PAR Proteins Research Articles

Abstract B46: Roles for LKB1 at the immune synapse during B-cell activation and antitumor responses

Abstract Defense from infection or cancer is provided by coordinated actions of the innate and adaptive immune system, including B lymphocytes that generate antibodies (Abs) against pathogens or tumor antigens. B cells establish immune synapses with antigen-presenting cells during activation to initiate Ab-production, which may include entrance into a germinal center (GC) reaction to generate very high-affinity Abs. Establishment of an immune synapse requires polarity proteins to coordinate uptake and processing of external antigens. For this, there is a highly conserved family of partitioning defective (Par) proteins that mediates cell polarity in multiple cell types. In particular, Par-3, a member of the Par polarity complex (Par-3, aPKC, Par-6), is a key protein that localizes to the immune synapse and is required for B cell polarization and immune synapse formation. Recently, we showed that LKB1, the mammalian homolog of Par-4 and a regulator of energy metabolism, controls B cell activation, GC formation and differentiation into Ab-producing plasma cells. However, roles for LKB1 in B cell polarity, immune synapse formation and anti-tumor immunity are unknown. Based on previous studies showing that LKB1 induces cell polarity and co-localizes with Par-3, we hypothesize that LKB1 is required for Par complex assembly and controls immune synapse formation during B cell activation. Subcellular analysis of LKB1 by immunofluorescence microscopy in mouse primary B cells reveals LKB1 co-localization with aPKC in membrane protrusions required for immune synapse formation. Interestingly, B cell specific LKB1 knock out (BKO) mice exhibit mislocalization of aPKC/Par complex, loss of lymphoid tissue polarity and spontaneous B cell activation that results in the formation of giant GCs and secretion of proinflammatory cytokines and chemokines. Similar inflammatory profiles with T cell chemoattractants are known to recruit CD8+ cytotoxic T lymphocytes (CTLs) into solid tumors, such as melanoma. These similarities suggest that LKB1 inactivated B cells in the tumor microenvironment could stimulate T cell infiltrates into solid tumors. Our studies suggest that physiologic inactivation of LKB1 in B cells controls B cell activation, GC formation and potentially infiltration of T cells in the tumor microenvironment during anti-tumor responses. We are currently utilizing an exciting genetic mouse melanoma model to examine the role of LKB1 in promoting anti-tumor immunity. Together, our studies assess LKB1 as a target for augmenting adaptive immunity against cancer. Citation Format: Laura Jimenez, Lynnea Waters, Diane N.H. Kim, Nicole Walsh, Michael A. Teitell. Roles for LKB1 at the immune synapse during B-cell activation and antitumor responses [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B46.

Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli.

Protein/RNA clusters arise frequently in spatially regulated biological processes, from the asymmetric distribution of P granules and PAR proteins in developing embryos to localized receptor oligomers in migratory cells. This co-occurrence suggests that protein clusters might possess intrinsic properties that make them a useful substrate for spatial regulation. Here, we demonstrate that protein droplets show a robust form of spatial memory, maintaining the spatial pattern of an inhibitor of droplet formation long after it has been removed. Despite this persistence, droplets can be highly dynamic, continuously exchanging monomers with the diffuse phase. We investigate the principles of biophysical spatial memory in three contexts: a computational model of phase separation; a novel optogenetic system where light can drive rapid, localized dissociation of liquid-like protein droplets; and membrane-localized signal transduction from clusters of receptor tyrosine kinases. Our results suggest that the persistent polarization underlying many cellular and developmental processes could arise through a simple biophysical process, without any additional biochemical feedback loops.

Shared and independent functions of aPKC\u03bb and Par3 in skin tumorigenesis

The polarity proteins Par3 and aPKC are key regulators of processes altered in cancer. Par3/aPKC are thought to dynamically interact with Par6 but increasing evidence suggests that aPKC and Par3 also exert complex-independent functions. Whereas aPKCλ serves as tumor promotor, Par3 can either promote or suppress tumorigenesis. Here we asked whether and how Par3 and aPKCλ genetically interact to control two-stage skin carcinogenesis. Epidermal loss of Par3, aPKCλ, or both, strongly reduced tumor multiplicity and increased latency but inhibited invasion to similar extents, indicating that Par3 and aPKCλ function as a complex to promote tumorigenesis. Molecularly, Par3/aPKCλ cooperate to promote Akt, ERK and NF-κB signaling during tumor initiation to sustain growth, whereas aPKCλ dominates in promoting survival. In the inflammatory tumorigenesis phase Par3/aPKCλ cooperate to drive Stat3 activation and hyperproliferation. Unexpectedly, the reduced inflammatory signaling did not alter carcinogen-induced immune cell numbers but reduced IL-4 Receptor-positive stromal macrophage numbers in all mutant mice, suggesting that epidermal aPKCλ and Par3 promote a tumor-permissive environment. Importantly, aPKCλ also serves a distinct, carcinogen-independent role in controlling skin immune cell homeostasis. Collectively, our data demonstrates that Par3 and aPKCλ cooperate to promote skin tumor initiation and progression, likely through sustaining growth, survival, and inflammatory signaling.

PAR3-PAR6-atypical PKC polarity complex proteins in neuronal polarization.

Polarity is a fundamental feature of cells. Protein complexes, including the PAR3-PAR6-aPKC complex, have conserved roles in establishing polarity across a number of eukaryotic cell types. In neurons, polarity is evident as distinct axonal versus dendritic domains. The PAR3, PAR6, and aPKC proteins also play important roles in neuronal polarization. During this process, either aPKC kinase activity, the assembly of the PAR3-PAR6-aPKC complex or the localization of these proteins is regulated downstream of a number of signaling pathways. In turn, the PAR3, PAR6, and aPKC proteins control various effector molecules to establish neuronal polarity. Herein, we discuss the many signaling mechanisms and effector functions that have been linked to PAR3, PAR6, and aPKC during the establishment of neuronal polarity.

217 Dual role of the polarity protein atypical kinase Cλ in skin carcinogenesis

Altered polarity is a hallmark of cancer. The atypical kinase C (aPKC)λ, a central regulator of cell polarity, is considered a tumor promoter but its mechanisms of action are largely unknown. Previously, we showed that Par3, an aPKCλ binding partner, inhibits Ras-driven skin carcinogenesis. To address how aPKCλ controls skin cancer and whether it acts in concert with Par3 we performed two-stage DMBA-TPA skin carcinogenesis. Loss of either aPKCλ, Par3 or both, similarly inhibited papilloma formation. Molecularly, Par3/aPKCλ enhance Akt, ERK, NF-kB, and Stat3 signaling. Thus, Par3 and aPKCλ cooperate to promote the outgrowth of Ras-driven skin tumors, likely through sustaining growth, survival and inflammation. As loss or mutation of p53 is a major driver of human squamous cell carcinoma (SCC), we also inactivated aPKCλ together with p53. To our surprise, these mice spontaneously develop squamous cell carcinomas much earlier (p90) than single epidermal p53 knockout mice (p238). Further analysis revealed a 2-3 fold increase in proliferation and a 10-fold increase in macrophage numbers in aPKCλ/p53 epidermal dko mice accompanied by activation of Stat3. Proteomic analysis revealed that aPKCλ interacts with p53. Thus, aPKCλ can also function as a tumor suppressor in SCC. Together, our results indicate a context dependent role for the polarity proteins aPKCλ and Par3 in non-melanoma skin cancer, which, in part may depend on differential regulation of the tumor micro-environment.

Self-organization principles of intracellular pattern formation.

Dynamic patterning of specific proteins is essential for the spatio-temporal regulation of many important intracellular processes in prokaryotes, eukaryotes and multicellular organisms. The emergence of patterns generated by interactions of diffusing proteins is a paradigmatic example for self-organization. In this article, we review quantitative models for intracellular Min protein patterns in Escherichia coli, Cdc42 polarization in Saccharomyces cerevisiae and the bipolar PAR protein patterns found in Caenorhabditis elegans. By analysing the molecular processes driving these systems we derive a theoretical perspective on general principles underlying self-organized pattern formation. We argue that intracellular pattern formation is not captured by concepts such as ‘activators’, ‘inhibitors’ or ‘substrate depletion’. Instead, intracellular pattern formation is based on the redistribution of proteins by cytosolic diffusion, and the cycling of proteins between distinct conformational states. Therefore, mass-conserving reaction–diffusion equations provide the most appropriate framework to study intracellular pattern formation. We conclude that directed transport, e.g. cytosolic diffusion along an actively maintained cytosolic gradient, is the key process underlying pattern formation. Thus the basic principle of self-organization is the establishment and maintenance of directed transport by intracellular protein dynamics.This article is part of the theme issue ‘Self-organization in cell biology’.

The establishment of methods for free PAR generation and PAR reader detection

Poly (ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor that catalyzes the poly (ADP-ribose) (PAR) onto a variety of target proteins, such as histones, DSB repair factors and PARP1 itself under consumption of NAD+. Besides, PARP1 can affect a variety of proteins in noncovalent modification manner to carry out specific cellular functions. Here, we established a method to generate non-radiolabeled free PAR by PARG moderately cleaving PAR from autoPARylated PARP1, and utilized dot-blot assay to determine the interaction between free PAR and interested proteins. The methods to generate free PAR and detect the noncovalent interactions between proteins and free PAR are nonradioactive and convenient, which will facilitate the studies to explore the significance of PAR reading in various biological processes.

Dynamics of cortical domains in early Drosophila development.

Underlying the plasma membrane of eukaryotic cells is an actin cortex that includes actin filaments and associated proteins. A special feature of all polarized and epithelial cells are cortical domains, each of which is characterized by specific sets of proteins. Typically, an epithelial cell contains apical, subapical, lateral and basal domains. The domain-specific protein sets contain evolutionarily conserved proteins, as well as cell-type-specific factors. Among the conserved proteins are, the Par proteins, Crumbs complex and the lateral proteins Scribbled and Discs large 1. Organization of the plasma membrane into cortical domains is dynamic and depends on cell type, differentiation and developmental stage. The dynamics of cortical organization is strikingly visible in early Drosophila embryos, which increase the number of distinct cortical domains from one, during the pre-blastoderm stage, to two in syncytial blastoderm embryos, before finally acquiring the four domains that are typical for epithelial cells during cellularization. In this Review, we will describe the dynamics of cortical organization in early Drosophila embryos and discuss the processes and mechanisms underlying cortical remodeling.

Rap1, Canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor.

ABSTRACTIn Drosophila epithelial cells, apical exclusion of Bazooka (the Drosophila Par3 protein) defines the position of the zonula adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. Here, we show that the small GTPase Rap1, its effector Canoe (Cno) and the Cdc42 effector kinase Mushroom bodies tiny (Mbt), converge in regulating epithelial morphogenesis by coupling stabilization of the adherens junction (AJ) protein E-Cadherin and Bazooka retention at the ZA. Furthermore, our results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating that their functions during ZA morphogenesis are interlinked. In this context, we find the Rap1-GEF Dizzy is enriched at the ZA and our results suggest that it promotes Rap1 activity during ZA morphogenesis. Altogether, we propose the Dizzy, Rap1 and Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis.

Redundant regulation of localization and protein stability of DmPar3.

Apical-basal polarity is an important characteristic of epithelia and Drosophila neural stem cells. The conserved Par complex, which consists of the atypical protein kinase C and the scaffold proteins Baz and Par6, is a key player in the establishment of apical-basal cell polarity. Membrane recruitment of Baz has been reported to be accomplished by several mechanisms, which might function in redundancy, to ensure the correct localization of the complex. However, none of the described interactions was sufficient to displace the protein from the apical junctions. Here, we dissected the role of the oligomerization domain and the lipid-binding motif of Baz in vivo in the Drosophila embryo. We found that these domains function in redundancy to ensure the apical junctional localization of Baz: inactivation of only one domain is not sufficient to disrupt the function of Baz during apical-basal polarization of epithelial cells and neural stem cells. In contrast, mutation of both domains results in a strongly impaired protein stability and a phenotype characterized by embryonic lethality and an impaired apical-basal polarity in the embryonic epithelium and neural stem cells, resembling a baz-loss of function allele. Strikingly, the binding of Baz to the transmembrane proteins E-Cadherin, Echinoid, and Starry Night was not affected in this mutant protein. Our findings reveal a redundant function of the oligomerization and the lipid-binding domain, which is required for protein stability, correct subcellular localization, and apical-basal cell polarization.

Poly(ADP-ribose) mediates asymmetric division of mouse oocyte.

Before fertilization, mammalian oocyte undergoes an asymmetric division which depends on eccentric positioning of the spindle at the oocyte cortex to form a polar body and an egg. Since the centriole is absent and, as a result, the polar array microtubules are not fully developed in oocytes, microtubules have seldom been considered as required for eccentric positioning of the spindle, while actin-related forces have instead been proposed to be primarily responsible for this process. However, the existing models are largely conflicting and the underlying mechanism of asymmetric division is still elusive. Here we show that poly(ADP-ribose) (PAR) is enriched at mouse oocyte cortical area throughout meiosis. Specific removal of cortical PAR results in an ectopic spindle and a failure of asymmetric division. During spindle migration, when the spindle deviates from the center of oocyte by a pushing force of cytoplasmic actin, the short polar array microtubules emanating from the juxtacortical spindle pole extend to the cortex and penetrate into cortical PAR, docking and stabilizing the spindle at the cortex which facilitates the asymmetric division. This process depends on the affinity between PAR and microtubule-associated proteins such as Spindly, which contributes to a physical link for cortical PAR and the spindle. Notably, fusing a PAR-binding domain to end-binding protein 3, a plus-end tracking protein at the polar array microtubules, restores the asymmetric division of oocytes with Spindly knockdown. Thus, our work demonstrates a comprehensive mechanism for oocyte spindle positioning and asymmetric division.

Structural basis for the interaction between the cell polarity proteins Par3 and Par6

Polarity is a fundamental property of most cell types. The Par protein complex is a major driving force in generating asymmetrically localized protein networks and consists of atypical protein kinase C (aPKC), Par3, and Par6. Dysfunction of this complex causes developmental abnormalities and diseases such as cancer. We identified a PDZ domain-binding motif in Par6 that was essential for its interaction with Par3 in vitro and for Par3-mediated membrane localization of Par6 in cultured cells. In fly embryos, we observed that the PDZ domain-binding motif was functionally redundant with the PDZ domain in targeting Par6 to the cortex of epithelial cells. Our structural analyses by x-ray crystallography and NMR spectroscopy showed that both the PDZ1 and PDZ3 domains but not the PDZ2 domain in Par3 engaged in a canonical interaction with the PDZ domain-binding motif in Par6. Par3 thus has the potential to recruit two Par6 proteins simultaneously, which may facilitate the assembly of polarity protein networks through multivalent PDZ domain interactions.

P077 Aberrant Muc1 and Muc13 expression in association with Par polarity complex and Vip dysfunction in a colitis T-cell transfer model

There is emerging evidence that loss of the gastrointestinal mucosal barrier integrity is a significant contributor to the pathophysiology of inflammatory bowel disease (IBD). Mucins represent the major components of the mucus barrier and inappropriate overexpression of transmembrane mucins can affect the barrier integrity by disrupting cell polarity and cell-cell interactions resulting in tight junction dysfunction.1 Aberrant expression of Muc1, Muc4, and Muc13, in particular, is observed in colonic tissue samples of IBD patients.2, 3 Nevertheless, the role of mucins in mucosal barrier disruption in IBD remains elusive. In this study, we investigated the expression patterns of transmembrane mucins and the mediators involved in barrier homeostasis during the progression of colitis in mice. Colitis was induced in immunocompromised SCID mice by the adoptive transfer of CD4+CD25-CD62L+ T cells from the spleens of BALB/c donor mice. At 2, 4, and 6 weeks post-colitis, animals were sacrificed and colonic tissue collected for RNA and protein analysis of membrane-bound mucins (Muc1, Muc4, and Muc13), polarity proteins, and the vasointestinal polypeptide (Vip). This latter protein is implicated in the regulation of tight junction and mucin expression (Hokari et al., 2005). Apoptotic cells in tissue sections were detected using a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Our data showed that Muc1 expression was significantly increased after 4 weeks of colitis (Figure a). Interestingly, a significant decrease of Muc13 expression was observed during acute colitis (2 weeks) whereas a remarkable turnover in expression was noted during chronic colitis (4–6 weeks; Figure b). Normal intestinal MUC13 expression plays crucial roles in epithelial proliferation, differentiation and apoptosis but inappropriate overexpression of this mucin could lower the level of protection. Indeed, a higher number of apoptotic cells was seen in tissue of chronic colitis-induced mice (Figure d). Furthermore, the polarity proteins Par3 and aPKC as well as Vip (Figure c) were significantly reduced in expression from week 2 onwards suggesting a disturbance in cell polarity. Relative expression of Muc1 (a), Muc13 (b), Vip (c) in the colon of mice with acute (2w) and chronic (4–6w) colitis. (d) Number of apoptotic cells in healthy and chronic colitis mice. Asterisk indicates significance (one-way ANOVA; p < 0.05). The findings of this study highlight a potential role for Muc1 and Muc13 in mucosal barrier disruption in IBD and the underlying mechanisms involved necessitate further investigation. 1. Kufe D. Mucins in cancer: function, prognosis and therapy. Nat Rev Cancer, 2009. 2. Sheng YH, Lourie R, Lindén SK, et al.. The MUC13 cell-surface mucin protects against intestinal inflammation by inhibiting epithelial cell apoptosis. Gut, 2011. 3. Maaike V, Tim V, Ricard F, et al. Genetic and transcriptomic bases of intestinal epithelial barrier dysfunction in inflammatory bowel disease. Inflamm Bowel Dis. 2017.

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na+-K+-ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP2) staining was most prominent in the luminal membrane domain along with the PIP3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

Germ Layer Specific Regulation of Cell Polarity and Adhesion: Insight into the Evolution of Mesoderm

In triploblastic animals, Par proteins regulate cell-polarity and adherens junctions of both ectodermal and endodermal epithelia. But, in embryos of the diploblastic cnidarian Nematostella vectensis, Par proteins are eliminated altogether in the bifunctional endomesodermal epithelia. Using immunohistochemistry, CRISPR/Cas9, and overexpression of specific mRNAs, we describe the functional association between Par proteins, s- catenin, and snail genes in N. vectensis embryos. We demonstrate that the aPKC/Par complex regulates the localization of s-catenin in the ectoderm by directing its role in cell adhesion, and that endomesodermal epithelia are organized by a different cell adhesion system. We also show that snail genes, which are mesodermal markers in bilaterians, are sufficient to downregulate Par proteins and translocate s-catenin from the junctions to the cytoplasm in N. vectensis. These data provide insight into the evolution of epithelial structure and the evolution of mesoderm in metazoan embryos.

PI(4,5)P2 forms dynamic cortical structures and directs actin distribution as well as polarity in Caenorhabditis elegans embryos.

Asymmetric division is crucial for embryonic development and stem cell lineages. In the one-cell Caenorhabditis elegans embryo, a contractile cortical actomyosin network contributes to asymmetric division by segregating partitioning-defective (PAR) proteins to discrete cortical domains. In the current study, we found that the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) localizes to polarized dynamic structures in C. elegans zygotes, distributing in a PAR-dependent manner along the anterior-posterior (A-P) embryonic axis. PIP2 cortical structures overlap with F-actin, and coincide with the actin regulators RHO-1 and CDC-42, as well as ECT-2. Particle image velocimetry analysis revealed that PIP2 and F-actin cortical movements are coupled, with PIP2 structures moving slightly ahead of F-actin. Importantly, we established that PIP2 cortical structure formation and movement is actin dependent. Moreover, we found that decreasing or increasing the level of PIP2 resulted in severe F-actin disorganization, revealing interdependence between these components. Furthermore, we determined that PIP2 and F-actin regulate the sizing of PAR cortical domains, including during the maintenance phase of polarization. Overall, our work establishes that a lipid membrane component, PIP2, modulates actin organization and cell polarity in C. elegans embryos.

The protein kinase SIK downregulates the polarity protein Par3.

The multifunctional cytokine transforming growth factor β (TGFβ) controls homeostasis and disease during embryonic and adult life. TGFβ alters epithelial cell differentiation by inducing epithelial-mesenchymal transition (EMT), which involves downregulation of several cell-cell junctional constituents. Little is understood about the mechanism of tight junction disassembly by TGFβ. We found that one of the newly identified gene targets of TGFβ, encoding the serine/threonine kinase salt-inducible kinase 1 (SIK), controls tight junction dynamics. We provide bioinformatic and biochemical evidence that SIK can potentially phosphorylate the polarity complex protein Par3, an established regulator of tight junction assembly. SIK associates with Par3, and induces degradation of Par3 that can be prevented by proteasomal and lysosomal inhibition or by mutation of Ser885, a putative phosphorylation site on Par3. Functionally, this mechanism impacts on tight junction downregulation. Furthermore, SIK contributes to the loss of epithelial polarity and examination of advanced and invasive human cancers of diverse origin displayed high levels of SIK expression and a corresponding low expression of Par3 protein. High SIK mRNA expression also correlates with lower chance for survival in various carcinomas. In specific human breast cancer samples, aneuploidy of tumor cells best correlated with cytoplasmic SIK distribution, and SIK expression correlated with TGFβ/Smad signaling activity and low or undetectable expression of Par3. Our model suggests that SIK can act directly on the polarity protein Par3 to regulate tight junction assembly.

Decreased Expression of the Polarity Regulatory PAR Complex Predicts Poor Prognosis of the Patients with Colorectal Adenocarcinoma

Partitioning defective (Par) proteins regulate cell polarity and differentiation. Par3, Par6β, and protein kinase Cζ (PKCζ), which are PAR complex members, have been shown to be associated with oncogenesis and progression. Herein, we report the expression pattern and clinical relevance of Par3, Par6β, and PKCζ in colorectal adenocarcinoma (CRAC). A total of 393 primary CRACs, 41 primary-metastatic CRAC pairs, 41 adenomas with low-grade dysplasia, and 41 nontumor colorectal tissue samples were examined by immunohistochemistry and Western blot assays for Par3, Par6β, and PKCζ protein expressions. The association Par3, Par6β, and PKCζ expressions and clinicopathologic factors, including patient survival, was evaluated. Primary CRACs and adenomas demonstrated higher levels of Par3, Par6β, and PKCζ than in nontumor colorectal epithelia. The expressions of Par3, Par6β, and PKCζ were higher in primary CRACs as compared to adenomas or in metastatic CRACs. Among primary CRACs, decreased Par3 expression was found to correlate with a high proliferation rate and poor histologic differentiation, decreased PKCζ expression was correlated with pathologic TNM stage (I-II vs III-IV) and lymph node metastasis, and decreased Par6β and PKCζ expressions were correlated with shortened overall survivals. In metastatic CRACs, decreased PKCζ expression was correlated with a shortened metastasis-free survival. While increased Par3, Par6β, and PKCζ expressions were implicated in tumorigenesis, decreased expressions of Par3, Par6β, and PKCζ were found to be associated with worse clinicopathologic factors in CRAC. In particular, the results of our study suggest that PKCζ down-expression is an independent poor prognostic and metastatic factor for CRAC.

Loss of polarity alters proliferation and differentiation in low-grade endometrial cancers by disrupting Notch signaling.

Cell adhesion and apicobasal polarity together maintain epithelial tissue organization and homeostasis. Loss of adhesion has been described as a prerequisite for the epithelial to mesenchymal transition. However, what role misregulation of apicobasal polarity promotes tumor initiation and/or early progression remains unclear. We find that human low-grade endometrial cancers are associated with disrupted localization of the apical polarity protein Par3 and Ezrin while, the adhesion molecule E-cadherin remains unchanged, accompanied by decreased Notch signaling, and altered Notch receptor localization. Depletion of Par3 or Ezrin, in a cell-based model, results in loss of epithelial architecture, differentiation, increased proliferation, migration and decreased Notch signaling. Re-expression of Par3 in endometrial cancer cell lines with disrupted Par3 protein levels blocks proliferation and reduces migration in a Notch dependent manner. These data uncover a function for apicobasal polarity independent of cell adhesion in regulating Notch-mediated differentiation signals in endometrial epithelial cells.

Cell Migration in Microfabricated 3D Collagen Microtracks is Mediated through the Prometastatic Protein Girdin.

In vivo, cancer cells can utilize tube-like microtracks formed within the extracellular matrix (ECM) of the stroma as 'highways' to escape the primary tumor, however very little is known about the molecular mechanisms that govern cell migration through these microtracks. Cell polarization and actin organization are both essential for efficient cell migration and cells are known to migrate very unidirectionally in confined spaces. In this study, we focused on understanding the role of Girdin during unidirectional migration. Girdin is a prometastatic protein known to be involved in cell polarity by directly interacting with the cell polarity protein Par-3 (Partitioning defective-3) and also known as an actin binding protein. We utilized a microfabricated platform to recreate these microtracks in vitro using collagen and used siRNA to knockdown Girdin in MDA-MB-231 cells. Our data indicate that knockdown of Girdin results in decreased cell speed during 3D collagen microtrack migration. Loss of Girdin also results in altered cell morphology and cell orientation. Moreover, Girdin-depletion impairs actin organization and stress fiber formation, which can be restored by upregulating the GTPase RhoA. Activation of RhoA induces actin stress fiber formation, restores elongated migratory cell shape and partial cell migration in 3D collagen microtracks in the absence of Girdin. Our data suggest that Girdin helps directional migration in collagen microtracks by promoting actin cytoskeletal organization and maintaining morphological cell polarity.