A novel PAK1/Notch-1 axis contributes to intestinal inflammation and cancer

Background: PAK1 belongs to a family of serine-threonine kinase and contributes to various oncogenic pathways like MAPK and Wnt-β catenin. Our lab recently linked activation of p21-activated kinase (PAK1) to IBD as well as colitis-associated cancer (Khare V, Inflamm Bowel Dis 2015). Here we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL-10 KO mice.

Methods: IL-10 KO mice were crossed with Pak1 KO mice to generate double-knockout mice (DKO). Inflammation was triggered with piroxicam (200ppm mixed to chow for 14 d). After 12 wks mice were euthanized, intestines were prepared and length was measured. Paraffin embedded Swiss rolls were stained with H&E and PAS. IHC was performed with antibodies against Ki67, ERK1/2, activated Notch1, γH2AX, p65/NFκB, lysozyme and LGR5.

Results: Unexpectedly, DKO mice displayed higher disease activity [DAI: 2.448 ± 0.5 vs. 1.095 ± 0.34], higher grades of inflammation [3.08 ± 0.22 vs. 1.25 ± 0.5] and more dysplastic lesions [6.5 ± 1.8 vs. 0.25 ± 0.25] compared to IL-10 KO mice. Interestingly, despite a higher degree of inflammation DKO mice had longer colons compared to IL-10 KO [10.83 cm ± 0.57 vs. 8.50 cm ± 0.46]. IHC (Table 1) revealed increase in Ki67 positive nuclei (indicating proliferation); MAPK pathway activation, as shown by increased pERK1/2 staining and an increase in γH2AX positive nuclei, indicating more DNA double-strand breaks. A reduced differentiation of goblet cells and Paneth cells was marked by PAS- and Lysozyme staining, together with an expansion of the stem cell compartment (shown by increased activated Notch1 and LGR5 staining) in the colonic crypts of DKOs. Interestingly NFκB was found to be reduced in DKO mice.

	IL-10 KO	DKO	P value
Ki-67 pERK1/2 (MAPK) γH2AX PAS	0.34 ± 0.01 0.33 ± 0.03 0.29 ± 0.02 17.6 ± 1.06	0.66 ± 0.03 0.59 ± 0.02 0.50 ± 0.025 5.50 ± 0.45	< 0.001 < 0.001 < 0.001 < 0.001
Lysozyme Activated Notch p65/NFκB LGR5	45.72 ± 3.44 0.39 ± 0.02 0.53 ± 0.02 25.00 ± 2.07	8.08 ± 1.96 0.63 ± 0.03 0.18 ± 0.01 53.08 ± 4.40	0.0063 < 0.001 < 0.001 < 0.001

Conclusion: Our data suggest a novel interaction of PAK-1 with the Notch pathway in regulating crypt cell stemness, proliferation and differentiation. Lack of mature goblet cells and mucus can exacerbate colitis in IL-10 KO mice. Hyperproliferation may promote dysplastic lesions independent of NFκB.