NF-κB Signaling in Targeting Tumor Cells by Oncolytic Viruses—Therapeutic Perspectives

In recent years, oncolytic virotherapy became a promising therapeutic approach, leading to the introduction of a novel generation of anticancer drugs. However, despite evoking an antitumor response, introducing an oncolytic virus (OV) to the patient is still inefficient to overcome both tumor protective mechanisms and the limitation of viral replication by the host. In cancer treatment, nuclear factor (NF)-κB has been extensively studied among important therapeutic targets. The pleiotropic nature of NF-κB transcription factor includes its involvement in immunity and tumorigenesis. Therefore, in many types of cancer, aberrant activation of NF-κB can be observed. At the same time, the activity of NF-κB can be modified by OVs, which trigger an immune response and modulate NF-κB signaling. Due to the limitation of a monotherapy exploiting OVs only, the antitumor effect can be enhanced by combining OV with NF-κB-modulating drugs. This review describes the influence of OVs on NF-κB activation in tumor cells showing NF-κB signaling as an important aspect, which should be taken into consideration when targeting tumor cells by OVs.


Introduction
Since its discovery in 1986, nuclear factor (NF)-κB has been widely studied and is well known as a pleiotropic transcription factor, which orchestrates inflammation, innate and adaptive immune responses, cell growth, and apoptosis. Therefore, deregulation of NF-κB activation pathways may result in autoimmune diseases and tumorigenesis. Importantly, NF-κB and its target genes have been shown to play a role in malignant transformation, proliferation, and survival of cancer cells, as well as angiogenesis and invasion/metastasis. Moreover, aberrant NF-κB signaling is associated with aggressiveness, recurrence, and therapeutic resistance of tumors. Therefore, NF-κB is considered a good candidate for the therapeutic target, becoming an important focus in cancer research [1][2][3][4].
Chemotherapy, which is widely used for cancer treatment, is mainly aimed at apoptosis, necrosis, autophagy, and mitotic catastrophe. Importantly, cellular senescence, resulting from chemotherapy treatment, involves NF-κB activation. This suggests that blockage of NF-κB would prevent this outcome. Unfortunately, due to the pleiotropic nature of NF-κB, the use of NF-κB inhibitors in cancer therapy may not always be beneficial. Apoptotic defects of cancer cells are responsible for chemoresistance. Therefore, these factors should be considered when targeting NF-κB in cancer treatment [1].

OVs
OVs, belonging to new generation of cancer immunotherapeutics, are natural or genetically modified pathogens, which infect and replicate in cancer cells but not in non-transformed cells, and trigger both antiviral and antitumor responses [22]. Upon administration of OV, the virus infects tumor cells resulting in their lysis. As a consequence of tumor-derived antigens' (TDAs) release, antigen-presenting cells (APCs) uptake and process TDAs to activate and prime T cells. Thus, the effector cells localize to, infiltrate, and eventually kill the tumor cells. Afterward, released TDAs are processed by APCs [23]. Nevertheless, using OVs as monotherapy may not be efficient due to the limited replication of the virus in the host, tumor resistance to the response generated, and immunosuppression within the tumor microenvironment [22].
In oncolytic virotherapy, one of the main concerns is the presence of neutralizing antibodies, which can already be found in patients vaccinated or previously treated with OVs [24,25]. This effect can be observed in MM patients treated with systemically administered measles virus armed with human thyroidal sodium iodide symporter (MV-NIS) [24]. Upon intravenous delivery of OV, both antibodies and complement promote Fc receptor-linked clearance of the virus by Kupfer cells and splenic macrophages [25]. However, such administration is not always beneficial. For instance, oncolytic herpes simplex virus type 1 (HSV)-1, which spreads from cell to cell, and is used for melanoma treatment, is more effective when administered intralesionally [24]. Nevertheless, intratumoral injection of an OV may not be efficient in the treatment of disseminated tumors, whereas systemic administration of a drug in trans with OV delivery may result in toxicity and increases the costs. Adversely, delivery of therapeutic gene product or a single therapeutic in cis may not be efficient when sustained expression is required [26]. Therefore, many therapeutic approaches based on OVs are under clinical trials. Nevertheless, the United States Food and Drug Administration (FDA) approved Talimogene laherparepvec (T-VEC), a modified HSV, in metastatic melanoma treatment [22,23,27,28]. In clinical trials, metastatic melanoma patients are given intralesional injections of T-VEC combined with intravenous pembrolizumab (anti-programmed death [PD]-1). This treatment represents a strategy of switching immunologically "cold" tumor, which is characterized by the absence or low tumor-infiltrating lymphoid cells (TILs), into "hot." The latter is defined by the presence of TILs in their microenvironment due to induction of proinflammatory response and activation of DCs and NK cells [22,29].
TILs are represented by subpopulations of CD3 + CD4 + helper and CD3 + CD8 + cytotoxic T cells [30], Tregs, NK cells, B cells, DCs, macrophages, and MDSCs [31]. TILs comprise epithelial, stromal, and peritumoral lymphocytes located in cancer cell nests, central stroma, and invasive margins, respectively. Although TILs may display higher reactivity toward tumor cells compared with non-infiltrating lymphocytes, they may promote tumor growth. Importantly, the presence and immunophenotype of TILs serve as a prognostic factor in melanoma, breast cancer, ovarian cancer, lung cancer, and renal cell carcinoma (RCC). For instance, in advanced melanoma, TILs Treg CD4 + CD25 + are linked to progression of the disease [30].
TILs obtained from resected tumors are used as effector cells in adoptive T-cell therapy (ATC), which can be combined with chemotherapy and immunotherapeutic drugs to maintain TILs proliferation and proper function to target tumor-associated antigens (TAAs). After ex vivo propagation and activation, TILs and IL-2, a T-cell growth factor, are administered autologously to lymphodepleted patients [30,32,33]. To facilitate virus entry to the tumor, TILs may be genetically modified with C-X-C chemokine receptor type 2 (CXCR2) [34]. The improvement of antitumor activity in vitro can also be obtained by blockade of transforming growth factor (TGF)-β signaling [35]. With durable responses being observed, TILs have been implemented in late-stage metastatic melanoma in clinical studies [33]. Clinical studies are currently carried on TILs combined with anti-CTL-associated protein (CTLA)-4 monoclonal antibodies (mAbs) (ipilimumab) (NCT01701674), peginterferon α-2b upregulating human leukocyte antigen (HLA) expression on tumor cells (NCT02379195), and nivolumab (anti-PD-1 mAbs) with or without IFN-α (NCT03638375) for metastatic melanoma treatment. Other clinical trials implementing TILs are focused on the treatment of nasopharyngeal carcinoma (NCT02421640), malignant pleural mesothelioma (NCT02414945), cervical carcinoma (NCT03108495), and squamous cell carcinoma of the head and neck (NCT03083873). Also, TILs along with pembrolizumab and other drugs are tested against digestive tract, urothelial, breast, ovarian/endometrial tumors (NCT01174121).
The expression of PD-1 on T cells is important in ATC. PD-1 binds tumor-associated PD-L1 and PD-L2, which interfere with T-cell activation, major histocompatibility complex (MHC), and TCR interaction, and induces T-cell apoptosis. Other immune checkpoint protein, CTLA-4 suppresses T-cell activation. Therefore, to induce the immune response, CTLA-4 or PD-1 interactions with ligands can be disrupted by mAbs in cancer immunotherapy [30].
In metastatic melanoma, injection of T-VEC results in local inflammation, the presence of type I IFNs, and granulocyte-macrophage-colony-stimulating factor (GM-CSF)-attracted DCs, leading to subsequent cell killing. Anti-PD-1 blocks the interaction between PD-1 on activated T cells and PDL-1 on tumor cells. Thus, T cells are reactivated to destroy the tumor [29]. Another target in antitumor therapy, CTLA-4, which can be expressed on tumor cells, infiltrating Tregs as well as on exhausted conventional T cells, is regarded as an immunosuppressive factor, but its role as a prognostic factor is unclear [36]. In advanced melanoma stage IIIc and IV treated with intratumoral injections of coxsackievirus A21 (CVA21), which infects ICAM-1 expressing cancer cells, antitumor response has been shown in clinical trials. Importantly, in preclinical studies on a mouse model, the use of anti-PD-1 or anti-CTLA-4 mAbs together with CVA improved anti-tumor response [37].
In tumor cells, the cellular response toward oncolytic DNA ( Figure 2) and RNA viruses ( Figure  3) is influenced by NF-κB. Since NF-κB is a key player in tumorigenesis, NF-κB activation level may indicate the effectiveness of oncolytic virotherapy.

AV
AVs, belonging to Adenoviridae family, are species-specific double-stranded (ds) DNA viruses, which infect humans and other vertebrates [38]. When used as OVs, AVs may induce unwanted dominant antiviral immune response [25,38,39]. Importantly, the stroma of target tumor blocks the diffusion of the virus, and neutralizing antibody production can be observed even after the intratumoral injection of the virus. Therefore, AV-based oncolytic virotherapies need improvement [25,38].

AV
AVs, belonging to Adenoviridae family, are species-specific double-stranded (ds) DNA viruses, which infect humans and other vertebrates [38]. When used as OVs, AVs may induce unwanted dominant antiviral immune response [25,38,39]. Importantly, the stroma of target tumor blocks the diffusion of the virus, and neutralizing antibody production can be observed even after the intratumoral injection of the virus. Therefore, AV-based oncolytic virotherapies need improvement [25,38].
Another AV, DNX-2401 (DNAtrix), with partial E1A gene deletion in retinoblastoma (Rb)-binding domain and integrin receptor arginine-glycine-aspartic acid (RGD)-4C insertion, which may act against glioma, was designed by FDA as an orphan drug. ONCOS-102 (Oncos therapeutics) with ∆24 deletion within Rb-binding E1A gene, insertion of GM-CSF-encoding gene and replacement of serotype 3 AV knob protein was also granted by FDA as an orphan drug against ovarian cancer, glioma, and malignant mesothelioma [42][43][44]. ONCOS-102 was tested in preclinical peritoneal mesothelioma model [45], and its efficiency in phase I clinical trials in the treatment of ovarian cancer has been demonstrated [46]. ONCOS-102 expressing GM-CSF is currently under phase I of clinical trials on malignant solid tumors treatment and is likely to prolong the survival of patients with ovarian carcinoma and malignant pleural mesothelioma [47].
Oncolytic AV type 5, dl922-947 mutant with deletions in E1A-conserved region 2 (CR2) and subsequent Rb-binding deficiency [50], is a candidate for anaplastic thyroid carcinoma treatment. In 8505-c and BHT101-5 human thyroid carcinoma anaplastic cell lines and TPC1 papillary cells, dl922-947 decreased C-X-C motif chemokine ligand 8 (CXCL8) promoter binding by p65. In addition, dl922-947 reduced CCL2 promoter binding by NF-κB in 8505-c and TPC1 cells by displacing p65 from the promoters. In vitro experiments have shown that the reduction of CXCL8 and CCL2 expression is linked to impaired tumor-induced angiogenesis and reduced chemotaxis. In vivo experiment on anaplastic thyroid carcinoma xenograft mouse model revealed that dl922-947 reduces CXCL8 expression and angiogenesis [51].
Conditionally replicating AV, AduPARE1A, harboring urokinase-type plasminogen activator receptor (UPAR) promoter, which controls E1A region, may act against pancreatic cancer [52]. In human primary pancreatic adenocarcinoma BxPC-3 and pancreas ductal adenocarcinoma PANC-1 cell lines, the synergistic antitumor effect of gemcitabine and AduPARE1A on NF-κB was demonstrated. The UPAR promoter is activated by NF-κB, which, in turn, is stimulated by gemcitabine. Therefore, NF-κB induces UPAR-controlled transgenes, and increase in E1A expression is observed. Competition between the adenoviral promoter and cellular promoters of NF-κB-regulated anti-apoptotic genes may lead to cell sensitization to gemcitabine-induced apoptosis [53].
Another important AV factor, E1B 19K, is a counterpart of mammalian B-cell lymphoma (Bcl)-2, which counteracts E1A-induced apoptosis and interferes with Bad and Bax proapoptotic proteins [54]. It has been shown that E1-deleted replication-defective AV harboring nitroreductase (NR)-encoding gene (RAd-NR) activates IκBα phosphorylation and NF-κB p65/p50 heterodimers in SKOV3 human ovarian carcinoma cell line. In addition, inhibition of NF-κB resulted in restored chemosensitivity of SKOV3 cells because of increased apoptosis. Also, RAd-NR induced NF-κB-dependent mRNA levels of c-IAP1 and c-IAP2, as well as proinflammatory IL-6 secretion. Moreover, conditionally replicating oncolytic E1B-attenuated dl1529 or CR-NR induced NF-κB activation and increased apoptotic threshold in HeLa cells. Importantly, inhibition of NF-κB in SKOV3 cells resulted in enhanced cytotoxic effect and increased apoptosis upon RAd-NR/CB1954 virus-directed enzyme prodrug therapy with the delivery of CB1954 prodrug-activating NR [55].
The E1A 243R and p53-binding E1B 55K AV proteins are known as inhibitors of inflammation upon virus infection, whereas E1B 19K counteracts inflammation in the presence of the two E1A 243R and E1B 55K [56]. Moreover, it has been demonstrated that cytopathic effect (CPE) resulting from wild-type (wt) AV infection is nonapoptotic. However, human lung adenocarcinoma A549 cells that underwent apoptosis upon infection with Ad5 mutant with E1B 19K-encoding gene deletion (H5dl337) were unable to inhibit NF-κB activation and proinflammatory cytokine responses in macrophages [57].
In order to enhance the antitumor effect, NF-κB activation can be induced via TLR9 by oncolytic Ad5D24-CpG, containing cytosine:guanine (CpG) islands within the genome. Importantly, stimulation of TLR9 induces DCs to type I IFN-mediated activation and proinflammatory IL-12 secretion. These events, in turn, promote NK cell cytotoxicity and IFN-γ secretion to induce antitumor responses. Such observations have been made in A549 lung cancer xenografts model. Also, a syngeneic model of melanoma showed an enhanced tumor response, including a profound drop in both total number and activation of MDSCs [58]. The efficacy of Ad5D24-CpG was also tested in mouse model of melanoma over-expressing chicken ovalbumin (OVA) [59]. Ad5D24-CpG delivering antiproliferative L-carnosine reduced lung and colon tumor growth in mouse xenograft model [60]. Also, in mouse xenograft model of human lung cancer, tumor growth was reduced by using Ad5D24-CpG in combination with paclitaxel [61].

HSV
HSV-1, belonging to family Herpesviridae of neurotropic dsDNA viruses, is a causative agent of a self-limiting disease. Due to its cytolytic replication cycle and easily modified large genome, HSV-1 is beneficial as an oncolytic agent [62,63]. Moreover, HSV-1 infects different cell types, and the presence of a separate attachment and fusion glycoproteins within its envelope is beneficial for modification to improve tumor targeting [63].
However, the enhancement of HSV-1 oncolytic activity can be obtained by combining HSV-1 with trichostatin A (TSA) [66]. TSA is an antitumor agent belonging to a histone deacetylase (HDAC) inhibitors, which change chromatin structure and act against skin cancer cells [67] and induce apoptosis [68]. For γ 1 34.5 gene-deficient HSV-1 mutant R849 with lacZ encoding bacterial β-galactosidase substitution [69], it has been demonstrated that TSA, which enhances p65 acetylation and nuclear accumulation, promotes viral replication in oral squamous cell carcinoma (SCC) SAS cells. This effect results from TSA-driven enhancement of DNA binding by NF-κB during HSV-1 infection. As a consequence, the oncolytic activity of HSV-1 toward SCC can be improved [66].
The improvement of oncolytic HSV activity is also needed for malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive and may be associated with neurofibromatosis type 1 (NF1). Therefore, preclinical models are being developed [70]. Some therapeutic approaches may utilize NF-κB inhibitors in MPNSTs, where NF-κB is constitutively active and cells are resistant to oncolytic HSV. The possible role of NF-κB signaling in IFN-stimulated genes (ISGs) expression has led to the conclusion that using NF-κB inhibitors, such as TPCA-1, which decrease ISGs expression, may improve productive infection of oncolytic HSV ∆γ 1 34.5 recombinants, which are devoid of ICP34.5 neurovirulence factor and whose replication is limited by protein kinase R (PKR) [71].
Another antitumor approach is based on UV-inactivated HSV-1 (UV-HSV-1), which is proposed for use as an adjuvant in donor mononuclear or NK cell infusions in acute myeloid leukemia therapy. UV-HSV-1 may induce cytolytic activity of human PBMCs and NK cells via TLR2/protein kinase C (PKC)/NF-κB, resulting in p65 nuclear translocation. This treatment results in leukemic cell killing [72].

PV
H-1 protoparvovirus (H-1PV), a member of Parvoviridae family, is a single-stranded (ss) DNA rodent pathogen and a promising OV, which can be administered by different routes. The main advantage of H-1PV is that it can cross the blood-brain barrier. H-1PV, whose replication is S-phase-dependent, evokes proinflammatory responses and is a hope for therapies of central nervous system tumors, including glioblastoma [73]. H-1PV armed with IL-2 can be used against lymphoma, glioma, colon, gastric cancer, neuroectodermal, pancreatic cancer, and others [25,73].
H-1PV's role in oncolytic therapy could rely on immune priming and influencing maturation of DCs to exert antitumor immunity [74]. Human interdigitating DCs (iDCs), incubated with H-1PV-induced tumor cell lysates, exhibited increased expression of TLR3, TLR9, and NF-κB and produced higher amounts of TNF-α compared to uninfected human melanoma (SK29Mel) cells in ex vivo model [75].
In oncolytic therapy of pancreatic cancer, strategies of gene transfer involve introduction of a suicide therapeutic gene, which is missing or underexpressed in tumor cells. This approach allows obtaining a bystander effect of antitumor response toward neighboring cells [76]. Recombinant parvovirus rPVH1-yCD expressing the suicide gene yCD (yeast cytosine deaminase), which converts the prodrug 5-fluorocytosine (5-FC) into 5-FU, was tested as a novel strategy in gene-directed enzyme prodrug therapy against pancreatic cancer. In Panc1 and AsPc1 chemoresistant pancreatic cancer cells, wt H-1PV diminished the constitutive NF-κB activity. Moreover, NF-κB DNA binding and transcriptional activity were significantly reduced upon rPVH1-yCD/5-FC treatment. Importantly, the antitumor activity of wt H-1PV and rPVH1-yCD/5-FC is likely to be associated with attenuation of both NF-κB and Akt/phosphatidylinositol 3-kinase (PI3K) activity. Therefore, it is assumed that the oncolytic activity of H-1PV is linked to NF-κB. These observations suggest that inhibitors of these signaling pathways may increase the effectiveness of pancreatic cancer therapy [77].

Encephalomyocarditis Virus (EMCV)
Encephalomyocarditis virus (EMCV), belonging to Picornaviridae family, is a mammalian non-enveloped, positive sense-(+)ssRNA virus, which rarely infects humans [78,79]. In preclinical studies, EMCV oncolytic properties were implemented in the treatment of patients with CCRCC, which is well known for its high resistance to chemotherapy and radiation [79].
Importantly, both CCRCC and 786-O cells are characterized by von Hippel-Lindau (VHL), a tumor suppressor protein, inactivation. The loss of VHL results in hypoxia-inducible factor (HIF)-2α stabilization at tumor enhancers. Thus, CCRCC genes linked to tumorigenesis are upregulated [81]. VHL-null 786-O cells infected with EMCV did not show IFN-mediated antiviral response, but enhanced survival signaling under control of NF-κB and elevated viral replication. Since VHL is a negative regulator of HIF, observed events can be a result of HIF-mediated promotion of NF-κB activation of the survival pathway. Thus, infected cells can be sensitized to virally induced cytotoxicity [79]. Importantly, the cross-talk between NF-κB signaling and IKK-regulated type I IFN gene expression is an obvious fact that implements the use of IKK inhibitors in oncolytic virotherapy. IKK-inhibitors, BMS-345541 [82] and TPCA-1 [83], counteract cell proliferation, p65 nuclear translocation, and NF-κB-regulated CXCL8 gene expression in glioma cells. Both inhibitors, when combined with OVs, such as EMCV, block IFN-regulated antiviral responses since the reversion of IFN-induced anticythopathic effect and antiviral effect by IKK inhibitors in glioma cells can be observed. Particularly, BMS-345541 inhibits the action of IFN against EMCV, which exert a CPE in U87 cells. Importantly, the correlation between this effect and suppression of NF-κB by BMS-345541 was observed [84].
This effect may result from the activation of exchange protein directly activated by cAMP isoform 1 (Epac1), which may either stabilize IκB or upregulate cellular oncogene Fos (c-Fos). c-Fos, in turn, interacts with NF-κB p65 and impairs its transcriptional activity [90]. Therefore, M1-induced ISGs expression can be abolished. Together, NF-κB has been shown to be involved in preventing type I IFN response by H89 [91].

Newcastle Disease Virus (NDV)
The Malaysian field outbreak isolate, NDV strain AF2240, is an avian pathogen and a member of ss(-)RNA Paramyxoviridae family. AF2240 is regarded as a good and efficient candidate in oncolytic virotherapy due to its high immunogenicity and selectivity toward tumors [92]. In tumor cells, NDV triggers innate and adaptive immune responses and exerts cytotoxic effect via caspase activation resulting in apoptosis [85]. What is important, ER stress upon NDV treatment induces antitumor immunity [64].
AF2240 triggers p38 mitogen-activated protein kinase phosphorylation upstream of IκBα processing and NF-κB nuclear translocation in 786-O cells. NF-κB, in turn, regulates IFN-β production in the early phase of NDV infection. In 786-O cells harboring VHL gene, the activity of NF-κB, IFN-β secretion, and killing effect was elevated upon NDV infection in comparison with 786-O cells. Interestingly, in NDV-infected 786-O cells treated with JSH-23, a canonical NF-κB signaling inhibitor [80], NF-κB activation was not completely abolished. Therefore, a cross-talk is assumed between canonical and non-canonical NF-κB signaling upon oncolytic respiratory syncytial virus (RSV) infection suggesting other regulatory mechanisms [93].

RV
RV is a human pathogen and enterotropic dsRNA virus belonging to Reoviridae family. RV is regarded as a good candidate for oncolytic treatment as an easily manufactured therapeutic agent, which has a stable genome and displays minimal toxicity for humans [94]. The ability of RV to use certain signaling pathways in transformed cells targeting constitutive Ras signaling makes it a good candidate as a drug against prostate cancer, glioma, MM, and pancreatic adenocarcinoma [25]. Indeed, an RV-based oncolytic Reolysin (Oncolytics Biotech Inc., Calgary, AB, Canada) has been approved as an orphan drug by FDA in ovarian, fallopian tube cancers, glioma, gastric, and pancreatic cancer [44].
In HeLa cells, RV activates p65/p50 complexes [95]. Further studies have shown that at late times, post infection RV inhibits NF-κB activation. This type of dynamics of NF-κB regulation in RV-infected cells is required for TNF-α-and TRAIL-induced apoptosis [96].
RV activates NF-κB p65 nuclear translocation not only in p53 +/+ HCT116 cells, but also in cells lacking p53. Since NF-κB inhibition has a profound effect on reduction of cell death following RV infection, the enhancement of NF-κB activation to increase cell death was evaluated by combining RV and actinomycin-D (ActD) or etoposide (Etp) treatment of p53 +/+ cells. Increased oncolysis observed may also be a consequence of upregulation of p21-encoding cyclin-dependent kinase inhibitor 1 (CDKN1A) gene [105], whose regulation depends on NF-κB [106].
Although RV-induced oncolysis has been implemented in clinical trials, the mechanisms of this process are still not fully elucidated. In breast cancer treatment studies, NF-κB has been investigated as a factor, which either promotes or suppresses apoptosis of tumor cells [107]. RV infection of MCF7 and hypotriploid HTB133 cells upregulated p65 NF-κB expression, as well as its nuclear translocation and DNA binding. NF-κB inhibition, in turn, resulted in oncolytic protection and downregulation of p53-upregulated modulator of apoptosis (PUMA) expression. This study suggests that in breast cancer therapy, NF-κB acts proapoptotically, mediates RV-induced oncolysis, and may serve as a predictive indicator of the sensitivity to RV treatment [108].

RSV
RSV, a member of Paramyxoviridae family, is an ss(-)RNA virus, which is regarded as an oncolytic agent. However, the data on the effect of RSV on apoptosis in individual cell types are, in fact, conflicting. Nevertheless, the oncolytic potential of RSV toward certain tumors, including skin neoplasm, has been demonstrated [109].
As shown in human prostate tumor xenografts, RSV leads to oncolysis of prostatic cancer cells in vitro and in vivo. In PC-3 cells, RSV-induced apoptosis via an intrinsic pathway that involves caspase-3. NF-κB downregulation, observed upon RSV infection, results in the upregulation of pro-apoptotic genes and downregulation of anti-apoptotic genes. Consequently, apoptosis and oncolysis can be observed [110].
Further studies demonstrated that RSV-induced modulation of innate antiviral response is linked to androgen dependence and/or androgen receptor expression on target cells. PC-3 cells, which are androgen-independent, are susceptible to RSV-induced apoptosis despite functional IFN-mediated antiviral signaling, but a defective NF-κB response. Importantly, defective NF-κB activation could not be observed in androgen-sensitive LNCaP and RWPE-1 prostate cancer cells [111].

VSV
VSV is a non-segmented ss(-)RNA virus, belonging to Rhabdoviridae family, infecting insects and livestock animals. Preexisting immunity against VSV in humans, as well as cell-cycle-independent cytoplasmic replication cycle without risk of host cell transformation in various cell lines, renders VSV a promising tool for oncolytic virotherapy [112]. Importantly, high sensitivity of VSV to IFN response favors it as a candidate for the treatment of tumors with aberrant IFN signaling. Nevertheless, VSV may also target tumors with abnormal function of Ras, p53, and myelocytomatosis (Myc) proteins [85].
The importance of IKK/NF-κB signaling in type I IFN-mediated antiviral response has been demonstrated in glioma cell lines. Both TPCA-1, an IKKβ inhibitor, which prevents IκBα phosphorylation [83], and a selective IKKα/IKKβ inhibitor, BMS-345541 [82], have been proposed in combination with VSV for anticancer therapies. In glioma cells, TPCA-1 and BMS-345541 inhibited NF-κB activation and CXCL8 gene expression, as well as IFN-activated gene expression. BMS-345541 has been demonstrated to counteract antiviral IFN effect against VSV in glioma cells since VSV is considered as a promising tool in oncolytic therapies of malignant brain tumors. Importantly, this effect correlated with decreased NF-κB activity [84].
The oncolytic potential of VSV can also be modulated by vorinostat [113], a histone deacetylase inhibitor [114], which modulates NF-κB-dependent genes [115]. Vorinostat increases p65 NF-κB nuclear accumulation, DNA binding, and acetylation, while inhibiting IFN signaling in PC-3 cells. Subsequent NF-κB activation also induces certain genes linked to autophagy. Together, vorinostat enhances VSV replication in PC-3 cells and oncolysis. The effect of vorinostat on NF-κB signaling, autophagy, and oncolysis in VSV-infected cells was also demonstrated in DU145 prostate cell line and HCT116 cells [113]. Other studies have shown the synergy of curcumin, an anti-inflammatory agent and NF-κB inhibitor [116] and VSV infection in enhancing oncolysis of PC-3 cells. When treated with curcumin and then infected with recombinant M protein mutant VSV, rM51R-M, the cells became sensitized to VSV-induced apoptosis and showed the reduction on phospho (p)-NF-κB expression. At the same time, the decrease in NF-κB-dependent anti-apoptotic Bcl-extra-large (xL) protein content was observed [117].
NF-κB signaling is involved in MM development [2]. NF-κB activity is also induced by VSV infection of myeloma cells and may promote apoptosis and virus spread. On the contrary, NF-κB plays a prosurvival role during VSV infection to ensure viral replication. Inhibition of NF-κB by BMS-345541 led to attenuation of VSV replication in U266 and 5TGM1 MM cell lines. Bortezomib, a proteasomal inhibitor [118], which inhibits IκBα degradation and p65 nuclear accumulation [119], counteracted virally induced p65 nuclear protein expression in U266 nuclear extracts. Therefore, antagonistically acting bortezomib may not be profitable for the virus spread. Despite no effect on the increase of VSV titer upon NF-κB in vitro, in vivo studies in mouse MM model have shown that bortezomib is not able to inhibit intratumoral replication of VSV. Moreover, bortezomib enhances the therapeutic effect of oncolytic VSV [120] armed with murine IFN-β and NIS VSV-IFNβ-NIS [121].

Conclusions
Oncolytic virotherapy is a new promising therapeutic approach, which needs improvement to enhance the antitumor effect. Therefore, combined therapies using immunomodulating drugs, including NF-κB regulators, are considered in the treatments. The importance of the complexity of NF-κB activation in tumor cells and the interplay between OVs and NF-κB signaling is shown in this review. Since NF-κB is a key player in tumorigenesis, which shapes the cellular response and plays a role in balancing the immune system, NF-κB activation level should be considered in oncolytic treatment. Thus, NF-κB activation level can indicate the efficacy and effectiveness of novel therapeutic approaches and help obtain the desired therapeutic effect.

Conflicts of Interest:
The authors declare no conflict of interest.