Epithelial morphological reversion drives Profilin-1-induced elevation of p27(kip1) in mesenchymal triple-negative human breast cancer cells through AMP-activated protein kinase activation.
ABSTRACT: Profilin-1 (Pfn1) is an important regulator of actin polymerization that is downregulated in human breast cancer. Previous studies have shown Pfn1 has a tumor-suppressive effect on mesenchymal-like triple-negative breast cancer cells, and Pfn1-induced growth suppression is partly mediated by upregulation of cell-cycle inhibitor p27(kip1) (p27). In this study, we demonstrate that Pfn1 overexpression leads to accumulation of p27 through promoting AMPK activation and AMPK-dependent phosphorylation of p27 on T198 residue, a post-translational modification that leads to increased protein stabilization of p27. This pathway is mediated by Pfn1-induced epithelial morphological reversion of mesenchymal breast cancer through cadherin-mediated restoration of adherens junctions. These findings not only elucidate a potential mechanism of how Pfn1 may inhibit proliferation of mesenchymal breast cancer cells, but also highlight a novel pathway of cadherin-mediated p27 induction and therefore cell-cycle control in cells.
Project description:The tumor suppressor gene p27(Kip1) plays a fundamental role in human cancer progression. Its expression and/or functions are altered in almost all the different tumor histotype analyzed so far. Recently, it has been demonstrated that the tumor suppression function of p27 resides not only in the ability to inhibit Cyclins/CDKs complexes through its N-terminal domain but also in the capacity to modulate cell motility through its C-terminal portion. Particular interest has been raised by the last amino-acid, (Threonine 198) in the regulation of both protein stability and cell motility.Here, we describe that the presence of Threonine in position 198 is of primary importance for the regulation of the protein stability and for the control of cell motility. However, while the control of cell motility is dependent on the phosphorylation of T198, the stability of the protein is specifically controlled by the steric hindrance of the last amino acid. The effects of T198 modification on protein stability are not linked to the capacity of p27 to bind Cyclins/CDKs complexes and/or the F-box protein Skp2. Conversely, our results support the hypothesis that conformational changes in the disordered structure of the C-terminal portion of p27 are important in its ability to be degraded via a proteasome-dependent mechanism. On the other hand T198 phosphorylation favors p27/stathmin interaction eventually contributing to the regulation of cell motility, supporting the hypothesis that the presence of T198 is fundamental for the regulation of p27 functions.
Project description:Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, has gained interest in epithelial-derived cancer because of its downregulation in expression in various adenocarcinoma. Pfn1 overexpression impairs tumorigenic ability of human breast cancer xenografts thus suggesting that Pfn1 could be a tumor-suppressor protein. The objective of the present study was to determine how Pfn1 overexpression affects cell-cycle progression of breast cancer cells. We show that Pfn1 overexpression in MDA-MB-231 breast cancer cells causes cell-cycle arrest in G1 phase and dramatically reduced proliferation in culture. Pfn1 overexpression results in increased protein stability of p27(kip1) (p27-a major cyclin-dependent kinase inhibitor) and marked elevation in the overall cellular level of p27. Proliferation defect of Pfn1 overexpressers can be partly rescued by silencing p27 expression thus suggesting a critical role of p27 in Pfn1-induced growth inhibition of MDA-MB-231 cells. Finally, Pfn1 overexpression was found to sensitize MDA-MB-231 cells to apoptosis in response to cytotoxic stimulus thus suggesting for the first time that survival of breast cancer cells can also be negatively influenced by Pfn1 upregulation. These findings may provide novel insights underlying Pfn1's tumor-suppressive action.
Project description:The p27/kip1 (p27) tumor suppressor inhibits cyclin/cyclin-dependent kinase (CDK) complexes and halts cell cycle progression. p27 further regulates invasion and migration in cancer cells, suggesting p27 also functions as an oncoprotein. Using a human osteosarcoma tissue microarray we identified high expression of cytoplasmic p27 in metastatic tumors. We demonstrated a positive correlation between mRNA and protein expression of p27 and expression of key metastatic markers, vimentin, snail-2, β-catenin and stathmin-1 (STMN1) in patient tumors. Our results show that T198 phosphorylation of p27 controls the interaction between p27 and STMN1 that regulates microtubule stabilization and the invasion and migration of osteosarcoma cells. We found that anti-tumoral activity of gemcitabine and the Wee1 kinase inhibitor AZD1775 in osteosarcoma cells, was dependent on drug sequencing that relied on p27 stabilization. Gemcitabine activated caspase-3 and synergized with AZD1775 through caspase-mediated cleavage of p27, that dissociated from STMN1 and effectively induced apoptosis. Further, blockage of nuclear export of p27 by inhibition of Exportin-1 (XPO1) promoted growth arrest, demonstrating that the biological effects of agents relied on the expression and localization of p27. Together, these data provide a rationale for combining chemotherapy with agents that promote p27 tumor suppressor activity for the treatment of osteosarcoma.
Project description:p27 restrains normal cell growth, but PI3K-dependent C-terminal phosphorylation of p27 at threonine 157 (T157) and T198 promotes cancer cell invasion. Here, we describe an oncogenic feedforward loop in which p27pT157pT198 binds Janus kinase 2 (JAK2) promoting STAT3 (signal transducer and activator of transcription 3) recruitment and activation. STAT3 induces TWIST1 to drive a p27-dependent epithelial-mesenchymal transition (EMT) and further activates AKT contributing to acquisition and maintenance of metastatic potential. p27 knockdown in highly metastatic PI3K-activated cells reduces STAT3 binding to the TWIST1 promoter, TWIST1 promoter activity and TWIST1 expression, reverts EMT and impairs metastasis, whereas activated STAT3 rescues p27 knockdown. Cell cycle-defective phosphomimetic p27T157DT198D (p27CK-DD) activates STAT3 to induce a TWIST1-dependent EMT in human mammary epithelial cells and increases breast and bladder cancer invasion and metastasis. Data support a mechanism in which PI3K-deregulated p27 binds JAK2, to drive STAT3 activation and EMT through STAT3-mediated TWIST1 induction. Furthermore, STAT3, once activated, feeds forward to further activate AKT.
Project description:Progesterone (P4) was demonstrated to inhibit migration in vascular smooth muscle cells (VSMCs), but to enhance migration in T47D breast cancer cells. To investigate the mechanism responsible for this switch in P4 action, we examined the signaling pathway responsible for the P4-induced migration enhancement in breast cancer cell lines, T47D and MCF-7. Here, we demonstrated that P4 activated the cSrc/AKT signaling pathway, subsequently inducing RSK1 activation, which in turn increased phosphorylation of p27 at T198 and formation of the p27pT198-RhoA complex in the cytosol, thereby preventing RhoA degradation, and eventually enhanced migration in T47D cells. These findings were confirmed in the P4-treated MCF-7. Comparing the P4-induced molecular events in between breast cancer cells and VSMCs, we found that P4 increased p27 phosphorylation at T198 in breast cancer cells through RSK1 activation, while P4 increased p27 phosphorlation at Ser10 in VSMCs through KIS activation. P27pT198 formed the complex with RhoA and prevented RhoA degradation in T47D cells, whereas p-p27Ser10 formed the complex with RhoA and caused RhoA degradation in VSMCs. The results of this study highlight the molecular mechanism underlying P4-enhanced breast cancer cell migration, and suggest that RSK1 activation is responsible for the P4-induced migration enhancement in breast cancer cells.
Project description:p90 ribosomal S6 kinase (RSK1) is an effector of both Ras/MEK/MAPK and PI3K/PDK1 pathways. We present evidence that RSK1 drives p27 phosphorylation at T198 to increase RhoA-p27 binding and cell motility. RSK1 activation and p27pT198 both increase in early G(1). As for many kinase-substrate pairs, cellular RSK1 coprecipitates with p27. siRNA to RSK1 and RSK1 inhibition both rapidly reduce cellular p27pT198. RSK1 overexpression increases p27pT198, p27-cyclin D1-Cdk4 complexes, and p27 stability. Moreover, RSK1 transfectants show mislocalization of p27 to cytoplasm, increased motility, and reduced RhoA-GTP, phospho-cofilin, and actin stress fibers, all of which were reversed by shRNA to p27. Phosphorylation by RSK1 increased p27pT198 binding to RhoA in vitro, whereas p27T157A/T198A bound poorly to RhoA compared with WTp27 in cells. Coprecipitation of cellular p27-RhoA was increased in cells with constitutive PI3K activation and increased in early G(1). Thus T198 phosphorylation not only stabilizes p27 and mislocalizes p27 to the cytoplasm but also promotes RhoA-p27 interaction and RhoA pathway inhibition. These data link p27 phosphorylation at T198 and cell motility. As for other PI3K effectors, RSK1 phosphorylates p27 at T198. Because RSK1 is also activated by MAPK, the increased cell motility and metastatic potential of cancer cells with PI3K and/or MAPK pathway activation may result in part from RSK1 activation, leading to accumulation of p27T198 in the cytoplasm, p27:RhoA binding, inhibition of RhoA/Rock pathway activation, and loss of actomyosin stability.
Project description:In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacologic activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKK?-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis.
Project description:Functional inactivation of the tumor suppressor p27(kip1) in human cancer occurs either through loss of expression or through phosphorylation-dependent cytoplasmic sequestration. Here we demonstrate that dysregulation of the PI3K/AKT pathway is important in thyroid carcinogenesis and that p27(kip1) is a key target of the growth-regulatory activity exerted by this pathway in thyroid cancer cells. Using specific PI3K inhibitors (LY294002, wortmannin, and PTEN) and a dominant active AKT construct (myrAKT), we demonstrated that the PI3K/AKT pathway controlled thyroid cell proliferation by regulating the expression and subcellular localization of p27. Results obtained with phospho-specific antibodies and with transfection of nonphosphorylable p27(kip1) mutant constructs demonstrated that PI3K/AKT-dependent regulation of p27(kip1) mislocalization in thyroid cancer cells occurred via phosphorylation of p27(kip1) at T157 and T198 (but not at S10 or T187). Finally, we evaluated whether these results were applicable to human tumors. Analysis of 100 thyroid carcinomas indicated that p27(kip1) phosphorylation at T157/T198 and cytoplasmic mislocalization were preferentially associated with activation of the PI3K/AKT pathway. Thus the PI3/AKT pathway and its effector p27(kip1) play major roles in thyroid carcinogenesis.
Project description:Entry of cells into the cell division cycle requires the coordinated activation of cyclin-dependent kinases (cdks) and the deactivation of cyclin kinase inhibitors. Degradation of p27kip1 is known to be a central component of this process as it allows controlled activation of cdk2-associated kinase activity. Turnover of p27 at the G1/S transition is regulated through phosphorylation at T187 and subsequent SCF(skp2)-dependent ubiquitylation. However, detailed analysis of this process revealed the existence of additional pathways that regulate the abundance of the protein in early G1 and as cells exit quiescence. Here, we report on a molecular mechanism that regulates p27 stability by phosphorylation at T198. Phosphorylation of p27 at T198 prevents ubiquitin-dependent degradation of free p27. T198 phosphorylation also controls progression through the G1 phase of the cell cycle by regulating the association of p27 with cyclin-cdk complexes. Our results unveil the molecular composition of a pathway, which regulates the abundance and activity of p27kip1 during early G1. They also explain how the T187- and the T198-dependent turnover systems synergize to allow cell cycle progression in G1.
Project description:Basal-like breast cancers are among the most aggressive cancers and effective targeted therapies are still missing. In order to identify new therapeutic targets, we performed Methyl-Seq and RNA-Seq of 10 breast cancer cell lines with different phenotypes. We confirmed that breast cancer subtypes cluster the RNA-Seq data but not the Methyl-Seq data. Basal-like tumor hypermethylated phenotype was not confirmed in our study but RNA-Seq analysis allowed to identify 77 genes significantly overexpressed in basal-like breast cancer cell lines. Among them, 48 were overexpressed in triple negative breast cancers of TCGA data. Some molecular functions were overrepresented in this candidate gene list. Genes involved in antioxydation, such as SOD1, MGST3 and PRDX or cadherin-binding genes, such as PFN1, ITGB1 and ANXA1, could thus be considered as basal like breast cancer biomarkers. We then sought if these genes were linked to BRCA1, since this gene is often inactivated in basal-like breast cancers. Nine genes were identified overexpressed in both basal-like breast cancer cells and BRCA1 mutated cells. Amongst them, at least 3 genes code for proteins implicated in epithelial cell migration and epithelial to mesenchymal transition (VIM, ITGB1 and RhoA). Our study provided several potential therapeutic targets for triple negative and BRCA1 mutated breast cancers. It seems that migration and mesenchymal properties acquisition of basal-like breast cancer cells is a key functional pathway in these tumors with a high metastatic potential.