The development of IKKα inhibitors as novel anti- pancreatic cancer drugs

Background: Pancreatic cancer is the 7th most common cancer in world and 8th among all cancers with regards to death rate. The main feature is very poor prognosis; the survival rate at 5 years from diagnosis is less than 5% (Jemal et al., 2008). Nuclear Factor kappa B (NFκB) is a transcription factor which plays a key role in many cellular processes related to cancer, for example, cell growth and survival (Gamble et al., 2012). To date there have been limited studies into the role of NFκB signalling in pancreatic cancer; however is emerging that the IKKα-regulated non-canonical NFκB pathway may be strongly linked to progression of pancreatic ductal adenocarcinomas (PDAC) (Wharry et al., 2009 ). Therefore, this study aims to characterise NFκB signalling in the pancreatic cancer setting examining the potential effects of novel IKKalpha small molecule kinase inhibitors.

Methods: All experiments were conducted in pancreatic cancer cell lines (Panc-1 cells). Panc-1 cells were grown on 6 or 12 well plates. Cells were pre-treated with different concentration of IKKα inhibitors and then stimulated with suitable agonist for activation NFκB pathways. Western blotting was used to determine the changing in the NFκB pathways (non-canonical; pp100, p52, canonical; IkBα, pp65). IKKα-selective small molecule kinase inhibitors were synthesised in-house as part of a CRUK small molecule drug discovery programme (PI Prof. S. MacKay).

Results: In Panc-1 cells, LTα1β2 stimulated the IKKα-dependent non-canonical NFκB pathway, inducing phosphorylation of p100 after 4h of stimulation (fold increase 27.75± 2.11, p<0.01), while the maximum activation of p52 formation was between 24 (33.96 ± 1.79 fold, p< 0.001) and 48h (24.82 ± 1.43 fold, p<0.001) compared with untreated sample. TNFα and FCS were without effect. TNFα and LTα1β2 stimulated the IKKβ-dependent canonical NFκB pathway. The results showed that the phosphorylation of p65 was evident after 5 min of stimulation (31.2 ± 2.12 fold, p< 0.001) and 15 min (26.38 ± 1.16 fold, p< 0.001), whilst cellular IκBα degradation was observed between 15 and 30 min of stimulation, the percentage of degradation was 97.2 ± 0.95, p<0.001 and 98.1 ± 0.88, p< 0.001 respectively compared with untreated sample. A number of novel IKKα inhibitors were also tested against both the non-canonical and canonical NFκB pathways. Five different compounds with varying IKKα versus IKKβ selectivity were examined; SU1087 (Ki: 4 vs 10nM), SU1261 (Ki:10 vs. 680nM); SU1392 (67 vs. 10000nM); SU1411 (50 vs. 840) and SU1436 (Ki: 93 vs. 3100nM)

Treatment with SU compounds inhibited LTα1β2-stimulated p100/52 processing to varying degrees, all in in a concentration-dependent manner; SU1261 - IC50 2.09μM, n=3; SU1411 - IC50 2.08μM, n=3; SU1087 IC50 2.11 μM; SU1436 - IC50 2.034 μM. SU compounds with higher potency against IKKβ inhibited TNFα-stimulated IκBα degradation; SU1087 - IC50 2.13 μM, n=3; SU1436 - IC50 4.68 μM. SU1392 had no effect on both canonical and non-canonical NFκB pathways.

Conclusion: In conclusion it has been shown that the IKK -dependent non-canonical pathway is active in pancreatic cells and can be inhibited by novel IKKα-selective small molecule kinase inhibitors.

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Wharry et al . 2009; 8(16): 1567-1576.