Chapter Three - IKK/Nuclear Factor-kappaB and Oncogenesis: Roles in Tumor-Initiating Cells and in the Tumor Microenvironment
Introduction
First described almost 30 years ago as a DNA-binding activity involved in the regulation of immunoglobulin κ light-chain gene expression (Sen & Baltimore, 1986), mammalian nuclear factor-kappaB pathway (NF-κB) is a family of five highly conserved transcription factors (RelA/p65, RelB, c-Rel, p50 [NF-κB1/p105 precursor], and p52 [NF-κB2/p100 precursor]) that form different homo- and heterodimers to regulate target gene expression (Baldwin, 2012, Hayden and Ghosh, 2012; Fig. 3.1). These proteins contain an approximate 300 amino acid conserved Rel homology domain that contains sequences for nuclear localization, DNA binding, dimerization, and interaction with the inhibitor of kappaB (IκB) proteins (Baldwin, 2012, Hayden and Ghosh, 2012). RelA, RelB, and c-Rel have C-terminal domains that contain transcriptional activation domains, while full-length p100 and p105 contain IκB-like ankyrin repeats (DiDonato, Mercurio, & Karin, 2012). NF-κB proteins, p50 and p52, are produced by proteolytic cleavage of precursors p105 and p100, respectfully. c-Rel is the cellular homologue of v-Rel, the transforming gene of avian retinculoendotheliosis virus. In Drosophila, there are three NF-κB family members (Dorsal, Dif, and Relish) that promote dorsoventral patterning in early development and innate immune signaling.
Two distinct regulatory pathways are known to control NF-κB activation: the canonical and noncanonical pathways (Fig. 3.2). The canonical pathway is controlled through an IKK complex which consists of the catalytic subunits, IKKβ and IKKα, and the regulatory and scaffold subunit, NEMO (IKKγ) (Ghosh et al., 1998, Israel, 2000, Karin and Ben-Neriah, 2000). Under resting conditions, the RelA/p50 heterodimer is held inactive by the IκB proteins. IκB physically blocks the nuclear localization sequence of RelA/p65 which leads to inactivation of the heterodimer. Various stimuli including lipopolysaccharide (LPS) and cytokines such as IL-1β and TNF trigger a signaling cascade through receptor-induced signaling to activate the IKK complex, with IKKβ functioning as the dominant kinase in this cascade (DiDonato et al., 2012). This leads to phosphorylation of IκBα on Ser32/Ser36, resulting in rapid IκBα ubiquitination and proteasome-dependent degradation. Once RelA/p50 is free from IκBα inhibition, it is able to accumulate in the nucleus and bind to kappaB sites within promoters and regulatory regions of genes that regulate apoptosis, the inflammatory response, and cell proliferation (DiDonato et al., 2012; Fig. 3.3). Along with phosphorylation and degradation of IκBα, posttranslational modifications of RelA/p65, including acetylation and methylation, can modulate NF-κB activity (Yang, Tajkhorshid, & Chen, 2010). Additionally, NF-κB activates transcription of the gene encoding its own inhibitor, IκBα, thus providing a negative feedback loop for additional control.
The noncanonical pathway, however, is controlled through an IKKα complex and leads to activation of the p52–RelB heterodimer (Hayden and Ghosh, 2004, Hayden and Ghosh, 2012). Ligands of BAFF, CD40, and LTβ-R ligands activate the noncanonical pathway by activating NIK which phosphorylates and activates IKKα homodimers, leading to phosphorylation and subsequent proteolytic processing of p100 to p52 (Senftleben et al., 2001). The transcriptionally active RelB–p52 heterodimer can then enter the nucleus to promote transcription of a variety of genes involved in B cell homeostasis and cellular development and differentiation (see Fig. 3.3).
While IKK is critical for activation of NF-κB complexes downstream of cytokine signaling and through oncoprotein expression, evidence has been presented that IKK can phosphorylate and regulate critical regulatory proteins involved in distinct signaling pathways. For example, it was reported that IKKβ can phosphorylate the tumor suppressor p53 leading to its destabilization (Xia et al., 2009). This finding is consistent with a prosurvival function for IKK signaling, blocking the potential apoptotic functions of p53. IKKβ was shown to phosphorylate the forkhead transcription factor Foxo3a, promoting its ubiquitination and proteolysis (Hu et al., 2004). IKKα phosphorylates the p27/Kip1 cdk inhibitor to promote tumor-initiating cells (TICs) in ErbB2-driven breast cancer (Zhang et al., 2013). Downstream of activated Akt, in a variety of cancers and in response to insulin exposure, IKKα interacts with mTORC1 to drive its activity (Dan, Adli, & Baldwin, 2007). Interestingly, this interaction reciprocally promotes IKK activity to activate NF-κB (Dan et al., 2008). These functions of IKK are clearly relevant to oncogenesis as blocking p53 function, suppressing p27 activity, and driving mTORC1 activity should promote tumorigenic potential of most cancers.
NF-κB is activated in a variety of cancers as detected by phosphorylation of IκBα and RelA, elevated levels of nuclear canonical and/or noncanonical forms of NF-κB, phosphorylation of IKK, and elevated expression of NF-κB target genes such as IL-6 and a variety of chemokines/cytokines. Most oncoproteins, such as oncogenic Ras alleles, and growth factor receptors (such as EGFR and ErbB2/Her2) are known to lead to NF-κB signaling (Finco et al., 1997, Merkhofer et al., 2010). In fact, IKK/NF-κB is required for efficient cell transformation and tumorigenesis induced by oncogenic Ras (Basseres et al., 2010, Ling et al., 2012, Mayo et al., 1997, Meylan et al., 2009, Xia et al., 2012). Loss of tumor suppressors such as PTEN and p53 have been shown to lead to elevated NF-κB activity. Interestingly, loss of p53 was shown to lead to a glucose-driven modification of IKK by O-GlcNac to increase its activity (Kawauchi et al., 2008, Kawauchi et al., 2009). Thus, many oncogenic signaling pathways lead to activation of IKK and NF-κB (see Table 3.1).
In most solid tumors, members of the IKK/NF-κB pathway are rarely mutated, presumably because this pathway functions downstream of oncogenic signaling pathways that are found mutated. These upstream activating mutations typically activate IKK/NF-κB along with additional signaling cascades such as Akt or MEK/Erk. In this regard, Akt signaling can promote IKK/NF-κB signaling. Importantly, both canonical and noncanonical NF-κB have been shown to be important for K-Ras-driven pancreatic cancer cell growth and survival. Downstream of oncogenic K-Ras, GSK-3α coordinates the stability of the TAK1/TAB complex to drive canonical IKK activation and promotes p100 to p52 processing to drive noncanonical NF-κB activation (Bang, Wilson, Ryan, Yeh, & Baldwin, 2013).
Genes encoding NF-κB pathway members have been found to be amplified in a variety of cancers and include TRAF6, IKBKB, IKBKG, IRAK1, and RIPK1 (Beroukhim et al., 2010). IKKβ and IKKγ, members of the important IKK complex, are also amplified in certain cancers as is the IKK-related kinase IKKɛ (Beroukhim et al., 2010, Boehm et al., 2007). In hematologic malignancies such as multiple myeloma and diffuse large B cell lymphoma, both canonical and noncanonical signaling pathways exhibit activating mutations (Annunziata et al., 2007, Keats et al., 2007, Lenz et al., 2008).
In cancer, NF-κB is proposed to contribute to oncogenesis through the induction of genes encoding proteins involved in suppressing apoptosis, promoting invasion and angiogenesis, and enhancing epithelial–mesenchymal transition (EMT) (Baldwin, 2001, Basseres and Baldwin, 2006). As described earlier, IKK can promote oncogenic phenotypes separate from its ability to promote NF-κB activation. NF-κB can have both tumor suppressive (Perkins & Gilmore, 2006) and oncogenic (Basseres & Baldwin, 2006) properties and is at the forefront of studies on inflammation and cancer (Greten et al., 2004, Pikarsky et al., 2004). As the topic of NF-κB in cancer is very expansive, we will focus on two areas of research in this review that have not been extensively reviewed: NF-κB involvement in the tumor microenvironment and in TICs.
Section snippets
Background on the tumor microenvironment
Tumors are heterogenic and are composed of tumor cells, which are heterogenous themselves, and noncancerous cells that are recruited into the tumor. A wide array of noncancerous cells associated with tumors make up the tumor stroma/microenvironment including macrophages, fibroblasts, T cells, B cells, neutrophils, etc. Research on the tumor microenvironment has seen an explosion of interest in recent years as it has become apparent that an environment rich in cells of the immune system such as
TICs/Cancer Stem Cells
Solid tumors are typically heterogeneous, being comprised of cells with different phenotypic and signaling properties. One subset of cells, known as tumor-initiating cells (TICs) or cancer stem cells, exhibit properties of self-renewal and tumorigenic potential when delivered to immunodeficient mice recipients (Charafe-Jauffret et al., 2009, D'Angelo and Wicha, 2010, Ginestier et al., 2010; Fig. 3.6). Additionally, these cells exhibit an EMT phenotype, are chemo/radioresistant, and are
Conclusions
The NF-κB pathway plays a dynamic role in various cancers and is becoming more understood as a key player in stromal interactions and in TIC function within the tumor. The influence of NF-κB can be seen in the form of tumor progression and other aggressive properties like TIC expansion and invasion. This highlights the fact that NF-κB is a viable target for therapeutic options for both cancer and stromal cells.
Acknowledgments
We thank members of the Baldwin lab for insightful discussion pertaining to this review, and we thank Scott Bradford for assisting with the figures. The authors’ work is funded by NCI Grants CA73756 and CA75080, NIH Grants AI35098 and F32 CA162628-01, and the Waxman Cancer Research Foundation. The authors declare no conflict of interest.
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