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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., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Interferon-γ (IFNγ) is a pleiotropic cytokine that has a crucial role in immune response and tumor immunity. Because of its anti-tumor effects, IFNγ has been used in cancer treatment. However, IFNγ also has tumor-promoting functions that are less well understood. Here, we show that IFNγ induces expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) in ovarian cancer (OC) cells. The IFNγ-induced IL-8 expression is dependent on JAK1, STAT1, and p65 NFκB, and is associated with an increased occupancy of K314/315 acetylated p65 NFκB and Ser-727 phosphorylated STAT1 at the IL-8 promoter. Neutralization of IL-8 using anti-IL-8 antibody reduces IFNγ-induced migration of OC cells, and their invasion ability in 3D spheroids. Together, these findings identify IL-8 as a novel target induced by IFNγ/JAK1/STAT1/p65 NFκB signaling, and indicate that the IFNγ-induced IL-8 contributes to IFNγ pro-tumorigenic effects in ovarian cancer cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) induces proliferation and invasion of solid tumor cells. In many types of solid cancer, including triple-negative breast cancer (TNBC), the IL-8 expression is induced by proteasome inhibition. In this chapter, we describe a protocol for the analysis of TNBC cell invasion induced by IL-8 in response to proteasome inhibition by bortezomib (BZ). Using this approach, we show that BZ increases the invasion ability of TNBC cells, and that the BZ-increased TNBC cell invasion is suppressed by IκB kinase (IKK) inhibition, which also decreases the IL-8 expression. The experimental protocol includes the cell invasion assay, microscopic evaluation of the invading cells, and quantitative analysis of the obtained images. This protocol should be applicable also for measurement of chemokine-induced tumor cell invasion in other types of cancer cells.

Ovarian cancer (OC) is the most common cause of cancer deaths among gynecological malignancies. OC ascites contain multicellular spheroid aggregates, which exhibit increased pro-survival signaling, invasive behavior, and chemotherapeutic resistance. OC cells are characterized by an increased expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which increases their survival and migration, thus contributing to OC metastasis and angiogenesis. While previous studies have shown that IL-8 increases proliferation of OC cells grown in monolayer cultures, the effect of IL-8 on proliferation of OC cells grown in 3D spheroids has not been investigated. The spheroid 3D culture assays have been particularly useful in translational research since they allow cell-to-cell interactions that resemble tumor growth in vivo, while allowing easy cell manipulations and visualization. Here, we used the 3D spheroid culture assay to investigate the effect of IL-8 on OC cell proliferation. Using this assay, our results show that IL-8 significantly increases proliferation of OC cells grown in 3D spheroids.

Triple negative breast cancer (TNBC) cells express increased levels of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which promotes their proliferation and migration. Because TNBC patients are unresponsive to current targeted therapies, new therapeutic strategies are urgently needed. While proteasome inhibition by bortezomib (BZ) or carfilzomib (CZ) has been effective in treating hematological malignancies, it has been less effective in solid tumors, including TNBC, but the mechanisms are incompletely understood. Here we report that proteasome inhibition significantly increases expression of IL-8, and its receptors CXCR1 and CXCR2, in TNBC cells. Suppression or neutralization of the BZ-induced IL-8 potentiates the BZ cytotoxic and anti-proliferative effect in TNBC cells. The IL-8 expression induced by proteasome inhibition in TNBC cells is mediated by IκB kinase (IKK), increased nuclear accumulation of p65 NFκB, and by IKK-dependent p65 recruitment to IL-8 promoter. Importantly, inhibition of IKK activity significantly decreases proliferation, migration, and invasion of BZ-treated TNBC cells. These data provide the first evidence demonstrating that proteasome inhibition increases the IL-8 signaling in TNBC cells, and suggesting that IKK inhibitors may increase effectiveness of proteasome inhibitors in treating TNBC., Competing Interests: The authors have declared that no competing interests exist.

Overexpression of the pro-angiogenic chemokine IL-8 (CXCL8) is associated with a poor prognosis in several solid tumors, including epithelial ovarian cancer (EOC). Even though histone deacetylase (HDAC) inhibition has shown remarkable antitumor activity in hematological malignancies, it has been less effective in solid tumors, including EOC. Here we report results that may explain the decreased efficiency of HDAC inhibition in EOC, based on our data demonstrating that HDAC inhibition specifically induces expression of IL-8/CXCL8 in SKOV3, CAOV3, and OVCAR3 cells. Suppression or neutralization of vorinostat-induced IL-8/CXCL8 potentiates the vorinostat inhibitory effect on cell viability and proliferation. The IL-8/CXCL8 expression induced by vorinostat in EOC cells is dependent on IκB kinase (IKK) activity and associated with a gene-specific recruitment of IKKβ and IKK-dependent recruitment of p65 NFκB to the IL-8/CXCL8 promoter. In addition, HDAC inhibition induces acetylation of p65 and histone H3 and their IL-8/CXCL8 promoter occupancy. In vivo results demonstrate that combining vorinostat and the IKK inhibitor Bay 117085 significantly reduces tumor growth in nude mice compared with control untreated mice or either drug alone. Mice in the combination group had the lowest IL-8/CXCL8 tumor levels and the lowest tumor expression of the murine neutrophil [7/4] antigen, indicating reduced neutrophil infiltration. Together, our results demonstrate that HDAC inhibition specifically induces IL-8/CXCL8 expression in EOC cells and that the mechanism involves IKK, suggesting that using IKK inhibitors may increase the effectiveness of HDAC inhibitors when treating ovarian cancer and other solid tumors characterized by increased IL-8/CXCL8 expression., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)