IFNγ induces Bcl3 expression by JAK1/STAT1/p65 signaling, resulting in increased IL‐8 expression in ovarian cancer cells

We have recently shown that IFNγ, produced during cancer therapy, induces expression of the Bcl3 proto‐oncogene in ovarian cancer (OC) cells, resulting in their increased proliferation, migration, and invasion, but the mechanisms are unknown. Here, we demonstrate that the IFNγ‐induced Bcl3 expression is dependent on JAK1 and STAT1 signaling, and on p65 NFκB. Furthermore, the IFNγ‐induced Bcl3 expression is associated with an increased occupancy of Ser‐727 phosphorylated STAT1 and acetylated histone H3 at the Bcl3 promoter. Our data indicate that Bcl3 promotes expression of the pro‐inflammatory chemokine interleukin‐8 (IL‐8) in OC cells. These findings identify Bcl3 as a novel target of IFNγ/JAK1/STAT1 signaling and suggest that targeting the JAK1/STAT1 pathway may suppress IFNγ‐induced Bcl3 expression in OC.

As other members of IjB family, the BCL-3 transcription is regulated by NFjB signaling and upregulated in response to stimulation with TNFa, interleukin (IL)-1, IL-6, and other pro-inflammatory cytokines [10,15,[23][24][25][26][27]. We have recently shown that the Bcl3 expression is increased in OC cells and tissues, and promotes expression of the immune checkpoint PD-L1 [9]. In addition, the Bcl3 expression in OC cells can be further increased by interferon-c (IFNc), resulting in increased survival, proliferation, and migration of OC cells [9]. However, the mechanisms of how IFNc induces the Bcl3 expression are unknown.
Interferon-c is a pleiotropic cytokine that can have, depending on the cellular and molecular context, both antitumor and pro-tumorigenic functions [28][29][30][31]. Because of its antitumor potential, IFNc has been used in cancer treatment [32][33][34][35]. In addition, IFNc expression can be induced in response to radiation therapy or immune checkpoint blockade (ICB) used in cancer treatment [36][37][38]. Considering the strong oncogenic potential of Bcl3, in this study, we have investigated the mechanisms by which IFNc induces the Bcl3 expression.
Our results demonstrate that the IFNc-induced Bcl3 expression in OC cells is dependent on JAK1 and STAT1 signaling, as well as on p65 NFjB. In addition, our data show that the Bcl3 expression promotes expression of the pro-angiogenic and pro-inflammatory chemokine IL-8/CXCL8 in OC cells. These findings identify Bcl3 as a novel target of IFNc/JAK1/STAT1 signaling and inducer of IL-8 expression, and suggest that targeting the JAK1/STAT1 pathway may suppress the IFNc-induced Bcl3 expression in OC.

Quantitative real-time RT-PCR
Total RNA was isolated using RNeasy Mini Kit (Qiagen, Germantown, MD, USA), and quantitative real-time RT-PCR (qRT-PCR) was performed as described [40]. The primers for quantification of human Bcl3, JAK1, STAT1, p65, IL-8, and actin mRNA were from Qiagen. The mRNA values are expressed as a percentage of control untreated (UT) samples, which were set as 100%.

Statistical analysis
The results represent at least three experiments and are presented as means AE SE. Data were analyzed by using INSTAT software package (GraphPad, San Diego, CA, USA). Statistical significance was evaluated by using one-way ANOVA Tukey post hoc test, and P < 0.05 was considered significant.

IFNc-induced Bcl3 expression in OC cells is mediated by JAK1
We have recently shown that IFNc induces Bcl3 expression, but the responsible mechanisms are unknown [9]. Since the main signaling pathway by which IFNc induces gene expression is via the canonical JAK1-STAT1 signaling pathway [28][29][30], we investigated whether the IFNc induced Bcl3 expression is dependent on JAK1/STAT1 signaling. To this end, we employed two commonly used OC cell lines: the highly invasive non-serous SKOV3 cells and the high-grade serous OVCAR3 cells, since both cell lines exhibit high levels of Bcl3 expression, which is further increased by IFNc [9].
To determine whether the IFNc-induced Bcl3 expression in OC cells is dependent on JAK activity, we first analyzed Bcl3 gene and protein levels in IFNctreated SKOV3 and OVCAR3 cells pretreated with the JAK pharmacological inhibitor, ruxolitinib (Rux). As shown in Fig. 1, 100 nM and 200 nM Rux significantly reduced Bcl3 mRNA levels in IFNc-treated SKOV3 (Fig. 1A) and OVCAR3 (Fig. 1B) cells. Furthermore, 100 and 200 nM Rux decreased the protein levels of Bcl3 in both cell types (Fig 1C,D), indicating that the IFNc-induced Bcl3 expression in OC cells is dependent on JAK activity.
To ascertain whether the IFNc-induced Bcl3 expression is specifically dependent on JAK1, we analyzed the Bcl3 levels in SKOV3 cells transfected with control and JAK1-specific siRNAs. As shown in Fig

IFNc-induced Bcl3 expression in OC cells is mediated by STAT1
In canonical IFNc signaling, activation of JAK1 leads to phosphorylation and activation of STAT1, which then translocates to the nucleus and induces  transcription of IFNc-stimulated genes (ISGs) [28][29][30].
To determine whether the IFNc-induced Bcl3 expression is dependent on STAT1, we analyzed Bcl3 levels in SKOV3 cells transfected with control and STAT1specific siRNAs. As shown in Figs 3A,C, IFNc increased mRNA and total protein levels of STAT1 in SKOV3 cells, and the IFNc-induced STAT1 expression was suppressed in STAT1 siRNA transfected cells. Importantly, suppression of STAT1 significantly reduced the IFNc-induced Bcl3 mRNA (Fig. 3B) and total protein (Figs 3C,D) levels, indicating that the IFNc-induced Bcl3 expression in OC cells is mediated by STAT1.

IFNc-induced Bcl3 expression in OC cells is mediated by p65 NFjB
Even though the JAK/STAT pathway is the main mechanism responsible for the expression of ISGs [28][29][30], several studies have shown that the IFNc-induced expression of NFjB-dependent genes can be regulated also by p65 NFjB [42,43]. To examine the possibility that the IFNc-induced Bcl3 expression in OC cells might be dependent on p65 NFjB, we analyzed the Bcl3 expression in IFNc-treated SKOV3 cells transfected with p65 siRNA. As shown in Fig. 4, IFNc increased mRNA (Fig. 4A) and protein (Fig. 4C) levels of p65 NFjB in SKOV3 cells, and the IFNcincreased p65 expression was suppressed in cells transfected with p65 specific siRNA (Figs. 4A,C). Importantly, suppression of p65 significantly reduced both mRNA (Fig. 4B) and protein (Figs 4C,D) levels of Bcl3 in SKOV3 cells treated with IFNc. These results indicate that the IFNc-induced Bcl3 expression in OC cells is also mediated by p65 NFjB.
IFNc increases Bcl3 promoter occupancy by Ser727-pSTAT1 and ac-histone H3 In most cell types, IFNc induces gene expression by STAT1 promoter binding, and in many cases, STAT1 phosphorylation at Ser727 is required for its full transcriptional and biological activity [29]. Using MEME Suite and JASPAR databases, we have identified a potential STAT1 binding site in human Bcl3 promoter, located -418 nucleotides from the transcription start site (TSS) (Fig. 5A). Thus, using ChIP, we analyzed whether IFNc increases STAT1 and Ser727 pSTAT1 (pSTAT1) occupancy at this site. In addition, since the IFNc-induced Bcl3 expression was dependent on p65 NFjB (Fig. 4), we analyzed whether IFNc induces a direct recruitment of p65 or its transcriptionally active K314/315 acetylated form (ac-p65) to the previously identified p65 NFjB binding site located -289 nucleotides from TSS [8]. As shown in Fig. 5B, IFNc increased recruitment of pSTAT1, but not unphosphorylated STAT1 to Bcl3 promoter, while neither p65 nor ac-p65 were recruited. However, IFNc increased Bcl3 promoter occupancy by Lys9 acetylated histone H3 (ac-H3) (Fig. 5B), consistent with active transcription. The increased promoter occupancy by pSTAT1 and ac-H3 (Fig. 5B) suggested that IFNc might increase STAT1 phosphorylation and histone H3 acetylation; alternatively, IFNc could just promote Bcl3 promoter occupancy by pSTAT1 and ac-H3, without increasing their total cellular levels. To distinguish between these two possibilities, we analyzed STAT1, p65, and histone H3 total cellular levels by western blotting. As shown in Fig. 5C, IFNc increased total cellular levels of pSTAT1 as well as unphosphorylated STAT1. In addition, as previously observed [40], IFNc increased the protein expression of p65, but not its K314/315 acetylation. Neither total histone H3 protein levels nor its Lys9 acetylation were affected by IFNc treatment in OC cells (Fig. 5C). Together, these data indicate that IFNc induces the Bcl3 expression in OC cells by promoting the STAT1 expression and its Ser727 phosphorylation and recruitment to Bcl3 promoter, which is facilitated by a concomitant IFNc-induced promoter acetylation.

Bcl3 promotes expression of IL-8/CXL8 in OC cells
We have previously shown that IFNc induces PD-L1 expression that is dependent on Bcl3 in OC cells [9]. In addition, we have recently found that IFNc induces expression of the pro-inflammatory and proangiogenic chemokine IL-8/CXCL8, resulting in increased migration and invasion of OC cells [40]; however, the mechanisms that regulate the IFNcinduced IL-8 expression remain largely unknown. Since the levels of PD-L1 and IL-8 correlate in cancer patients [44][45][46], we hypothesized that the IFNcinduced IL-8 expression in OC cells might be also mediated by Bcl3. Indeed, Bcl3 suppression by siRNA significantly decreased both constitutive and IFNcinduced IL-8 mRNA expression and cytokine release in OC cells (Fig. 6), indicating that Bcl3 promotes the IL-8 expression.

Discussion
Increased Bcl3 expression in cancer cells promotes their proliferation, migration, and metastatic potential, but the mechanisms that regulate the Bcl3 expression in solid tumors are incompletely understood. We have recently shown that the Bcl3 expression is induced by IFNc in OC cells, resulting in their increased proliferation and PD-L1 expression [9]. In this study, we show that the IFNc-induced Bcl3 expression is dependent on JAK1/STAT1 and p65 NFjB, and promotes expression of the pro-inflammatory chemokine IL-8. To our knowledge, this is the first study that identifies Bcl3 as a target of the JAK1/STAT1 signaling, and inducer of the IL-8 expression.
Our data indicate that IFNc induces the Bcl3 expression by increasing acetylation of the Bcl3 promoter, with a simultaneous promoter recruitment of Ser727 pSTAT1. However, even though the IFNcinduced Bcl3 expression in OC cells is also dependent on p65 NFjB, we did not observe p65/ac-p65 recruitment to the -289 p65 NFjB binding site in Bcl3 promoter. It is possible that p65 NFjB might mediate the IFNc induced Bcl3 expression through its binding to other NFjB binding site(s) in Bcl3 promoter, or to a downstream enhancer. This hypothesis is supported by a previous study that showed that NFjB induces Bcl3 transcription through an intronic enhancer [10]. Alternatively, p65 NFjB might facilitate Bcl3 transcription through a cooperative interaction with STAT1. Indeed, several studies have demonstrated a crosstalk between NFjB and STAT1 signaling in the regulation of IFNc-induced inflammatory genes [30,[47][48][49].
Interestingly, in the immunosuppressive cutaneous T-cell lymphoma cells, Bcl3 inhibits expression and release of the pro-inflammatory chemokine IL-8 [8]. However, our present findings indicate that Bcl3 induces the IL-8 expression in OC cells. The IL-8 expression is increased in OC and other solid tumors, where it induces cancer progression through its induction of tumor cell proliferation, migration, invasion, and immune escape [50][51][52][53][54]. Increased IL-8 serum levels reflect tumor burden and correlate with poor prognosis in OC and other solid tumors [44,45,54]. Since both Bcl3 and IL-8 are IFNc-inducible genes that promote tumor cell proliferation, migration, and invasion [9,40], the positive regulation of IL-8 by Bcl3 suggests that the Bcl3 oncogenic function in OC cells might be partly mediated by its upregulation of IL-8.
Because of its anticancer properties, IFNc has been used in cancer treatment [32][33][34][35]. In addition, IFNc expression is induced by radiation and ICB in cancer treatment [36][37][38]. However, IFNc also has important tumor-promoting functions that include the induced PD-L1 and IL-8 expression resulting in increased cancer cell proliferation and immune escape, but the mechanisms are much less understood [9,40]. Understanding the tumor-promoting mechanisms and molecular targets of IFNc is crucial to minimize its tumor-promoting functions in IFNc-based therapies, and in cancer treatments associated with IFNc increase.
Our present findings are the first to demonstrate that the IFNc-induced Bcl3 expression is dependent of JAK1/STAT1 and p65 NFjB signaling. In addition, our in vitro data show that Bcl3 promotes the IL-8 expression in OC cells. Together, these results suggest that targeting the IFNc-induced JAK1/STAT1/p65 NFjB signaling may suppress the IFNc-induced Bcl3 and IL-8 expression in OC. Future studies should extend these in vitro findings and determine whether IFNc induces the Bcl3 and IL-8 expression in animal models and clinical samples.