5. What is the role of PAK1 in
intestinal inflammation?
Khare, Dammann et al. IBD. 2015
Proliferation Apoptosis
6. • PAK1 is a target of anti-inflammatory drug 5-ASA
– 5-ASA reduced tumorigenesis, PAK1 overexpression, and β-
catenin signaling in polyps in APCmin mice (Khare et al.
Biochem pharmacol. 2013)
• Does 5-ASA’s effect on tumorigenesis and Wnt
signalling require PAK1 inhibition?
– PAK1 deletion in 2 models of CRC
• APCmin and APCmin/ PAK1-/- mice
• AOM/DSS model in WT and PAK1-/- mice
– 1 I.P. injection of 10 mg/kg AOM and 4 cycles of 1.7% DSS
– ± 500mg/kg 5-ASA chow
What is the role of PAK1 in CRC?
14. Conclusion
• PAK1 overexpression and activation in IBD likely
drives tumorigenesis
– Proliferation-> AKT-β-catenin
• PAK1 deletion impairs tumor-initiation in APCmin
and AOM/DSS mice
– Downregulation of AKT-β-catenin signaling
• 5-ASA’s chemopreventive effects likely mediate
a PAK1-AKT-β-catenin axis
15. Thank you
• Supervisor: Univ. Prof. Dr. Christoph Gasche
• Co-supervisor: Dr. Vineeta Khare
• Colleagues and friends
– Mag. Michaela Lang
– Dr. Kristine Jimenez
– Dr. Rayko Evstatiev
– Felix Harpain
– Dr. Adrian Frick
– Tina Austerlitz
– Max Baumgartner
Editor's Notes
Talk about the microarray.
We also showed that PAK1 interacts with Beta catenin in both CRC and normal colon epithelial cells.
Figure 2 (A-C) PAK1 deletion impairs Wnt/ β-catenin signaling in APCmin adenomas. (A) Immunhistochemical (IHC) staining and corresponding dot-plots of PAK1, β-catenin, and p-β-catenin (Ser 675) in tumors of APCmin and APCmin/ PAK1-/- mice. PAK1 was expressed throughout the tumor in APCmin mice and absent in APCmin/ PAK1-/- mice. β-catenin and p-β-catenin (Ser 675) expression were significantly reduced in adenomas of APCmin/ PAK1-/- mice. (B) IHC staining of two targets downstream of β-catenin, Cyclin D1 and C-Myc. PAK1 deletion did affect Cyclin D1 expression levels, but significantly reduced expression of C-Myc in APCmin polyps. (C) IHC staining of p-AKT (Thr 308) demonstrating that p-AKT is reduced in tumors of APCmin/ PAK1-/- mice. IHC data are at least (n=5) mice per group. Independent samples T-test, *p<0.05, **p<0.01, *p<0.001.
Figure 2 (A-C) PAK1 deletion impairs Wnt/ β-catenin signaling in APCmin adenomas. (A) Immunhistochemical (IHC) staining and corresponding dot-plots of PAK1, β-catenin, and p-β-catenin (Ser 675) in tumors of APCmin and APCmin/ PAK1-/- mice. PAK1 was expressed throughout the tumor in APCmin mice and absent in APCmin/ PAK1-/- mice. β-catenin and p-β-catenin (Ser 675) expression were significantly reduced in adenomas of APCmin/ PAK1-/- mice. (B) IHC staining of two targets downstream of β-catenin, Cyclin D1 and C-Myc. PAK1 deletion did affect Cyclin D1 expression levels, but significantly reduced expression of C-Myc in APCmin polyps. (C) IHC staining of p-AKT (Thr 308) demonstrating that p-AKT is reduced in tumors of APCmin/ PAK1-/- mice. IHC data are at least (n=5) mice per group. Independent samples T-test, *p<0.05, **p<0.01, *p<0.001.
Figure 3 (A-D) The effect of PAK1 deletion on tumorigenesis in an AOM/DSS model. (A) Weight curves of male C57BL/6J WT and PAK1-/- mice with normal or 500 mg/kg 5-ASA chow throughout a 12 week azoxymethane/dextran sodium sulfate (AOM/DSS) model. Mice received one intraperitoneal injection of AOM and four cycles of 1.7% DSS in drinking water. DSS cycles were followed by two weeks of normal water. (B) Representative images of tumor size were taken during colonoscopy directly before euthanasia. Small tumor <25%, medium <50%, and large >50% of the colon circumference. The control (Con) image was taken from an untreated mouse without AOM/DSS. (C) Boxplots are colonoscopy data demonstrating total tumor number, small, medium, and large tumors in all four groups. PAK1 deletion reduced total tumor number but did not alter tumor size. 5-ASA reduced total tumor number and size. (D) Microscopic analysis of total tumor number, dysplastic lesions, and carcinoma in situ. PAK1 deletion and 5-ASA reduced both the total tumor number and the number of dysplastic lesions. Neither PAK1 deletion nor 5-ASA altered the number of carcinoma in situ. All data are (n=8) WT, (n=13) PAK1-/-, (n=16) WT + 5-ASA , and (n=13) PAK1-/- + 5-ASA mice. Statistical analysis were performed by ANOVA using LSD post hoc analysis, *p<0.05, **p<0.01, ***p<0.001.
Figure 4 (A-D) Inhibition of PAK1 impairs AKT/ β-catenin signaling in AOM/DSS tumorigenesis. (A) Immunohistochemistry (IHC) of PAK1 in control (Con) and 5-ASA treated mice. PAK1 is overexpressed in tumors in comparison to normal mucosa, and 5-ASA treatment reduced PAK1 overexpression. The dot plot demonstrates the mean immunoreactivity score (IRS) generated from tumors in control (n=11) and 5-ASA (n=9) treated WT mice. Statistical analysis were performed using the independent samples Mann Whitney U test, *p<0.05. (B) IHC images and IRS dot plots of β-catenin and phospho β-catenin (Ser 675). PAK1 deletion and 5-ASA treatment reduced expression of both total and phospho β-catenin. (C) IHC of p-AKT (Thr 308) and p-AKT (Ser 473). PAK1 deletion and 5-ASA reduced p-AKT (Thr 308) expression. The effect of 5-ASA on p-AKT (Thr 308) was blocked in PAK1-/- mice. 5-ASA blocked p-AKT (Ser 473) independent of PAK1. (D) IHC of p-mTOR (Ser 2448). 5-ASA reduced mTOR phosporylation, and this effect was reduced in PAK1-/- mice. Statistical analysis were performed by ANOVA using LSD post hoc analysis at least 5 mice per group, *p<0.05, **p<0.01, ***p<0.001.
Figure 4 (A-D) Inhibition of PAK1 impairs AKT/ β-catenin signaling in AOM/DSS tumorigenesis. (A) Immunohistochemistry (IHC) of PAK1 in control (Con) and 5-ASA treated mice. PAK1 is overexpressed in tumors in comparison to normal mucosa, and 5-ASA treatment reduced PAK1 overexpression. The dot plot demonstrates the mean immunoreactivity score (IRS) generated from tumors in control (n=11) and 5-ASA (n=9) treated WT mice. Statistical analysis were performed using the independent samples Mann Whitney U test, *p<0.05. (B) IHC images and IRS dot plots of β-catenin and phospho β-catenin (Ser 675). PAK1 deletion and 5-ASA treatment reduced expression of both total and phospho β-catenin. (C) IHC of C-Myc in AOM/DSS tumors. PAK1 deletion and 5-ASA reduced nuclear expression of C-Myc. (D) IHC of p-AKT (Thr 308). PAK1 deletion and 5-ASA reduced p-AKT (Thr 308) expression. The effect of 5-ASA on p-AKT (Thr 308) was blocked in PAK1-/- mice. Statistical analysis were performed by ANOVA using LSD post hoc analysis at least 4 mice per group, *p<0.05, **p<0.01, ***p<0.001.
