Autocrine laminin-5 ligates {alpha}6{beta}4 integrin and activates RAC and NF{kappa}B to mediate anchorange-independent survival of mammary tumors

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express α6β4 integrin. Here, we show that autocrine LM-5 mediates anchorage independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail–truncated α6β4 integrin. α6β4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of β4 integrin is necessary for basal and epidermal growth factor–induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional β1 and β4 integrin through activation of NFκB, and overexpression of NFκB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5–α6β4 integrin–RAC–NFκB signaling

Autocrine laminin-5 ligates α6β4 integrin and activates RAC and NFκB to mediate anchorage-independent survival of mammary tumors

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express α6β4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail–truncated α6β4 integrin. α6β4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of β4 integrin is necessary for basal and epidermal growth factor–induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional β1 and β4 integrin through activation of NFκB, and overexpression of NFκB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5–α6β4 integrin–RAC–NFκB signaling

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several Kras-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby Stat3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial Stat3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 and mechanics as key drivers of this phenotype

Tissue Mechanics Orchestrate Wnt-Dependent Human Embryonic Stem Cell Differentiation

Regenerative medicine is predicated on understanding the mechanisms regulating development and applying these conditions to direct stem cell fate. Embryogenesis is guided by cell-cell and cell-matrix interactions, but it is unclear how these physical cues influence stem cells in culture. We used human embryonic stem cells (hESCs) to examine whether mechanical features of the extracellular microenvironment could differentially modulate mesoderm specification. We found that, on a hydrogel-based compliant matrix, hESCs accumulate β-catenin at cell-cell adhesions and show enhanced Wnt-dependent mesoderm differentiation. Mechanistically, Src-driven ubiquitination of E-cadherin by Cbl-like ubiquitin ligase releases P120-catenin to facilitate transcriptional activity of β-catenin, which initiates and reinforces mesoderm differentiation. By contrast, on a stiff hydrogel matrix, hESCs show elevated integrin-dependent GSK3 and Src activity that promotes β-catenin degradation and inhibits differentiation. Thus, we found that mechanical features of the microenvironmental matrix influence tissue-specific differentiation of hESCs by altering the cellular response to morphogens

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development

Mechanical Tension Promotes Formation of Gastrulation-like Nodes and Patterns Mesoderm Specification in Human Embryonic Stem Cells

Embryogenesis is directed by morphogens that induce differentiation within a defined tissue geometry. Tissue organization is mediated by cell-cell and cell-extracellular matrix (ECM) adhesions and is modulated by cell tension and tissue-level forces. Whether cell tension regulates development by modifying morphogen signaling is less clear. Human embryonic stem cells (hESCs) exhibit an intrinsic capacity for self-organization, which motivates their use as a tractable model of early human embryogenesis. We engineered patterned substrates that recapitulate the biophysical properties of the early embryo and mediate the self-organization of "gastrulation-like" nodes in cultured hESCs. Tissue geometries that generated local nodes of high cell-adhesion tension directed the spatial patterning of the BMP4-dependent "gastrulation-like" phenotype by enhancing phosphorylation and junctional release of β-catenin to promote Wnt signaling and mesoderm specification. Furthermore, direct force application via mechanical stretching promoted BMP-dependent mesoderm specification, confirming that tissue-level forces can directly regulate cell fate specification in early human development

Evidence that clusterin has discrete chaperone and ligand binding sites

Clusterin is the first identified extracellular mammalian chaperone and binds to a wide variety of partly unfolded, stressed proteins. Clusterin also binds to many different unstressed ligands including the cell surface receptor low density lipoprotein receptor-related protein-2 (LRP-2). It is unknown whether clusterin binds to all of these many ligands via one or more binding sites. Furthermore, the region(s) of clusterin involved in these many binding interactions remain to be identified. As part of an investigation of these issues, we expressed recombinant human clusterin in the yeast Pichia pastoris. The resultant protein had variable proteolytic truncations of the C-terminal region of the a-chain and the N-terminal region of the b-chain. We compared the chaperone and ligand binding activities of this recombinant product with those of clusterin purified from human serum. We also tested whether the binding of clusterin to ligands could be inhibited by competitive binding with other clusterin ligands or by anti-clusterin monoclonal antibodies. Collectively, our results indicate that (i) clusterin has three independent classes of binding sites for LRP-2, stressed proteins and unstressed ligands, respectively, and (ii) the binding sites for LRP-2 and stressed proteins are likely to be in parts of the molecule other than the C-terminal region of the alpha-chain or the N-terminal region of the b-chain. It has been suggested that, in vivo, clusterin binds to toxic molecules in the extracellular environment and carries these to cells expressing LRP-2 for uptake and degradation. This hypothesis is supported by our demonstration that clusterin has discrete binding sites for LRP-2 and other (potentially toxic) molecules

Autocrine laminin-5 ligates alpha6beta4 integrin and activates RAC and NFkappaB to mediate anchorage-independent survival of mammary tumors.

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express alpha6beta4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail-truncated alpha6beta4 integrin. alpha6beta4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of beta4 integrin is necessary for basal and epidermal growth factor-induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional beta1 and beta4 integrin through activation of NFkappaB, and overexpression of NFkappaB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5-alpha6beta4 integrin-RAC-NFkappaB signaling

Autocrine laminin-5 ligates alpha6beta4 integrin and activates RAC and NFkappaB to mediate anchorage-independent survival of mammary tumors.

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express alpha6beta4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail-truncated alpha6beta4 integrin. alpha6beta4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of beta4 integrin is necessary for basal and epidermal growth factor-induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional beta1 and beta4 integrin through activation of NFkappaB, and overexpression of NFkappaB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5-alpha6beta4 integrin-RAC-NFkappaB signaling