Misregulation of the canonical Wnt/β-catenin pathway and aberrant activation of Wnt signaling target genes are common in colorectal cancer and contribute to cancer progression. Altered expression of HEF1 (Human Enhancer of Filamentation 1, also known as NEDD9 or Cas-L) has been implicated in progression of melanoma, breast, and colorectal cancer. However, the regulation of HEF1 and the role of HEF1 in colorectal cancer tumorigenesis are not fully understood. We here identify HEF1 as a novel Wnt signaling target. The expression of HEF1 was up-regulated by Wnt3a, β-catenin, and Dvl2 in a dose-dependent fashion, and was suppressed following β-catenin down-regulation by shRNA. In addition, elevated HEF1 mRNA and protein levels were observed in colorectal cancer cell lines and primary tumor tissues, as well as in the colon and adenoma polyps of Apcmin/+ mice. Moreover, HEF1 levels in human colorectal tumor tissues increased with the tumor grade. Chromatin immunoprecipitation (ChIP) assays and HEF1 promoter analyses revealed three functional TCF-binding sites in the promoter of HEF1 responsible for HEF1 induction by Wnt signaling. Ectopic expression of HEF1 increased cell proliferation and colony formation, while down-regulation of HEF1 in SW480 cells by shRNA had the opposite effects and inhibited the xenograft tumor growth. Furthermore, overexpression of HEF1 in SW480 cells promoted cell migration and invasion. Together, our results determined a novel role of HEF1 as a mediator of the canonical Wnt/β-catenin signaling pathway for cell proliferation, migration, and tumorigenesis, as well as an important player in colorectal tumorigenesis and progression. HEF1 may represent an attractive candidate for drug targeting in colorectal cancer.
HEF1; Wnt signaling; colorectal cancer; tumorigenesis
Human enhancer of filamentation 1 (HEF1, also known as NEDD9 or Cas-L) is a scaffolding protein that is implicated in regulating diverse cellular processes, such as cellular attachment, motility, cell cycle progression, apoptosis and inflammation. Here, we identify HEF1 as a novel hypoxia-inducible factor-1α (HIF-1α) regulated gene and reveal that HEF1 mediates hypoxia-induced migration of colorectal carcinoma cells. HEF1 is highly expressed in cultured colorectal carcinoma cells exposed to hypoxia and in the hypoxic areas of human colorectal cancer specimens. Moreover, our data demonstrates that HIF-1α mediates the effects of hypoxia on induction of HEF1 expression via binding to a hypoxia-responsive element (HRE) of the HEF1 promoter in CRC cells. Importantly, the induction of HEF1 expression significantly enhances hypoxia-stimulated HIF-1α transcriptional activity by modulating the interaction between HIF-1α and its transcriptional cofactor p300. Inhibition of HEF1 expression also reduced the levels of hypoxia-inducible genes, including those that regulate cell motility. Cell migration was reduced dramatically following knockdown of HEF1 expression under hypoxic conditions. Thus, this positive feedback loop may contribute to adaptive responses of carcinoma cells in encountering hypoxia during cancer progression.
Human enhancer of filamentation 1 (HEF1); hypoxia; HIF-1α; colorectal cancer; carcinoma cell migration
Upregulation of the scaffolding protein HEF1, also known as NEDD9 and Cas-L, has recently been identified as a pro-metastatic stimulus in a number of different solid tumors, and has also been strongly associated with pathogenesis of BCR-Abl-dependent tumors. As the evidence mounts for HEF1/NEDD9/Cas-L as a key player in metastatic cancer, it is timely to review the molecular regulation of HEF1/NEDD9/Cas-L. Most of the mortality associated with cancer arises from uncontrolled metastases, thus a better understanding of the properties of proteins specifically associated with promotion of this process may yield insights that improve cancer diagnosis and treatment. In this review, we summarize the extensive literature regarding HEF1/NEDD9/CAS-L expression and function in signaling relevant to cell attachment, migration, invasion; cell cycle; apoptosis; and oncogenic signal transduction. The complex function of HEF1/NEDD9/CAS-L revealed by this analysis leads us to propose a model in which alleviation of cell cycle checkpoints and acquired resistance to apoptosis is permissive for a HEF1/NEDD9/CAS-L-promoted pro-metastatic phenotype.
HEF1/NEDD9/CAS-L; NEDD9; Cas-L; metastasis; scaffolding adaptor protein; invasion; mitosis; apoptosis; signal transduction
For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.
HEF1 (human enhancer of filamentation 1) is a member of a docking protein family that includes p130Cas and Efs. Through assembly of multiple protein interactions at focal adhesion sites, these proteins activate signaling cascades in response to integrin receptor binding of the extracellular matrix. The HEF1 protein is cell cycle regulated, with full-length forms cleaved in mitosis at a caspase consensus site to generate an amino-terminal 55-kDa form that localizes to the mitotic spindle. The identification of a caspase cleavage site in HEF1 led us to investigate whether HEF1 belongs to a select group of caspase substrates cleaved in apoptosis to promote the morphological changes characteristic of programmed cell death. Significantly, inducing expression of HEF1 in MCF-7 or HeLa cells causes extensive apoptosis, as assessed by multiple criteria. Endogenous HEF1 is cleaved into 65- and 55-kDa fragments and a newly detected 28-kDa form in response to the induction of apoptosis, paralleling cleavage of poly(ADP-ribose) polymerase and focal adhesion kinase (FAK); the death-promoting activity of over-expressed HEF1 is associated with production of the 28-kDa form. While the generation of the cleaved HEF1 forms is caspase dependent, the accumulation of HEF1 forms is further regulated by the proteasome, as the proteasome inhibitors N-acetyl-l-leucinyl-l-leucinyl-l-norleucinyl and lactacystin enhance their stability. Finally, the induction of HEF1 expression also increases Jun N-terminal protein kinase (JNK) activation, and activated JNK colocalizes with HEF1, implicating this pathway in HEF1 action. Based on these results, we propose that dysregulation of HEF1 and its family members along with FAK may signal the destruction of focal adhesion sites and regulate the onset of apoptosis.
HEF1, p130Cas, and Efs/Sin constitute a family of multidomain docking proteins that have been implicated in coordinating the regulation of cell adhesion. Each of these proteins contains an SH3 domain, conferring association with focal adhesion kinase; a domain rich in SH2-binding sites, phosphorylated by or associating with a number of oncoproteins, including Abl, Crk, Fyn, and others; and a highly conserved carboxy-terminal domain. In this report, we show that the HEF1 protein is processed in a complex manner, with transfection of a single cDNA resulting in the generation of at least four protein species, p115HEF1, p105HEF1, p65HEF1, and p55HEF1. We show that p115HEF1 and p105HEF1 are different phosphorylation states of the full-length HEF1. p55HEF1, however, encompasses only the amino-terminal end of the HEF1 coding sequence and arises via cleavage of full-length HEF1 at a caspase consensus site. We find that HEF1 proteins are abundantly expressed in epithelial cells derived from breast and lung tissue in addition to the lymphoid cells in which they have been predominantly studied to date. In MCF-7 cells, we find that expression of the endogenous HEF1 proteins is cell cycle regulated, with p105HEF1 and p115HEF1 being rapidly upregulated upon induction of cell growth, whereas p55HEF1 is produced specifically at mitosis. While p105HEF1 and p115HEF1 are predominantly cytoplasmic and localize to focal adhesions, p55HEF1 unexpectedly is shown to associate with the mitotic spindle. In support of a role at the spindle, two-hybrid library screening with HEF1 identifies the human homolog of the G2/M spindle-regulatory protein Dim1p as a specific interactor with a region of HEF1 encompassed in p55HEF1. In sum, these data suggest that HEF1 may directly connect morphological control-related signals with cell cycle regulation and thus play a role in pathways leading to the progression of cancer.
Epithelial to mesenchymal transition (EMT) plays an important role in many biological processes. The latest studies revealed that aggressive breast cancer, especially the triple-negative breast cancer (TNBC) subtype was frequently associated with apparent EMT, but the mechanisms are still unclear. NEDD9/HEF1/Cas-L is a member of the Cas protein family and was identified as a metastasis marker in multiple cancer types. In this study, we wished to discern the role of NEDD9 in breast cancer progression and to investigate the molecular mechanism by which NEDD9 regulates EMT and promotes invasion in triple-negative breast cancer. We showed that expression of NEDD9 was frequently upregulated in TNBC cell lines, and in aggressive breast tumors, especially in TNBC subtype. Knockdown of endogenous NEDD9 reduced the migration, invasion and proliferation of TNBC cells. Moreover, ectopic overexpression of NEDD9 in mammary epithelial cells led to a string of events including the trigger of EMT, activation of ERK signaling, increase of several EMT-inducing transcription factors and promotion of their interactions with the E-cadherin promoter. Data presented in this report contribute to the understanding of the mechanisms by which NEDD9 promotes EMT, and provide useful clues to the evaluation of the potential of NEDD9 as a responsive molecular target for TNBC chemotherapy.
The focal adhesion docking protein NEDD9/HEF1/Cas-L regulates cell migration and cancer invasion. NEDD9 is a member of the Cas family of proteins that share conserved overall protein-protein interaction domain structure, including a substrate domain that is characterized by extensive tyrosine (Y) phosphorylation. Previous studies have suggested that phosphorylation of Y253 in the substrate domain of the Cas family protein p130Cas is specifically required for p130Cas function in cell migration. While it is clear that tyrosine phosphorylation of the NEDD9 substrate domain is similarly required for the regulation of cell motility, whether individual NEDD9 tyrosine residues have discrete function in regulating motility has not previously been reported. In the present study we have used a global sequence alignment of Cas family proteins to identify a putative NEDD9 equivalent of p130Cas Y253. We find that NEDD9 Y189 aligns with p130Cas Y253 and that it is conserved among NEDD9 vertebrate orthologues. Expression of NEDD9 in which Y189 is mutated to phenylalanine results in increased rates of cell migration and is correlated with increased disassembly of GFP.NEDD9 focal adhesions. Conversely, mutation to Y189D significantly inhibits cell migration. Our previous data has suggested that NEDD9 stabilizes focal adhesions and the present data therefore suggests that phosphorylation of Y189 NEDD9 is required for this function. These findings indicate that the individual tyrosine residues of the NEDD9 substrate domain may serve discrete functional roles. Given the important role of this protein in promoting cancer invasion, greater understanding of the function of the individual tyrosine residues is important for the future design of approaches to target NEDD9 to arrest cancer cell invasion.
Chemokine signaling is critical for T cell function during homeostasis and inflammation, and directs T cell polarity and migration through the activation of specific intracellular pathways. Here, we uncovered a previously uncharacterized role for the Abl family tyrosine kinases Abl and Arg in the regulation of T cell-dependent inflammatory responses, and we showed that the Abl kinases were required for chemokine-induced T cell polarization and migration. Our data demonstrated that Abl and Arg were activated downstream of chemokine receptors and controlled the chemokine-induced tyrosine phosphoylation of human enhancer of filamentation 1(HEF1), an adaptor protein that modulates the activity of the guanosine triphosphatase (GTPase) Rap1. Furthermore, Abl-mediated tyrosine phosphorylation of HEF1 and activation of Rap1 were required for chemokine-induced T cell migration. T cells isolated from conditional knockout mice lacking Abl and Arg exhibited defective homing to lymph nodes and impaired migration to sites of inflammation. These findings suggest that Abl family kinases are potential therapeutic targets for the treatment of T cell–dependent immune disorders that are characterized by chemokine-mediated inflammation.
Former vascular endothelial growth factor (VEGF) - head and neck squamous cell carcinoma (HNSCC) studies have focused on VEGF’s contributions toward tumor-associated angiogenesis. Previously, we have shown that HNSCC cells produce high levels of VEGF. We therefore hypothesized that VEGF serves a biphasic role i.e. proangiogenic and protumorigenic in HNSCC pathogenesis. Western blots confirmed the presence of VEGF’s primary mitogenic receptors, VEGFR-2/KDR and VEGFR-1/Flt-1 in cultured HNSCC cells. Subsequent studies evaluated VEGF’s effects on HNSCC intracellular signaling, mitogenesis, invasive capacities and matrix metalloproteinases (MMPs) activities. Introduction of hrVEGF165 initiated ROS-mediated intracellular signaling, resulting in kinase activation and phosphorylation of KDR and Erk1/2. As high endogenous VEGF production rendered HNSCC cells refractory to exogenous VEGF’s mitogenic effects, siRNA was employed, inhibiting endogenous VEGF production for up to 96h. Relative to transfection vector matched controls, siRNA treated HNSCC cells showed a significant decrease in proliferation at both 30nM and 50nM siRNA doses. Addition of exogenous hrVEGF165 (30ng/ml and 50ng/ml) to siRNA-silenced HNSCC cells resulted in dose-dependent increases in cell proliferation. Cell invasion assays showed VEGF is a potent HNSCC chemoattractant and demonstrated that VEGF pretreatment enhanced invasiveness of HNSCC cells. Conditioned media from VEGF challenged HNSCC cells showed a moderate increase in gelatinase activity. Our results demonstrate, for the first time, that HNSCC cells are both targets and effectors for VEGF. These data introduce the prospect that VEGF targeted therapy has the potential to fulfill both anti-angiogenic and anti-tumorigenic functions.
VEGF; KDR; Flt-1; Head and neck squamous cell carcinoma; intracellular signaling
Mammalian Cas proteins regulate cell migration, division and survival, and are often deregulated in cancer. However, the presence of four paralogous Cas family members in mammals (BCAR1/p130Cas, EFS/Sin1, NEDD9/HEF1/Cas-L, and CASS4/HEPL) has limited their analysis in development. We deleted the single Drosophila Cas gene, Dcas, to probe the developmental function of Dcas. Loss of Dcas had limited effect on embryonal development. However, we found that Dcas is an important modulator of the severity of the developmental phenotypes of mutations affecting integrins (If and mew) and their downstream effectors Fak56D or Src42A. Strikingly, embryonic lethal Fak56D-Dcas double mutant embryos had extensive cell polarity defects, including mislocalization and reduced expression of E-cadherin. Further genetic analysis established that loss of Dcas modified the embryonal lethal phenotypes of embryos with mutations in E-cadherin (Shg) or its signaling partners p120- and β-catenin (Arm). These results support an important role for Cas proteins in cell-cell adhesion signaling in development.
Proteins of the CAS (Crk-Associated Substrate) family (BCAR1/p130Cas, NEDD9/HEF1/Cas-L, EFS/SIN and CASS4/HEPL) are integral players in normal and pathological cell biology. CAS proteins act as scaffolds to regulate protein complexes controlling migration and chemotaxis, apoptosis, cell cycle, and differentiation, and have more recently been linked to a role in progenitor cell function. Reflecting these complex functions, over-expression of CAS proteins has now been strongly linked to poor prognosis and increased metastasis in cancer, as well as resistance to first-line chemotherapeutics in multiple tumor types including breast and lung cancers, glioblastoma, and melanoma. Further, CAS proteins have also been linked to additional pathological conditions including inflammatory disorders, Alzheimer’s and Parkinson’s disease, as well as developmental defects. This review will explore the roles of the CAS proteins in normal and pathological states in the context of the many mechanistic insights into CAS protein function that have emerged in the past decade.
CAS; BCAR1; NEDD9; scaffold; cancer; invasion; metastasis; mitosis
Budding in Saccharomyces cerevisiae follows a genetically programmed pattern of cell division which can be regulated by external signals. On the basis of the known functional conservation between a number of mammalian oncogenes and antioncogenes with genes in the yeast budding pathway, we used enhancement of pseudohyphal budding in S. cerevisiae by human proteins expressed from a HeLa cDNA library as a morphological screen to identify candidate genes that coordinate cellular signaling and morphology. In this report, we describe the isolation and characterization of human enhancer of filamentation 1 (HEF1), an SH3-domain-containing protein that is similar in structure to pl30cas, a recently identified docking protein that is a substrate for phosphorylation by a number of oncogenic tyrosine kinases. In contrast to p130cas, the expression of HEF1 appears to be tissue specific. Further, whereas p130cas is localized predominantly at focal adhesions, immunofluorescence indicates that HEF1 localizes to both the cell periphery and the cell nucleus and is differently localized in fibroblasts and epithelial cells, suggesting a more complex role in cell signalling. Through immunoprecipitation and two-hybrid analysis, we demonstrate a direct physical interaction between HEF1 and p130cas, as well as an interaction of the SH3 domain of HEF1 with two discrete proline-rich regions of focal adhesion kinase. Finally, we demonstrate that as with p130cas, transformation with the oncogene v-abl results in an increase in tyrosine phosphorylation on HEF1, mediated by a direct association between HEF1 and v-Abl. We anticipate that HEF1 may prove to be an important linking element between extracellular signalling and regulation of the cytoskeleton.
The Cas scaffolding proteins (NEDD9/HEF1/CAS-L, BCAR1/p130Cas, EFSSIN, and HEPL/CASS4) regulate cell migration, division and survival, and are often deregulated in cancer. High BCAR1 expression is linked to poor prognosis in breast cancer patients, while upregulation of NEDD9 contributes to the metastatic behavior of melanoma and glioblastoma cells. Our recent work knocking out the single Drosophila Cas protein, Dcas, identified a genetic interaction with E-cadherin. As E-cadherin is often downregulated during epithelial-mesenchymal transition (EMT) prior to metastasis, if such an activity was conserved in mammals it might partially explain how Cas proteins promote aggressive tumor behavior. We here establish that Cas proteins negatively regulate E-cadherin expression in human mammary cells. Cas proteins do not affect E-cadherin transcription, but rather, BCAR1 and NEDD9 signal through SRC to promote E-cadherin removal from the cell membrane and lysosomal degradation. We also find mammary tumors arising in MMTV-polyoma virus T-antigen mice have enhanced junctional E-cadherin in a Nedd9−/− background. Cumulatively, these results suggest a new role for Cas proteins in cell-cell adhesion signaling in cancer.
The dynamic regulation of focal adhesions is implicated in cellular processes of proliferation, differentiation, migration, and apoptosis. The focal adhesion-associated docking protein HEF1 is cleaved by caspases during both mitosis and apoptosis. Common to both of these cellular processes is the loss of focal adhesions, transiently during mitosis and permanently during apoptosis. The proteolytic processing of HEF1 during both mitosis and apoptosis therefore posits a general role for HEF1 as a sensor of altered adhesion states. In this study, we find that HEF1 undergoes proteolytic processing specifically in response to cellular detachment, while HEF1 proteolysis is prevented by specific integrin receptor ligation and focal adhesion formation. We show that overexpression of a C-terminal caspase-derived 28-kDa HEF1 peptide causes cellular rounding that is demonstrably separable from apoptosis. Mutation of the divergent helix-loop-helix motif found in 28-kDa HEF1 significantly reduces the induction of apoptosis by this peptide, while deletion of the amino-terminal 28 amino acids of 28-kDa HEF1 completely abrogates the induction of apoptosis. Conversely, these mutations have no effect on the rounding induced by 28-kDa HEF1. Finally, we detect a novel focal adhesion targeting domain located in the C terminus of HEF1 and show that this activity is necessary for HEF1-induced cell spreading. Together, these data suggest that proteolytic and other posttranslational modifications of HEF1 in response to loss of adhesion serve to modulate the disassembly of focal adhesions.
The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events.
Elevated expression of cyclooxygenase-2 (COX-2) and one of its downstream enzymatic products, prostaglandin E2 (PGE2) have been directly linked to colorectal carcinogenesis in a number of ways. Among which, PGE2 promotes cell proliferation, cell cycle progression, and thus tumor growth. All of the mechanism(s) by which PGE2 signaling regulates cell growth are not completely understood. Here, we demonstrate that PGE2 treatment induces human enhancer of filamentation 1 (HEF1) expression and its link with cell cycle machinery in colorectal cancer cells. PGE2 rapidly stimulated the expression of HEF1 mRNA and protein in colorectal cancer cells. Both PGE2 treatment and HEF1 overexpression resulted in similar effects on cell proliferation, cell cycle progression, and tumor growth. Moreover, knockdown of HEF1 using shRNA suppressed PGE2-driven cell proliferation and cell cycle progression. Cell cycle alterations involved HEF1 fragmentation as well as co-distribution of HEF1 and Aurora A along spindle asters during cell division. Furthermore, HEF1 co-immunoprecipitated with and activated Aurora A. Intriguingly, HEF1 expression was increased in 50% of human colorectal cancers compared with expression in paired normal tissue. These data suggest that PGE2 induces HEF1 expression, which in turn promotes cell cycle progression through its interaction and activation of Aurora A. Clearly, HEF1 is a downstream mediator of PGE2 action during colorectal carcinogenesis.
PGE2; HEF1/NEDD9/Cas-L; colorectal cancer; cell proliferation; cell cycle
The effect of vascular endothelial growth factor (VEGF) ligands and cediranib on tumor cell proliferation, migration, and invasion was determined. It has recently been suggested that autocrine signaling through the VEGF receptor (VEGFR) pathway may play a role in tumor cell survival, invasion, and migration. The purpose of the present study was to determine the expression of VEGFRs and VEGFR ligands in a panel of gastrointestinal carcinoma cells. Additionally, we evaluated the effects of VEGF autocrine signaling on tumor cell proliferation, migration, and invasion utilizing cediranib (AZD2171), a pan-VEGFR inhibitor. Five colorectal, three pancreatic, and two hepatocellular carcinoma cell lines were screened for VEGFR and VEGF expression by several methods. Expression of VEGFR-1 and VEGFR-3 was cell line–dependent, whereas VEGFR-2 was not detected. Secretion of VEGF-A was detected in the supernatants of all cell lines whereas VEGF-C secretion was detected in the Panc-1,MiaPaca2, and Hep1 cells only. Tumor cells showed increased migratory activity, but not proliferation, when stimulated with VEGFs. The pan-VEGFR inhibitor cediranib (100 nmol/L) inhibited tumor cell migration and invasion, with no effects on proliferation. Cediranib decreased VEGFR-1 and VEGFR-3 phosphorylation as well as activation of downstream effectors. VEGFR-1 and VEGFR-3 expression was detected in all the gastrointestinal carcinoma cells evaluated. Although activation of the VEGF pathway did not affect cell proliferation, our data indicate that this pathway seems to play a role in tumor cell migration and invasion in these cell lines. Therefore, inhibition of VEGFR by cediranib may represent a clinically relevant treatment option for gastrointestinal tumors.
The multi-functional adaptor protein NEDD9/HEF1/Cas-L regulates cell motility, invasion and cell cycle progression, and plays key roles in cancer progression and metastasis. NEDD9 is localized to the centrosome and is required for activation of Aurora A kinase in mitosis. Here we demonstrate that the HECT-WW protein Smurf2 physically associates with NEDD9 and is required for the stability of NEDD9 protein. Smurf2 depletion results in a marked decrease in NEDD9 protein levels, by facilitating polyubiquitination and proteasomal degradation of NEDD9. Conversely, forced overexpression of Smurf2 results in upregulation of endogenous NEDD9 protein, confirming the role for Smurf2 in NEDD9 stability. Cells with Smurf2 depletion fail to activate Aurora A at the G2/M boundary, leading to a marked delay in mitotic entry. These observations suggest that the stable complex of Smurf2 and NEDD9 is required for timely entry into mitosis via Aurora A activation.
Elevated Src kinase activity is linked to the progression of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Src regulates HNSCC proliferation and tumor invasion, with the Src-targeted small molecule inhibitor saracatinib displaying potent anti-invasive effects in preclinical studies. However, the pro-invasive cellular mechanism(s) perturbed by saracatinib are unclear. The anti-proliferative and anti-invasive effects of saracatinib on HNSCC cell lines were therefore investigated in pre-clinical cell and mouse model systems. Saracatinib treatment inhibited growth, cell cycle progression and transwell Matrigel invasion in HNSCC cell lines. Dose-dependent decreases in Src activation and phosphorylation of the invasion-associated substrates focal adhesion kinase, p130 CAS and cortactin were also observed. While saracatinib did not significantly impact HNSCC tumor growth in a mouse orthotopic model of tongue squamous cell carcinoma, impaired perineural invasion and cervical lymph node metastasis was observed. Accordingly, saracatinib treatment displayed a dose-dependent inhibitory effect on invadopodia formation, extracellular matrix degradation and matrix metalloprotease 9 activation. These results suggest that inhibition of Src kinase by saracatinib impairs the pro-invasive activity of HNSCC by inhibiting Src substrate phosphorylation important for invadopodia formation and associated matrix metalloprotease activity.
Saracatinib; Src; Head and Neck cancer; Invadopodia; Invasion; MMP
Although HEF1 has a well-defined role in integrin-dependent attachment signaling at focal adhesions, it relocalizes to the spindle asters at mitosis. We report here that overexpression of HEF1 causes increase in centrosome numbers and multipolar spindles, resembling defects induced by manipulation of the mitotic regulatory kinase Aurora A (AurA). We show that HEF1 associates with and controls activation of AurA. We also show HEF1 depletion causes centrosomal splitting, monoastral spindles, and hyperactivation of Nek2, implying additional action earlier in cell cycle. These results provide new insights into the role of an adhesion protein in coordination of cell attachment and division.
HEF1; centrosome; spindle; mitosis; Aurora-A; Nek2
Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells. We show that depletion of FAK induces the formation of active invadopodia but impairs invasive cell migration. FAK-deficient MTLn3 breast cancer cells display enhanced assembly and dynamics of invadopodia that are rescued by expression of wild-type FAK but not by FAK that cannot be phosphorylated at tyrosine 397. Moreover, our findings demonstrate that FAK depletion switches phosphotyrosine-containing proteins from focal adhesions to invadopodia through the temporal and spatial regulation of c-Src activity. Collectively, our findings provide novel insight into the interplay between FAK and Src to promote invasion.
During the last few years, several studies have pointed to a surprising link between environmental pollutants cellular signaling and important cell functions such as plasticity, adhesion and migration. This unexpected link could be related to endogenous functions of pollutants receptors that may be disrupted by environmental factors, which is supported by observations in invertebrate species. It could also reveal novel toxic end-points and mechanisms of those pollutants, such as teratogenesis and cancer metastasis that are highly relevant from a public health point of view. In the present short article, we will review our recent observations on the aryl hydrocarbon receptor and its new molecular and cellular targets. We identified HEF1/NEDD9/CAS-L, a multifunctional protein involved in integrin-based signaling as a transcriptional target of the receptor, and showed that its induction was critical for cell plasticity mediated by environmental pollutants. We will put our studies in perspective with other observations made by several groups.
aryl hydrocarbon receptor; toxicity; environmental pollutants; dioxins; aromatic hydrocarbons; Hef1/Nedd9/Cas-L; tumor progression; cell migration; development
The hemagglutinin, esterase, and fusion (HEF) glycoprotein of influenza C virus possesses receptor binding, receptor destroying, and membrane fusion activities. The HEF cDNAs from influenza C/Ann Arbor/1/50 (HEF-AA) and influenza C/Taylor/1223/47 (HEF-Tay) viruses were cloned and expressed, and transport of HEF to the cell surface was monitored by susceptibility to cleavage by exogenous trypsin, indirect immunofluorescence microscopy, and flow cytometry. Previously it has been found in studies with the C/Johannesburg/1/66 strain of influenza C virus (HEF-JHB) that transport of HEF to the cell surface is severely inhibited, and it is thought that the short cytoplasmic tail, Arg-Thr-Lys, is involved in blocking HEF cell surface expression (F. Oeffner, H.-D. Klenk, and G. Herrler, J. Gen. Virol. 80:363–369, 1999). As the cytoplasmic tail amino acid sequences of HEF-AA and HEF-Tay are identical to that of HEF-JHB, the data indicate that cell surface expression of HEF-AA and HEF-Tay is not inhibited by this amino acid sequence. Furthermore, the abundant cell surface transport of HEF-AA and HEF-Tay indicates that their cell surface expression does not require coexpression of another viral protein. The HEF-AA and HEF-Tay HEF glycoproteins bound human erythrocytes, promoted membrane fusion in a low-pH and trypsin-dependent manner, and displayed esterase activity, indicating that the HEF glycoprotein alone mediates all three known functions at the cell surface.
Greater understanding of metastasis is required to improve cancer treatment outcomes. Recently, changes in expression of the scaffold protein HEF1/CAS-L/NEDD9 were found to be a potent pro-metastatic stimulus in melanoma and other cancers. Mechanistic studies suggest diverse cellular roles of HEF1 and highlight its importance in the response to extracellular cues that drive invasion and metastasis. As a metastatic “hub” for signaling in cancer, HEF1 may provide a useful target for drug discovery efforts.