Abstract
NF-κB is a pro-inflammatory transcription factor that critically regulates immune responses and other distinct cellular pathways. However, many NF-κB-mediated pathways for cell survival and apoptosis signaling in cancer remain to be elucidated. Cell cycle and apoptosis regulatory protein 1 (CARP-1 or CCAR1) is a perinuclear phosphoprotein that regulates signaling induced by anticancer chemotherapy and growth factors. Although previous studies have reported that CARP-1 is a part of the NF-κB proteome, regulation of NF-κB signaling by CARP-1 and the molecular mechanism(s) involved are unclear. Here, we report that CARP-1 directly binds the NF-κB-activating kinase IκB kinase subunit γ (NEMO or NF-κB essential modulator) and regulates the chemotherapy-activated canonical NF-κB pathway. Importantly, blockade of NEMO-CARP-1 binding diminished NF-κB activation, indicated by reduced phosphorylation of its subunit p65/RelA by the chemotherapeutic agent adriamycin (ADR), but not NF-κB activation induced by tumor necrosis factor α (TNFα), interleukin (IL)-1β, or epidermal growth factor. High-throughput screening of a chemical library yielded a small molecule inhibitor of NEMO-CARP-1 binding, termed selective NF-κB inhibitor 1 (SNI)-1). We noted that SNI-1 enhances chemotherapy-dependent growth inhibition of a variety of cancer cells, including human triple-negative breast cancer (TNBC) and patient-derived TNBC cells in vitro, and attenuates chemotherapy-induced secretion of the pro-inflammatory cytokines TNFα, IL-1β, and IL-8. SNI-1 also enhanced ADR or cisplatin inhibition of murine TNBC tumors in vivo and reduced systemic levels of pro-inflammatory cytokines. We conclude that inhibition of NEMO-CARP-1 binding enhances responses of cancer cells to chemotherapy.
Highlights
NF-B is a pro-inflammatory transcription factor that critically regulates immune responses and other distinct cellular pathways
We previously found that TNF␣, adriamycin, or CARP-1 functional mimetic (CFM)-4 compound caused increased transcriptional activation of NF-B in human TNBC cells, whereas knockdown of CARP-1 attenuated activation of NF-B by these agents (30)
Because adriamycin or epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor Iressa inhibited HBC growth in part by inducing CARP-1 expression (1, 3), and CARP-1 was found to be a part of the NF-B proteome (29), we investigated whether and how CARP-1 regulates NF-B signaling
Summary
NF-B is a pro-inflammatory transcription factor that critically regulates immune responses and other distinct cellular pathways. CARP-1 co-activated tumor suppressor p53 to transduce the DNA damage–induced transcriptional increase of cyclin-dependent kinase inhibitor p21WAF1 in breast cancer cells (12) Chemotherapeutics such as ADR induce double-strand breaks (DSBs), whereas phosphorylation of H2AX at serine 139 (␥-H2AX) by ATM/ATR functions to repair DSBs (16 –18). The phosphorylated NEMO is mono-ubiquitinated, which triggers its nuclear export and IKK activation in the cytoplasm (28) This therapyinduced activation of canonical NF-B promotes production of pro-inflammatory cytokines, cell growth, and survival signaling and contributes to therapy resistance. Because CARP-1 is a regulator of cell growth and survival signaling (1, 3, 12) and a component of the NF-B proteome (29), and CARP-1 depletion inhibited transcriptional activation of NF-B by ADR, TNF␣, or an experimental CARP-1 functional mimetic (CFM) compound (30), we investigated the molecular mechanism of CARP-1– dependent regulation of NF-B signaling. Pharmacological inhibition of NEMO–CARP-1 binding enhances cisplatin efficacy in part by impacting levels of circulating pro-inflammatory cytokines in immunocompetent mice bearing subcutaneous tumors of murine breast cancer cells
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