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Introduction: Anti-myeloma BCMA-specific chimeric antigen receptor (CAR) T-cell therapies represent a promising new treatment strategy, with high response rates observed in the early stages of therapy. However, the responses are not durable. One known mechanism of relapse has been traced to the loss of BCMA expression following long-term CAR-T therapy. Another potential reason is that while BCMA CAR-T cells eliminate the bulk of BCMA-positive MM cells, a small subset of BCMA-negative, very drug-resistant MM cells, such as tumor-initiating cells (TICs) survive and seed relapses. There is a strong correlation between the presence of MM TICs with minimal residual disease, acquired drug resistance and relapse. This suggests that TIC-targeted therapies could improve outcomes. We have previously demonstrated that MM cells expressing CD24 also exhibit features of TICs, e.g. self-renewal, increased expression of embryonic stem cell genes and drug resistance. We have generated bispecific CAR-T cells which recognize both BCMA and CD24 antigens and have tested their therapeutic efficacy in MM cells in vitro and in vivo models. Methods: We constructed a bispecific BCMA-CD24 CAR vector, with 2 complete CAR units: BCMA CAR and CD24 CAR. P2A was inserted between these two CARs. The BCMA CAR contains a safety switch in the hinge region, and a CD28 co-activation domain with CD3ζ. The CD24 CAR contained a 4-1BB co-activation domain with CD3ζ. To decrease the risk of severe immunological side effects, we integrated RQR8, an immunological safety switch with epitopes for CD34 and CD20 as a suicide molecule into the hinge region. Lentivirus particles were used to transduce primary human T cells. CAR-T cells were detected on day 7 by flow cytometry using antibodies to CD34. We performed co-culture killing assays, detected the T cell activation marker CD69 and measured the cytokines in the supernatant. We determined whether BCMA-CD24 CAR-T cells targeted the TIC population by flow cytometry and microscopy. The NOD. Cg-Prkdc scidIl2rg tm1Wjl/SzJ (NSG) xenograft mouse model was used for in vivo studies. 8-week-old NSG mice were administered 2 × 10 6 MM cells by intravenous injection. On day 7 after MM cells injection, 1 × 10 6 CAR-T cells were administered. Mice were weighed and monitored for signs of distress every two days. Bioluminescence images were acquired 10 min after D-luciferin injection. Myeloma progression was monitored every 7 days until the mice develop hind limb paralysis or the bioluminescence signal (ROI) is more than 2 × 10 10. Results: CAR-T cells were detected by flow cytometry using the RQR8-specific CD34 antibody. The BCMA-CD24 CAR was found to be expressed on roughly 13% of T-cells. To determine the selective lysis by the CAR-T cells, we performed co-culture killing assays in which MM cell lines over-expressing CD24 (ARP-1 CD24OE or OCI CD24OE cells) were incubated with CAR-T cells. When the CAR-T: MM ratio was 5:1, the lysis percentage of target cells was 99% (ARP-1 CD24OE) and 89% (OCI CD24OE). CAR-T cell activation was determined by increased CD69 expression and IL-2 production. As expected, exposure to CD24 + MM cells resulted in strong activation of CAR-T cells, and CAR-T cells did target and kill the TIC population. Bioluminescence imaging showed CAR-T mediated antitumor activity, yielding near-complete tumor clearance. Additionally, mice treated with CAR-T cells exhibited increased survival compared with mice in the control groups. Conclusion and Significance: This study developed a BCMA-CD24 CAR-T, a novel MM immunotherapy. We have demonstrated strong cytotoxic activity and selectivity for MM cells in vitro and in vivo. Future studies will be aimed at determining if BCMA-CD24 CAR-T can target TIC-mediated relapses. Disclosures No relevant conflicts of interest to declare.

Introduction The development of new treatments for high-risk multiple myeloma (HRMM) are needed. The PD-1/PD-L1 axis is one of the chief inhibitory immune checkpoints in antitumor immunity. Despite the success of PD-1 (PDCD1) / PD-L1 (CD274) blockade in some neoplasms, use of it as a monotherapy has failed to improve outcome in RRMM. We have previously demonstrated that the cell-cycle-regulated serine-threonine kinase, NEK2 is elevated in HRMM and that inhibition of NEK2 can overcome drug-resistance and prolong survival of xenografted MM cells. Here, we aimed to investigate the possible role of NEK2 in regulating the immune checkpoint response in MM and development of possible anti-PD1/PDL1 combination therapies. Methods Gene expression profiles and pathway enrichment analyses were conducted on oligonucleotide microarray gene expression profiles from over 1000 primary MM samples to evaluate the correlation of NEK2 and immune checkpoint expression levels. To elucidate the underlying mechanism, we used Nek2 -/- mice crossed with EμMyc mice to generate B cell tumor mouse model with NEK2 deficiency. RNA-sequencing analyses of premalignant B cells was compared between EμMyc/Nek2 WT and EμMyc/Nek2 -/- mice. The hub molecular regulators in the NEK2 correlated pathways were further determined by western blot using NEK2 overexpressing and knockdown cell lines and then verified by co-immunoprecipitation with a NEK2 antibody. Lastly, to establish its clinic significance, the efficacy of INH1 (small compound NEK2 inhibitor), (D)-PPA 1 (peptide-based PD-1/PD-L1 interaction inhibitor) or a PD-L1 (monoclonal antibody) was tested in bone marrow BM mononuclear cells from primary MM patients in-vitro as well as in MM xenografts. Tumor burden and T cell immune responses were monitored by M-spike and mass cytometry. Results Gene expression profiles demonstrated that CD274 expression was significantly higher in the non-proliferative hyperdiploid (HY) subtype of MM, representing between 25-35% of all MM. NEK2 was negatively correlated with CD274 gene expression across all 7 MM subtypes. Gene set enrichment analysis showed that the IFN-γ signaling pathway, which can induce CD274 expression, was significantly enriched in the HY subtype as well as premalignant B cells from EμMyc/Nek2 -/- mice. Elevated expression of EZH2, a histone methyltransferase gene, is also highly correlated wirth NEK2 levels in primary MM. We found that NEK2 inhibition increases CD274 expression as well as reduced EZH2 expression and H3K27me3 levels in MM cell lines. In contrarst, myeloma cells overexpressing NEK2 showed increased expression and activity of EZH2 and H3K27me3 levels. Thus, NEK2 appears to regulate CD274/PD-L1 expression through EZH2-mediated histone methylation. Next we demonstrated that NEK2 and EZH2 directly interact and that overexpression of NEK2 leads to increased methylation of the CD274/PD-L1 gene. We treated BM mononuclear cells from primary MM with PD-1/PD-L1 inhibitor with and without a NEK2 inhibitor. The combination was most effective at eliminating CD138 + myeloma cells while having no effects on T, B and myeloid cell populations. Conclusion Our study showed that expression of CD274/PD-L1 is suppressed in primary HRMM and that CD274/PD-L1 expression is negatively regulated by NEK2 via EZH2-mediated methylation. Inhibition of NEK2 sensitizes myeloma cells to PD-1/PD-L1 blockade, showing either a synergistic or an additive effect in MM cell cytotoxicity. Disclosures No relevant conflicts of interest to declare.

Bone destruction is a major complication of multiple myeloma (MM). Healthy bone is constantly remodeled through bone resorption by osteoclasts and bone formation by osteoblasts. New bone formation in MM is virtually non-existent, because differentiation of osteoblasts is inhibited by DKK1, a Wnt-β-catenin signaling inhibitor secreted by MM cells, reported by our group in NEJM, 2003. MM in its early stages is totally dependent on its microenvironment and for the hyperdiploid type MM this dependence is perpetual. Based on concordant gene expression signatures, predominantly driven by recurrent translocations and hyperdiploidy, we have classified MM into 7 distinct molecular entities. One subgroup, with significantly less bone disease and superior event-free and overall survival following high-dose therapy and stem cell transplantation than the other subgroups, defined as the Low Bone (LB) disease subgroup. Consistent with the LB phenotype, we have observed a strong inverse correlation between DKK1 and CST6 expression and by analyzing gene expression profiling (GEP) and RNA-sequencing data of more than 1,000 myeloma patients, we identified CST6 as the most upregulated gene in the LB subgroup. The aim of the present study was to determine the role of Cystatin E/M (CST6) in MM biology and to apply this knowledge to prevent both bone disease and MM cell growth. CST6, a 14-17 kD secretory protein, is a lysosomal protease inhibitor and suggested tumor suppressor gene. We showed that overexpression of CST6 in human MM cell lines prevents MM cell growth in vitro and in vivo. Also, purified CST6 protein from conditioned media of CST6-overexpressing MM cells significantly inhibits MM cell growth (p Disclosures van Rhee: CDCN: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotech: Consultancy; EUSA: Consultancy.

Feedback regulation of transcription factor NF-κB by its inhibitor IκBα plays an essential role in control of NF-κB activity. To understand the biological significance of IκBα-mediated feedback regulation of NF-κB, we generated mice harboring mutated κB enhancers in the promoter of the IκBα gene ( IκBα M/M ) to inhibit NF-κB–regulated IκBα expression. Here, we report that these mutant mice are defective in NF-κB–induced expression of IκBα. This defective feedback regulation of NF-κB by IκBα not only altered activity of NF-κB, but also the expression of NF-κB–regulated genes. As a result, IκBα M/M , the homozygous knock-in mice with mutated κB enhancers in the IκBα promoter, acquire shorten life span, hypersensitivity to septic shock, abnormal T-cell development and activation, and Sjögren ’ s Syndrome. These findings therefore demonstrate that the IκBα-mediated feedback regulation of NF-κB has an essential role in controlling T-cell development and functions, provide mechanistic insight into the development of Sjögren ’ s Syndrome, and suggest the potential of NF-κB signaling as a therapeutic target for Sjögren ’ s Syndrome and other autoimmune diseases.

Inhibition of peroxisome proliferator-activated receptor gamma (PPARgamma) function by TNF-alpha contributes to glucose and fatty acid metabolic disorders in inflammation and cancer, although the molecular mechanism is not fully understood. In this study, we demonstrate that nuclear translocation of HDAC3 is regulated by TNF-alpha, and this event is required for inhibition of transcriptional activity of PPARgamma by TNF-alpha. HDAC3 is associated with IkappaBalpha in the cytoplasm. After IkappaBalpha degradation in response to TNF-alpha, HDAC3 is subject to nuclear translocation, leading to an increase in HDAC3 activity in the nucleus. This event leads to subcellular redistribution of HDAC3. Knock-out of IkappaBalpha, but not p65 or p50, leads to disappearance of HDAC3 in the cytoplasm, which is associated with HDAC3 enrichment in the nucleus. These data suggest that inhibition of PPARgamma by TNF-alpha is not associated with a reduction in the DNA binding activity of PPARgamma. Rather, these results suggest that IkappaBalpha-dependent nuclear translocation of HDAC3 is responsible for PPARgamma inhibition by TNF-alpha.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.

We previously reported that NF-kappaB is constitutively activated in most human pancreatic cancer tissues and cell lines but not in normal pancreatic tissues and immortalized pancreatic ductal epithelial cells. IkappaBalphaM-mediated inhibition of constitutive NF-kappaB activity in human pancreatic cancer cells suppressed tumorigenesis and liver metastasis in an orthotopic nude mouse model, suggesting that constitutive NF-kappaB activation plays an important role in pancreatic tumor progression and metastasis. However, the underlying mechanism by which NF-kappaB is activated in pancreatic cancer remains to be elucidated. In this study, we found that an autocrine mechanism accounts for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1alpha was the primary cytokine secreted by these cells that activates NF-kappaB. Neutralization of interleukin-1alpha activity suppressed the constitutive activation of NF-kappaB and the expression of its downstream target gene, urokinase-type plasminogen activator, in metastatic pancreatic cancer cell lines. Our results demonstrate that regulation of interleukin-1alpha expression is primarily dependent on AP-1 activity, which is in part induced by signaling pathways that are epidermal growth factor receptor-dependent and -independent. In conclusion, our findings suggest a possible mechanism for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cells and a possible missing mechanistic link between inflammation and cancer.

The Rel/NF-kappaB transcription factors play a key role in the regulation of apoptosis and in tumorigenesis by controlling the expressions of specific genes. To determine the role of the constitutive activity of RelA in tumorigenesis, we generated pancreatic tumor cell lines that express a dominant negative mutant of IkappaBalpha (IkappaBalphaM). In this report, we show that the inhibition of constitutive NF-kappaB activity, either by ectopic expression of IkappaBalphaM or by treating the cells with a proteasome inhibitor PS-341 which blocks intracellular degradation of IkappaBalpha proteins, downregulates the expression of bcl-xl. We identified two putative NF-kappaB binding sites (kappaB/A and B) in the bcl-xl promoter and found that these two sites interact with different NF-kappaB proteins. p65/p50 heterodimer interacts with kappaB/A site whereas p50/p50 homodimer interacts with kappaB/B. The bcl-xl promoter reporter gene assays reveal that NF-kappaB dependent transcriptional activation is mainly mediated by kappaB/A site, indicating that bcl-xl is one of the downstream target genes regulated by RelA/p50. Both IkappaBalphaM and PS-341 completely abolish NF-kappaB DNA binding activity; however, PS-341, but not ectopic expression of IkappaBalphaM, sensitized cells to apoptosis induced by Taxol. This is due to the Taxol-mediated reactivation of RelA through phosphorylation and degradation of IkappaBbeta and the re-expression of NF-kappaB regulated bcl-xl gene in these cancer cells as ectopic expression of the bcl-xl gene confers resistance to Taxol-induced apoptosis in PS-341 sensitized cells. These results demonstrate the important function of various NF-kappaB/IkappaB complexes in regulating anti-apoptotic genes in response to apoptotic stimuli, and they raise the possibility that NF-kappaB : IkappaBalpha and NF-kappaB : IkappaBbeta complexes are regulated by different upstream activators, and that NF-kappaB plays a key role in pancreatic tumorigenesis.

Expression of embryonic stem cells (ESCs) markers (SOX2, OCT4, Nanog and Nestin) is crucial for progression of various human malignancies. The purpose of this study was to investigate the expression and prognostic impact of these molecules in nasopharyngeal carcinoma (NPC) patients by immunohistochemistry and immunofluorescence. In the present study, we found that the expression levels of SOX2, OCT4 and Nanog were highly expressed in NPC compared with the non-tumorous tissues. Furthermore, these proteins correlated significantly with several clinicalpathological factors and epithelial-mesenchymal transition (EMT)-associated indicators (E-cadherin/N-cadherin and Snail). In multivariate analyses, high expression of OCT4 (P = 0.013) and Nanog (P = 0.040), but not that of SOX2, was associated with worse survival and had strongly independent prognostic effects. Of note, OCT4 and Nanog were more frequently located at the invasive front of tumors, and correlated significantly with various aggressive behaviors including T classification, N classification, M classification and clinical stage. Furthermore, patients with co-expression of OCT4 and Nanog in the invasive front had significantly worse survival (P = 0.005). Interestingly, at the invasive front, these molecules correlated significantly with Nestin expression in endothelial cells (P

Constitutive Kras and NF-κB activation is identified as signature alterations in pancreatic ductal adenocarcinoma (PDAC). However, how NF-κB is activated in PDAC is not yet understood. Here, we report that pancreas-targeted IKK2/β inactivation inhibited NF-κB activation and PDAC development in Kras(G12D) and Kras(G12D);Ink4a/Arf(F/F) mice, demonstrating a mechanistic link between IKK2/β and Kras(G12D) in PDAC inception. Our findings reveal that Kras(G12D)-activated AP-1 induces IL-1α, which, in turn, activates NF-κB and its target genes IL-1α and p62, to initiate IL-1α/p62 feedforward loops for inducing and sustaining NF-κB activity. Furthermore, IL-1α overexpression correlates with Kras mutation, NF-κB activity, and poor survival in PDAC patients. Therefore, our findings demonstrate the mechanism by which IKK2/β/NF-κB is activated by Kras(G12D) through dual feedforward loops of IL-1α/p62.

Feedback regulation of transcription factor NF-kappaB by its inhibitor IkappaBalpha plays an essential role in control of NF-kappaB activity. To understand the biological significance of IkappaBalpha-mediated feedback regulation of NF-kappaB, we generated mice harboring mutated kappaB enhancers in the promoter of the IkappaBalpha gene (IkappaBalpha(M/M)) to inhibit NF-kappaB-regulated IkappaBalpha expression. Here, we report that these mutant mice are defective in NF-kappaB-induced expression of IkappaBalpha. This defective feedback regulation of NF-kappaB by IkappaBalpha not only altered activity of NF-kappaB, but also the expression of NF-kappaB-regulated genes. As a result, IkappaBalpha(M/M), the homozygous knock-in mice with mutated kappaB enhancers in the IkappaBalpha promoter, acquire shorten life span, hypersensitivity to septic shock, abnormal T-cell development and activation, and Sjögren's Syndrome. These findings therefore demonstrate that the IkappaBalpha-mediated feedback regulation of NF-kappaB has an essential role in controlling T-cell development and functions, provide mechanistic insight into the development of Sjögren's Syndrome, and suggest the potential of NF-kappaB signaling as a therapeutic target for Sjögren's Syndrome and other autoimmune diseases.

Transcription factor nuclear factor-κB (NF-κB) is constitutively activated in most pancreatic cancer tissues and cell lines but not in normal pancreas nor in immortalized/nontumorigenic human pancreatic ductal epithelial cells. Inhibition of constitutive NF-κB activation in pancreatic cancer cell lines suppresses tumorigenesis and tumor metastasis. Recently, we identified autocrine secretion of proinflammatory cytokine interleukin (IL)-1α as the mechanism of constitutive NF-κB activation in metastatic pancreatic cancer cell lines. However, the role of IL-1α in determining the metastatic potential of pancreatic tumor remains to be further investigated. In the current study, we stably expressed IL-1α in the nonmetastatic, IL-1α–negative MiaPaCa-2 cell lines. Our results showed that the secretion of IL-1α in MiaPaCa-2 cells constitutively activated NF-κB and increased the expression of NF-κB downstream genes involved in the different steps of the metastatic cascade, such as urokinase-type plasminogen activator, vascular endothelial growth factor, and IL-8. MiaPaCa-2/IL-1α cells showed an enhanced cell invasion in vitro compared with parental MiaPaCa-2 cells and induced liver metastasis in an orthotopic mouse model. The metastatic phenotype induced by IL-1α was inhibited by the expression of phosphorylation-defective IκB (IκB S32, 36A), which blocked NF-κB activation. Consistently, silencing the expression of IL-1α by short hairpin RNA in the highly metastatic L3.6pl pancreatic cancer cells completely suppressed their metastatic spread. In summary, these findings showed that IL-1α plays key roles in pancreatic cancer metastatic behavior through the constitutive activation of NF-κB. Our findings further support the possible link between inflammation and cancer and suggest that IL-1α may be a potential therapeutic target for treating pancreatic adenocarcinoma. (Mol Cancer Res 2009;7(5):624–33)

BACKGROUND Constitutive activation of nuclear factor-κB (NF-κB) is a frequent molecular alteration in pancreatic cancer and a number of studies have suggested that constitutive NF-κB activity plays a key role in the aggressive behavior of this disease. In an attempt to identify an effective therapeutic agent for pancreatic cancer, the authors studied the role of FUT-175, a synthetic serine protease inhibitor, in the inhibition of NF-κB activation and the induction of apoptotic responses. METHODS To examine the effect of FUT-175 on the inhibition of NF-κB and the induction of apoptosis in pancreatic cancer cell lines, Western and Northern blot analyses, electromobility shift (EMSA), luciferase reporter gene, DNA fragmentation, immunoprecipitation, in vitro kinase, small interfering RNA (siRNA), and chromatin immunoprecipitation (ChIP) assays were performed. RESULTS In a time-dependent and dose-dependent manner, FUT-175 inhibited IκBα phosphorylation and NF-κB activation, thereby inhibiting the antiapoptotic activity of NF-κB. Simultaneously, FUT-175 up-regulated the expression of tumor necrosis factor receptor-1 (TNFR1), which in turn activated the proapoptotic caspase-8 and Bid pathways and induced apoptosis in pancreatic cancer cells. FUT-175-induced activation of Fas-associated death domain (FADD) and caspase-8 was suppressed by RNA interference-mediated inhibition of TNFR1 expression. Furthermore, expression of the transcription factor PEA3 was up-regulated by FUT-175 and was involved in FUT-175–mediated TNFR1 expression. CONCLUSIONS These results suggested a possible mechanism by which FUT-175 may disrupt interconnected signaling pathways by both suppressing the NF-κB antiapoptotic activity and inducing TNFR-mediated apoptosis. Supported by this unique function as a NF-κB inhibitor and apoptosis inducer, this well-established synthetic serine protease inhibitor with as-of-yet poorly understood mechanisms of actions appears to be a potentially therapeutic agent for pancreatic cancer. Cancer 2007. © 2007 American Cancer Society.

Keratinocyte growth factor (KGF)/fibroblast growth factor-7 (FGF-7) is a paracrine- and epithelium-specific growth factor produced by cells of mesenchymal origin. It acts exclusively through FGF-7 receptor (FGFR2/IIIb), which is expressed predominantly by epithelial cells, but not by fibroblasts, suggesting that it might function as a paracrine mediator of mesenchymal-epithelial interactions. KGF/FGF-7 plays an essential role in the growth of epithelial cells and is frequently overexpressed in cancers of epithelial origin such as pancreatic cancer, switching paracrine stimulation of KGF/FGF-7 to an autocrine loop. Less is known, however, about the signaling pathways by which KGF/FGF-7 regulates the response of epithelial cells. To delineate the signaling pathways activated by KGF/FGF-7 and examine cellular response to KGF/FGF-7 stimulation, we performed functional analysis of KGF/FGF-7 action. In this report, we show that KGF/FGF-7 activated nuclear factor kappaB (NF-kappaB), which in turn induced expression of VEGF, MMP-9, and urokinase-type plasminogen activator and increased migration and invasion of KGF/FGF-7-stimulated human pancreatic ductal epithelial cells. Expression of phosphorylation-defective IkappaBalpha (IkappaBalphaS32A,S36A), which blocked NF-kappaB activation, inhibited KGF/FGF-7-induced gene expression and cell migration and invasion. Our results demonstrate for the first time that KGF/FGF-7 induces NF-kappaB activation and that NF-kappaB plays an essential role in regulation of KGF/FGF-7-inducible gene expression and KGF/FGF-7-initiated cellular responses. Thus, these findings identify one signaling pathway for KGF/FGF-7-regulated cell migration and invasion and suggest that paracrine sources of KGF/FGF-7 are one of the malignancy-contributing factors from tumor stroma.

The silicon clathrate superconductor is uncommon as its structure is dominated by strong Si-Si covalent bonds, rather than the metallic bond, that are more typical of traditional superconductors. To understand the influence of large magnetic moment of Eu on superconductivity for type-I clathrates, a series of samples with the chemical formula Ba8−xEuxAl6Si40 (x = 0, 0.5, 1, and 2) were synthesised in which Eu occupied Ba sites in cage center. With the increase of Eu content, the cubic lattice parameter decreases monotonically signifying continuous shrinkage of the constituting (Ba/Eu)@Si20 and (Ba/Eu)@Si24 cages. The temperature dependence of magnetization at low temperature revealed that Ba8Al6Si40 is superconductive with transition temperature at TC = 5.6 K. The substitution of Eu for Ba results in a strong superconductivity suppression; Eu-doping largely decreases the superconducting volume and transition temperature TC. Eu atoms enter the clathrate lattice and their magnetic moments break paired elect...

RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-kappaB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding site in the promoter of Plk3, and this kappaB site is directly involved in its induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-kappaB-regulated proapoptotic responses.

BACKGROUND Currently, pancreatic adenocarcinoma is the fourth leading cause of cancer-related death in the United States. Despite the advances in pancreatic carcinoma research, patients with this devastating disease have a very poor prognosis. To identify the gene expression profile of pancreatic carcinoma, an important step in the process of developing new diagnostic and therapeutic strategies, the authors investigated the alteration of gene expression in this disease. METHODS The authors analyzed a public serial analysis of gene expression (SAGE) database and examined in greater detail the expression of synuclein-γ mRNA in several pancreatic carcinoma cell lines and tumor tissue samples by reverse transcriptase–polymerase chain reaction (RT-PCR) analysis and Northern blot analysis. The expression of synuclein-γ protein was investigated further by immunohistochemical and Western blot analyses using tumor cell lines, tumor tissue, and serum samples. RESULTS Synuclein-γ mRNA was overexpressed in 11 of 12 pancreatic carcinoma cell lines, including AsPc-1, MDAPanc28, Capan-1, Capan-2, PANC-1, HS766T, MDAPanc3, MDAPanc48, Colo357FG, MiaPaCa2, CFPac1, and BxPc3. The expression of synuclein-γ protein was detectable in 8 of 12 pancreatic carcinoma cell lines (67%) and in 22 of 32 pancreatic tumor tissue samples (69%) by Western blot analysis. On immunohistochemical staining, synuclein-γ protein was present in 61% of the tumor tissue samples examined from patients with Stage I and II pancreatic carcinoma. The overexpression of synuclein-γ is correlated with perineural and lymph node invasion. Synuclein-γ protein also was detectable by Western blot in serum samples from 21 of 56 patients (38%) with pancreatic carcinoma. CONCLUSIONS Synuclein-γ, which initially was described as a breast carcinoma–specific gene involved in invasion, metastasis, and chemotherapy resistance, was frequently overexpressed in pancreatic carcinoma. Overexpression of synuclein-γ may play a role in pancreatic carcinoma invasion. Further studies will be necessary to determine the role of synuclein-γ in pancreatic carcinoma. Cancer 2004. © 2004 American Cancer Society.

Both pro- and antiapoptotic activities of NF-kappaB transcription factor have been observed; however, less is known about the mechanism by which NF-kappaB induces apoptosis. To elucidate how NF-kappaB regulates proapoptotic signaling, we performed functional analyses using wild-type, ikk1(-/-), ikk2(-/-), rela(-/-) murine fibroblasts, MDAPanc-28/Puro, MDAPanc-28/IkappaBalphaM, and HCT116/p53(+/+) and HCT116/p53(-/-) cells with investigational anticancer agent doxycycline as a superoxide inducer for generating apoptotic stimulus. In this report, we show that doxycycline increased superoxide generation and subsequently activated NF-kappaB, which in turn up-regulated p53 expression and increased the stability and DNA binding activity of p53. Consequently, NF-kappaB-dependent p53 activity induced the expression of p53-regulated genes PUMA and p21(waf1) as well as apoptosis. Importantly, lack of RelA, IKK, and p53 as well as expression of a dominant negative IkappaBalpha (IkappaBalphaM) inhibited NF-kappaB-dependent p53 activation and apoptosis. The doxycycline-induced NF-kappaB activation was not inhibited in HCT116/p53(-/-) cells. Our results demonstrate that NF-kappaB plays an essential role in activation of wild-type p53 tumor suppressor to initiate proapoptotic signaling in response to overgeneration of superoxide. Thus, these findings reveal a mechanism of NF-kappaB-regulated proapoptotic signaling.

The transcription factor NF-kappaB regulates genes involved in innate and adaptive immune response, inflammation, apoptosis, and oncogenesis. Proinflammatory cytokines induce the activation of NF-kappaB in both transient and persistent phases. We investigated the mechanism for this biphasic NF-kappaB activation. Our results show that MEKK3 is essential in the regulation of rapid activation of NF-kappaB, whereas MEKK2 is important in controlling the delayed activation of NF-kappaB in response to stimulation with the cytokines TNF-alpha and IL-1alpha. MEKK3 is involved in the formation of the IkappaBalpha:NF-kappaB/IKK complex, whereas MEKK2 participates in assembling the IkappaBbeta:NF-kappaB/IKK complex; these two distinct complexes regulate the proinflammatory cytokine-induced biphasic NF-kappaB activation. Thus, our study reveals a novel mechanism in which different MAP3K and IkappaB isoforms are involved in specific complex formation with IKK and NF-kappaB for regulating the biphasic NF-kappaB activation. These findings provide further insight into the regulation of cytokine-induced specific and temporal gene expression.

The tumor suppressor gene Smad4/DPC4, a key transcription factorin transforming growth factor beta (TGF-beta) signaling cascades,is inactivated in 50% of pancreatic adenocarcinomas. We seek to determine the role of Smad4/DPC4 in the suppression of tumor cell growth and in the regulation of TGF-beta-mediated expression of cell-cycle regulatory genes p15(ink4b) and p21(waf1).Smad4/DPC4 is overexpressed by adenoviral infection in CFPac-1 pancreatic cancer cells, in which the Smad4/DPC4 is homozygously deleted, and in Capan-1 pancreatic cancer cells, in which Smad4/DPC4 is not expressed. Expression of the TGF-beta downstream target gene p21(waf1), regulation of the p15(ink4b) promoter, anchorage-independent growth, and tumorigenesis were examined.We demonstrate that expression of Smad4/DPC4 in Capan-1 cells reduced anchorage-independent growth by more than 50%, and inhibited xenograft tumor growth. However, overexpression of Smad4/DPC4 did not inhibit CFPac-1 cell growth. Interestingly, Smad4/DPC4 induced expression of p15(ink4b), p21(waf1), and TGF-beta-responsive reporter gene in Capan-1 but not in CFPac-1 cells. Furthermore, we found a previously unidentified Smad4 binding element (SBE) located in the region between -356 and -329 bp of the p15(ink4b) promoter. The p15(ink4b) promoter reporter gene assays revealed that Smad4-dependent transcriptional activation is mediated by this SBE, which indicates that p15(ink4b) is one of the downstream target genes regulated by Smad/DPC4.These results explain the role of Smad4/DPC4 in TGF-beta-mediated inhibition of cell proliferation in vitro and in vivo. Moreover, these results suggest that Smad4/DPC4-mediated tumor suppression and induction of TGF-beta-regulated cell-cycle-inhibitory genes may depend on additional factors that are absent in CFPac-1 cells.