Asymmetric cell division and cell polarization are fundamental traits required for both embryonic development and tissue remodeling. Maintenance of tissue architecture requires preservation of cell polarity, accurate asymmetric cell division and directional cell migration. Disturbance of these features of normal tissue organization is a hallmark of malignancy. In epithelial tissues, tight junctions (TJs), adherens junctions (AJs), gap junctions and desmosomes are all essential complexes which mediate cell-cell interactions required to maintain epithelial cell polarity. TJs, the most apical component of cell-cell junctional complexes create a physical barrier which functions to selectively regulate paracellular solute trafficking through epithelial sheets, prevent lateral migration of lipids and proteins between apical and basolateral membrane domains, thereby maintaining compartmentalization and tissue homeostasis.
The physical interaction of polarized epithelial cells with the basement membrane also ensures correct positioning and acts as a survival factor for epithelial cells. Cells that lose contact with the basement membrane undergo apoptosis or anoikis [1
] however, in carcinomas, this positional control and homeostasis is disrupted or absent. Dissolution of epithelial TJs, AJs and desmosomes, reorganization of the actin cytoskeleton and loss of apical-basal polarity are events indicative of neoplastic transition from polarized epithelial cells to depolarized mesenchymal cell phenotypes. Reduced expression of several structural components of both AJs and TJs in progressively poorly differentiated breast cancers implies a tumor suppressive role for these junctional complexes. Loss of E-cadherin, the prominent molecule localized at AJs is linked to tumor progression in epithelial cancers including lobular breast cancer [4
]. ZO-1 and claudin-7, both components of TJs show decreased staining in several invasive breast cancer cell lines and in poorly differentiated breast carcinomas [5
] and are becoming increasingly viewed as prognostic biomarkers and predictors of tumor grade. Loss of heterozygosity of ZO-1 in a proportion of breast tumors suggests ZO-1 may play a direct role in malignant progression [6
]. A large number of studies have contributed significantly to our understanding of the complex protein networks involved in assembly and stability of TJs. The topological role these complexes play in regulating TJs provides mechanistic insight into how deregulation or loss of stoichiometric co-ordination between key components required for maintaining epithelial cell polarity and plasticity might contribute to tumorigenisis providing a novel avenue for the investigation of tumor biology.
There has been considerable progress in elucidating the biochemical mechanisms underlying these processes in model organisms and in 2D and 3D mammalian cell culture models such as Martin-Darby canine kidney cells (MDCK), NMuNG, EpH4 and EMT-6 mouse mammary tumor cells [7
]. This body of work has made it clear that proteins involved in apico-basal polarity and TJ formation are functionally connected in a complex network with several other pathways. Three evolutionarily conserved molecular complexes regulate epithelial cell polarization and TJ polymerization (for review, refer to [8
]): 1) integral membrane proteins that constitute TJ strands, including occludins, claudins and Junctional Adhesion Molecule; 2) Peripherally associated cytoplasmic proteins that have the ability to organize the integral membrane TJ components, connecting them to actin filaments and/or other cytoplasmic proteins, completing formation of the TJ molecular complex; and 3) signaling proteins required for junctional assembly, regulation of barrier function and regulation of gene transcription. A series of molecules now recognized as indispensable participants in establishment of apico-basal asymmetry and TJ assembly are PAR6, atypical protein kinase C (aPKC), the RHO guanosine triphosphatase (GTPase) CDC42 and PAR3, a PDZ (PSD95/Dlg/ZO-1)-domain-containing membrane-scaffold adapter protein. This highly conserved quaternary complex functionally regulates asymmetric cell division from worms to mammals, by affecting cytoskeleton re-organization, post-golgi vesicle trafficking and microtubule network polarization [9
]. In mammalian epithelial cells, interference of PAR3, PAR6 or aPKC have been shown to inhibit TJ but not AJ [11
]. PAR6 interacts directly with aPKC isoforms ι/λ and ζ forming the core of this complex [14
]. Binding of CDC42•GTP to inactive PAR6-aPKC complex induces a conformational change in PAR6, leading to phosphorylation and activation of aPKC, and subsequent binding of this complex to PAR3 (reviewed in [15
]). CDC42•GTP is required for maintaining this polarization but alone is not sufficient to establish polarity. Studies in D. melanogaster have shown the par3 homologue bazooka, to be the pivotal component controlling asymmetric cell division [16
]. Two additional binding partners for PAR6 have now been identified as necessary for maintenance of cell asymmetry: STARDUST-CRUMBS-PATJ [17
] and DLG-SCRIB-LGL [19
]. There are 3 PARD6 gene family members in mammalian cells (homologous to a single pard6 member in Drosophila and C-elegans), PARD6A, PARD6B and PARD6G [11
]. Expression of each PAR6 isoform is tissue specific [21
], with apparent differences in function, localization and effector interactions, which have not yet been well defined.
PAR6 has recently risen as central to the coordinated maintenance of cell polarity by physically linking several of the multi-component complexes described above, converging CDC42- and aPKC-mediated signal transduction (for review see [22
]). Recent studies have identified an important association between PAR6 and TGFβ receptors, regulating interaction of PAR6 with SMURF1, an E3 ubiquitin ligase which targets RHO GTPases for degradation leading to TJ dissolution, TGFβ -induced EMT and metastasis [7
]. Cai et al have demonstrated that G-protein-activated plospholipase C-β (PLCβ) interacts with both PAR6 and PAR3, mediating downstream signal transduction by hydrolysis of phosphatidylinositol-4,5-bisphosphate, releasing intracellular calcium and diacylglycerol, all important second messengers required for cell polarity and asymmetric cell division processes [25
]. Signaling networks impinging on the PAR6 complex have been further expanded to signaling through TIAM1 guanine nucleotide exchange factor, which is a regulator of cell motility and invasion [26
]. In addition, activation of several oncogenes (and oncogenic signaling mechanisms), including ERBB2, KRAS, RAF, FOS, JUN, VSRC, RHO and RAC have been shown to disrupt apical-basal polarity by altering the localization of apical membrane markers [27
] and references therein). While the mechanisms by which this occurs are still under investigation, Aranda and colleagues have shown that ERBB2 disrupts apical-basal polarity by associating directly with the PAR6-aPKC core complex [27
]. These studies further highlight the need to understand how extracellular effectors signal to the polarity machinery to maintain correct cell architecture in an appropriate biochemical and biophysical context. Moreover, molecular aberrations contributing to loss of stoichiometric equilibrium of molecules governing cell morphology are likely to play a major role in progression of epithelial tumors [28
Our laboratory and others have extensively investigated genomic DNA copy number alterations as key genetic events in the development and progression of human cancers. The advent of microarray-based Comparative Genome Hybridization (aCGH) has vastly increased the resolution at which we can view and map regions of chromosomal gain and loss in tumors that are likely to harbor oncogenes and tumor suppressor genes [29
]. aCGH-based studies in breast cancer [30
] have provided a comprehensive view of regions of DNA gain at chr20q11-13 a common region of amplification in breast cancer. In this study, a significant peak in amplification was observed at chromosome 20q13.13, a region containing the BCAS4 gene, previously reported to be highly amplified in breast cancer cell lines [32
]. The gene immediately centromeric to BCAS4 is PARD6B. In this study, we find amplification and overexpression of PARD6B in a proportion of breast cancer cell lines. Given that PAR6 plays a central role in TJ assembly, maintenance of cell polarity, and is implicated in breast tumor progression [7
], including overexpression in premalignant neoplasms [33
], we have investigated the requirement of PAR6, CDC42 and aPKCζ to promote TJ polymerization breast cancer cells. We also examine in situ PAR6 expression and localization in normal polarized epithelia and epithelial breast tumors. Our observations uncover a previously unreported mechanism for dysregulation of PAR6 expression in breast cancer, and highlight the importance of further investigating the disturbances of the mechanisms that control cell polarity in cancer.