Dysregulation of cell polarity proteins synergize with oncogenes or the microenvironment to induce invasive behavior in epithelial cells

Changes in expression and localization of proteins that regulate cell and tissue polarity are frequently observed in carcinoma. However, the mechanisms by which changes in cell polarity proteins regulate carcinoma progression are not well understood. Here, we report that loss of polarity protein expression in epithelial cells primes them for cooperation with oncogenes or changes in tissue microenvironment to promote invasive behavior. Activation of ErbB2 in cells lacking the polarity regulators Scribble, Dlg1 or AF-6, induced invasive properties. This cooperation required the ability of ErbB2 to regulate the Par6/aPKC polarity complex. Inhibition of the ErbB2-Par6 pathway was sufficient to block ErbB2-induced invasion suggesting that two polarity hits may be needed for ErbB2 to promote invasion. Interestingly, in the absence of ErbB2 activation, either a combined loss of two polarity proteins, or exposure of cells lacking one polarity protein to cytokines IL-6 or TNFα induced invasive behavior in epithelial cells. We observed the invasive behavior only when cells were plated on a stiff matrix (Matrigel/Collagen-1) and not when plated on a soft matrix (Matrigel alone). Cells lacking two polarity proteins upregulated expression of EGFR and activated Akt. Inhibition of Akt activity blocked the invasive behavior identifying a mechanism by which loss of polarity promotes invasion of epithelial cells. Thus, we demonstrate that loss of polarity proteins confers phenotypic plasticity to epithelial cells such that they display normal behavior under normal culture conditions but display aggressive behavior in response to activation of oncogenes or exposure to cytokines.     

Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures

The three-dimensional culture of MCF-10A mammary epithelial cells on a reconstituted basement membrane results in formation of polarized, growth-arrested acini-like spheroids that recapitulate several aspects of glandular architecture in vivo. Oncogenes introduced into MCF-10A cells disrupt this morphogenetic process, and elicit distinct morphological phenotypes. Recent studies analyzing the mechanistic basis for phenotypic heterogeneity observed among different oncogenes (e.g., ErbB2, cyclin D1) have illustrated the utility of this three-dimensional culture system in modeling the biological activities of cancer genes, particularly with regard to their ability to disrupt epithelial architecture during the early aspects of carcinoma formation. Here we provide a collection of protocols to culture MCF-10A cells, to establish stable pools expressing a gene of interest via retroviral infection, as well as to grow and analyze MCF-10A cells in three-dimensional basement membrane culture.     

Assembly of epithelial tight junctions is negatively regulated by Par6

Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts-claudins, occludin, and junctional adhesion molecule (JAM)-plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Here, we show that Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca(2+) depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs, but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells.     

Par6b Regulates the Dynamics of Apicobasal Polarity during Development of the Stratified Xenopus Epidermis

During early vertebrate development, epithelial cells establish and maintain apicobasal polarity, failure of which can cause developmental defects or cancer metastasis. This process has been mostly studied in simple epithelia that have only one layer of cells, but is poorly understood in stratified epithelia. In this paper we address the role of the polarity protein Partitioning defective-6 homolog beta (Par6b) in the developing stratified epidermis of Xenopus laevis. At the blastula stage, animal blastomeres divide perpendicularly to the apicobasal axis to generate partially polarized superficial cells and non-polarized deep cells. Both cell populations modify their apicobasal polarity during the gastrula stage, before differentiating into the superficial and deep layers of epidermis. Early differentiation of the epidermis is normal in Par6b-depleted embryos; however, epidermal cells dissociate and detach from embryos at the tailbud stage. Par6b-depleted epidermal cells exhibit a significant reduction in basolaterally localized E-cadherin. Examination of the apical marker Crumbs homolog 3 (Crb3) and the basolateral marker Lethal giant larvae 2 (Lgl2) after Par6b depletion reveals that Par6b cell-autonomously regulates the dynamics of apicobasal polarity in both superficial and deep epidermal layers. Par6b is required to maintain the “basolateral” state in both epidermal layers, which explains the reduction of basolateral adhesion complexes and epidermal cells shedding.     

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell–cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.     

Establishing epithelial glandular polarity: interlinked roles for ARF6, Rac1, and the matrix microenvironment

Epithelial cysts comprise the structural units of the glandular epithelium. Although glandular inversion in epithelial tumors is thought to be a potential mechanism for the establishment of metastatic disease, little is known about the morphogenic cues and signaling pathways that govern glandular polarity and organization. Using organotypic cultures of Madin-Darby canine kidney cells in reconstituted basement membrane, we show that cellular depletion of the small GTP-binding protein ARF6 promotes the formation of inverted cysts, wherein the apical cell membrane faces the cyst exterior, and the basal domain faces the central lumen, while individual cell polarity is maintained. These cysts are also defective in interactions with laminin at the cyst–matrix interface. This inversion of glandular orientation is accompanied by Rac1 inactivation during early cystogenesis, and temporal activation of Rac1 is sufficient to recover the normal cyst phenotype. In an unnatural collagen I microenvironment, ARF6-depleted, inverted epithelial cysts exhibit some loss of cell polarity, a marked increase in Rho activation and Rac1 inactivation, and striking rearrangement of the surrounding collagen I matrix. These studies demonstrate the importance of ARF6 as a critical determinant of glandular orientation and the matrix environment in dictating structural organization of epithelial cysts.     

Multiple splice variants of Par3 and of a novel related gene,Par3L,produce proteins with different binding properties

The partitioning-defective 3 (par3) gene encodes a protein with three postsynaptic density90/DiscslargeA/ZO-1 (PDZ) domains that is required for cell polarity establishment in metazoans. Par3 is a component of a protein complex that can include Cdc42-GTP, Par6 and atypical protein kinase Cs (aPKCs). We now describe the identification of a related human gene, Par3L. Both Par3L and Par3 are expressed as numerous alternatively spliced variants. Although Par3 expression appears to be ubiquitous, that of Par3L is more restricted. Multiple variants are often expressed simultaneously within a specific cell type or tissue. Although all of the Par3L/Par3 isoforms can associate with tight junctions in epithelial cells, they show different binding properties. No Par3L isoforms and only a subset of Par3 isoforms detectably bind aPKCs. These data suggest that aPKC binding or phosphorylation is not required for targeting of Par3/Par3L to cell-cell contacts. Par3L isoforms also show differential binding to Par6. Despite these differences, the N-terminal region of Par3L, like that of Par3, can disrupt the formation of tight junctions when ectopically expressed in Madin-Darby canine kidney (MDCK) cells.     

Nucleotide exchange factor ECT2 interacts with the polarity protein complex Par6/Par3/protein kinase Czeta (PKCzeta) and regulates PKCzeta activity

Regulation of cell polarity is an important biological event that governs diverse cell functions such as localization of embryonic determinants and establishment of tissue and organ architecture. The Rho family GTPases and the polarity complex Par6/Par3/atypical protein kinase C (PKC) play a key role in the signaling pathway, but the molecules that regulate upstream signaling are still not known. Here we identified the guanine nucleotide exchange factor ECT2 as an activator of the polarity complex. ECT2 interacted with Par6 as well as Par3 and PKCzeta. Coexpression of Par6 and ECT2 efficiently activated Cdc42 in vivo. Overexpression of ECT2 also stimulated the PKCzeta activity, whereas dominant-negative ECT2 inhibited the increase in PKCzeta activity stimulated by Par6. ECT2 localization was detected at sites of cell-cell contact as well as in the nucleus of MDCK cells. The expression and localization of ECT2 were regulated by calcium, which is a critical regulator of cell-cell adhesion. Together, these results suggest that ECT2 regulates the polarity complex Par6/Par3/PKCzeta and possibly plays a role in epithelial cell polarity.     

PATJ regulates directional migration of mammalian epithelial cells

Directional migration is important in wound healing by epithelial cells. Recent studies have shown that polarity proteins such as mammalian Partitioning-defective 6 (Par6), atypical protein kinase C (aPKC) and mammalian Discs large 1 (Dlg1) are crucial not only for epithelial apico-basal polarity, but also for directional movement. Here, we show that the protein associated with Lin seven 1 (PALS1)-associated tight junction protein (PATJ), another evolutionarily conserved polarity protein, is also required for directional migration by using a wound-induced migration assay. In addition, we found that aPKC and Par3 localize to the leading edge during migration of epithelia and that PATJ regulates their localization. Furthermore, our results show that microtubule-organizing centre orientation is disrupted in PATJ RNA interference (RNAi) MDCKII (Madin-Darby canine kidney II) cells during migration. Together, our data indicate that PATJ controls directional migration by regulating the localization of aPKC and Par3 to the leading edge. The migration defect in PATJ RNAi cells seems to be due to the disorganization of the microtubule network induced by mislocalization of polarity proteins.     

The Par-Tiam1 complex controls persistent migration by stabilizing microtubule-dependent front-rear polarity

BACKGROUND: The establishment and maintenance of cell polarity is crucial for many biological functions and is regulated by conserved protein complexes. The Par polarity complex consisting of Par3, Par6, and PKCzeta, in conjunction with Tiam1-mediated Rac signaling, controls apical-basal cell polarity in contacting epithelial cells. Here we tested the hypothesis that the Par complex, in conjunction with Tiam1, controls "front-rear" polarity during the persistent migration of freely migrating keratinocytes. RESULTS: Wild-type (WT) epidermal keratinocytes lacking cell-cell contacts are stably front-rear polarized and migrate persistently. In contrast, Tiam1-deficient (Tiam1 KO) and (si)Par3-depleted keratinocytes are generally unpolarized and migrate randomly because front-rear polarity is short lived. Immunoprecipitation experiments show that in migrating keratinocytes, Tiam1 associates with Par3 and PKCzeta. Moreover, Par3, PKCzeta, and Tiam1 proteins are enriched at the leading edges of polarized keratinocytes. Tiam1 KO keratinocytes are impaired in chemotactic migration toward growth factors, whereaes haptotactic migration is similar to WT. Par3 depletion or the blocking of PKCzeta signaling in WT keratinocytes impairs chemotaxis but has no additional effect on Tiam1 KO cells. The migratory and morphological defects in keratinocytes with impaired Par-Tiam1 function closely resemble cells with pharmacologically destabilized microtubules (MTs). Indeed, MTs in Tiam1 KO keratinocytes and WT cells treated with a PKCzeta inhibitor are unstable, thereby negatively influencing directional but not random migration. CONCLUSIONS: We conclude that the Par-Tiam1 complex stabilizes front-rear polarization of noncontacting migratory cells, thereby stimulating persistent and chemotactic migration, whereas in contacting keratinocytes, the same complex controls the establishment of long-lasting apical-basal polarity. These findings underscore a remarkable flexibility of the Par polarity complex that, depending on the biological context, controls distinct forms of cellular polarity.     

The WD40 protein Morg1 facilitates Par6–aPKC binding to Crb3 for apical identity in epithelial cells

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6–aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morg1 (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6–aPKC in Madin-Darby canine kidney epithelial cells. Morg1 also interacts with the apical transmembrane protein Crumbs3 to promote Par6–aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morg1 disrupted both tight junction development in monolayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6–aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells.     

Novel insights into epithelial polarity proteins in Drosophila

Apical-basal polarity is a basic organizing principle of epithelial cells. Consequently, defects in polarity are associated with numerous human pathologies, including many forms of cancer. Recent work in Drosophila has identified novel roles for, or has greatly enhanced our understanding of, functional modules within the epithelial polarity network. A series of recent papers have highlighted the key function of the scaffolding protein Bazooka/Par3 as an early polarity landmark, and its crucial role in dynamic segregation of the apical membrane from the adherens junction. Moreover, novel polarity modules have recently been discovered; the Yurt/Coracle group supports the basolateral membrane during a defined time window of development, while a second module, including the kinases LKB1 and AMP-activated protein kinase, is required for polarity when epithelial cells experience metabolic stress. These new findings emphasize unforeseen complexities in the regulation of epithelial polarity, and raise new questions about the mechanisms of epithelial tissue organization and function.     

PAR3 is essential for cyst-mediated epicardial development by establishing apical cortical domains

Epithelial cysts are one of the fundamental architectures for mammalian organogenesis. Although in vitro studies using cultured epithelial cells have revealed proteins required for cyst formation, the mechanisms that orchestrate the functions of these proteins in vivo remain to be clarified. We show that the targeted disruption of the mouse Par3 gene results in midgestational embryonic lethality with defective epicardial development. The epicardium is mainly derived from epicardial cysts and essential for cardiomyocyte proliferation during cardiac morphogenesis. PAR3-deficient epicardial progenitor (EPP) cells do not form cell cysts and show defects in the establishment of apical cortical domains, but not in basolateral domains. In PAR3-deficient EPP cells, the localizations of aPKC, PAR6beta and ezrin to the apical cortical domains are disturbed. By contrast, ZO1 and alpha4/beta1 integrins normally localize to cell-cell junctions and basal domains, respectively. Our observations indicate that EPP cell cyst formation requires PAR3 to interpret the polarity cues from cell-cell and cell-extracellular matrix interactions so that each EPP cell establishes apical cortical domains. These results also provide a clear example of the proper organization of epithelial tissues through the regulation of individual cell polarity.     

The interaction of mPar3 with the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity

The Par polarity complex consisting of the evolutionarily conserved proteins mPar3, mPar6, and aPKC regulates cell polarity in many cell types including neurons. Here we show that mPar3 is required for the establishment of neuronal polarity and links the Smurf2 to Kinesin-2. The HECT domain E3 ubiquitin ligase Smurf2 ensures that neurons extend only a single axon by initiating the degradation of inactive Rap1B through the ubiquitin/proteasome system. Its interaction with mPar3 is required to localize Smurf2 to growth cones and restrict Rap1B to the axon. Interfering with the binding of mPar3 to Kinesin-2 or Smurf2 to mPar3 and knockdown of mPar3 by RNAi disrupt the establishment of neuronal polarity through the failure to restrict Rap1B to a single neurite.     

Expression, location, and interactions of ErbB2 and its intramembrane ligand Muc4 (sialomucin complex) in rat mammary gland during pregnancy

Muc4 (also called Sialomucin complex) is a heterodimeric glycoprotein complex consisting of a peripheral O-glycosylated subunit ASGP-1 (ascites sialoglycoprotein-1) tightly but non-covalently bound to an N-glycosylated transmembrane subunit ASGP-2. Muc4/SMC can act as an intramembrane ligand for ErbB2 via an EGF-like domain present in the transmembrane subunit. The complex is developmentally regulated in normal rat mammary gland and overexpressed in a number of mammary tumors. Overexpression of Muc4/SMC has been shown to block cell-cell and cell-matrix interactions, protect tumor cells from immune surveillance, promote metastasis, and protect from apoptosis. We have investigated whether Muc4/SMC and ErbB2 are co-expressed and co-localized in normal rat mammary gland and whether Muc4/SMC-ErbB2 complex formation is developmentally regulated in this tissue. Muc4/SMC and ErbB2 have different expression patterns and regulatory mechanisms in the developing rat mammary gland, but both are maximally expressed during late pregnancy and lactation. The two proteins form a complex in lactating mammary gland which is not detected in the virgin gland. Moreover, this complex does not contain ErbB3. ErbB2 is co-localized with Muc4/SMC at the apical surfaces of ductal and alveolar cells in lactating gland; however, another form of ErbB2, recognized by a different antibody, localizes to the basolateral surfaces of these cells. ErbB2 phosphorylated on Tyr 1248 co-localized with Muc4/SMC at the apical surface but not at the basolateral surfaces of these cells. To investigate the function of Muc4 in the mammary gland, transgenic mice were derived using an MMTV-Muc4 construct. Interestingly, mammary gland development in the transgenic mice was aberrant, exhibiting a bifurcated pattern, including invasion down the blood vessel, similar to that exhibited by transgenic mice inappropriately expressing activated ErbB2 in the mammary gland. These data provide further evidence of the ability of Muc4/SMC to interact with ErbB2 and influence its behavior in normal epithelia.     

A Highlights from MBoC Selection: Par6α Interacts with the Dynactin Subunit p150Glued and Is a Critical Regulator of Centrosomal Protein Recruitment

The centrosome contains proteins that control the organization of the microtubule cytoskeleton in interphase and mitosis. Its protein composition is tightly regulated through selective and cell cycle–dependent recruitment, retention, and removal of components. However, the mechanisms underlying protein delivery to the centrosome are not completely understood. We describe a novel function for the polarity protein Par6α in protein transport to the centrosome. We detected Par6α at the centrosome and centriolar satellites where it interacted with the centriolar satellite protein PCM-1 and the dynactin subunit p150^Glued. Depletion of Par6α caused the mislocalization of p150^Glued and centrosomal components that are critical for microtubule anchoring at the centrosome. As a consequence, there were severe alterations in the organization of the microtubule cytoskeleton in the absence of Par6α and cell division was blocked. We propose a model in which Par6α controls centrosome organization through its association with the dynactin subunit p150^Glued.     

Similar requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in diverse cell types

During animal development, a complex of Par3, Par6 and atypical protein kinase C (aPKC) plays a central role in cell polarisation. The small G protein Cdc42 also functions in cell polarity and has been shown in some cases to act by regulating the Par3 complex. However, it is not yet known whether Cdc42 and the Par3 complex widely function together in development or whether they have independent functions. For example, many studies have implicated Cdc42 in cell migrations, but the Par3 complex has only been little studied, with conflicting results. Here we examine the requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in a range of different developmental events. We found similar requirements in all tissues examined, including polarised growth of vulval precursors and seam cells, migrations of neuroblasts and axons, and the development of the somatic gonad. We also propose a novel role for primordial germ cells in mediating coalescence of the Caenorhabditis elegans gonad. These results indicate that CDC-42 and the PAR-3/PAR-6/aPKC complex function together in diverse cell types.     

The receptor-like tyrosine phosphatase lar is required for epithelial planar polarity and for axis determination within drosophila ovarian follicles.

The follicle cell monolayer that encircles each developing Drosophila oocyte contributes actively to egg development and patterning, and also represents a model stem cell-derived epithelium. We have identified mutations in the receptor-like transmembrane tyrosine phosphatase Lar that disorganize follicle formation, block egg chamber elongation and disrupt Oskar localization, which is an indicator of oocyte anterior-posterior polarity. Alterations in actin filament organization correlate with these defects. Actin filaments in the basal follicle cell domain normally become polarized during stage 6 around the anterior-posterior axis defined by the polar cells, but mutations in Lar frequently disrupt polar cell differentiation and actin polarization. Lar function is only needed in somatic cells, and (for Oskar localization) its action is autonomous to posterior follicle cells. Polarity signals may be laid down by these cells within the extracellular matrix (ECM), possibly in the distribution of the candidate Lar ligand Laminin A, and read out at the time Oskar is localized in a Lar-dependent manner. Lar is not required autonomously to polarize somatic cell actin during stages 6. We show that Lar acts somatically early in oogenesis, during follicle formation, and postulate that it functions in germarium intercyst cells that are required for polar cell specification and differentiation. Our studies suggest that positional information can be stored transiently in the ECM. A major function of Lar may be to transduce such signals.     

Neph-Nephrin proteins bind the Par3-Par6-atypical protein kinase C (aPKC) complex to regulate podocyte cell polarity

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their foot processes and the interposed slit diaphragm. So far, very little is known about the guidance cues and polarity signals required to regulate proper development and maintenance of the glomerular filtration barrier. We now identify Par3, Par6, and atypical protein kinase C (aPKC) polarity proteins as novel Neph1-Nephrin-associated proteins. The interaction was mediated through the PDZ domain of Par3 and conserved carboxyl terminal residues in Neph1 and Nephrin. Par3, Par6, and aPKC localized to the slit diaphragm as shown in immunofluorescence and immunoelectron microscopy. Consistent with a critical role for aPKC activity in podocytes, inhibition of glomerular aPKC activity with a pseudosubstrate inhibitor resulted in a loss of regular podocyte foot process architecture. These data provide an important link between cell recognition mediated through the Neph1-Nephrin complex and Par-dependent polarity signaling and suggest that this molecular interaction is essential for establishing the three-dimensional architecture of podocytes at the kidney filtration barrier.