Classically, the proteins considered to be the core of the Par complex include the two Par PDZ domain-containing proteins Par3 and Par6, with the addition of atypical protein kinase C aPKC , and the Par complex has been shown to be required for the establishment and the maintenance of apical—basal polarity and apical domain development in epithelial cells. Additionally, the functions of the Par complex are regulated by phosphorylation.
The activity of the Par complex is further regulated by the dynamic nature of Par3's association with the stable Par6—aPKC complex. A compelling study in Drosophila demonstrated that Par3 is in fact excluded from the apical domain by the Par6—aPKC complex. This correlates with the observation that in many epithelial tissues, including in mammals, Par3 and the Par6—aPKC complex do not colocalise. On one hand, aPKC phosphorylates Par3 on Ser in mammalian Par3 to decrease their affinity for each other while, on the other hand, Crumbs and Stardust compete with Par3 to interact with the same domain of Par6 Figure 1.
Further investigation is required, but the existing evidence suggests that the observations outlined above may be generalised to epithelial tissues in mammals. The three members of the Scribble complex have been shown to interact genetically, 16 with Dlg and Scribble physically interacting through a protein called GUK-holder in Drosophila neuronal synapses.
More recently, Scribble and Lgl2 have been reported to interact directly in polarised mammalian epithelial cells, although this interaction has not yet been reported in other experimental systems. These membrane domains consequently acquire unique identities that set the basis for the establishment of apical—basal polarity in epithelial cells. Asymmetric cell division is pivotal for the maintenance of epithelial tissue homeostasis.
When a stem cell divides asymmetrically, it generates two daughter cells: one with an identical cell fate and the other with a different one. Asymmetric cell division relies on the asymmetric distribution of cell fate determinants Numb, Pros, Brat, Pon and Mira and, as a result, core polarity proteins and the correct orientation of mitotic spindles. Moreover, deletion of both Drosophila APC genes results in asymmetric stem cell division defects as a result of mitotic spindle misorientation.
Loss of cell polarity in epithelial stem cells can lead to asymmetric division defects, thereby favouring tumour initiation.
Apical—basal polarity is fundamental to the asymmetric segregation of cell fate determinants. Thick yellow and green lines represent cell fate determinants. Red dots represent centrosomes. In the absence of apical—basal polarity, this segregation is defective, potentially leading to an excess in symmetric divisions and an accumulation of cells with proliferative potential. In mammals, probably as a consequence of their redundancy, the function of core polarity proteins in the regulation of asymmetric cell division has been harder to elucidate.
However, Lgl1 knockout mice present some defects in the asymmetric division of neural progenitors, that may result in overproliferation and a lack of differentiation. The transition from an epithelial to mesenchymal phenotype that occurs during EMT has been associated with metastatic progression; apical—basal polarity is lost during this process and cell—cell junctions are weakened and disrupted. Several lines of evidence suggest that core polarity proteins are important for the formation and maintenance of the AJC, suggesting that their loss could induce or at least contribute to EMT Figure 3.
For example, it has been shown that Par3 depletion in mammalian epithelial cells disrupts the formation of tight junctions. Epithelial cell polarity represents a barrier to the later stages of tumour development.
Apical—basal polarity is involved in the formation and maintenance of the AJC. Decreased expression of core polarity proteins is linked to weakening or disruption of the AJC, thereby leading to EMT and potential malignancy. Underlying their potential role as tumour suppressors, core polarity proteins are often targeted and disrupted by oncogenic signalling.
Polarity defects could collaborate with oncogenic pathways to induce tumour formation. For example, Scrib-deficient mutants cooperate with oncogenes to mediate transformation in Drosophila.
Normally, Scrib-deficient mutant clones in the eye imaginal discs are eliminated by JNK-dependent apoptosis. However, in the presence of activated oncogenic pathways such as Ras or Notch, apoptosis is inhibited and neoplastic tumours occur.
A number of viral oncogenes have been found to directly interact with polarity proteins, suggesting that disruption of polarity is important. Moreover, it seems that viral proteins have evolved additional ways to target cell polarity, strongly suggesting that its disruption is necessary for malignant transformation. In addition to viral oncogenes, core cell polarity mechanisms are targeted by abnormally activated growth factor signalling pathways.
Scrib, dlg and lgl were identified as tumour suppressors in Drosophila , in screens for mutations causing cancerous overgrowth of the larval imaginal discs and brain. In particular, the deregulation of Scrib has been reported to promote the transformation of mammary epithelial cells in vitro and in vivo , by disrupting morphogenesis and cell polarity and by inhibiting myc-induced apoptosis, thus providing novel insight into how core polarity proteins regulate cell transformation.
The deregulation of core polarity proteins in cancer is not limited to Scrib, Dlg and Lgl, however, as almost every protein involved in the core apical—basal polarity machinery of epithelial cells has been shown to be affected in some way. This study will undoubtedly be complemented by data from ongoing cancer genome sequencing efforts such as the Cancer Genome Atlas.
The Par6—aPKC complex has also been shown to be deregulated in cancer. For example, deregulation of Par6 was observed in ER-positive breast tumours.
Over the past decade, a number of tumour suppressor pathways have been directly linked to epithelial cell polarity, suggesting that the integrity of apical—basal polarity is crucial for the prevention of tumour development.
Although LKB1 deficiency does not cause a gross defect in cell polarity in the intestine, for example, 70 it may regulate cell polarity through its ability to phosphorylate members of the AMPK-related kinase ARK family in other tissues.
Germline mutations in the LKB1 gene cause Peutz—Jeghers syndrome and predispose patients to develop colon cancer. In agreement with this, a growing list of tumour suppressors has been identified that regulate cell polarity.
For example, the tumour suppressor von Hippel-Lindau exerts its regulation on polarity at several levels: it is able to directly interact with aPKC and mediate its ubiquitination and subsequent degradation, and its interaction with the Par complex is involved in the regulation of polarised microtubule growth and formation of primary cilia.
However, as apical accumulation of phosphatidylinositol 4,5 biphosphate is dependent on apical targeting of PTEN, and as membrane targeting of Par3 is mediated by direct binding to phosphoinositide lipids, PTEN may be instrumental in the apical localisation of Par3. For example, hDlg can interact with the APC tumour suppressor, and their interaction negatively regulates cell cycle progression.
Recently, apical—basal polarity in epithelial cells has been linked to another tumour suppressor, with the discovery that ASPP2 is a new binding partner of Par3. In vivo , ASPP2 deficiency results in defects arising during the development of the central nervous system, characterised by a loss of apical—basal polarity and an expansion of neural progenitor cells.
Therefore, it will be of great interest to investigate the extent to which ASPP2's regulation of the localisation of Par3 and the apical—basal polarity of epithelial cells contributes to its tumour suppressive function, and how this interplays with its role in regulating p53, the most mutated tumour suppressor in human cancers, and p63, a key regulator of epithelial stratification.
There are a number of studies that also suggest that mitotic spindle orientation is crucial for asymmetric cell division in mammals. For instance, p63 has been shown to be important for mitotic spindle orientation during asymmetric cell division of epidermal stem cells.
For example, the tumour suppressor p53 has recently been linked with the regulation of asymmetric divisions of mammary stem cells. Taken together, these studies emphasise the role and importance of known tumour suppressors in the control of epithelial cell polarity. Many of those tumour suppressors regulate the functions of core polarity proteins through direct interactions suggesting that, in addition to their better-known roles in the control of cellular proliferation, these roles are crucial for the prevention of tumour development.
In this review, we have highlighted how epithelial cell polarity may contribute to tumour suppression Figure 4 , through its role in controlling asymmetric cell division and the integrity of the AJC. Loss of cell polarity is a hallmark of cancer, however, studies in transgenic mouse models have so far been unable to clearly answer the question of whether core polarity proteins are tumour suppressors or not.
Due to the vital importance of core polarity proteins in maintaining tissue homeostasis, redundancy mechanisms have evolved in mammals that may make this issue too complex to address. Nonetheless, it is emerging that an increasing number of well-known tumour suppressors have a pivotal role in regulating cell polarity. Hence, regulators of polarity may themselves represent a new class of tumour suppressors. Oncogene ; 27 : — Lee M, Vasioukhin V. Cell polarity and cancer--cell and tissue polarity as a non-canonical tumor suppressor.
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For instance strains of MCF10A cells that notoriously lack the capability to form apically located tight junctions under usual culture conditions, as shown by fluorescence immunostaining and structural analysis by electron microscopy [ 8 , 9 ], have been used to confirm the critical role of genes, like Crumbs3 in the establishment of apical polarity. Non-neoplastic HMT S1 cells capable of acquiring basoapical polarity represent a very useful model to develop studies that require well-differentiated tissue structures.
With these cells we could identify certain 3D culture conditions necessary for the production of basoapically polarized structures. This culture condition might be of interest to better understand the role of collagen IV in epithelial differentiation, notably, as suggested by our data with the HTP culture, in the establishment of apical polarity.
The molecular mechanism underlying the lack of apical polarity formation in CBL culture remains to be understood.
Possibly, chemical factors necessary for apical polarity formation might be absent from CBL extracts. More excitingly, there is also a possibility that the mechanical environment of the cells might influence apical polarity.
Indeed extracellular matrix stiffness was shown to affect the morphogenesis of mammary epithelial cells [ 28 ]. It would be interesting to develop an experimental design to specifically assess the effect matrix stiffness on apical polarity.
Our experiments with function blocking antibodies directed toward integrins underscore the importance of the tissue context for the response to ECM alterations. This is because such previous experiments were performed before acinar morphogenesis, while our experiments were conducted on already formed acini.
These experiments illustrate the impact of tissue architecture resulting from acinar morphogenesis that not only affects the organization of the signaling network [ 29 ], but also structural organization all the way to the cell nucleus [ 30 ] and thus, influences how cells respond to changes in their microenvironment.
With the HTP culture it should become possible to obtain large numbers of polarized tissue structures necessary to perform measurements that require large amounts of cellular material. Importantly, the HTP culture method will be useful, in combination with microscopy techniques, for rapid screening of the effects of environmental factors and drugs on breast epithelium homeostasis e. The large number of individual tissue structures produced under these conditions will allow for meaningful statistical analyses.
Our findings demonstrate an important role for the BM in the establishment of apical polarity, suggesting that any extracellular factor that perturbs either cell-BM connections or the stability of the BM via collagen IV would rapidly affect the functional integrity of the epithelium and possibly its homeostasis since apical polarity proteins have been linked to proliferation control and cancer development [ 32 ].
Due to the prominent role of apical polarity in early stages of cancer development, it is paramount to have a model that permits the identification of factors that control such polarity. We believe that the HTP culture developed here for breast epithelial cells could provide an important tool for high content screening of risk and protective factors of epithelial architecture and thus, homeostasis.
Unless otherwise specified, at day 7 or day 8 , cells were induced to exit the cell cycle upon culture without addition of epidermal growth factor EGF during 72 or 48 hours.
Important considerations to maintain full differentiation capabilities of the S1 cell line: Firstly, immortalized non-neoplastic cells are a heterogeneous population in which some cells have better differentiation capabilities than others. In order to keep a high percentage of differentiation-capable cells, a defined concentration of cells is used for plating for monolayer and 3D cultures and cells are kept in culture for 10 passages maximum, with passage 60 as the latest possible passage used for 3D culture assays.
Secondly, for propagation of cells as a monolayer i. If cells are detached from the surface of the flask too early, the population will be enriched in cells that had attached rapidly, which might ultimately impair proper acini formation. EGF was omitted from the culture medium 48 to 72 hours prior to the end of the culture period. Statistical comparisons were performed using GraphPad Prism 3. Nonpaired t -test was used for comparison of two groups, and one-way Anova test for comparison of more than two groups.
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