Endometrial hyperplasia with loss of APC in a novel population of Lyz2-expressing mouse endometrial epithelial cells.

Loss of heterozygosity and promoter hypermethylation of APC is frequently observed in human endometrial cancer, which is the most common gynecological cancer in the USA, but its carcinogenic driver status in the endometrial epithelium has not been confirmed. We have identified a novel population of progenitor endometrial epithelial cells (EECs) in mice that express lysozyme M (LysM) and give rise to approximately 15% of all EECs in adult mice. LysM is a glycoside hydrolase that is encoded by Lyz2 and functions to protect cells from bacteria as part of the innate immune system. Its expression has been shown in a subset of hematopoietic stem cells and in specialized lung and small intestinal epithelial cells. Conditional deletion of Apc in LysM + EECs results in significantly more epithelial cells compared to wild-type mice. At 5 months of age, the ApccKO mice have enlarged uterine horns with pathology that is consistent with endometrial hyperplasia with cystic endometrial glands, non-villous luminal papillae and nuclear atypia. Nuclear accumulation of ß-catenin and ERα, both of which are known to induce endometrial hyperplasia, was observed in the EECs of the ApccKO mice. These results confirm that loss of APC in EECs can result in a phenotype similar to endometrial hyperplasia.

Targeting the mDia Formin-Assembled Cytoskeleton Is an Effective Anti-Invasion Strategy in Adult High-Grade Glioma Patient-Derived Neurospheres.

High-grade glioma (HGG, WHO Grade III⁻IV) accounts for the majority of adult primary malignant brain tumors. Failure of current therapies to target invasive glioma cells partly explains the minimal survival advantages: invasive tumors lack easily-defined surgical margins, and are inherently more chemo- and radioresistant. Much work centers upon Rho GTPase-mediated glioma invasion, yet downstream Rho effector roles are poorly understood and represent potential therapeutic targets. The roles for the mammalian Diaphanous (mDia)-related formin family of Rho effectors have emerged in invasive/metastatic disease. mDias assemble linear F-actin to promote protrusive cytoskeletal structures underlying tumor cell invasion. Small molecule mDia intramimic (IMM) agonists induced mDia functional activities including F-actin polymerization. mDia agonism inhibited polarized migration in Glioblastoma (WHO Grade IV) cells in three-dimensional (3D) in vitro and rat brain slice models. Here, we evaluate whether clinically-relevant high-grade glioma patient-derived neuro-sphere invasion is sensitive to formin agonism. Surgical HGG samples were dissociated, briefly grown as monolayers, and spontaneously formed non-adherent neuro-spheres. IMM treatment dramatically inhibited HGG patient neuro-sphere invasion, both at neuro-sphere embedding and mid-invasion assay, inducing an amoeboid morphology in neuro-sphere edge cells, while inhibiting actin- and tubulin-enriched tumor microtube formation. Thus, mDia agonism effectively disrupts multiple aspects of patient-derived HGG neuro-sphere invasion.

Differential Toxicity of mDia Formin-Directed Functional Agonists and Antagonists in Developing Zebrafish.

The mammalian Diaphanous-related (mDia) formins are cytoskeletal regulators that assemble and, in some cases, bundle filamentous actin (F-actin), as well as stabilize microtubules. The development of small molecule antagonists and agonists that interrogate mDia formin function has allowed us to investigate the roles of formins in disease states. A small molecule inhibitor of FH2 domain (SMIFH2) inhibits mDia-dependent actin dynamics and abrogates tumor cell migration and cell division in vitro and ex vivo tissue explants. mDia formin activation with small molecule intramimics IMM01/02 and mDia2-DAD peptides inhibited glioblastoma motility and invasion in vitro and ex vivo rat brain slices. However, SMIFH2, IMMs, and mDia2 DAD efficacy in vivo remains largely unexplored and potential toxicity across a range of developmental phenotypes has not been thoroughly characterized. In this study, we performed an in vivo screen of early life-stage toxicity in Danio rerio zebrafish embryos 2 days post-fertilization (dpf) in response to SMIFH2, IMM01/02, and mDia2 DAD. SMIFH2 at concentrations ≥5-10 µM induced significant defects in developing zebrafish, including shorter body lengths, tail curvature and defective tail cellularity, craniofacial malformations, pericardial edema, absent and/or compromised vasculature function and flow, depressed heart rates and increased mortality. Conversely, IMM and mDia2 DAD peptides were minimally toxic at concentrations up to 10-20 and 50 µM, respectively. SMIFH2's therapeutic potential may therefore be limited by its substantial in vivo toxicity at functional concentrations. mDia formin agonism with IMMs and mDia2 DADs may therefore be a more effective and less toxic anti-invasive therapeutic approach.

The Formin, DIAPH1, is a Key Modulator of Myocardial Ischemia/Reperfusion Injury.

The biochemical, ionic, and signaling changes that occur within cardiomyocytes subjected to ischemia are exacerbated by reperfusion; however, the precise mechanisms mediating myocardial ischemia/reperfusion (I/R) injury have not been fully elucidated. The receptor for advanced glycation end-products (RAGE) regulates the cellular response to cardiac tissue damage in I/R, an effect potentially mediated by the binding of the RAGE cytoplasmic domain to the diaphanous-related formin, DIAPH1. The aim of this study was to investigate the role of DIAPH1 in the physiological response to experimental myocardial I/R in mice. After subjecting wild-type mice to experimental I/R, myocardial DIAPH1 expression was increased, an effect that was echoed following hypoxia/reoxygenation (H/R) in H9C2 and AC16 cells. Further, compared to wild-type mice, genetic deletion of Diaph1 reduced infarct size and improved contractile function after I/R. Silencing Diaph1 in H9C2 cells subjected to H/R downregulated actin polymerization and serum response factor-regulated gene expression. Importantly, these changes led to increased expression of sarcoplasmic reticulum Ca2+ ATPase and reduced expression of the sodium calcium exchanger. This work demonstrates that DIAPH1 is required for the myocardial response to I/R, and that targeting DIAPH1 may represent an adjunctive approach for myocardial salvage after acute infarction.

Lack of Diaph3 relaxes the spindle checkpoint causing the loss of neural progenitors.

The diaphanous homologue Diaph3 (aka mDia2) is a major regulator of actin cytoskeleton. Loss of Diaph3 has been constantly associated with cytokinesis failure ascribed to impaired accumulation of actin in the cleavage furrow. Here we report that Diaph3 is required before cell fission, to ensure the accurate segregation of chromosomes. Inactivation of the Diaph3 gene causes a massive loss of cortical progenitor cells, with subsequent depletion of intermediate progenitors and neurons, and results in microcephaly. In embryonic brain extracts, Diaph3 co-immunoprecipitates with BubR1, a key regulator of the spindle assembly checkpoint (SAC). Diaph3-deficient cortical progenitors have decreased levels of BubR1 and fail to properly activate the SAC. Hence, they bypass mitotic arrest and embark on anaphase in spite of incorrect chromosome segregation, generating aneuploidy. Our data identify Diaph3 as a major guard of cortical progenitors, unravel novel functions of Diaphanous formins and add insights into the pathobiology of microcephaly.

Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments.

Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function.

Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells.

Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA-mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42-Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4-MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.

Small-molecule agonists of mammalian Diaphanous-related (mDia) formins reveal an effective glioblastoma anti-invasion strategy.

The extensive invasive capacity of glioblastoma (GBM) makes it resistant to surgery, radiotherapy, and chemotherapy and thus makes it lethal. In vivo, GBM invasion is mediated by Rho GTPases through unidentified downstream effectors. Mammalian Diaphanous (mDia) family formins are Rho-directed effectors that regulate the F-actin cytoskeleton to support tumor cell motility. Historically, anti-invasion strategies focused upon mDia inhibition, whereas activation remained unexplored. The recent development of small molecules directly inhibiting or activating mDia-driven F-actin assembly that supports motility allows for exploration of their role in GBM. We used the formin inhibitor SMIFH2 and mDia agonists IMM-01/-02 and mDia2-DAD peptides, which disrupt autoinhibition, to examine the roles of mDia inactivation versus activation in GBM cell migration and invasion in vitro and in an ex vivo brain slice invasion model. Inhibiting mDia suppressed directional migration and spheroid invasion while preserving intrinsic random migration. mDia agonism abrogated both random intrinsic and directional migration and halted U87 spheroid invasion in ex vivo brain slices. Thus mDia agonism is a superior GBM anti-invasion strategy. We conclude that formin agonism impedes the most dangerous GBM component-tumor spread into surrounding healthy tissue. Formin activation impairs novel aspects of transformed cells and informs the development of anti-GBM invasion strategies.

Homozygous loss of DIAPH1 is a novel cause of microcephaly in humans.

The combination of family-based linkage analysis with high-throughput sequencing is a powerful approach to identifying rare genetic variants that contribute to genetically heterogeneous syndromes. Using parametric multipoint linkage analysis and whole exome sequencing, we have identified a gene responsible for microcephaly (MCP), severe visual impairment, intellectual disability, and short stature through the mapping of a homozygous nonsense alteration in a multiply-affected consanguineous family. This gene, DIAPH1, encodes the mammalian Diaphanous-related formin (mDia1), a member of the diaphanous-related formin family of Rho effector proteins. Upon the activation of GTP-bound Rho, mDia1 generates linear actin filaments in the maintenance of polarity during adhesion, migration, and division in immune cells and neuroepithelial cells, and in driving tangential migration of cortical interneurons in the rodent. Here, we show that patients with a homozygous nonsense DIAPH1 alteration (p.Gln778*) have MCP as well as reduced height and weight. diap1 (mDia1 knockout (KO))-deficient mice have grossly normal body and brain size. However, our histological analysis of diap1 KO mouse coronal brain sections at early and postnatal stages shows unilateral ventricular enlargement, indicating that this mutant mouse shows both important similarities as well as differences with human pathology. We also found that mDia1 protein is expressed in human neuronal precursor cells during mitotic cell division and has a major impact in the regulation of spindle formation and cell division.

The formin DIAPH1 (mDia1) regulates megakaryocyte proplatelet formation by remodeling the actin and microtubule cytoskeletons.

Megakaryocytes are highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rho-associated protein kinase (Rock)/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that 2 major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly, but also in the regulation of microtubule stability and dynamics during PPF.

A novel role for p115RhoGEF in regulation of epithelial plasticity.

Epithelial plasticity plays a critical role during physiological processes, such as wound healing and tissue regeneration, and dysregulation of epithelial plasticity can lead to pathological conditions, such as cancer. Cell-cell junctions are a critical feature of epithelial cells and loss of junctions is associated with acquisition of mesenchymal features, such as enhanced protrusion and migration. Although Rho has been implicated in regulation of junctions in epithelial cells, the role of Rho signaling in the regulation of epithelial plasticity has not been understood. We show that members of the RGS RhoGEFs family play a critical role in regulation of epithelial cell-cell junctions in breast epithelial cells. We identify a novel role for p115RhoGEF in regulation of epithelial plasticity. Loss of p115RhoGEF leads to decreased junctional E-cadherin and enhanced protrusiveness and migration. Conversely, overexpression of p115RhoGEF enhanced junctional E-cadherin and inhibited cell protrusion and migration. siRNA screen of 23 Rho effectors showed that members of the Diaphanous-Related Formin (DRF) family are required for p115RhoGEF-mediated changes in epithelial plasticity. Thus, our data indicates a novel role for p115RhoGEF in regulation of epithelial plasticity, which is dependent on Rho-DRF signaling module.

Mammalian diaphanous-related formin 1 regulates GSK3ß-dependent microtubule dynamics required for T cell migratory polarization.

The mammalian diaphanous-related formin (mDia1), a Rho-regulated cytoskeletal modulator, has been shown to promote T lymphocyte chemotaxis and interaction with antigen presenting cells, but the mechanisms underpinning mDia1 roles in these processes have not been defined. Here we show that mDia1(-/-) T cells exhibit impaired lymphocyte function-associated antigen 1 (LFA-1)-mediated T cell adhesion, migration and in vivo trafficking. These defects are associated with impaired microtubule (MT) polarization and stabilization, altered MT dynamics and reduced peripheral clustering of the MT plus-end-protein, adenomatous polyposis coli (APC) in migrating T cells following LFA-1-engagement. Loss of mDia1 also leads to impaired inducible inactivation of the glycogen synthase kinase (GSK) 3ß as well as hyperphosphorylation and reduced levels of APC in migrating T cells. These findings identify essential roles for the mDia1 formin in modulating GSK3ß-dependent MT contributions to induction of T-cell polarity, adhesion and motility.

Small-molecule intramimics of formin autoinhibition: a new strategy to target the cytoskeletal remodeling machinery in cancer cells.

Although the cancer cell cytoskeleton is a clinically validated target, few new strategies have emerged for selectively targeting cell division by modulating the cytoskeletal structure, particularly ways that could avoid the cardiotoxic and neurotoxic effects of current agents such as taxanes. We address this gap by describing a novel class of small-molecule agonists of the mammalian Diaphanous (mDia)-related formins, which act downstream of Rho GTPases to assemble actin filaments, and their organization with microfilaments to establish and maintain cell polarity during migration and asymmetric division. GTP-bound Rho activates mDia family members by disrupting the interaction between the DID and DAD autoregulatory domains, which releases the FH2 domain to modulate actin and microtubule dynamics. In screening for DID-DAD disruptors that activate mDia, we identified two molecules called intramimics (IMM-01 and -02) that were sufficient to trigger actin assembly and microtubule stabilization, serum response factor-mediated gene expression, cell-cycle arrest, and apoptosis. In vivo analysis of IMM-01 and -02 established their ability to slow tumor growth in a mouse xenograft model of colon cancer. Taken together, our work establishes the use of intramimics and mDia-related formins as a new general strategy for therapeutic targeting of the cytoskeletal remodeling machinery of cancer cells.

Formin mDia1 mediates vascular remodeling via integration of oxidative and signal transduction pathways.

RATIONALE: The mammalian diaphanous-related formin (mDia1), governs microtubule and microfilament dynamics while functioning as an effector for Rho small GTP-binding proteins during key cellular processes such as adhesion, cytokinesis, cell polarity, and morphogenesis. The cytoplasmic domain of the receptor for advanced glycation endproducts binds to the formin homology 1 domain of mDia1; mDia1 is required for receptor for advanced glycation endproducts ligand-induced cellular migration in transformed cells. OBJECTIVE: Because a key mechanism in vascular remodeling is the induction of smooth muscle cell migration, we tested the role of mDia1 in this process. METHODS AND RESULTS: We report that endothelial denudation injury to the murine femoral artery significantly upregulates mDia1 mRNA transcripts and protein in the injured vessel, particularly in vascular smooth muscle cells within the expanding neointima. Loss of mDia1 expression significantly reduces pathological neointimal expansion consequent to injury. In primary murine aortic smooth muscle cells, mDia1 is required for receptor for advanced glycation endproducts ligand-induced membrane translocation of c-Src, which leads to Rac1 activation, redox phosphorylation of AKT/glycogen synthase kinase 3ß, and consequent smooth muscle cell migration. CONCLUSIONS: We conclude that mDia1 integrates oxidative and signal transduction pathways triggered, at least in part, by receptor for advanced glycation endproducts ligands, thereby regulating pathological neointimal expansion.

Identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory response.

The antimalaria drug chloroquine has been used as an anti-inflammatory agent for treating systemic lupus erythematosus and rheumatoid arthritis. We report that chloroquine promoted the transrepression of proinflammatory cytokines by the glucocorticoid receptor (GR). In a mouse collagen-induced arthritis model, chloroquine enhanced the therapeutic effects of glucocorticoid treatment. By inhibiting lysosome function, chloroquine synergistically activated glucocorticoid signaling. Lysosomal inhibition by either bafilomycin A1 (an inhibitor of the vacuolar adenosine triphosphatase) or knockdown of transcription factor EB (TFEB, a master activator of lysosomal biogenesis) mimicked the effects of chloroquine. The abundance of the GR, as well as that of the androgen receptor and estrogen receptor, correlated with changes in lysosomal biogenesis. Thus, we showed that glucocorticoid signaling is regulated by lysosomes, which provides a mechanistic basis for treating inflammation and autoimmune diseases with a combination of glucocorticoids and lysosomal inhibitors.

The role of formins in human disease.

Formins are a conserved family of proteins that play key roles in cytoskeletal remodeling. They nucleate and processively elongate non-branched actin filaments and also modulate microtubule dynamics. Despite their significant contributions to cell biology and development, few studies have directly implicated formins in disease pathogenesis. This review highlights the roles of formins in cell division, migration, immunity, and microvesicle formation in the context of human disease. In addition, we discuss the importance of controlling formin activity and protein expression to maintain cell homeostasis.

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.

Ubiquitin-mediated degradation of the formin mDia2 upon completion of cell division.

Formins assemble non-branched actin filaments and modulate microtubule dynamics during cell migration and cell division. At the end of mitosis formins contribute to the generation of actin filaments that form the contractile ring. Rho small GTP-binding proteins activate mammalian diaphanous-related (mDia) formins by directly binding and disrupting an intramolecular autoinhibitory mechanism. Although the Rho-regulated activation mechanism is well characterized, little is known about how formins are switched off. Here we reveal a novel mechanism of formin regulation during cytokinesis based on the following observations; 1) mDia2 is degraded at the end of mitosis, 2) mDia2 is targeted for disposal by post-translational ubiquitin modification, 3) forced expression of activated mDia2 yields binucleate cells due to failed cytokinesis, and 4) the cytokinesis block is dependent upon mDia2-mediated actin assembly as versions of mDia2 incapable of nucleating actin but that still stabilize microtubules have no effect on cytokinesis. We propose that the tight control of mDia2 expression and ubiquitin-mediated degradation is essential for the completion of cell division. Because of the many roles for formins in cell morphology, we discuss the relevance of mDia protein turnover in other processes where ubiquitin-mediated proteolysis is an essential component.