Our study reveals that FAK is amplified and overexpressed in a large fraction of primary human breast cancers. Elimination of FAK does not obviously affect mammary gland development, but it suppresses tumor initiation and progression in the PyMT model of breast cancer. In cell culture, FAK does not appear to be necessary for normal cell survival and proliferation. However, it supports Ras- and PI3K-dependent neoplastic transformation by orchestrating multiple core functions, including proliferation, survival, and avoidance of senescence. In addition, FAK is necessary for tumor invasion and metastasis. We conclude that FAK exerts critical functions at multiple steps of mammary tumorigenesis. The exquisite dependency of Ras- and PI3K-transformed mammary tumor cells on FAK signaling has broad biological implications and identifies a vulnerability that could be exploited therapeutically.
The gene encoding human FAK resides distal to MYC
at 8q23 within a chromosomal segment that is characterized by frequent aberrations in breast cancer (31
). FISH analysis of 79 primary tumors collected at MSKCC has revealed that FAK
is commonly amplified in breast cancer: 50% of the samples examined were found to contain copy number gains and 10% contained high-level amplifications. Amplification at the FAK
locus correlated with increased expression of FAK but not MYC in these samples, suggesting that FAK is capable of driving expansion of the distal amplicon detected at 8q23 (32
). Two lines of evidence suggest that overexpression of FAK is of clinical significance. First, elevated expression of FAK is inversely correlated with metastasis-free survival in the large cohort of patients from the NKI dataset. Second, multivariate analysis indicates that elevated FAK is an independent predictor of poor outcome and it outperforms many commonly used clinical parameters, such as lymph node involvement, ER negativity, and poor differentiation, as assessed by histology. These findings indicate that FAK
is frequently amplified in breast cancer and suggest that FAK overexpression negatively affects the clinical course of the disease.
Conditional deletion of FAK does not obviously affect mammary gland development, but it suppresses tumor initiation and progression in the PyMT model of breast cancer. Strikingly, virtually all the adenocarcinomas that nonetheless arose in the mutant background were composed of cells expressing FAK, and in mice bred into the Rosa26 reporter (Rosa26R) strain, adenocarcinomas were found to have originated from cells that had escaped Cre-mediated recombination of FAK
. To determine whether FAK is necessary for tumor initiation, we have focused on MIN lesions, which constitute the first morphologically recognizable neoplastic lesions arising in the mammary gland (39
). We found that greater than 80% of the MIN lesions arising in FAK mutant mice had originated from cells that had escaped Cre-mediated recombination. In fact, even the earliest MIN lesions budding out of otherwise seemingly normal ducts or the smallest intraductal lesions consisted almost exclusively of cells expressing FAK but not Cre. These results document a stringent requirement for FAK during mammary tumor initiation.
Muller and colleagues have argued instead that deletion of FAK does not affect PyMT-mediated transformation of the mammary gland (26
). Although they have not estimated the percentage of MIN lesions lacking FAK in their MMTV-Cre
mice, they have noticed several early adenomas lacking FAK in these mice. How do we explain this apparent discrepancy? We believe that the major difference between our compound mice and theirs lies in the efficiency of Cre-mediated deletion. Since the Cre
transgene we have employed deletes in more than 95% of mammary epithelial cells, it is more likely that this transgene is activated early in the developmental hierarchy that gives rise to differentiated progeny in the mammary gland. In other words, we suspect that our Cre
transgene is activated and thereby deletes FAK
in stem or progenitor cells. In contrast, their transgene may not be activated in these cells, allowing for their transformation. Since tumor progenitor cells are estimated to constitute a minority of cells in tumors (46
), the bulk of more-differentiated cells in their early adenomas are likely to have undergone Cre
-mediated deletion of FAK
and thereby fail to express the protein. Yet they may have originated from progenitor cells expressing FAK.
To examine the cellular mechanisms through which FAK supports mammary oncogenesis, we used genetic methods to inactivate FAK in mouse mammary tumor cells transformed by PyMT, activated Ras, or Neu. Provocatively, loss of FAK caused growth arrest followed by apoptosis in tumor cells transformed by PyMT and senescence in those transformed by activated Ras. Although elimination of FAK exerted a more moderate effect in ErbB2-transformed cells, it induced those carrying a deletion of the β4 signaling domain to undergo growth arrest and apoptosis, suggesting that FAK cooperates with the β4 integrin to sustain these cancer cells. Similarly, inactivation of FAK induced growth arrest followed by apoptosis in human breast cancer cells carrying mutant PIK3CA or PTEN, induced senescence in those harboring mutant RAS, and exerted more moderate inhibitory effects in those carrying HER2 amplifications. These results suggest that FAK is required to maintain neoplastic transformation in mammary tumor cells carrying oncogenic mutations that potently activate Ras or PI3K and that it cooperates with the β4 integrin to promote ErbB2-mediated tumorigenesis.
To examine the role of FAK in mammary tumor invasion and metastasis, we used a lung colonization assay. Bioluminescence imaging indicated that loss of FAK completely suppresses metastasis in this assay. Confocal microscopy followed by 3D reconstruction revealed that FAK-deficient mammary tumor cells are not metastatic for 2 major reasons: they survive poorly in the microvascular compartment of the lung, and they are unable to extravasate into the parenchyma of the organ. In agreement with this conclusion, we observed that loss of FAK increases sensitivity to anoikis and inhibits Matrigel invasion in mammary tumor cells transformed by various oncogenes. Together with the correlation between FAK expression and poor metastasis-free survival observed in the NKI dataset, these results suggest that FAK plays a broad prometastatic role in breast cancer.
What is the mechanism through which FAK promotes mammary tumorigenesis? The FAK/SFK complex has multiple substrates (13
), but our mutational analysis suggests that the ability of FAK to support oncogenesis specifically depends on the integrity of Pro712 and Pro713, which mediate FAK’s interaction with the SH3 domain of p130Cas
). In addition, knockdown of p130Cas
phenocopies the effect of loss of FAK in human breast cancer cell lines carrying multiple, distinct, clinically relevant oncogenic mutations, suggesting that FAK supports mammary tumorigenesis largely through p130Cas
. Interestingly, p130Cas
is necessary for morphological transformation of fibroblasts by the viral oncogene Src (47
), and overexpression of p130Cas
promotes hyperplasia and accelerates ErbB2-mediated tumorigenesis in the mammary gland of transgenic mice (48
Whereas the mechanisms through which p130Cas
regulates cell migration and tumor cell invasion are understood to a significant detail, those which may enable p130Cas
to promote cell survival and proliferation and thereby tumorigenicity are not clear (41
). It is intriguing, however, that a major target-effector of p130Cas
, the adaptor protein Crk, can transform fibroblasts in vitro when it is deregulated by viral fusion or a mutation that impairs auto-inhibition (49
). In addition, Crk can interact with several potentially pro-oncogenic proteins, including c-Abl, SOS, and JNK (41
). Adding more complexity, the substrate domain of p130Cas
undergoes mechanical extension and is primed for phosphorylation by FAK/SFK in response to reinforcement of integrin-cytoskeletal linkages (50
). Since mammary tumor cells acquire a contractile phenotype upon adhering to a fibrotic, rigid stroma (51
), it is possible that p130Cas
undergoes extension and multisite phosphorylation under these conditions, resulting in enhanced signaling. Together, these observations suggest the possibility that the FAK/Src complex and its target-effector p130Cas
are integral components of the mechanoregulatory circuit that controls tensional homeostasis in the mammary gland (51
) and that amplification and overexpression of FAK contributes to disruption of this regulatory circuit during mammary tumorigenesis. Our results, however, do not exclude the possibility that in addition to p130Cas
, other FAK/SFK substrates contribute to mammary oncogenesis.
The observation that loss of FAK allows activation of a DNA damage response followed by induction of senescence in cells transformed by activated RAS
is unexpected. Hyperactivation of Ras signaling causes senescence in primary fibroblasts and epithelial cells in vitro and in transgenic models of various cancers, including breast cancer (53
), but loss of p53 or Rb function can counteract this effect of Ras to allow transformation. Since inactivation of FAK also induces senescence in MDA-MB231 cells, which carry mutant P53
, FAK does not appear to oppose senescence by inhibiting p53 or p16 function. The observation that activated Ras signaling also induces senescence independently of Rb or p53 in normal mammary epithelial cells (54
) suggests that loss of FAK allows reactivation of a Ras-dependent senescence program. The ability of FAK to prevent senescence in cells fully transformed by mutant RAS
suggests that these cells do not conclusively avert the risk of undergoing senescence through the acquisition of additional oncogenic mutations, but they require continuous FAK signaling to maintain their replicative potential.
Our findings indicate that FAK is an integral and necessary component in the network of signaling interactions that initiate and support mammary tumorigenesis. The observation that FAK
is frequently amplified in human breast cancer suggests the possibility that FAK may function as a classical oncogene in mammary epithelium. Formal proof that this is the case will require demonstration that overexpression of FAK can initiate tumorigenesis in this tissue in vivo. Alternatively, it is possible that FAK is a potent, general modifier of oncogenesis, akin to the heat shock protein HSP90 and the transcription factor heat shock factor 1 (HSF1) (55
). In either case, the requirement for FAK illustrated by our results suggests new therapeutic opportunities. In particular, the observation that loss of FAK does not affect the ability of normal mammary epithelial cells to survive and proliferate, both in vitro and in vivo, but exerts striking inhibitory effects in mammary tumor cells carrying oncogenic mutations, which activate Ras or PI3K, suggest that these cells may be addicted to FAK signaling, as they are to oncogene signaling (1
). Since FAK appears to operate largely in parallel with classical oncogenic signaling, combined inhibition of FAK and either PI3K or Ras signaling may exhibit broad therapeutic efficacy in breast cancer.