The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis.

The family of Abelson interactor (Abi) proteins is a component of WAVE regulatory complex (WRC) and a downstream target of Abelson (Abl) tyrosine kinase. The fact that Abi proteins also interact with diverse membrane proteins and intracellular signaling molecules places these proteins at a central position in the network that controls cytoskeletal functions and cancer cell metastasis. Here, we identified a motif in Abi proteins that conforms to consensus sequences found in a cohort of receptor and non-receptor tyrosine kinases that bind to Cbl-tyrosine kinase binding domain. The phosphorylation of tyrosine 213 in this motif is essential for Abi degradation. Double knockout of c-Cbl and Cbl B in Bcr-Abl-transformed leukemic cells abolishes Abi1, Abi2, and WAVE2 degradation. Moreover, knockout of Abi1 reduces Src family kinase Lyn activation in Bcr-Abl-positive leukemic cells and promotes EGF-induced EGF receptor downregulation in breast cancer cells. Importantly, Abi1 depletion impeded breast cancer cell invasion in vitro and metastasis in mouse xenografts. Together, these studies uncover a novel mechanism by which the WRC and receptor/non-receptor tyrosine kinases are regulated and identify Abi1 as a potential therapeutic target for metastatic breast cancer.

CRISPR/CAS9-mediated knockout of Abi1 inhibits p185Bcr-Abl-induced leukemogenesis and signal transduction to ERK and PI3K/Akt pathways.

BACKGROUND: Abl interactor 1 (Abi1) is a downstream target of Abl tyrosine kinases and a component of the WAVE regulatory complex (WRC) that plays an important role in regulating actin cytoskeleton remodeling and membrane receptor signaling. While studies using short hairpin RNA (shRNA) have suggested that Abi1 plays a critical role in Bcr-Abl-induced leukemogenesis, the mechanism involved is not clear. METHODS: In this study, we knocked out Abi1 expression in p185Bcr-Abl-transformed hematopoietic cells using CRISPR/Cas9-mediated gene editing technology. The effects of Abi1 deficiency on actin cytoskeleton remodeling, the Bcr-Abl signaling, IL-3 independent growth, and SDF-induced chemotaxis in these cells were examined by various in vitro assays. The leukemogenic activity of these cells was evaluated by a syngeneic mouse transplantation model. RESULTS: We show here that Abi1 deficiency reduced the IL3-independent growth and SDF-1α-mediated chemotaxis in p185Bcr-Abl-transformed hematopoietic cells and inhibited Bcr-Abl-induced abnormal actin remodeling. Depletion of Abi1 also impaired the Bcr-Abl signaling to the ERK and PI3 kinase/Akt pathways. Remarkably, the p185Bcr-Abl-transformed cells with Abi1 deficiency lost their ability to develop leukemia in syngeneic mice. Even though these cells developed drug tolerance in vitro after prolonged selection with imatinib as their parental cells, the imatinib-tolerant cells remain incapable of leukemogenesis in vivo. CONCLUSIONS: Together, this study highlights an essential role of Abi1 in Bcr-Abl-induced leukemogenesis and provides a model system for dissecting the Abi1 signaling in Bcr-Abl-positive leukemia.

G-protein coupled receptor 34 activates Erk and phosphatidylinositol 3-kinase/Akt pathways and functions as alternative pathway to mediate p185Bcr-Abl-induced transformation and leukemogenesis.

Tyrosine 177 and the Src homology 2 (SH2) domain play important roles in linking p185Bcr-Abl to downstream pathways critical for cell growth and survival. However, a mutant p185(Y177FR552L) (p185(YR)), in which tyrosine 177 and arginine 552 in the SH2 domain are mutated, is still capable of transforming hematopoietic cells in vitro. Transplant of these cells into syngeneic mice also leads to leukemogenesis, albeit with a phenotype distinct from that produced by wild-type p185Bcr-Abl (p185(wt))-transformed cells. Here we show that G-protein coupled receptor 34 (Gpr34) expression is markedly up-regulated in p185(YR)-transformed cells compared to those transformed by p185(wt). Knockdown of Gpr34 in p185(YR) cells is sufficient to suppress growth factor-independent proliferation and survival in vitro and attenuate leukemogenesis in vivo. The Erk and phosphatidylinositol 3-kinase/Akt pathways are activated in p185(YR) cells and the activation is dependent on Gpr34 expression. These studies identify Gpr34 as an alternative pathway that may mediate p185Bcr-Abl-induced transformation and leukemogenesis.

Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease.

BACKGROUND: Polycystic Kidney Disease (PKD) kidneys exhibit increased extracellular matrix (ECM) collagen expression and metalloproteinases (MMPs) activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. METHODS: We examined the role of type I collagen (collagen I) and membrane bound type 1 MMP (MT1-MMP) on cyst development using both in vitro 3 dimensional (3D) collagen gel culture and in vivo PCK rat model of PKD. RESULTS: We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. CONCLUSIONS: Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

Knockdown of VEGF receptor-1 (VEGFR-1) impairs macrophage infiltration, angiogenesis and growth of clear cell renal cell carcinoma (CRCC).

Angiogenesis is essential for tumor growth and metastasis. VEGF has been shown to be a central player in this process. The biological activity of VEGF is mainly mediated by two tyrosine kinase receptors, VEGFR-1 and VEGFR-2. While increasing evidence suggests that VEGF/VEGFR-1 signaling is crucial for tumor angiogenesis, its molecular mechanism is not well understood. Here we show that VEGFR-1 knockdown dramatically inhibits tumor growth. This inhibition is associated with significant decrease of tumor VEGF levels and tumor angiogenesis as well as an increased tumor necrosis. Moreover, we demonstrate that VEGF in CRCC tumors is mainly produced by tumor stromal cells instead of the tumor cells themselves. It has been shown that macrophages constitute a significant part of tumor stromal cells and produce a large amount of VEGF. We therefore examined the macrophage infiltration in the xenograft tumors. Remarkably, VEGFR-1 knockdown attenuates the tumor macrophages infiltration. To understand the mechanism, we investigated the impact of VEGFR-1 knockdown on the expression of monocyte chemoattractant protein-1 (MCP-1), one of the main chemoattractants for macrophages. Significantly, VEGFR-1 knockdown inhibits MCP-1 expression of CRCC cells. Taken together, these data indicate that VEGF/VEGFR-1 signaling plays an essential role in initiating tumor angiogenesis by regulating MCP-1 expression, which in turn, attracts macrophages infiltration and VEGF production. Thus, these studies suggest that blockade of VEGFR-1 function may provide a tumor-specific, VEGF-based therapeutic strategy for treatment of CRCC.

CDK1-mediated phosphorylation of Abi1 attenuates Bcr-Abl-induced F-actin assembly and tyrosine phosphorylation of WAVE complex during mitosis.

Coordinated actin remodeling is crucial for cell entry into mitosis. The WAVE regulatory complex is a key regulator of actin assembly, yet how the WAVE signaling is regulated to coordinate actin assembly with mitotic entry is not clear. Here, we have uncovered a novel mechanism that regulates the WAVE complex at the onset of mitosis. We found that the Bcr-Abl-stimulated F-actin assembly is abrogated during mitosis. This mitotic inhibition of F-actin assembly is accompanied by an attenuation of Bcr-Abl-induced tyrosine phosphorylation of the WAVE complex. We identified serine 216 of Abi1 as a target of CDK1/cyclin B kinase that is phosphorylated in cells at the onset of mitosis. The Abi1 phosphorylated on serine 216 displayed greatly reduced tyrosine phosphorylation in the hematopoietic cells transformed by Bcr-Abl. Moreover, a phosphomimetic mutation of serine 216 to aspartic acid in Abi1 was sufficient to attenuate Bcr-Abl-induced tyrosine phosphorylation of the WAVE complex and F-actin assembly. Ectopic expression of Abi1 with serine 216 mutations interfered with cell cycle progression. Together, these data show that CDK1-mediated phosphorylation of serine 216 in Abi1 serves as a regulatory mechanism that may contribute to coordinated actin cytoskeleton remodeling during mitosis.

Inhibition of class II phosphoinositide 3-kinase gamma expression by p185(Bcr-Abl) contributes to impaired chemotaxis and aberrant homing of leukemic cells.

The expression of p185(Bcr-Abl) in Ba/F3 cells inhibits the chemotactic response of these cells to SDF1alpha. A mutant p185(Bcr-Abl) with deletion of amino acids from 176 to 426 (p185(Delta176-426)) is deficient in suppressing SDF1alpha-stimulated chemotaxis. Comparison of the gene expression profiles among parental Ba/F3 cells and cells transformed by p185(Bcr-Abl) and p185(Delta176-426) reveals that class II phosphoinositide 3-kinase gamma (PI3KC2gamma) expression is markedly down-regulated by p185(Bcr-Abl) but not p185(Delta176-426). Furthermore, knockdown of PI3KC2gamma expression in p185(Delta176-426) cells is sufficient to suppress SDF1alpha-stimulated chemotaxis and to promote infiltration of these cells into the liver. Together, these studies suggest that inhibition of PI3KC2gamma expression may represent a mechanism by which Bcr-Abl suppresses SDF1alpha-induced chemotaxis and induces abnormal homing of leukemic cells.

Abl interactor 1 regulates Src-Id1-matrix metalloproteinase 9 axis and is required for invadopodia formation, extracellular matrix degradation and tumor growth of human breast cancer cells.

Abl interactor 1 (Abi1) is a key regulator of actin polymerization/depolymerization. The involvement of Abi1 in the development of abnormal cytoskeletal functions of cancer cells has recently been reported. It remains unclear, however, how Abi1 exerts its effects in tumor cells and whether it contributes to tumor progression in vivo. We report here a novel function for Abi1 in the regulation of invadopodia formation and Src-inhibitor of differentiation protein 1 (Id1)-matrix metalloproteinase (MMP)-9 pathway in MDA-MB-231 human breast cancer cells. Abi1 is found in the invadopodia of MDA-MB-231 cells. Epigenetic silencing of the Abi1 gene by short hairpin RNA in MDA-MB-231 cells impaired the formation of invadopodia and resulted in downregulation of the Src activation and Id1/MMP-9 expression. The decreased invadopodia formation and MMP-9 expression correlate with a reduction in the ability of these cells to degrade extracellular matrix. Remarkably, the knockdown of Abi1 expression inhibited tumor cell proliferation and migration in vitro and slowed tumor growth in vivo. Taken together, these results indicate that the Abi1 signaling plays a critical role in breast cancer progression and suggest that this pathway may serve as a therapeutic target for the treatment of human breast cancer.

Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration in vitro and leukemogenesis in vivo.

Abl interactor (Abi) 1 was first identified as the downstream target of Abl tyrosine kinases and was found to be dysregulated in leukemic cells expressing oncogenic Bcr-Abl and v-Abl. Although the accumulating evidence supports a role of Abi1 in actin cytoskeleton remodeling and growth factor/receptor signaling, it is not clear how it contributes to Bcr-Abl-induced leukemogenesis. We show here that Abi1 gene silencing by short hairpin RNA attenuated the Bcr-Abl-induced abnormal actin remodeling, membrane-type 1 metalloproteinase clustering and inhibited cell adhesion and migration on fibronectin-coated surfaces. Although the knock down of Abi1 expression did not affect growth factor-independent growth of Bcr-Abl-transformed Ba/F3 cells in vitro, it impeded competitive expansion of these cells in non obese diabetic (NOD)/ severe combined immuno-deficiency (SCID) mice. Remarkably, the knock down of Abi1 expression in Bcr-Abl-transformed Ba/F3 cells impaired the leukemogenic potential of these cells in NOD/SCID mice. Abi1 contributes to Bcr-Abl-induced leukemogenesis in part through Src family kinases, as the knock down of Abi1 expression attenuates Bcr-Abl-stimulated activation of Lyn. Together, these data provide for the first time the direct evidence that supports a critical role of Abi1 pathway in the pathogenesis of Bcr-Abl-induced leukemia.

Bcr-Abl induces abnormal cytoskeleton remodeling, beta1 integrin clustering and increased cell adhesion to fibronectin through the Abl interactor 1 pathway.

Hematopoietic cells isolated from patients with Bcr-Abl-positive leukemia exhibit multiple abnormalities of cytoskeletal and integrin function. These abnormalities are thought to play a role in the pathogenesis of leukemia; however, the molecular events leading to these abnormalities are not fully understood. We show here that the Abi1 pathway is required for Bcr-Abl to stimulate actin cytoskeleton remodeling, integrin clustering and cell adhesion. Expression of Bcr-Abl induces tyrosine phosphorylation of Abi1. This is accompanied by a subcellular translocation of Abi1/WAVE2 to a site adjacent to membrane, where an F-actin-enriched structure containing the adhesion molecules such as beta1-integrin, paxillin and vinculin is assembled. Bcr-Abl-induced membrane translocation of Abi1/WAVE2 requires direct interaction between Abi1 and Bcr-Abl, but is independent of the phosphoinositide 3-kinase pathway. Formation of the F-actin-rich complex correlates with an increased cell adhesion to fibronectin. More importantly, disruption of the interaction between Bcr-Abl and Abi1 by mutations either in Bcr-Abl or Abi1 not only abolished tyrosine phosphorylation of Abi1 and membrane translocation of Abi1/WAVE2, but also inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, integrin clustering and cell adhesion to fibronectin. Together, these data define Abi1/WAVE2 as a downstream pathway that contributes to Bcr-Abl-induced abnormalities of cytoskeletal and integrin function.