Anoikis resistance is a key to the survival of cells in malignant transformation and metastasis [18, 19]. It may also be a key to the adaptation of cells to suspension culture and spheroids growth used in engineering. For epithelial cells, suppression of anoikis upon detachment seems to be induced when cell-cell contacts are formed. For example, cadherin-mediated homotypic interactions maintain the survival of human prostate carcinoma DU-145 cells in the absence of extracellular matrix (ECM) attachments . Also, disruption of E-cadherin cell-cell contacts showed more important for suppressing anoikis of normal enterocytes after detachment from villus epithelium, as compared to cell-ECM disruption . Growth as spheroids renders tumor cells less sensitive to exogenous apoptotic stimuli, and spheroids have greater drug resistance than the corresponding monolayers ; and some cells used in engineering also grow as spheroids in suspension. Thus, elucidating the mechanisms by which spheroids survive through cell-cell contacts has potentially profound value for understanding survival mechanism of such cells and may also be applied to the control of growth of cells used in biomedical engineering. However, much less is known about how survival pathways are activated under anchorage-independent matrix-deficient conditions in these cells.
Recently, Marieb et al. found that the adhesion molecule CD147 promotes anchorage-independent, hyaluronan-dependent growth of human breast carcinoma cells . CD147 is also reported to regulate cell-matrix adhesion . However, it is not clear whether CD147-mediated cell-cell contact has a role in anoikis resistance or the growth of spheroids of cells used in engineering. Using our sophisticated model, which we established from anoikis-resistant HEK293ar and the parental HEK293 cells, we found that HAb18G/CD147 cell-cell adhesion suppresses anoikis in an E-cadherin-dependent manner. In addition, we have shown that cell-cell adhesion-based survival signals arising from adjacent HEK292ar cells may inhibit anoikis in a PI3K/AKT-dependent manner (Figs. 6A, B, C, D). In all, our results indicate that HAb18G/CD147-mediated cell-cell contacts mediate anoikis resistance specifically through an E-cadherin-dependent pathway, and that anoikis suppression mediated by cell-cell contacts in HEK293ar cells involves the PI3K/Akt pathway.
Although the data suggest that elevated HAb18G/CD147 expression is correlated with the progression and invasion potentials of human hepatoma cells [23, 24], the role of HAb18G/CD147-mediated cell-cell contacts in acquiring resistance to anoikis remains obscure. Earlier evidence suggested that CD147 promotes cancer cell survival by regulating intercellular contacts and inhibiting anoikis ; in that study, cells transfected with the CD147 gene and expressing different levels of CD147 were used. In our study, we first found evidence that elevated endogenous expression of HAb18G/CD147 contributes to cell-cell adhesion and subsequently confers resistance to anoikis under suspension conditions (Figs. 2A, B, C, E, F). Our anoikis-resistant HEK293ar cell model would be more suitable for investigating cell-cell contact-directed anoikis suppression, since it is closer to the natural in vivo status of physiological models and cells used in engineering. In addition, owing to the use of HEK293 cells in biotechniques, our cell model and the elucidation of the cell spheroid mechanism may be relevant to bioengineering.
Cell-cell contact-triggered cell survival also involves anti-apoptotic signalling through E-cadherin-, Src-, and PI3K/Akt-dependent pathways [15, 25]. For instance, E- cadherin, a classical cadherin that promotes calcium-dependent cell-cell adhesion, suppresses anoikis in squamous carcinoma and normal proximal tubular cells [25, 26]. In Ewing tumor spheroids, E-cadherin cell-cell contacts may activate the ErbB4 RTK signal pathway . Homophilic E-cadherin binding is also involved in activating Akt kinase, which ultimately inhibits caspase-3 activity and prevents anoikis . We found that E-cadherin expression was elevated in the anoikis-resistant HEK293ar cells (Figs. 3A, B, C) after 24 h suspension culture. Also, high levels of E-cadherin expression inhibited anoikis by mediating cell-cell contacts upon cell-matrix detachment, which was required by HAb18G/CD147, functioning as an anoikis suppressor. Likewise, knockdown of HAb18G/CD147 disrupted cell-cell contacts and anoikis ensued (Figs. 2C, D, E, F). In addition, as comparison of E-cadherin expression between the HEK293ar and its parental cell focused on the contrast of this protein after 24 h suspension culture, this time course should be long enough for the stabilisation of ligated E-cadherin and this may preclude the E-cadherin increase from the stabilisation of ligated E-cadherin at sites of adhesion and therefore help in valuing the function of this protein accurately in anoikis resistance. An important conclusion is that HAb18G/CD147 may have a more prominent role in suppressing anoikis than E-cadherin, and E-cadherin may be a downstream effector of CD147 in the development of anoikis resistance, although the precise mechanisms by which E-cadherin complexes are remodeled and degraded remain to be determined.
The pronounced effect of HAb18G/CD147 knockdown on cell survival may result from a direct decrease in E-cadherin expression and subsequent loss of cell-cell contacts (Figs. 4A, B, C). In addition, our more recent data have shown that LY294002, a specific PI3K inhibitor, significantly reduced the effect of HAb18G/CD147 on cell adhesion and metastatic invasion (p < 0.01) of human hepatoma cells . E-cadherin, predominantly expressed at cell-cell contacts, is stably bound to the PI3K complex; this protein expression is necessary and sufficient for activating the PI3K/AKT pathway . We have also demonstrated that HAb18G/CD147-mediated cell-cell contacts can mediate cell survival under anchorage-independent condition in an E-cadherin-dependent manner; and that cell-cell contacts mediated resistance of anoikis involves PI3K pathway in our model. Based on all these results, we may, at least in part, infer that HAb18G/CD147 conferred anoikis resistance through E-cadherin mediated cell-cell contacts, which may activate the PI3K/AKT pathway and promote spheroids formation by establishing cell-cell contacts. However, we do not wish to imply HAb18G/CD147 or E-cadherin stimulates survival via PI3K stimulation, since this does not occur directly in response to CD147 and E-cadherin upregulation in our model. And how HAb18G/CD147 affects E-cadherin expression needs further investigation.