Addressing Reported Pro-Apoptotic Functions of NF-κB: Targeted Inhibition of Canonical NF-κB Enhances the Apoptotic Effects of Doxorubicin

The ability of the transcription factor NF-κB to upregulate anti-apoptotic proteins has been linked to the chemoresistance of solid tumors to standard chemotherapy. In contrast, recent studies have proposed that, in response to doxorubicin, NF-κB can be pro-apoptotic through repression of anti-apoptotic target genes. However, there is little evidence analyzing the outcome of NF-κB inhibition on the cytotoxicity of doxorubicin in studies describing pro-apoptotic NF-κB activity. In this study, we further characterize the activation of NF-κB in response to doxorubicin and evaluate its role in chemotherapy-induced cell death in sarcoma cells where NF-κB is reported to be pro-apoptotic. Doxorubicin treatment in U2OS cells induced canonical NF-κB activity as evidenced by increased nuclear accumulation of phosphorylated p65 at serine 536 and increased DNA–binding activity. Co-treatment with a small molecule IKKβ inhibitor, Compound A, abrogated this response. RT–PCR evaluation of anti-apoptotic gene expression revealed that doxorubicin-induced transcription of cIAP2 was inhibited by Compound A, while doxorubicin-induced repression of other anti-apoptotic genes was unaffected by Compound A or siRNA to p65. Furthermore, the combination of doxorubicin and canonical NF-κB inhibition with Compound A or siRNA to p65 resulted in decreased cell viability measured by trypan blue staining and MTS assay and increased apoptosis measured by cleaved poly (ADP-ribose) polymerase and cleaved caspase 3 when compared to doxorubicin alone. Our results demonstrate that doxorubicin-induced canonical NF-κB activity associated with phosphorylated p65 is anti-apoptotic in its function and that doxorubicin-induced repression of anti-apoptotic genes occurs independent of p65. Therefore, combination therapies incorporating NF-κB inhibitors together with standard chemotherapies remains a viable method to improve the clinical outcomes in patients with advanced stage malignancies

Honokiol Arrests Cell Cycle, Induces Apoptosis, and Potentiates the Cytotoxic Effect of Gemcitabine in Human Pancreatic Cancer Cells

Survival rates for patients with pancreatic cancer are extremely poor due to its asymptomatic progression to advanced and metastatic stage for which current therapies remain largely ineffective. Therefore, novel therapeutic agents and treatment approaches are desired to improve the clinical outcome. In this study, we determined the effects of honokiol, a biologically active constituent of oriental medicinal herb Magnolia officinalis/grandiflora, on two pancreatic cancer cell lines, MiaPaCa and Panc1, alone and in combination with the standard chemotherapeutic drug, gemcitabine. Honokiol exerted growth inhibitory effects on both the pancreatic cancer cell lines by causing cell cycle arrest at G1 phase and induction of apoptosis. At the molecular level, honokiol markedly decreased the expression of cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4), and caused an increase in Cdk inhibitors, p21 and p27. Furthermore, honokiol treatment led to augmentation of Bax/Bcl-2 and Bax/Bcl-xL ratios to favor apoptosis in pancreatic cancer cells. These changes were accompanied by enhanced cytoplasmic accumulation of NF-κB with a concomitant decrease in nuclear fraction and reduced transcriptional activity of NF-κB responsive promoter. This was associated with decreased phosphorylation of inhibitor of kappa B alpha (IκB-α) causing its stabilization and thus increased cellular levels. Importantly, honokiol also potentiated the cytotoxic effects of gemcitabine, in part, by restricting the gemcitabine-induced nuclear accumulation of NF-κB in the treated pancreatic cancer cell lines. Altogether, these findings demonstrate, for the first time, the growth inhibitory effects of honokiol in pancreatic cancer and indicate its potential usefulness as a novel natural agent in prevention and therapy

TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms

The huge majority of myeloma cell lines express TNFR2 while a substantial subset of them failed to show TNFR1 expression. Stimulation of TNFR1 in the TNFR1-expressing subset of MM cell lines had no or only a very mild effect on cellular viability. Surprisingly, however, TNF stimulation enhanced cell death induction by CD95L and attenuated the apoptotic effect of TRAIL. The contrasting regulation of TRAIL- and CD95L-induced cell death by TNF could be traced back to the concomitant NFκB-mediated upregulation of CD95 and the antiapoptotic FLIP protein. It appeared that CD95 induction, due to its strength, overcompensated a rather moderate upregulation of FLIP so that the net effect of TNF-induced NFκB activation in the context of CD95 signaling is pro-apoptotic. TRAIL-induced cell death, however, was antagonized in response to TNF because in this context only the induction of FLIP is relevant. Stimulation of TNFR2 in myeloma cells leads to TRAF2 depletion. In line with this, we observed cell death induction in TNFR1-TNFR2-costimulated JJN3 cells. Our studies revealed that the TNF-TNF receptor system adjusts the responsiveness of the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms that generate a highly context-dependent net effect on myeloma cell survival

TBK1 Kinase Addiction in Lung Cancer Cells Is Mediated via Autophagy of Tax1bp1/Ndp52 and Non-Canonical NF-kappa B Signalling

K-Ras dependent non-small cell lung cancer (NSCLC) cells are 'addicted' to basal autophagy that reprograms cellular metabolism in a lysosomal-sensitive manner. Here we demonstrate that the xenophagy-associated kinase TBK1 drives basal autophagy, consistent with its known requirement in K-Ras-dependent NSCLC proliferation. Furthermore, basal autophagy in this context is characterised by sequestration of the xenophagy cargo receptor Ndp52 and its paralogue Tax1bp1, which we demonstrate here to be a bona fide cargo receptor. Autophagy of these cargo receptors promotes non-canonical NF-κB signalling. We propose that this TBK1-dependent mechanism for NF-κB signalling contributes to autophagy addiction in K-Ras driven NSCLC

RIP4 inhibits STAT3 signaling to sustain lung adenocarcinoma differentiation.

Loss of epithelial differentiation and extracellular matrix (ECM) remodeling are known to facilitate cancer progression and are associated with poor prognosis in patients with lung cancer. We have identified Receptor-interacting serine/threonine protein kinase 4 (RIP4) as a regulator of tumor differentiation in lung adenocarcinoma (AC). Bioinformatics analyses of human lung AC samples showed that poorly differentiated tumors express low levels of RIP4, whereas high levels are associated with better overall survival. In vitro, lung tumor cells expressing reduced RIP4 levels showed enhanced activation of STAT3 signaling and had a greater ability to invade through collagen. In contrast, overexpression of RIP4 inhibited STAT3 activation, which abrogated interleukin-6-dependent induction of lysyl oxidase, a collagen cross-linking enzyme. In an autochthonous mouse model of lung AC initiated by Kras(G12D) expression with loss of p53, Rip4 knockdown tumors progressed to a poorly differentiated state marked by an increase in Hmga2, reduced Ttf1, and enrichment of genes regulating extracellular remodeling and Jak-Stat signaling. Tail vein injections of cells overexpressing Rip4 showed a reduced potential to invade and form tumors, which was restored by co-expression of Stat3. Altogether, our work has identified that loss of RIP4 enhances STAT3 signaling in lung cancer cells, promoting the expression of ECM remodeling genes and cancer dedifferentiation

Transcriptional Activation of TINF2, a Gene Encoding the Telomere-Associated Protein TIN2, by Sp1 and NF-κB Factors

The expression of the telomere-associated protein TIN2 has been shown to be essential for early embryonic development in mice and for development of a variety of human malignancies. Recently, germ-line mutations in TINF2, which encodes for the TIN2 protein, have been identified in a number of patients with bone-marrow failure syndromes. Yet, the molecular mechanisms that regulate TINF2 expression are largely unknown. To elucidate the mechanisms involved in human TINF2 regulation, we cloned a 2.7 kb genomic DNA fragment containing the putative promoter region and, through deletion analysis, identified a 406 bp region that functions as a minimal promoter. This promoter proximal region is predicted to contain several putative Sp1 and NF-κB binding sites based on bioinformatic analysis. Direct binding of the Sp1 and NF-κB transcription factors to the TIN2 promoter sequence was demonstrated by electrophoretic mobility shift assay (EMSA) and/or chromatin immunoprecipitation (ChIP) assays. Transfection of a plasmid carrying the Sp1 transcription factor into Sp-deficient SL2 cells strongly activated TIN2 promoter-driven luciferase reporter expression. Similarly, the NF-κB molecules p50 and p65 were found to strongly activate luciferase expression in NF-κB knockout MEFs. Mutating the predicted transcription factor binding sites effectively reduced TIN2 promoter activity. Various known chemical inhibitors of Sp1 and NF-κB could also strongly inhibit TIN2 transcriptional activity. Collectively, our results demonstrate the important roles that Sp1 and NF-κB play in regulating the expression of the human telomere-binding protein TIN2, which can shed important light on its possible role in causing various forms of human diseases and cancers

Disruption of STAT3 signaling promotes KRAS induced lung tumorigenesis

STAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased KrasG12D-driven AC initiation and malignant progression leading to markedly reduced survival. Knockdown of STAT3 in xenografted human AC cells increases tumour growth. Clinically, low STAT3 expression levels correlate with poor survival and advanced malignancy in human lung AC patients with smoking history, which are prone to KRAS mutations. Consistently, KRAS mutant lung tumours exhibit reduced STAT3 levels. Mechanistically, we demonstrate that STAT3 controls NF-B-induced IL-8 expression by sequestering NF-B within the cytoplasm, thereby inhibiting IL-8-mediated myeloid tumour infiltration and tumour vascularization and hence tumour progression. These results elucidate a novel STAT3NF-BIL-8 axis in KRAS mutant AC with therapeutic and prognostic relevance.P 25599(VLID)183891

Activated K-ras and INK4a/Arf Deficiency Cooperate During the Development of Pancreatic Cancer by Activation of Notch and NF-κB Signaling Pathways

BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, suggesting that novel strategies for the prevention and treatment of PDAC are urgently needed. K-ras mutations are observed in >90% of pancreatic cancer, suggesting its role in the initiation and early developmental stages of PDAC. In order to gain mechanistic insight as to the role of mutated K-ras, several mouse models have been developed by targeting a conditionally mutated K-ras(G12D) for recapitulating PDAC. A significant co-operativity has been shown in tumor development and metastasis in a compound mouse model with activated K-ras and Ink4a/Arf deficiency. However, the molecular mechanism(s) by which K-ras and Ink4a/Arf deficiency contribute to PDAC has not been fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS:To assess the molecular mechanism(s) that are involved in the development of PDAC in the compound transgenic mice with activated K-ras and Ink4a/Arf deficiency, we used multiple methods, such as Real-time RT-PCR, western blotting assay, immunohistochemistry, MTT assay, invasion, EMSA and ELISA. We found that the deletion of Ink4a/Arf in K-ras(G12D) expressing mice leads to PDAC, which is in part mediated through the activation of Notch and NF-κB signaling pathways. Moreover, we found down-regulation of miR-200 family, which could also play important roles in tumor development and progression of PDAC in the compound transgenic mice. CONCLUSIONS/SIGNIFICANCE:Our results suggest that the activation of Notch and NF-κB together with the loss of miR-200 family is mechanistically linked with the development and progression of PDAC in the compound K-ras(G12D) and Ink4a/Arf deficient transgenic mice

ARHGAP10, a novel human gene coding for a potentially cytoskeletal Rho-GTPase activating protein

Rho-GTPase activating proteins (Rho-GAPs) are negative regulators of Rho-GTPase signaling pathways related to actin cytoskeleton dynamics, cell proliferation, and differentiation. We have identified a novel human gene, termed ARHGAP10, that codes for a 1957-aminoacid Rho-GAP, containing a PDZ, a PH, and a Rho-GAP domain. The cDNA is 7118 bp long and has an open reading frame of 5874 bp. A computational analysis located this gene on chromosome 10 band 10p12.32 suggesting that it is composed of 25 exons. Northern analysis revealed that it is widely expressed, with high levels in brain and muscle, Real-time quantitative PCR analysis confirmed an increase in ARHGAP10 expression during differentiation of HL-60 cells with all-transretinoic acid and hematopoietic stem cells with erythropoietin, suggesting that this gene could play a role in normal hematopoiesis. The fact that this gene is highly expressed in muscle and brain, which are highly differentiated tissues, further supports the hypothesis that ARHGAP10 is important for cell differentiation. (C) 2002 Elsevier Science (USA). All rights reserved.294357958