5970

RELA

tumor-initiating cells (TICs) are a sub-population of cells that exhibit a robust ability to self-renew and contribute to the formation of primary tumors, the relapse of previously treated tumors and the development of metastases. TICs have been identified in various tumors including those of the breast, and are particularly enriched in the basal-like and claudin-low subtypes of breast cancer. The signaling pathways that contribute to the function and maintenance of TICs are under intense study. We explored the potential involvement of the nuclear factor-kappaB (NF-kappaB) family of transcription factors in TICs in cell lines that are representative of basal-like and claudin-low breast cancer. NF-kappaB was found to be activated in breast cancer cells that form tumorspheres efficiently. Moreover, both canonical and non-canonical NF-kappaB signaling is required for these cells to self-renew in vitro and to form xenograft tumors efficiently in vivo using limiting dilutions of cells. Consistent with this fact, canonical and non-canonical NF-kappaB signaling is activated in TICs isolated from breast cancer cell lines. Experimental results indicate that NF-kappaB promotes the function of TICs by stimulating epithelial-to-mesenchymal transition and by upregulating the expression of the inflammatory cytokines interleukin-1beta and interleukin-6. The results suggest the use of NF-kappaB inhibitors for clinical therapy of certain breast cancers.

Non-neuronal cholinergic system is involved in lung physiology and lung cancer. However, the biochemical events downstream acetylcholine (ACh) receptor activation leading to carcinogenesis and tumor progression are not fully understood. Our previous work has shown that non-neuronal ACh acts as an autoparacrine growth factor to stimulate cell proliferation and promote epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) via activation of M2 muscarinic receptor (M2R). The aim of the present study was to delineate the underlying mechanisms linking M2R and lung tumor progression, which may provide potential therapeutic targets to delay lung cancer progression. Inhibition of M2R by antagonist or siRNA suppresses NSCLC cell migratory and invasive capacities, reverses EMT and simultaneously inactivates PI3K/Akt, MAPK ERK and NF-kappaB p65. On the other hand, M2R activation stimulates NSCLC migration and invasion and promotes EMT via NF-kappaB p65 activation. Moreover, NF-kappaB p65 activation induced by M2R activation was partially inhibited by either Akt or ERK inhibitor. Taken together, these results demonstrated for the first time that NF-kappaB p65 activation is essential in NSCLC progression associated with non-neuronal cholinergic system. Our data suggest that M2R/ERK/Akt/NF-kappaB axis could be a potential target for NSCLC treatment.

The epithelial-mesenchymal transition (EMT) plays an essential role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and tumor progression. However, the mechanisms underlying this process are poorly understood. Many signaling pathways, including the NF-kappaB signaling pathway, trigger EMT during development and differentiation. In the present study, we report that N-Myc interactor (NMI) inhibits EMT progression by suppressing transcriptional activities of NF-kappaB in human gastric cancer cells. We show that the expression of NMI is significantly reduced in invasive gastric cancer cells and gastric cancer tissues. Overexpression of NMI inhibited cell migration and invasion, and this inhibition was enhanced after TNF-alpha stimulation. tumorigenicity assay in nude mice support the notion that NMI inhibits EMT in cancer cells. Mechanistically, NMI promotes the interaction between NF-kappaB/p65 and histone deacetylases (HDACs) and inhibits the acetylation and transcriptional activity of p65. The expression of p65 rescues NMI-mediated inhibition of EMT and the inhibition of the acetylation of p65 mediated by NMI is HDACs-dependent. Taken together, these findings suggest that NMI can suppress tumor invasion and metastasis by inhibiting NF-kappaB pathways, providing an alternative mechanism for EMT inhibition in stomach neoplasm.CI - Copyright (c) 2016 Elsevier Ireland Ltd. All rights reserved.

epithelial-mesenchymal transition (EMT) is an essential process in breast cancer metastasis. The aim of the present study was to determine the role of secretions of tumor-associated leukocytes (TALs) isolated from negative and positive lymph nodes (nLNs and pLNs, respectively) breast cancer patients in regulating EMT mechanism and the associated signaling pathways. We found an increased infiltration of TALs, which was associated with downregulation of E-cadherin and over-expression of vimentin in the breast carcinoma tissues of pLNs as compared to nLNs patients and normal breast tissues obtained from healthy volunteers during mammoplasty. Furthermore, TALs isolated from pLNs breast cancer patients secreted an elevated panel of cytokines by up to 2-5-fold when compared with those isolated from nLNs patients. Secretome of TALs of pLNs possessed higher TARC, IGF-1, IL-3, TNF-beta, IL-5, G-CSF, IL-4, and IL-1alpha with more than a fivefold compared to those of nLNs. Using the human breast cancer cell lines MCF-7 and MDA-MB-231, we found that cytokines secreted by TALs isolated from nLNs and pLNs breast cancer patients promoted EMT via upregulation of TGF-beta and vimentin and downregulation of E-cadherin at messenger RNA (mRNA) levels in both cell lines and at protein level in MCF-7. While TGF-beta is over-expressed by MDA-MB-231 seeded in media conditioned by secretome of TALs isolated from nLNs and pLNs breast cancer patients. The downstream TGF-beta signaling transcription factors, Snail, Slug, and Twist, known to be associated with EMT mechanism were over-expressed by MCF-7 and MDA-MB-231 seeded in media conditioned by secretome of TALs isolated from nLNs and pLNs breast cancer patients. Acquisition of EMT in MCF-7 cells is mechanistically attributed to the activation of EGFR((Tyr845)) and NF-kappaB/p65((Ser276)) signaling which are significantly highly expressed by MCF-7 cells seeded in media conditioned by secretome of TALs isolated from pLNs compared to nLNs patients. Overall, this study provides implications of secretome of TALs and activated EGFR((Tyr845)) and NF-kappaB/p65((Ser276)) in EMT process that may be considered a therapeutic strategy to inhibit lymph node metastasis in breast cancer patients.

The aim of this study was to investigate the expression of T-cell immunoglobulin mucin domain molecule-3 (Tim-3) in osteosarcoma tissues, and analyze its effect on cell proliferation and metastasis in an osteosarcoma cell line. Tim-3 mRNA and protein expression in osteosarcoma tissue was detected by reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Additionally, the cell viability, apoptosis rate, and invasive ability of the osteosarcoma cell line MG-63 were tested using the methyl thiazolyl tetrazolium assay, Annexin V-propidium iodide flow cytometry, and a Transwell assay, respectively, following Tim-3 interference using small interfering RNA (siRNA). We also analyzed the expression of Snail, E-cadherin, vimentin, and nuclear factor (NF)-kB in the cells by western blot. We observed that Tim-3 mRNA and protein was significantly overexpressed in osteosarcoma tissues, compared to the adjacent normal tissue (P < 0.01). Moreover, MG-63 cells transfected with the Tim-3 siRNA presented lower cell viability, a greater number of apoptotic cells, and decreased invasive ability (P < 0.01), compared to control cells. Additionally, we observed a decrease in Snail and vimentin expression, an increase in the E-cadherin level, and an increase in NF-kB p65 phosphorylation (P < 0.01) in Tim-3 siRNA-transfected MG-63 cells. Based on these results, we concluded that Tim-3 is highly expressed in osteosarcoma tissue. Moreover, we speculated that interfering in Tim-3 expression could significantly suppress osteosarcoma cell (MG-63) proliferation and metastasis via the NF-kB/Snail signaling pathway and epithelial-mesenchymal transition.

Anterior gradient 2 (AGR2), an endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI), is associated with cancer development and malignant progression. Here, we show that high level of AGR2 promotes the aggressive phenotype of prostate cancer (PCa) mouse models developed by either patient-derived xenografts or surgical intra-prostate implantation of PCa cells, associated with enrichment of the blood vessel network in tumor tissues. Angiogenesis markers VEGFR2 and CD34, accompanied with the invasive marker Vimentin, were predominantly stained in metastatic liver tissues. Secreted AGR2 was defined to enhance VEGFR2 activity as evidenced by physical interaction of purified recombinant human AGR2 (rhAGR2) with rhVEGFA through the formation of a disulfide bond. Mutant or deleted thioredoxin motif in rhAGR2 was also unable to bind to rhVEGFA that led to the significant abolishment in the vessel formation, but partially affecting the aggressive process, implicating alternative mechanisms are required for AGR2-conferring metastasis. Cytosolic AGR2 contributed to cell metastasis ascribed to its stabilizing effect on p65 protein, which subsequently activated the NF-kappaB and facilitated epithelial to mesenchymal transition (EMT). Importantly, GSH and cabozantinib, but not bevacizumab, effectively blocked the pro-angiogenic effect of rhAGR2 in vitro and in vivo, providing evidence that secreted AGR2 acts as a predictive biomarker for selection of angiogenesis-targeting therapeutic drugs based on its levels in the circular system.CI - Copyright (c) 2018 Elsevier B.V. All rights reserved.

Aberrant expression of long noncoding RNAs (lncRNAs) is associated with cancer tumorigenesis and progression. It has been suggested that lncRNAs may be potential clinical diagnostic and prognostic biomarkers, and therapeutic targets. In the present study, the expression levels of small nucleolar RNA host gene 15 (SNHG15) were significantly upregulated in renal cell carcinoma (RCC) tissues and cell lines compared with in adjacent tissues and a proximal tubule epithelial cell line, as determined by reverse transcriptionquantitative polymerase chain reaction. Subsequently, knockdown of SNHG15 expression with small interfering RNA inhibited RCC proliferation, invasion and migration, was determined by western blotting and Transwell assays. Furthermore, the present study suggested that SNHG15 may be involved in the nuclear factorkappaB signaling pathway, induce the epithelialmesenchymal transition process, and promote RCC invasion and migration.

The epithelial-mesenchymal transition (EMT) is a multistep dedifferentiation program important in tissue repair. Here, we examined the role of the transcriptional regulator NF-kappaB in EMT of primary human small airway epithelial cells (hSAECs). Surprisingly, transforming growth factor beta (TGFbeta) activated NF-kappaB/RELA proto-oncogene, NF-kappaB subunit (RELA) translocation within 1 day of stimulation, yet induction of its downstream gene regulatory network occurred only after 3 days. A time course of TGFbeta-induced EMT transition was analyzed by RNA-Seq in the absence or presence of inducible shRNA-mediated silencing of RELA. In WT cells, TGFbeta stimulation significantly affected the expression of 2,441 genes. Gene set enrichment analysis identified WNT, cadherin, and NF-kappaB signaling as the most prominent TGFbeta-inducible pathways. By comparison, RELA controlled expression of 3,138 overlapping genes mapping to WNT, cadherin, and chemokine signaling pathways. Conducting upstream regulator analysis, we found that RELA controls six clusters of upstream transcription factors, many of which overlapped with a transcription factor topology map of EMT developed earlier. RELA triggered expression of three key EMT pathways: 1) the WNT/beta-catenin morphogen pathway, 2) the JUN transcription factor, and 3) the Snail family transcriptional repressor 1 (SNAI1). RELA binding to target genes was confirmed by ChIP. Experiments independently validating WNT dependence on RELA were performed by silencing RELA via genome editing and indicated that TGFbeta-induced WNT5B expression and downstream activation of the WNT target AXIN2 are RELA-dependent. We conclude that RELA is a master transcriptional regulator of EMT upstream of WNT morphogen, JUN, SNAI1-ZEB1, and interleukin-6 autocrine loops.CI - (c) 2018 Tian et al.

BACKGROUND/AIMS: TNF-alpha and TGF-beta associated epithelial-mesenchymal transition (EMT) occurs via NF-kappaB-dependent transcriptional upregulation of Twist1.Chrysin (ChR) is a major active ingredient ofpropolis, which inhibits various cancer cells and possesses anti-inflammatory activities. This study aimed to assess whether and how ChR inhibits proinflammatory cytokine-induced EMT phenotype and cancer stem-like cell (CSLC) features in the HeLa cell line. METHODS: HeLa cells were co-administered TNF-alpha (10.0 ng/mL) and TGF-beta (5.0 ng/mL) for 24h following TGF-beta (5.0 ng/mL) alone for 6 d in the presence or absence of ChR (5.0, 10.0 and 20.0muM). Then, the levels of EMT-related factors, multi-potential transcription factors, and stem cell markers were analyzed by immunoblot. Wound healing and tumor sphere formation assays were performed to assess the migration and self-renewal capabilities of cells, respectively. Overexpression and/or knockdown of NF-kappaBp65 and/or Twsit1 were used to explore the molecular mechanisms. RESULTS: The results showed that ChR inhibited EMT and CSLC properties in HeLa cells administered TNF-alpha after prolonged TGF-beta treatment, in a concentration-dependent fashion. NF-kappaBp65 knockdown and ChR(10.0muM) cooperatively enhanced the inhibition of NF-kappaBp65 and Twist1 expression, EMT, and CSLC properties. Conversely, overexpression of NF-kappaBp65 combined with ChR(10.0muM) antagonized such activities. Meanwhile, Twist1silencing or overexpression combined with ChR treatment did not affect NF-kappaBp65 levels, but also reduced or enhanced EMT and CSLC properties. Importantly, overexpressing Twist1 combined with ChR reversed the effects of NF-kappaBp65 knockdown and ChR. CONCLUSION: ChR inhibits proinflammatory cytokine-induced EMT and CSLC features in HeLa cells by blocking the NF-kappaB/Twist axis.CI - (c) Copyright by the Author(s). Published by Cell Physiol Biochem Press.