Previously, our laboratory showed that expression of different cadherin family members by squamous epithelial cells markedly effected morphology (Islam et al. 1996
), i.e., when oral squamous epithelial cells expressed N-cadherin, they converted to a fibroblastic phenotype concurrent with decreased cell–cell adhesion. Thus, when we turned our attention to breast cancer cells for the present study, we were interested not only in the expression of various cadherins by these cells, but also in whether these cadherins influenced the morphology of the cells. We were not surprised to find that breast cancer cells endogenously expressing N-cadherin displayed a fibroblastic phenotype with tenuous cell–cell contacts, whereas breast cancer cells endogenously expressing E-cadherin displayed a typical epithelial morphology. We were, however, surprised to find that transfection of N-cadherin into the E-cadherin–expressing BT-20 breast cancer cell line had no effect on morphology, even though it had a dramatic effect on cell behavior. Equally surprising was the fact that forced expression of E-cadherin had no effect on the morphology of the fibroblastic N-cadherin–expressing MDA-MB-435 cells. Thus, the breast cancer cell lines examined in this study behaved very differently from the oral squamous epithelial lines that we characterized previously. Interestingly, the oral squamous epithelial cells downregulated E-cadherin when they were forced to express N-cadherin, suggesting an inverse relationship between these cadherins. In contrast, the breast cancer cells continued to express their endogenous cadherin(s) when transfected with a different cadherin. The continued expression of endogenous cadherin may account for the lack of morphological change in the transfectants. Thus, the breast cancer cells differ from the oral squamous epithelial cells in two very important ways: first, the oral squamous epithelial cells appear to coregulate cadherins in an inverse manner, whereas these cadherins are independently regulated in breast cancer cells; and second, expression of E-cadherin by the oral squamous epithelial cells is sufficient for epithelial morphology, whereas epithelial morphology in the breast cancer cells appears to depend on other factors, in addition to E-cadherin.
In the present study, we have demonstrated that N-cadherin (or cadherin-11) expression in human breast carcinoma cells promotes an invasive phenotype. By transfecting the BT-20 cells with these nonepithelial cadherins, we have provided evidence for a direct role of these cadherins in cell motility and invasion. Previous studies have correlated the expression of N-cadherin or cadherin-11 with invasion in breast cancer cells. However, in this study, we took the important next step and used transfection studies to show that a cell line that has a low invasion rate could be converted to a highly invasive cell by expression of N-cadherin or cadherin-11. The BT-20 breast cancer cell line provided an important tool for these studies since they did not downregulate E-cadherin when forced to express N-cadherin. Thus, we can conclude that, even in cells expressing high levels of E-cadherin, N-cadherin (or cadherin-11) can promote motility, suggesting that, in this regard, both N-cadherin and cadherin-11 are dominant over E-cadherin. A study by Sommers et al. 1994
supports this idea. These authors showed that transfection of E-cadherin into the E-cadherin–negative breast cancer cell lines, BT549 and HS578, did not decrease the invasive capacity of these cells. These authors suggested that the transfected E-cadherin was not functional; however, these authors were unaware of the fact that the BT549 and HS578 cell lines express N-cadherin.
A previous study using MDA-MB-435 cells showed that transfection of E-cadherin into these cells reduced their capacity to form tumors when injected into the foot pads of nude mice (Meiners et al. 1998
). In contrast to our study, these authors showed that E-cadherin–transfected clones of MDA-MB-435 cells underwent a morphological change upon E-cadherin expression. In addition, they showed that E-cadherin–transfected clones were less tumorigenic in their assay than the parental cells. One difference in the study of Meiners et al. 1998
and ours is that they did not assay for N-cadherin expression in their E-cadherin–positive clones of MDA-MB-435 transfectants. Our study clearly demonstrates that N-cadherin influences the behavior of the cells, and that cells retaining N-cadherin do not undergo a morphological or behavioral change upon expression of E-cadherin. Thus, one possible explanation for the difference between these two studies is that the cells in the Meiners' study did not express N-cadherin. The point of our study was to determine if N-cadherin was capable of influencing the behavior of epithelial cells, even if they expressed E-cadherin, thus, we were particularly careful to select cell lines that retained N-cadherin expression after transfection with E-cadherin ().
One puzzling aspect of cell lines derived from metastatic tumors is that they often express E-cadherin and appear to be relatively normal epithelial cells. A possibility suggested by the present study is that such cells may have upregulated the expression of N-cadherin during the process of metastasis. Our results suggest that expression of N-cadherin would confer on these cells the capacity to invade, even though they continued to express E-cadherin. In this regard, expression of an inappropriate cadherin like N-cadherin (or other related cadherins) may be a better gauge of the clinical state of a tumor than is decreased expression of E-cadherin.
Some of the E-cadherin–negative breast cancer cells expressed endogenous P-cadherin. These cells had a fibroblastic morphology similar to that of the N-cadherin–expressing cells; however, they were not highly invasive, suggesting that P-cadherin confers upon breast cancer cells characteristics different from those conferred by either E- or N-cadherin. P-cadherin is expressed in the myoepithelial cells surrounding the lumenal epithelial cells of the mammary gland. Radice et al. 1997
recently showed that P-cadherin deficient mice develop age-dependent hyperplasia and dysplasia of the mammary epithelium, and suggested that P-cadherin may play a role in maintaining the normal phenotype of breast epithelial cells. One possibility is that the P-cadherin–expressing tumor cells were derived from the myoepithelium, rather than from the true epithelium.
E-cadherin has been termed an invasion suppressor because transfection of this protein into some E-cadherin–negative invasive carcinoma cells resulted in decreased invasive capacity. Our prediction is that at least some of these cell lines cells expressed a cadherin, like N-cadherin or cadherin-11, and overexpression of E-cadherin resulted in downregulation of the endogenous cadherin, as we saw with the oral squamous epithelial cells. Thus, we hypothesize that the invasion suppressor role of E-cadherin arises in part from its ability to decrease the level of N-cadherin in certain, but not all, tumors. In the present study, cell lines that did not express any classical cadherins, as evidenced by lack of β-catenin protein, as well as lack of detectable cadherin, had low invasion rates. Our hypothesis, that loss of E-cadherin alone does not necessarily increase invasive capacity in breast carcinoma cells, is supported by the observation that function-blocking antibodies against E-cadherin did not confer a highly motile, invasive phenotype on MCF-7 cells, a breast cancer cell line that is E-cadherin–positive and N-cadherin–negative (Sommers et al. 1991
). The current study suggests that, in some carcinoma cells, expression of N-cadherin, or a similar cadherin such as cadherin-11, may actually be necessary for increased motility and invasion. A recent clinical study suggested that inactivation of E-cadherin is an early event in the progression of lobular breast carcinomas (Vos et al. 1997
). We might suggest that a subsequent event would be activation of the expression of an inappropriate cadherin, such as N-cadherin or cadherin-11.
Understanding the mechanism by which N-cadherin promotes motility in epithelial cells is important if we are to develop treatments that will decrease the invasiveness of tumor cells. A number of studies have shown that epithelial cells can be induced to scatter in response to growth factors, such as hepatocyte growth factor and members of the FGF, EGF, and TGF families (Blay and Brown 1985
; Vallés et al., 1990; Behrens et al. 1991
; Geimer and Bade 1991
; Gherardi and Stoker 1991
; Rosen et al. 1991
; Miettinen et al. 1994
; Savagner et al. 1994
, Savagner et al. 1997
). Walsh, Doherty, and coworkers have established, through extensive studies on FGF receptor and cell adhesion molecules, that N-cadherin and the FGF receptor cooperate to induce neurite outgrowth in cerebellar neurons (reviewed in Doherty and Walsh 1996
; Walsh and Doherty 1997
). These authors have proposed a scheme for activation of the kinase activity of the FGF receptor through cis interactions with N-cadherin, via an HAV domain in the FGF receptor and an HAV interaction domain in the fourth extracellular domain of N-cadherin (Doherty and Walsh 1996
). In addition, it has been proposed that the cadherins form lateral dimers in the plane of the membrane (Shapiro et al. 1995
; Takeda et al. 1999
), which could result in dimerization of the FGF receptor, and subsequent activation of the signal transduction pathway. We based the studies presented herein on the model presented by Walsh and Doherty, and proposed that interaction of N-cadherin with the FGF receptor in N-cadherin–expressing epithelial cells may result in increased motility, similar to that seen by treating epithelial cells with growth factors. To test this hypothesis, we interfered with the N-cadherin–dependent FGF receptor signal transduction pathway proposed by Walsh and Doherty by inhibiting a downstream enzyme, diacylglycerol lipase. We showed that inhibiting diacylglycerol lipase decreased motility of N-cadherin–expressing cells in a dose-dependent manner while having no effect on the motility of N-cadherin–negative cells. Thus, our data strongly support the notion that N-cadherin promotes motility in breast cancer cells by activating growth factor receptor signal transduction pathways. Continued efforts in our laboratory are aimed at further defining the signal transduction pathway(s) that mediate cadherin-dependent motility in epithelial cells.
At first glance, it might seem unlikely that expression of an additional cell adhesion molecule would confer a motile and invasive phenotype upon an epithelial cell. However, motile cells, such as fibroblasts and myoblasts, express N-cadherin (Knudsen et al. 1995
; Huttenlocher et al. 1998
) and a switch from E- to N-cadherin occurs in the chick embryo when epiblast cells ingress through the primitive streak to form the mesoderm (Edelman et al. 1983
; Hatta and Takeichi 1986
). Another interesting cadherin switch occurs during establishment of the human placenta, where fetal cytotrophoblast cells invade the vasculature of the uterus. During this invasive process, the cytotrophoblast cells downregulate the expression of E-cadherin and upregulate vascular/endothelial (VE) cadherin (Zhou et al. 1997
). Thus, it is feasible to suggest that increased expression of a nonepithelial cell cadherin, such as N-cadherin, could increase the invasive potential of tumor cells. Ongoing studies in our laboratory are designed to determine how N-cadherin differs from E-cadherin in its ability to induce cell motility. We hypothesize that E-cadherin does not have the ability to interact with the relevant growth factor receptors, and we are preparing chimeric molecules between E- and N-cadherin to test this hypothesis.
An important message from the present studies is that cadherins may not function identically in different cell types. The fact that cadherins may act differently in different cell types is particularly evident when comparing the current study with earlier studies showing that mouse L cells or S180 fibroblasts attained an epithelial morphology when transfected with either E- or N-cadherin (Nagafuchi et al. 1987
; Hatta et al. 1988
; Matsuzaki et al. 1990
). It will be important in future studies to consider the cellular makeup, as well as the complement of cadherin family members, when interpreting data on cellular morphology and behavior.