Nhi Huynh

P21-activated kinase 1 stimulates colon cancer cell growth and migration/invasion via ERK- and AKT-dependent pathways

The p21-activated kinase (PAK) family of serine/threonine kinases plays an important role in cell proliferation, survival and motility, as well as in cell transformation and tumor progression. PAK1 promotes transformation through facilitating the ERK/MAPK pathway and enhances cell migration and survival by stimulating AKT. PAK1 expression increases with the progression of colorectal cancer (CRC). In this study, we have investigated the importance of PAK1 in the biology of colon cancer cells. Reduction of PAK1 expression decreased the activities of ERK and AKT leading to decreased cell proliferation, migration/invasion, and survival. Dual inhibition of ERK and AKT suppressed these cellular processes to levels comparable to those achieved by reduction of PAK1 expression, whereas inactivation of either the ERK or AKT pathway alone partially inhibited cell migration/invasion and survival and had no effect on proliferation. We conclude that PAK1 stimulates colon cancer cell proliferation, migration/invasion, and survival via ERK- and AKT-dependent pathways. These findings establish the central importance of PAK1 in CRC signal transduction and clarify the mechanism by which PAK1 regulates CRC growth and migration. Instead of simultaneously inhibiting both ERK and AKT, the PAK1 convergence point could be an alternative target for CRC therapy.

P-21 activated kinase 1 knockdown inhibits β-catenin signalling and blocks colorectal cancer growth

The p21-activated kinase 1 (PAK1) plays important roles in cell growth, motility, and transformation. The aims of this study were to delineate the signalling mechanisms downstream of PAK1, and to investigate the importance of PAK1 for colorectal cancer (CRC) growth and metastasis in vivo. PAK1 knockdown in human CRC cell lines inhibited β-catenin expression, β-catenin/TCF4 transcriptional activity, and the expression of c-Myc. In mouse models PAK1 knockdown suppressed the growth and metastasis of human CRC cells by decreasing proliferation and increasing apoptosis. Our findings demonstrate for the first time the crucial role of PAK1 in CRC progression in vivo.

Glaucarubinone and gemcitabine synergistically reduce pancreatic cancer growth via down-regulation of P21-activated kinases.

Pancreatic cancer is one of the most lethal of human malignancies. Nearly 100% cases of pancreatic cancer carry mutations in KRas. P-21-activated kinases (PAKs) are activated by and act downstream of KRas. Glaucarubinone, a natural product first isolated from the seeds of the tree Simarouba glauca, was originally developed as an antimalarial drug, and has more recently been recognised as an anticancer agent. The aims of this study were to determine whether glaucarubinone, alone or in combination with the front-line chemotherapeutic agent gemcitabine, would inhibit the growth of pancreatic cancer cells in vitro or in vivo and the mechanism involved. Growth of the human pancreatic cancer cell lines PANC-1 and MiaPaCa-2 was measured by (3)H-thymidine incorporation in vitro, and by volume as xenografts in SCID mice. The expression and activities of the two serine/threonine kinases PAK1 and PAK4, which are key regulators of cancer progression, were measured by Western blotting. Here we report that glaucarubinone decreased proliferation and migration of pancreatic cancer cells in vitro, and reduced their growth as xenografts in vivo. Treatment with glaucarubinone and gemcitabine reduced proliferation in vitro and tumor growth in vivo more than treatment with either glaucarubinone or gemcitabine alone. Treatment with glaucarubinone reduced PAK1 and PAK4 activities, which were further decreased by the combination of glaucarubinone and gemcitabine. These results indicate that glaucarubinone reduced pancreatic cancer cell growth at least in part via inhibition of pathways involving PAK1 and PAK4. The synergistic inhibition by glaucarubinone and gemcitabine observed both in vitro and in vivo suggests that glaucarubinone may be a useful adjunct to current regimes of chemotherapy.

P21-activated kinase 1 promotes colorectal cancer survival by up-regulation of hypoxia-inducible factor-1α.

P21 activated kinase 1 (PAK1) enhances colorectal cancer (CRC) progression by stimulating Wnt/β-catenin and Ras oncogene, which promote CRC survival via stimulation of hypoxia-inducible factor 1α (HIF-1α). The aim of this study was to assess the mechanism involved in the stimulation by PAK1 of CRC survival. PAK1 promoted CRC cell survival by up-regulation of HIF-1α. PAK1 was over-expressed and hyper-activated in tumors of ApcΔ(14/+) mice, which was correlated with over-expression of HIF-1α and β-catenin. Inhibition of PAK1 decreased tumor growth and the expression of HIF-1α and β-catenin in tumors of ApcΔ(14/+) mice, and suppressed xenograft tumor survival in SCID mice. These findings indicate that PAK1 stimulates CRC survival by up-regulation of HIF-1α.

p-21-Activated kinase 1 mediates gastrin-stimulated proliferation in the colorectal mucosa via multiple signaling pathways

Gastrins, including amidated (Gamide) and glycine-extended (Ggly) forms, function as growth factors for the gastrointestinal mucosa. The p-21-activated kinase 1 (PAK1) plays important roles in growth factor signaling networks that control cell motility, proliferation, differentiation, and transformation. PAK1, activated by both Gamide and Ggly, mediates gastrin-stimulated proliferation and migration, and activation of β-catenin, in gastric epithelial cells. The aim of this study was to investigate the role of PAK1 in the regulation by gastrin of proliferation in the normal colorectal mucosa in vivo. Mucosal proliferation was measured in PAK1 knockout (PAK1 KO) mice by immunohistochemistry. The expression of phosphorylated and unphosphorylated forms of the signaling molecules PAK1, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT), and the expression of β-catenin and its downstream targets c-Myc and cyclin D1, were measured in gastrin knockout (Gas KO) and PAK1 KO mice by Western blotting. The expression and activation of PAK1 are decreased in Gas KO mice, and these decreases are associated with reduced activation of ERK, AKT, and β-catenin. Proliferation in the colorectal mucosa of PAK1 KO mice is reduced, and the reduction is associated with reduced activation of ERK, AKT, and β-catenin. In compensation, antral gastrin mRNA and serum gastrin concentrations are increased in PAK1 KO mice. These results indicate that PAK1 mediates the stimulation of colorectal proliferation by gastrins via multiple signaling pathways involving activation of ERK, AKT, and β-catenin.

Glaucarubinone inhibits colorectal cancer growth by suppression of hypoxia-inducible factor 1α and β-catenin via a p-21 activated kinase 1-dependent pathway.

p-21-Activated kinase 1 (PAK1) enhances colorectal cancer (CRC) progression by stimulating Wnt/β-catenin, ERK and AKT pathways. PAK1 also promotes CRC survival via up-regulation of hypoxia-inducible factor 1α (HIF-1α), a key player in cancer survival. Glaucarubinone, a quassinoid natural product, inhibits pancreatic cancer growth by down-regulation of PAK1. The aim of this study was to investigate the effect of glaucarubinone on CRC growth and metastasis, and the mechanism involved. Cell proliferation was measured in vitro by [(3)H]-thymidine incorporation and in vivo by volume of tumor xenografts. Protein concentrations were measured by Western blotting of cell extracts. We report here that glaucarubinone inhibited CRC growth both in vitro and in vivo. The potency of glaucarubinone as an inhibitor of cell proliferation was negatively correlated to PAK1 expression in CRC cells. Glaucarubinone suppressed the expression of HIF-1α and β-catenin. Knockdown of PAK1 by shRNA enhanced inhibition by glaucarubinone while constitutively active PAK1 blocked the inhibitory effect. Our findings indicate that glaucarubinone inhibited CRC growth by down-regulation of HIF-1α and β-catenin via a PAK1-dependent pathway.

Depletion of p21-activated kinase 1 up-regulates the immune system of APC∆14/+ mice and inhibits intestinal tumorigenesis

BackgroundP21-activated kinase 1 (PAK1) stimulates growth and metastasis of colorectal cancer (CRC) through activation of multiple signalling pathways. Up-regulation of CRC stem cell markers by PAK1 also contributes to the resistance of CRC to 5-fluorouracil. The aim of this study was to investigate the effect of PAK1 depletion and inhibition on the immune system and on intestinal tumour formation in APC∆14/+ mice.MethodsThe PAK1 KO APC∆14/+ mice were generated by cross-breeding of PAK1 KO mice with APC∆14/+ mice. Splenic lymphocytes were analysed by flow cytometry, and immunohistochemical staining. The numbers of intestinal tumours were counted. Blood cells were also counted.ResultsCompared to APC+/+ mice, the numbers of both T- and B- lymphocytes were reduced in the spleen of APC∆14/+ mice. Depletion of PAK1 in APC∆14/+ mice increased the numbers of splenic T- and B- lymphocytes and decreased the numbers of intestinal tumours. Treatment of APC∆14/+ mice with PF-3758309, a PAK inhibitor reduced the numbers of intestinal tumours and increased the numbers of blood lymphocytes.ConclusionDepletion of active PAK1 up-regulates the immune system of APC∆14/+ mice and suppresses intestinal tumour development. These observations suggest an important role for PAK1 in the immune response to tumours.

Up-regulation of stem cell markers by P21-activated kinase 1 contributes to 5-fluorouracil resistance of colorectal cancer

ABSTRACT Cancer stem cells (CSC) are tumorigenic and resistant to chemotherapy. In colorectal cancer (CRC), CSCs have been identified by the expression of specific markers, including CD44, Bmi1 and Nanog. Although p21-activated kinase 1 (PAK1), acting downstream of Ras, stimulates Wnt/β-catenin signaling and is known to play an important role in CRC development and progression, the role of PAK1 in the expression of CSC markers has not previously been investigated. The effect of PAK1 over-expression, knockdown or inhibition on the expression or alteration (in the case of CD44) of CSC markers in human CRC cell lines was measured by immunofluorescence and Western blotting. The effect of PAK1 modulation on tumorigenesis, and on resistance to treatment with 5-fluorouracil (5-FU), was measured by sphere formation in vitro and by growth of xenografted tumors in vivo. The results show that PAK1 activity correlated with the expression of CSC markers and the CD44 isoform profile, and with tumor growth both in vitro and in vivo. Furthermore PAK overexpression partially overcame the inhibition of CRC growth by 5-FU, and PAK inhibition was synergistic with 5-FU treatment. Our findings lay the foundation for a combination therapy in which PAK1 inhibitors targeting CSCs may be combined with conventional 5-FU-based chemotherapy for the treatment of CRC.

Demonstration and biological significance of a gastrin‐P21‐activated kinase 1 feedback loop in colorectal cancer cells

Gastrins, including amidated gastrin17 and glycine‐extended gastrin17, are important growth factors in colorectal cancer (CRC). The p21‐activated kinase 1 (PAK1) plays key roles in cellular processes including proliferation, survival, and motility, and in cell transformation and tumor progression. PAK1 expression increases with the progression of CRC, and knockdown of PAK1 blocks CRC cell growth and metastasis both in vitro and in vivo. The aim of this study was to determine the interaction between PAK1 and gastrins in CRC cells. PAK1 expression and activation were assayed by Western blots, and concentrations of gastrin mRNA and peptides by real‐time PCR and radioimmunoassay, respectively. Proliferation of CRC cells was measured by 3H‐thymidine incorporation, and vascular endothelial growth factor (VEGF) secretion was measured by ELISA. Gastrins activated PAK1 via PI3K‐dependent pathways. Activated PAK1 in turn mediated gastrin‐stimulated activation of β‐catenin and VEGF secretion in CRC cells, as knockdown of PAK1 blocked stimulation of these cellular processes by gastrins. Downregulation of gastrin reduced the expression and activity of PAK1, but in contrast there was a compensatory increase in gastrins either when PAK1 was downregulated, or after treatment with a PAK inhibitor. Our results indicate that PAK1 is required for the stimulation of CRC cells by gastrins, and suggest the existence of an inhibitory feedback loop by which PAK1 downregulates gastrin production in CRC cells.

Glaucarubinone Combined with Gemcitabine Improves Pancreatic Cancer Survival in an Immunocompetent Orthotopic Murine Model

ABSTRACT Background: Pancreatic cancer continues to have a poor survival rate with an urgent need for improved treatments. Glaucarubinone, a natural product first isolated from the seeds of the tree Simarouba glauca, has recently been recognized as having anti-cancer properties that may be particularly applicable to pancreatic cancer. Methods: The effect of glaucarubinone on the growth and migration of murine pancreatic cancer cells was assessed by 3H-thymidine incorporation assay. The survival impact of glaucarubinone alone and in combination with gemcitabine chemotherapy was assessed using an immunocompetent orthotopic murine model of pancreatic cancer. Results: Glaucarubinone inhibited the growth of the murine pancreatic cancer cell lines LM-P and PAN02. Treatment with either glaucarubinone or gemcitabine reduced proliferation in vitro and the combination was synergistic. The combination treatment improved survival two-fold compared to gemcitabine treatment alone (p = 0.046) in PAN02 cells. Conclusions: The synergistic inhibition by glaucarubinone and gemcitabine observed in vitro and the improved survival in vivo suggest that glaucarubinone may be a useful adjunct to current chemotherapy regimens.