role in autoimmunity, but the mechanisms are uncertain. Although the induction of autoimmunity by IFN-α is consistent with primarily immunomodulatory effects, the high frequency of nonautoimmune inflammation suggests other mechanisms. We used thyroiditis as a model to dissect these possibilities. IFN-α treatment of cultured thyrocytes increased expression of thyroid differentiation markers, thyroglobulin, thyroid-stimulating hormone receptor, thyroid peroxidase, and sodium iodide transporter. RNAseq analysis demonstrated that pathways of Ag presentation, pattern recognition receptors, and cytokines/chemokines were also stimulated. These changes were associated with markedly increased nonapoptotic thyroid cell death, suggesting direct toxicity. To corroborate these in vitro findings, we created transgenic mice with thyroid-specific overexpression of IFN-α under control of the thyroglobulin promoter. Transgenic mice developed marked inflammatory thyroid destruction associated with immune cell infiltration of thyroid and surrounding tissues leading to profound hypothyroidism, findings consistent with our in vitro results. In addition, transgenic mice thyroids showed upregulation of pathways similar to those observed in cultured thyrocytes. In particular, expression of granzyme B, CXCL10, a subset of the tripartite motif-containing family, and other genes involved in recruitment of bystander cytotoxic immune responses were increased. Pathways associated with apoptosis and autophagy were not induced. Taken together, our data demonstrate that the induction of tissue inflammation and autoimmunity by IFN-α involves direct tissue toxic effects as well as provocation of destructive bystander immune responses.

Endogenous expression of Hras(G12V) induces developmental defects and neoplasms with copy number imbalances of the oncogene.

We developed mice with germline endogenous expression of oncogenic Hras to study effects on development and mechanisms of tumor initiation. They had high perinatal mortality, abnormal cranial dimensions, defective dental ameloblasts, and nasal septal deviation, consistent with some of the features of human Costello syndrome. These mice developed papillomas and angiosarcomas, which were associated with Hras(G12V) allelic imbalance and augmented Hras signaling. Endogenous expression of Hras(G12V) was also associated with a higher mutation rate in vivo. Tumor initiation by Hras(G12V) likely requires augmentation of signal output, which in papillomas and angiosarcomas is achieved via increased Hras-gene copy number, which may be favored by a higher mutation frequency in cells expressing the oncoprotein.

HCV E2 protein binds directly to thyroid cells and induces IL-8 production: a new mechanism for HCV induced thyroid autoimmunity.

HCV infection is well-known to be associated with autoimmune thyroiditis. However, the mechanisms by which HCV triggers thyroiditis are unknown. We hypothesized that HCV envelope proteins could induce thyroidal inflammation directly, thereby triggering thyroiditis by a bystander activation mechanism. To test this hypothesis we examined whether the HCV receptor CD81 was expressed and functional on thyroid cells. We found significant levels of CD81 mRNA by QPCR analysis, as well as CD81 protein by flow cytometric (FACS) analysis. Incubation of thyroid cells with HCV envelope glycoprotein E2 resulted in E2 binding to thyroid cells and activation of IL-8, an important pro-inflammatory cytokine. Intriguingly, thyroid cells incubated with E2 continued to proliferate normally and did not undergo apoptosis, as was reported in hepatocytes. We conclude that: (1) HCV envelope glycoprotein E2 can bind to CD81 receptors which are expressed on thyroid cells and induce a cascade of signaling pathway leading to IL-8 release; and (2) HCV may trigger thyroiditis in genetically susceptible individuals by bystander activation mechanisms.

RET/PTC-induced cell growth is mediated in part by epidermal growth factor receptor (EGFR) activation: evidence for molecular and functional interactions between RET and EGFR.

RET/PTC rearrangements are one of the genetic hallmarks of papillary thyroid carcinomas. RET/PTC oncoproteins lack extracellular or transmembrane domains, and activation takes place through constitutive dimerization mediated through coiled-coil motifs in the NH(2) terminus of the chimeric protein. Based on the observation that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 decreased RET/PTC kinase autophosphorylation and activation of downstream effectors in thyroid cells, despite lacking activity on the purified RET kinase, we proceeded to examine possible functional interactions between RET/PTC and EGFR. Conditional activation of RET/PTC oncoproteins in thyroid PCCL3 cells markedly induced expression and phosphorylation of EGFR, which was mediated in part through mitogen-activated protein kinase signaling. RET and EGFR were found to coimmunoprecipitate. The ability of RET to form a complex with EGFR was not dependent on recruitment of Shc or on their respective kinase activities. Ligand-induced activation of EGFR resulted in phosphorylation of a kinase-dead RET, an effect that was entirely blocked by PKI166. These effects were biologically relevant, as the EGFR kinase inhibitors PKI166, gefitinib, and AEE788 inhibited cell growth induced by various constitutively active mutants of RET in thyroid cancer cells as well as NIH3T3 cells. These data indicate that EGFR contributes to RET kinase activation, signaling, and growth stimulation and may therefore be an attractive therapeutic target in RET-induced neoplasms.

Interferon alpha treatment and thyroid dysfunction.

Interferon alpha (IFN alpha) is the cornerstone therapeutic agent for chronic hepatitis C virus (HCV) infection. Prospective studies have shown that up to 15% of HCV patients receiving IFN alpha develop clinical thyroid disease, and up to 40% become thyroid antibody positive. In some cases IFN-induced thyroiditis (IIT) may result in discontinuation of interferon therapy; thus, IIT represents a major clinical problem for hepatitis C patients receiving IFN alpha therapy. Recently, the mechanisms leading to the development of IIT have begun to be unraveled. It is now clear that HCV itself plays a role in the disease. Moreover, recent data suggest the IFN alpha precipitates thyroiditis by both immune modulatory mechanisms and direct thyroid toxic effects. Genetic factors also play a major role in the etiology of IIT. IIT can manifest both as clinical autoimmune thyroiditis (ie, Hashimoto's thyroiditis and Graves' disease) and as nonautoimmune thyroiditis (ie, destructive thyroiditis). Early detection and therapy of these conditions are important to avoid complications of thyroid disease such as cardiac arrhythmias. This article reviews the epidemiology and clinical manifestations of IIT and the mechanisms causing IIT, focusing on the role of HCV.

BRAF mediates RET/PTC-induced mitogen-activated protein kinase activation in thyroid cells: functional support for requirement of the RET/PTC-RAS-BRAF pathway in papillary thyroid carcinogenesis.

In human papillary thyroid cancers (PTCs), mutations of RET/PTC, NTRK, RAS, or BRAF are found in about two thirds of cases with practically no overlap, providing genetic evidence that constitutive signaling along RET-RAS-BRAF-MAPK is key to their development. The requirement for BRAF in RET/PTC-mediated MAPK activation and gene expression has not been tested functionally. There are three RAF isoforms: ARAF, BRAF, and CRAF. Compared with the others, ARAF is a much weaker stimulator of MAPK. To determine the key RAF isoform mediating RET/PTC-induced ERK phosphorylation, we stably transfected doxycycline-inducible RET/PTC3-expressing thyroid PCCL3 cells with small interfering RNA vectors to induce selective knockdown of BRAF or CRAF. Conditional RET/PTC3 expression induced comparable ERK phosphorylation in CRAF knockdown and control cells but negligible ERK phosphorylation in BRAF knockdown cells. Selective knockdown of BRAF prevented RET/PTC-dependent down-regulation of the sodium iodide symporter, a gene that confers key biological effects of RET/PTC in PTCs. Moreover, microarray analysis revealed numerous RET/PTC-regulated genes showing requirement of BRAF for appropriate expression. These data indicate that BRAF is required for RET/PTC-induced MAPK activation in thyroid cells and support the notion that BRAF inactivation may be an attractive target for PTCs.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9.

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1alpha-dependent mechanism.

Osteoblast-specific expression of insulin-like growth factor-1 in bone of transgenic mice induces insulin-like growth factor binding protein-5.

The activities of insulin-like growth factors (IGFs) in bone are modulated by a family of binding proteins (IGFBPs) whose physiological roles remain poorly understood. We have previously shown that targeted overexpression of IGF-I in osteoblasts of transgenic (OC-IGF-I) mice stimulates bone formation. In this model, bone formation is markedly but transiently increased in an age-dependent manner, raising the possibility that IGF-I may be influencing IGFBPs to in turn modulate its paracrine actions within bone. We sought to characterize the IGFBPs in normal mouse bone during development and to determine whether osteoblast-targeted overexpression of IGF-I influenced bone IGFBP abundance in vivo. Femoral bone IGFBP content was assessed in control nontransgenic and OC-IGF-I mice by I125-IGF-I ligand and immunoblotting. Bone IGFBP-5 and IGF-I mRNA abundance was determined using real-time reverse transcription (RT)-PCR. Ligand blot of bone extract showed a 30-kDa band, identified as IGFBP-5 by immunoblot, predominated. The abundance of IGFBP-5 declined with age in both control and transgenic bone. Ligand and immunoblot analysis revealed a 5-fold increase in IGFBP-5 protein levels at 3 weeks in transgenic bone (P<0.0001). The elevated IGFBP-5 protein levels were associated with a similar increase in IGF-I mRNA abundance (4-fold, P<0.01) and a significant increase in IGFBP-5 mRNA abundance (1.5-fold). Despite the age-related decline at 6 weeks, IGFBP-5 remained significantly (P<0.01) more abundant in transgenic bone compared to controls. In contrast, bone IGFBP-4 abundance was relatively unchanged by either age or IGF-I overexpression. These studies demonstrate a distinctive developmental pattern of IGFBP-5 content in mouse bone and show that osteoblast-derived IGF-I determines skeletal IGFBP-5 abundance, at least in part by inducing its synthesis. In that IGFBP-5 is thought to stimulate bone formation, directly or via IGF-I action, such changes in bone IGFBP-5 may be important to ensure robust bone acquisition in the early postnatal period.

Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization.

To examine the local actions of IGF signaling in skeletal tissue in a physiological context, we have used Cre-mediated recombination to disrupt selectively in mouse osteoblasts the gene encoding the type 1 IGF receptor (Igf1r). Mice carrying this bone-specific mutation were of normal size and weight but, in comparison with normal siblings, demonstrated a striking decrease in cancellous bone volume, connectivity, and trabecular number, and an increase in trabecular spacing. These abnormalities correlated with a striking decrease in the rate of mineralization of osteoid that occurred despite an unexpected osteoblast and osteoclast hyperactivity, detected from the significant increments in both osteoblast and erosion surfaces. Our findings indicate that IGF1 is essential for coupling matrix biosynthesis to sustained mineralization. This action is likely to be particularly important during the pubertal growth spurt when rapid bone formation and consolidation are required.

Induction of vascular endothelial growth factor by IGF-I in osteoblast-like cells is mediated by the PI3K signaling pathway through the hypoxia-inducible factor-2alpha.

IGF-I is known to stimulate the expression of oxygen- and nutrient-sensitive genes in several cell types. In this study we investigated the signaling pathways and transcriptional mechanisms that mediate IGF-I induction of vascular endothelial growth factor (VEGF) expression in human osteoblast-like cells. IGF-I (50 ng/ml) induced a rapid increase (3-fold) in VEGF mRNA in osteoblasts that was accompanied by an increase in the level of hypoxia-inducible factor-2alpha (HIF-2alpha) protein without changes in HIF-2alpha mRNA expression. These effects were mimicked by chemical inhibition of proteosomal degradation of HIF-2alpha. Transcriptional activation of a proximal VEGF promoter-luciferase construct was greatly enhanced by cotransfection with an HIF-2alpha, but not an HIF-1alpha, construct. IGF-I acutely stimulated Akt phosphorylation, which was abolished by pretreatment of cells with the PI3K inhibitor LY294002. Pretreatment of the cells with LY294002 also greatly attenuated IGF-I induction of HIF-2alpha and blunted IGF-I-induced VEGF promoter activity. Finally, forced expression of a constitutively active PI3K expression construct induced VEGF promoter to levels similar to those observed with IGF-I alone. These data indicate that IGF-I, by activation of the PI3K pathway, induces VEGF expression in osteoblasts through a transcriptional control mechanism common to those that activate VEGF and other hypoxia response genes.