Introduction
The activity of Rho-like GTPases in response to receptor stimulation is strictly controlled to stimulate, locally and temporally, specific downstream signaling pathways in cells. The regulators of the activity of Rho GTPases consist of three classes of proteins: guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). To date, over 70 Rho GEFs have been identified (Schmidt and Hall
2002). In earlier studies, we have identified the
Tiam1 gene (T-cell invasion and metastasis gene 1), which encodes a specific GEF and thus activator of the Rho-like GTPase Rac (Habets et al.
1994; Michiels et al.
1995). Rho GTPases are best characterized for their regulation of actin cytoskeleton dynamics, but they also control various other processes including apoptosis, cell proliferation and gene transcription (Bishop and Hall
2000). It is therefore not surprising that Rho GTPases and their regulators may contribute to various aspects of tumorigenicity (Malliri et al.
2002a; Sahai and Marshall
2002).
The activators of Rho GTPases not only catalyze GDP/GTP exchange but also contribute to RhoGTPase downstream signaling by connecting active GTPases to specific scaffold and effector proteins (Mertens et al.
2003; Rossman et al.
2005). Scaffold proteins that complex Tiam1 with components of specific Rac effector pathways include IB2 and spinophilin, which direct Tiam1-mediated Rac activation towards the p38 MAPK and p70S6 K cascades implicated in transcription and translation, respectively (Buchsbaum et al.
2002,
2003). Tiam1 also binds to different components of the Par polarity complex and thereby regulates polarity processes in various cell types. Tiam1 associates with Par3 and PKCξ and connects Tiam1-mediated Rac signaling to the establishment of apical–basal cell polarity in contacting epithelial cells as well as front–rear polarity in freely migrating epithelial cells (Chen and Macara
2005; Mertens et al.
2005; Pegtel et al.
2007). Tiam1 also associates with activated Rap1 and in conjunction with the Par polarity complex controls chemokine-induced T-cell polarization (Gerard et al.
2007). The association of Tiam1 with IRSp53 and p21Arc provides a direct link between Tiam1-mediated Rac activation and Arp2/3 complex-controlled actin polarization, which is required for cytoskeletal dynamics during cell migration and cell polarization (Connolly et al.
2005; ten Klooster et al.
2006). In receptor signaling, activated Ras may activate Rac by direct binding to Tiam1 (Lambert et al.
2002) or indirectly by activation of phosphoinositide 3-kinase (PI3-kinase) that may recruit and activate Tiam1 and thereby Rac downstream of Ras (Fleming et al.
2000; Zondag et al.
2000).
Tiam1 is expressed in human and rodent tumor cells of different tissue origin and has been shown to affect various aspects of tumorigenicity (Habets et al.
1995; Minard et al.
2004). The influence of Tiam1 on different stages of tumor development is illustrated in previous studies using mouse tumor model systems and Tiam1 knockout (
Tiam1
−/−) mice. Tiam1-deficiency inhibits Ras-induced mouse skin tumor initiation and growth in a two-stage DMBA/TPA carcinogenesis model (Malliri et al.
2002b). These skin tumors arise by DMBA-induced Ras mutations in keratinocytes of the epidermis.
Tiam1
−/− mice are more resistant to the Ras-induced skin tumor development because epidermal keratinocytes are more susceptible for Ras-induced apoptosis during tumor initiation. Although the few Ras-induced skin tumors that do occur in
Tiam1
−/− mice grow much slower than wild type tumors, they convert more frequently to a malignant phenotype, presumably as a result of the function of Tiam1 in maintenance of E-cadherin-based cell–cell adhesions (Malliri et al.
2002b,
2004). Such a bifunctional effect of Tiam1-deficiency on tumor formation and progression was also found for intestinal tumors in APC Min (multiple intestinal neoplasia) mutant mice (Malliri et al.
2006). Tiam1 is a Wnt-responsive gene in colon cells and its deficiency reduces the formation and growth of polyps in APC Min mutant mice but promotes invasion of progressed malignant intestinal tumors (Malliri et al.
2006). These in vivo data indicate that Tiam1 functions downstream of at least two independent oncogenic signaling pathways, i.e., the Ras and the Wnt pathway.
Tiam1 is thus a potential therapeutic target, and chemical inhibitors have been developed to inhibit the function of Tiam1 and Rac in tumors in vivo (Gao et al.
2004; Shutes et al.
2007). In this context, it is important to decipher the specificity of Tiam1 as a modifier of tumor development and progression in the context of different oncogenic signaling pathways and of tumor cell types. Therefore, we investigated the function of Tiam1 in mammary tumorigenesis induced by two alternative oncogenic signaling pathways. We crossed
Tiam1
−/− mice with two strains of breast cancer prone transgenic mice that express oncogenic Myc or Neu under the control of the mouse mammary tumor virus (MMTV) promoter. Interestingly, we found that Tiam1-deficiency did not influence Myc-induced tumorigenesis but specifically impaired c-
neu induced mammary tumor formation in mice, illustrating that Tiam1-mediated Rac signaling is required for only specific oncogenic signaling pathways that lead to tumorigenesis.
Discussion
We crossed Tiam1
−/− mice with MMTV-c-myc and MMTV-c-neu transgenic mice to study the consequences of Tiam1-deficiency in Myc-induced and Neu-induced mammary tumorigenesis. Our analyses revealed that Tiam1 is required for oncogenic signaling induced by Neu but not by Myc. More specifically, we found that Tiam1-deficiency delays the initiation of Neu-induced mammary tumors but does not affect the growth of these tumors. Initiation and growth of Myc-induced mammary tumors was independent of the expression of Tiam1.
Besides the dramatic delay in the onset of the first detectable Neu-induced mammary tumors, also the total number of tumors per mouse was lower in
Tiam1
−/− mice than in
Tiam1
+/+ animals. This is consistent with the findings in skin and intestinal tumors, where the latency of tumor onset and the number of tumors per mouse were dramatically decreased in a Tiam1-deficient background (Malliri et al.
2002b,
2006). In the skin tumor model, we found that
Tiam1
−/− mice produced less Ras-induced tumors, because keratinocytes in the basal layer of the epidermis are more susceptible to apoptosis (Malliri et al.
2002b). Recently, we found that the Tiam1/Rac-mediated survival pathway in keratinocytes acts through ROS-mediated activation of the ERK pathway (Rygiel et al.
2008). Also in APC Min mice, the decreased initiation of intestinal tumors by aberrant β-catenin signaling in Tiam1-deficient mice compared to wild type mice was attributed to increased apoptosis susceptibility (Malliri et al.
2006). Consistent with this, Tiam1 expression levels correlate with apoptosis susceptibility in human colon tumor cells (Minard et al.
2006).
It is likely that Tiam1-deficiency in the MMTV-c-
neu model affects tumor initiation by increasing the susceptibility to apoptosis of the targeted mammary epithelial cells. We have attempted to study apoptosis sensitivity in vivo, but we could not find significant differences in apoptosis between established Neu-induced tumors produced in
Tiam1
+/+ and
Tiam1
−/− mice (data not shown). Analysis of apoptosis in tumor samples of MMTV-c-
neu tumors by TUNEL and Caspase 3 stainings appeared difficult because of the heterogeneity of these tumors. Moreover, apoptosis resistance is most likely essential during the initiating events of tumorigenicity, which is difficult to study in vivo. Once tumors have been formed, differences in apoptosis sensitivity in
Tiam1
+/+ and
Tiam1
−/− tumors are presumably not detectable anymore, as Tiam1-independent events have rescued the tumor-initiating cells from apoptosis. We performed therefore in vitro studies using MDA-MB-361 breast cancer cells with high Neu expression and found that the survival signaling of these tumor cells is dependent on the presence of Tiam1. Similarly, as found in DMBA-induced skin tumors and β-catenin-induced intestinal tumors, the presence of Tiam1 seems to be required to prevent apoptosis during initiation of mammary tumors by the Neu oncogene. Tiam1-mediated Rac-signaling might prevent apoptosis by activating various well-known survival signaling pathways including the NFkappaB and ERK pathways (Joneson and Bar-Sagi
1999; Rygiel et al.
2008; Zahir et al.
2003).
Although less mammary tumors were produced in
Tiam1
−/− mice, the growth of the tumors in
Tiam1
+/+ and
Tiam1
−/− mice was the same once they were formed. In contrast, we showed in skin and intestinal tumors a function of Tiam1 in both initiation and growth of these tumors (Malliri et al.
2002b,
2006). Interestingly, in DMBA (Ras)-induced skin tumors, decreased growth of tumors in
Tiam1
−/− mice was found only when proliferation of tumors was promoted by TPA treatment. Skin tumors that were generated by treatment with DMBA only grew equally well in the presence or absence of Tiam1 (Malliri et al.
2002b), indicating that TPA-induced but not DMBA-induced proliferation depends on Tiam1-mediated Rac activation. TPA is able to induce cyclin D1 expression, a regulator of cell proliferation (Yan and Wenner
2001), suggesting that Tiam1 is required for TPA-induced proliferation by influencing cyclin D1 levels. Moreover, both the Ras and Neu oncogenes are absolutely dependent on cyclin D1 expression for mammary tumor formation in MMTV-
ras and MMTV-c-
neu transgenic mice (Yu et al.
2001). Cyclin D1-deficient mice are resistant to Ras-induced and Neu-induced mammary tumors, while they remain fully sensitive to other oncogenic pathways (Yu et al.
2001). However, the fact that the proliferation of Neu-induced tumors is independent of Tiam1 suggests that Tiam1 does not regulate cyclin D1 levels in mammary tumors. Indeed, tumor lysates from MMTV-c-
neu;
Tiam1
+/+
and MMTV-c-
neu;
Tiam1
−/− mice revealed a large variation in cyclin D1 levels independent of the presence of Tiam1 (data not shown). As Neu predominantly signals through Ras and the growth of DMBA-only treated skin tumors is independent of Tiam1, it is unlikely that Tiam1–Rac signaling contributes to Ras-controlled proliferation of tumors. Interestingly, it has been shown that Neu-mediated protection from apoptosis is dependent on its association with the Par polarity complex, while Neu-mediated proliferation is not (Aranda et al.
2006). Tiam1 associates with the Par polarity complex and is able to activate this complex (Mertens et al.
2006), providing a possible mechanism by which Tiam1 could interfere in initiation but not growth of Neu-induced mammary tumors.
A higher number of metastases was found in the MMTV-c-
neu;
Tiam1
+/+
mice when compared to MMTV-c-
neu;
Tiam1
−/− mice, suggesting that Tiam1 promotes metastasis of breast tumors. Studies in human tumors also show a positive correlation between Tiam1 expression and progression and invasiveness of mammary, colon and prostate tumors (Adam et al.
2001; Engers et al.
2006; Liu et al.
2007; Minard et al.
2005,
2006). This is in contrast to the findings in skin and intestinal tumors, where progression was associated with loss of Tiam1 (Malliri et al.
2002b,
2006). The latter could be explained by a function of Tiam1 in the formation and maintenance of intercellular adhesions (Engers et al.
2001; Hordijk et al.
1997; Mertens et al.
2005; Uhlenbrock et al.
2004). In the MMTV-c-
neu mice, the metastases appear in pulmonary blood vessels as tight tumor emboli that are thought to arise from circulating cell clumps that get stocked in the veins of the lungs. As in human inflammatory breast cancers (IBC), such circulating tumor emboli might benefit from strong E-cadherin-mediated cell–cell interactions favoring passive dissemination in distinct organs (Kleer et al.
2001; Tomlinson et al.
2001). However, we could not find significant differences in E-cadherin expression between Neu-induced tumors in
Tiam1
+/+ and
Tiam1
−/− mice that could support such a mechanism. Alternatively, the higher number of metastases found in the MMTV-c-
neu;
Tiam1
+/+
mice could be the result of the increased number of tumors found in these mice.
In conclusion, the effects of Tiam1-mediated Rac signaling on tumorigenesis appear oncogene-dependent and tumor cell type-dependent and either positively or negatively correlate with tumor progression. As Tiam1-mediated Rac activation controls different signaling pathways that may influence initiation, growth and progression of tumors, the cellular outcome of altered Tiam1 expression may depend on a balance between factors that promote or inhibit the formation and progression of tumors.