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Esophageal adenocarcinoma (EAC) is a highly aggressive malignancy that is characterized by resistance to chemotherapy and a poor clinical outcome. The overexpression of the receptor tyrosine kinase AXL is frequently associated with unfavorable prognosis in EAC. Although it is well documented that AXL mediates cancer cell invasion as a downstream effector of epithelial-to-mesenchymal transition, the precise molecular mechanism underlying this process is not completely understood. Herein, we demonstrate for the first time that AXL mediates cell invasion through the regulation of lysosomes peripheral distribution and cathepsin B secretion in EAC cell lines. Furthermore, we show that AXL-dependent peripheral distribution of lysosomes and cell invasion are mediated by extracellular acidification, which is potentiated by AXL-induced secretion of lactate through AKT-NF-κB-dependent MCT-1 regulation. Our novel mechanistic findings support future clinical studies to evaluate the therapeutic potential of the AXL inhibitor R428 (BGB324) in highly invasive EAC.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
The discovery of selective inhibitors of biological target proteins is the primary goal of many drug discovery campaigns. However, this goal has proven elusive, especially for inhibitors targeting the well-conserved orthosteric adenosine triphosphate (ATP) binding pocket of kinase enzymes. The human kinome is large and it is rather difficult to profile early lead compounds against around 500 targets to gain an upfront knowledge on selectivity. Further, selectivity can change drastically during derivatization of an initial lead compound. Here, we have introduced a computational model to support the profiling of compounds early in the drug discovery pipeline. On the basis of the extensive profiled activity of 70 kinase inhibitors against 379 kinases, including 81 tyrosine kinases, we developed a quantitative structure-activity relation (QSAR) model using artificial neural networks, to predict the activity of these kinase inhibitors against the panel of 379 kinases. The model's performance in predicting activity ranges from 0.6 to 0.8 depending on the kinase, from the area under the curve (AUC) of the receiver operating characteristics (ROC). The profiler is available online at http://www.meilerlab.org/index.php/servers/show?s_id=23.
Cranial neural crest cells are a multipotent cell population that generate all the elements of the pharyngeal cartilage with differentiation into chondrocytes tightly regulated by temporal intracellular and extracellular cues. Here, we demonstrate a novel role for miR-27, a highly enriched microRNA in the pharyngeal arches, as a positive regulator of chondrogenesis. Knock down of miR-27 led to nearly complete loss of pharyngeal cartilage by attenuating proliferation and blocking differentiation of pre-chondrogenic cells. Focal adhesion kinase (FAK) is a key regulator in integrin-mediated extracellular matrix (ECM) adhesion and has been proposed to function as a negative regulator of chondrogenesis. We show that FAK is downregulated in the pharyngeal arches during chondrogenesis and is a direct target of miR-27. Suppressing the accumulation of FAK in miR-27 morphants partially rescued the severe pharyngeal cartilage defects observed upon knock down of miR-27. These data support a crucial role for miR-27 in promoting chondrogenic differentiation in the pharyngeal arches through regulation of FAK.
Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs) in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs) in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPμ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPμ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPμ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5) and postnatal (P0, P3, P7) kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage), and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPμ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1) VE-PTP and PTPμ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2) their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3) peritubular capillary VE-PTP expression is down-regulated in adult kidneys, suggesting a role of VE-PTP in the development of peritubular capillaries.
Dysregulation of the oncogenic transcription factor MYC induces B-cell transformation and is a driver for B-cell non-Hodgkin lymphoma (B-NHL). MYC overexpression in B-NHL is associated with more aggressive phenotypes and poor prognosis. Although genomic studies suggest a link between MYC overexpression and B-cell receptor (BCR) signaling molecules in B-NHL, signaling pathways essential to Myc-mediated B-cell transformation have not been fully elucidated. We utilized intracellular phospho-flow cytometry to investigate the relationship between Myc and BCR signaling in pre-malignant B cells. Utilizing the Eμ-myc mouse model, where Myc is overexpressed specifically in B cells, both basal and stimulated BCR signaling were increased in precancerous B lymphocytes from Eμ-myc mice compared with wild-type littermates. B cells overexpressing Myc displayed constitutively higher levels of activated CD79α, Btk, Plcγ2 and Erk1/2. Notably, Myc-overexpressing B cells maintained elevated BCR signaling despite treatment with ibrutinib, a Bruton's tyrosine kinase inhibitor. Furthermore, PI3K/Akt pathway signaling was also increased in Eμ-myc B cells, and this increase was partially suppressed with ibrutinib. In addition, experiments with Btk-null B cells revealed off-target effects of ibrutinib on BCR signaling. Our data show that in pre-malignant B cells, Myc overexpression is sufficient to activate BCR and PI3K/Akt signaling pathways and further enhances signaling following BCR ligation. Therefore, our results indicate that precancerous B cells have already acquired enhanced survival and growth capabilities before transformation, and that elevated MYC levels confer resistance to pharmacologic inhibitors of BCR signaling, which has significant implications for B-NHL treatment.
Kinases downstream of B-cell antigen receptor (BCR) represent attractive targets for therapy in non-Hodgkin lymphoma (NHL). As clinical responses vary, improved knowledge regarding activation and regulation of BCR signaling in individual patients is needed. Here, using phosphospecific flow cytometry to obtain malignant B-cell signaling profiles from 95 patients representing 4 types of NHL revealed a striking contrast between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) tumors. Lymphoma cells from diffuse large B-cell lymphoma patients had high basal phosphorylation levels of most measured signaling nodes, whereas follicular lymphoma cells represented the opposite pattern with no or very low basal levels. MCL showed large interpatient variability in basal levels, and elevated levels for the phosphorylated forms of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 were associated with poor outcome. CLL tumors had elevated basal levels for the phosphorylated forms of BCR-signaling nodes (Src family tyrosine kinase, spleen tyrosine kinase [SYK], phospholipase Cγ), but had low α-BCR-induced signaling. This contrasted MCL tumors, where α-BCR-induced signaling was variable, but significantly potentiated as compared with the other types. Overexpression of CD79B, combined with a gating strategy whereby signaling output was directly quantified per cell as a function of CD79B levels, confirmed a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signaling levels. Furthermore, α-BCR-induced signaling strength was variable across patient samples and correlated with BCR subunit CD79B expression, but was inversely correlated with susceptibility to Bruton tyrosine kinase (BTK) and SYK inhibitors in MCL. These individual differences in BCR levels and signaling might relate to differences in therapy responses to BCR-pathway inhibitors.
© 2017 by The American Society of Hematology.
Targeting vascular endothelial growth factor (VEGF) is a common treatment strategy for neovascular eye disease, a major cause of vision loss in diabetic retinopathy and age-related macular degeneration. However, the decline in clinical efficacy over time in many patients suggests that monotherapy of anti-VEGF protein therapeutics may benefit from adjunctive treatments. Our previous work has shown that through decreased activation of the cytoskeletal protein paxillin, growth factor-induced ischemic retinopathy in the murine oxygen-induced retinopathy model could be inhibited. In this study, we demonstrated that VEGF-dependent activation of the Src/FAK/paxillin signalsome is required for human retinal endothelial cell migration and proliferation. Specifically, the disruption of focal adhesion kinase (FAK) and paxillin interactions using the small molecule JP-153 inhibited Src-dependent phosphorylation of paxillin (Y118) and downstream activation of Akt (S473), resulting in reduced migration and proliferation of retinal endothelial cells stimulated with VEGF. However, this effect did not prevent the initial activation of either Src or FAK. Furthermore, topical application of a JP-153-loaded microemulsion affected the hallmark features of pathologic retinal angiogenesis, reducing neovascular tuft formation and increased avascular area, in a dose-dependent manner. In conclusion, our results suggest that using small molecules to modulate the focal adhesion protein paxillin is an effective strategy for treating pathologic retinal neovascularization. To our knowledge, this is the first paradigm validating modulation of paxillin to inhibit angiogenesis. As such, we have identified and developed a novel class of small molecules aimed at targeting focal adhesion protein interactions that are essential for pathologic neovascularization in the eye.
Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells-MDCK-within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.
Triple-negative breast cancer (TNBC) and other molecularly heterogeneous malignancies present a significant clinical challenge due to a lack of high-frequency "driver" alterations amenable to therapeutic intervention. These cancers often exhibit genomic instability, resulting in chromosomal rearrangements that affect the structure and expression of protein-coding genes. However, identification of these rearrangements remains technically challenging. Using a newly developed approach that quantitatively predicts gene rearrangements in tumor-derived genetic material, we identified and characterized a novel oncogenic fusion involving the MER proto-oncogene tyrosine kinase (MERTK) and discovered a clinical occurrence and cell line model of the targetable FGFR3-TACC3 fusion in TNBC. Expanding our analysis to other malignancies, we identified a diverse array of novel and known hybrid transcripts, including rearrangements between noncoding regions and clinically relevant genes such as ALK, CSF1R, and CD274/PD-L1 The over 1,000 genetic alterations we identified highlight the importance of considering noncoding gene rearrangement partners, and the targetable gene fusions identified in TNBC demonstrate the need to advance gene fusion detection for molecularly heterogeneous cancers. Cancer Res; 76(16); 4850-60. ©2016 AACR.
©2016 American Association for Cancer Research.