FuiBoon Kai

Force Matters: Biomechanical Regulation of Cell Invasion and Migration in Disease

Atherosclerosis, cancer, and various chronic fibrotic conditions are characterized by an increase in the migratory behavior of resident cells and the enhanced invasion of assorted exogenous cells across a stiffened extracellular matrix (ECM). This stiffened scaffold aberrantly engages cellular mechanosignaling networks in cells, which promotes the assembly of invadosomes and lamellae for cell invasion and migration. Accordingly, deciphering the conserved molecular mechanisms whereby matrix stiffness fosters invadosome and lamella formation could identify therapeutic targets to treat fibrotic conditions, and reducing ECM stiffness could ameliorate disease progression.

NUP98-HOXA9-transgenic zebrafish develop a myeloproliferative neoplasm and provide new insight into mechanisms of myeloid leukaemogenesis.

NUP98‐HOXA9 [t(7;11) (p15;p15)] is associated with inferior prognosis in de novo and treatment‐related acute myeloid leukaemia (AML) and contributes to blast crisis in chronic myeloid leukaemia (CML). We have engineered an inducible transgenic zebrafish harbouring human NUP98‐HOXA9 under the zebrafish spi1(pu.1) promoter. NUP98‐HOXA9 perturbed zebrafish embryonic haematopoiesis, with upregulated spi1expression at the expense of gata1a. Markers associated with more differentiated myeloid cells, lcp1, lyz, and mpx were also elevated, but to a lesser extent than spi1, suggesting differentiation of early myeloid progenitors may be impaired by NUP98‐HOXA9. Following irradiation, NUP98‐HOXA9‐expressing embryos showed increased numbers of cells in G2‐M transition compared to controls and absence of a normal apoptotic response, which may result from an upregulation of bcl2. These data suggest NUP98‐HOXA9‐induced oncogenesis may result from a combination of defects in haematopoiesis and an aberrant response to DNA damage. Importantly, 23% of adult NUP98‐HOXA9‐transgenic fish developed a myeloproliferative neoplasm (MPN) at 19–23 months of age. In summary, we have identified an embryonic haematopoietic phenotype in a transgenic zebrafish line that subsequently develops MPN. This tool provides a unique opportunity for high‐throughput in vivo chemical modifier screens to identify novel therapeutic agents in high risk AML.

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix

Mammary tumor cells adopt a basal-like phenotype when invading through a dense, stiffened, 3D matrix. These cells exert higher integrin-mediated traction forces, consistent with a physical motor-clutch model, display an altered molecular organization at the nanoscale, and recruit a suite of paxillin-associated proteins implicated in metastasis.

Golgi complex-plasma membrane trafficking directed by an autonomous, tribasic Golgi export signal.

The first example of a cytosolic, membrane-proximal, tribasic motif required for Golgi export to the plasma membrane is identified and characterized. This novel Golgi export signal can also mediate trafficking of a heterologous Golgi-resident protein, indicating that it functions as an autonomous Golgi export signal.

Prostate cancer cell migration induced by myopodin isoforms is associated with formation of morphologically and biochemically distinct actin networks

Myopodin is an actin‐binding protein that promotes cancer cell migration in response to serum stimulation and is associated with invasive tumor development. To determine whether enhanced migration reflects changes in actin cytoskeleton remodeling, fluorescence confocal microscopy was used to examine the composition and morphology of filamentous actin structures in mock‐transduced cells vs. stably transduced PC3 cells expressing human myopodin isoforms, and the chemokinetic response of cells was quantified using transwell assays. The same approaches were used to analyze the effects of external migration stimuli, actin polymerization inhibitors or deletion of the isoform‐specific amino‐ and/or carboxy termini on cell migration and actin bundle formation. Results indicate that the termini of the myopodin isoforms differentially alter the formation of morphologically distinct F‐actin networks that also differ in their myosin and myopodin staining patterns. Furthermore, enhanced cell migration was reduced by > 50% when actin bundle formation was impaired by myopodin‐truncation, low concentrations of an actin polymerization inhibitor, or in the absence of an external migration stimulus. Human myopodin isoforms are therefore potent regulators of stress fiber formation, inducing the formation of biochemically and morphologically distinct F‐actin networks in the cell body whose presence directly correlates with increased cell migration.—Kai, F., Duncan, R., Prostate cancer cell migration induced by myopodin isoforms is associated with formation of morphologically and biochemically distinct actin networks. FASEB J. 27, 5046–5058 (2013). www.fasebj.org

Myopodin isoforms alter the chemokinetic response of PC3 cells in response to different migration stimuli via differential effects on Rho-ROCK signaling pathways

The gene encoding myopodin, an actin binding protein, is commonly deleted in invasive, but not in indolent, prostate cancers. There are conflicting reports on the effects of myopodin expression on prostate cancer cell migration and invasion. The recent recognition that myopodin is expressed as four different isoforms further complicates our understanding of how this potentially important invasive prostate cancer biomarker affects tumor cell migration and invasion. We now show that myopodin affects the chemokinetic, rather than the chemotactic, properties of PC3 prostate cancer cells. Furthermore, all myopodin isoforms can either increase or decrease PC3 cell migration in response to different chemokinetic stimuli. These migration properties were reflected by differences in cell morphology and the relative dependence on Rho-ROCK signaling pathways induced by the environmental stimuli. Truncation analysis determined that a unique 9-residue C-terminal sequence in the shortest isoform and the conserved, PDZ domain-containing N-terminal region of the long isoforms both contribute to the ability of myopodin to alter the response of PC3 cells to chemokinetic stimuli. Matrigel invasion assays also indicated that myopodin primarily affects the migration, rather than the invasion, properties of PC3 cells. The correlation between loss of myopodin expression and invasive prostate cancer therefore reflects complex myopodin interactions with pathways that regulate the cellular migration response to diverse signals that may be present in a tumor microenvironment.

A bulky glycocalyx fosters metastasis formation by promoting G1 cell cycle progression

Metastasis depends upon cancer cell growth and survival within the metastatic niche. Tumors which remodel their glycocalyces, by overexpressing bulky glycoproteins like mucins, exhibit a higher predisposition to metastasize, but the role of mucins in oncogenesis remains poorly understood. Here we report that a bulky glycocalyx promotes the expansion of disseminated tumor cells in vivo by fostering integrin adhesion assembly to permit G1 cell cycle progression. We engineered tumor cells to display glycocalyces of various thicknesses by coating them with synthetic mucin-mimetic glycopolymers. Cells adorned with longer glycopolymers showed increased metastatic potential, enhanced cell cycle progression, and greater levels of integrin-FAK mechanosignaling and Akt signaling in a syngeneic mouse model of metastasis. These effects were mirrored by expression of the ectodomain of cancer-associated mucin MUC1. These findings functionally link mucinous proteins with tumor aggression, and offer a new view of the cancer glycocalyx as a major driver of disease progression.

Genetically Encoded Toolbox for Glycocalyx Engineering: Tunable Control of Cell Adhesion, Survival, and Cancer Cell Behaviors

The glycocalyx is a coating of protein and sugar on the surface of all living cells. Dramatic perturbations to the composition and structure of the glycocalyx are frequently observed in aggressive cancers. However, tools to experimentally mimic and model the cancer-specific glycocalyx remain limited. Here, we develop a genetically encoded toolkit to engineer the chemical and physical structure of the cellular glycocalyx. By manipulating the glycocalyx structure, we are able to switch the adhesive state of cells from strongly adherent to fully detached. Surprisingly, we find that a thick and dense glycocalyx with high O-glycan content promotes cell survival even in a suspended state, characteristic of circulating tumor cells during metastatic dissemination. Our data suggest that glycocalyx-mediated survival is largely independent of receptor tyrosine kinase and mitogen activated kinase signaling. While anchorage is still required for proliferation, we find that cells with a thick glycocalyx can dynamically attach to a matrix scaffold, undergo cellular division, and quickly disassociate again into a suspended state. Together, our technology provides a needed toolkit for engineering the glycocalyx in glycobiology and cancer research.

A tension-mediated glycocalyx–integrin feedback loop promotes mesenchymal-like glioblastoma

Glioblastoma multiforme (GBMs) are recurrent lethal brain tumours. Recurrent GBMs often exhibit mesenchymal, stem-like phenotypes that could explain their resistance to therapy. Analyses revealed that recurrent GBMs have increased tension and express high levels of glycoproteins that increase the bulkiness of the glycocalyx. Studies showed that a bulky glycocalyx potentiates integrin mechanosignalling and tissue tension and promotes a mesenchymal, stem-like phenotype in GBMs. Gain- and loss-of-function studies implicated integrin mechanosignalling as an inducer of GBM growth, survival, invasion and treatment resistance, and a mesenchymal, stem-like phenotype. Mesenchymal-like GBMs were highly contractile and expressed elevated levels of glycoproteins that expanded their glycocalyx, and they were surrounded by a stiff extracellular matrix that potentiated integrin mechanosignalling. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignalling and a bulky glycocalyx, implying a causal link towards a mesenchymal, stem-like phenotype in GBMs. Strategies to ameliorate GBM tissue tension offer a therapeutic approach to reduce mortality due to GBM.Barnes et al. report a dynamic and reciprocal crosstalk between tissue tension and glycocalyx bulkiness that promotes a mesenchymal, stem-like phenotype in GBM.