BACKGROUND/AIM: Although genetic factors are presumed to account only for a part of the inter-individual variation in lung cancer susceptibility, the results are conflicting and there are no data available regarding the Polish population. We, therefore, performed a case-control study to investigate the association of seven selected single nucleotide polymorphisms (SNPs), in genes coding for excision repair cross-complimentary group 1 (ERCC1: rs11615, rs3212986, rs2298881), nuclear factor ĸB (NFKB2: rs7897947, rs12769316), bone morphogenetic protein 4 (BMP4: rs1957860), complement receptor 1 (CR1: rs7525160) and del/ins polymorphism in the family hypoxia inducible factor 2 gene (EGLN2: rs10680577), with non-small cell lung cancer (NSCLC) risk.
MATERIALS AND METHODS: Real-time PCR with melting curve analysis was used for genotyping of NSCLC patients and healthy individuals of Polish origin.
RESULTS: The ERCC1 rs11615 T allele and rs3212986 GG homozygosity were found to be associated with a higher risk of developing NSCLC. In addition, NFKB2 rs12769316 GG homozygosity was more frequently detected among male patients than controls, while no significant differences were found between the five polymorphisms.
CONCLUSION: ERCC1 polymorphisms may affect NSCLC risk in the Polish population, while the NFKB2 variant may be a possible marker of the disease in males.

Mechanism of PAH mixtures, using granulosa tumour cells, was investigated. Cells were exposed to a mixture of all 16 priority PAHs (M1) or a mixture of five PAHs not classified as human carcinogens (M2). The effect of siAHR, siAHRR and siNFKB2 on the expression of CYP1A1, CYP1B1, GSTM1, ERα, AR and cell proliferation was described. M1 decreased AhR and CYP1A1, while increased AhRR and ARNT expression. M2 also decreased AhR and CYP1A1 but had no effect on AhRR expression. siAHRR reversed the inhibitory effect of M1 on AhR and CYP1A1,while inhibitory effect of M2 was still observed. siNFKB2 reversed inhibitory effect of both mixtures on AhR and CYP1A1 expression and stimulatory effect of M1 on AhRR expression. siAHR reversed stimulatory effect of both mixtures on ERα expression. Stimulatory effect of M1 on cell proliferation was not observed in siAHR, was still observed in siESR1 cells. M2 had no effect on cell proliferation, however stimulatory effect was appeared in siAHR and siESR1cells. In conclusion: M1 by activation of AhRR and NFkB p52, but M2 only by activation of NFκB attenuated AhR signalling and ligand-induced CYP1A1 expression. Interaction between AhR and ER following M1 and M2 exposure is primarily initiated through AhR.

Alternative splicing plays an important role in numerous cellular processes and aberrant splice decisions are associated with cancer. Although some studies point to a regulation of alternative splicing and its effector mechanisms in a time-dependent manner, the extent and consequences of such a regulation remains poorly understood. In the present work, we investigated the time-dependent production of isoforms in two Hodgkin lymphoma cell lines of different progression stages (HD-MY-Z, stage IIIb and L-1236, stage IV) compared to a B lymphoblastoid cell line (LCL-HO) with a focus on tumour necrosis factor (TNF) pathway-related elements. For this, we used newly generated time-course RNA-sequencing data from the mentioned cell lines and applied a computational pipeline to identify genes with isoform-switching behaviour in time. We analysed the temporal profiles of the identified events and evaluated in detail the potential functional implications of alterations in isoform expression for the selected top-switching genes. Our data indicate that elements within the TNF pathway undergo a time-dependent variation in isoform production with a putative impact on cell migration, proliferation and apoptosis. These include the genes

Although overexpression of the non-canonical NFκB subunit p52 has been observed in several tumors, the function and mechanism of p52 in bladder cancer (BC) are less well understood. Here, we aimed at understanding the role and mechanism underlying p52 regulation of BC invasion. Human p52 was stably knockdown with shRNA targeting p52 in two bladder cancer cell lines (T24 and UMUC3). Two constitutively expressing constructs, p52 and p100, were stably transfected in to T24 or UMUC3, respectively. The stable transfectants were used to determine function and mechanisms responsible for p52 regulation of BC invasion. We demonstrate that p52 mediates human BC invasion. Knockdown of p52 impaired bladder cancer invasion by reduction of rhogdiβ mRNA stability and expression. Positively regulation of rhogdiβ mRNA stability was mediated by p52 promoting AUF1 protein degradation, consequently resulting in reduction of AUF1 binding to rhogdiβ mRNA. Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1. Moreover, we found that p52 upregulated miR-145, which directly bound to the 3'-UTR of sp1 mRNA, leading to downregulation of Sp1 protein translation. Our results reveal a comprehensive pathway that p52 acts as a positive regulator of BC invasion by initiating a novel miR-145/Sp1/USP8/AUF1/RhoGDIβ axis. These findings provide insight into the understanding of p52 in the pathology of human BC invasion and progression, which may be useful information in the development of preventive and therapeutic approaches for using p52 as a potential target.

Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-β (LTβ), receptor LTβR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTβR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTβ, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTβR, NIK, and RELB proteins. Recombinant LTβ, and siRNA depletion of endogenous LTβR and NIK, modulated expression of LTβR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTβ induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTβ-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTβ is expressed. Our findings show that LTβ/LTβR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.

BACKGROUND: MMP-1 (Matrix metalloproteinase-1) promotes carcinogenesis and distant metastasis in different cancers. Regulation of MMP-1 could occur at multiple levels: epigenetically, post-transcriptionally, or post-translationally. An increasing body of evidence supports that the cytoplasmic transcription factor STAT3 (signal transducer and activator of transcription 3) is activated constitutively in a variety of cancers wherein it significantly affects the growth of tumors and also facilitates metastasis. In addition, STAT3 has been found to regulate nuclear activity pro-inflammatory transcriptional factor, NF-κB signaling, especially, the alternative one (RelB/p100) by directly interacting with them METHOD AND RESULTS: In this proof of concept study, we tested the hypothesis that STAT3 interacts with RelB to promote tumor invasion by positively regulating MMP-1 in colon cancer. We found that RelB and STAT3 were constitutively localized in the nucleus of colon cancer in surgically-resected specimens with use of Western blot analysis, which was further confirmed by immunofluorescence (IF) staining in colon carcinoma cell line HT29. We further observed that STAT3/RelB knockdown resulted in reduced MMP-1. Our results from chromatin immunoprecipitation studies further established that association between RelB and MMP-1 promoter decreased when STAT3 was depleted, and conversely, STAT3 association with MMP-1 decreased with the knockdown of RelB.
CONCLUSION: These results suggest that STAT3 and ReB constitute a minimal activator complex for positive regulation of MMP-1 in colon cancer.

Multiple studies have shown that chronic inflammation is closely related to the occurrence and development of colorectal cancer (CRC). Classical NF-κB signaling, the key factor in controlling inflammation, has been found to be of great importance to CRC development. However, the role of alternative NF-κB signaling in CRC is still elusive. Here, we found aberrant constitutive activation of alternative NF-κB signaling both in CRC tissue and CRC cells. Knockdown of RelB downregulates c-Myc and upregulates p27

BACKGROUND: Metastatic breast cancer carries a poor prognosis despite the success of newly targeted therapies. Treatment options remain especially limited for the subtype of triple negative breast cancer (TNBC). Several signaling pathways, including NF-κB, are altered in TNBC, and the complexity of this disease implies multi-faceted pathway interactions. Given that IKKε behaves as an oncogene in breast cancer, we hypothesized that IKKε regulates NF-κB signaling to control diverse oncogenic functions in TNBC.
METHODS: Vector expression and RNA interference were used to investigate the functional role of IKKε in triple-negative breast cancer cells. Viability, protein expression, NF-κB binding activity, invasion, anoikis, and spheroid formation were examined in cells expressing high or low levels of IKKε, in conjunction with p52 RNA interference or MEK inhibition.
RESULTS: This study found that non-canonical NF-κB p52 levels are inversely proportional to ΙΚΚε, and growth of TNBC cells in anchorage supportive, high-attachment conditions requires IKKε and activated MEK. Growth of these cells in anchorage resistant conditions requires IKKε and activated MEK or p52. In this model, IKKε and MEK cooperate to support overall viability whereas the p52 transcription factor is only required for viability in low attachment conditions, underscoring the contrasting roles of these proteins.
CONCLUSIONS: This study illustrates the diverse functions of IKKε in TNBC and highlights the adaptability of NF-κB signaling in maintaining cancer cell survival under different growth conditions. A better understanding of the diversity of NF-κB signaling may ultimately improve the development of novel therapeutic regimens for TNBC.

Long noncoding RNAs (lncRNAs) have been associated with hepatocellular carcinoma (HCC), but the underlying molecular mechanisms of their specific association with hepatocarcinogenesis have not been fully explored.

Gastric cancer (GC) is one of the most lethal malignant tumors; the resistance of this type of tumor is the main source of GC treatment failure. In this study, we used bioinformatics analysis to verify differences in resistant GCs and identify an effective method for reversing drug resistance in GC. Microarray data [gene and microRNA (miRNA)] were analyzed using GEO2R software, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were applied to further enrich the genetic data. miRNA-gene interactions were determined using Cytoscape (v.3.5.1). Online software was used to analyze protein interactions and predict network structure. The Cancer Genome Atlas (TCGA) database was used to verify the expression levels of genes in GC resistance. miR-604 expression levels were verified by real-time PCR in GC cell lines. We screened 3981 GC resistance-associated genes and 244 miRNAs using bioinformatics methods. Six hub genes were identified and verified in the TCGA database, including five up-regulated genes, POLR2L, POLR2C, POLR2F, APRT, and LMAN2, and a down-regulated gene, NFKB2. The up-regulated genes POLR2L, POLR2C, APRT, and LMAN2 interact with miR-604; therefore, we focused on miR-604, which has low expression in drug-resistant GC. The results of this study indicate that through bioinformatics technologies, we have determined the hub genes and hub miRNAs related to drug resistance in GC. Among them, miR-604 could become a new indicator in the diagnosis of drug-resistant GC and may be used to investigate the pathogenesis of resistance in GC.

Nuclear factor‑κB (NF‑κB) is widely involved in various lymphoid malignancies. However, its exact functional role and potential regulatory mechanisms in Hodgkin's lymphoma (HL) remains unclear. The present study aimed to investigate the regulatory mechanism of NF‑κB in HL by analysis of a gene expression profile that was obtained from HL cells with or without NF‑κB subunit 2 (NFKB2) knockdown. The GSE64234 dataset containing 6 HL cell line specimens transfected with small interfering (si)RNA against NFKB2 and 6 control specimens transfected with non‑targeting siRNA sequences was downloaded from the Gene Expression Omnibus database. Based on these data, differentially expressed genes (DEGs) were screened for following data preprocessing. Functional enrichment analysis was subsequently conducted among the identified upregulated and downregulated DEGs. Additionally, a protein‑protein interaction (PPI) network was constructed and module analyses were performed. Finally, microRNAs (miRNAs/miRs) targeting the identified DEGs were predicted for the construction of a miRNA‑target regulatory network. A total of 253 DEGs were identified, consisting of 109 upregulated and 144 downregulated DEGs. Pathway enrichment analysis revealed that B‑cell lymphoma 2‑like 1 (BCL2L1) was significantly enriched in the NF‑κB signaling pathway, and colony‑stimulating factor 2 (CSF2) and BCL2L1 were enriched in the Jak‑signal transducer and activator of transcription (STAT) signaling pathway. BCL2L1 and CSF2 were determined to be hub genes in the PPI network. A total of 6 miRNAs, including let‑7a‑5p, miR‑9‑5p, miR‑155‑5p, miR‑135a‑5p, miR‑17‑5p and miR‑375, were identified in the miRNA‑target regulatory network. The results of the present study indicated that NFKB2 may be involved in HL development through regulation of BCL2L1, CSF2, miR‑135a‑5p, miR‑155‑5p and miR‑9‑5p expression, as well as the modulation of Jak‑STAT and NF‑κB signaling pathways.

Cullin 4B, a member of the Cullins, which serve as scaffolds to facilitate the assembly of E3 ligase complexes, is aberrantly expressed in many cancers, including osteosarcoma. Recently, we observed that CUL4B forms the CRL4B

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest Kras

BACKGROUND Papillary thyroid carcinoma (PTC) is associated with mutations of BRAFV600E and RET/PTC and high levels of expression of nuclear factor-κB (NF-κB). However, few studies have focused on the association between NF-κB expression and mutations in BRAFV600E and RET/PTC, especially regarding PTC cell proliferation and migration. The aim of this in vitro study was to investigate the effect of BRAFV600E or RET/PTC on NF-κB expression, cell proliferation and cell migration in four established PTC cell lines. MATERIAL AND METHODS Four cell lines included TPC-1 (BRAFWT/WT), BCPAP (BRAFV600E/V600E), PCCL3, and PTC3-5 (RET/PTC), were grown in culture in vitro with or without suppression of NF-κB using pyrrolidine dithiocarbamate (PDTC), and cell proliferation, and cell migration were evaluated. RESULTS Expression of the BRAF gene was increased in the BCPAP cell line when compared with the TPC-1 cells. Expression of the RET gene was increased in the PTC3-5 cell line when compared with the PCCL3 cells. In the BCPAP and PTC3-5 cell lines, the relative expression of NF-κB protein, including phosphorylated p100/52, phosphorylated p65, phosphorylated IKKa/b, phosphorylated IκBα, and p65 nuclear translocation were increased compared with the TPC-1 and PCCL3 cells. Proliferation and migration of BCPAP and PTC3-5 cells were increased compared with the TPC-1 and PCCL3 cells. Suppression of NF-κB reduced NF-κB protein expression and inhibited the proliferation of cells in the TPC-1, BCPAP, PCCL3 and PTC3-5 cell lines, and migration of the BCPAP and PTC3-5 cells. CONCLUSIONS BRAFV600E and RET/PTC and the expression of NF-κB promote the proliferation and migration of papillary thyroid carcinoma cells in vitro.

CONTEXT: Little is known about the phenotypic and molecular characteristics associated with various domains of quality of life (QOL) in women after breast cancer surgery.
OBJECTIVES: In a sample of women with breast cancer (n = 398), purposes were as follows: to identify latent classes with distinct trajectories of QOL from before surgery through six months after surgery and to evaluate for differences in demographic and clinical characteristics, as well as for polymorphisms in cytokine genes, between these latent classes.
METHODS: Latent class analyses were done to identify subgroups of patients with distinct QOL outcomes. Candidate gene analyses were done to identify cytokine gene polymorphisms associated with various domains of QOL (i.e., physical, psychological, spiritual, social).
RESULTS: One latent class was identified for the psychological and spiritual domains. Two latent classes were identified for the social domain and overall QOL scores. Three latent classes were identified for the physical domain. For the physical and social domains, as well as for the overall QOL scores, distinct phenotypic characteristics (i.e., younger age, poorer functional status, higher body mass index, and receipt of adjuvant chemotherapy) and a number of cytokine gene polymorphisms (CXCL8, NFKB2, TNFSF, IL1B, IL13, and NFKB1) were associated with membership in the lower QOL classes.
CONCLUSIONS: Findings suggest that women experience distinctly different physical well-being, social well-being, and total QOL outcomes during and after breast cancer surgery. The genetic associations identified suggest that cytokine dysregulation influences QOL outcomes. However, specific QOL domains may be impacted by different cytokines.

Despite great advancements in the treatment of non-Hodgkin lymphoma (NHL), sensitivity of different subtypes to therapy varies. Targeting the aberrant activation NF-κB signaling pathways in lymphoid malignancies is a promising strategy. Here, we report that 11(13)-dehydroivaxillin (DHI), a natural compound isolated from the Carpesium genus, induces growth inhibition and apoptosis of NHL cells. Multiple signaling cascades are influenced by DHI in NHL cells. PI3K/AKT and ERK are activated or inhibited in a cell type dependent manner, whereas NF-κB signaling pathway was inhibited in all the NHL cells tested. Applying the cellular thermal shift assay, we further demonstrated that DHI directly interacts with IKKα/IKKβ in NHL cells. Interestingly, DHI treatment also reduced the IKKα/IKKβ protein level in NHL cells. Consistent with this finding, knockdown of IKKα/IKKβ inhibits cell proliferation and enhances DHI-induced proliferation inhibition. Overexpression of p65, p52 or RelB partially reverses DHI-induced cell growth inhibition. Furthermore, DHI treatment significantly inhibits the growth of NHL cell xenografts. In conclusion, we demonstrate that DHI exerts anti-NHL effect in vitro and in vivo, through a cumulative effect on NF-κB and other pathways. DHI may serve as a promising lead compound for the therapy of NHL.

CONTEXT: Most of the reviews on the biological basis for symptom clusters suggest that inflammatory processes are involved in the development and maintenance of the symptom clusters. However, no studies have evaluated for associations between genetic polymorphisms and common symptom clusters (e.g., mood disturbance, sickness behavior).
OBJECTIVES: Examine the associations between cytokine gene polymorphisms and the severity of three distinct symptom clusters (i.e., mood-cognitive, sickness-behavior, treatment-related) in a sample of patients with breast and prostate cancer (n = 157) at the completion of radiation therapy.
METHODS: Symptom severity was assessed using the Memorial Symptom Assessment Scale. Symptom clusters were created using exploratory factor analysis. The associations between cytokine gene polymorphisms and the symptom cluster severity scores were evaluated using regression analyses.
RESULTS: Polymorphisms in C-X-C motif chemokine ligand 8 (CXCL8), interleukin (IL13), and nuclear factor kappa beta 2 (NFKB2) were associated with severity scores for the mood-cognitive symptom cluster. In addition to interferon gamma (IFNG1), the same polymorphism in NFKB2 (i.e., rs1056890) that was associated with the mood-cognitive symptom cluster score was associated with the sickness-behavior symptom cluster. Polymorphisms in interleukin 1 receptor 1 (IL1R1), IL6, and NFKB1 were associated with severity factor scores for the treatment-related symptom cluster.
CONCLUSION: Our findings support the hypotheses that symptoms that cluster together have a common underlying mechanism and the most common symptom clusters in oncology patients are associated polymorphisms in genes involved in a variety of inflammatory processes.

An increasing number of researchers are focusing on the influence of local peptide hormones such as angiotensin II (Ang II) and relaxin 2 (RLN2) in the regulation of inflammation and carcinogenesis. The interaction between the renin‑angiotensin system (RAS) and relaxin family peptide system (RFPS) is known to influence the proliferation, adhesion and migration of normal and cancer prostate cell lines. The aim of the present study was to evaluate changes in the expression of nuclear factor‑κB subunit 1 (NFKB1), nuclear factor‑κB subunit 2 (NFKB2), REL proto‑oncogene nuclear factor‑κB p65 subunit (REL), RELA proto‑oncogene nuclear factor‑κB subunit (RELA) and RELB proto‑oncogene nuclear factor‑κB subunit (RELB) mRNA caused by Ang II and RLN2. The members of NF‑kB family are involved in many processes associated with cancer development and metastasis. Reverse transcription‑quantitative polymerase chain reaction analysis identified that both peptide hormones have an influence on the relative expression of nuclear factor‑κB. Following treatment with either peptide, NFKB1 expression was downregulated in all prostate cancer cell lines (LNCaP, DU‑145 and PC3), but not in normal epithelial cells (PNT1A). Conversely, RELB mRNA was enhanced only in non‑cancerous prostate cells. RELA expression was strongly stimulated in the most aggressive cell line, whereas REL mRNA was unchanged. In many cases, the effect was strictly dependent on the cell line and/or the type of peptide: Ang II increased expression of both RELA and REL genes in the androgen‑dependent cell line while RLN2 enhanced NFKB2 and RELA mRNA in androgen‑independent cells (DU‑145). Further research is needed to understand the regulation of NF‑κB family members by key renin‑angiotensin system and RFPS peptides in prostate cancer cells; however, prostate carcinogenesis appears to be influenced by the balance between the cross‑regulation of nuclear factor‑κB (NF‑κB) and androgen receptor pathways by Ang II and relaxin 2.

Diffuse large B-cell lymphoma (DLBCL) has been categorized into two molecular subtypes that have prognostic significance, namely germinal center B-cell like (GCB) and activated B-cell like (ABC). Although ABC-DLBCL has been associated with NF-κB activation, the relationships between activation of specific NF-κB signals and DLBCL phenotype remain unclear. Application of novel gene expression classifiers identified two new DLBCL categories characterized by selective p100 (NF-κB2) and p105 (NF-κB1) signaling. Interestingly, our molecular studies showed that p105 signaling is predominantly associated with GCB subtype and histone mutations. Conversely, most tumors with p100 signaling displayed ABC phenotype and harbored ABC-associated mutations in genes such as MYD88 and PIM1. In vitro, MYD88 L265P mutation promoted p100 signaling through TAK1/IKKα and GSK3/Fbxw7a pathways, suggesting a novel role for this protein as an upstream regulator of p100. p100 signaling was engaged during activation of normal B cells, suggesting p100's role in ABC phenotype development. Additionally, silencing p100 in ABC-DLBCL cells resulted in a GCB-like phenotype, with suppression of Blimp, IRF4 and XBP1 and upregulation of BCL6, whereas introduction of p52 or p100 into GC cells resulted in differentiation toward an ABC-like phenotype. Together, these findings identify specific roles for p100 and p105 signaling in defining DLBCL molecular subtypes and posit MYD88/p100 signaling as a regulator for B-cell activation.

Triple negative breast cancer (TNBC) typically exhibits rapid progression, high mortality and faster relapse rates relative to other breast cancer subtypes. In this report we examine the combination of taxanes (paclitaxel or docetaxel) with a breast cancer stem cell (CSC)-targeting agent sulforaphane for use against TNBC. We demonstrate that paclitaxel or docetaxel treatment induces IL-6 secretion and results in expansion of CSCs in TNBC cell lines. Conversely, sulforaphane is capable of preferentially eliminating CSCs, by inhibiting NF-κB p65 subunit translocation, downregulating p52 and consequent downstream transcriptional activity. Sulforaphane also reverses taxane-induced aldehyde dehydrogenase-positive (ALDH+) cell enrichment, and dramatically reduces the size and number of primary and secondary mammospheres formed. In vivo in an advanced treatment orthotopic mouse xenograft model together with extreme limiting dilution analysis (ELDA), the combination of docetaxel and sulforaphane exhibits a greater reduction in primary tumor volume and significantly reduces secondary tumor formation relative to either treatment alone. These results suggest that treatment of TNBCs with cytotoxic chemotherapy would be greatly benefited by the addition of sulforaphane to prevent expansion of and eliminate breast CSCs.

PURPOSE: Nuclear factor-kappa B (NF-κB) signalling has been shown to regulate properties of breast cancer stem cells. However, the specific contribution of the non-canonical NF-κB pathway, components of which are elevated in aggressive breast cancer has not been addressed.
METHODS: Through shRNA silencing of the Nfkb2 gene, the role of p100/p52 in 4T1 and N202.1A cell lines were assessed by NF-κB reporter, invasion, tumoursphere and orthotopic transplantation assays. The processing of p100 into p52 was also inhibited with a p97 ATPase inhibitor, NMS-873, and its effects on tumoursphere formation was assessed.
RESULTS: Knockdown of Nfkb2 led to opposing changes in NF-κB-dependent transcription. NF-κB activity was elevated in 4T1 cells and this resulted in increased motility, cancer stem cell (CSC) activity and tumourigenicity in vivo. Conversely, depletion of Nfkb2 in N202.1a cells decreased NF-κB activity, CSC properties and tumourigenicity in vivo. By selectively overexpressing the p52 subunit in Nfkb2 depleted cells, we found that the increased malignancy in 4T1 cells could not be reverted in the presence of p52, whereas the decreased tumourigenicity of N202.1a cells could be rescued by p52. These results indicate that p100 and its subunit p52 have opposing effects on breast CSC activity. Accordingly, inhibition of an upstream regulator of p100 processing was effective in reducing tumoursphere formation of N202.1A and SKBR3 (ErbB2
CONCLUSION: These findings indicate that inhibiting the processing of p100 may be a potential therapeutic strategy to suppress CSC activity in a subset of breast tumours.

A causal link between overexpression of aryl hydrocarbon receptor (AHR) and its target cytochrome P450 1A1 (CYP1A1) and metastatic outgrowth of various cancer entities has been established. Nevertheless, the mechanism how AHR/CYP1A1 support metastasis formation is still little understood. In vitro we discovered a potential mechanism facilitating tumour dissemination based on the production of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Utilising a three-dimensional lymph endothelial cell (LEC) monolayer & MDA-MB231 breast cancer cell spheroid co-culture model in combination with knock-down approach allowed elucidation of the molecular/biochemical basis of AHR/CYP1A1-induced tumour breaching through the LEC barrier. Enzyme immunoassay evidenced the potential of recombinant CYP1A1 to synthesise 12(S)-HETE in vitro and qPCR and Western blotting measured gene and protein expression in specific experimental settings. In detail, AHR induced CYP1A1 expression and 12(S)-HETE secretion in tumour spheroids, which caused LEC junction retraction thereby forming large discontinuities allowing transmigration of the tumour. This was enforced by the activating AHR ligand 6-formylindolo (3,3-b)carbazole (FICZ), or inhibited by the AHR antagonist 3,3’-diindolylmethane (DIM) as well as by siRNA against AHR and CYP1A1. AHR and NF-κB were negatively cross talking and therefore, the inhibition of AHR (but not CYP1A1) induced RELA, RELB, NFKB1, NFKB2 and the NF-κB target MMP1, which itself promotes tumour intravasation by a mechanism that is different from 12(S)-HETE. Conversely, the inhibition of NFKB2 induced AHR, CYP1A1 and 12(S)-HETE synthesis. The approved clinical drugs guanfacine and vinpocetine, which inhibit CYP1A1 and NF-κB, respectively, significantly inhibited LEC barrier breaching in vitro indicating an option to reduce metastatic dissemination.

Tudor staphylococcal nuclease (TSN, also known as Tudor-SN, SND1 or p100) is an evolutionarily conserved protein with invariant domain composition, represented by tandem repeat of staphylococcal nuclease domains and a tudor domain. Conservation along significant evolutionary distance, from protozoa to plants and animals, suggests important physiological functions for TSN. It is known that TSN is critically involved in virtually all pathways of gene expression, ranging from transcription to RNA silencing. Owing to its high protein-protein binding affinity coexistent with enzymatic activity, TSN can exert its biochemical function by acting as both a scaffolding molecule of large multiprotein complexes and/or as a nuclease. TSN is indispensible for normal development and stress resistance, whereas its increased expression is closely associated with various types of cancer. Thus, TSN is an attractive target for anti-cancer therapy and a potent tumor marker. Considering ever increasing interest to further understand a multitude of TSN-mediated processes and a mechanistic role of TSN in these processes, here we took an attempt to summarize and update the available information about this intriguing multifunctional protein.

Owing to activation of several resistance-mediating pathways including NF-κB signaling, metastasized melanoma is almost universally resistant against chemotherapy. Given that blocking of NF-κB either by proteasome-, pan-IKK- or selective IKKβ-inhibitors may increase the susceptibility of melanoma cells to chemotherapy, we have assessed the role of the second kinase within the IKK complex, IKKα. While expression of IKKα and overall activation of NF-κB were heterogeneous, the IKKα-specific p100/p52 processing was detected in a small subset of melanomas (1/9 primary and 1/12 metastatic melanomas) as well as in 1/8 melanoma cell lines. Down-modulation of IKKα by siRNA resulted in diminution of doxorubicin-induced NF-κB activation, constitutive and TNFα-stimulated expression of CXCL8 and ICAM-1, and cell migration. In contrast, overexpression of IKKα in melanoma cells did not significantly affect progression-related functions. Thus, IKKα may be a worthwhile target only in selected individualized therapies but not in general melanoma therapy.

Environmental drug resistance constitutes a serious impediment for therapeutic intervention in multiple myeloma. Tumor-promoting cytokines, such as tumor necrosis factor (TNF), induce nuclear factor-κB (NFκB)- driven expression of pro-survival factors, which confer resistance in myeloma cells to apoptotic insults from TNF-related apoptosis-inducing ligand (TRAIL) and other chemotherapeutic drugs. It is thought that RelA:p50 dimer, activated from IκBα-inhibited complex in response to TNF-induced canonical NFκB signal, mediates the pro-survival NFκB function in cancerous cells. Myeloma cells additionally acquire gain-of-function mutations in the non-canonical NFκB module, which induces partial proteolysis of p100 into p52 to promote RelB:p52/NFκB activation from p100-inhibited complex during immune cell differentiation. However, role of non-canonical NFκB signaling in the drug resistance in multiple myeloma remains unclear. Here we report that myeloma-associated non-canonical aberrations reinforce pro-survival TNF signaling in producing a protracted TRAIL-refractory state. These mutations did not act through a typical p52 NFκB complex, but completely degraded p100 to reposition RelB under IκBα control, whose degradation during TNF signaling induced an early RelB:p50 containing NFκB activity. More so, autoregulatory RelB synthesis prolonged this TNF-induced RelB:p50 activity in myeloma cells harboring non-canonical mutations. Intriguingly, TNF-activated RelB:p50 dimer was both necessary and sufficient, and RelA was not required, for NFκB-dependent pro-survival gene expressions and suppression of apoptosis. Indeed, high RelB mRNA expressions in myeloma patients correlated with the augmented level of pro-survival factors and resistance to therapeutic intervention. In sum, we provide evidence that cancer-associated mutations perpetuate TNF-induced pro-survival NFκB activity through autoregulatory RelB control and thereby exacerbate environmental drug resistance in multiple myeloma.

IκBζ, an atypical member of the nuclear IκB family of proteins, is expressed at low levels in most resting cells, but is induced upon stimulation of Toll-like/IL-1 receptors through an IRAK1/IRAK4/NFκB-dependent pathway. Like its homolog Bcl3, IκBζ can regulate the transcription of a set of inflamatory genes through its association with the p50 or p52 subunits of NF-κB. Long studied as a key component of the immune response, IκBζ emerges as an important regulator of inflammation, cell proliferation and survival. As a result, growing evidence support the role of this transcription factor in the pathogenesis number of human hematological and solid malignancies.

We investigated gene expression profiles of the NF-κB pathway in patients with triple-negative breast cancer (TNBC) receiving adjuvant chemotherapy to determine the prognostic value of NF-κB pathway genes according to chemotherapeutic regimen. We used the nCounter expression assay to measure expression of 11 genes (NFKB1, NFKB2, RELA, RELB, REL, TP53, FOXC1, TBP, SP1, STAT3 and IRF1 genes) belonging to the NF-κB pathway using mRNA extracted from paraffin-embedded tumor tissues from 203 patients diagnosed with TNBC. Of the 203 patients, 116 were treated with a chemotherapeutic regimen containing doxorubicin. As revealed by the expression profiles of the 11 genes, increased expression of SP1 was associated with poor prognosis in TNBC patients treated with adjuvant doxorubicin chemotherapy (5-year distant recurrence-free survival [5Y DRFS], low vs. high expression [cut-off: median]: 92.3% vs. 71.6%, P = 0.001). In a multivariate Cox regression model, SP1 expression was a useful marker for predicting long-term prognosis in TNBC patients receiving doxorubicin treatment, and we thus suggest that SP1 expression could serve as a prognostic marker in these patients.

Human BRCA1 mutation carriers and BRCA1-deficient mouse mammary glands contain an abnormal population of mammary luminal progenitors that can form 3D colonies in a hormone-independent manner. The intrinsic cellular regulatory defect in these presumptive breast cancer precursors is not known. We have discovered that nuclear factor kappaB (NF-κB) (p52/RelB) is persistently activated in a subset of BRCA1-deficient mammary luminal progenitors. Hormone-independent luminal progenitor colony formation required NF-κB, ataxia telangiectasia-mutated (ATM), and the inhibitor of kappaB kinase, IKKα. Progesterone (P4)-stimulated proliferation resulted in a marked enhancement of DNA damage foci in Brca1(-/-) mouse mammary. In vivo, NF-κB inhibition prevented recovery of Brca1(-/-) hormone-independent colony-forming cells. The majority of human BRCA1(mut/+) mammary glands showed marked lobular expression of nuclear NF-κB. We conclude that the aberrant proliferative capacity of Brca1(-/-) luminal progenitor cells is linked to the replication-associated DNA damage response, where proliferation of mammary progenitors is perpetuated by damage-induced, autologous NF-κB signaling.

Although the precursor protein of NFκB2 (p100) is thought to act as a tumor suppressor in mammalian cells, the molecular mechanism of its anti-tumor activity is far from clear. Here, we are, for the first time, to report that p100 protein expression was dramatically decreased in bladder cancers of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice and human patients. Knockdown of p100 in cultured human bladder cancer cells promoted anchorage-independent growth accompanied with elevating abundance of cell-cycle-related proteins and accelerated cell-cycle progression. Above effects could be completely reversed by ectopically expression of p100, but not p52. Mechanistically, p100 inhibited Cyclin D1 protein translation by activating the transcription of LARP7 and its hosted miR-302d, which could directly bind to 3'-UTR of cyclin d1 mRNA and inhibited its protein translation. Furthermore, p100 suppressed the expression of PHLPP2 (PH domain and leucine-rich repeat protein phosphatases 2), thus promoting CREB phosphorylation at Ser133 and subsequently leading to miR-302d transcription. Taken together, our studies not only for the first time establish p100 as a key tumor suppressor of bladder cancer growth, but also identify a novel molecular cascade of PHLPP2/CREB/miR-302d that mediates the tumor suppressive function of p100.

Sézary syndrome is a leukemic form of cutaneous T-cell lymphoma with an aggressive clinical course. The genetic etiology of the disease is poorly understood, with chromosomal abnormalities and mutations in some genes being involved in the disease. The goal of our study was to understand the genetic basis of the disease by looking for driver gene mutations and fusion genes in 15 erythrodermic patients with circulating Sézary cells, 14 of them fulfilling the diagnostic criteria of Sézary syndrome. We have discovered genes that could be involved in the pathogenesis of Sézary syndrome. Some of the genes that are affected by somatic point mutations include ITPR1, ITPR2, DSC1, RIPK2, IL6, and RAG2, with some of them mutated in more than one patient. We observed several somatic copy number variations shared between patients, including deletions and duplications of large segments of chromosome 17. Genes with potential function in the T-cell receptor signaling pathway and tumorigenesis were disrupted in Sézary syndrome patients, for example, CBLB, RASA2, BCL7C, RAMP3, TBRG4, and DAD1. Furthermore, we discovered several fusion events of interest involving RASA2, NFKB2, BCR, FASN, ZEB1, TYK2, and SGMS1. Our work has implications for the development of potential therapeutic approaches for this aggressive disease.