Kevin McCool

DNA damage-dependent NF-κB activation: NEMO turns nuclear signaling inside out.

Summary:  The dimeric transcription factor nuclear factor κB (NF‐κB) functions broadly in coordinating cellular responses during inflammation and immune reactions, and its importance in the pathogenesis of cancer is increasingly recognized. Many of the signal transduction pathways that trigger activation of cytoplasmic NF‐κB in response to a broad array of immune and inflammatory stimuli have been elaborated in great detail. NF‐κB can also be activated by DNA damage, though relatively less is known about the signal transduction mechanisms that link DNA damage in the nucleus with activation of NF‐κB in the cytoplasm. Here, we focus on the conserved signaling pathway that has emerged that promotes NF‐κB activation following DNA damage. Post‐translational modification of NF‐κB essential modulator (NEMO) plays a central role in linking the cellular DNA damage response to NF‐κB via the ataxia telangiectasia mutated (ATM) kinase. Accumulating evidence suggests that DNA damage‐dependent NF‐κB activation may play significant biological roles, particularly during lymphocyte differentiation and progression of human malignancies.

The Role of Histone Acetylation in Regulating Early Gene Expression Patterns during Early Embryonic Stem Cell Differentiation

We have examined the role of histone acetylation in the very earliest steps of differentiation of mouse embryonic stem cells in response to withdrawal of leukemia inhibitory factor (LIF) as a differentiation signal. The cells undergo dramatic changes in morphology and an ordered program of gene expression changes representing differentiation to all three germ layers over the first 3-5 days of LIF withdrawal. We observed a global increase in acetylation on histone H4 and to a lesser extent on histone H3 over this time period. Treatment of the cells with trichostatin A (TSA), a histone deacetylase inhibitor, induced changes in morphology, gene expression, and histone acetylation that mimicked differentiation induced by withdrawal of LIF. We examined localized histone acetylation in the regulatory regions of genes that were transcriptionally either active in undifferentiated cells, induced during differentiation, or inactive under all treatments. There was striking concordance in the histone acetylation patterns of specific genes induced by both TSA and LIF withdrawal. Increased histone acetylation in local regions correlated best with induction of gene expression. Finally, TSA treatment did not support the maintenance or progression of differentiation. Upon removal of TSA, the cells reverted to the undifferentiated phenotype. We concluded that increased histone acetylation at specific genes played a role in their expression, but additional events are required for maintenance of differentiated gene expression and loss of the pluripotent state.

Dynamic changes in histone H3 phosphoacetylation during early embryonic stem cell differentiation are directly mediated by mitogen- and stress-activated protein kinase 1 via activation of MAPK pathways

Embryonic stem (ES) cells are pluripotent cells capable of unlimited self-renewal and differentiation into the three embryonic germ layers under appropriate conditions. Mechanisms for control of the early period of differentiation, involving exit from the pluripotent state and lineage commitment, are not well understood. An emerging concept is that epigenetic histone modifications may play a role during this early period. We have found that upon differentiation of mouse ES cells by removal of the cytokine leukemia inhibitory factor, there is a global increase in coupled histone H3 phosphorylation (Ser-10)-acetylation (Lys-14) (H3 phosphoacetylation). We show that this occurs through activation of both the extracellular signal-regulated kinase (ERK) and p38 MAPK signaling pathways. Early ES cell differentiation is delayed using pharmacological inhibitors of the ERK and p38 pathways. One common point of convergence of these pathways is the activation of the mitogen- and stress-activated protein kinase 1 (MSK1). We show here that MSK1 is the critical mediator of differentiation-induced H3 phosphoacetylation using both the chemical inhibitor H89 and RNA interference. Interestingly, inhibition of H3 phosphoacetylation also alters gene expression during early differentiation. These results point to an important role for both epigenetic histone modifications and kinase pathways in modulating early ES differentiation.

Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling.

The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced by TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO-IKKβ interaction and was a poor direct IKKβ inhibitor, but prevented the formation of TNF-induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMO(Y308S) mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo.

A PAR-SUMOnious mechanism of NEMO activation.

In this issue of Molecular Cell, Stilmann et al. (2009) demonstrate a new mode of prosurvival NF-kappaB activation through the formation of a PARP-1-poly(ADP-ribose) signaling scaffold in response to DNA damage.

IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation

Background: Nuclear import of NEMO is critical for DNA damage-dependent NF-κB signaling, but the mechanism remains unknown. Results: IPO3 binds NEMO, promotes its nuclear import, and is critical for DNA damage-dependent NF-κB activation. Conclusion: IPO3 is a nonclassical nuclear import receptor for NEMO. Significance: IPO3 is a new player in DNA damage-dependent NF-κB signaling with implications in cancer therapy. Activation of IκB kinase (IKK) and NF-κB by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-κB essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-κB signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-κB activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin α and β family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin β family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-κB activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-κB signaling arm of the mammalian DNA damage response.

Neoadjuvant chemotherapy is associated with more anemia and perioperative blood transfusions than primary debulking surgery in women with advanced stage ovarian cancer

OBJECTIVE The purpose of this case-controlled study was to determine the prevalence of anemia and incidence of perioperative blood transfusions in patients undergoing treatment for advanced ovarian cancer with neoadjuvant chemotherapy (NACT) or primary debulking surgery (PDS). METHODS We performed a single institution review of patients diagnosed with stage IIIB-IVB epithelial ovarian cancer between 2010 and 2013 undergoing either NACT or PDS. Anemia was defined as a hemoglobin (Hgb) concentration of ≤11.5 g/dL. Continuous variables were compared by student t-test and binary variables compared via chi square analysis. RESULTS One hundred thirty-one women were included, 66 treated with NACT and 65 treated with PDS. Average Hgb prior to surgery was lower in women who received NACT (10.7 g/dL vs 12.8 g/dL, p < 0.0001). Women treated with NACT had a decrease in mean Hgb during chemotherapy treatment (11.8 g/dL at diagnosis to 10.7 g/dL preoperatively). Seventy-seven percent of NACT patients were anemic prior to surgery compared to 15% of patients prior to PDS (p < 0.001). Mean EBL at debulking was higher in patients selected for PDS (871 mL) than NACT (544 mL); however, the perioperative transfusion rate was higher during interval debulking surgeries (NACT 77% vs PDS 56%, p = 0.01). CONCLUSION Women selected for NACT were more likely to be anemic at diagnosis and became progressively anemic during NACT. Despite less blood loss during debulking surgery, NACT patients receive more blood transfusions perioperatively than patients undergoing PDS. This represents a potential opportunity for therapeutic intervention during NACT to correct anemia prior to interval debulking surgery.

Quantification of cellular NEMO content and its impact on NF-κB activation by genotoxic stress.

NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5). We determined that the C5 cell clone has an average of 4 x 105 molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6–6x105 molecules per cell) yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies.

Peri-operative allogeneic blood transfusion is associated with poor overall survival in advanced epithelial ovarian Cancer; potential impact of patient blood management on Cancer outcomes

BACKGROUND Transfusion related immune modulation associated with red blood cell (RBC) transfusion is thought to result in decreased cancer survival. Results in epithelial ovarian cancer (EOC) have been mixed however most suggest worse oncologic outcomes in patients who were transfused at the time of debulking surgery. The impact of restrictive transfusion strategies on this patient population is currently not known. METHODS We conducted a retrospective study of women with EOC. The study population was divided into two groups based on whether they were transfused RBCs during the peri-operative period or not. Clinical characteristics and prognosticators were compared between groups. Overall survival was compared between groups based on transfusion status and other known prognostic factors. Cox proportional hazard modeling was used to examine the association between the prognostic factors and the study endpoint. RESULTS Sixty-six percent of women were transfused. Transfusion was associated with CA125, the use of neoadjuvant chemotherapy (NACT), surgical blood loss, and anemia. The mean pre-transfusion Hgb was 7.8 + 0.6 g/dL and 94% had a hemoglobin level greater than the transfusion threshold of 7 g/dL. RBC transfusion, suboptimal debulking, anemia, and NACT were associated with decreased survival. Only RBC transfusion and suboptimal debulking status remained significant in a multivariate model. CONCLUSIONS Peri-operative RBC transfusion compromises survival in ovarian cancer supporting the need to minimize the use of transfusion at the time of debulking surgery. Adherence to evidence-based transfusion guidelines offers an opportunity to reduce transfusion rates in this population with a resulting positive influence on survival.