We have previously described the AP20187-dependent iFGFR1 dimerization system that was developed and applied to a transgenic mouse model (Welm et al., 2002
). Although a transgenic mouse model is critical for understanding certain aspects of epithelial tumorigenesis, such as the roles of the inflammatory response and angiogenesis, the complexity of the in vivo environment makes it difficult to delineate the molecular mechanisms involved in the early stages of oncogenesis. Therefore, we analyzed the molecular mechanisms of iFGFR1 in 3D cultures of HC11 mouse mammary epithelial cells to mimic the conditions under which iFGFR1 is activated in vivo.
There are two distinct stages of phenotypic progression observed in this 3D culture model: the first stage includes rapid loss of polarity, reinitiation of proliferation, and reduction of luminal cell apoptosis (observed 24 h after iFGFR1 activation). The second stage involves invasion of cells into the surrounding matrix and EMT (observed 4–5 d after iFGFR1 activation; A).
Figure 9. Model for iFGFR1-induced cell invasion and EMT in HC11 3D culture system. (A) Two distinct stages of phenotypic progression were observed in an HC11 3D culture system after iFGFR1 activation. The dashed line suggests that the phenotypes are still progressing, (more ...)
Although several RTKs, including colony-stimulating factor receptor (CSF-1R), ErbB2, and Met, have been studied in MCF10A 3D cultures (Shaw et al., 2004
), the present study is the first time that FGFR1 signaling has been examined in a 3D culture system. In the HC11 model system, iFGFR1 activation is inducible and allows us to study the consequences of iFGFR1 signaling with time on cell proliferation and invasion in the preformed and growth-arrested HC11 acinar structures. The only other example of using this AP20187-inducible system in a 3D culture system is the activation of ErbB2 in MCF10A mammary acini, which resulted in the formation of structures containing multiple acinar units with filled lumens but without invasive properties. Cooperation of the ErbB2 and TGFβ signaling pathways was required for the sustained, elevated activation of ERK and the induction of migration and invasion of MCF10A cells (Muthuswamy et al., 2001
; Seton-Rogers et al., 2004
). This result suggests that coordination of these two signal transduction pathways is required to promote cell invasion. Thus, it is surprising that FGFR1 signaling can promote both proliferation and invasion in the absence of other oncogenic events, and this may be a unique property of this RTK.
Another RTK, CSF-1R, when co-overexpressed with its ligand in MCF10A 3D cultures, resulted in a cell invasion phenotype similar to that observed after FGFR1 activation characterized by loss of E-cadherin from plasma membrane and release of individual cells into the matrix. However, in this case no reduction in total E-cadherin was observed. Thus, it was suggested that other mechanisms, such as an increased rate of endocytosis or a defect in the recycling of internalized molecules, may be involved in the loss of E-cadherin from the plasma membrane (Wrobel et al., 2004
). Therefore, FGFR1 and CSF-1R appear to regulate cell invasion through distinct mechanisms. Although RTKs can activate a similar array of downstream signaling effectors, including ERK and phosphatidylinositol-3 kinase, their phenotypes as determined in 3D cultures appear to be quite distinct (Shaw et al., 2004
). The current study of FGFR1 signaling in 3D cultures supports this observation.
To understand how iFGFR1 regulates cell invasion, we first demonstrated that iFGFR1 activation markedly disrupted cell–cell contacts characterized by the loss of E-cadherin and β-catenin at the cell membrane. Adherens junctions represent a powerful invasion suppressor complex in normal mammary as well as other epithelial cells (Herrenknecht et al., 1991
; McCrea and Gumbiner, 1991
Multiple mechanisms of inactivation of the E-cadherin–catenin complex in normal development and tumor formation have been reported, including genetic mutation, gene inactivation through methylation, repression of expression by Snail and other family members, and cleavage by MMPs (Berx et al., 1996
; Bracke et al., 1996
; Lochter et al., 1997
; Ciruna and Rossant, 2001
). Although it has been proposed that Snail expression downstream of FGFR1 is required for the normal repression of E-cadherin expression during early embryonic development (Ciruna and Rossant, 2001
), we did not detect induction of Snail expression or inhibition of E-cadherin expression by quantitative RT-PCR in our system (unpublished data). This finding suggests that in the HC11 model during cell invasion, loss of E-cadherin is regulated through iFGFR1 by other mechanisms. Interestingly, after iFGFR1 activation, we observed a rapid increase in Twist expression, a known transcriptional repressor of E-cadherin during EMT (Yang et al., 2004
). Because Twist has also been suggested to function as a transcriptional activator (Yang et al., 2004
), it may be involved in inducing vimentin expression downstream of iFGFR1 signaling.
MMP-3 is one MMP that has been suggested to lead directly or indirectly to the cleavage of E-cadherin (Lochter et al., 1997
; Noe et al., 2001
) and is involved in promoting EMT and invasion in mammary tumor cells (Sternlicht et al., 1999
). It has also been suggested to function through Rac1b and reactive oxygen species to induce expression of Snail and EMT (Radisky et al., 2005
). That the effects of iFGFR1 signaling on cell invasion are dependent on MMP-3 activity is supported by several observations. First, quantitative RT-PCR revealed a rapid and dramatic increase in MMP-3 expression on FGFR1 activation, which was followed by E-cadherin cleavage, detected by immunoblotting. Therefore, in the HC11 model system, the decrease in E-cadherin after iFGFR1 activation is not regulated at the transcriptional level, which is dependent on increased Snail expression but is likely the result of cleavage by MMP-3. Second, the introduction of MMP-3 siRNA, not MMP-9 siRNA, into iFGFR1 cells inhibited the cleavage of E-cadherin, thus demonstrating the important role of MMP-3 during iFGFR1-induced EMT.
In iFGFR1-activated acini, the induction of SMA and vimentin expression was also detected. Expression of these mesenchymal markers together with the loss of E-cadherin and β-catenin from cell–cell contacts as well as the invasive behavior of the cells suggest that the HC11 mammary epithelial cells were converted from an epithelial to a mesenchymal phenotype after the activation of iFGFR1. Although it has been known that FGFR1 can stimulate EMT in mesoderm during gastrulation, it is intriguing to find that FGFR1 activation can also induce EMT in mammary epithelial cells. EMT is a hallmark of tumor progression associated with the acquisition of invasive and metastatic features of breast carcinomas (Thompson et al., 1994
; Birchmeier and Birchmeier, 1995
). Abolishment of these phenotypes by the pan MMP inhibitor GM6001 further suggests that MMP activity is required for iFGFR1 signaling to promote EMT and invasive ability of mammary epithelial cells.
In addition to MMP-3, we also observed induction of MMP-9 expression and activity after iFGFR1 activation (Welm et al., 2002
). Thus, MMP-9 may function to activate local growth factors, stimulate angiogenesis in vivo, and degrade the ECM during cell invasion, whereas MMP-3 is critical for the cleavage of E-cadherin. This is another potential mechanism through which FGFR1 may act to promote cell invasion.
It is still not clear how cell polarity, proliferation, and survival are regulated by iFGFR1. Because the changes in these cellular processes upon iFGFR1 activation were not blocked by the addition of the MMP inhibitor (unpublished data), it is possible that they are regulated by independent signaling cascades downstream of iFGFR1. Therefore, these data suggest that MMP-3 induction may play an important role downstream of iFGFR1 during invasion and cell migration but was not required for iFGFR1-mediated effects on cell proliferation, cell polarity, and cell survival.
Based on these and previous studies that have reported the ability of MMP-3 to cleave E-cadherin and promote EMT (Lochter et al., 1997
; Noe et al., 2001
), we propose a model whereby iFGFR1 dimerization activates several downstream signaling pathways that lead to cell polarity disruption, acinar growth, and an invasive phenotype ( B). Multiple transcription factors can be activated and bind to the MMP-3 promoter to induce its expression, eventually resulting in increased MMP-3 activity in mammary epithelial cells. Subsequently, MMP-3 cleaves E-cadherin at the cell membrane, resulting in cell invasion and EMT. This may also provide one potential mechanism by which FGFR1 functions during ductal morphogenesis to regulate the invasion of mammary epithelium into the stromal fat pad. Aberrant regulation of this pathway may lead to early tumor progression. Thus, these results provide new insight into the possible mechanisms through which FGFR1 may contribute to breast cancer progression.
Although the iFGFR1 HC11 3D culture system has been proven to be an excellent system for studying some aspects of the FGFR1 signaling pathway (e.g., cell invasion and proliferation), inflammation and angiogenesis, two processes that are involved in tumor initiation and progression within the iFGFR1 transgenic mouse model, cannot be studied in this monotypic HC11 3D culture system. Although it may be possible for some of these processes to be modeled in organotypic 3D cultures, further analysis of iFGFR1 in transgenic mice will most likely be required to characterize the molecular mechanisms that regulate these processes.