Abstract SY06-04: Regulation of cancer cell proliferation and survival by NF-κB
Authors: Perkins Neil 
Published: May 2015

In mammalian cells the NF-κB family of transcription factors consists of five subunits, RelA (p65), c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52), which form a wide variety of homodimeric and heterodimeric complexes. In most normal, unstimulated cells, NF-κB complexes are held in an inactive form, bound to one of a family of inhibitory proteins, termed IκBs. The precursor proteins p100 and p105 can also function as IκBs, prior to their processing to p52 and p50, which function as nuclear regulatory subunits. There are, broadly speaking, two major pathways leading to induction of NF-κB subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes and involves the degradation of IκBα in a manner dependent on IκB kinase (IKK) β and the IKK regulatory subunit NEMO (IKKγ). The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of p52/RelB containing complexes, and is dependent on IKKα and NF-κB inducing kinase (NIK).

Aberrantly active NF-κB is associated with many diseases, including cancer. The ability to both respond to and induce inflammatory stimuli is an important component of NF-κB's role in disease. NF-κB also has other functions and can contribute to tumorigenesis through inducing proliferation, metastasis as well as resistance to apoptosis. However, NF-κB can also exhibit apparently contradictory functions, more akin to those of a tumor suppressor. These include pro-apoptotic activity in response to some stimuli and induction of cellular senescence. NF-κB is also associated with the senescence associated secretory phenotype (SASP), which can exhibit tumor-promoting properties but also contribute to the effectiveness of cancer therapy.

My laboratory is interested in how NF-κΒ subunits are regulated by oncogenes, tumor suppressors and stimuli associated with cancer development and therapy. A theme emerging from these studies has been the importance of context for NF-κΒ activity and how subunits can both repress as well as activate gene targets, leading to alterations in cell fate. We have demonstrated the importance of post-translational modifications in controlling these activities and argued that the activity of parallel signaling pathways have a critical role in determining NF-κΒ dependent transcriptional output. Based on this work, together with that from other researchers, we propose that the concerted action of tumor suppressors functions to keep the oncogenic activities of NF-κΒ subunits in check and that loss of tumor suppressor activity during tumor development is required to unleash these anti apoptotic and pro-metastatic activities in malignant cancer cells.

The role of c-Rel in the pathogenesis of B-cell lymphoma. Deregulation of the c-Rel subunit is common in haematological malignancies with 50% of some types of B-cell lymphoma reported to have gain or amplification of the region containing the REL gene. However, studies assessing the functional role of c-Rel in the pathogenesis of lymphoma are sparse. To address this, we have used the Eμ-Myc mouse model, which develop spontaneous B-cell lymphoma at age 3-6 months.

In vivo imaging of Eμ-Myc mice incorporating an NF-κB luciferase reporter transgene confirmed that NF-κB activity was greater in lymphoid organs of pre-malignant Eμ-myc/NF-κB reporter animals. Surprisingly, Eμ-myc/crel-/- mice demonstrated significantly shorter overall survival and accelerated disease progression compared with Eμ-myc mice, suggesting that c-Rel loss can, contrary to current models, drive lymphomagenesis. However, consistent with the known role of c-Rel as a regulator of cell death, we observed that Eμ-myc/crel-/- tumors grown ex vivo underwent apoptosis more rapidly. Therefore, to determine whether these effects were due to the host microenvironment, Eμ-myc tumors were implanted into either WT or crel-/- animals and tumor development monitored. By contrast with spontaneous, lymphoma development, we found that Eμ-myc tumors developed faster but with similar kinetics in both WT and crel-/- mice, suggesting that the effects on survival are not due to differences in stroma. Data will be presented on the mechanism/s behind faster lymphoma development in Eμ-myc/crel-/- mice. These studies confirm the importance, and complexity of c-Rel in the pathogenesis of B-cell lymphoma, in which it may be acting as both a tumour promoter and suppressor.

The RelA(p65) T505A Knock-in Mouse. We have previously demonstrated that RelA function in cancer cells can be regulated by phosphorylation of Thr505 within the C-terminal RelA transactivation domain. Phosphorylation at this site by Chk1 in response to certain DNA damage agents and in S-phase following DNA-replication checkpoint activation, regulates divergent NF-κB functions, including apoptotic cell death. We have created a RelA T505A knock-in mouse to investigate the in vivo functional link between RelA and DNA damage activated checkpoint kinase signaling.

Adult RelA T505A mice are healthy with no overt immunological alterations but exhibit some abnormalities such as heavier spleens and enlarged lymph nodes. Significantly, analysis of RelA T505A immortalized mouse embryonic fibroblasts, confirmed the importance of T505 as a mediator of pro-apoptotic RelA functions in response to certain inducers of DNA replication stress, such as the chemotherapeutic drug cisplatin. Moreover, T505A mice exhibit faster regrowth and an enhanced proliferative response in models of liver regeneration, liver partial hepatectomy and acute CCl4 treatments. Previous data from our group had predicted a tumor suppressor role for T505 phosphorylation. To this end, RelA T505A mice were crossed onto Eμ-Myc mice that spontaneously develop B-cell lymphoma. Significantly, T505A homozygous mice showed accelerated lymphoma onset compared to wildtypes.

These studies suggest that RelA T505 negatively regulates NF-κB function under genotoxic stress and modulation of RelA activity by checkpoint kinase signaling pathways can play an important role in determining NF-κB function during tumorigenesis.

Research in the Perkins lab is funded by Cancer Research UK (grant C1443/A12750), Leukaemia and Lymphoma Research (grant 11022), Association of International Cancer Research (grant 13-1150) and the Wellcome Trust (grant 094409).

Citation Format: Neil Perkins. Regulation of cancer cell proliferation and survival by NF-κB. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY06-04. doi:10.1158/1538-7445.AM2014-SY06-04