Yuu Taguchi

NF-κB non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype

Patients with triple-negative breast cancer display the highest rates of early relapse of all patients with breast cancer. The basal-like subtype, a subgroup of triple-negative breast cancer, exhibits high levels of constitutively active NF-κB signalling. Here we show that NF-κB activation, induced by inflammatory cytokines or by epigenetically dysregulated NIK expression, cell-autonomously upregulates JAG1 expression in non-cancer stem cells. This upregulation stimulates NOTCH signalling in cancer stem cells in trans, leading to an expansion of cancer stem cell populations. Among breast cancers, the NF-κB-dependent induction of JAG1 and the NOTCH-dependent expansion of the cancer stem cell population occur only in the basal-like subtype. Collectively, our results indicate that NF-κB has a non-cell-autonomous role in regulating cancer stem cell populations by forming intratumoural microenvironments composed of JAG1-expressing non-cancer stem cells with a basal-like subtype.

A unique domain in RANK is required for Gab2 and PLCγ2 binding to establish osteoclastogenic signals

TRAF6 is essential for osteoclastogenesis and for both RANK‐ and CD40‐mediated activation of IKK and MAPKs. RANK, but not CD40, can promote osteoclastogenesis because only RANK induces NFATc1 activation through PLCγ2‐induced Ca2+ oscillations together with the co‐stimulatory signals emanating from immune receptors linked to ITAM‐containing adaptors. These previous data suggest that RANK harbors a unique domain that functions in concert with the TRAF6‐binding site in osteoclastogenesis. Here we identify such a domain, highly conserved domain in RANK (HCR), which is dispensable for the early phase of RANK and ITAM signaling but is essential for their late‐phase signaling, including sustained activation of NF‐κB and PLCγ2 leading to NFATc1 activation. HCR recruits an adaptor protein, Gab2, which further associates with PLCγ2 in the late phase. Formation of the HCR‐mediated signaling complex could account for the sustained activation of NF‐κB and PLCγ2. The present study identifies HCR as a unique domain that plays a critical role in the long‐term linkage between RANK and ITAM signals, providing a molecular basis for therapeutic strategies.

Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation in Relb-Venus knock-in mice

RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb(Venus/Venus) mice developed without any abnormalities observed in the Relb(-/-) mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb(Venus/Venus) DC analyses revealed that DCs consist of RelB(-), RelB(low) and RelB(high) populations. The RelB(high) population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB(low) population was in the cytoplasm. Although both the RelB(low) and RelB(-) populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB(low) than in the RelB(-) splenic conventional DCs. Taken together, our results identify the RelB(low) population as a possible novel intermediate maturation stage of cDCs and the Relb(Venus/Venus) mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway.

Identification and characterization of anti-osteoclastogenic peptides derived from the cytoplasmic tail of receptor activator of nuclear factor kappa B

Pathological bone resorption by osteoclasts is primarily treated with bisphosphonates. Because the administration of bisphosphonates is associated with a risk for multiple adverse symptoms, a precise understanding of the mechanisms underlying osteoclastogenesis is required to develop drugs with minimal side-effects. Osteoclastogenesis depends on receptor activator of nuclear factor kappa B (RANK) signaling mediated by TRAF6. We previously identified a highly conserved domain in the cytoplasmic tail of RANK (HCR), which did not share any significant homology with other proteins and was essential for osteoclastogenesis. HCR acts as a platform for the formation of Gab2- and Vav3-containing signal complexes, and ectopic expression of the HCR peptide inhibits osteoclastogenesis. Here, we uncover the mechanisms of HCR peptide-mediated inhibition of osteoclastogenesis. Expression of either the amino- or carboxyl-terminal half of the HCR peptide (N- or C-peptide) independently inhibited RANK signaling prior to cell–cell fusion. In contrast, expression of the GY-peptide, which is a part of the C-peptide, did not significantly affect prefusion RANK signaling, but did inhibit cell–cell fusion to prevent formation of multinucleated mature osteoclasts. Moreover, Gab2, which is involved in RANK signaling by binding TRAF6, bound the C-peptide but not the N-peptide, suggesting that the C- and the N-peptides sequester TRAF6 in a Gab2-dependent and Gab2-independent manner, respectively. In contrast, the GY-peptide did not bind Gab2 but could bind Vav3, which mediates signaling for cell–cell fusion. Collectively, we propose that the HCR peptide inhibits osteoclastogenesis through two modes of action—inhibition of (1) prefusion RANK signaling and (2) cell–cell fusion by blocking TRAF6- and Vav3-mediated signaling, respectively.

cIAP1/2 negatively regulate RANKL-induced osteoclastogenesis through the inhibition of NFATc1 expression.

Receptor activator of nuclear factor κB (RANK) is a member of the tumor necrosis factor receptor superfamily (TNFRSF) and triggers osteoclastogenesis by inducing the expression of NFATc1 through the activation of the NF‐κB and MAPK pathways. Cellular inhibitors of apoptosis proteins 1 and 2 (cIAP1/2), which are ubiquitin E3 ligases, are involved in the activation of the NF‐κB and MAPK pathways by various members of the TNFRSF. However, the involvement of cIAP1/2 in RANK signaling has remained largely unknown. In this study, we reveal the involvement of cIAP1/2 in RANK ligand (RANKL)‐induced osteoclastogenesis. The over‐expression of cIAP1 or cIAP2 in the mouse monocytic cell line Raw264.7 resulted in the significant suppression of RANKL‐induced NFATc1 mRNA expression and osteoclastogenesis, whereas the activation of the NF‐κB and MAPK pathways was barely changed by these over‐expressions. The depletion of endogenous cIAP1/2 by their specific inhibitor MV1 or their siRNA‐mediated knockdown resulted in enhanced RANKL‐induced NFATc1 expression and osteoclastogenesis without affecting the activation of the NF‐κB and MAPK pathways. In combination, these results indicate that cIAP1/2 negatively regulate osteoclastogenesis by inhibiting NFATc1 mRNA expression in a manner that is distinct from the previously identified functions of cIAP1/2.

A Dithiol Compound Binds to the Zinc Finger Protein TRAF6 and Suppresses its Ubiquitination

Despite various inhibitors targeting the zinc center(s) of enzymes, drugs that target zinc fingers have not been examined in detail. We previously developed a dithiol compound named SN‐1 that has an inhibitory effect on the function of zinc finger transcription factors, but its mechanism of action has not yet been elucidated. To establish a general principle for new drugs, the details of the action of SN‐1 against a zinc finger protein were examined. As a zinc‐finger‐containing protein, we focused on TRAF6, which is related to cancer and inflammation. Binding of SN‐1 to TRAF6 and its effect on TRAF6 ubiquitination were examined in vitro, and the binding mode was calculated by computational methodology. Furthermore, ubiquitination of TRAF6 and downstream signaling was examined by cell‐based experiments. The results show that SN‐1 binds to TRAF6, inhibiting its auto‐ubiquitination and downstream NF‐κB signaling. Docking studies indicate that SN‐1 binds directly to the first zinc finger of TRAF6. This binding disrupts the neighboring structure, that is, the RING finger domain, to suppress the ubiquitin ligase activity of TRAF6. Taken together, this study provides a platform for developing new small molecules that target zinc finger proteins.

NF-κB Signaling in Osteoclastogenesis

Osteoclasts are unique multinucleated cells that can resorb bone. Bone mass is determined by a tightly regulated balance between osteoclasts and osteoblasts, which generate bones. Thus, the excessive formation of osteoclasts leads to the pathological bone resorption observed in postmenopausal osteoporosis, rheumatoid arthritis, Paget’s disease, and bone tumor metastases. During osteoclast differentiation, NF-κB is activated by TRAF6-mediated signals from RANK expressed on the surface of osteoclast progenitor cells upon RANKL stimulation, activating NFATc1, a master transcription factor in osteoclastogenesis. However, in contrast regular NF-κB activation, sufficient NFATc1 activation requires long-term activation of NF-κB, which can be induced uniquely by RANK but not by CD40, a receptor that also uses TRAF6 to activate NF-κB. Through analysis of various RANK mutants, we identified the 60-amino-acid HCR domain (mouse RANK) in the cytoplasmic tail of RANK. HCR is highly conserved among vertebrates and is crucial for long-term NF-κB activation. Interestingly, when HCR was attached to the cytoplasmic tail of CD40, the chimeric receptor promoted osteoclast formation, even though CD40 itself cannot. In this chapter, we explore the molecular mechanisms of HCR-mediated signals and the possible application of the HCR peptide as an anti-bone-resorptive drug.