Oncogenic activation of NF-kappaB

Abstract

Recent genetic evidence has established a pathogenetic role for NF-kappaB signaling in cancer. NF-kappaB signaling is engaged transiently when normal B lymphocytes respond to antigens, but lymphomas derived from these cells accumulate genetic lesions that constitutively activate NF-kappaB signaling. Many genetic aberrations in lymphomas alter CARD11, MALT1, or BCL10, which constitute a signaling complex that is intermediate between the B-cell receptor and IkappaB kinase. The activated B-cell-like subtype of diffuse large B-cell lymphoma activates NF-kappaB by a variety of mechanisms including oncogenic mutations in CARD11 and a chronic active form of B-cell receptor signaling. Normal plasma cells activate NF-kappaB in response to ligands in the bone marrow microenvironment, but their malignant counterpart, multiple myeloma, sustains a variety of genetic hits that stabilize the kinase NIK, leading to constitutive activation of the classical and alternative NF-kappaB pathways. Various oncogenic abnormalities in epithelial cancers, including mutant K-ras, engage unconventional IkappaB kinases to activate NF-kappaB. Inhibition of constitutive NF-kappaB signaling in each of these cancer types induces apoptosis, providing a rationale for the development of NF-kappaB pathway inhibitors for the treatment of cancer.

Figure 1.

Role of BCR signaling to NF-κB in ABC DLBCL. (A) Schematic of BCR signaling to NF-κB. Recurrent genetic alterations in ABC DLBCL that result in constitutive NF-κB activation are indicated by the gray boxes. Proximal signaling by the BCR is initiated by SRC-family kinases (SFK; e.g., LYN, FYN, FGR, and BLK), which phosphorylate the ITAM motifs in the CD79A and CD79B components of the BCR receptor. SYK is recruited to the phosphorylated ITAMs and activated to phosphorylate many downstream proteins. The PI(3) kinase pathway is activated by SRC-family kinase phosphorylation of the BCR coreceptor CD19. The generation of PIP3 by PI(3) kinase recruits BTK and associated BLNK and phospholipase Cγ2 (PLCγ2) to the plasma membrane. PLCγ2 generates inositol triphosphate (IP3), which leads to opening of the capacitative calcium channel, thereby activating the NF-AT pathway. Diacylglycerol (DAG) is also generated, which activates protein kinase Cβ (PKCβ). PKCβ phosphorylates the latent form of CARD11 (CARD-off) in the cytoplasm, causing it to adopt an active conformation (CARD11-on), translocate to the plasma membrane, and recruit the signaling adapters BCL10 and MALT1. MALT1 binds TRAF6, causing TRAF6 to catalyze K63-linked polyubiquitination of the IKKγ subunit, MALT1 and itself. TAB2 recognizes the polyubiquitin chains on TRAF6, leading to phosphorylation of IKKβ in its activation loop by TAK1 kinase. Ubiquitination of IKKγ and phosphorylation of IKKβ activate IKKβ to phosphorylate IκBα. Phosphorylated IκBα is ubiquitinated by the ubiquitin ligase βTrCP, leading to its proteasomal degradation. Nuclear NF-κB heterodimers activate multiple target genes, including A20. A20 terminates NF-κB signaling by removing K63-linked ubiquitin chains from IKKγ, TRAF6, and MALT1, and attaching K48-linked ubiquitin chains. (B) The role of chronic active BCR signaling in the pathogenesis of ABC DLBCL. Genetic mutations in CD79B and CD79A ITAM regions in ABC DLBCLs suggest that BCR signaling is key to the pathogenesis of ABC DLBCL. CD79B and CD79A ITAM mutations in the mouse cause hyperactive BCR signaling, suggesting that CD79 ITAM mutations in ABC DLBCL may amplify antigen-stimulated BCR signaling. CD79 ITAM mutations increase surface BCR expression and decrease activation of LYN kinase, a negative regulator of BCR signaling, potentially resulting in increased signaling to NF-κB and greater clonal expansion. A separate step leading to chronic active BCR signaling in ABC DLBCL is the acquisition of spontaneous BCR clustering, which is not caused by the ITAM mutations. The BCR clustering phenotype could theoretically be acquired either before or after the CD79 mutations. Finally, ABC DLBCLs must acquire additional oncogenic hits to become fully malignant.

Figure 2.

Pathogenesis of MALT lymphoma. (A) Schematic of the c-IAP2-MALT1 fusion oncoprotein produced by the t (11;18) translocation in MALT lymphomas. Shown is one common fusion breakpoint (dashed line), but other breakpoints occur less frequently (for review, see Isaacson et al. 2004). (B) Molecular mechanisms of NF-κB activation by the c-IAP2-MALT1 fusion protein. c-IAP2-MALT1 forms multimers by heterotypic interactions between its BIR and MALT1 regions. c-IAP2-MALT1 binds TRAF6, activating the K63-linked ubiquitin ligase activity of TRAF6 for IKKγ and itself. Polyubiquitated TRAF6 binds TAB2, thereby activating the associated TAK1 kinase to phosphorylate IKKβ. The ubiquitin-binding domain of c-IAP2-MALT1 stabilizes its interaction with ubiquitinated IKK. The proteolytic cleavage of a substrate protein, potentially A20, by the paracaspase domain of MALT1 is required for the function of c-IAP2-MALT1. TRAF2 is also required for c-IAP2-MALT1 activity, but its substrate is unknown.

Figure 3.

Constitutive NF-κB pathway activation in multiple myeloma. (A) Genetic abnormalities that activate NF-κB in multiple myeloma. Gray boxes highlight recurrent genetic aberrations in multiple myeloma involving NF-κB pathway components. The kinase NIK is tethered to the ubiquitin ligases c-IAP1 and/c-IAP2 (c-IAP1/2) by TRAF2 and TRAF3, leading to rapid turnover of NIK protein because of c-IAP1/2-catalyzed K46-linked polyubiquitination. On recruitment to a subset of TNF receptor-family proteins—notably CD40, TACI, and lymphotoxin-β receptor (LTBR)—TRAF2 ubiquitin ligase activity is induced, leading to K63-linked polyubiquitination of c-IAP1/2. The K46-linked ubiquitin ligase activity of c-IAP1/2 is directed toward TRAF3, leading to its proteasomal degradation. NIK is liberated and stabilized in the process. NIK overexpression stimulates both the classical and alternative NF-κB pathways. The deubiquitinase CYLD negative regulates this signaling pathway by removing K63-linked polyubiquitin chains from IKKγ. (B) Model of NF-κB activation during the genesis of multiple myeloma. Normal plasma cells receive signals from BAFF and APRIL, two TNF family ligands in the bone marrow microenvironment. Their receptors, TACI and BCMA, are highly expressed in multiple myeloma and signal to NF-κB through both the classical and alternative NF-κB pathways. Initial transformation is often caused by oncogenic translocations, but the myeloma cell may still remain dependent on the bone marrow microenvironment to receive prosurvival NF-κB signals. Myeloma cells that acquire mutations that cause constitutive NF-κB pathway activation are selected because they allow the malignant cells to survive and proliferate without being limited by the bone marrow microenvironment.

Figure 4.

Solid tumors activate the NF-κB pathway to maintain cell survival. (A) IKKε is frequently overexpressed in breast cancer in association with a gain or amplification of its genomic locus. IKKε phosphorylates a serine residue in CYLD, thereby lowering its deubiquitinase activity toward the IKKγ subunit, leading to a more active IKK enzyme. In addition, IKKε can directly phosphorylate serine-46 of IκBα. (B) Regulation of NF-κB by TBK1 downstream of mutant K-ras in lung cancer. Mutant K-ras signals to several downstream pathways, including the MAP kinase, PI(3) kinase, and RAL GTPase pathways. K-ras-associated RAL guanine nucleotide exchange factors (Ral-GEFs) promote the active, GTP-bound state of the RAL proteins. RalB interacts with Sec5, which in turn recruits TBK1, causing kinase activation. TBK1 triggers classical NF-κB pathway activation, as judged by the accumulation of nuclear p50/c-rel heterodimers. Because TBK1 can only phosphorylate one serine in IκBα, an as yet unknown kinase must cooperate with TBK1 to achieve dual IκBα phosphorylation.