Richard A. West

The Wnt co-receptor Lrp6 is required for normal mouse mammary gland development.

Canonical Wnt signals are transduced through a Frizzled receptor and either the LRP5 or LRP6 co-receptor; such signals play central roles during development and in disease. We have previously shown that Lrp5 is required for ductal stem cell activity and that loss of Lrp5 delays normal mammary development and Wnt1-induced tumorigenesis. Here we show that canonical Wnt signals through the Lrp6 co-receptor are also required for normal mouse mammary gland development. Loss of Lrp6 compromises Wnt/β-catenin signaling and interferes with mammary placode, fat pad, and branching development during embryogenesis. Heterozygosity for an inactivating mutation in Lrp6 is associated with a reduced number of terminal end buds and branches during postnatal development. While Lrp6 is expressed in both the basal and luminal mammary epithelium during embryogenesis, Lrp6 expression later becomes restricted to cells residing in the basal epithelial layer. Interestingly, these cells also express mammary stem cell markers. In humans, increased Lrp6 expression is associated with basal-like breast cancer. Taken together, our results suggest both overlapping and specific functions for Lrp5 and Lrp6 in the mammary gland.

T Cell Responses in Mammalian Diaphanous-related Formin mDia1 Knock-out Mice

Activated T cells rapidly assemble filamentous (F-) actin networks in response to ligation of the T cell receptor or upon interaction with adhesive stimuli in order to facilitate cell migration and the formation of the immune synapse. Branched filament assembly is crucial for this process and is dependent upon activation of the Arp2/3 complex by the actin nucleation-promoting factor Wiskott-Aldrich Syndrome protein (WASp). Genetic disruption of the WAS gene has been linked to hematopoietic malignancies and various cytopenias. Although the contributions of WASp and Arp2/3 to T cell responses are fairly well characterized, the role of the mammalian Diaphanous (mDia)-related formins, which both nucleate and processively elongate non-branched F-actin, has not been demonstrated. Here, we report the effects on T cell development and function following the knock out of the murine Drf1 gene encoding the canonical formin p140mDia1. Drf1-/- mice develop lymphopenia characterized by diminished T cell populations in lymphoid tissues. Consistent with a role for p140mDia1 in the regulation of the actin cytoskeleton, isolated Drf1-/- splenic T cells adhered poorly to extracellular matrix proteins and migration in response to chemotactic stimuli was completely abrogated. Both integrin and chemokine receptor expression was unaffected by Drf1-/- targeting. In response to proliferative stimuli, both thymic and splenic Drf1-/- T cells failed to proliferate; ERK1/2 activation was also diminished in activated Drf1-/- T cells. These data suggest a central role for p140mDia1 in vivo in dynamic cytoskeletal remodeling events driving normal T cell responses.

MET Kinase Inhibitor SGX523 Synergizes with Epidermal Growth Factor Receptor Inhibitor Erlotinib in a Hepatocyte Growth Factor–Dependent Fashion to Suppress Carcinoma Growth

The hepatocyte growth factor (HGF)-MET pathway supports several hallmark cancer traits, and it is frequently activated in a broad spectrum of human cancers (http://www.vai.org/met/). With the development of many cancer drugs targeting this pathway, there is a need for relevant in vivo model systems for preclinical evaluation of drug efficacy. Here, we show that production of the human HGF ligand in transgenic severe combined immunodeficient mice (hHGF(tg)-SCID mice) enhances the growth of many MET-expressing human carcinoma xenografts, including those derived from lung, breast, kidney, colon, stomach, and pancreas. In this model, the MET-specific small-molecule kinase inhibitor SGX523 partially inhibits the HGF-dependent growth of lung, breast, and pancreatic tumors. However, much greater growth suppression is achieved by combinatorial inhibition with the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. Together, these results validate the hHGF(tg)-SCID mouse model for in vivo determination of MET sensitivity to drug inhibition. Our findings also indicate that simultaneously targeting the MET and EGFR pathways can provide synergistic inhibitory effects for the treatment of cancers in which both pathways are activated.

Myeloproliferative Defects following Targeting of the Drf1 Gene Encoding the Mammalian Diaphanous–Related Formin mDia1

Rho GTPase-effector mammalian diaphanous (mDia)-related formins assemble nonbranched actin filaments as part of cellular processes, including cell division, filopodia assembly, and intracellular trafficking. Whereas recent efforts have led to thorough characterization of formins in cytoskeletal remodeling and actin assembly in vitro, little is known about the role of mDia proteins in vivo. To fill this knowledge gap, the Drf1 gene, which encodes the canonical formin mDia1, was targeted by homologous recombination. Upon birth, Drf1+/- and Drf1-/- mice were developmentally and morphologically indistinguishable from their wild-type littermates. However, both Drf1+/- and Drf1-/- developed age-dependent myeloproliferative defects. The phenotype included splenomegaly, fibrotic and hypercellular bone marrow, extramedullary hematopoiesis in both spleen and liver, and the presence of immature myeloid progenitor cells with high nucleus-to-cytoplasm ratios. Analysis of cell surface markers showed an age-dependent increase in the percentage of CD11b+-activated and CD14+-activated monocytes/macrophages in both spleen and bone marrow in Drf1+/- and Drf1-/- animals. Analysis of the erythroid compartment showed a significant increase in the proportion of splenic cells in S phase and an expansion of erythroid precursors (TER-119+ and CD71+) in Drf1-targeted mice. Overall, knocking out mDia1 expression in mice leads to a phenotype similar to human myeloproliferative syndrome (MPS) and myelodysplastic syndromes (MDS). These observations suggest that defective DRF1 expression or mDia1 function may contribute to myeloid malignancies and point to mDia1 as an attractive therapeutic target in MDS and MPS.

5q- myelodysplastic syndromes: chromosome 5q genes direct a tumor-suppression network sensing actin dynamics

Complete loss or interstitial deletions of chromosome 5 are the most common karyotypic abnormality in myelodysplastic syndromes (MDSs). Isolated del(5q)/5q– MDS patients have a more favorable prognosis than those with additional karyotypic defects, who tend to develop myeloproliferative neoplasms (MPNs) and acute myeloid leukemia. The frequency of unbalanced chromosome 5 deletions has led to the idea that 5q harbors one or more tumor-suppressor genes that have fundamental roles in the growth control of hematopoietic stem/progenitor cells (HSCs/HPCs). Cytogenetic mapping of commonly deleted regions (CDRs) centered on 5q31 and 5q32 identified candidate tumor-suppressor genes, including the ribosomal subunit RPS14, the transcription factor Egr1/Krox20 and the cytoskeletal remodeling protein, α-catenin. Although each acts as a tumor suppressor, alone or in combination, no molecular mechanism accounts for how defects in individual 5q candidates may act as a lesion driving MDS or contributing to malignant progression in MPN. One candidate gene that resides between the conventional del(5q)/5q– MDS-associated CDRs is DIAPH1 (5q31.3). DIAPH1 encodes the mammalian Diaphanous-related formin, mDia1. mDia1 has critical roles in actin remodeling in cell division and in response to adhesive and migratory stimuli. This review examines evidence, with a focus on mouse gene-targeting experiments, that mDia1 acts as a node in a tumor-suppressor network that involves multiple 5q gene products. The network has the potential to sense dynamic changes in actin assembly. At the root of the network is a transcriptional response mechanism mediated by the MADS-box transcription factor, serum response factor (SRF), its actin-binding myocardin family coactivator, MAL, and the SRF-target 5q gene, EGR1, which regulate the expression of PTEN and p53-family tumor-suppressor proteins. We hypothesize that the network provides a homeostatic mechanism balancing HPC/HSC growth control and differentiation decisions in response to microenvironment and other external stimuli.

Mechanical contributions to astrocyte adhesion using a novel in vitro model of catheter obstruction.

Drainage and diversion of cerebrospinal fluid (CSF) through shunt systems is the most common treatment for hydrocephalus, but complications due to tissue obstruction of the catheter occur in up to 61% of patients. Although shunt systems have undergone limited technological advancements to resist mammalian cell adhesion, there is a need to further reduce adhesion that can exacerbate obstruction. The high intrinsic variability in clinical studies and an inability to predict chronic adhesion of host cells in vitro while maintaining the environmental conditions observed in hydrocephalus have impeded progress. We designed the hydrocephalus shunt catheter bioreactor (HSCB) to measure inflammatory cell adhesion under experimentally manipulated conditions of CSF pressure, pulsation rate, and flow rates. For a 20-h period, astrocytes were perfused through the pulsatile flow system, and adhesion on silicone catheters was recorded. These results were compared with those obtained under static cell culture conditions. Astrocyte adhesion was significantly increased under conditions of increased flow rate (0.25 and 0.30 mL/min), and a trend toward increased adhesion was observed under conditions of elevated pressure and pulsation rate. Because the HSCB represents physiologic conditions more accurately than static cell culture, our results suggest that standard static cell culturing techniques are insufficient to model inflammatory cell adhesion on catheters used in the treatment of hydrocephalus and that changes to the ventricular microenvironment can alter the mechanisms of cellular adhesion. The HSCB represents a relevant test system and is an effective model system for the analysis of cellular adhesion and occlusion of shunt catheters.

Reduction of protein adsorption and macrophage and astrocyte adhesion on ventricular catheters by polyethylene glycol and N‐acetyl‐L‐cysteine

Cellular obstruction of poly(dimethyl)siloxane (PDMS) catheters is one of the most prevalent causes of shunt failure in the treatment of hydrocephalus. By modifying PDMS using short- and long-chain mono-functional polyethylene glycol (PEG604 and PEG5K, respectively) and N-acetyl-L-cysteine via adsorption and covalent binding (NAC and NAC/EDC/NHS, respectively), we increased surface wettability. We hypothesized that these surface modifications would inhibit protein adsorption and decrease host macrophage and astrocyte adhesion. Tested in a bioreactor set to mimic physiological flow, all modified surfaces significantly decreased albumin adsorption compared with PDMS (p < 0.05) except for PEG604-modified PDMS (p = 0.14). All four modification strategies significantly reduced (p < 0.01) fibronectin adsorption. PEG604, PEG5K, NAC, and NAC/EDC/NHS reduced the average level of macrophage adhesion by 53%, 63%, 40%, and 58% (p <.0.05 except when comparing PDMS with NAC) and astrocyte adhesion by 47%, 83%, 91%, and 72% (p < 0.05 except when comparing PDMS with PEG604), respectively. Combined with saline soak results which suggest that the surface wettability is stable over 30 days for each modification, our results are consistent with the hypothesis that these modifications decrease cell adhesion on catheters in vitro for the treatment of hydrocephalus.

Loss of RhoB Expression Enhances the Myelodysplastic Phenotype of Mammalian Diaphanous-Related Formin mDia1 Knockout Mice

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis and hyperplastic bone marrow. Complete loss or interstitial deletions of the long arm of chromosome 5 occur frequently in MDS. One candidate tumor suppressor on 5q is the mammalian Diaphanous (mDia)-related formin mDia1, encoded by DIAPH1 (5q31.3). mDia-family formins act as effectors for Rho-family small GTP-binding proteins including RhoB, which has also been shown to possess tumor suppressor activity. Mice lacking the Drf1 gene that encodes mDia1 develop age-dependent myelodysplastic features. We crossed mDia1 and RhoB knockout mice to test whether the additional loss of RhoB expression would compound the myelodysplastic phenotype. Drf1 −/− RhoB −/− mice are fertile and develop normally. Relative to age-matched Drf1 −/− RhoB +/− mice, the age of myelodysplasia onset was earlier in Drf1 −/− RhoB −/− animals—including abnormally shaped erythrocytes, splenomegaly, and extramedullary hematopoiesis. In addition, we observed a statistically significant increase in the number of activated monocytes/macrophages in both the spleen and bone marrow of Drf1 −/− RhoB −/− mice relative to Drf1 −/− RhoB +/− mice. These data suggest a role for RhoB-regulated mDia1 in the regulation of hematopoietic progenitor cells.

Effects of surface wettability, flow, and protein concentration on macrophage and astrocyte adhesion in an in vitro model of central nervous system catheter obstruction

While silicone devices have vastly improved an array of medical treatments, reactions at the tissue-substrate interface often impede their functionality. Insertion of a poly(dimethyl)siloxane (PDMS) catheter into the cerebral ventricles to drain excess cerebrospinal fluid (CSF) is the most common treatment of hydrocephalus, but shunting often fails because inflammatory tissue, choroid plexus cells, and debris grow into these central nervous system catheters and obstruct flow. We hypothesized that plasma oxidation of PDMS would inhibit macrophage and astrocyte adhesion under flow (0 to 0.3 mL/min) and protein (20.8 to 240 mg/dL) conditions similar to those observed in the physiological state. Oxidation (to increase wettability) had an inhibitory effect on macrophage cell binding (yielding a significant 88% change) that was generally more pronounced than the effect of flow (22% change) or protein concentration (3% change). In contrast, greater flow increased binding of astrocytes in most cases (yielding a significant 97% change); plasma oxidation (19% change), and protein concentration (60% change) had less pronounced effects. This study is the initial indicator that plasma oxidation of PDMS catheters may inhibit macrophage adhesion during CSF outflow but may not be as effective at inhibiting astrocyte binding.

Retrovirus-induced lymphomagenesis: a correlation between disease pathogenesis and flow cytometric analysis.

Perinatal infection with a temperature-sensitive mutant (ts-1) of Moloney murine leukemia virus (MoMuLV) results in massive splenomegaly and thymomegaly in mice and development of lymphoma in >55 % of infected pups. Previous flow cytometry studies showed a decrease in CD4(+) cells in perinatally infected pups, but cell population changes in infected animals with lymphoma compared with infected animals without lymphoma has not yet been reported. In the current study, BALB/c mice were infected with ts-1 through breast milk transmission and observed until development of clinical signs and symptoms of lymphoma and/or symptomatic ts-1 infection. Flow cytometry studies were performed on blood, spleen and thymus samples and correlated with gross morphology and histological changes, resulting from the development of lymphoma. Infected animals with lymphoma had significant decreases in CD4(+) and CD8(+) cell counts in blood and spleen compared with controls. The spleens of infected animals without lymphoma showed a decrease in CD4(+) and CD8(+) cell counts, but this was not significant compared with controls. In the thymus, CD4(+) and CD8(+) cell counts also decreased, but this was not significant in infected animals with and without lymphoma compared with controls. Markers of myeloid cell dysfunction increased in the thymus of animals with infection with and without lymphoma compared with controls. Thus, immunosuppression and CD4(+)/CD8(+) cell decreases in the spleen and thymus are associated with malignant transformation and development of lymphoma in this animal model.