Ack1 Expression Research Articles

Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer.

Androgen receptor (AR) antagonism is exacerbated by HOXB13 in castration-resistant prostate cancers (CRPC). However, it is unclear when and how HOXB13 primes CRPCs for AR antagonism. By mass-spectrometry analysis of CRPC extract, we uncovered a novel lysine 13 (K13) acetylation in HOXB13 mediated by CBP/p300. To determine whether acetylated K13-HOXB13 is a clinical biomarker of CRPC development, we characterized its role in prostate cancer biology. We identified tumor-specific acK13-HOXB13 signal enriched super enhancer (SE)-regulated targets. We analyzed the effect of loss of HOXB13K13-acetylation on chromatin binding, SE proximal target gene expression, self-renewal, enzalutamide sensitivity, and CRPC tumor growth by employing isogenic parental and HOXB13K13A mutants. Finally, using primary human prostate organoids, we evaluated whether inhibiting an acK13-HOXB13 target, ACK1, with a selective inhibitor (R)-9b is superior to AR antagonists in inhibiting CRPC growth. acK13-HOXB13 promotes increased expression of lineage (AR, HOXB13), prostate cancer diagnostic (FOLH1), CRPC-promoting (ACK1), and angiogenesis (VEGFA, Angiopoietins) genes early in prostate cancer development by establishing tumor-specific SEs. acK13-HOXB13 recruitment to key SE-regulated targets is insensitive to enzalutamide. ACK1 expression is significantly reduced in the loss of function HOXB13K13A mutant CRPCs. Consequently, HOXB13K13A mutants display reduced self-renewal, increased sensitivity to enzalutamide, and impaired xenograft tumor growth. Primary human prostate tumor organoids expressing HOXB13 are significantly resistant to AR antagonists but sensitive to (R)-9b. In summary, acetylated HOXB13 is a biomarker of clinically significant prostate cancer. Importantly, PSMA-targeting agents and (R)-9b could be new therapeutic modalities to target HOXB13-ACK1 axis regulated prostate cancers.

Chronologically modified androgen receptor in recurrent castration-resistant prostate cancer and its therapeutic targeting.

Resistance to second-generation androgen receptor (AR) antagonists such as enzalutamide is an inevitable consequence in patients with castration-resistant prostate cancer (CRPC). There are no effective therapeutic options for this recurrent disease. The expression of truncated AR variant 7 (AR-V7) has been suggested to be one mechanism of resistance; however, its low frequency in patients with CRPC does not explain the almost universal acquisition of resistance. We noted that the ability of AR to translocate to nucleus in an enzalutamide-rich environment opens up the possibility of a posttranslational modification in AR that is refractory to enzalutamide binding. Chemical proteomics in enzalutamide-resistant CRPC cells revealed acetylation at Lys609 in the zinc finger DNA binding domain of AR (acK609-AR) that not only allowed AR translocation but also galvanized a distinct global transcription program, conferring enzalutamide insensitivity. Mechanistically, acK609-AR was recruited to the AR and ACK1/TNK2 enhancers, up-regulating their transcription. ACK1 kinase-mediated AR Y267 phosphorylation was a prerequisite for AR K609 acetylation, which spawned positive feedback loops at both the transcriptional and posttranslational level that regenerated and sustained high AR and ACK1 expression. Consistent with these findings, oral and subcutaneous treatment with ACK1 small-molecule inhibitor, (R)-9b, not only curbed AR Y267 phosphorylation and subsequent K609 acetylation but also compromised enzalutamide-resistant CRPC xenograft tumor growth in mice. Overall, these data uncover chronological modification events in AR that allows prostate cancer to evolve through progressive stages to reach the resilient recurrent CRPC stage, opening up a therapeutic vulnerability.

ACK1 Contributes to the Pathogenesis of Inflammation and Autoimmunity by Promoting the Activation of TLR Signaling Pathways.

Toll-like receptors (TLRs) are the first line of defense in the immune system, whose activation plays a key role in the pathogenesis of inflammation and autoimmunity. TLRs can activate a variety of immune cells such as macrophages and dendritic cells, which produce proinflammatory cytokines, chemokines, and co-stimulatory molecules that lead to the development of inflammation and autoimmune diseases. As a nonreceptor tyrosine kinase, ACK1 is involved in multiple signaling pathways and physiological processes. However, the roles of ACK1 in the activation of TLR pathways and in the pathogenesis of inflammation and autoimmune diseases have not yet been reported. We found that the expression of ACK1 could be upregulated by TLR pathways in vivo and in vitro. Intriguingly, overexpression of ACK1 significantly promoted the activation of TLR4, TLR7, and TLR9 pathways, while knockdown of ACK1 or the use of the ACK1 inhibitor AIM-100 significantly inhibited the activation of TLR4, TLR7, and TLR9 pathways. In vivo studies showed that the inhibition of ACK1 activity by AIM-100 could significantly protect mice from the TLR4 agonist lipopolysaccharide (LPS)-mediated endotoxin shock and alleviate the condition of imiquimod-mediated lupus-prone mice and MRL/lpr mice. In summary, ACK1 participates in TLR-mediated inflammation and autoimmunity and has great potential in controlling inflammation and alleviating autoimmune diseases.

LncRNA LINC00963 Promotes Tumorigenesis and Radioresistance in Breast Cancer by Sponging miR-324-3p and Inducing ACK1 Expression

Upregulation of long non-coding RNA LINC00963 has been observed in several cancer types. In this study, we analyzed the clinical and biological significance of LINC00963 in breast cancer. The key microRNA (miR) mediating the action of LINC00963 was identified. We show that LINC00963 upregulation is correlated with aggressive parameters of breast cancer. Silencing of LINC00963 suppresses the proliferation and tumorigenesis of breast cancer cells, whereas LINC00963 overexpression exerts an opposite effect. Knockdown of LINC00963 enhances DNA damage and oxidative stress and sensitizes breast cancer cells to radiation. Mechanistically, LINC00963 antagonizes the repressive activity of miR-324-3p on ACK1 expression. Clinically, there is a negative correlation between miR-324-3p and LINC00963 expression in breast cancer tissues. Overexpression of LINC00963 or ACK1 rescues the inhibitory effects of miR-324-3p on breast cancer cell proliferation and radiosensitivity. In addition, knockdown of ACK1 attenuates LINC00963-dependent breast cancer growth and tumorigenesis. Taken together, LINC00963 promotes tumorigenesis and radioresistance in breast cancer through interplay with miR-324-3p and derepression of ACK1. LINC00963 may represent a potential target for the treatment of breast cancer.

Knockdown of activated Cdc42-associated kinase inhibits human extravillous trophoblast migration and invasion and decreases protein expression of pho-Akt and matrix metalloproteinase

Introduction: The sufficient invasion and migration of human extravillous trophoblast (EVTs) cells are crucial for placentation. Inadequate invasion of trophoblasts may correlate with the development of preeclampsia. Many studies have suggested that activated Cdc42-associated kinase (ACK1) is associated with tumor metastasis and invasion. This study investigated the ACK1 expression and its function in trophoblasts during placental development.Methods: ACK1 expression in human placentas was determined through immunofluorescence. We investigated the migration/invasion of the immortalized human first-trimester EVT cell line HTR8/SVneo. Hypoxia–reoxygenation (H/R) conditions were applied to mimic preeclampsia model in vitro. Lentiviral vector-based short-hairpin RNA directed against the sequence of ACK1 (ACK1 shRNA) was used to knock down ACK1 expression in HTR8/SVneo cells. Cell apoptosis and proliferation were determined through flow cytometry and cell counting Kit-8 (CCK-8) assays, respectively. The expression of matrix metalloproteinase (MMP) 2/9 and tissue inhibitors of metalloproteinase (TIMP) 1/2 was measured by western blotting.Results: ACK1 localized within trophoblasts of human placental villi, decidual cells in the maternal decidua. ACK1 levels in preeclampsia (PE) placentas were significantly lower than those in controls. ACK1 shRNA significantly inhibited HTR8/SVneo cells migration and invasion but did not affect their apoptosis and proliferation. ACK1 knockdown decreased MMP2/9 and increased TIMP1/2 expression, as well as downregulated the phosphorylation of AKt (p-Akt). In addition, ACK1 and MMP2/9 were downregulated following treatment with LY294002, whereas ACK1 shRNA had no effect on phosphorylation of PI3K(p-PI3K). After exposed in H/R condition, ACK1 expression, MMP2/9 protein, and p-Akt were also significantly decreased.Discussion and conclusions: ACK1 expression is lowered in preeclamptic placentas and promotes trophoblast cell invasion, migration. H/R conditions decrease ACK1 expression and appear to decouple the positive relationship between ACK1 expression and Akt activation.

MiR-24 represses metastasis of human osteosarcoma cells by targeting Ack1 via AKT/MMPs pathway

The expression levels of the protein tyrosine kinase Ack1 has been reported to be dysregulated in various cancers and involve in oncogenesis and progression. However, the expression and role of Ack1 in osteosarcoma remains unknown. In this study, we found that Ack1 were evidently upregulated in human osteosarcoma tissues and cell lines. In addition, the clinical data showed that high expression level of Ack1 is closely associated with clinical stage and positive distant metastasis, and negatively correlated with overall survival. Then, bioinformatics prediction and luciferase reporter assay indicated Ack1 as a direct target of miR-24, and Ack1 could be downregulated by miR-24 at both the mRNA and protein expression levels. Moreover, Ack1 expression levels were inversely correlated with that of miR-24 in osteosarcoma tissues. Furthermore, functional assay showed that miR-24 significantly suppressed osteosarcoma progression partially mediated by inhibiting Ack1 expression. Finally, western bolt assay revealed that miR-24 regulate AKT/MMPs pathway via Ack1 in osteosarcoma cells. In conclusion, our study demonstrated the suppression of miR-24 on osteosarcoma metastasis by targeting Ack1 via AKT/MMPs pathways, providing a novel strategy for the diagnosis and treatment of osteosarcoma patients.

Ack1 overexpression promotes metastasis and indicates poor prognosis of hepatocellular carcinoma.

Despite the substantial data supporting the oncogenic role of Ack1, the predictive value and biologic role of Ack1 in hepatocellular carcinoma (HCC) metastasis remains unknown. In this study, both correlations of Ack1 expression with prognosis of HCC, and the role of Ack1 in metastasis of HCC were investigated in vitro and in vivo. Our results showed that Ack1 was overexpressed in human HCC tissues and cell lines. High Ack1 expression was associated with HCC metastasis and determined as a significant and independent prognostic factor for HCC after liver resection. Ack1 promoted HCC invasion and metastasis in vitro and in vivo. Mechanistically, we confirmed that Ack1 enhanced invasion and metastasis of HCC via EMT by mediating AKT phosphorylation. In conclusion, our study shows Ack1 is a novel prognostic biomarker for HCC and promotes metastasis of HCC via EMT by activating AKT signaling.

ACK1 promotes hepatocellular carcinoma progression via downregulating WWOX and activating AKT signaling.

Several studies have revealed that ACK1 is upregulated in various cancers and promotes tumor progression. However, the role and mechanism of ACK1 in hepatocellular carcinoma (HCC) remains unknown. In this study, the expression of ACK1 was assessed in several cell lines and 150 pairs of HCC and adjacent noncancerous liver tissues. The protein expression of p-ACK1 and WWOX were detected by immunohistochemistry to evaluate their correlation with ACK1. Flow cytometry, caspase 3/7 activity assay, BrdU cell proliferation assay, MTT assay and Transwell assay were used to detect apoptosis, proliferation, invasion and migration of HCC cells. The regulatory effect of ACK1 on WWOX, AKT, p-AKT, MMP2 and MMP9 in HCC cells was confirmed by immuno-blotting. We found that ACK1 was more highly expressed in HCC tissues than in non-HCC tissues, and over-expression of ACK1 was correlated with clinicopathological features of poor prognosis. Clinical analysis demonstrated that ACK1 is an independent prognostic marker for predicting overall survival and disease-free survival of HCC patients. Pearson's correlation coefficient analysis indicated that ACK1 was positively associated with p-ACK1 and was negatively associated with WWOX expression. In vitro studies showed that knockdown of ACK1 promoted HCC cell apoptosis and repressed HCC cells invasion, migration and proliferation. Furthermore, knockdown of ACK1 resulted in upregulation of WWOX and inactivation of AKT signaling. In this study, we also found that knockdown of ACK1 resulted in the downregulation of MMP2 and MMP9 in HCC. Our results indicate that ACK1 is an independent prognostic marker and promotes HCC progression via downregulating WWOX and activating AKT signaling.

Overexpression of activated Cdc42-associated kinase1 (Ack1) predicts tumor recurrence and poor survival in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers in China. Recent research suggested that activated Cdc42-associated kinase 1 (Ack1) played an important role in facilitating tumorigenesis, tumor invasion and metastasis. However, the role of Ack1 in HCC is not clear. Herein, the expression level of Ack1 mRNA in 30 fresh HCC specimens (carcinoma, peri-carcinoma and distal-carcinoma tissues) was detected by reverse transcription-polymerase chain reaction (RT-PCR), while the expression of Ack1 protein in 18 fresh HCC specimens (carcinoma, peri-carcinoma and distal-carcinoma tissues) was analyzed by Western blotting. Immunohistochemical (IHC) staining was also employed to assess both the expression level and distribution of Ack1 protein in HCC tissues collected from 173 lesions, so as to study the correlations between Ack1 protein expression and other HCC-related clinicopathologic parameters. The results showed that both Ack1 mRNA and protein were significantly over-expressed in HCC tissues than that of either peri-carcinoma or distal-carcinoma tissues (P<0.001, P=0.012, respectively), while there was no significant difference between peri-carcinoma and distal-carcinoma tissues. Furthermore, the results of IHC indicated that the rates of Ack1 expressions in the patients with capsular invasion, hepatic vessel involvement and recurrence were higher than without above three conditions (P=0.037, P=0.036, P=0.019, respectively), whereas the patients with overexpression of Ack1 protein had low survival rate (P=0.007). Ack1 expression, tumor size and recurrence were independently related to survival (P=0.014, P=0.018, P<0.001, respectively). Thus, the level of Ack1 is associated with tumor invasion potential, and the expression of Ack1 plays an important role as predictor of recurrence and poor outcome in HCC patients.

Bosutinib inhibits migration and invasion via ack1 in kras mutant non-small cell lung cancer

The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers for accurate patient stratification. Here, we describe the role of ACK1, a non-receptor tyrosine kinase in abrogating migration and invasion in KRAS mutant lung adenocarcinoma. Bosutinib, which inhibits ACK1 at 2.7 nM IC50, was found to inhibit cell migration and invasion but not viability in a panel of non-small cell lung cancer (NSCLC) cell lines. Knockdown of ACK1 abrogated bosutinib-induced inhibition of cell migration and invasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafish metastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate that cytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue. Interestingly, ACK1 expression in “normal” tissue adjacent to tumour, but not tumour, was independently associated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinib attenuates migration and invasion in the context of KRAS mutant NSCLC and may fulfil a therapeutic niche through combinatorial treatment approaches.

Abstract A143: A preclinical in vivo model to assess pharmacodynamic inhibition of ACK1.

Abstract Introduction: Activated Cdc42-associated kinase (ACK1, TNK2) has been reported to be over-expressed in a range of tumor types including prostate and lung cancer, and has been suggested as a potential novel anticancer target. A number of kinase inhibitors currently in clinical development show inhibition of ACK1 biochemical activity in enzyme assays, yet their ability to inhibit ACK1 in vivo is unknown. This study describes the development of a pharmacodynamic model to evaluate ACK1 target coverage in vivo. Methods: MDA-MB-231 cells were engineered to over-express ACK1 under the control of a doxycycline-inducible promoter (MDA-MB-231-ACK1). MDA-MB-231-ACK1 cells were grown as subcutaneous xenografts in MF-1 nude mice and ACK1 expression induced by dosing doxycycline by oral gavage. Saracatinib/AZD-0530 (100mg/kg qd) or ASP-3026 (50mg/kg bid) were dosed for four consecutive days to reach steady state exposures at approximately MTD. Pharmacodynamic inhibition of ACK1 autophosphorylation was assessed using an in-house MSD assay and confirmed by Western blotting, in order to evaluate target inhibition in vivo. Results: ASP-3026 and saracatinib showed inhibition of cellular pACK1 of MDA-MB-231-ACK1 cells in vitro, with IC50s of 0.12 μM and 1.1 μM, respectively. Using the same cell line in vivo, expression of total and phosphorylated ACK1 was induced by daily oral dosing of doxycycline. We used this model to evaluate PK-PD correlations of ASP-3026 and saracatinib using pACK1 as a marker of target inhibition. Good plasma and tumor exposure of ASP-3026 was achieved at 50mg/kg bid and no drug-drug interactions were observed in combination with doxycycline. ASP-3026 showed 50-80% inhibition of pACK1 in vivo consistent with unbound plasma concentrations in excess of in vitro IC50 and in good agreement with predictions. Good exposure of saracatinib was observed at 100 mg/kg qd with total plasma Cmax of &amp;gt;4μM in excess of maximally tolerated clinical exposures (0.82μM). Tumor exposures of saracatinib were up to 10 x higher than plasma exposures (Cmax &amp;gt;40 μM), and in considerable excess of the cellular IC50, yet no ACK1 inhibition was observed in vivo. Conclusions: A robust PD model was established to evaluate ACK1 inhibition in vivo. Up to 80% inhibition of ACK1 in vivo was observed with ASP-3026, falling to approximately 50% at Cmin, suggesting that this agent does not completely inhibit ACK1 in vivo at steady state. In contrast, saracatinib did not significantly inhibit ACK1 at any of the exposures achieved, suggesting that it has limited in vivo activity against this target. Unbound plasma exposures predicted in vivo PD responses very well, whereas tumor exposures of these agents were not predictive of target inhibition. This model can be incorporated into a screening cascade for prioritising in vivo active ACK1 inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A143. Citation Format: Mark Albertella, Shilina Roman, Mike Briggs, Aaron Cranston, Ralph Graeser, George Hynd, David Jones, Peter Lockey, Akbor Malik, Stephen Price, Susanna Stimpson, Patrizia Tisselli, Janine Arts, Simon Green. A preclinical in vivo model to assess pharmacodynamic inhibition of ACK1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A143.

Abstract 4533: Interaction between androgen receptor and its corepressor SLIRP is regulated by Ack1 tyrosine kinase

Abstract Reactivation of androgen receptor (AR) is critical to castration resistant progression of prostate cancer. We previously demonstrated that Ack1 (activated cdc42-associated kinase) promotes growth of prostate xenograft tumors in castrated animals by activation of AR through phosphorylation of the Tyr-267 site. However, the molecular mechanisms underlying activation of AR by Ack1 need to be fully elucidated. Using the proteomics-based approach of Differential In-Gel Electrophoresis, we searched for proteins whose interaction with AR was regulated by Ack1 by characterizing proteins coimmunoprecipitating with AR in transfected 293T cells in the absence or in the presence of activated Ack1 kinase. SLIRP (SRA stem-loop interacting RNA binding protein) was identified as a candidate protein. We confirmed that the interaction between AR and SLIRP is abrogated by Ack1 expression and dihydrotestosterone (DHT) in 293T cells and in LNCaP prostate cancer cells. Additionally, treatment with heregulin, a ligand activating HER-2/HER-3 and Ack1 downstream, also led to dissociation of AR and SLIRP in LNCaP cells. Tyrosine kinase activity of Ack1 was shown to be required for its ability to dissociate AR and SLIRP through the use of a kinase-dead mutant in 293T cells and the use of an Ack1 inhibitor dasatinib in LNCaP cells treated with heregulin. However, disruption of interaction between AR and SLIRP by Ack1 cannot be attributed to any known AR tyrosine phosphorylation sites since Ack1 expression led to disruption of binding between SLIRP and AR Y267F, Y363F, and Y534F mutants. Since SLIRP does not contain tyrosine residues, the substrate of Ack1 involved in AR-SLIRP dissociation is not known yet. We showed that SLIRP functions as a physiologically relevant corepressor of AR in prostate cancer cells. Chromatin immunoprecipitation analysis demonstrated that SLIRP was bound to the androgen regulated enhancers of AR target genes in the absence of androgen and that androgen or heregulin treatment led to loss of SLIRP binding at the androgen regulated enhancers. AR-dependent reporter activity was inhibited by overexpression of SLIRP and was increased by SLIRP knockdown. SLIRP knockdown also increased the expression of endogenous AR target mRNA levels of PSA and hK2. In summary, our data suggest that Ack1 kinase inhibits the interaction between AR and the SLIRP corepressor and causes dissociation of SLIRP from the AR target gene enhancers and thereby enhance AR transcriptional activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4533. doi:10.1158/1538-7445.AM2011-4533

A single nucleotide polymorphism in activated cdc42 associated tyrosine kinase 1 influences the interferon therapy in hepatitis C patients

Cdc42 is a Rho family GTPase protein and was recently implicated in mediating hepatitis C virus (HCV) infectivity. This study examines the association between Cdc42-related gene and interferon (IFN) therapy in HCV patients. We analyzed the associations between the outcome of IFN therapy and 17 tagging single nucleotide polymorphisms (SNPs) within two genes involved in Cdc42 signaling (CDC42 and ACK1). A total of 295 out of the 409 study subjects were sustained responders (SR) and 114 were non-responders (NR). Replication was performed using an independent set of 794 IFN-treated patients. SNP rs2278034 [A/G] in intron 11 of activated Cdc42 associated tyrosine kinase (ACK) 1 was associated with the outcome of IFN therapy (p=6.4 × 10(-4)). Replication analysis confirmed the association (p=2.2 × 10(-3)) for patients treated with IFN monotherapy, but the association was not significant for pegylated-IFN-plus ribavirin therapy. Analysis using published HapMap expression data revealed that ACK1 expression correlates with IFN-stimulated gene (ISG) expression independently of ethnicity, but the relationship between rs2278034 and ACK1 expression was observed only within Asian populations. Over-expression of ACK1, but not the kinase-inactive mutant, increased ISG transcription in Huh7 cells. ACK1 expression enhanced the IFN-stimulated response element (ISRE) and interferon-γ-activated site (GAS) promoter activity through tyrosine phosphorylation of signal transducers and activators of transcription (STAT) 1. Furthermore, ACK1 over-expression in HCV-N replicon cells inhibited HCV replication. SNP rs2278034 in ACK1 is associated with IFN therapy outcome in patients with HCV. ACK1 may play a role in innate and IFN-induced antiviral action against HCV.

The effect of granulocyte-macrophage-stimulating factor on the gene expression in human trophoblast of early pregnancy

Objective: Granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitates mammalian embryonic development and implantation. However, its biological function after implantation is not elucidated. The aim of this study is to assess the changes of gene expression by GM-CSF in human trophoblast obtained in early pregnancy. Methods: Human trophoblast obtained in early pregnancy was cultured with or without GM-CSF. The difference of gene expression was evaluated with microarray and selected genes were reevaluated with real-time reverse transcription-polymerase chain reaction (RT-PCR). Results: Microarray analysis revealed that the expressions of 468 genes were increased while those of 40 genes were decreased by GM-CSF. These genes were evaluated according to the known biologic pathways. The regulation of actin cytoskeleton and focal adhesion pathways were mostly influenced by GM-CSF. Annexin A2, thymosin-like 3, vimentin, myogenin, ACK1, and tensin1 genes were selected for real-time RT-PCR. The increased expressions of of vimentin and ACK1, and decreased expressions of tensin1 were confirmed by real-time RT-PCR. Conclusion: GM-CSF activates focal adhesion pathway in human trophoblast by increasing the expression of vimentin and ACK1, and decreasing the expression of tensin1.

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast Cancer Cell Migration

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco- or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco- and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130(Cas) on Tyr-410 and an increase in the abundance of the Crk.p130(Cas) complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130(Cas) and disrupts the Crk.p130(Cas) complex. We further determined that suppression of expression of Cdc42, Ack1, p130(Cas), or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk.p130(Cas) complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.

Activated Cdc42-associated Kinase 1 Is a Component of EGF Receptor Signaling Complex and Regulates EGF Receptor Degradation

Cdc42-associated tyrosine kinase 1 (ACK1) is a specific down-stream effector of Cdc42, a Rho family small G-protein. Previous studies have shown that ACK1 interacts with clathrin heavy chain and is involved in clathrin-coated vesicle endocytosis. Here we report that ACK1 interacted with epidermal growth factor receptor (EGFR) upon EGF stimulation via a region at carboxy terminus that is highly homologous to Gene-33/Mig-6/RALT. The interaction of ACK1 with EGFR was dependent on the kinase activity or tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 was colocalized with EGFR on EEA-1 positive vesicles upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNAi inhibited ligand-induced degradation of EGFR upon EGF stimulation, suggesting that ACK1 plays an important role in regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin-association (Uba) domain at the carboxy terminus, ACK1 binds to both poly- and mono-ubiquitin. Overexpression of the Uba domain-deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Taken together, our studies suggest that ACK1 senses signal of EGF and regulates ligand-induced degradation of EGFR.

Expression pattern of ACK1 tyrosine kinase during brain development in the mouse

Ack1 is a non-receptor tyrosine kinase that is highly expressed in the adult central nervous system (CNS). Here, we studied the distribution of Ack1 mRNA throughout the development of mouse CNS. Expression was detected in all areas of the brain but especially high levels were observed in the neocortex, hippocampus, and cerebellum. Interestingly, expression levels were prominent in areas of proliferation such as the subventricular zone and areas that originate other structures such the pontine nucleus and the ganglionic eminence. During development, several areas showed an increase in Ack1 expression, especially the dentate gyrus and CA3 in the hippocampus, layer V in the neocortex, and the Purkinje cell layer in the cerebellum. These results demonstrate that this kinase is up-regulated during development and that it is expressed in proliferative areas and in migratory pathways in the developing brain.

Requirement of Activated Cdc42-Associated Kinase for Survival of v-Ras-Transformed Mammalian Cells

Activated Cdc42-associated kinase (ACK) has been shown to be an important effector molecule for the small GTPase Cdc42. We have shown previously an essential role for Cdc42 in the transduction of Ras signals for the transformation of mammalian cells. In this report, we show that the ACK-1 isoform of ACK plays a critical role in transducing Ras-Cdc42 signals in the NIH 3T3 cells. Overexpression of a dominant-negative (K214R) mutant of ACK-1 inhibits Ras-induced up-regulation of c-fos and inhibits the growth of v-Ras-transformed NIH 3T3 cells. Using small interfering RNA, we knocked down the expression of ACK-1 in both v-Ha-Ras-transformed and parental NIH 3T3 cells and found that down-regulation of ACK-1 inhibited cell growth by inducing apoptosis only in v-Ha-Ras-transformed but not parental NIH 3T3 cells. In addition, we studied the effect of several tyrosine kinase inhibitors and found that PD158780 inhibits the kinase activity of ACK-1 in vitro. We also found that PD158780 inhibits the growth of v-Ha-Ras-transformed NIH 3T3 cells. Taken together, our results suggest that ACK-1 kinase plays an important role in the survival of v-Ha-Ras-transformed cells, suggesting that ACK-1 is a novel target for therapies directed at Ras-induced cancer.

Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42, Ack-1 and p130cas.

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-alpha4 beta1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p130 cas, an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization.