Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer.
ABSTRACT: Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon and pancreatic cancers combined. Current research suggests that standard chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway-involving AKT, CREB, Bcl-xL, survivin, and Bcl-2-downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that short hairpin RNA (shRNA) knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl have complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl.
Project description:Genome-wide association studies have implicated the TAM receptor tyrosine kinase (RTK) Mer in liver disease, yet our understanding of the role that Mer and its related RTKs Tyro3 and Axl play in liver homeostasis and the response to acute injury is limited. We find that Mer and Axl are most prominently expressed in hepatic Kupffer and endothelial cells and that as mice lacking these RTKs age, they develop profound liver disease characterized by apoptotic cell accumulation and immune activation. We further find that Mer is critical to the phagocytosis of apoptotic hepatocytes generated in settings of acute hepatic injury, and that Mer and Axl act in concert to inhibit cytokine production in these settings. In contrast, we find that Axl is uniquely important in mitigating liver damage during acetaminophen intoxication. Although Mer and Axl are protective in acute injury models, we find that Axl exacerbates fibrosis in a model of chronic injury. These divergent effects have important implications for the design and implementation of TAM-directed therapeutics that might target these RTKs in the liver.
Project description:Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with those of asbestos-associated disease. We previously demonstrated co-activation of multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET, and AXL in mesothelioma cell lines, suggesting that these kinases could serve as novel therapeutic targets. Although clinical trials have not shown activity for EGFR inhibitors in mesothelioma, concurrent inhibition of various activated RTKs has pro-apoptotic and anti-proliferative effects in mesothelioma cell lines. Thus, we hypothesised that a coordinated network of multi-RTK activation contributes to mesothelioma tumorigenesis.Activation of PI3K/AKT/mTOR, Raf/MAPK, and co-activation of RTKs were evaluated in mesotheliomas. Effects of RTK and downstream inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, apoptosis, activation of signalling intermediates, expression of cell-cycle checkpoints, and cell-cycle alterations.We demonstrate activation of the PI3K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, but not in non-neoplastic mesothelial cells. The AKT activation, but not MAPK activation, was dependent on coordinated activation of RTKs EGFR, MET, and AXL. In addition, PI3K/AKT/mTOR pathway inhibition recapitulated the anti-proliferative effects of concurrent inhibition of EGFR, MET, and AXL. Dual targeting of PI3K/mTOR by BEZ235 or a combination of RAD001 and AKT knockdown had a greater effect on mesothelioma proliferation and viability than inhibition of individual activated RTKs or downstream signalling intermediates. Inhibition of PI3K/AKT was also associated with MDM2-p53 cell-cycle regulation.These findings show that PI3K/AKT/mTOR is a crucial survival pathway downstream of multiple activated RTKs in mesothelioma, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention.
Project description:Astrocytomas account for the majority of malignant brain tumors diagnosed in both adult and pediatric patients. The therapies available to treat these neoplasms are limited, and the prognosis associated with high-grade lesions is extremely poor. Mer (MerTK) and Axl receptor tyrosine kinases (RTK) are expressed at abnormally high levels in a variety of malignancies, and these receptors are known to activate strong antiapoptotic signaling pathways that promote oncogenesis. In this study, we found that Mer and Axl mRNA transcript and protein expression were elevated in astrocytic patient samples and cell lines. shRNA-mediated knockdown of Mer and Axl RTK expression led to an increase in apoptosis in astrocytoma cells. Apoptotic signaling pathways including Akt and extracellular signal-regulated kinase 1/2, which have been shown to be activated in resistant astrocytomas, were downregulated with Mer and Axl inhibition whereas poly(ADP-ribose) polymerase cleavage was increased. Furthermore, Mer and Axl shRNA knockdown led to a profound decrease of astrocytoma cell proliferation in soft agar and a significant increase in chemosensitivity in response to temozolomide, carboplatin, and vincristine treatment. Our results suggest Mer and Axl RTK inhibition as a novel method to improve apoptotic response and chemosensitivity in astrocytoma and provide support for these oncogenes as attractive biological targets for astrocytoma drug development.
Project description:The anticoagulant factor protein S (PS) protects neurons from hypoxic/ischemic injury. However, molecular mechanisms mediating PS protection in injured neurons remain unknown. Here, we show mouse recombinant PS protects dose-dependently mouse cortical neurons from excitotoxic NMDA-mediated neuritic bead formation and apoptosis by activating the phosphatidylinositol 3-kinase (PI3K)-Akt pathway (EC(50) = 26 ± 4 nm). PS stimulated phosphorylation of Bad and Mdm2, two downstream targets of Akt, which in neurons subjected to pathological overstimulation of NMDA receptors (NMDARs) increased the antiapoptotic Bcl-2 and Bcl-X(L) levels and reduced the proapoptotic p53 and Bax levels. Adenoviral transduction with a kinase-deficient Akt mutant (Ad.Akt(K179A)) resulted in loss of PS-mediated neuronal protection, Akt activation, and Bad and Mdm2 phosphorylation. Using the TAM receptors tyrosine kinases Tyro3-, Axl-, and Mer-deficient neurons, we showed that PS protected neurons lacking Axl and Mer, but not Tyro3, suggesting a requirement of Tyro3 for PS-mediated protection. Consistent with these results, PS dose-dependently phosphorylated Tyro3 on neurons (EC(50) = 25 ± 3 nm). In an in vivo model of NMDA-induced excitotoxic lesions in the striatum, PS dose-dependently reduced the lesion volume in control mice (EC(50) = 22 ± 2 nm) and protected Axl(-/-) and Mer(-/-) transgenic mice, but not Tyro3(-/-) transgenic mice. Using different structural PS analogs, we demonstrated that the C terminus sex hormone-binding globulin-like (SHBG) domain of PS is critical for neuronal protection in vitro and in vivo. Thus, our data show that PS protects neurons by activating the Tyro3-PI3K-Akt pathway via its SHGB domain, suggesting potentially a novel neuroprotective approach for acute brain injury and chronic neurodegenerative disorders associated with excessive activation of NMDARs.
Project description:KRAS mutation is frequently seen in a subtype of ovarian cancer categorized as type 1. The KRAS-MAPK pathway, which is closely involved in type 1 cancer progression, is under the regulation of receptor tyrosine kinases (RTKs). AXL, one of the RTKs, has been reported to be overexpressed in ovarian cancer and contributes to the poor prognosis. However, there is no useful target-based agent against such gene profiles. We examined the combined effect of the dual RAF/MEK inhibitor CH5126766 and AXL inhibitor R428 on the growth of ovarian cancer HEY-T30 and OVCAR-5 cell lines, both of which bear KRAS mutation and express AXL at a high level, using the WST-8 assay and the colony formation assay. The synergistic effect of the combination was evaluated by the combination index. The apoptotic cells were analyzed by flow cytometry. The expression of apoptotic proteins and the phosphorylation of MAPK and AKT pathway proteins were investigated by western blotting. We found that CH5126766 and R428 suppressed the phosphorylation of ERK and AKT, respectively, and their combination synergistically inhibited the growth of both cell lines with enhancement of apoptosis accompanied by the Bim upregulation. Combined treatment with CH5126766 and R428 is expected as the novel therapeutic option for KRAS-mutated ovarian cancer with high expression of AXL.
Project description:B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite aggressive therapeutic approaches. We previously found that Axl receptor tyrosine kinase (RTK) plays a critical role in CLL B-cell survival. Here, we explored the possibility of using a high-affinity Axl inhibitor as a single agent or in combination with Bruton's tyrosine kinase (BTK) inhibitors for future clinical trial to treat patients with CLL.Expression/activation status of other members of the TAM (e.g., Tyro3, Axl, and MER) family of RTKs in CLL B cells was evaluated. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 with or without the presence of CLL bone marrow stromal cells (BMSCs). Inhibitory effects of TP-0903 on the Axl signaling pathway were also evaluated in CLL B cells. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination.CLL B cells overexpress Tyro3, but not MER. Of interest, Tyro3 remains as constitutively phosphorylated and forms a complex with Axl in CLL B cells. TP-0903 induces massive apoptosis in CLL B cells with LD50 values of nanomolar ranges. Importantly, CLL BMSCs could not protect the leukemic B cells from TP-0903-induced apoptosis. A marked reduction of the antiapoptotic proteins Mcl-1, Bcl-2, and XIAP and upregulation of the proapoptotic protein BIM in CLL B cells was detected as a result of Axl inhibition. Finally, combination of TP-0903 with BTK inhibitors augments CLL B-cell apoptosis.Administration of TP-0903 either as a single agent or in combination with BTK inhibitors may be effective in treating patients with CLL.
Project description:Axl, a member of the TAM (Tyro3, AXL, Mer) receptor tyrosine kinase family, plays critical roles in cell growth, proliferation, apoptosis, and migration. In the present study, we demonstrated that the anti-cancer activity of bufalin, a major bioactive component of the Chinese traditional medicine Chan Su, is mediated by the down-regulation of Axl in non-small-cell lung cancer (NSCLC) cells. We observed the inhibitory effect of bufalin on the proliferation of A549 and H460 NSCLC cells and the clonogenicity of these cells was reduced by bufalin treatment in a dose-dependent manner. Next, we found that the protein level of Axl was decreased in proportion to the concentration of bufalin in both A549 and H460 cells. Moreover, the promoter activity of the Axl gene was decreased by bufalin in a dose- and time-dependent manner, indicating that bufalin down-regulates Axl gene expression at the transcriptional level. We further examined if the anti-proliferative property of bufalin is influenced by Axl at the protein level. Axl overexpression attenuated the effect of bufalin in inhibiting cell proliferation and colony formation and inducing apoptosis in H460 cells, while knockdown of Axl gene expression induced the opposite effect. Taken together, our data indicate that the anti-proliferative and pro-apoptotic effects of bufalin were associated with the protein level of Axl, suggesting that Axl is a potent therapeutic target of bufalin in suppressing proliferation and inducing apoptosis in NSCLC cells.
Project description:TAM receptors (Tyro3, Axl, and Mer) are receptor tyrosine kinases (RTKs) that are expressed by multiple immune cells including NK cells. Although RTKs typically enhance cellular functions, TAM receptor ligation blocks NK-cell activation. The mechanisms by which RTKs block NK-cell signaling downstream of activating receptors are unknown. In this report, we demonstrate that TAM receptors attenuate NK cell responses via the activity of E3 ubiquitin ligase Casitas B lineage lymphoma b (Cbl-b). Specifically, we show that Tyro3, Axl, and Mer phosphorylate Cbl-b, and Tyro3 ligation activates Cbl-b by phosphorylating tyrosine residues 133 and 363. Ligation of TAM receptors by their ligand Gas6 suppresses activating receptor-stimulated NK-cell functions such as IFN-? production and degranulation, in a TAM receptor kinase- and Cbl-b-dependent manner. Moreover, Gas6 ligation induces the degradation of LAT1, a transmembrane adaptor protein required for NK cell activating receptor signaling, in WT but not in Cbl-b knock-out NK cells. Together, these results suggest that TAM receptors may attenuate NK-cell function by phosphorylating Cbl-b, which in turn dampens NK-cell activation signaling by promoting the degradation of LAT1. Our data therefore support a mechanism by which RTKs attenuate, rather than stimulate, signaling pathways via the activation of ubiquitin ligases.
Project description:Mer is a receptor tyrosine kinase (RTK) with oncogenic properties that is often overexpressed or activated in various malignancies. Using both immunohistochemistry and microarray analyses, we demonstrated that Mer was overexpressed in both tumoral and stromal compartments of about 70% of non-small cell lung cancer (NSCLC) samples relative to surrounding normal lung tissue. This was validated in freshly harvested NSCLC samples; however, no associations were found between Mer expression and patient features. Although Mer overexpression did not render normal lung epithelial cell tumorigenic in vivo, it promoted the in vitro cell proliferation, clonogenic colony formation and migration of normal lung epithelial cells as well as NSCLC cells primarily depending on MAPK and FAK signaling, respectively. Importantly, Mer overexpression induced resistance to erlotinib (EGFR inhibitor) in otherwise erlotinib-sensitive cells. Furthermore, Mer-specific inhibitor rendered erlotinib-resistant cells sensitive to erlotinib. We conclude that Mer enhances malignant phenotype and pharmacological inhibition of Mer overcomes resistance of NSCLC to EGFR-targeted agents.
Project description:Epidermal growth factor receptor (EGFR)-mutation-positive non-smallcell lung cancer (NSCLC) is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs). We investigated receptor tyrosine kinases (RTKs), Src family kinases and focal adhesion kinase (FAK) as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2) of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2) inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1) was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p=0.0407) and overall survival (hazard ratio of 2.23, p=0.0192). A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing.