ABSTRACT
Long-lived quiescent mammary stem cells (MaSCs) are presumed to coordinate the dramatic expansion of ductal epithelium that occurs through the different phases of postnatal development, but little is known about the molecular regulators that underpin their activation. We show that ablation of the transcription factor Foxp1 in the mammary gland profoundly impairs ductal morphogenesis, resulting in a rudimentary tree throughout life. Foxp1-deficient glands were highly enriched for quiescent Tspan8hi MaSCs, which failed to become activated even in competitive transplantation assays, thus highlighting a cell-intrinsic defect. Foxp1 deletion also resulted in aberrant expression of basal genes in luminal cells, inferring a role in cell-fate decisions. Notably, Foxp1 was uncovered as a direct repressor of Tspan8 in basal cells, and deletion of Tspan8 rescued the defects in ductal morphogenesis elicited by Foxp1 loss. Thus, a single transcriptional regulator Foxp1 can control the exit of MaSCs from dormancy to orchestrate differentiation and development.
Subject(s)
Adult Stem Cells/metabolism , Cell Differentiation , Forkhead Transcription Factors/metabolism , Mammary Glands, Human/growth & development , Morphogenesis , Repressor Proteins/metabolism , 3T3 Cells , Adult Stem Cells/cytology , Animals , Cells, Cultured , Female , Forkhead Transcription Factors/genetics , HEK293 Cells , Humans , Mammary Glands, Human/cytology , Mammary Glands, Human/metabolism , Mice , Mice, Inbred C57BL , Repressor Proteins/genetics , Tetraspanins/genetics , Tetraspanins/metabolismABSTRACT
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 (3)H-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 ß-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.
ABSTRACT
P21 activated kinase 1 (PAK1) enhances colorectal cancer (CRC) progression by stimulating Wnt/ß-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 ß-catenin. Inhibition of PAK1 decreased tumor growth and the expression of HIF-1α and ß-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.
Subject(s)
Colorectal Neoplasms/enzymology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , p21-Activated Kinases/physiology , Animals , Cell Hypoxia , Cell Survival , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , Enzyme Activation , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , HCT116 Cells , HT29 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, SCID , RNA, Small Interfering/genetics , Tumor Burden , Up-Regulation , Vascular Endothelial Growth Factor A/metabolism , beta Catenin/metabolism , p21-Activated Kinases/antagonists & inhibitorsABSTRACT
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 ß-catenin expression, ß-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.
Subject(s)
Cell Proliferation , Colorectal Neoplasms/enzymology , Gene Knockdown Techniques , beta Catenin/metabolism , p21-Activated Kinases/deficiency , Animals , Apoptosis , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , HCT116 Cells , HT29 Cells , Humans , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Liver Neoplasms/secondary , Mice , Mice, SCID , Neoplasm Invasiveness , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , Signal Transduction , Time Factors , Transcription Factor 4 , Transcription Factors/metabolism , Transfection , Tumor Burden , Xenograft Model Antitumor Assays , beta Catenin/genetics , p21-Activated Kinases/geneticsABSTRACT
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.
Subject(s)
Carcinoma/pathology , Cell Movement , Cell Proliferation , Colonic Neoplasms/pathology , Extracellular Signal-Regulated MAP Kinases/physiology , Oncogene Protein v-akt/physiology , p21-Activated Kinases/physiology , Carcinoma/genetics , Carcinoma/metabolism , Cell Movement/drug effects , Cell Movement/genetics , Cell Movement/physiology , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , HCT116 Cells , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Models, Biological , Neoplasm Invasiveness , Oncogene Protein v-akt/metabolism , RNA, Small Interfering/pharmacology , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , Tumor Cells, Cultured , p21-Activated Kinases/antagonists & inhibitors , p21-Activated Kinases/geneticsABSTRACT
Gastrins, including amidated gastrin (Gamide) and glycine-extended gastrin (Ggly), are known to accelerate the growth of gastric and colorectal cancer cells by stimulation of proliferation and inhibition of apoptosis. Gamide controls apoptosis by regulation of proteins of the Bcl-2 family and by regulation of the activation of caspases. However the interactions between Ggly and proteins of the Bcl-2 family and caspases are not known. Since in other systems G proteins of the Rho family inhibit apoptosis via interaction with proteins of the Bcl-2 family, leading to changes in caspase activities, we have compared the role of Rho family G proteins in regulation of Bcl-2-like (Bad/Bax/Bcl-xl) protein expression and caspase 3 activation by Ggly and Gamide. The effects of the specific inhibitors C3 (for Rho) and Y-27632 (for ROCK), and of dominant negative mutants of Rac, Cdc42 and PAK, were investigated in the gastric epithelial cell line IMGE-5. Apoptosis was induced by serum starvation and confirmed by annexin V staining and caspase 3 activation. Ggly inhibits caspase 3 activation via a Bcl-2-like protein-mediated pathway which requires activation of both Rho/ROCK and Rac/Cdc42/PAK. Gamide inhibits caspase 3 activation via redundant Bcl-2-like protein-mediated pathways which involve alternative activation of Rac/Cdc42/PAK and Rho/ROCK. Gamide and Ggly differentially activate members of Rho family G proteins which in turn regulate different proteins of the Bcl-2 family leading to changes in caspase 3 activity. The findings offer potential targets for blocking the growth-stimulating effects of these gastrins.