Search

Background: Breast cancer (BC) has a marked tendency to spread to the bone, resulting in significant skeletal complications and mortality. Recently, circular RNAs (circRNAs) have been reported to contribute to cancer initiation and progression. However, the function and mechanism of circRNAs in BC bone metastasis (BC-BM) remain largely unknown., Methods: Bone-metastatic circRNAs were screened using circRNAs deep sequencing and validated using in situ hybridization in BC tissues with or without bone metastasis. The role of circIKBKB in inducing bone pre-metastatic niche formation and bone metastasis was determined using osteoclastogenesis, immunofluorescence and bone resorption pit assays. The mechanism underlying circIKBKB-mediated activation of NF-κB/bone remodeling factors signaling and EIF4A3-induced circIKBKB were investigated using RNA pull-down, luciferase reporter, chromatin isolation by RNA purification and enzyme-linked immunosorbent assays., Results: We identified that a novel circRNA, circIKBKB, was upregulated significantly in bone-metastatic BC tissues. Overexpressing circIKBKB enhanced the capability of BC cells to induce formation of bone pre-metastatic niche dramatically by promoting osteoclastogenesis in vivo and in vitro. Mechanically, circIKBKB activated NF-κB pathway via promoting IKKβ-mediated IκBα phosphorylation, inhibiting IκBα feedback loop and facilitating NF-κB to the promoters of multiple bone remodeling factors. Moreover, EIF4A3, acted acting as a pre-mRNA splicing factor, promoted cyclization of circIKBKB by directly binding to the circIKBKB flanking region. Importantly, treatment with inhibitor eIF4A3-IN-2 reduced circIKBKB expression and inhibited breast cancer bone metastasis effectively., Conclusion: We revealed a plausible mechanism for circIKBKB-mediated NF-κB hyperactivation in bone-metastatic BC, which might represent a potential strategy to treat breast cancer bone metastasis., (© 2021. The Author(s).)

Lymph node metastasis (LNM) is a critical cause for disease progression and treatment failure in cervical cancer. However, the mechanism underlying cervical cancer LNM remains unclear. In this study, HN1 was found to be dramatically upregulated in cervical cancer and patients with higher HN1 expression are more likely to exhibit a higher rate of LNM and lower survival rate. Univariate and multivariate Cox-regression analyses showed that HN1 is an independent prognostic factor in cervical cancer. Meanwhile, HN1 promotes lymphangiogenesis of cervical cancer in vitro. The in vivo experiment also indicates that HN1 enhances LNM in cervical cancer. Furthermore, we also found that HN1 activated the NF-κB signaling pathway to enhance the expression of downstream genes. Taken together, our study suggests that HN1 plays a crucial role in promoting LNM and acts as a prognostic biomarker in cervical cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Non-small-cell lung cancer (NSCLC), in which the NF-κB pathway is constitutively activated, is one of the most common malignancies. Herein, we identify an E3 ubiquitin ligase, tripartite motif-containing 37 (TRIM37), participating in the K63 polyubiquitination of TRAF2, which is a significant step in the activation of NF-κB signaling. Both the mRNA and the protein expression levels of TRIM37 were much higher in NSCLC cell lines and tissues than in normal bronchial epithelial cells and matched adjacent non-tumor tissues. TRIM37 expression correlated closely with clinical stage and poor survival in NSCLC. Overexpression of TRIM37 antagonized cisplatin-induced apoptosis, induced angiogenesis and proliferation, and increased the aggressiveness of NSCLC cells in vitro and in vivo, whereas inhibition of TRIM37 led to the opposite effects. Gene set enrichment analysis (GSEA) showed that TRIM37 expression significantly correlated with NF-κB signaling. Furthermore, we found that TRIM37 bound to TRAF2 and promoted K63-linked ubiquitination of TRAF2, sustaining the eventual activation of the NF-κB pathway. Mutation in the ring finger domain of TRIM37, a hallmark of E3 ubiquitin ligases, led to loss of the ability to promote K63 polyubiquitination of TRAF2 and activate NF-κB signaling. Taken together, our findings provide evidence that TRIM37 plays an important role in constitutive NF-κB pathway activation and could serve as a prognostic factor and therapeutic target in NSCLC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: The primary issue arising from prostate cancer (PCa) is its high prevalence to metastasize to bone, which severely affects the quality of life and survival time of PCa patients. miR-210-3p is a well-documented oncogenic miRNA implicated in various aspects of cancer development, progression and metastasis. However, the clinical significance and biological roles of miR-210-3p in PCa bone metastasis remain obscure., Methods: miR-210-3p expression was evaluated by real-time PCR in 68 bone metastatic and 81 non-bone metastatic PCa tissues. The biological roles of miR-210-3p in the bone metastasis of PCa were investigated both in vitro by EMT and Transwell assays, and in vivo using a mouse model of left cardiac ventricle inoculation. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to discern and examine the relationship between miR-210-3p and its potential targets. RT-PCR was performed to identify the underlying mechanism of miR-210-3p overexpression in bone metastasis of PCa. Clinical correlation of miR-210-3p with its targets was examined in human PCa and metastatic bone tissues., Results: miR-210-3p expression is elevated in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Overexpression of miR-210-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Upregulating miR-210-3p enhances, while silencing miR-210-3p represses the EMT, invasion and migration of PCa cells in vitro. Importantly, silencing miR-210-3p significantly inhibits bone metastasis of PC-3 cells in vivo. Our results further demonstrate that miR-210-3p maintains the sustained activation of NF-κB signaling via targeting negative regulators of NF-κB signaling (TNF-α Induced Protein 3 Interacting Protein 1) TNIP1 and (Suppressor Of Cytokine Signaling 1) SOCS1, resulting in EMT, invasion, migration and bone metastasis of PCa cells. Moreover, our results further indicate that recurrent gains (amplification) contribute to miR-210-3p overexpression in a small number of PCa patients. The clinical correlation of miR-210-3p with SOCS1, TNIP1 and NF-κB signaling activity is verified in PCa tissues., Conclusion: Our findings unravel a novel mechanism for constitutive activation of NF-κB signaling pathway in the bone metastasis of PCa, supporting a functional and clinical significance of epigenetic events in bone metastasis of PCa.

The strength and duration of NF-κB signaling is tightly controlled at multiple levels under physiologic conditions, but the mechanism underlying constitutive activation of the NF-κB pathway in cancer remains unclear. In this study, we investigated miRNA-mediated regulation of the NF-κB cascade in breast cancer. We report that miR-892b expression was significantly downregulated in human breast cancer specimens and correlated with poor patient survival. Overexpression of miR-892b in breast cancer cells significantly decreased tumor growth, metastatic capacity, and the ability to induce angiogenesis, whereas miR-892b depletion enhanced these properties, in vitro and in vivo. Furthermore, we demonstrate that miR-892b attenuated NF-κB signaling by directly targeting and suppressing multiple mediators of NF-κB, including TRAF2, TAK1, and TAB3, and thus, miR-892b silencing in breast cancer cells sustains NF-κB activity. Moreover, miR-892b downregulation was attributed to aberrant hypermethylation of its promoter. Taken together, our results provide insight into a new mechanism by which NF-κB signaling becomes constitutively activated in breast cancer and suggest a tumor-suppressive role for miR-829b, prompting further investigation into miRNA mimics for cancer therapy., (©2016 American Association for Cancer Research.)

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, in which the NF-κB pathway plays an important role and is constitutively activated. Better understanding of the molecular pathogenesis of HCC and the NF-κB pathway are needed to improve patient outcomes. Herein, we identified an unappreciated protein involved in NF-κB-induced activation, Golgi phosphoprotein 3 (GOLPH3). The mRNA and protein expression levels of GOLPH3 were frequently up-regulated in HCC and GOLPH3 expression correlated closely with clinical stage and survival in both the testing and validation cohorts. Ectopic over-expression of GOLPH3 in PLC/PRF/5 (PLC) and Huh7 HCC cells protected against cisplatin-induced apoptosis, promoted angiogenesis and proliferation and increased the aggressiveness of HCC cells in vitro and in vivo, whereas inhibition of GOLPH3 led to decreased aggressiveness. Through analysis of two published HCC patient profiles, GOLPH3 expression significantly correlated with NF-κB signalling. Furthermore, we demonstrated that GOLPH3 promoted K63-linked polyubiquitination of tumour necrosis factor receptor-associated factor 2 (TRAF2), receptor interacting protein (RIP) and NF-κB essential modulator (NEMO) and substantially sustained the activation of NF-κB in HCC cells. Taken together, our findings provided evidence that GOLPH3 is a prognostic and/or potential therapeutic biomarker for HCC patients and plays an important role in activation of the NF-κB pathway during HCC progression., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

High levels of angiogenesis and resistance to apoptosis are major clinical features of hepatocellular carcinoma (HCC), a lethal disease with a high incidence worldwide. However, the precise mechanisms underlying these malignant properties remain unclear. Here, we demonstrated that acylglycerol kinase (AGK) is markedly overexpressed in HCC cell lines and clinical tissues. Immunohistochemical analysis of 245 clinical HCC specimens revealed patients with high levels of AGK expression had poorer overall survival compared to patients with low AGK expression. Furthermore, overexpressing AGK significantly enhanced angiogenesis and inhibited apoptosis in vitro and promoted the tumorigenicity of HCC cells in vivo; silencing endogenous AGK had the opposite effects. Importantly, AGK enhanced angiogenesis and inhibited apoptosis in HCC in part via activation of NF-κB signaling. Our findings provide new evidence that AGK plays an important role in promoting angiogenesis and providing resistance to apoptosis, thus AGK may represent a novel therapeutic target for HCC.

Purpose: Constitutive activation of NF-κB signaling plays vital roles in esophageal squamous cell carcinoma (ESCC) progression. The aim of this study was to evaluate the effect of miR-138 on NF-κB activation and ESCC progression., Experimental Design: Expression of miR-138 in ESCC cell lines, ESCC tissues, and 205 archived ESSC specimens was determined using real-time PCR analysis. Anchorage-independent growth, chicken chorioallantoic membrane, Transwell matrix invasion and Annexin V-binding assays, and a xenograft tumor model were used to determine the role of miR-138 in ESCC progression. The effect of miR-138 on NF-κB activation was investigated using IKK in vitro kinase, electrophoretic mobility shift, lipid raft isolation, and luciferase reporter assays., Results: miR-138 was downregulated and inversely correlated with tumor progression and patient survival in ESCCs. Downregulation of miR-138 enhanced, whereas upregulation of miR-138 reduced, the aggressive phenotype of ESCC cells both in vitro and in vivo. Silencing miR-138 promoted K63-linked polyubiquitination of the NF-κB signaling intermediaries TRAF2 and RIP1 and sustained NF-κB activation. Furthermore, downregulation of miR-138 induced lipid raft formation via upregulating multiple components of lipid rafts, including FLOT1, FLOT2, and caveolin-1. Importantly, the in vitro analysis was consistent with a significant inverse correlation between miR-138 expression and NF-κB hyperactivation in a cohort of human ESCC specimens., Conclusion: Our results show that miR-138 functions as a tumor-suppressive miRNA and that downregulation of miR-138 contributes to constitutive NF-κB activation and ESCC progression., (©2013 AACR.)

Deubiquitinases, such as CYLD, A20 and Cezanne, have emerged as important negative regulators that balance the strength and the duration of NF-κB signaling through feedback mechanisms. However, how these serial feedback loops are simultaneously disrupted in cancers, which commonly exhibit constitutively activated NF-κB, remains puzzling. Herein, we report that miR-486 directly suppresses NF-κB-negative regulators, CYLD and Cezanne, as well as multiple A20 activity regulators, including ITCH, TNIP-1, TNIP-2 and TNIP-3, resulting in promotion of ubiquitin conjugations in NF-κB signaling and sustained NF-κB activity. Furthermore, we demonstrate that upregulation of miR-486 promotes glioma aggressiveness both in vitro and in vivo through activation of NF-κB signaling pathway. Importantly, miR-486 levels in primary gliomas significantly correlate with NF-κB activation status. These findings uncover a novel mechanism for constitutive NF-κB activation in gliomas and support a functionally and clinically relevant epigenetic mechanism in cancer progression.

Angiogenesis in glioma is associated with the poor prognosis of the disease and closely correlates with the highly invasive phenotype of glioma cells, which represents the most challenging impediment against the currently glioma treatments. Bmi-1, an onco-protein, has been implicated in the progression of various human cancers, including gliomas, whereas its role in glioma angiogenesis remains unclear. Our current study examined the effects of Bmi-1 on glioma angiogenesis in vitro as well as in vivo. We found that overexpression of Bmi-1 enhanced, whereas knockdown of Bmi-1 diminished, the capability of glioma cells to induce tubule formation and migration of endothelial cells and neovascularization in chicken chorioallantoic membrane. In vivo, Bmi-1 overexpression and knockdown, respectively, promoted and inhibited angiogenesis in orthotopically transplanted human gliomas. Furthermore, NF-κB activity and VEGF-C expression was induced by Bmi-1 overexpression, whereas Bmi-1 knockdown attenuated NF-κB signaling and decreased VEGF-C expression. Additionally suppression of NF-κB activity using a specific chemical inhibitor abrogated the NF-κB activation and the pro-angiogenic activities of glioma cells. Together, our data suggest that Bmi-1 plays an important role in glioma angiogenesis and therefore could represent a potential target for anti-angiogenic therapy against the disease.

Background & Aims: The flotillin family of proteins, including flotillin-1 (FLOT1 or Reggie-2), are lipid raft proteins that initiate receptor kinase signaling and are up-regulated in several tumor types. We investigated the role of FLOT1 signaling and activation of the transcription factor nuclear factor (NF)-κB in esophageal squamous cell carcinoma (ESCC) cells., Methods: We used immunoblot and immunochemical analyses to determine levels of the lipid raft-associated protein FLOT1 in ESCC cell lines and 432 ESSC samples from patients; primary normal esophageal epithelial cells and matched adjacent nontumor tissues were used as controls. We determined the ability of FLOT1 to activate NF-κB using kinase, electrophoretic mobility shift, and luciferase reporter assays. We measured the effects of FLOT1 overexpression and knockdown with short hairpin RNAs in ESCC cell lines using colony formation, anchorage-independent growth, chicken chorioallantoic membrane, transwell matrix penetration, and Annexin V-binding assays. We analyzed growth of ESCC xenograft tumors in nude mice., Results: Levels of FLOT1 were increased in ESCC cell lines and samples from patients, compared with controls; protein levels correlated with disease stage and survival time. Overexpression of FLOT1 in Kyse30 and Kyse510 ESCC cell lines increased proliferation, anchorage-independent growth, and invasive activity and protected them from apoptosis. FLOT1-transduced ESCC cells formed larger tumors in nude mice than control cells (transduced with only the vector). FLOT1 facilitated recruitment of the tumor necrosis factor-α receptor to lipid rafts; promoted K63-linked polyubiquitination of the signaling intermediaries tumor necrosis factor receptor associated factor 2, receptor interacting protein, and NEMO; and sustained the activation of NF-κB. Levels of FLOT1 correlated with activation of NF-κB in ESCC samples from patients., Conclusions: The lipid raft protein FLOT1 is up-regulated in ESCC cell lines and samples from patients and promotes ESCC cell proliferation and tumor growth in mice. FLOT1 activates tumor necrosis factor-α receptor signaling and sustains activation of NF-κB in ESCC cells., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The strength and duration of NF-κB signaling are tightly controlled by multiple negative feedback mechanisms. However, in cancer cells, these feedback loops are overridden through unclear mechanisms to sustain oncogenic activation of NF-κB signaling. Previously, we demonstrated that overexpression of miR-30e* directly represses IκBα expression and leads to hyperactivation of NF-κB. Here, we report that miR-182 was overexpressed in a different set of gliomas with relatively lower miR-30e* expression and that miR-182 directly suppressed cylindromatosis (CYLD), an NF-κB negative regulator. This suppression of CYLD promoted ubiquitin conjugation of NF-κB signaling pathway components and induction of an aggressive phenotype of glioma cells both in vitro and in vivo. Furthermore, we found that TGF-β induced miR-182 expression, leading to prolonged NF-κB activation. Importantly, the results of these experiments were consistent with an identified significant correlation between miR-182 levels with TGF-β hyperactivation and activated NF-κB in a cohort of human glioma specimens. These findings uncover a plausible mechanism for sustained NF-κB activation in malignant gliomas and may suggest a new target for clinical intervention in human cancer.

Background: The prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear., Methods: A172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay., Results: Ectopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples., Conclusions: Bmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF-kappaB/MMP-9 pathway and therefore might represent a novel therapeutic target for glioma.

Stomatin-like protein 2 (STOML2), a member of the stomatin family, has been reported to be up-regulated in several types of human cancers. The clinical significance and biological role of STOML2 in gliomas remain largely unknown. Here, we describe the significantly up-regulated expression of STOML2 in glioma cell lines and glioma tissues at both the transcriptional and translational levels. Silencing endogenous STOML2 in glioma cells and primary glioma cells drastically reduced their migratory speed and invasive ability, associated with induction of matrix metallopeptidase 9 (MMP-9). We also demonstrated that knockdown of STOML2 significantly inhibited the transcriptional activity of NF-κB and repressed the expression levels of NF-κB target genes, including MMP-9. A luciferase reporter assay revealed that the impact of STOML2 on MMP-9 expression is NF-κB-dependent. Immunohistochemical analysis showed that the up-regulation of STOML2 was significantly correlated with the WHO histological grade of gliomas (p < 0.001). Patients with higher STOML2 expression levels had an overall shorter survival time, whereas patients with lower expression of STOML2 had a longer survival time. A multivariate analysis revealed that STOML2 expression might be an independent prognostic indicator for the survival of glioma patients. Taken together, our results suggest that overexpression of STOML2 is associated with glioma aggressiveness and may represent an independent prognostic factor for the outcome of glioma patients., (Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Constitutive activation of NF-κB is a frequent event in human cancers, playing important roles in cancer development and progression. In nontransformed cells, NF-κB activation is tightly controlled by IκBs. IκBs bind NF-κB in the cytoplasm, preventing it from translocating to the nucleus to modulate gene expression. Stimuli that activate NF-κB signaling trigger IκB degradation, enabling nuclear translocation of NF-κB. Among the genes regulated by NF-κB are those encoding the IκBs, providing a negative feedback loop that limits NF-κB activity. How transformed cells override this NF-κB/IκB negative feedback loop remains unclear. Here, we report in human glioma cell lines that microRNA-30e* (miR-30e*) directly targets the IκBα 3ι-UTR and suppresses IκBα expression. Overexpression of miR-30e* in human glioma cell lines led to hyperactivation of NF-κB and enhanced expression of NF-κB-regulated genes, which promoted glioma cell invasiveness in in vitro assays and in an orthotopic xenotransplantation model. These effects of miR-30e* were shown to be clinically relevant, as miR-30e* was found to be upregulated in primary human glioma cells and correlated with malignant progression and poor survival. Hence, miR-30e* provides an epigenetic mechanism that disrupts the NF-κB/IκBα loop and may represent a new therapeutic target and prognostic marker.

Purpose: The present study was to examine the effect of sphingosine kinase-1 (SPHK1) on chemotherapeutics-induced apoptosis in non-small cell lung cancer (NSCLC) cells, which is relatively insensitive to chemotherapy, and its clinical significance in NSCLC progression., Experimental Design: The correlation of SPHK1 expression and clinical features of NSCLC was analyzed in 218 paraffin-embedded archived NSCLC specimens by immunohistochemical analysis. The effect of SPHK1 on apoptosis induced by chemotherapeutics was examined both in vitro and in vivo, using Annexin V staining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assays. Western blotting and luciferase analysis were performed to examine the impact of SPHK1 on the PI3K/Akt/NF-κB signaling., Results: The expression of SPHK1 was markedly increased in NSCLC and correlated with tumor progression and poor survival of patients with NSCLC. Upregulation of SPHK1 significantly inhibited doxorubicin- or docetaxel-induced apoptosis, associated with induction of antiapoptotic proteins Bcl-xl, c-IAP1, c-IAP2, and TRAF1. In contrast, silencing SPHK1 expression or inhibiting SPHK1 activity with specific inhibitor, SK1-I, significantly enhanced the sensitivity of NSCLC cells to apoptosis induced by chemotherapeutics both in vitro and in vivo. Moreover, we demonstrated that upregulation of SPHK1 activated the PI3K/Akt/NF-κB pathway, and that inhibition of the PI3K/Akt/NF-κB pathway abrogated the antiapoptotic effect of SPHK1 on NSCLC cells., Conclusions: Our results suggest that SPHK1 is a potential pharmacologic target for the treatment of NSCLC and inhibition of SPHK1 expression or its kinase activity might represent a novel strategy to sensitize NSCLC to chemotherapy.

Abstract: Aberrant activation of nuclear factor-kappa B (NF-κB) pathway has been proven to play important roles in the development and progression of cancers. Activation of NF-κB via the classical pathway is modulated by IκBs kinase (IKK-β). However, the mechanism underlying the epigenetic regulation of IKK-β/NF-κB pathway remains largely unknown. In this study, we found that the expression level of miR-218 was markedly downregulated in glioma cell lines and in human primary glioma tissues. Upregulation of miR-218 dramatically reduced the migratory speed and invasive ability of glioma cells. Furthermore, we showed that ectopically expressing miR-218 in glioma cells resulted in downregulation of matrix metalloproteinase-9 (MMP-9) and reduction in NF-κB transactivity at a transcriptional level, but inhibition of miR-218 enhanced the expression of MMP-9 and transcriptional activity of NF-κB. Moreover, we showed that miR-218 inactivated the NF-κB pathway through downregulating IKK-β expression by directly targeting the 3′-untranslated region (3′-UTR) of IKK-β. Taken together, our results suggest that miR-218 plays an important role in preventing the invasiveness of glioma cells, and our results present a novel mechanism of miRNA-mediated direct suppression of IKK-β/NF-κB pathway in gliomas. [ABSTRACT FROM AUTHOR]