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Although microRNAs (miRs) have been implicated in the pathogenesis of various human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In this study, array and quantitative reverse transcriptase-PCR techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in cultured EAC cells (EACC)/primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. RNA crosslink immunoprecipitation, quantitative reverse transcriptase-PCR (qRT-PCR) and immunoblot experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Chromatin and methylated DNA immunoprecipitation experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression and downregulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and upregulation of KLK7 in IEE and EACC. Overexpression of miR-217 significantly decreased, whereas overexpression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via upregulation of KLK7 and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies.

The inflammatory microenvironment promotes skin tumorigenesis. However, the mechanisms of how cells protect themselves from inflammatory signals have yet to be revealed. Downregulation of IKKα promotes skin tumor progression from papillomas to squamous cell carcinomas, which is frequently accompanied by genomic instability, including aneuploid chromosomes and extra centrosomes. In this study, we found that IKKα promoted oligomerization of nucleophosmin (NPM), a negative centrosome duplication regulator, which further enhanced NPM and centrosome association, inhibited centrosome amplification, and maintained genome integrity. Levels of NPM hexamers and IKKα were conversely associated with skin tumor progression. Importantly, pro-inflammatory cytokine-induced IKKα activation promoted the formation of NPM oligomers and reduced centrosome numbers in mouse and human cells, whereas kinase-dead IKKα blocked this connection. Therefore, our findings suggest a previously unknown mechanism in which an IKKα-NPM axis may use the inflammatory signal to suppress centrosome amplification, promote genomic integrity, and prevent tumor progression.

Here, we report that kinase-dead IKKα knockin mice develop spontaneous lung squamous cell carcinomas (SCCs) associated with IKKα downregulation and marked pulmonary inflammation. IKKα reduction upregulated the expression of p63, Trim29, and keratin 5 (K5), which serve as diagnostic markers for human lung SCCs. IKKα(low)K5(+)p63(hi) cell expansion and SCC formation were accompanied by inflammation-associated deregulation of oncogenes, tumor suppressors, and stem cell regulators. Reintroducing transgenic K5.IKKα, depleting macrophages, and reconstituting irradiated mutant animals with wild-type bone marrow (BM) prevented SCC development, suggesting that BM-derived IKKα mutant macrophages promote the transition of IKKα(low)K5(+)p63(hi) cells to tumor cells. This mouse model resembles human lung SCCs, sheds light on the mechanisms underlying lung malignancy development, and identifies targets for therapy of lung SCCs.

Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of pancreatic insulin-producing β cells by autoreactive T cells early in life. Despite daily insulin injections, patients typically develop cardiovascular and other complications; and intensive efforts are being directed toward identifying therapeutic targets to prevent the disease without directly impinging on the host defense. Fas ligand (FasL) is one potential target. Fas-FasL interactions primarily regulate T-cell homeostasis, not activation. Nevertheless, spontaneous gene mutation of Fas (called lpr mutation) or FasL (called the gld mutation) prevents autoimmune diabetes in nonobese diabetic (NOD) mice, the widely used model for T1D. Furthermore, although homozygous gld mutations cause age-dependent lymphoproliferation, limiting the gld mutation to one allele (NOD-gld/+) or treating NOD–wild-type mice with FasL-neutralizing monoclonal antibody completely prevents the disease development without causing lymphoproliferation or immune suppression. Herein, we show that the heterozygous gld mutation inhibits the accumulation of diabetogenic T cells in the pancreas, without interfering with their proliferation and expansion in the draining pancreatic lymph nodes. Pancreata from NOD-gld/+ mice contained B cells that expressed CD5 and produced IL-10, which was critical for maintenance of the disease resistance because its neutralization with an IL-10 receptor–blocking monoclonal antibody allowed accumulation of CD4 T cells in the pancreas and led to insulitis development. The results provide novel insights into the pathogenesis of T1D that could have important therapeutic implications.

Inflammation contributes to the pathogenesis of ischemic acute kidney injury (AKI), and T cells mediate the early phase of ischemia-reperfusion injury (IRI). The Fas/Fas ligand (FasL) pathway modulates the balance of T cell subsets in the peripheral circulation as well as multiple inflammatory responses, suggesting that FasL may mediate ischemic AKI. Here, we induced bilateral renal IRI in mice bearing a loss-of-function mutation of FasL (the gld mutation) and in wild-type mice. Compared with wild-type mice, serum creatinine was lower in gld mice (1.4 ± 0.9 mg/dl versus 2.6 ± 0.4) at 24 hours after IRI (P < 0.05). In addition, gld mice had fewer TNF-α-producing T lymphocytes in the kidneys and renal lymph nodes. Furthermore, pharmacologic blockade of FasL protected the kidneys of wild-type mice from IRI. Analysis of bone marrow chimeric mice suggested that the pathogenic effect of FasL involves leukocytes; reconstitution of wild-type mice with gld splenocytes attenuated IRI. In contrast, reconstitution of gld mice with wild-type splenocytes enhanced IRI. These data demonstrate that FasL, particularly on leukocytes, mediates ischemic AKI.

We report a link between Cullin5 (Cul5) E3 ubiquitin ligase and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the folding of its client proteins into their functional conformation. Many Hsp90 clients have been reported to be aberrantly expressed in a number of cancers. We demonstrate Cul5 interaction with members of the Hsp90 chaperone complex as well as the Hsp90 client, ErbB2. We observed recruitment of Cul5 to the site of ErbB2 at the plasma membrane and subsequent induction of polyubiquitination and proteasomal degradation. We also demonstrate Cul5 involvement in regulation of another Hsp90 client, Hif-1α. We observed Cul5 degradation of ErbB2 to occur independently of ElonginB-ElonginC function. The involvement of Cul5 in Hsp90 client regulation has implications in the effectiveness of Hsp90 targeted chemotherapy, which is currently undergoing clinical trials. The link between Cul5 and Hsp90 client regulation may represent an avenue for cancer drug development.

Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.

Human immunodeficiency virus tyoe 1 (HIV-1) Vif counteracts host restriction cytidine deaminase (APOBEC3G) A3G by co-opting the cellular ubiquitin-proteasome machinery. Vif utilizes a viral-specific BC-box to recruit ElonginB-ElonginC and a novel zinc-binding HCCH motif to recruit Cullin5 (Cul5) to form an E3 ubiquitin ligase targeting A3G for polyubiquitination and subsequently proteasomal degradation. To determine the structural requirements in HIV-1 Vif HCCH motif for Cul5 binding and Vif function, we investigated the arrangement of the His and Cys residues, the role of the spacing between them, and the requirement for the conserved residues. Our data demonstrate that exchanging Cys for His and vice versa in the highly conserved Zn-coordinating HCCH motif disrupted Vif function and interaction with Cul5. Moreover, the maintenance of both conserved residues and spacing within the HCCH motif is critical for Vif function. We have identified a "viral Cul5 box" with consensus Hx2YFxCFx4Phix2APhix7-8Cx5H that is required for Cul5 selection and subsequent A3G degradation. This novel motif may represent a potential new target for anti-viral drug development.

The adenovirus protein E4orf6 targets p53 for polyubiquitination and proteasomal degradation and is known to form a complex with the Cul5-ElonginB-ElonginC E3 ubiquitin ligase. However, whether Cul5 is directly responsible for the E4orf6-mediated degradation of p53 remains unclear. By using a dominant-negative mutant of Cul5 and silencing Cul5 expression through RNA interference, we have now demonstrated that E4orf6-mediated p53 degradation requires Cul5. Furthermore, we have identified a lentiviral Vif-like BC-box motif in E4orf6 that is highly conserved among adenoviruses from multiple species. More importantly, we have shown that this Vif-like BC-box is essential for the recruitment of Cul5-ElonginB-ElonginC E3 ubiquitin ligase by E4orf6 and is also required for E4orf6-mediated p53 degradation. E4orf6 selectively recruited Cul5 despite the lack of either a Cul5-box, which is used by cellular substrate receptors to recruit Cul5, or a newly identified HCCH zinc-binding motif, which is used by primate lentiviral Vif to recruit Cul5. Therefore, adenovirus E4orf6 molecules represent a novel family of viral BC-box proteins the cellular ancestor of which is as yet unknown.

APOBEC3 proteins are cellular antiviral proteins that are targeted for proteasomal degradation by primate lentiviral Vif proteins. Vif acts as a substrate receptor for the Cullin5 (Cul5) E3 ubiquitin ligase, specifically interacting with Cul5 through a novel H-(x5)-C-(x17-18)-C-(x3-5)-H zinc binding motif. Using the membrane-permeable zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), we demonstrated a requirement for zinc for Vif function in vivo. Treatment with TPEN at an IC50 of 1.79 microM inhibits Cul5 recruitment and APOBEC3G (A3G) degradation. Zinc chelation prevented Vif function in infectivity assays, allowing the virus to become sensitive to the antiviral activity of A3G. Zinc chelation had no effect on cellular Cul5-SOCS3 E3 ligase assembly, suggesting that zinc-dependent E3 ligase assembly may be unique to HIV-1 Vif, representing a new target for novel drug design.

APOBEC3G (A3G) and related cytidine deaminases are potent inhibitors of retroviruses. HIV-1 Vif hijacks the cellular Cul5-E3 ubiquitin ligase to degrade APOBEC3 proteins and render them ineffective against these viruses. Here, we report that HIV-1 Vif is a novel zinc-binding protein containing an H-x5-C-x17–18-C-x3–5-H motif that is distinct from other recognized classes of zinc fingers. Zinc-binding stabilized a conserved hydrophobic interface within the HCCH motif that is critical for Vif-Cul5 E3 assembly and Vif function. An N-terminal region in the first Cullin repeat of Cul5, which is dispensable for adaptor ElonginC binding, was required for interaction with Vif. This region is the most divergent sequence between Cul2 and Cul5, a factor that may contribute to the selection of Cul5 and not Cul2 by Vif. This is the first example of a zinc-binding substrate receptor responsible for the assembly of a Cullin-RING ligase, representing a new target for antiviral development.

Cullin–Ring E3 ubiquitin ligases target substrates for ubiquitin-dependent, proteasome-mediated degradation and regulate critical cellular processes. These cullins assemble with cellular substrate receptor proteins through specific adaptor molecules. F-box- and BC-box-containing receptors use Skp1, ElonginB, and ElonginC as adaptors to recruit Cul1/Cul7 and Cul2/Cul5, respectively. At present, the determinants of Cul2 vs. Cul5 specificity for the BC-box-containing receptors are poorly defined. Here, we demonstrate that primate lentiviral Vif (virion infectivity factor) proteins represent previously uncharacterized substrate receptor proteins that contain divergent BC-box motifs. These molecules selectively assemble with a Cul5–E3 ligase to suppress the antiviral activity of autologous cytidine deaminase APOBEC3G. A previously unrecognized Hx 5 Cx 17-18 Cx 3-5 H motif that is highly conserved among all primate lentiviral Vif proteins was found to be critical for the selective assembly and activity of Vif–Cul5–E3 ligase. Non-primate lentiviral Vif proteins, which lack this HCCH motif, displayed reduced interaction with Cul5. These data suggest that in addition to target protein specificity, substrate receptor proteins play important roles in cullin selection and functional assembly of cullin-Ring E3 ligases. The discovery of these viral substrate receptor molecules that recruit Cul5 through distinct mechanisms from cellular proteins may facilitate the identification of additional cellular factors that regulate cellular functions through Cul5–E3 ligase. Motifs in Vif that are absent from cellular proteins could also be targets for the development of innovative therapeutics.

APOBEC3G, which induces hypermutations in newly synthesized viral DNA, is suppressed by HIV-1 Vif, acting through Cul5-ElonginB-ElonginC E3 ubiquitin ligase. We have now characterized a novel SOCS box in HIV-1 Vif that mediates its interaction with ElonginC. In this SOCS box, alanine replaces the consensus cysteine in the previously identified SOCS box. This new motif was necessary but insufficient for interaction with Cul5-ElonginB-ElonginC, as two highly conserved Cys residues outside the SOCS box were required to interact with Cul5 but not ElonginC. Therefore, selective assembly with Cul5 versus Cul2 E3 may require protein interfaces besides the SOCS-box-ElonginC interaction.

(Abstract) Macrophage depletion has been used extensively to study autoimmune disease and more recently in tumor models. The clodronate-containing liposomes will be recognized as foreign particles and get engulfed by macrophages upon dosing into the animal by the chosen routes. Consequently, macrophages that have engulfed liposomes will all be destroyed by the liposomal. In the protocol presented here the clodronate-containing liposomes were used to systemically deplete macrophages in mice.

Following our previous findings that IKKα reduction promotes carcinogen-induced skin carcinogenesis, somatic IKKα deletion induces spontaneous skin papillomas and squamous cell carcinomas (SCCs) in Ikkα-floxed mice, and overexpressed IKKα represses carcinogen-induced skin carcinogenesis [1-3], we recently reported that kinase-dead Ikkα knockin (IkkαKA/KA) mice develop spontaneous lung, forestomach, and skin SCCs, which are associated with IKKα downregulation and inflammation [4]. We further studied the pathogenesis of lung SCCs after stabilizing the skin condition by introducing the transgenic Lori.IKKα into the skin of IkkαKA/KA (L-IkkαKA/KA) mice (Figure ​(Figure11). Figure 1 IKKα downregulation dysregulates the expression of multiple oncogenes and tumor suppressors in K5+ lung epithelial cells The human Ikkα gene is located at 10q24.31, close to Pten (10q23). Many studies have suggested that alterations within the 10q22-10q26 region may be involved in human SCC development. IKKα downregulation and Ikkα mutations and deletions have been reported in various types of human SCCs. A point mutation-mediated IKKα deletion has been identified in a lethal human syndrome, in which multiple organs are malformed [5]. Chemical carcinogen treatment induces Ikkα mutations and deletions in mouse skin SCCs [1]. These findings underscore the importance of IKKα in the pathogenesis of these human diseases. Lung cancer is the leading cause of cancer mortality worldwide. Lung SCC, one of major types of lung cancers, is strongly associated with smoking that contains carcinogens. Smoking can also act as an inflammatory irritator. It is thought that SCCs, frequently found in the upper and central regions of the lungs, are derived from the basal cells (a type of squamous epithelial cell) of the pseudostratified epithelial layer of bronchi; whereas adenocarcinomas (ADCs) are derived from the epithelial cells of alveoli. Many molecular alterations, such as deregulated EGFR, PIK3CA, p53, Rb, and c-Myc, have been identified in human lung SCCs, but mice overexpressing or lacking these genes do not well recapitulate the development of lung SCCs, although a proportion of K-rasG12D;Lkb1−/− mice develop mixed SCCs and ADCs in the lungs [4,6]. Biomarkers of increased p63, Trim29, and keratin 5 (K5) are used to distinguish human lung SCCs from ADCs. We also have demonstrated increased p63, Trim29, and K5 levels, deregulated RhoV, CDK1, IGF, ROS1, c-Myc, p53, Rb, and LKB1 expression, and elevated EGFR, ERK, and p38 activity in lung SCCs derived from L-IkkαKA/KA mice [4]. Therefore, the mouse lung SCCs resemble human cancers. Interestingly, many human lung SCCs express elevated Sox2; however, the mouse lung SCCs express elevated Nanog and Oct3/4, but not Sox2 (our unpublished data), suggesting that human and mouse lung SCCs may express a different spectrum of stem cell regulators [4]. The nuclear IKKα has been shown to regulate the expression of many genes in keratinocytes through an epigenetic mechanism [2]. We further revealed that IKKα downregulation upregulates the level of H3K4me3, a positive transcription mark, and reduces the level of H3K27me3, a negative transcription mark, on the loci of p63 and Trim29 in the IkkαKA/KA SCC cell line and IkkαKA/KA MEFs compared to wild-type cells. Reintroducing IKKα reverses the levels of H3K4me3 and H3K27me3, represses the expression of ΔNp63 and Trim29 in IkkαKA/KA cells, and inhibits the proliferation of the IkkαKA/KA SCC cell line [4]. A similar regulatory mechanism for the expression of ΔNp63 and Trim29 was detected in the human lung SCC cell line. Thus, the IKKα-mediated epigenetic regulation plays a role in the pathogenesis of lung SCCs. In addition, the elevated ΔNp63 level, increased EGFR and ERK activity, and K5+ cell expansion have been detected in the epidermis of Ikkαf/f/K5.CreER and Ikkα−/− mice (our unpublished result) and in the L-IkkαKA/KA lungs [4,7], suggesting that some similar molecular mechanisms may be involved in the pathogenesis of skin and lung SCCs in these different IKKα mutant mice. Previously, we showed that IKKα inactivation interrupts early hematopoietic cell development, resulting in increased myeloid cells in IkkαKA/KA mice [8]. We further demonstrated that these increased macrophages are necessary for promoting lung SCC formation in L-IkkαKA/KA mice by increasing DNA damage to deregulate tumor suppressors, oncogenes, and stem cell regulators (Figure ​(Figure1)1) [4]. We detected markedly increased macrophages, T cells, and multiple mediators in L-IkkαKA/KA lungs compared to wild-type lungs. NF-κB activity is increased in CD45− cells isolated from L-IkkαKA/KA lungs as well as in a cell line derived from the L-IkkαKA/KA lung SCC [4]. The increased NF-κB activity may contribute to multiple elevated mediator products and anti-apoptotic activities in lung epithelial cells. Importantly, we detected increased macrophages, and reduced IKKα levels in human lung SCCs and their adjacent lung tissues, indicating that IKKα reduction may not only promote lung epithelial cell malignant transformation, but also enhance inflammatory responses. We have identified similar multi-molecular alterations in mouse and human lung SCCs [4]. Our immediate goal is to identify specific molecules, cells, or pathways that can be targeted for therapy, which will benefit human cancer patients. Using this model, we will investigate conditions for lung SCC metastasis and essential pulmonary inflammatory cell development, as well as the relationship between lung SCC and ADC pathogenesis.

Spontaneous mutation of Fas (lpr) or FasL (gld) completely protects nonobese diabetic mice from autoimmune diabetes but also causes massive double-negative T-cell lymphoproliferation. In this study, we used bone marrow chimeras and adoptive transfer analysis to investigate further the role of FasL in the pathogenesis of autoimmune diabetes and to determine whether gld-induced tolerance and double-negative T-cell lymphoproliferation can be uncoupled from each other. We show that FasL expressed on hematopoietic and nonhematopoietic compartments plays nonredundant roles in the pathogenesis of autoimmune diabetes. Mutation of FasL in either compartment interferes with the autoimmune process and prevents onset of diabetes, but FasL expressed in the hematopoietic compartment is the dominant regulator of T-cell homeostasis. Furthermore, pathogenesis of diabetes is dependent on normal FasL expression in both compartments, whereas only minimal FasL function is required to maintain T-cell homeostasis. Consequently, partial disruption of FasL protects from autoimmune diabetes without causing T-cell lymphoproliferation. This is demonstrated genetically in nonobese diabetic-gld/+ mice and pharmacologically by using FasL-neutralizing antibody. These results have important implications for understanding the role of the Fas pathway in pathogenesis of autoimmune diseases and for designing novel FasL-modulating therapies.

APOBEC3G (A3G) is a single-stranded DNA cytidine deaminase that targets retroviral minus-strand DNA and has potent antiviral activity against diverse retroviruses. However, the mechanisms of A3G antiviral functions are incompletely understood. Here we demonstrate that A3G, A3F, and, to a lesser extent, the noncatalytic A3GC291S block human immunodeficiency virus type 1 (HIV-1) replication by interfering with proviral DNA formation. In HIV-1 virions, A3G interacted with HIV-1 integrase and nucleocapsid, key viral factors for reverse transcription and integration. Unlike A3G, the weak antiviral A3C cytidine deaminase did not interact with either of these factors and did not affect viral reverse transcription or proviral DNA formation. Thus, multiple steps of the HIV-1 replication cycle, most noticeably the formation of proviral DNA, are inhibited by both cytidine deamination-dependent and -independent mechanisms.

B cells serve both pathogenic and regulatory roles in Type 1 diabetes (T1D). Whereas the regulatory role is mainly limited to IL-10-producing (IL-10pos) B cells, little is known about the characteristics of pathogenic B cells. Here we identified a novel population of FasLhiCD5+ B cells that is significantly increased in T1D as compared to non-diabetic autoantibody positive (Ab+) subjects. Both the FasLhi and IL-10pos sets are localized within the CD5+ B subpopulation. However, FasLhiCD5+ B cells were significantly expanded and IL-10posCD5+ B cells decreased in T1D as compared to Ab+ subjects. FasLhiCD5+ B cells do not produce IL-10 and are largely spared from activation-induced apoptosis, whereas IL-10posCD5+ B cells are prone to Fas-mediated apoptosis. Causally, using NOD mouse model, we identified FasL-expressing B cells as negative regulators of IL-10pos B cells and facilitators of islet infiltration by diabetogenic T cells. These results identify FasLhiCD5+ B cells as an elevated subpopulation in T1D subjects and initiator of the diabetogenic process in NOD mice.

Meeting abstracts Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer after hepatocellular carcinoma (HCC). The risk of ICC is higher in patients with primary sclerosing cholangitis (PSC). To date, the cellular and molecular mechanism underlying the pathological

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer after hepatocellular carcinoma (HCC). The risk of ICC is higher in patients with primary sclerosing cholangitis (PSC). To date, the cellular and molecular mechanism underlying the pathological progression of PSC and ICC is poorly understood. IKKα is part of the IκB kinase (IKK) complex, which plays an important role in regulating inflammation- associated carcinogenesis through both NF-κB-dependent and independent pathways. Here, we show that IKKα mutant mice developed very serious PSC as early as four weeks of age. The ALT/AST and bilirubin levels were significantly increased in the serum of IKKα mutant mice along with lymphocytic and eosinophilic infiltration into the liver. Liver inflammation in the IKKα mutant mice, mediated by macrophages, neutrophils and CD4+ T cells, was associated with the death of cholangiocytes and hepatocytes, and obstruction of intrahepatic and extrahepatic bile ducts, which impeded bile flow and ultimately led to biliary fibrosis and cirrhosis. Additionally, upon activation of NOTCH signaling in the liver via hydrodynamic shear, we observed that NOTCH-induced ICC, with the PSC, developed significantly faster in IKKα mutant mice. To identify whether intrinsic IKKα dysfunction in hepatocytes promotes the NOTCH-induced ICC in IKKα mutant mice, we generated IKKα hep KO mice, in which IKKα is conditionally knocked out in hepatocytes. No biliary disease or liver injury was observed in these mice. We then established an accelerated ICC model utilizing hydrodynamic delivery of NICD and AKT expression vectors. Unexpectedly, ICC development was remarkably slower in the IKKα hep KO mice compared to the IKKα floxed control mice, and this delayed ICC development was associated with reduced activation or levels of AKT, NOTCH, MAPK/ERK and c-Myc. These data suggest that IKKα may play a protective role in PSC, while promote ICC derived from hepatocytes. In contrast to ICC, we also established the cMET/β-catenin-induced HCC model in the IKKα hep KO mice, which did not reveal any difference in tumor development between IKKα hep KO and control mice. Taken together, our findings suggest that IKKα plays complicated and important roles in PSC and ICC pathological progression. Citation Format: Qun Jiang, Zuoxiang Xiao, Timothy Back, Anthony Scarzello, Scott Roan, Jami Willette-Brown, Feng Zhu, Yinling Hu, Robert H. Wiltrout. Ikkα modulates primary sclerosing cholangitis and intrahepatic cholangiocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3618. doi:10.1158/1538-7445.AM2014-3618

We recently reported that kinase-dead IKKα knockin mice, in which an ATP binding site of IKKα, Lys 44 was replaced by alanine (IkkαKA/KA), develop spontaneous lung SCCs that were closely associated with lung inflammatory microenvironment. This mouse model resembles human lung SCCs, sheds light on the mechanisms underlying lung malignancy development, and identifies targets for therapy of lung SCCs (Xiao et al. Cancer Cell 23(4):527-40, 2013). To further elucidate what and how the inflammatory stimuli contribute to the tumorigenesis, we generated bitransgenic mice harboring IkkαKA/KA and tnfrsf1-/- (KALTFR). It was identified that the KALTFR mice display lung tumor-free even the lung inflammations are still severe, while almost all of the littermate IkkαKA/KA mice developed lung SCCs. Interestingly, we found a noticeably increased TNFR1 expression in the lung SCCs of IkkαKA/KA mice compared to normal lung tissues. It has also been detected that the TNFR1 expressions are significantly higher in both transcript and protein levels in human lung SCCs with the compare to their adjacent tissues. These findings indicate that TNFR1 may play a crucial role in the development of lung SCC in either IkkαKA/KA mice or humans. Further investigation demonstrated that TNFR1 is a critical factor to regulate nuclear transcriptional factors and oncogenes including C-Myc, through one bromodomain (BRD) proteins- containing complex. These findings broaden our understanding about how inflammatory microenvironment contributes to tumorigenesis through TNFα-TNFRs engagements. Citation Format: Zuoxiang Xiao, Jami Willette-Brown, Yinling Hu, Sichuan Xi. TNFR1 is indispensable in the development of spontaneous lung squamous cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 603. doi:10.1158/1538-7445.AM2014-603

APOBEC3G exerts its antiviral activity by targeting to retroviral particles and inducing viral DNA hypermutations in the absence of Vif. However, the mechanism by which APOBEC3G is packaged into virions remains unclear. We now report that viral genomic RNA enhances but is not essential for human APOBEC3G packaging into human immunodeficiency virus type 1 (HIV-1) virions. Packaging of APOBEC3G was also detected in HIV-1 Gag virus-like particles (VLP) that lacked all the viral genomic RNA packaging signals. Human APOBEC3G could be packaged efficiently into a divergent subtype HIV-1, as well as simian immunodeficiency virus, strain mac, and murine leukemia virus Gag VLP. Cosedimentation of human APOBEC3G and intracellular Gag complexes was detected by equilibrium density and velocity sucrose gradient analysis. Interaction between human APOBEC3G and HIV-1 Gag was also detected by coimmunoprecipitation experiments. This interaction did not require p6, p1, or the C-terminal region of NCp7. However, the N-terminal region, especially the first 11 amino acids, of HIV-1 NCp7 was critical for HIV-1 Gag and APOBEC3G interaction and virion packaging. The linker region flanked by the two active sites of human APOBEC3G was also important for efficient packaging into HIV-1 Gag VLP. Association of human APOBEC3G with RNA-containing intracellular complexes was observed. These results suggest that the N-terminal region of HIV-1 NC, which is critical for binding to RNA and mediating Gag-Gag oligomerization, plays an important role in APOBEC3G binding and virion packaging.

Although alterations in microRNA (miRNA; miR) expression have been implicated in the pathogenesis of a variety of human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In order to examine this issue, array and RT-PCR techniques were used to examine miRNA expression profiles in immortalized esophageal epithelial cells (Het-1A), as well as NCI-SB-EsC1 (EsC1), NCI-SB-EsC2 (EsC-2), OE19, and OE33 esophageal cancer cells cultured in normal media (NM) with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression (1.5-9.2 fold) in esophageal cancer cells and Het-1A cells. Endogenous miR-217 expression levels in cultured esophageal cancer cells were significantly lower than those observed in Het-1A cells. Consistent with these findings, miR-217 was significantly down-regulated (3.5-23.5 fold) in resected esophageal cancers relative to adjacent normal esophageal tissues. Software-guided analysis revealed that miR-217 potentially targeted KLK7, encoding a kallikrein family member implicated in invasion and metastasis of several cancers. Constitutive over-expression of miR-217 inhibited expression, whereas depletion of endogenous miR-217 enhanced expression of KLK7 in Het-1A, EsC1 and EsC2 cells. RNA cross-link immunoprecipitation (CLIP) experiments confirmed direct interaction of miR-217 with KLK7 transcripts. Methylated DNA Immunoprecipitation (MeDIP) and chromatin immunoprecipitation (ChIP) experiments demonstrated that CSC increased DNA methylation and decreased H3K4me3 levels within the miR-217 genomic locus in Het-1A, EsC1 and EsC2 cells. Deoxyazacytidine (DAC) induced miR-217 expression in EsC1 and EsC2 cells but not Het-1A cells, and markedly attenuated CSC-mediated miR-217 repression in these cells. Over-expression of miR-217 significantly decreased proliferation of esophageal cancer and Het-1A cells, as well as invasion of esophageal cancer cells. Depletion of miR-217 increased growth of Het-1A cells, but not EsC1 and EsC2 cells presumably due to lower endogenous miR-217 expression in these cancer cells. Experiments are currently in progress to examine the effects of miR-217 and KLK7 expression on tumorigenicity of esophageal cancer cells. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of esophageal carcinomas, and suggest that restoration of miR-217 expression may be a novel strategy for therapy of these malignancies. Citation Format: Sichuan Xi, Suzanne Inchauste, Zuoxiang Xiao, Jigui Shan, Mary Zhang, Julie A. Hong, Manish T Raiji, David G. Beer, David S. Schrump. Epigenetic repression of miR-217 contributes to tobacco-induced esophageal carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3071. doi:10.1158/1538-7445.AM2013-3071 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

During the last decades lung cancer has become the leading cause of cancer deaths in the world, and the need to develop better diagnostic techniques and therapies is urgent. To advance the understanding of this disease, various genetically engineered and chemical induced mouse models have been established. However, there are no robust animal models of lung squamous cell carcinomas (SCCs), one of the major types of lung cancer. Here, we generated Ikkα-KA/KA knock-in mice (KA/KA) in which an ATP binding site of IKKα, Lys 44 was replaced by alanine. All the knock-in mice at 4 to 6 months of age developed spontaneous lethal lung SCCs associated with markedly increased leukocyte infiltration and expression of cytokines, and chemokines in the lungs. Furthermore, we identified deregulated c-myc, Nanog, Oct3/4, p53, Rb, EGFR, MAPK, Jun-B, p63, Trim29, Rhov, CDK1, and IGF1 in mouse lung SCCs, and identified reduced IKKα and IκBα and increased ROS1 in mutant lungs and SCCs, some of which were found in human lung SCCs. Lung cancers were prevented by reintroducing epithelial-cell IKKα, depleting macrophages or depleting lymphocytes. This study not only provides a novel model for studying the pathogenesis, treatment, early detection, and prevention of human lung SCCs, but also demonstrates how a single mutation in IKKα elicits malignancy through the combined epithelial-cell-autonomous and immune mechanisms. Citation Format: Zuoxiang Xiao, Qun Jiang, Jami Willette-Brown, Feng Zhu, Sichuan Xi, Sandra Burkett, Fanching Lin, Timothy Back, Mahesh Datla, Zhonghe Sun, Romina Goldszmid, Xiaolin Wu, David Schrump, Howard Young, Georgio Trinchieri, Robert Wiltrout, Yinling Hu. The pivotal role of IKKalpha in the development of spontaneous lung squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1083. doi:10.1158/1538-7445.AM2013-1083

Lung cancer is the leading cause of cancer deaths worldwide. To advance the understanding of this disease, various genetically engineered and chemical induced mouse models have been established. However, most animal models resemble human lung adenocarcinoma, and spontaneous lung squamous cell carcinomas (SCCs) mouse models are very rare. Here, we generated Ikkα-KA/KA knock-in mice (KA/KA) in which an ATP binding site of IKKα, Lys 44 was replaced by alanine. The knock-in mice develop severe skin lesions and begin to die after 6 months. We found lung SCCs in some of the mice. To study lung SCC development, we decided to stabilize the skin condition by reintroducing transgenic IKKα by crossing KA/KA with Lori.IKKα transgenic mice to generate KA/KA/Lori.IKKα (KA/KAL) mice. Almost all the KA/KAL (100%) mice at 4 to 6 months of age developed spontaneous lethal lung SCCs. The endogenous IKKα protein level generally markedly declined in an age dependent manner in these IKKα mutant mice. Progenitor cell related markers Sox2, OCT3/4 and Nanog were only increased in the lung SCC tissue but not in the tumor adjacent tissues, implying the involvement of cancer stem cells in lung SCC. Furthermore, we detected substantial increases in Ras and CyclinD1 levels and EGFR, ERK and p38 activities in lung SCCs. On the other hand, we detected reduction in tumor suppressor gene Rb and IαBβ accompanying with reduced IKKα levels in KA/KAL lungs as well as in lung SCCs. Importantly, we observed a similar alteration pattern in mouse and human lung SCCs. Finally, reintroducing IKKα into lung epithelial cells prevented lung SCC development in mice. Collectively, our study supports the tumor suppressing role of IKKα in lung tumorigenesis. This novel lung SCC mouse model may facilitate investigations in pathogenesis, diagnosis, and treatment of human lung SCC disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2560. doi:1538-7445.AM2012-2560

NF-kB plays critical roles in linking inflammation and carcinogenesis. The activation of NF-kB is regulated by the IkB Kinase Complex (IKK), composed of IKKα, IKKα, and NF-κB essential modulator (NEMO). We have established a mouse model of lung squamous cell carcinoma (SCC) by inactivating IKKα in vivo. However, IKKα's attributes relating to linkage between inflammation and lung SCC have not yet been well-defined. We now show that inactivation of IKKα in mice causes systemic chronic inflammation, which consequently promotes lung SCC initiation and progression. In mice with mutated IKKα, CD4 T cells, dendritic cells, and macrophages are all significantly increased in the spleen and lung. Proinflammatory cytokines and chemokines are also remarkably elevated in the lungs of these mice. An M2 macrophage phenotype is observed only in the lungs of old IKKα mutant mice, not in younger mice, suggesting the M1/M2 polarization may be driven by the proinflammatory microenvironment in these mice. Production of Th2 cytokines and IL-10 are elevated in CD4 T cells from the lungs of mice with mutated IKKα, and may contribute to M2 polarization during the chronic inflammation. In addition, depletion of macrophages in these mice can significantly reduce Th2 cytokine production, oxidative DNA damage, and shut down SCC development in the lungs, suggesting their important roles in inflammation-associated lung carcinogenesis. Together, our data suggest that IKKα may play important roles in linking inflammation and lung SCC tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2561. doi:1538-7445.AM2012-2561

Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects beyond addiction. However, the underlying mechanism by which microglia in response to opioids remains largely unknown. Here we show that morphine induces the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, TLR9 deficiency significantly inhibited morphine-induced apoptosis in microglia. Similar results were obtained when endogenous TLR9 expression was suppressed by the TLR9 inhibitor CpGODN. Inhibition of p38 MAPK by its specific inhibitor SB203580 attenuated morphine-induced microglia apoptosis in wild type microglia. Morphine caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type microglia, but not in TLR9 deficient microglia. In addition, morphine treatment failed to induce an increased levels of phosphorylated p38 MAPK and MAP kinase kinase 3/6 (MKK3/6), the upstream MAPK kinase of p38 MAPK, in either TLR9 deficient or µ-opioid receptor (µOR) deficient primary microglia, suggesting an involvement of MAPK and µOR in morphine-mediated TLR9 signaling. Moreover, morphine-induced TLR9 expression and microglia apoptosis appears to require μOR. Collectively, these results reveal that opioids prime microglia to undergo apoptosis through TLR9 and µOR as well. Taken together, our data suggest that inhibition of TLR9 and/or blockage of µOR is capable of preventing opioid-induced brain damage.

A key role for the Fas pathway in promoting organ-specific-autoimmunity is indicated by resistance of mice bearing the lpr mutation of Fas or gld mutation of Fas ligand (FasL) to several organ-specific autoimmune diseases, including autoimmune diabetes and multiple sclerosis. However, the underlying mechanism remains elusive. Here we show that FasL expressed on B cells plays a key role in facilitating activation of diabetogenic T cells in pancreatic lymph nodes (PLNs) and pancreatic islet accumulation resulting in initiation of insulitis in the autoimmune diabetes-prone NOD mice. Expression of FasL on diabetogenic T cells themselves is not sufficient to mediate its diabetogenic role. By contrast, expression of FasL on B cells leads to deletion of localized IL-10-producing B cells, the presence of which in PLNs and pancreatic islets is associated with suppression of diabetogenic T cell priming and islet accumulation, respectively. Consequently, FasL inactivation by gld mutation or antibody blockade preserves the local IL-10-producing B cells and protects islets from diabetogenic T cells by a mechanism that is reversed by IL-10R blockade in vivo. The results provide novel insights into how FasL orchestrates the pathogenic relationship among B cell subsets in its implications for diabetogenic T cells.

Beside CD4 and CD8 αβT cells, several minor but important T cell subsets develop in the thymus. These include NKT cells, which are selected on MHC class 1b molecule CD1d. NKT cells are broadly divided into type 1 NKT cells, which express an invariant TCR and bind αGalCer-CD1d tetramer and type 2 NKT cells, which express non-invariant TCRs and do not bind αGalCer-CD1d tetramer. Significance advances in development of type 1 NKT cells have been made by using αGalCer-CD1d tetramer for visualization of their thymic development. However, very little is known about development of type 2 NKT cells due to the lack of a specific marker for their identification in the thymus. Here we show that the highly conserved heparan sulphate proteoglycan syndecan-1 (sdc1) is selectively expressed by a small subset of TCRαβ thymocytes. Unlike mainstream i.e sdc1-TCRβ+ thymocytes, the majority of sdc1+ TCRβ+ thymocytes are CD4-CD8-(DN) T cells whose development is CD1d-dependent and are not reactive to αGalCer-CD1d tetramer and hence are type 2 NKT cells. Functional maturity of this subset is indicated by their production of Th2 cytokines in vitro stimulation with CD3/CD28 or CD1d beads. Sdc1 appears to play an important role in regulation of cytokine production and homing of type 2 NK T cells. Identification of sdc1 as a specific marker for type 2 NKT cells in the thymus will facilitate understanding their development.

Type-1 diabetes (T1D) is an organ specific autoimmune disease that affects children. T, B and dendritic cells infiltrate islets in the early stage of disease leading to insulitis followed by islet destruction and hyperglycemia. Development of T1D in non-obese diabetic (NOD) mice is completely inhibited by gld or lpr mutations of Fas and FasL, respectively, even though mutant mice develop T cell lymphoproliferation and the Fas pathway is not essential for T cell activation. Recently, we have shown heterozygous gld mutation (gld/+) provides complete protection without causing lymphoproliferation. Using NOD-gld/+ mouse as a model, we found that the protection is not due to deletion of autoreactive T cells or switching of T helper polarization. Islet-reactive BDC2.5 TCR transgenic CD4 T cells transferred into NOD-gld/+ mice were efficiently primed in the pancreatic lymph nodes but failed to accumulate in the pancreas. FasL deficiency selectively reduced expression of CD40 and frequency of B cells in the pancreas. Transfer of wild type B cells into NOD-gld/+ mice results in mobilization of endogenous T cells particularly CD8 T cells and of co-transferred BDC2.5 T cells resulting in severe insulitis. These results provide the first evidence that B cells play an essential role in gld-mediated protection from T1D and that expression of functional FasL on B cells is key for initiation insulitis. Supported by grants from AHA and JDRF.

Identification of specific markers of thymocytes that develop into specialized lineages is important for understanding thymic differentiation and lineage commitment. We have recently shown that double-negative (DN) but not conventional T cells express syndecan 1 (sdc1), a major heparan sulphate proteoglycan previously known to be expressed by intestinal epithelia and plasma cells. Here we show a highly restricted expression of sdc1 by a small subset of cells of the αβ lineage during thymic development. Unlike mainstream (sdc1-) thymocytes, sdc1+ thymocytes developed mainly into CD4-CD8-TCRαβ + DN T cells including NKT cells. Sdc1 expression on this small subset was first detected at the DN4 stage, indicating that it was not induced by TCR-MHC interactions. Distribution of sdc1+ thymocytes among the double-positive (DP) thymocyte pool was limited to distinct (TCRβlowCD4lowCD8low) cells. β2m-deficiency blocked maturation of sdc1+TCRαβ + DN cells without affecting the immature subset development. A few sdc1+ thymocytes were selected on MHC class II into CD4 T cells. The existence of phenotypically definable thymocytes such as sdc1-expressing cells within the immature (DN4 and DP) pool suggests that early thymocytes include unique lineage committed precursors that have not been previously identified due to the lack of specific markers. Funding: NIH DK069279 and DK066039.

The Fas pathway is not required for T cell activation yet loss-of-function mutation that inactivates Fas (lpr) or FasL (gld) prevents autoimmune diabetes in NOD mice without interfering with normal immune responses. However, the Fas pathway has not been considered a viable therapeutic option because these mutations also cause double negative (DN) T cell lymphoproliferation and lupus-like syndrome. Using bone marrow chimeras and adoptive transfer, we show that gld mutation on either hemopoietic or nonhemopoietic compartment is equally protective from autoimmune diabetes; however, gld mutation on hemopoietic cells is responsible for most of DN T cell lymphoproliferation. Therefore, limiting gld mutation to the nonhemopoietic tissue completely controls diabetogenicity of wild type T cells without causing lymphoproliferation. The protection is recapitulated therapeutically by treating NOD-wt mice with FasL neutralizing antibody. Anti-FasL treatment curtailed insulitis and did not lead to lupus-like disease or DN T cell lymphoproliferation. These results suggest the potential of generating a new class of immunotherapy that is based on targeting T cell apoptosis inducing ligand rather than costimulatory pathways.