The HEF1 gene is conserved in all vertebrates, and localizes at chromosome 6p25-24 in humans, and chromosome 13 A3.3 in mice. The HEF1 protein is predominantly cytoplasmic, and concentrates at focal adhesions during interphase in adherent cells, and at centrosomes and other parts of the mitotic apparatus during G2/M. HEF1 lacks any known enzymatic function, but contains many functional modules for protein interaction, leading to its classification as a scaffolding protein (2
). Validated interaction sequences include () an SH3 domain, at least 15 SH2 domain-binding sites, and an evolutionarily well-conserved carboxy-terminal domain of unknown structure. Proteins that functionally and/or physically interact with HEF1 () include many with direct roles in promoting tumor invasion, as discussed below. Vertebrate HEF1 has two paralogs, p130Cas/BCAR1 and Efs/Sin, which conserve domain structure and many but not all functional interactions (6
): together, HEF1, p130Cas, and Efs are referred to as members of the Cas family.
A. Schematic of structure of the HEF1/NEDD9/CasL protein. Human HEF1 is 834 amino acids; key functional domains include an amino-terminal SH3 domains, which binds FAK; a “substrate domain” (SD) containing multiple embedded SH2 binding (more ...)
Significantly elevated levels of HEF1 mRNA (10–70 fold) and protein exist in >35% of the metastatic melanomas produced by “escaper” tumors in Tyr-rtTA+;Tet-RAS+;Ink4a/Arf−/−
mutant mice, as well as in a similar percentage of human metastatic melanomas (5
). Importantly, elevated HEF1 protein expression was essential for the metastatic properties of the involved tumors both in vitro
and in vivo
). As HEF1 is a scaffolding protein, its action involves regulated assembly of protein complexes. Consequently, the effect of altered HEF1 expression is dependent on the relative stoichiometry and availability of other complex constituents. Hence, moderate overexpression may drive the assembly of functional complexes, causing constitutive activation of downstream effector pathways. Conversely, excessive HEF1 over-expression may be equivalent to loss of HEF1 expression, if either condition induces complex disruption. It is perhaps significant that one study has now identified reduced
HEF1 expression as part of a signature for metastatic breast cancers ((9
), discussed further below). Notably, both overexpression and depletion of HEF1 cause mitotic defects in cultured cells (10
). While the exact mechanism of HEF1 action in metastasis requires further investigation, to date, studies of HEF1 overexpression, depletion, and genetic deletion have revealed the following metastasis-relevant properties:
1] HEF1 positively regulates the Src-FAK-Crk “migratory switch”
The initial reports identifying HEF1 established that this protein interacts directly with FAK, Src, and Crk (2
). FAK is commonly constitutively activated in melanomas, and an important target of cancer drug development. The consensus of work by many groups suggests a mechanism in which cell attachment triggers the interaction of Src, HEF1, and FAK: overexpression or mutational activation of one of these proteins can also drive complex formation. These interactions enhance the activation of Src and FAK, and lead to extensive tyrosine phosphorylation of HEF1, creating binding sites for effector proteins with SH2 domains: the most important of these are Crk and Crk-L. A Cas-Crk complex has been described as a “master switch” for cell migration; through Crk-L and DOCK180, HEF1 activates Rac and other components of the cell migration machinery (reviewed in (8
2] HEF1 activates machinery for tumor growth, invasion and homing
Tumor progression depends on the activation of essential effector kinases that mediate proliferation and survival in an expanding hypoxic tumor mass. Tumor invasion depends on the increased expression and activity of extracellular proteases that degrade or remodel basement membranes to allow cellular movement, while activating latent growth factors that promote tumor progression. Related to this, HEF1 overexpression may induce molecules involved in tissue remodeling and invasion (12
). HEF1 overexpression also induces the activation of ERK, p38, and JNK kinases through interactions with intermediary signaling effectors (reviewed in (6
)). For example, overexpressed HEF1 transcriptionally induces ErbB2/HER2/neu, a growth factor receptor at the top of the Ras > Raf > MEK > ERK pathway, while the HEF1 interactor FAK directly binds Shc, which influences activation of Ras through its binding partners EGFR and Grb2.
HEF1 also binds proteins of the AND-34/CHAT family (13
), which activate JNK and ERK by signaling through Rap1 (14
). Activated Rap1 is also an important intermediate in “inside-out” cell signaling, in which internally-derived signals activate integrin-ligand binding in response to upstream cues that typically involve chemokine stimulation. This process, much studied in consideration of the migration and “homing” of lymphoid cells, is now appreciated as playing a role in tumor invasion and targeting of metastases. Lymphoid cells from HEF1 null mice, or with depleted HEF1, are greatly impaired for chemokine response, migration, and homing, accompanied by failed activation of CHAT-H and Rap1 (14
). Critically, these defects involve the CXCR4-CXCL12 targeting system, which is important not only for targeting of lymphocytes to secondary organs, but is also a major contributor to tumor metastasis (16
). By inference, CXCR4 signaling is expected to be hyperactivated in cells with overexpressed HEF1.
3] HEF1 conditions TGF-β responses
One of the enigmas of tumor progression is how tumor interpretation of TGF-β signals modulates over time. Extrinsic TGF-β inhibits the growth of early tumors; however, TGF-β promotes the growth of later stage invasive tumors (17
), downregulating E-cadherin and promoting mesenchymal transformation. Intriguingly, HEF1 binds directly to TGF-β pathway effectors and inhibitors, including multiple SMADs (e.g., (18
), and discussed in (6
)). Via these interactions, HEF1 induces negative feedback for aspects of TGF-β-dependent signaling. Intriguingly, the TGF-β pathway signaling molecule SMAD7 has very recently been shown to inhibit melanoma metastasis to bone (19
); HEF1 overexpression would be predicted to limit SMAD7 activity, thus promoting metastasis.
4] HEF1 activates RhoA and Aurora A: counter-pressures for tumor growth
Given the extensive biology linking HEF1 to invasion signaling pathways, recent observations that this protein also regulates cell cycle progression through mitosis were unexpected (6
). In MCF-7 cells and other epithelial cell lines, HEF1 functions at two discrete points during cytokinesis. At the centrosome, HEF1 interacts with and activates Aurora A kinase during mitotic entry. Overexpressed HEF1 hyper-activates Aurora A, inducing failure of cytokinesis. Separately, HEF1 positively regulates RhoA activation and elevated HEF1 expression leads to abnormally persistent RhoA activity throughout cytokinesis, preventing normal cellular reattachment to surrounding matrix, and providing a second stimulus for deficient cytokinesis. HEF1-overexpressing cells exhibiting defective cytokinesis then arrest in G1, and subsequently undergo apoptosis at high frequency, implying the triggering of cell division checkpoints.
The recognition that HEF1 overexpression triggers cell division checkpoints and apoptosis may be particularly important in understanding why HEF1 overexpression is associated with later (rather than early) stages of tumor progression. In the Tyr-rtTA+;Tet-RAS+;Ink4a/Arf−/− melanoma mouse model, both Rb- and p53-dependent cell division checkpoints have been disabled and concurrently constitutive Ras overexpression provides a strong stimulus towards continued proliferation. Such prior changes may be essential for cells to tolerate pro-apoptotic effects of sustained HEF1 overexpression. In this context, it is interesting that early metastatic melanomas are often characterized by genomic rearrangements and aneuploidy, and manifest a high level of apoptosis relative to pre-metastatic tumors.
5] HEF1 and Aurora A regulate ciliary disassembly
Cilia are small organelles that protrude from the surface of many mammalian cell types and act as cellular “antennas”, with growth factor receptors localized at cilia sensing extracellular cues to regulate cell growth. Defects in ciliary structural integrity or associated signaling induce numerous developmental syndromes, are a primary cause of polycystic kidney disease, and have in the past year been strongly linked to cancer development (reviewed in (20
)). Very recently, HEF1 activation of Aurora A at the ciliary basal body was shown to trigger a ciliary resorption pathway involving the tubulin deacetylase HDAC6 as an effector protein (21
). This unexpected finding suggests a totally new mechanism by which overexpression of HEF1 can influence the growth properties of metastatic cancers: much more work is necessary to understand the importance of this observation.