MLLT11/AF1q boosts oncogenic STAT3 activity through Src-PDGFR tyrosine kinase signaling.
ABSTRACT: Constitutive STAT3 activation by tyrosine phosphorylation of mutated or amplified tyrosine kinases (pYSTAT3) is critical for cancer initiation, progression, invasion, and motility of carcinoma cells. We showed that AF1q is associated with STAT3 signaling in breast cancer cells. In xenograft models, enhanced AF1q expression activated STAT3 and promoted tumor growth and metastasis in immunodeficient NSG mice. The cytokine secretory phenotype of MDA-MB-231LN breast cancer cells with altered AF1q expression revealed changes in expression of platelet-derived growth factor subunit B (PDGF-B). AF1q-induced PDGF-B stimulated motility, migration, and invasion of MDA-MB-231LN cells, and AF1q up-regulated platelet-derived growth factor receptor (PDGFR) signaling. Further, AF1q-induced PDGFR signaling enhanced STAT3 activity through Src kinase activation, which could be blocked by the Src kinase inhibitor PP1. Moreover, AF1q up-regulated tyrosine kinase signaling through PDGFR signaling, which was blockable by imatinib. In conclusion, we demonstrated that enhanced AF1q expression contributes to persistent and oncogenic pYSTAT3 levels in invasive carcinoma cells by activating Src kinase through activation of the PDGF-B/PDGFR cascade. Therefore, AF1q plays an essential role as a cofactor in PDGF-B-driven STAT3 signaling.
Project description:Protein ubiquitination controls protein stability and subcellular localization of tyrosine kinase receptors, hence affecting signaling both quantitatively and qualitatively. In this report, we demonstrate that, after ligand stimulation, the PDGF ? receptor (PDGFR?) becomes ubiquitinated in a manner requiring both the c-Cbl and Cbl-b ubiquitin ligases. Simultaneous depletion of c-Cbl and Cbl-b resulted in reduced ligand-induced PDGFR? clearance from the cell surface because of reduced endocytosis of the receptor. Cbl-b formed a complex with c-Cbl, as well as with the PDGFR?, in response to PDGF-BB stimulation. We were unable to find a direct interaction between the receptor and c-Cbl, raising the possibility that Cbl-b is necessary for c-Cbl to interact with PDGFR?. Phosphorylated Tyr-1021 in PDGFR? was the primary interaction site for Cbl-b, with some contribution from Tyr-1009. Depletion of c-Cbl and Cbl-b led to an increased ligand-induced tyrosine phosphorylation of the receptor. Several tyrosine residues with elevated phosphorylation (i.e. Tyr-579, Tyr-581, Tyr-1009, and Tyr-1021) have previously been shown to interact with Src kinases and PLC?. Indeed, in cells depleted of c-Cbl and Cbl-b, both Src and PLC? phosphorylation were enhanced, whereas activation of other pathways, such as Erk1/2 MAP kinase and Akt, were not affected. In addition, Stat3 phosphorylation, which has been connected to Src activity, was also elevated in cells lacking c-Cbl and Cbl-b. Functionally, we found that cells depleted of c-Cbl and Cbl-b were more prone to migrate toward PDGF-BB, whereas no reproducible effect on cell proliferation could be observed. In conclusion, internalization as well as signaling via PDGFR? are controlled by ubiquitination.
Project description:Dynamin 2 (Dyn2), a large GTPase, is involved in receptor tyrosine kinase (RTK)-promoted cell migration. However, the molecular mechanisms by which Dyn2 regulates RTK-induced cell migration have not been established. Recently, we reported that tyrosine-protein phosphatase non-receptor type 11 (SHP-2) and phosphatidylinositol 3-kinase (PI3K) mediate platelet-derived growth factor receptor-? (PDGFR?)-promoted glioma tumor growth and invasion. Here, we show that Dyn2 is an effector downstream of the PDGFR?-PI3K/SHP-2 signaling in glioma cells. Depletion of endogenous Dyn2 by short hairpin RNAs (shRNAs) inhibited PDGFR?-stimulated phosphorylation of Akt, Erk1/2, Rac1 and Cdc42 activities, glioma cell migration and survival in vitro and tumor growth and invasion in the brains of mice. Dyn2 binds to SHP-2 and PI3K and colocalizes with PDGFR? at the invasive fronts in PDGF-A-stimulated glioma cells. Inhibition of SHP-2 by siRNA knockdown abrogated Dyn2 association with activated PDGFR? and PDGFR? activation of Rac1 and Cdc42, and glioma cell migration, thereby establishing a link between SHP-2 interaction with Dyn2 and the PDGFR? signaling. Furthermore, a dominant-negative SHP-2 C459S mutant inhibited PDGF-A-stimulated glioma cell migration, phosphorylation of Dyn2 and concomitantly blocked PDGFR?-induced Src activation. Inhibition of Src by Src inhibitors attenuated PDGF-A-stimulated phosphorylation of Akt and Dyn2 and glioma cell migration. Additionally, mutations of binding sites to PI3K, SHP-2 or Src of PDGFR? impaired PDGFR?-stimulated phosphorylation of Akt and Dyn2, and Dyn2 association with activated PDGFR?. Taken together, this study identifies Dyn2 as an effector that mediates PDGFR?-SHP-2-induced glioma tumor growth and invasion, suggesting that targeting the PDGFR?-SHP-2-Dyn2 pathway may be beneficial to patients with malignant glioblastomas.
Project description:Salivary gland malignancies of the head and neck form a heterogeneous group. Adenoid cystic carcinomas are an aggressive entity of salivary gland malignancies characterized by frequent distant metastases and poor response to radio- and chemotherapy. AF1Q is a MLL fusion partner, which can activate Wnt and STAT3 signaling. Recently, overexpression of AF1q has been identified as a poor prognosticator in patients of different malignancies. A total of 46 patients with adenoid cystic carcinoma were immunohistochemically evaluated for expression of AF1q and clinical outcome was analyzed in this context. Additionally, STAT3 and the Wnt downstream target CD44 were investigated and correlated with AF1q. AF1q was overexpressed in 52.2%. Overexpression of AF1q was associated with poorer overall survival (p?=?0.03). Additionally, lymph node metastases and solid tumor parts were more frequently observed in AF1qhigh patients (p?=?0.07 and 0.05, respectively). AF1q did not influence the occurrence of distant metastases. Expression of AF1q was associated with higher levels of STAT3 and CD44 (p?=?0.003 and 0.006, respectively). AF1q is a novel prognostic marker for poor overall survival in adenoid cystic carcinoma patients. The deleterious effects on survival may be a result of promotion of the STAT3 and Wnt pathway.
Project description:c-Src non-receptor tyrosine kinase is an important component of the platelet-derived growth factor (PDGF) receptor signaling pathway. c-Src has been shown to mediate the mitogenic response to PDGF in fibroblasts. However, the exact components of PDGF receptor signaling pathway mediated by c-Src remain unclear. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to identify Src-family kinase substrates involved in PDGF signaling. Using SILAC, we were able to detect changes in tyrosine phosphorylation patterns of 43 potential c-Src kinase substrates in PDGF receptor signaling. This included 23 known c-Src kinase substrates, of which 16 proteins have known roles in PDGF signaling while the remaining 7 proteins have not previously been implicated in PDGF receptor signaling. Importantly, our analysis also led to identification of 20 novel Src-family kinase substrates, of which 5 proteins were previously reported as PDGF receptor signaling pathway intermediates while the remaining 15 proteins represent novel signaling intermediates in PDGF receptor signaling. In validation experiments, we demonstrated that PDGF indeed induced the phosphorylation of a subset of candidate Src-family kinase substrates - Calpain 2, Eps15 and Trim28 - in a c-Src-dependent fashion.
Project description:We tested the hypothesis whether selective blunting of platelet-derived growth factor (PDGF)-dependent vascular smooth muscle cell (VSMC) proliferation and migration is sufficient to prevent neointima formation after vascular injury.To prevent neointima formation and stent thrombosis after coronary interventions, it is essential to inhibit VSMC proliferation and migration without harming endothelial cell function. The role of PDGF-a potent mitogen and chemoattractant for VSMC that does not affect endothelial cells-for neointima formation remains controversial.To decipher the signaling pathways that control PDGF beta receptor (?PDGFR)-driven VSMC proliferation and migration, we characterized 2 panels of chimeric CSF1R/?PDGFR mutants in which the binding sites for ?PDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase [PI3K], GTPase activating protein of ras, SHP-2, phospholipase C? 1 [PLC?]) were individually mutated. Based on in vitro results, the importance of PDGF-initiated signals for neointima formation was investigated in genetically modified mice.Our results indicate that the chemotactic response to PDGF requires the activation of Src, PI3K, and PLC?, whereas PDGF-dependent cell cycle progression is exclusively mediated by PI3K and PLC?. These 2 signaling molecules contribute to signal relay of the ?PDGFR by differentially regulating cyclin D1 and p27(kip1). Blunting of ?PDGFR-induced PI3K and PLC? signaling by a combination mutant (F3) completely abolished the mitogenic and chemotactic response to PDGF. Disruption of PDGF-dependent PI3K and PLC? signaling in mice expressing the F3 receptor led to a profound reduction of neointima formation after balloon injury.Signaling by the activated ?PDGFR, particularly through PI3K and PLC?, is crucial for neointima formation after vascular injury. Disruption of these specific signaling pathways is sufficient to attenuate pathogenic processes such as vascular remodeling in vivo.
Project description:RhoB is a small GTPase localized at the plasma membrane and endosomes that participates in the regulation of endocytic trafficking of the epidermal growth factor (EGF) receptor and the nonreceptor kinases Src and Akt. This study was performed to determine whether RhoB plays a critical role in trafficking and signaling by the platelet-derived growth factor receptor-beta (PDGFR-beta) in vascular smooth muscle cells.Cells derived from RhoB knockout mice failed to proliferate in response to PDGF, and downstream signaling was compromised as reflected by reduced phosphorylation of the effector kinases Akt and ERK1/2. In normal cells, PDGF stimulated trafficking of PDGFR-beta into a perinuclear late endosomal compartment and triggered entry of Src, Akt, extracellular signal-regulated kinase (ERK) into the cell nucleus. In contrast, PDGF treatment of RhoB null cells resulted in neither PDGFR-beta trafficking to late endosomes nor nuclear localization of Src, Akt, or ERK. In support of an essential function in these processes, restoring expression of RhoB in null cells rescued these defects and restored cell proliferation in response to PDGF.Our findings establish RhoB as a critical regulator of PDGFR-beta trafficking and signaling in vascular smooth muscle cells.
Project description:PDGF-BB/PDGFR-?? signaling plays very crucial roles in the process of many diseases such as liver fibrosis. However, drug candidates with selective affinities for PDGF-B/PDGFR-? remain deficient. Here, we identified a natural cyclopeptide termed destruxin A5 that effectively inhibits PDGF-BB-induced PDGFR-? signaling. Interestingly and importantly, the inhibitory mechanism is distinct from the mechanism of tyrosine kinase inhibitors because destruxin A5 does not have the ability to bind to the ATP-binding pocket of PDGFR-?. Using Biacore T200 technology, thermal shift technology, microscale thermophoresis technology and computational analysis, we confirmed that destruxin A5 selectively targets the PDGF-B/PDGFR-? interaction interface to block this signaling. Additionally, the inhibitory effect of destruxin A5 on PDGF-BB/PDGFR-?? signaling was verified using in vitro, ex vivo and in vivo models, in which the extent of liver fibrosis was effectively alleviated by destruxin A5. In summary, destruxin A5 may represent an efficacious and more selective inhibitor of PDGF-BB/PDGFR-?? signaling.
Project description:The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated- protein-kinase (p42/p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric G-protein Gi) induced an approx. 40-50% decrease in the activation of c-Src and p42/p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PP1, which abolished p42/p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and p42/p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, Gi, to regulate c-Src and subsequent p42/p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in p42/p44 MAPK activation. This is confirmed here by experiments which showed that PI3K inhibitors (wortmannin and LY294002) reduced the activation of p42/p44 MAPK by PDGF. PI3K activity was increased in Grb-2 immunoprecipitates from PDGF-stimulated cells and was decreased by pretreating these cells with PTX. These findings show that Gi might also promote Grb-2-PI3K complex formation and that Grb-2 may be a site at which PI3K is integrated into the p42/p44 MAPK cascade. In conclusion, our results demonstrate that Gi enables the PDGFR to signal more efficiently to p42/p44 MAPK, and this appears to be achieved through the regulation of c-Src and Grb-2/PI3K, which are intermediates in the p42/p44 MAPK cascade.
Project description:Platelet-derived growth factor (PDGF) receptor (PDGFR) expression correlates with metastatic medulloblastoma. PDGF stimulation of medulloblastoma cells phosphorylates extracellular signal-regulated kinase (ERK) and promotes migration. We sought to determine whether blocking PDGFR activity effectively inhibits signaling required for medulloblastoma cell migration and invasion. DAOY and D556 human medulloblastoma cells were treated with imatinib mesylate (Gleevec), a PDGFR tyrosine kinase inhibitor, or transfected with small interfering RNA (siRNA) to PDGFRB to test the effects of blocking PDGFR phosphorylation and expression, respectively. PDGFR cell signaling, migration, invasion, survival, and proliferation following PDGF-BB stimulation, with and without PDGFR inhibition, were measured. PDGF-BB treatment of cells increased PDGFRB, Akt and ERK phosphorylation, and transactivated epidermal growth factor receptor (EGFR), which correlated with enhanced migration, survival, and proliferation. Imatinib (1 ?mol/L) treatment of DAOY and D556 cells inhibited PDGF-BB- and serum-mediated migration and invasion at 24 and 48 h, respectively, and concomitantly inhibited PDGF-BB activation of PDGFRB, Akt, and ERK but increased PTEN expression and activity. Imatinib treatment also induced DAOY cell apoptosis at 72 h and inhibited DAOY and D556 cell proliferation at 48 h. siRNA silencing of PDGFRB similarly inhibited signaling, migration, and survival and both siRNA and imatinib treatment inhibited PDGF-BB-mediated EGFR transactivation, indicating that the effects of imatinib treatment are specific to PDGFRB target inhibition. These results indicate that PDGFRB tyrosine kinase activity is critical for migration and invasion of medulloblastoma cells possibly by transactivating EGFR; thus, imatinib may represent an important novel therapeutic agent for the treatment of medulloblastoma.
Project description:Platelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the beta(2)-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.