Focal adhesion kinase contributes to proliferative potential of ErbB2 mammary tumour cells but is dispensable for ErbB2 mammary tumour induction in vivo

Elevated expression of FAK has been associated with highly invasive human breast cancers [1, 2]. In particular, several groups have reported a correlation between FAK and human epithelial growth factor receptor (ErbB2, Neu) overexpression in ErbB2-positive human breast cancer [3‐5]. Activation of FAK has also been observed in human breast cancer cell lines expressing elevated levels of ErbB2 [6, 7]. Moreover, recent studies have indicated that FAK and the related kinase Pyk2 are expressed in ErbB2-positive breast cancer and contribute to the proliferative and invasive potential of breast cancer cell lines [8, 9].

Direct evidence for the in vivo importance of FAK in tumourigenesis derives from several recent studies in which components of the integrin signaling pathway were selectively ablated in the germline of mice. For example, mice heterozygous for a FAK null allele exhibit a dramatic delay in tumour induction in a chemically-induced skin carcinogenesis model [10]. Because germline deletion of FAK results in embryonic lethality [10, 11], it is difficult to assess whether complete ablation of FAK could impact on chemical skin carcinogenesis. To circumvent this limitation, the same group demonstrated that conditional ablation of FAK in the skin resulted in an absolute block in the progression of benign papilloma lesions to malignant carcinomas in this model [12]. More recently, it has been shown that prostate-specific ablation of FAK in an SV40 T antigen mouse model resulted in the inability of prostate tumours to progress to the aggressive neuroendocrine phenotype [13].

Although these studies have largely focused on tissues such as skin, there is compelling evidence suggesting that activation of FAK is directly involved in the induction of mammary tumours in vivo. In the polyomavirus middle T (PyVmT) model of mammary tumour progression, mammary epithelial disruption of FAK prevented the transition of mammary hyperplastic growths into mammary adenocarcinomas [14]. This result was subsequently confirmed by a number of independent laboratories [15‐17]. By contrast, another group claimed that FAK function in PyVmT tumour progression played a critical role in the initial progression of primary epithelium to the hyperplastic state [17]. The minor difference between these groups likely reflects the timing at which FAK-deficient lesions were monitored. Another suggested explanation is that the block in PyVmT tumour progression incurred by abrogation of FAK signaling was due to a deficit in the tumour-initiating cell population [15]. Taken together, these observations confirm that FAK plays a critical role in converting PyVmT mammary epithelial hyperplasias into the malignant phenotype.

Given the dramatic impact of FAK deletion on PyVmT tumour induction, we evaluated whether deletion of FAK in an activated ErbB2 mouse model resulted in a comparable phenotype. To accomplish this, we first intercrossed the mouse mammary tumour virus (MMTV)-activated ErbB2 strain (NDL2-5) to separate strains of mice bearing the MMTV-Cre and conditional FAK alleles and monitored virgin female cohorts for the development of mammary tumours. Although a delay in mammary tumour onset was observed, all animals developed mammary tumours that were highly metastatic. Genetic and biochemical analyses revealed that the majority of these tumours had escaped Cre-mediated recombination of the conditional FAK alleles and were proficient for FAK expression, indicating a strong selection for retention of FAK signaling. Cre-mediated deletion of FAK in established ErbB2 mammary tumour cells resulted in a profound delay in tumour growth that was further associated with a proliferative defect. Finally, using a transgenic mouse model that co-expressed ErbB2 and Cre recombinase, we demonstrated that while mammary epithelial ablation of FAK delayed tumour onset and reduced the number of neoplastic lesions, animals developed mammary tumours with 100% penetrance. Collectively these observations argue that while FAK contributes to ErbB2 tumour cell proliferation, it is ultimately dispensable for ErbB2 mammary tumour initiation and progression.

Elevated expression of FAK has been previously implicated as an important determinant in both the initiation and the progression of ErbB2-positive breast cancers [6, 7]. Consistent with these observations, several in vitro studies with ErbB2-transformed mammary tumour cells have identified FAK and its closely related family member Pyk2 as critical signals in ErbB2-dependent proliferation [8, 9, 17]. Although these studies provide compelling data supporting a role for FAK in ErbB2 tumour progression, whether FAK is required in vivo for ErbB2 mammary tumour progression has not been addressed; this question is imperative given that the integrin/FAK signalling output from ErbB2 tumour cells is highly dependent on the microenvironment within the mammary fat pad. Here we demonstrate using two well-characterized transgenic mouse models of ErbB2 breast cancer that while the presence of functional FAK confers a proliferative advantage to ErbB2 tumour cells, metastatic ErbB2 mammary tumours can be generated in the complete absence of FAK expression.

To evaluate the role of FAK in ErbB2 mammary tumour progression, we initially interbred the MMTV-activated ErbB2 strain (NDL2-5) [20] with separate strains of mice bearing MMTV-Cre and conditional FAK alleles [12, 18]. Although a delay in mammary tumour onset was observed (Figure 1), the majority of tumour epithelia maintained expression of FAK due to loss of Cre expression (Figure 2). This phenomenon persisted throughout the progression to metastasis since we could not detect any FAK-deficient lesions in the lungs of FAK^flx/flx tumour-bearing mice (Figure 3). One potential explanation for the presence of these escapee populations of tumour cells that have not undergone Cre-mediated recombination is that the few FAK-ablated tumour epithelia detected were at a proliferative disadvantage relative to their FAK-proficient counterparts (Figure 2). This observation is consistent with results from similar experiments in the PyVmT tumour model system where FAK was shown to influence the proliferative status of these tumour cells [14, 16, 17]. Likewise, mammary disruption of β1-integrin or c-Src signalling pathways (which are immediately upstream of FAK) also impairs PyVmT tumour cell proliferation [22, 23]. Collectively these data argue that the integrin/c-Src/FAK signalling network is a key regulator of the proliferative capacity of both ErbB2 and PyVmT mammary tumour cells.

While these studies indicate the importance of FAK in the initiation phase of ErbB2 mammary tumour progression, acute deletion of FAK in established tumour cells also impacts on their ability to proliferate in both in vitro and in vivo settings (Figure 4) (Figure S2a in Additional file 2). In fact, from a population of tumour cells that were largely FAK-deficient (Figure 4a), the tumours that eventually arose regained FAK expression (Figure S2c in Additional file 2). The presence of FAK protein suggests that these tumour cells presumably derived from the small population of cells that had not undergone productive excision of the FAK conditional allele; alternatively, it could also represent FAK from the stromal compartment of the mammary gland which was the case in the FAK-null/MMTV-NIC tumours (Figure 6a and 6b). Again these data suggest that retention of a functional FAK gene confers a strong proliferative advantage to ErbB2 tumour cells over their FAK-deleted counterparts.

Although these data demonstrate that FAK activation is involved in the induction and maintenance of ErbB2 tumours, our studies with the MMTV-NIC model indicate that ErbB2 tumours completely lacking FAK can develop eventually. By contrast, deletion of FAK in the PyVmT model prevented tumour progression entirely [14]. One possible explanation for this difference is that the tumour-initiating cell population in the PyVmT model is absolutely reliant on FAK, whereas this population in the ErbB2 model can function independently of FAK signalling. Consistent with this hypothesis, FAK function has been reported to be important in tumour-initiating cells in the PyVmT model [15].

Examination of downstream signaling pathways revealed that loss of FAK resulted in a reduction in tyrosine phosphorylation of p130Cas (Figure 6). However, in contrast to complete loss of tyrosine phosphorylation of p130Cas in an ErB2 model with abrogation of either β1-integrin or integrin linked kinase (ILK) [26, 27], FAK-deficient tumours still retained a basal level of tyrosine-phosphorylated p130Cas that was further correlated with its ability to couple with the FAK-related kinase Pyk2 (Figure S4 in Additional file 4 and Figure S5d in Additional file 5). Although it has been proposed that FAK was critical for migratory behaviour whereas Pyk2 was involved in the proliferative phase of ErbB2 tumour induction in vitro [8], the observation that FAK-null ErbB2 tumours can form metastatic lesions argues that at least in the in vivo setting, Pyk2 can functionally substitute for FAK. One of the main targets for Pyk2 is likely p130Cas, as it has been reported that p130Cas is essential for ErbB2 tumour progression [28]. While these data implicate the Pyk2/FAK/p130Cas signalling complex as an important signalling element in ErbB2 tumour progression, both ILK- and β-1 integrin-ablated ErbB2 tumours lack detectable tyrosine phosphorylated p130Cas [26, 27] indicating that tyrosine phosphorylation of the p130Cas scaffold is not essential for ErbB2 tumour induction. However, tumours from both these models are poorly metastatic [26, 27] suggesting that engagement of Pyk2/FAK/p130Cas signalling likely plays a more important role in the metastatic phase of ErbB2 tumour induction. Consistent with this concept, upregulation of Pyk2 has been observed in metastatic foci derived from PyVmT cancer stem cells devoid of FAK function [25].

One of the most interesting findings of these studies was the observation that acute knockout of FAK from established ErbB2 tumour cells (Figure 4) was more deleterious than early abrogation of FAK function in the MMTV-NIC model (Figure 6). A hypothesis for this difference in response between these two models is that established ErbB2 tumour cells have become addicted to the requirement for FAK function whereas MMTV-NIC-initiated tumour cells have evolved to circumvent the FAK signalling network through compensation by Pyk2. Indeed, the phenomenon of oncogene addiction has been observed in a number of human cancers [29].

Collectively these observations suggest that targeting both Pyk2 and FAK will be required to effectively eliminate ErbB2-dependent mammary tumours. Indeed, it has been reported that small molecule inhibitors specific to FAK tyrosine kinases have both in vitro and in vivo efficacy in interfering with mammary tumour progression [30, 31]. PF-562-271, a dual FAK/Pyk2 inhibitor with anti-tumour activity, has been successfully used in preclinical studies [25, 32]. The future clinical application of therapeutic strategies against FAK/Pyk2 signalling has great potential for the treatment of primary human breast cancer.