Inflammatory mechanisms influence tumor development and metastatic progression1. Of interest is the role of such mechanisms in metastatic spread of tumors whose etiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. We found that prostate cancer metastasis is associated with lymphocyte infiltration into advanced tumors and elevated expression of the tumor necrosis factor (TNF) family members receptor activator of NF-κB (RANK) ligand (RANKL) and lymphotoxin (LT)2. But the source of RANKL and its role in metastasis were not established. RANKL and its receptor RANK control proliferation of mammary lobuloalveolar cells during pregnancy3 through activation of IκB kinase α (IKKα)4, a protein kinase that is required for self-renewal of mammary cancer progenitors5 and prostate cancer metastasis2. We therefore examined whether RANKL, RANK and IKKα are also involved in mammary/breast cancer metastasis. Indeed, RANK signaling in mammary carcinoma cells that overexpress the ErbB2 (c-Neu) proto-oncogene6, which is frequently amplified in metastatic human breast cancers7,8, was important for pulmonary metastasis. Metastatic spread of ErbB2-transformed carcinoma cells was also dependent on CD4+CD25+ T cells, whose major pro-metastatic function appeared to be RANKL production. RANKL-producing T cells were mainly FoxP3+ and found in close proximity to smooth muscle actin (SMA)-positive stromal cells in mouse and human breast cancers. The T cell-dependence of pulmonary metastasis was replaced by administration of exogenous RANKL, a procedure that also stimulated pulmonary metastasis of RANK-positive human breast carcinoma cells. These results are consistent with the adverse prognostic impact of tumor-infiltrating CD4+ or FoxP3+ T cells on human breast cancer9,10 and suggest that targeting of RANKL-RANK signaling can be used in conjunction with other therapies to prevent subsequent metastatic disease.

Z. are inventors of two pending patent applications for use of BCL-X L PROTACs as senolytic and antitumor agents. R.H., G.Z., and D.Z. are co-founders of and have equity in Dialectic Therapeutics, which develops BCL-X L PROTACs to treat cancer.

Metastasis, the cardinal feature of malignant tumors, is an important clinical variable in patient prognosis. To understand the basis for metastasis, we systematically selected for highly invasive cells from breast cancer cell lines, MCF7 and MDA-MB-453, with moderate to low invasive ability using Boyden chamber invasion assay. The four-cycle selected invasive lines, named MCF7-I4 and MDA-MB-453-I4, respectively, displayed epithelial-mesenchymal transition (EMT) and dramatically enhanced invasive ability. EMT changes were corroborated with decreased level of E-cadherin and increased vimentin, fibronectin, and B 1 integrin. Twist, a basic helix-loop-helix transcription factor, and AKT2, a known proto-oncogene, were found to be elevated in the invasive cells compared with the parental. Ectopic expression and knockdown of Twist by short interference RNA resulted in significant increase and reduction, respectively, of AKT2 protein and mRNA expression. Twist bound to E-box elements on AKT2 promoter and enhanced its transcriptional activity. Moreover, silencing AKT2 decreased Twist-promoted migration, invasion, and paclitaxel resistance. Reintroducing AKT2 largely rescued the phenotype resulted from knockdown of Twist in I4 cells, suggesting that AKT2 is a downstream target and functional mediator of Twist. Finally, we observed a 68.8% correlation of elevated Twist and AKT2 expression in latestage breast cancers as oppose to 13% in early-stage breast cancers. Our study identifies Twist as a positive transcriptional regulator of AKT2 expression, and Twist-AKT2 signaling is involved in promoting invasive ability and survival of breast cancer cells.

Balanced production of type I interferons (IFN) and proinflammatory cytokines upon engagement of Toll-like receptors (TLRs), which signal via adaptors containing a Toll-IL-1-Receptor (TIR) domain, such as MyD88 and TRIF, has been proposed to control the pathogenesis of autoimmune disease and tumor responses to inflammation. Here we show that TRAF3, a ubiquitin ligase that interacts with both MyD88 and TRIF, differentially regulated production of IFN and proinflammatory cytokines. Degradative TRAF3 ubiquitination during MyD88-dependent TLR signaling was essential for activation of mitogen-activated protein kinases (MAPKs) and inflammatory cytokine production. By contrast, TRIF-dependent signaling triggered non-canonical TRAF3 self-ubiquitination that activated the IFN response. Inhibition of degradative TRAF3 ubiquitination prevented expression of all proinflammatory cytokines without impacting the IFN response.

SummaryCytokine signaling is thought to require assembly of multi-component signaling complexes at cytoplasmic segments of membrane-embedded receptors, in which receptor-proximal protein kinases are activated. Indeed, CD40, a tumor necrosis factor receptor (TNFR) family member, forms a complex containing adaptor molecules TRAF2 and TRAF3, ubiquitin conjugating enzyme Ubc13, cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/2), IκB kinase regulatory subunit IKKγ (also called NEMO) and mitogen-activated protein kinase (MAPK) kinase kinase MEKK1 upon ligation. TRAF2, Ubc13, and IKKγ were required for complex assembly and activation of MEKK1 and MAPK cascades. However, these kinases were not activated unless the multi-component signaling complex translocated from CD40 to the cytosol upon c-IAP1/2-induced degradation of TRAF3. This twostage signaling mechanism may apply to other innate immune receptors, accounting for spatial and temporal separation of MAPK and IKK signaling.Cytoplasmic segments of cytokine, growth factor and antigen receptors serve as assembly sites for complexes containing adaptor proteins, protein kinases, and other signaling factors (1,2). Ligand-induced assembly of such complexes is thought to concentrate and subsequently activate protein kinases on the receptor, thereby triggering a plethora of effector pathways that control metabolism, proliferation and survival. Amongst cytokine receptors, the tumor necrosis factor receptor (TNFR) family contains members of biomedical importance that bind trimeric ligands (3,4). TNFR engagement results in assembly of multi-component receptor-associated signaling complexes that activate inhibitor of NF-κB (IκB) kinase (IKK) and mitogen-or stressactivated protein kinase (MAPK/SAPK) cascades (3,5). Key players in TNFR signaling are TNF receptor-associated factors (TRAF), serving as E3 ubiquitin ligases and adaptors that recruit and activate protein kinases that act at the apex of effector pathways (3,5-7). These protein kinases include transforming growth factor-β activated kinase 1 (TAK1) and MAP or extracellular signal-regulated kinase kinase kinase 1 (MEKK1), which in turn activate IKK (8), and the MAPK/SAPKs JNK and p38 (5,9). Most TRAF proteins contain an N-terminal RING finger required for signal transduction and formation of K63-linked polyubiquitin chains conjugated through the C-terminal glycine of the added ubiquitin moiety and K63 of the †To whom correspondence should be addressed.