IκB kinase-ε-mediated phosphorylation triggers IRF-1 degradation in breast cancer cells.

Interferon Regulatory Factors (IRFs) are key regulators of immunity, cell survival and apoptosis. IRF transcriptional activity and subcellular localization are tightly regulated by posttranscriptional modifications including phosphorylation. The IκB kinase family member IKK-ε is essential in regulating antiviral innate immunity mediated by IRFs but is now also recognized as an oncoprotein amplified and overexpressed in breast cancer cell lines and patient-derived tumors. In the present study, we report that the tumor suppressor IRF-1 is a specific target of IKK-ε in breast cancer cells. IKK-ε-mediated phosphorylation of IRF-1 dramatically decreases IRF-1 protein stability, accelerating IRF-1 degradation and quenching IRF-1 transcriptional activity. Chemical inhibition of IKK-ε activity, fully restores IRF-1 levels and function and positively correlates with inhibition of cell growth and proliferation of breast cancer cells. By using a breast cancer cell line stably expressing a dominant negative version of IRF-1 we were able to demonstrate that IKK-ε preferentially exerts its oncogenic potential in breast cancer through the regulation of IRF-1 and point to the IKK-ε-mediated phosphorylation of IRF-1 as a therapeutic target to overcome IKK-ε-mediated tumorigenesis.

Alternate NF-κB-Independent Signaling Reactivation of Latent HIV-1 Provirus.

Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 from infected individuals because of the establishment of proviral latency in long-lived cellular reservoirs. The shock-and-kill approach aims to reactivate viral replication from the latent state (shock) using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells (kill) by specific therapeutics. The NF-κB RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat (LTR). Nevertheless, prolonged NF-κB activation contributes to the development of various autoimmune, inflammatory, and malignant disorders. In the present study, we established a cellular model of HIV-1 latency in J-Lat CD4+ T cells that stably expressed the NF-κB superrepressor IκB-α 2NΔ4 and demonstrate that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-κB activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors or specific short hairpin RNAs, c-Jun was identified to be an essential factor binding to the LTR enhancer κB sites and mediating the combined synergistic reactivation effect. Furthermore, acetylsalicylic acid (ASA), a potent inhibitor of the NF-κB activator kinase IκB kinase ß (IKK-ß), did not significantly diminish reactivation in a primary CD4+ T central memory (TCM) cell latency model. The present work demonstrates that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-κB, with the potential to avoid unwanted autoimmune- and or inflammation-related side effects associated with latency-reversing strategies.IMPORTANCE The shock-and-kill approach consists of the reactivation of HIV-1 replication from latency using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-κB is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-κB in HIV-1-infected patients resulting from the combined administration of different LRAs could represent a potential risk, especially in the case of a prolonged treatment. We demonstrate here that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivate HIV-1 from latency, even under conditions where NF-κB activation is repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-κB in patients under combination antiretroviral therapy during the shock-and-kill approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs.

A model of the three-dimensional structure of human interferon responsive factor 1 and its modifications upon phosphorylation or phosphorylation-mimicking mutations.

Interferon responsive factor 1 (IRF-1) is a pleiotropic transcription factor, possessing non-redundant biological activities that depend on its interaction with different protein partners and multiple post-translational modifications including phosphorylation. In particular, a 5'-SXXXSXS-3' motif of the protein represents the target of the IκB-related kinases, TANK-binding kinase (TBK)-1 and inhibitor of nuclear factor kappa-B kinase (IKK)-ε. Here, a 3D model of human IRF-1 was determined by using multi-template comparative modeling and molecular dynamics approaches. Models obtained through either phosphorylation or aspartate mutation of residues 215, 219 and 221 were also calculated and compared to the wild type. Calculations indicated that each of these modifications mainly induces a rigidification of the protein structure and only slightly changes in electrostatics and hydrophobicity of IRF-1 surface, resulting in the impairment of the capacity of IRF-1 containing as partate mutations (S221D and S215D/S219D/S221D) to synergize with tumour necrosis factor (TNF)-α stimulation in inducing interferon (IFN) promoter-mediated reporter gene activation. Therefore, these changes are qualitatively correlated to the amount of negative charge located on the 215-221 segments of IRF-1 by phosphorylation or aspartate mutation. Hypotheses on the structural mechanism that governs the phosphorylation-related damping of IRF-1 activity were also drawn. Communicated by Ramaswamy H. Sarma.