Preclinical and clinical studies of the NEDD9 scaffold protein in cancer and other diseases

Abstract

Cancer progression requires a significant reprogramming of cellular signaling to support the essential tumor-specific processes that include hyperproliferation, invasion (for solid tumors) and survival of metastatic colonies. NEDD9 (also known as CasL and HEF1) encodes a multi-domain scaffolding protein that assembles signaling complexes regulating multiple cellular processes relevant to cancer. These include responsiveness to signals emanating from the T and B cell receptors, integrins, chemokine receptors, and receptor tyrosine kinases, as well as cytoplasmic oncogenes such as BCR-ABL and FAK- and SRC-family kinases. Downstream, NEDD9 regulation of partners including CRKL, WAVE, PI3K/AKT, ERK, E-cadherin, Aurora-A (AURKA), HDAC6, and others allow NEDD9 to influence functions as pleiotropic as migration, invasion, survival, ciliary resorption, and mitosis. In this review, we summarize a growing body of preclinical and clinical data that indicate that while NEDD9 is itself non-oncogenic, changes in expression of NEDD9 (most commonly elevation of expression) are common features of tumors, and directly impact tumor aggressiveness, metastasis, and response to at least some targeted agents inhibiting NEDD9-interacting proteins. These data strongly support the relevance of further development of NEDD9 as a biomarker for therapeutic resistance. Finally, we briefly discuss emerging evidence supporting involvement of NEDD9 in additional pathological conditions, including stroke and polycystic kidney disease.

Keywords: CAS family protein; CAS-L; HEF1; Human malignancy; NEDD9; Prognosis; Protein function; Signal transduction.

Figure 1. NEDD9 gene and protein

A. Schematic representation of NEDD9 gene (transcript variant NM_006403, 7 exons) and mRNA. Relative sizes of promoter, exons and introns are not to scale. Inside the red arrow are factors inducing transcription of NEDD9, with upstream regulatory factors indicated to right. The RNA B2 homology region begins 10 bp upstream of exon 1, and ends at the intron between exons 4 and 5 of NEDD9. The intronic region between exons 6 and 7 contains a C>G SNP (rs760678), discussed in text. A miR-145 binding region is located at 3′UTR of the NEDD9 mRNA. The encoded protein is 834 aa in humans, arising from 7 coding exons (pale blue boxes): numbers of amino acids encoded by each exon are indicated within boxes, with functionally defined domains encoded by exons indicated below. B. Domain structure and upstream regulators of NEDD9 protein. SH3 - N-terminal SH3 domain, SD - substrate domain, SRR - serine rich region, FAT - focal adhesion targeting domain. The T-cell and B-cell receptors (TCR and BCR), integrins and chemokine receptors [108] provide upstream activation signals. In T cells and B cells, this process requires NEDD9 interaction with NSP family scaffold proteins via the NEDD9 C-terminal domain [111, 112]. NEDD9 coordinates signaling between integrins and RTKs such as EGFR through interactions with NSP proteins [111, 112] and the adaptor proteins SHC and GRB2 (reviewed in [27, 113]). Subsequent activation of FAK, SRC and ABL-family kinase (ABL1, ABL2) causes extensive phosphorylation of NEDD9 substrate domain that creates multiple binding sites for downstream effectors; among these sites, Y189, Y317, and Y279 have been functionally validated [41]. FAK phosphorylation of the DYDY (amino acids 628–631) motif in the NEDD9 C-terminus creates a binding site for SRC kinase, and is important for NEDD9 function in migration and dother signaling functions [114]{Tachibana, 1997 #193}{Iwata, 2005 #103}{Kondo, 2012 #79}. Y189 phosphorylation is implicated in focal adhesion; this is a proposed phosphorylation site for FAK and SRC kinases [114]. Phosphorylation of S296 by Aurora-A kinase is implicated in NEDD9 proteasomal degradation [115], cell spreading [116] and cell cycle control [30]. NEDD9 potentiates TGFβ signaling by recruiting the inhibitory SMAD6 and SMAD7 proteins, and preventing their interaction with TGFβ [63]. TGFβ induces NEDD9 mRNA [97]; conversely, the TGFβ receptor effector SMAD3 mediates NEDD9 degradation via the APC complex [34, 35]. ABL – Abelson murine leukemia viral oncogene homolog; AURKA – Aurora kinase A; RAFTK – protein tyrosine kinase 2 beta (PYK2, PTK2B); FAK – focal adhesion kinase; SRC – (short for sarcoma), proto-oncogene non-receptor tyrosine kinase Src; SHC1 – SH2-domain containing transforming protein; GRB2 – growth factor receptor-bound protein 2; SMAD – homolog of C. Elegans protein SMA (from gene sma for small body size) and Drosophila protein MAD (mothers against decapentaplegic); APC/C – anaphase-promoting complex/cyclosome.

Figure 2. Signaling by NEDD9 relevant to the pathogenesis of BCR-ABL positive malignancies

NEDD9 co-localizes and interacts with BCR-ABL and its adaptor protein CRKL [49, 51], likely serving as a hub [50] for an assembly of protein complexes between CRKL and its downstream effectors: SYK [53], C3G [42], BRAF, SAPK, JNK [47], and STAT5 [52]. SRC family kinase Syk induces the PI3K/AKT/MTOR pathway enhancing cell survival [117]; activation of the RAS/RAF/MEK/ERK pathway drives cell proliferation; transcription factors STAT5 and JNK activate transcription of a large number of target genes [47]. Signaling through B-cell receptor, chemokine receptor and/or integrins lead to NEDD9 activation by the FAK/SRC/PXN complex; active NEDD9, in turn, helps to support continuous activation of FAK and SRC [56]. Phosphorylated NEDD9 binds CRKL and promotes migration and chemotaxis through activation of WASP/ARP2/3 signaling (reviewed in [4, 54]). ABL-mediated tyrosine phosphorylation of NEDD9 and binding of C3G leads to activation of Rap1 and migration/chemotaxis of lymphoid cells [41]. ARP2/3 - protein complex regulating actin cytoskeleton; WASP - Wiskott-Aldrich syndrome family protein; CRKL - adaptor protein encoded by V-crk avian sarcoma virus CT10 oncogene homolog; PXN – paxillin; FAK – focal adhesion kinase; SRC – (short for sarcoma), proto-oncogene non-receptor tyrosine kinase Src; RAF – family of serine/threonine protein kinases controlling cell proliferation; activation of RAF kinases requires interaction with RAS-GTPases; SYK - spleen tyrosine kinase; JNK – c-Jun N-terminal kinase; SAPK – stress-activated protein kinase; STAT – signal transducers and activators of transcription.

Figure 3. NEDD9 signaling mechanisms analyzed in breast cancer models

NEDD9 is activated by FAK and SRC kinases, and also helps support continuous activation of these kinases [56]. NEDD9 mediates TGFβ effects through a positive feedback loop [61]. Transcription factors SRF (serum response factor) and SMAD induce NEDD9 expression; NEDD9 scaffolding connects TGFβ/SMAD and Rho-actin-SRF signals to coordinate the expression of genes involved in tumorigenesis [61]. NEDD9 binding to the DOCK3 GEF activates RAC, which signals through WAVE/SCAR to initiate ARP2/3-dependent actin branching, lamellipodia formation, and MMP activation [69]. NEDD9 and SRC negatively regulate expression of E-cadherin through upregulation of the SLUG and SNAIL transcription factors [60], and also by regulating SRC-dependent removal of E-cadherin from adherens junctions [68]. NEDD9 promotes trafficking of ligand-bound integrins from early to late endosomes and degradation of ligand/integrin complexes by inactivating caveolin (CAV1), which mediates recycling of ligand-bound integrins from early endosomes back to the plasma membrane [65]. NEDD9 regulates turnover of MMP14 and its enzymatic recovery through the late endosomes by suppressing Arf6 dependent trafficking of MMP14/TIMP2 complexes from early/sorting endosomes back to the surface. MMP14 cleaves pro-MMP2 to produce active MMP2, degrading type IV collagen of basal membrane and promoting invasion [66]. FAK – focal adhesion kinase; SRC – (short for sarcoma), proto-oncogene non-receptor tyrosine kinase Src; SRF - Serum response factor; SMAD – homolog of C. Elegans protein SMA (from gene sma for small body size) and Drosophila protein MAD (mothers against decapentaplegic); DOCK3 – Dedicator of cytokinesis 3; SLUG, SNAIL – members of SNAIL family zinc finger transcription factors; ARP2/3 – protein complex regulating actin cytoskeleton; WAVE member of the Wiskott-Aldrich syndrome family protein, activates ARP2/3 complex; MMP - matrix metalloproteinase TIMP2 – metallopeptidase inhibitor 2; ARF6 – ADP-ribosylation factor 6; CAV1 – caveolin 1.

Figure 4. Interaction of NEDD9 with Aurora-A (AURKA) mediates resorption of cilia and mitosis

In G1 or G0 cells, transient NEDD9 induction in response to serum, Ca2+-bound calmodulin (CaM), or serum-derived growth factors allow NEDD9-dependent activation of AURKA kinase [31, 106, 107]. This induces resorption of the primary cilium, based in part on AURKA phosphorylation and activation of HDAC6, which deacetylates tubulin in the ciliary axoneme [31]. In mitosis, NEDD9 colocalizes with and binds Aurora-A and CaM at the centrosomes: these interactions support Aurora-A activation. Later in mitosis, NEDD9 and CaM move with Aurora-A to the spindle, and NEDD9 protects AURKA from degradation [37]. AURKA – Aurora kinase A, HDAC6 - histone deacetylase 6.