Linking the Cell Cycle to Cell Adhesion Functions
By Sebastian Mueller, MD, and Axel H. Schönthal, PhD
Understanding the molecular mechanisms that control the proliferation of mammalian cells is required to develop efficient therapies for those instances when these controls are deranged (i.e., during tumor development and in cancer). Research on cell cycle-regulatory events has made great strides, and the components of these processes and their interactions are fairly well understood. More recently, cell surface proteins, which are required for the attachment of cells to their micro-environment (i.e., to the basal membrane or to other cells), have come to the forefront of ongoing research efforts. Of these receptors, the cadherins in particular have been implicated in the progression of normal cells to their highly malignant successors—although it remained unclear whether and how these molecules connected with the cell cycle machinery. Now, a few novel studies have provided the first few pieces to this puzzle by providing a functional link between cell surface cadherins and intracellular inhibitors of the cell cycle.
Background
The proliferation of all mammalian cells is strictly regulated by the sequential activity of various cyclin-dependent kinases (CDKs). This group of enzymes constitutes the "cell cycle engine" that executes the orderly progression of cells through the various stages of the cell cycle.1 Their enzymatic activity is tightly regulated by different mechanisms, one of which is the interaction with inhibitory proteins, the cyclin-dependent kinase inhibitors (CKIs), which results in inactive CDK complexes and subsequent cell cycle arrest. Two families of CKIs exist: p21 (also called WAF1, CIP1, or sdi1), p27 (Kip1), and p57 (Kip2), which bind to and inactivate most CDKs; and the INK4 family of p15, p16, p18, and p19, which only bind to CDK4 and CDK6.2 Furthermore, the CKI p21 extends its potent cell cycle-inhibitory effects by blocking the activity of proliferating cell nuclear antigen (PCNA), which is a subunit of DNA polymerase, an enzyme needed for DNA replication during S phase.3
p21 Expression Stimulated by p53
An exciting discovery was the finding that the expression of p21 could be stimulated by the tumor suppressor p53, a connection that greatly contributed to the current understanding of p53 function.4 It is generally assumed that the activation of p21 by p53 is a crucial event that secures the integrity of the genome in the defense against various adverse conditions. For instance, the p53 protein is activated in response to treatment of cells with different types of chemotherapeutic drugs or gamma-radiation, assaults that are known to cause DNA damage. The activation of p53 is followed by the increased expression of p21, inhibition of CDK activity, and subsequent growth arrest. Overall, the ensuing pause in proliferative activity allows the cells to repair their DNA damage, which ensures that defects are not replicated and transmitted to the next generation of cells. In that sense, p53 was dubbed the "guardian of the genome," and p21 was thought to be an essential executioner of this pathway.5 This scenario provided a convincing rationale to explain why loss of the p53 guardian, as seen in more than 50% of all human tumors, leads to genomic instability and cancerous growth. Surprisingly, however, despite p21’s major role as a cell cycle inhibitor and as one of the executioners of p53 function, inactivating mutations of this gene are very rarely found in tumor cells.6,7 This fact is quite intriguing, and novel insights into this phenomenon will be furnished further below.
E-cadherin is an abundant cell surface receptor of epithelial cells that mediates and secures cell-cell contacts (i.e., intercellular adhesion). This molecule not only plays a crucial role in normal tissue morphogenesis and organization, but presumably also in invasive processes and the metastatic spread of tumor cells.8,9 It is assumed that the loss of E-cadherin supports the progression of tumor cells toward the more malignant phenotype.10 However, studies in vitro have resulted in contradictory findings. For example, one study found that E-cadherin was required for the survival and growth of human HSC-3 oral squamous carcinoma cells in suspension culture.11 In contrast, another study found that E-cadherin caused growth suppression of EMT/6 mouse mammary carcinoma cells in suspension culture.12 The reason why E-cadherin appeared to be growth-suppressive in one study, but growth-stimulatory in the other, is unclear; however, it is quite likely that cell-type specific peculiarities affect the respective function of this cell surface receptor. Moreover, until recently it was completely unclear how E-cadherin function was connected to the cell cycle engine.
Connecting E-cadherin to the Cell Cycle Engine
Two recent reports have established a functional connection between E-cadherin expression and the activity of two CKIs, namely p21(WAF1) and p27(Kip1).12,13 Both studies analyzed tumor cells in vitro that were kept in suspension culture, rather than in monolayer culture (i.e., attached to a tissue culture dish). The advantage of this culture system is that the cells are restricted to cell-cell interactions (which are predominantly regulated by cadherins in these cells), rather than cell-matrix interactions (which are executed by the integrin family of cell surface receptors). In both studies, it was found that the expression of E-cadherin caused the cells to form dense multicellular aggregates. However, the implications of increased E-cadherin levels for cellular functions and cell cycle regulation were quite different in the two studies.
The work by St. Croix et al used various breast, lung, and colon carcinoma cell lines.12 They showed that the formation of compact spheroids: 1) was completely dependent on the action of E-cadherin; and 2) resulted in greatly reduced proliferation of cells. Indeed, the addition of anti-E-cadherin specific antibodies to the growth medium completely prevented aggregation and preserved the strong proliferation of cells. Moreover, all E-cadherin-mediated multicellular aggregates consistently contained elevated levels of p27(Kip1), which led to the down-regulation of CDK activity in these cells. The authors concluded from their observations that E-cadherin was a major growth suppressor, which exerted its negative effects on the cell cycle through the upregulation of p27. Thus, these results would suggest that the loss of E-cadherin expression—as seen in many tumor cells—might contribute to a more aggressive growth pattern of these cells.
A different approach, with quite unexpected results, was pursued by Mueller et al.13 This group analyzed a pair of HCT116 colon carcinoma cell lines that only differed in their p21(WAF1) status: one line contained wild type p21 (called HCT116), whereas the other was genetically engineered to lack both copies of the p21(WAF1) gene (called HCTp21-/-).14 While there was no difference in their growth rate under monolayer conditions, the two cell lines exhibited striking differences in suspension culture: HCT116 cells formed dense multicellular spheroids and grew well, whereas HCTp21-/- had lost the ability to form spheroids and eventually died through apoptosis. Thus, HCTp21-/- had lost their anchorage-independence—the cells could only grow under conditions where they were allowed to attach (anchor) to a tissue culture dish. Because, in general, the ability to grow anchorage independently correlates well with the tumorigenicity of cells, this result suggested that the HCTp21-/- cells had lost their tumorigenic phenotype. This could indeed be confirmed and led to the unexpected finding that the loss of a cell cycle inhibitor led to the reversion of these tumor cells to a more normal variant. Together with the above mentioned observation that mutations of the p21 gene are extremely rare in tumor cells, this might suggest that p21 could have some sort of growth-protective function, which could be attributable to its documented apoptosis-inhibitory properties.15 The latter was also supported by the finding that HCTp21-/- cells in suspension were much more chemosensitive toward various anticancer drugs than HCT116 cells.13
Intriguingly, the studies by Mueller et al further demonstrate that the growth-supportive effects of p21 were mediated via the induced expression of E-cadherin.13 In HCTp21-/- cells, E-cadherin was not induced unless the cells were transfected with a construct providing the p21 gene. Conversely, inhibition of p21 function in HCT116 cells by antisense constructs blocked E-cadherin expression and prevented their growth in suspension. Thus, these findings confirmed and extended those of others, that E-cadherin-mediated intercellular adhesions were essential for the growth and survival (i.e., anchorage-independence) of certain cancer cells.11 The study by Mueller et al introduced p21 as a requirement for this function of E-cadherin—at least in the HCT116 cell line.13 The exact chain of events that connects p21 activity to E-cadherin function, however, remains to be established.
Summary
In comparison, the above mentioned reports describe opposing directions for E-cadherin signaling. The first report indicated that activation of E-cadherin (through cell-cell contacts) induced expression of p27 and subsequent growth arrest.12 Thus, the events appear to proceed from the cell-surface receptor to p27 inside the cell. In contrast, in the second report, the order of the events and their consequences are reversed: the presence of p21 is relayed to E-cadherin and results in growth-support.13 The reason for this discrepancy is currently unclear, but might have to do with cell type-specific differences and diverse patterns of gene expression. In any case, the newly discovered connection between crucial cell cycle-regulatory components and a cell adhesion molecule that has been implicated in malignant tumorigenesis provides further insight into the interdependence of cellular growth regulation and the respective micro-environment. (Dr. Schönthal is Associate Professor, Department of Molecular Microbiology and Immunology, K. Norris Jr. Comprehensive Cancer Center, University of Southern California, Keck School of Medicine, Los Angeles, CA; and Dr. Mueller is Clinical Fellow, Department of Internal Medicine IV, University of Heidelberg, Heidelberg, Germany.)
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The INK4 family of CKIs:
a. includes p27 and p57.
b. only bind to CDK4 and CDK6.
c. inactivates most CDKs.
d. includes p21 and WAF1.
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