Impaired Formation Of Homotypic Cell-in-cell Structures In Human Tumor Cells Lacking Alpha-catenin Expression

BackgroundCell-in-cell (CIC), it refers to one or more cells in another cell cytoplasm, and remain intact cell membrane structure, from the discovery of more than 100 years of history. For its understanding also it experienced from the outset considered mere accident, but now admit that it does have a wide range of biological significance. Today, more and more people join into the CIC research, for the cognizance of CIC become more clearly and the methods has become more mature.Currently, according to the different cell types involved in CIC structure can be roughly divided into two categories:homogenous and heterogeneous CIC structure. CIC-called homogeneous structure, it means the same type of cell, for instance, epithelial cells and epithelial cells formed CIC. This phenomenon tends to occur between tumor cells. CIC heterogeneity formation refers to the of different types of cells derived from the CIC, as between the tumor cells and immune cells (e.g. NK cells, T cells, B cells), the immune cells go into the tumor cells, also called " drilling tumor "Some study found, in CIC internal structure cells often death, but the death mechanism is not fully understood, some of which is through non-apoptotic pathway, mainly the outside lysosomal participate cell death, which constitutes a new mechanism of death. On homogeneous CIC, the current study has discovered a new mechanism of death, the researchers named "entosis", refers to the CIC structure is first formed between the epithelial cells, the internal cells are enveloped by the cell membrane which are derived from outside, vesicle-like. Then the LC3 of external cell enrich in membrane vesicle surface, and lipid-based in molecular ATG5, ATG7, VPS34 and recruiting target cells lysosomal proteases and protein LAMP1 CathepsinB. These molecules move into vesicles, lysosome dissolves internal cells and leads to the interior cell death.It is reported that the form of CIC is associated with the immune tolerance. And our research shows that the CIC structure has a close relationship with the formation and development of tumors. Its importance is evident. For further understanding the biological function of the CIC, we need to study the mechanism of its formation. The study of the formation mechanism should follow the law of human cognition-awareness overall in the details, to grasp the details in the whole.For the level of detail needed to implement the specific gene or biological molecules. In previous studies, we found that the deletion of E-cadherin and P-cadherin significantly inhibit the formation of CIC structure between tumor cells; and expression of E-cadherin and P-cadherin, the CIC will re-form structure between tumor cells. As we all know, E-cadherin and E-cadherin as mutually intercellular adhesion molecule is important, which prompted us the adhesion and connection between cells seems to be related to the structure and the formation of CIC. Focus on intercellular adhesion molecule-related connection, we will examine targeting of a-catenin, which is located between the cell surface adhesion molecules and intracellular F-actin, through their own domain can connect both to form a stable connection between cells.After more than one hundred years of research, study on cell-in-cell is still in its infancy. Ideas are divided on how cell-in-cell structure is formulated. Some hold that this is a passive process and without consuming energy. Some argue that this happens actively and requires energy. Some think this is a drilling process led by inner cells. Others believe the outer cells take the lead in swallowing. This phenomenon is caused by complicated experiment data and obscure normal rules. Researchers cannot tap on experiment data without a comprehensive understanding of cell-in-cell structure. This imposes difficulty on cell-in-cell study. Therefore, a comprehensive, objective and reliable model is rather important for future research.Objective1. Study relationship between alpha-catenin and forming CIC structure (CICs).2. Discuss mechanism of forming CICs by alpha-catenin.3. Get the comprehensive biological model through induction and deduction.MethodsWe recently reported that tumor cells lacking expression of E- and P-cadherin were incapable of forming homotypic CICs by entosis, and re-expression of E- or P-cadherin was sufficient to induce CICs formation in these tumor cells. In this work, we found that homotypic CICs formation was impaired in some tumor cells expressing high level of E-cadherin due to loss expression of alpha-catenin. To further confirm the role of α-catenin in CICs formation, we established retroviral construct pQCXIP-EGFP-α-catenin, that was made by subcloning EGFP-α-catenin fusion gene from pEGFP-C1-α-catenin in between Age I and Mfe I sites of pQCXIP-EGFP-N1, which was constructed by replacing gene encoding GFP in pQCXIP-GFP-C1 with EGFP gene from pEGFP-N1. Retro viruses were made and used to infect target cells. Virus-infected MDA-MB-468 and H820 cells were selected with puromycin. And we depleted α-catenin expression by RNA interference in cells. Then we observed α-catenin, E-cadherin, Tubulin, F-actin by Immunostaining, in order to find the mechanism of forming CICs.Functions of living things are complicated. Biological molecules participating different process or even the same process vary a lot. Trying to understand biological functions entails biological experiment, which is uncertain and lopsided in nature. The establishment of a model will be complicated if it is based only on biological experiment. Researchers cannot ensure that the model is the right one. However, physical functions come from wildly acknowledged axiom or theorem and provide general explanations to physical phenomenon that can be applied to a wide range of things. Therefore, we start from physical viewpoint and set up general physical model of how cell-in-cell structure is formulated. Then, we fit the model to existing experiment results and relevant biological pathway. Finally, we get the comprehensive biological model through induction and deduction.Results1. Tumor cells lacking expression of α-catenin show impaired CICs formation.In our work, we found that some cancer cells such as MDA-MB-468, MDA-MB-436, ZR75-1, H820, H441 although expressed E-cadherin at levels comparable to that of MCF10A, displayed impaired CICs formation. That suggests that mechanisms other than loss of epithelial cadherins should be responsible for defects in CICs formation in these cells. In order to identify these molecules, we forced on adherens junctions (AJs). Through Western blot, we found α-catenin did not express in two of these cell lines, MDA-MB-468 and H820. Since α-catenin is a functional component of AJs, we therefore hypothesize that loss expression of α-catenin compromised AJs and subsequently CICs formation.2. Restored cell-cell adhesion upon α-catenin expression.As shown in results, for both MDA-MB-468 and H820 cells, while EGFP-expressing control cells are scattered and loosely interact with each other, cells expressing α-catenin stay together in a compact fashion, indicating that cell-cell adhesion is restored.3. Expression of α-catenin induces CICs formation.We examine CICs formation in MDA-MB-468 and H820 cells with restored cell-cell adhesion. As evidenced by western blot and EGFP expression, α-catenin was successfully expressed in these two cells. Upon suspension, CICs were readily detected in both cell lines. But CICs formation in H820 and MDA-MB-468 cells occurred in a relative slower pace as compared with MCF10A and MCF7 cells. H820 and MDA-MB-468 cells need 36 hours, while MCF10A and MCF7 need 6 hours.4. Depletion of α-catenin compromised cell-cell adhesion and CICs formation.To further confirm the role of α-catenin in CICs formation, we depleted α-catenin expression by RNA interference in MCF10A cells, which display high frequency of CICs. Transfection of three siRNA duplexes efficiently knocked down a-catenin as detected by Western Blot, with silencing efficiency of more than 80% for all three duplexes. Expectedly, cell-cell adhesions were disrupted as evidenced by loose cell-cell interaction and scattered morphology even when cultured in high density Meanwhile, CICs formation was consistently inhibited in three siRNAs-transfected cells. So expression of α-catenin, like E- and P-cadherin, is prerequisite for CICs formation of epithelium-originated cells.5. Remodeled cytoskeleton in alpha-catenin-expressing cells.Our previous work showed that cytoskeleton was remodeled during CICs formation induced by E-/P-cadherin expression, we therefore examined the distribution of AJs molecules and cytoskeleton upon α-catenin expression. For both adherent MDA-MB-468 and H820 cells expressing α-catenin, E-cadherin locates neatly at the cell-cell boundary, where a-catenin was also enriched. This pattern of E-cadherin is in sharp contrast to that in control cells, where few linear E-cadherin locates at cell-cell contact region as demonstrated by line-scan analysis and overall quantification.6. Expression of alpha-catenin in cells will enhance bundling actin filaments nearby cytomembrane.Meanwhile, actin filaments seem to be enhanced with more phalloidin-positive filaments detected in α-catenin-expressing MDA-MB-468 cells, whereas F-actin distribution displayed less complexity in control cells as demonstrated by pixel intensity profiling, which is consistent with α-catenin’s role in bundling actin filaments. The phenotype of enhanced F-actin bundling was even stronger in α-catenin-expressing H820 cells, where F-actin seems increased excessively and almost centralized on the cell cortex.ConclusionExpression of α-catenin in these tumor cells restored cell-cell adhesion and promoted CICs formation. That is to say, impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression. The mechanism of that may be expression of alpha-catenin in cells can remodel cytoskeleton, then enhance or restore cell-cell adhesion, while if cell-cell adhesion can’t be established, CIC formation will not be initiated.As a result, in addition to restoring AJs, excessive α-catenin forms homo-dimmer to promote F-actin bundling, which was thought to reduce actin dynamics. The increased bundling and reduced dynamics of F-actin by α-catenin might explain the phenotype that CICs formation in H820 and MDA-MB-468 cells occurred in a relative slower pace as compared with MCF10A and MCF7 cells, as active remodeling of actin cytoskeleton is required for efficient CICs formation.Model Outlined1. The acting ring, which allows two cells to formulate stable and flexible attachment to each other, is the determining factor in forming cell-in-cell structure.2. The unbalanced and hugely different contraction of myosin within one cell is the driving force behind the formulation of cell-in-cell structure.3. The active and passive transformation capacity of cells determines their roles, i.e. whether they are inner or outer cells.