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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.
Tumor vasculature is known to be more permeable than the vasculature found in healthy tissue, which in turn can lead to a more aggressive tumor phenotype and impair drug delivery into tumors. While the stiffening of the stroma surrounding solid tumors has been reported to increase vascular permeability, the mechanism of this process remains unclear. Here, we utilize an in vitro model of tumor stiffening, ex ovo culture, and a mouse model to investigate the molecular mechanism by which matrix stiffening alters endothelial barrier function. Our data indicate that the increased endothelial permeability caused by heightened matrix stiffness can be prevented by pharmaceutical inhibition of focal adhesion kinase (FAK) both in vitro and ex ovo. Matrix stiffness-mediated FAK activation determines Src localization to cell-cell junctions, which then induces increased vascular endothelial cadherin phosphorylation both in vitro and in vivo. Endothelial cells in stiff tumors have more activated Src and higher levels of phosphorylated vascular endothelial cadherin at adherens junctions compared to endothelial cells in more compliant tumors. Altogether, our data indicate that matrix stiffness regulates endothelial barrier integrity through FAK activity, providing one mechanism by which extracellular matrix stiffness regulates endothelial barrier function. Additionally, our work also provides further evidence that FAK is a promising potential target for cancer therapy because FAK plays a critical role in the regulation of endothelial barrier integrity.-Wang, W., Lollis, E. M., Bordeleau, F., Reinhart-King, C. A. Matrix stiffness regulates vascular integrity through focal adhesion kinase activity.
Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.
Carotid body glomus cells mediate essential reflex responses to arterial blood hypoxia. They are dopaminergic and secrete growth factors that support dopaminergic neurons, making the carotid body a potential source of patient-specific cells for Parkinson's disease therapy. Like adrenal chromaffin cells, which are also hypoxia-sensitive, glomus cells are neural crest-derived and require the transcription factors Ascl1 and Phox2b; otherwise, their development is little understood at the molecular level. Here, analysis in chicken and mouse reveals further striking molecular parallels, though also some differences, between glomus and adrenal chromaffin cell development. Moreover, histology has long suggested that glomus cell precursors are 'émigrés' from neighbouring ganglia/nerves, while multipotent nerve-associated glial cells are now known to make a significant contribution to the adrenal chromaffin cell population in the mouse. We present conditional genetic lineage-tracing data from mice supporting the hypothesis that progenitors expressing the glial marker proteolipid protein 1, presumably located in adjacent ganglia/nerves, also contribute to glomus cells. Finally, we resolve a paradox for the 'émigré' hypothesis in the chicken - where the nearest ganglion to the carotid body is the nodose, in which the satellite glia are neural crest-derived, but the neurons are almost entirely placode-derived - by fate-mapping putative nodose neuronal 'émigrés' to the neural crest.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
While many adhesion receptors are known to influence tumor progression, the mechanisms by which they dynamically regulate cell-cell adhesion remain elusive. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a clinically relevant driver of metastasis and hypothesized that a tunable mechanism of ectodomain shedding regulates its contribution to dissemination. To test this hypothesis, we examined an under-explored ALCAM splice variant (ALCAM-Iso2) and demonstrated that loss of the membrane-proximal region of ALCAM (exon 13) increased metastasis four-fold. Mechanistic studies identified a novel MMP14-dependent membrane distal cleavage site in ALCAM-Iso2, which mediated a ten-fold increase in shedding, thereby decreasing cellular cohesion. Importantly, the loss of cohesion is not limited to the cell capable of shedding because the released extracellular domain diminished cohesion of non-shedding cells through disruption of ALCAM-ALCAM interactions. ALCAM-Iso2-dominated expression in bladder cancer tissue, compared to normal bladder, further emphasizes that ALCAM alternative splicing may contribute to clinical disease progression. The requirement for both the loss of exon 13 and the gain of metalloprotease activity suggests that ALCAM shedding and concomitant regulation of tumor cell adhesion is a locally tunable process.
Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer cross-linked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.
The use of rodent models to evaluate efficacy during testing is accompanied by significant economic and regulatory hurdles which compound the costs of screening for promising drug candidates. Vasopermeation Enhancement Agents (VEAs) are a new class of biologics that are designed to increase the uptake of cancer therapeutics at the tumor site by modifying vascular permeability in the tumor to increase the therapeutic index of co-administered drugs. To evaluate the efficacy of a panel of VEA clinical candidates, we compared the rodent Miles assay to an equivalent assay in the ex ovo chicken embryo model. Both model systems identified the same candidate (PVL 10) as the most active promoter of vasopermeation in non-tumor tissues. An ex ovo chicken embryo system was utilized to test each candidate VEA in two human tumor models at a range of concentrations. Vasopermeation activity due to VEA was dependent on tumor type, with HEp3 tumors displaying higher levels of vasopermeation than MDA-MB-435. One candidate (PVL 10) proved optimal for HEp3 tumors and another (PVL 2) for MDA-MB-435. The use of the ex ovo chicken embryo model provides a rapid and less costly alternative to the use of rodent models for preclinical screening of drug candidates.
Transcriptional repression of E-cadherin is a hallmark of Epithelial-to-Mesenchymal Transition (EMT) and is associated with cancer cell invasion and metastasis. Understanding the mechanisms underlying E-cadherin repression during EMT may provide insights into the development of novel targeted therapeutics for cancer. Here, we report on the chemical probe, ML327, which de-represses E-cadherin transcription, partially reverses EMT, and inhibits cancer cell invasiveness and tumor cell migration in vitro and in vivo. Induction of E-cadherin mRNA expression by ML327 treatment does not require de novo protein synthesis. RNA sequencing analysis revealed that ML327 treatment significantly alters expression of over 2,500 genes within three hours in the presence of the translational inhibitor, cycloheximide. Network analysis reveals Hepatocyte Nuclear Factor 4-alpha (HNF4α) as the most significant upstream transcriptional regulator of multiple genes whose expressions were altered by ML327 treatment. Further, small interfering RNA-mediated depletion of HNF4α markedly attenuates the E-cadherin expression response to ML327. In summary, ML327 represents a valuable tool to understand mechanisms of EMT and may provide the basis for a novel targeted therapeutic strategy for carcinomas.
Directional cell movement through tissues is critical for multiple biological processes and requires maintenance of polarity in the face of complex environmental cues. Here we use intravital imaging to demonstrate that secretion of exosomes from late endosomes is required for directionally persistent and efficient in vivo movement of cancer cells. Inhibiting exosome secretion or biogenesis leads to defective tumour cell migration associated with increased formation of unstable protrusions and excessive directional switching. In vitro rescue experiments with purified exosomes and matrix coating identify adhesion assembly as a critical exosome function that promotes efficient cell motility. Live-cell imaging reveals that exosome secretion directly precedes and promotes adhesion assembly. Fibronectin is found to be a critical motility-promoting cargo whose sorting into exosomes depends on binding to integrins. We propose that autocrine secretion of exosomes powerfully promotes directionally persistent and effective cell motility by reinforcing otherwise transient polarization states and promoting adhesion assembly.
Receptor protein tyrosine phosphatase sigma (RPTPσ) regulates neuronal extension and acts as a presynaptic nexus for multiple protein and proteoglycan interactions during synaptogenesis. Unknown mechanisms govern the shift in RPTPσ function, from outgrowth promotion to synaptic organization. Here, we report crystallographic, electron microscopic and small-angle X-ray scattering analyses, which reveal sufficient inter-domain flexibility in the RPTPσ extracellular region for interaction with both cis (same cell) and trans (opposite cell) ligands. Crystal structures of RPTPσ bound to its postsynaptic ligand TrkC detail an interaction surface partially overlapping the glycosaminoglycan-binding site. Accordingly, heparan sulphate and heparin oligomers compete with TrkC for RPTPσ binding in vitro and disrupt TrkC-dependent synaptic differentiation in neuronal co-culture assays. We propose that transient RPTPσ ectodomain emergence from the presynaptic proteoglycan layer allows capture by TrkC to form a trans-synaptic complex, the consequent reduction in RPTPσ flexibility potentiating interactions with additional ligands to orchestrate excitatory synapse formation.