Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis.

Cellular senescence has been theorized to oppose neoplastic transformation triggered by activation of oncogenic pathways in vitro, but the relevance of senescence in vivo has not been established. The PTEN and p53 tumour suppressors are among the most commonly inactivated or mutated genes in human cancer including prostate cancer. Although they are functionally distinct, reciprocal cooperation has been proposed, as PTEN is thought to regulate p53 stability, and p53 to enhance PTEN transcription. Here we show that conditional inactivation of Trp53 in the mouse prostate fails to produce a tumour phenotype, whereas complete Pten inactivation in the prostate triggers non-lethal invasive prostate cancer after long latency. Strikingly, combined inactivation of Pten and Trp53 elicits invasive prostate cancer as early as 2 weeks after puberty and is invariably lethal by 7 months of age. Importantly, acute Pten inactivation induces growth arrest through the p53-dependent cellular senescence pathway both in vitro and in vivo, which can be fully rescued by combined loss of Trp53. Furthermore, we detected evidence of cellular senescence in specimens from early-stage human prostate cancer. Our results demonstrate the relevance of cellular senescence in restricting tumorigenesis in vivo and support a model for cooperative tumour suppression in which p53 is an essential failsafe protein of Pten-deficient tumours.

Role of Dok-1 and Dok-2 in leukemia suppression.

Chronic myelogenous leukemia (CML) is characterized by the presence of the chimeric p210bcr/abl oncoprotein that shows elevated and constitutive protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. Although several p210bcr/abl substrates have been identified, their relevance in the pathogenesis of the disease is unclear. We have identified a family of proteins, Dok (downstream of tyrosine kinase), coexpressed in hematopoietic progenitor cells. Members of this family such as p62dok (Dok-1) and p56dok-2 (Dok-2) associate with the p120 rasGTPase-activating protein (rasGAP) upon phosphorylation by p210bcr/abl as well as receptor and nonreceptor tyrosine kinases. Here, we report the generation and characterization of single and double Dok-1 or Dok-2 knockout (KO) mutants. Single KO mice displayed normal steady-state hematopoiesis. By contrast, concomitant Dok-1 and Dok-2 inactivation resulted in aberrant hemopoiesis and Ras/MAP kinase activation. Strikingly, all Dok-1/Dok-2 double KO mutants spontaneously developed transplantable CML-like myeloproliferative disease due to increased cellular proliferation and reduced apoptosis. Furthermore, Dok-1 or Dok-2 inactivation markedly accelerated leukemia and blastic crisis onset in Tec-p210bcr/abl transgenic mice known to develop, after long latency, a myeloproliferative disorder resembling human CML. These findings unravel the critical and unexpected role of Dok-1 and Dok-2 in tumor suppression and control of the hematopoietic compartment homeostasis.

c-Abl phosphorylates Dok1 to promote filopodia during cell spreading.

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.

Dok-1 independently attenuates Ras/mitogen-activated protein kinase and Src/c-myc pathways to inhibit platelet-derived growth factor-induced mitogenesis.

The Dok adaptor proteins play key regulatory roles in receptor and non-receptor kinase-initiated signaling pathways. Dok-1, the prototype member of this family, negatively regulates cell proliferation elicited by numerous growth factors, including platelet-derived growth factor (PDGF). However, how Dok-1 exerts its negative effect on mitogenesis has remained elusive. Using Dok-1 knockout cells and Dok-1 mutants deficient in binding to specific Dok-1-interacting proteins, we show that Dok-1 interferes with PDGF-stimulated c-myc induction and Ras/mitogen-activated protein kinase (MAPK) activation by tethering different signaling components to the cell membrane. Specifically, Dok-1 attenuates PDGF-elicited c-myc induction by recruiting Csk to active Src kinases, whereupon their activities and consequent c-myc induction are diminished. On the other hand, Dok-1 negatively regulates PDGF-induced MAPK activation by acting on Ras-GAP and at least one other Dok-1-interacting protein. Importantly, we demonstrate that Dok-1's actions on both of these signaling pathways contribute to its inhibitory effect on mitogenesis. Our data suggest a mechanistic basis for the inhibitory effect of Dok-1 on growth factor-induced mitogenesis and its role as a tumor suppressor.

Pten dose dictates cancer progression in the prostate.

Complete inactivation of the PTEN tumor suppressor gene is extremely common in advanced cancer, including prostate cancer (CaP). However, one PTEN allele is already lost in the vast majority of CaPs at presentation. To determine the consequence of PTEN dose variations on cancer progression, we have generated by homologous recombination a hypomorphic Pten mouse mutant series with decreasing Pten activity: Pten(hy/+) > Pten(+/-) > Pten(hy/-) (mutants in which we have rescued the embryonic lethality due to complete Pten inactivation) > Pten prostate conditional knockout (Pten(pc)) mutants. In addition, we have generated and comparatively analyzed two distinct Pten(pc) mutants in which Pten is inactivated focally or throughout the entire prostatic epithelium. We find that the extent of Pten inactivation dictate in an exquisite dose-dependent fashion CaP progression, its incidence, latency, and biology. The dose of Pten affects key downstream targets such as Akt, p27(Kip1), mTOR, and FOXO3. Our results provide conclusive genetic support for the notion that PTEN is haploinsufficient in tumor suppression and that its dose is a key determinant in cancer progression.

Dok-1 negatively regulates platelet integrin αIIbß3 outside-in signalling and inhibits thrombosis in mice.

Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin ß3, but the underlying effects of Dok-1 on αIIbß3 signalling, platelet activation and thrombosis remain to be elucidated. In the present study, using Dok-1-deficient (Dok-1-/-) mice, we determined the phenotypic role of Dok-1 in αIIbß3 signalling. We found that platelets from Dok-1-/- mice displayed normal aggregation, activation of αIIbß3 (assessed by binding of JON/A), P-selectin surface expression (assessed by anti-CD62P), and soluble fibrinogen binding. These findings indicate that Dok-1 does not affect "inside-out" platelet signalling. Compared with platelets from wild-type (WT) mice, platelets from Dok-1-/- mice exhibited increased clot retraction (p < 0.05 vs WT), increased PLCγ2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation (p < 0.01 vs WT), demonstrating that Dok-1 negatively regulates αIIbß3 "outside-in" signalling. Finally, we found that Dok-1-/- mice exhibited significantly shortened bleeding times and accelerated carotid artery thrombosis in response to photochemical injury (p < 0.05 vs WT mice). We conclude that Dok-1 modulates thrombosis and haemostasis by negatively regulating αIIbß3 outside-in signalling.

DOK2 inhibits EGFR-mutated lung adenocarcinoma.

Somatic mutations in the EGFR proto-oncogene occur in ~15% of human lung adenocarcinomas and the importance of EGFR mutations for the initiation and maintenance of lung cancer is well established from mouse models and cancer therapy trials in human lung cancer patients. Recently, we identified DOK2 as a lung adenocarcinoma tumor suppressor gene. Here we show that genomic loss of DOK2 is associated with EGFR mutations in human lung adenocarcinoma, and we hypothesized that loss of DOK2 might therefore cooperate with EGFR mutations to promote lung tumorigenesis. We tested this hypothesis using genetically engineered mouse models and find that loss of Dok2 in the mouse accelerates lung tumorigenesis initiated by oncogenic EGFR, but not that initiated by mutated Kras. Moreover, we find that DOK2 participates in a negative feedback loop that opposes mutated EGFR; EGFR mutation leads to recruitment of DOK2 to EGFR and DOK2-mediated inhibition of downstream activation of RAS. These data identify DOK2 as a tumor suppressor in EGFR-mutant lung adenocarcinoma.

Identification of DOK genes as lung tumor suppressors.

Genome-wide analyses of human lung adenocarcinoma have identified regions of consistent copy-number gain or loss, but in many cases the oncogenes and tumor suppressors presumed to reside in these loci remain to be determined. Here we identify the downstream of tyrosine kinase (Dok) family members Dok1, Dok2 and Dok3 as lung tumor suppressors. Single, double or triple compound loss of these genes in mice results in lung cancer, with penetrance and latency dependent on the number of lost Dok alleles. Cancer development is preceded by an aberrant expansion and signaling profile of alveolar type II cells and bronchioalveolar stem cells. In human lung adenocarcinoma, we identify DOK2 as a target of copy-number loss and mRNA downregulation and find that DOK2 suppresses lung cancer cell proliferation in vitro and in vivo. Given the genomic localization of DOK2, we propose it as an 8p21.3 haploinsufficient human lung tumor suppressor.

Downstream of tyrosine kinases-1 and Src homology 2-containing inositol 5'-phosphatase are required for regulation of CD4+CD25+ T cell development.

The adaptor protein, downstream of tyrosine kinases-1 (Dok-1), and the phosphatase SHIP are both tyrosine phosphorylated in response to T cell stimulation. However, a function for these molecules in T cell development has not been defined. To clarify the role of Dok-1 and SHIP in T cell development in vivo, we compared the T cell phenotype of wild-type, Dok-1 knockout (KO), SHIP KO, and Dok-1/SHIP double-knockout (DKO) mice. Dok-1/SHIP DKO mice were runted and had a shorter life span compared with either Dok-1 KO or SHIP KO mice. Thymocyte numbers from Dok-1/SHIP DKO mice were reduced by 90%. Surface expression of both CD25 and CD69 was elevated on freshly isolated splenic CD4(+) T cells from SHIP KO and Dok-1/SHIP DKO, suggesting these cells were constitutively activated. However, these T cells did not proliferate or produce IL-2 after stimulation. Interestingly, the CD4(+) T cells from SHIP KO and Dok-1/SHIP DKO mice produced higher levels of TGF-beta, expressed Foxp3, and inhibited IL-2 production by CD3-stimulated CD4(+)CD25(-) T cells in vitro. These findings suggest Dok-1 and SHIP function in pathways that influence regulatory T cell development.

Dok1 and SHIP act as negative regulators of v-Abl-induced pre-B cell transformation, proliferation and Ras/Erk activation.

The v-Abl tyrosine kinase activates several signaling pathways during transformation of bone marrow cells in mice. Because the SH2-containing inositol 5'-phosphatase (SHIP) and Downstream of tyrosine kinase 1 (Dok1) have been shown to interact with Abl, the effect of SHIP and Dok1 deficiency on v-Abl transformation was investigated. Bone marrow cells from either Dok1- or SHIP-deficient mice are more susceptible to transformation by v-Abl. v-Abl-transformed preB cells from these knockout mice show Abl kinase-dependent hyperproliferation and moderate resistance to apoptosis. Elevated activation of Ras, Raf-1, and Erk, but not of Akt, was observed in either SHIP(-/-) or Dok1(-/-) v-Abl-transformed cells. This activation is sensitive to treatment with STI571. Furthermore, treatment of these cells with either a farnesyltransferase inhibitor or a MEK1/2 inhibitor abrogates the increased proliferation of SHIP(-/-) or Dok1(-/-) cells in a dose-dependent manner. Complementation of SHIP(-/-) or Dok1(-/-) cells abrogates their hyperproliferation and intracellular Erk activation. These data indicate that both SHIP and Dok1 functionally regulate the activation of Ras-Erk pathway by v-Abl and affect the mitogenic activity of v-Abl transformed bone marrow cells.

Stromal cell-derived factor-1alpha/CXCL12-induced chemotaxis of T cells involves activation of the RasGAP-associated docking protein p62Dok-1.

Events mediating stromal cell-derived factor-1 (SDF-1alpha/CXCL12) chemotaxis of lymphocytes are not completely known. We evaluated intracellular signaling through RasGAP-associated protein p62Dok-1 (downstream of tyrosine kinase [Dok-1]) and associated proteins. SDF-1alpha/CXCL12 stimulated Dok-1 tyrosine phosphorylation and association with RasGAP, adaptor protein p46Nck, and Crk-L in Jurkat T cells. The phosphorylation of Dok-1 was blocked by pretreatment of cells with the src kinase inhibitor PP2. Src kinase family member Lck was implicated. SDF-1alpha/CXCL12 did not phosphorylate Dok-1 in J.CaM1.6 cells, a Jurkat derivative not expressing Lck, but did phosphorylate Dok-1 in J.CaM1.6 cells expressing Lck. SDF-1alpha/CXCL12 induced the tyrosine phosphorylation of Pyk2 and the association of Pyk2 with zeta chain-associated protein-70 kilodaltons (Zap-70) and Vav. SDF-1alpha/CXCL12 enhanced the association of RasGAP with Pyk2. CXCR4-expressing NIH3T3 and Baf3 cells transfected with full-length Dok-1 cDNA were suppressed in their responses to SDF-1alpha/CXCL12-induced chemotaxis; mitogen-activated protein (MAP) kinase activity was also decreased. Chemotaxis to SDF-1/CXCL12 was significantly enhanced in Dok-1(-/-) CD4+ and CD8+ splenic T cells. These results implicate Dok-1, Nck, Crk-L, and Src kinases-especially Lck, Pyk2, Zap-70, Vav, and Ras-GAP-in intracellular signaling by SDF-1alpha/CXCL12, and they suggest that Dok-1 plays an important role in SDF-1alpha/CXCL12-induced chemotaxis in T cells.

Downstream of kinase, p62(dok), is a mediator of Fc gamma IIB inhibition of Fc epsilon RI signaling.

The low-affinity receptor for IgG, Fc gamma RIIB, is expressed widely in the immune system and functions to attenuate Ag-induced immune responses. In mast cells, coaggregation of Fc gamma RIIB with the high-affinity IgE receptor, Fc epsilon RI, leads to inhibition of Ag-induced degranulation and cytokine production. Fc gamma RIIB inhibitory activity requires a conserved motif within the Fc gamma RIIB cytoplasmic domain termed the immunoreceptor tyrosine-based inhibition motif. When coaggregated with an activating receptor (e.g., Fc epsilon RI, B cell Ag receptor), Fc gamma RIIB is rapidly phosphorylated on tyrosine and recruits the SH2 domain-containing inositol 5-phosphatase (SHIP). However, the mechanisms by which SHIP mediates Fc gamma RIIB inhibitory function in mast cells remain poorly defined. In this report we demonstrate that Fc gamma RIIB coaggregation with Fc epsilon RI stimulates enhanced SHIP tyrosine phosphorylation and association with Shc and p62(dok). Concurrently, enhanced p62(dok) tyrosine phosphorylation and association with RasGAP are observed, suggesting that SHIP may mediate Fc gamma RIIB inhibitory function in mast cells via recruitment of p62(dok) and RasGAP. Supporting this hypothesis, recruitment of p62(dok) to Fc epsilon RI is sufficient to inhibit Fc epsilon RI-induced calcium mobilization and extracellular signal-regulated kinase 1/2 activation. Interestingly, both the amino-terminal pleckstrin homology and phosphotyrosine binding domains and the carboxyl-terminal proline/tyrosine-rich region of p62(dok) can mediate inhibition, suggesting activation of parallel downstream signaling pathways that converge at extracellular signal-regulated kinase 1/2 activation. Finally, studies using gene-ablated mice indicate that p62(dok) is dispensable for Fc gamma RIIB inhibitory signaling in mast cells. Taken together, these data suggest a role for p62(dok) as a mediator of Fc gamma RIIB inhibition of Fc epsilon RI signal transduction in mast cells.