IFNγ-Induced Bcl3, PD-L1 and IL-8 Signaling in Ovarian Cancer: Mechanisms and Clinical Significance

Simple Summary IFNγ is a multifunctional cytokine produced not only by activated lymphocytes but also in response to cancer immunotherapies; it has both antitumor and tumor-promoting effects. In ovarian cancer cells, the tumor-promoting effects of IFNγ are mediated by increased expression of the proto-oncogene Bcl3, the immune checkpoint PD-L1 and the proinflammatory cytokine IL-8. Recent studies have shown that IFNγ induces the expression of Bcl3, which then promotes the expression of PD-L1 and IL-8 in ovarian cancer cells, resulting in their increased proliferation and migration. In this review, we summarize the recent findings on the IFNγ tumor-promoting functions and on the mechanisms by which IFNγ induces Bcl3, PD-L1 and IL-8 expression in cancer cells, with a special focus on ovarian cancer. We also highlight the importance of a better understanding of these mechanisms to optimize the development of combinatorial approaches in cancer immunotherapies. Abstract IFNγ, a pleiotropic cytokine produced not only by activated lymphocytes but also in response to cancer immunotherapies, has both antitumor and tumor-promoting functions. In ovarian cancer (OC) cells, the tumor-promoting functions of IFNγ are mediated by IFNγ-induced expression of Bcl3, PD-L1 and IL-8/CXCL8, which have long been known to have critical cellular functions as a proto-oncogene, an immune checkpoint ligand and a chemoattractant, respectively. However, overwhelming evidence has demonstrated that these three genes have tumor-promoting roles far beyond their originally identified functions. These tumor-promoting mechanisms include increased cancer cell proliferation, invasion, angiogenesis, metastasis, resistance to chemotherapy and immune escape. Recent studies have shown that IFNγ-induced Bcl3, PD-L1 and IL-8 expression is regulated by the same JAK1/STAT1 signaling pathway: IFNγ induces the expression of Bcl3, which then promotes the expression of PD-L1 and IL-8 in OC cells, resulting in their increased proliferation and migration. In this review, we summarize the recent findings on how IFNγ affects the tumor microenvironment and promotes tumor progression, with a special focus on ovarian cancer and on Bcl3, PD-L1 and IL-8/CXCL8 signaling. We also discuss promising novel combinatorial strategies in clinical trials targeting Bcl3, PD-L1 and IL-8 to increase the effectiveness of cancer immunotherapies.


Introduction
Ovarian cancer (OC) is the most aggressive gynecologic cancer, with poor survival and high mortality rates.One of the key features of OC is its heterogeneity, partly explaining, together with the lack of specific symptoms and biomarkers for early detection, the lack of successful treatments and poor outcomes [1][2][3][4][5][6][7][8].The current treatment strategy includes tumor reduction surgery followed by platinum and taxane-based chemotherapy.However, since most patients relapse and develop chemoresistance, new therapeutic strategies are urgently needed.Immunotherapies have shown great promise in treating a variety of cancers, but in OC, the results have been disappointing.Further studies are needed to better understand the nature of immune signaling in OC and develop more effective therapeutic approaches to increase the effectiveness of cancer immunotherapies.
In this review, we summarize the current knowledge on the mechanisms by which IFNγ induces the expression of Bcl3, PD-L1 and IL-8/CXCL8 in cancer cells, with a special focus on ovarian cancer, and highlight the importance of a better understanding of these mechanisms to optimize the development of combinatorial approaches in cancer immunotherapies.

IFNγ Signaling in Ovarian Cancer
The biological effects of IFNγ are mediated by its binding to cell surface receptors IFNGR1 and IFNGR2, resulting in oligomerization of the receptor and phosphorylation and activation of the tyrosine Janus protein kinases JAK1 and JAK2 [9,52,53].Activated JAK1/JAK2 then phosphorylate the transcription factor STAT1 at Tyr-701, resulting in its dimerization and nuclear translocation [52,53].In addition to STAT1 phosphorylation at Tyr-701 by JAK1/2, STAT1 can be phosphorylated at Ser-727, which is required for its maximum transcriptional activity; however, the regulation of STAT1 phosphorylation on Ser-727 is cell specific [9,52].In some cells, IFNγ signaling may also activate IκB kinase (IKK) to promote the expression of NFκB-dependent genes [54][55][56].Furthermore, several studies have shown that IFNγ induces promoter histone acetylation by recruiting the histone acetyltransferases (HATs) p300 and CBP, resulting in increased promoter accessibility and transcription of interferon stimulated genes (ISGs) [57][58][59][60].
While early studies suggested that increased IFNγ levels correlate with improved clinical outcomes in patients with ovarian cancer [62], more recent studies have shown that high IFNγ expression in OC tissues promotes cancer progression and correlates with poor survival [36,46,63].This discrepancy might reflect the high heterogeneity of OC tissues, different analytical methods used and the low stability of endogenous IFNγ in human serum and tissues.[47,49,50].JAK1 phosphorylates STAT1 at Tyr-701, which is required for the nuclear translocation of STAT1.STAT1 is then phosphorylated at Ser-727, which promotes its recruitment to the Bcl3, PD-L1 and IL-8 promoters.In addition, IFNγ induces p300-mediated acetylation of p65 NFκB at K314/315, resulting in increased p65 transcriptional activity and recruitment to PD-L1 and IL-8 promoters [47,49].IFNγ also induces acetylation of histones (Ac) at the Bcl3, PD-L1 and IL-8 promoters in OC cells, thus facilitating transcription factor recruitment and transcription [47][48][49][50][51]. Created with BioRender.comwith granted permission and license.

Bcl3 Expression and Function in OC
Bcl3 expression is regulated by microRNA miR-125b, which is downregulated in OC tissues [94,95].Overexpression of miR-125b decreases the Bcl3 levels in OC cells, resulting in decreased cell proliferation and cell cycle arrest and decreased tumorigenesis when injected into nude mice [94].Bcl3 gene expression is increased in ovarian clear-cell adenocarcinoma, ovarian endometrioid adenocarcinoma, ovarian mucinous adenocarcinoma, ovarian serous adenocarcinoma, and ovarian serous surface papillary carcinoma [47].Interestingly, compared to other OC types, ovarian serous surface papillary carcinoma has the highest levels of Bcl3 [47]; since tumors are often highly heterogeneous, it is plausible that a subset of OC cells might express considerably higher Bcl3 levels.
In vitro studies demonstrated that Bcl3 suppression in OC cells induces their apoptosis and inhibits cell migration [47].In addition, Dai et al. [96] showed that Bcl3 promotes survival, migration and invasion of OC cells by inducing the expression of the coppercarrying plasma protein ceruloplasmin.Intriguingly, we found that Bcl3 expression in OC cells is further enhanced by IFNγ, thus identifying Bcl3 as a new member of the ISGs [47][48][49][50].IFNγ-induced Bcl3 facilitates the expression of IL-8 and PD-L1, linking the function of Bcl3 in OC to angiogenesis and immune escape [47][48][49][50][51]. Thus, the newly emerging functions of Bcl3 in ovarian cancer include the induction of OC cell proliferation, survival, migration and invasion and also angiogenesis and immune escape (Figure 3).

Mechanisms Regulating IFNγ-Induced Bcl3 Expression in OC
IFNγ-induced Bcl3 expression is facilitated by increased acetylation of the Bcl3 promoter and is dependent on both p65 NFκB and JAK1/STAT1 signaling [47,50] (Figure 2).IFNγ stimulation of OC cells increases acetylation of histones at the Bcl3 promoter, with a simultaneous promoter recruitment of Ser-727 pSTAT1, which is required for maximum transcriptional activity of STAT1 [50].Although IFNγ-induced Bcl3 expression in OC cells is also dependent on p65 NFκB, p65 NFκB is not directly recruited to the Bcl3 promoter in OC cells [50].However, p65 NFκB can mediate IFNγ-induced Bcl3 expression through binding to a downstream enhancer or through a cooperative interaction with STAT1.The latter scenario seems to be supported by several previous studies that reported crosstalk between NFκB and STAT1 in the regulation of IFNγ-induced inflammatory genes [9,[97][98][99].
Future studies are needed to analyze in detail the mechanisms by which IFNγ induces the Bcl3 expression.

PD-L1 Expression and Function in OC
Immunotherapies blocking the PD-1/PD-L1 interaction have demonstrated great promise in treating a variety of cancers, but most patients fail to respond or develop resistance [41,104,105,112].One of the factors determining the effectiveness of PD-1/PD-L1-blocking therapies is the surface expression of PD-L1.However, PD-L1 expression in tumors is highly heterogeneous, and varies among specific tumor types, cases, tissues and sampling times [36,104,112].One plausible explanation for the high variability in PD-L1 in cancer tissues may be its dependence on the expression of IFNγ, which rapidly upregulates PD-L1 on most tumor cells [36,104,107,108,112].In addition, the most widely used PD-L1 assay, immunohistochemistry, often measures mainly the surface levels of PD-L1 but not its intracellular levels; thus, additional mechanisms regulated by intracellular PD-L1 might be responsible for the limited responsiveness to PD-L1 targeted immunotherapies and the development of resistance.
Multiple causes are likely responsible for the limited effectiveness of PD-L1 as a clinical biomarker and therapeutic target in PD-L1 blocking therapies in OC; they may include the highly heterogeneous nature of OC tumors, the high inducibility and dependence of PD-L1 expression on IFNγ signaling and the incompletely understood functions of intracellular PD-L1, which is inaccessible to the currently used PD-1/PD-L1-blocking therapies.A better understanding of the mechanisms that regulate PD-L1 expression in OC cells and its intracellular functions should lead to the development of more effective PD-L1-targeting strategies for OC patients.

Mechanisms Regulating IFNγ-Induced PD-L1 Expression in OC
Although IFNγ is the main inducer of PD-L1 in most cells, the specific mechanisms and signaling pathways involved appear to be cell specific.The human PD-L1/CD274 promoter contains binding sites for the transcription factors NFκB, STAT1, IRF1 and hypoxiainducible transcription factor (HIF) [47,140,141].Consistent with the promoter multiple p65 NFκB binding sites, PD-L1 expression in OC cells treated with the chemotherapeutic drugs gemcitabine and paclitaxel is dependent on p65 NFκB signaling [110].IFNγ induces the expression of p65 NFκB, its Bc3-facilitated acetylation and recruitment to PD-L1 promoter in OC cells [47,49] (Figure 2).
In addition, IFNγ-induced PD-L1 expression in OC cells is regulated by the canonical pathway, which involves the JAK1/STAT1 signaling [49].IFNγ induces the expression of the transcription factor IRF1, which is then recruited to PD-L1 promoter and is required for maximum PD-L1 expression in OC cells [49].Thus, IFNγ-induced PD-L1 expression in OC cells is dependent on simultaneous IFNγ-induced and p300-dependent PD-L1 promoter acetylation and promoter recruitment of the transcription factors IRF1, Ser-727phosphorylated STAT1 and K314/315-acetylated p65 NFκB [44] (Figure 2).

Mechanisms Regulating IFNγ-Induced IL-8 Expression in OC
Since mice do not have a homolog of the IL-8/CXCL8 gene, which is present in other species, including humans [192], compared to other cytokines, our understanding of the mechanisms that regulate IL-8 expression in cancer cells has been lagging.The IL-8 transcription is regulated predominantly by the transcription factor NFκB; in addition, in some cells, the transcription factors AP-1 and C/EBP are required for maximum IL-8 expression [193][194][195][196][197][198][199].The human IL-8 promoter also contains a binding site for the transcription factor HIF-1α, which is adjacent to the NFκB site [199], consistent with hypoxia-induced IL-8 expression in OC cells [157].
In OC cells, IL-8 expression is regulated predominantly by p65 NFκB homodimers phosphorylated at serine 536 by IKK [173][174][175].In addition, the IL-8 expression in OC cells is facilitated by increased acetylation of the IL-8 promoter and recruitment of IKK [175,176].Inhibition of IKK suppresses IL-8 expression in vitro and increases the effectiveness of proteasome and HDAC inhibitors in vivo [175,176].In this context, several studies have correlated the use of nonsteroidal anti-inflammatory drugs targeting the IKK enzymes with a reduced risk of ovarian cancer [200,201].
In IFNγ-stimulated OC cells, IL-8 expression is dependent on both JAK1/STAT1 and p65/Bcl3 signaling and is facilitated by concomitant promoter acetylation by p300 and recruitment of Ser-727 pSTAT1 and K314/315 ac-p65 NFκB (Figure 2).Neutralization of IFNγ-induced IL-8 using an anti-IL-8 blocking antibody reduces IFNγ-induced migration of OC cells and their invasion ability in 3D spheroids [51], indicating that IFNγ-induced IL-8 expression contributes to the pro-tumorigenic effects of IFNγ in ovarian cancer cells.

Genomic Studies in OC
Considering that the expression of Bcl3, PD-L1 and IL-8 in ovarian cancer cells is induced by IFNγ and dependent on JAK1/STAT1 signaling [47][48][49][50][51], we examined the gene co-expression profiles of Bcl3, PD-L1/CD274 and IL-8/CXCL8 in OC tissues using The Cancer Genome Atlas (TCGA) database and the UCSC Xena Browser (https://xena.ucsc.edu(accessed on 1 April 2024) [202].Heatmap analysis of 379 OC samples from the GDC-TCGA database revealed a positive correlation between Bcl3, PD-L1/CD274 and IL-8/CXCL8 mRNA expression (Figure 5A).In addition, Pearson's and Spearman's correlation tests revealed positive correlations between Bcl3 and PD-L1/CD274, between Bcl3 and IL-8/CXCL8 and between IL-8/CXCL8 and PD-L1/CD274 expression in the GDC-TCGA database (Figure 5B).The positive correlation between IL-8 and PD-L1 in OC was also demonstrated by Wang et al., who showed that co-culture of ascites-derived ovarian cancer cells with CD8+ T cells resulted in increased gene and protein levels of IL-8 and PD-L1 in OC cells [203], and by Wu et al., who demonstrated IL-8 and PD-L1 co-expression in ovarian cancer organoids [204].We have also examined the TCGA datasets using the cBioPortal for Cancer Genomics (https://www.cbioportal.org(accessed on 14 July 2024) [205] to evaluate whether there is any difference in overall survival (OS) in OC patients with altered expression of Bcl3, PD-L1 and IL-8.Analysis of 1949 samples revealed a statistical difference (p < 0.05) in OS in OC patients with altered Bcl3 expression but not PD-L1 or IL-8 expression; this is consistent with a model in which OS is driven mainly by the expression of Bcl3.However, more studies are needed to confirm these genome-wide studies also on the protein level.
Furthermore, recent studies in other cancers have shown that increased systemic and tumor-associated IL-8 levels correlate with reduced clinical benefits of ICB therapies used in advanced melanoma, NSCLC, urothelial carcinoma, renal cell carcinoma, and glioma [210][211][212][213]. Since ICB therapies also enhance IFNγ expression [15][16][17]106], one of the underlying mechanisms may involve the IFNγ-induced IL-8 expression, resulting in increased cancer cell growth and resistance to ICB.These data suggest that targeting IL-8 signaling may increase the effectiveness of PD-L1-blocking therapies in ovarian cancer.
To this end, future studies should determine whether immunotherapies targeting the PD-1/PD-L1 axis in OC patients increase their serum levels of IL-8.

Targeting Bcl3 in OC
Bcl3 expression in OC cells promotes the expression of PD-L1 and IL-8, resulting in increased OC cell proliferation, migration, invasion and immune escape (Figure 6).Since Bcl3 gene expression is significantly increased in ovarian clear-cell adenocarcinoma, endometrioid adenocarcinoma, ovarian mucinous adenocarcinoma, ovarian serous adenocarcinoma and ovarian serous surface papillary carcinoma [47], Bcl3 might serve as a potential novel biomarker in OC.In this context, Bcl3 protein expression analyzed by immunohistochemistry is currently being tested as a potential biomarker to predict the response to alkylating chemotherapy in glioma patients (NCT03011671) [76,214].Future studies should determine whether Bcl3 protein levels are increased in OC tissues and whether they correlate with PD-L1 and IL-8 expression.
In contrast to NFκB knockouts, Bcl3 knockout mice are viable, indicating that suppression of Bcl3 may represent a possible novel anticancer strategy [73,215].Indeed, a recent study demonstrated that Bcl3-/-mice exhibit increased sensitivity to cisplatin chemotherapy in colorectal cancer, thus offering a rationale for targeting Bcl3 as an adjuvant to conventional therapies [216].In this regard, Soukupova et al. recently developed small-molecule Bcl3 inhibitors with promising antimetastatic activity and low toxicity in triple-negative breast cancer [81].Future studies are warranted to determine whether Bcl3 may serve as a potential biomarker in ovarian cancer and whether Bcl3 inhibition might be used as an antimetastatic strategy in ovarian cancer and other solid tumors.

Targeting PD-L1 in OC
In contrast to other solid tumors, such as NSCLC or melanoma, in ovarian cancer, surface PD-L1 expression has not been proven as a reliable biomarker to select patients for anti-PD-1/PD-L1 therapy and targeting the PD-1/PD-L1 signaling has produced disappointing results [106].The underlying mechanisms likely include the highly heterogeneous nature of OC tumors, the high dependence of PD-L1 expression on IFNγ signaling, and the incompletely understood functions of intracellular PD-L1, which is inaccessible to currently used PD-1/PD-L1-blocking therapies.Since stability and intracellular localization and functions of PD-L1 are regulated by PD-L1 post-translational modifications that include acetylation and glycosylation, targeting these post-translational modifications may improve the effectiveness of PD-L1-targeting immunotherapies [123,217,218].
HuMax-IL8 (BMS-986253) is a fully human monoclonal antibody that neutralizes IL-8 [230].A phase I clinical study evaluated the safety and tolerability of HuMax-IL8, as well as changes in IL-8 serum levels in patients with incurable metastatic or unresectable solid tumors [230].Although no objective tumor responses were observed, the antibody was safe and well-tolerated and associated with decreased serum IL-8 levels [230].The acceptable safety profile of HuMax-IL8 (BMS-986253) suggested its potential in combination immunotherapies [230].
There have been no clinical trials evaluating the efficacy of combined inhibition of IL-8 and PD-1/PD-L1 signaling in ovarian cancer.However, considering that IL-8 expression is increased in OC tissues and that the neutralizing IL-8 antibody HuMax-IL8 (BMS-986253) and the CXCR1/2 inhibitor SX-682 are well tolerated, future clinical studies should assess whether the combination of IL-8 and PD-1/PD-L1 blockade might increase the effectiveness of PD-1/PD-L1-targeting immunotherapies in ovarian cancer patients.

Conclusions and Perspectives
Bcl3, PD-L1 and IL-8 have long been known to have important cellular functions as a proto-oncogene, an immune checkpoint ligand and a neutrophil chemoattractant, respectively.However, overwhelming evidence shows that these three genes have tumorpromoting functions far beyond their originally identified functions in many types of cancer, including ovarian cancer.These tumor-promoting mechanisms include increased cancer cell proliferation, migration, invasion, angiogenesis, EMT, metastasis, cancer stemness, resistance to chemotherapy and immune escape.The expression of Bcl3, PD-L1 and IL-8 is induced by IFNγ, which is produced not only by activated T cells but also in response to PD-1/PD-L1-blocking cancer immunotherapies.Perhaps not surprisingly, recent studies have shown that the IFNγ-induced Bcl3, PD-L1 and IL-8 expression is regulated by the same JAK1/STAT1 signaling pathway: IFNγ first induces the expression of Bcl3, which then promotes the expression of PD-L1 and IL-8 in ovarian cancer cells (Figure 6).
Since increased systemic and tumor-associated IL-8 levels correlate with reduced clinical benefit of PD-1/PD-L1-blocking therapies in solid cancers, including melanoma, NSCLC and glioma [210][211][212][213], serum IL-8 levels have been used as a biomarker for responsiveness to PD-1/PD-L1-targeting immunotherapies in those cancers.Based on recent in vitro studies in OC cells, future clinical studies are warranted to determine whether serum IL-8 levels may serve as a prognostic biomarker for PD-1/PD-L1-targeting therapies in ovarian cancer patients.In addition, considering the tumor-promoting functions of IL-8 and the low toxicity of the IL-8-neutralizing human antibody HuMax-IL8 (BMS-986253), blocking IL-8 signaling with IL-8-neutralizing antibody may increase the effectiveness of PD-1/PD-L1 immunotherapies in ovarian cancer patients.
As the expression of Bcl3, PD-L1 and IL-8 is induced by IFNγ, the expression of which greatly varies in cancer tissues, it will be important to analyze the expression levels of Bcl3, PD-L1, IL-8 and IFNγ in tumor tissues at the single-cell level.Future investigations should also determine whether immunotherapies targeting PD-1/PD-L1 signaling and other therapies associated with induced IFNγ release increase IL-8 serum levels and Bcl3 expression in tumor tissues.
Further studies are needed to better understand the nature of immune signaling in the ovarian cancer TME.There are many outstanding questions to be addressed: What are the specific cells, in addition to TILs, responsible for IFNγ production, and what are the cellular and molecular targets of IFNγ in OC TME?What are the specific mechanisms that regulate the stability and the transcriptional activity of Bcl3 and its ability to promote PD-L1 and IL-8 expression in OC?What are the specific functions of nuclear and cytoplasmic PD-L1 and what are the mechanisms that regulate the intracellular localization of PD-L1 and its stability?A more thorough understanding of these mechanisms will lead to the development of more reliable biomarkers and PD-1/PD-L1-targeting cancer immunotherapies.In addition, future clinical trials are warranted to assess whether the combination of IL-8 and PD-1/PD-L1 blockade might increase the effectiveness of PD-1/PD-L1-targeting immunotherapies in ovarian cancer patients.
Author Contributions: Conceptualization: I.V.; Acquisition of data: S.U.R., F.Z.S., A.V. and I.V.; Funding acquisition: I.V.; Writing, review and editing: S.U.R., F.Z.S., A.V. and I.V.All authors have read and agreed to the published version of the manuscript.

Funding:
The work in I. Vancurova's laboratory is funded by the NIH grant R16GM149263.

Figure 4 .
Figure 4. Mechanisms inducing IL-8 expression in ovarian cancer and the tumor promoting effects of IL-8 in OC cells.

Figure 6 .
Figure 6.Model of how IFNγ induces Bcl3-dependent IL-8 and PD-L1 expression in OC cells.IFNγ induces first the expression of Bcl3 in OC cells, resulting in increased transcription of IL-8 and PD-L1.The increased expression of IL-8 and PD-L1 enhances OC cell proliferation, migration, invasion, epithelial-to-mesenchymal transition (EMT), metastasis, angiogenesis, cell stemness and immune escape.

Table 1 .
Antitumor and tumorigenic functions of IFNγ in ovarian cancer cells.

Table 2 .
Active clinical trials based on simultaneous inhibition of IL-8 and PD-1/PD-L1 signaling.