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. 2014 Sep 30;5(18):8778-89.
doi: 10.18632/oncotarget.2398.

p-21 activated kinase 4 promotes proliferation and survival of pancreatic cancer cells through AKT- and ERK-dependent activation of NF-κB pathway

Nikhil Tyagi  1 , Arun Bhardwaj  1 , Ajay P Singh  2 , Steven McClellan  1 , James E Carter  3 , Seema Singh  1
Affiliations

p-21 activated kinase 4 promotes proliferation and survival of pancreatic cancer cells through AKT- and ERK-dependent activation of NF-κB pathway

Nikhil Tyagi et al. Oncotarget. .

Abstract

Identification of novel molecular targets and understanding the mechanisms underlying the aggressive nature of pancreatic cancer (PC) remain prime focus areas of research. Here, we investigated the expression and pathobiological significance of p21-activated kinase 4 (PAK4), a gene that was earlier shown to be amplified in a sub-set of PC. Our data demonstrate PAK4 overexpression in PC tissues and cell lines with little or no expression in the normal pancreas. PAK4 silencing in two PC cell lines, MiaPaCa and T3M4, by RNA interference causes suppression of growth and clonogenic ability due to decreased cell cycle progression and apoptosis-resistance. PAK4-silenced PC cells exhibit altered expression of proliferation- and survival-associated proteins. Moreover, we observe decreased nuclear accumulation and transcriptional activity of NF-κB in PAK4-silenced PC cells associated with stabilization of its inhibitory protein, IκBα. Transfection of PAK4-silenced PC cells with constitutively-active mutant of IKKβ, an upstream kinase of IκBα, leads to restoration of NF-κB activity and PC cell growth. Furthermore, we show that PAK4-induced NF-κB activity is mediated through activation and concerted action of ERK and Akt kinases. Together, these findings suggest that PAK4 is a regulator of NF-κB pathway in PC cells and can serve as a novel target for therapy.

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Conflict of interest statement

Conflict of Interest

No potential conflict of interest to disclose.

Figures

Figure 1
Figure 1. PAK4 expression analysis in pancreatic cancer tissue specimens and cell lines
(A) Immunohistochemical assay was performed on paraffin embedded tissue microarray containing tissue sections (in triplicates) of normal pancreas (n=9) and pancreatic tumor (n=56) using PAK4 specific antibody. PAK4 expression was examined by immunoblot assays in (B) frozen pancreatic tissues [normal (n=7) and malignant n=21] specimens, (C) PC cell lines and (D) in vitro pancreatic cancer progression model (hTERT-HPNE and derived cell lines). β-actin was used as internal control. Bars (mean ± SEM, n=3) indicate the normalized expression levels of PAK4.
Figure 2
Figure 2. PAK4-overexpression is associated with increased growth and clonogenic potential of pancreatic cancer cells
(A) Total protein from the stable pooled population of PAK4-silenced (MiaPaCa-shPAK4 and T3M4-shPAK4) along with their respective control cells (MiaPaCa-NTScr and T3M4-NTScr) was isolated and PAK4 expression was determined by immunoblot analyses. β-actin was used as loading control. (B) Cells (1×104) were seeded in 6-well plates; growth was monitored by counting the cell number daily up to 8 days. The doubling time (dt) and percent inhibition in growth on 8th day was calculated as described in Materials & Methods. (C) For anchorage dependent clonogenicity assay, cells were seeded at low density (1×103cells/well) in regular media. After 2 weeks, colonies were stained with crystal violet, visualized, photographed and counted using imaging system. (D) For anchorage independent clonogenicity assay, cells (2.5×103 cells/mL) suspended in regular media containing 0.4 % agarose were seeded in 6-well plate having bottom layer of 0.8 % agar growth medium and allowed to form colonies for 3 weeks. After 3 weeks, colonies were visualized and counted using Nikon eclipse microscope. Data represent as mean ± SEM. n=3, *, p< 0.05.
Figure 3
Figure 3. PAK4 facilitates cell cycle progression and confers apoptosis resistance
(A) For cell cycle analysis, PAK4 silenced (MiaPaCa-shPAK4 and T3M4-shPAK4) cells along with their respective controls (MiaPaCa-NTScr and T3M4-NTScr) were synchronized by culturing them in serum-free media for 72 h, and then incubated in regular medium for 24 h. Subsequently, distribution of cells in different phases of cell cycle was analyzed by Propidium iodide (PI) staining followed by flow cytometry. (B) For apoptosis assay, PC cells were grown in regular media for 72 h and percentage of apoptotic cells was analyzed by PE Annexin V and 7AAD staining followed by flow cytometry.
Figure 4
Figure 4. Effect of PAK4 silencing on the expression of proteins associated with cell-cycle and apoptosis
Total protein from PAK4-silenced (MiaPaCa-shPAK4 and T3M4-shPAK4) cells along with their scrambled control (MiaPaCa-NTScr and T3M4-NTScr) was isolated and expression of various cell cycle and survival-associated proteins was examined by immunoblot analysis. β-actin was used as an internal control.
Figure 5
Figure 5. PAK4 induces transcriptional activity of NF-κB/p65 in human pancreatic cancer cells by promoting its nuclear translocation
(A) Sub-confluence level of MiaPaCa and T3M4 cells were co-transfected with NF-κB responsive luciferase reporter and TK-Renilla luciferase (control) plasmids. 48 h post-transfection, cells were harvested in passive lysis buffer and luciferase (Fire-fly; test and Renilla, transfection efficiency control) activity was assessed using a dual-luciferase assay system. Data is presented as normalized fold-change in luciferase activity (mean± SEM; n = 3, *, p < 0.05). (B) Total, nuclear and cytoplasmic extracts were prepared and expression of NF-κB/p65, p-IκBα (S32/36) and IκBα was determined by immunoblot analysis. Laminin (for nuclear fraction), α-tubulin (for cytoplasmic fraction) and β-actin (for total fraction) were used as loading controls.
Figure 6
Figure 6. NF-κB-mediates PAK4-induced proliferation and apoptosis resistance in pancreatic cancer cells
PAK4 silenced (MiaPaCa-shPAK4 and T3M4-shPAK4) cells were transfected with constitutively active IKKβ mutant (IKKβ-SSEE) or empty vector (pCMV). (A) After 48 h transfection, cells were again transfected with NF-κB -luciferase promoter-reporter constructs and NF-κB transcriptional activity was examined as described previously. Bars represent the mean of triplicates ± SEM, *, p < 0.05. (B) Nuclear and cytoplasmic fractions were prepared after 24 h of transfection and expression level of NF-κB was examined by immunoblot analysis. Laminin (for nuclear fraction), α-tubulin (for cytoplasmic fraction) and β-actin (for total fraction) were used as loading controls. (C) After 72 h of transfection, cell viability was examined by WST-1 assay. Bars represent the mean of triplicates ± SEM, *, p < 0.05. (D) Total protein lysate was prepared after 48 h of transfection and expression level of various cell cycle and survival-associated proteins were examined by immunoblot analysis. β-actin was used as loading control.
Figure 7
Figure 7. PAK4-activated Akt and ERK cooperatively promotes nuclear accumulation and transcriptional activity of NF-κB
(A) Total cellular protein was isolated from PAK4-silenced (MiaPaCa-shPAK4 and T3M4-shPAK4) and control (MiaPaCa-NTScr and T3M4-NTScr) cells and expression level of Akt, p-Akt, ERK, p-ERK was examined by immunoblot analysis. β-actin was used as loading control. (B) PAK4 expressing (MiaPaCa-NTScr and T3M4-NTScr) cells were treated with Akt inhibitor (LY294002, 20 μm) or ERK inhibitor (PD98059, 25 μm) 1 h prior to the transfection of NF-κB-luciferase promoter-reporter construct and NF-κB transcriptional activity was examined after 48 h as described previously. Bars represent the mean of triplicates ± SEM, *, p< 0.05. (C) Total and nuclear protein extracts from the MiaPaCa-NTScr and T3M4-NTScr cells treated with Akt and/or ERK inhibitors alone or in combination for 24 h were prepared and effects on NF-κB/p65 (in nuclear), p-IκBα, and IκBα (in total) were examined by immunoblot analysis. Laminin (for nuclear fraction) and β-actin (for total fraction) were used as loading.
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References

    1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29. - PubMed
    1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:11–30. - PubMed
    1. Abo A, Qu J, Cammarano MS, Dan C, Fritsch A, Baud V, Belisle B, Minden A. PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia. EMBO J. 1998;17:6527–40. - PMC - PubMed
    1. Kumar R, Gururaj AE, Barnes CJ. p21-activated kinases in cancer. Nat Rev Cancer. 2006;6:459–71. - PubMed
    1. Radu M, Semenova G, Kosoff R, Chernoff J. PAK signalling during the development and progression of cancer. Nat Rev Cancer. 2014;14:13–25. - PMC - PubMed

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