Non-autonomous Functions Research Articles

Targeting YAP-Dependent MDSC Infiltration Impairs Tumor Progression.

The signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo-YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP-TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5-CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer. We demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo-YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5-CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival.

Glial TDP-43 regulates axon wrapping, GluRIIA clustering and fly motility by autonomous and non-autonomous mechanisms.

Alterations in the glial function of TDP-43 are becoming increasingly associated with the neurological symptoms observed in Amyotrophic Lateral Sclerosis (ALS), however, the physiological role of this protein in the glia or the mechanisms that may lead to neurodegeneration are unknown. To address these issues, we modulated the expression levels of TDP-43 in the Drosophila glia and found that the protein was required to regulate the subcellular wrapping of motoneuron axons, promote synaptic growth and the formation of glutamate receptor clusters at the neuromuscular junctions. Interestingly, we determined that the glutamate transporter EAAT1 mediated the regulatory functions of TDP-43 in the glia and demonstrated that genetic or pharmacological compensations of EAAT1 activity were sufficient to modulate glutamate receptor clustering and locomotive behaviors in flies. The data uncovers autonomous and non-autonomous functions of TDP-43 in the glia and suggests new experimentally based therapeutic strategies in ALS.

Abstract 198: Aberrant Endothelial-myocardial Crosstalk Causes Hypertrophy in Noonan Syndrome With Multiple Lentigines

Congenital heart disease (CHD) is the most common birth defect worldwide; however, underlying mechanisms remain unknown. Loss-of-function mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP2, are implicated in CHD and cause Noonan Syndrome with Multiple Lentigines (NSML). NSML presents with multiple cardiac defects, including hypertrophy. Here, we found that the NSML-associated adult-onset cardiac hypertrophy stems from aberrant signaling originating from developing endocardium. Embryonic NSML hearts showed diminished trabeculation and valvular hyperplasia, defects recapitulated in endocardial-, but not myocardial- or neural crest-, specific NSML mice. NSML hearts also developed ventricular septal defects, a phenotype reproduced only in myocardial-specific NSML hearts, suggesting NSML mutations have both cell autonomous and non-autonomous functions in cardiac development. Importantly, endocardial-specific expression of NSML was sufficient to induce adult-onset cardiac hypertrophy. Mechanistically, we observed aberrant AKT activity in NSML embryos, with decreased downstream FOXP1/FGF and NOTCH1/EPHB2 signaling, two pathways necessary for reciprocal crosstalk between developing endocardium and myocardium. Taken together, our data provide the first functional and disease-based evidence to suggest that critical mechanisms exist to control endocardial-myocardial crosstalk, the aberrant regulation of which may lead to CHD and adult-onset cardiac disease.

Non-autonomous conformal iterated function systems and Moran-set constructions

We study non-autonomous conformal iterated function systems with finite or countable infinite alphabet alike. These differ from the usual (autonomous) iterated function systems in that the contractions applied at each step in time are allowed to vary. (In the case where all maps are affine similarities, the resulting system is also called a “Moran set construction”.) We shall show that, given a suitable restriction on the growth of the number of contractions used at each step, the Hausdorff dimension of the limit set of such a system is determined by an equation known as Bowen’s formula. We also give examples that show the optimality of our results. In addition, we prove Bowen’s formula for a class of infinite alphabet-systems and deal with Hausdorff measures for finite systems, as well as continuity of topological pressure and Hausdorff dimension for both finite and infinite systems. In particular we strengthen the existing continuity results for infinite autonomous systems. As a simple application of our results, we show that, for a transcendental meromorphic function f f , the Hausdorff dimension of the set of transitive points (i.e., those points whose orbits are dense in the Julia set) is bounded from below by the hyperbolic dimension of f f .

Autonomous and nonautonomous regulation of Wnt-mediated neuronal polarity by the C. elegans Ror kinase CAM-1

Wnts are a conserved family of secreted glycoproteins that regulate various developmental processes in metazoans. Three of the five Caenorhabditis elegans Wnts, CWN-1, CWN-2 and EGL-20, and the sole Wnt receptor of the Ror kinase family, CAM-1, are known to regulate the anterior polarization of the mechanosensory neuron ALM. Here we show that CAM-1 and the Frizzled receptor MOM-5 act in parallel pathways to control ALM polarity. We also show that CAM-1 has two functions in this process: an autonomous signaling function that promotes anterior polarization and a nonautonomous Wnt-antagonistic function that inhibits anterior polarization. These antagonistic activities can account for the weak ALM phenotypes displayed by cam-1 mutants. Our observations suggest that CAM-1 could function as a Wnt receptor in many developmental processes, but the analysis of cam-1 mutants may fail to reveal CAM-1's role as a receptor in these processes because of its Wnt-antagonistic activity. In this model, loss of CAM-1 results in increased levels of Wnts that act through other Wnt receptors, masking CAM-1's autonomous role as a Wnt receptor.

Subcellular transcriptome alterations in a cell culture model of spinal muscular atrophy point to widespread defects in axonal growth and presynaptic differentiation.

Neuronal function critically depends on coordinated subcellular distribution of mRNAs. Disturbed mRNA processing and axonal transport has been found in spinal muscular atrophy and could be causative for dysfunction and degeneration of motoneurons. Despite the advances made in characterizing the transport mechanisms of several axonal mRNAs, an unbiased approach to identify the axonal repertoire of mRNAs in healthy and degenerating motoneurons has been lacking. Here we used compartmentalized microfluidic chambers to investigate the somatodendritic and axonal mRNA content of cultured motoneurons by microarray analysis. In axons, transcripts related to protein synthesis and energy production were enriched relative to the somatodendritic compartment. Knockdown of Smn, the protein deficient in spinal muscular atrophy, produced a large number of transcript alterations in both compartments. Transcripts related to immune functions, including MHC class I genes, and with roles in RNA splicing were up-regulated in the somatodendritic compartment. On the axonal side, transcripts associated with axon growth and synaptic activity were down-regulated. These alterations provide evidence that subcellular localization of transcripts with axonal functions as well as regulation of specific transcripts with nonautonomous functions is disturbed in Smn-deficient motoneurons, most likely contributing to the pathophysiology of spinal muscular atrophy.

Ccm3, a gene associated with cerebral cavernous malformations, is required for neuronal migration

Loss of function of cerebral cavernous malformation 3 (CCM3) results in an autosomal dominant cerebrovascular disorder. Here, we uncover a developmental role for CCM3 in regulating neuronal migration in the neocortex. Using cell type-specific gene inactivation in mice, we show that CCM3 has both cell autonomous and cell non-autonomous functions in neural progenitors and is specifically required in radial glia and newly born pyramidal neurons migrating through the subventricular zone, but not in those migrating through the cortical plate. Loss of CCM3 function leads to RhoA activation, alterations in the actin and microtubule cytoskeleton affecting neuronal morphology, and abnormalities in laminar positioning of primarily late-born neurons, indicating CCM3 involvement in radial glia-dependent locomotion and possible interaction with the Cdk5/RhoA pathway. Thus, we identify a novel cytoplasmic regulator of neuronal migration and demonstrate that its inactivation in radial glia progenitors and nascent neurons produces severe malformations of cortical development.

Ccm3, a gene associated with cerebral cavernous malformations, is required for neuronal migration

Loss of function of cerebral cavernous malformation 3 (CCM3) results in an autosomal dominant cerebrovascular disorder. Here, we uncover a developmental role for CCM3 in regulating neuronal migration in the neocortex. Using cell type-specific gene inactivation in mice, we show that CCM3 has both cell autonomous and cell non-autonomous functions in neural progenitors and is specifically required in radial glia and newly born pyramidal neurons migrating through the subventricular zone, but not in those migrating through the cortical plate. Loss of CCM3 function leads to RhoA activation, alterations in the actin and microtubule cytoskeleton affecting neuronal morphology, and abnormalities in laminar positioning of primarily late-born neurons, indicating CCM3 involvement in radial glia-dependent locomotion and possible interaction with the Cdk5/RhoA pathway. Thus, we identify a novel cytoplasmic regulator of neuronal migration and demonstrate that its inactivation in radial glia progenitors and nascent neurons produces severe malformations of cortical development.

Abstract 3902: MDA-9/syntenin and IGFBP-2 promote angiogenesis in human melanoma.

Abstract Vascularization of tumors promotes not only their survival and growth, but also facilitates metastases from primary to distant sites. For that reason, understanding the molecular determinants controlling tumor angiogenesis is mandatory to develop clinically efficacious angiogenesis inhibitors for cancer therapy. Melanoma differentiation associated gene or mda-9, also known as syntenin, is a multifunctional scaffold protein that cross talks with a plethora of proteins and regulates diverse physiological and pathological processes, including tumor progression and metastasis. Since, tumor angiogenesis promotes metastasis, we hypothesized that in addition to augmenting invasion and migration, MDA-9/Syntenin might also promote angiogenesis thereby facilitating tumor progression and metastasis. Genetic (gain-of-function and loss-of-function) and pharmacological approaches were employed to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, chicken choriallantoic membrane assays (CAM assays) and xenograft tumor animal models. Employing both genetic and chemical approaches we now demonstrate that MDA-9/Syntenin expression correlates with in vitro and in vivo (tumorigenic/metastatic) transformed/invasive phenotypes of human melanoma. Additionally, our immunohistochemical and in vivo CAM assays confirm that angiogenesis is an essential component of MDA-9/Syntenin-induced tumor progression. To define how MDA-9/Syntenin regulates angiogenesis we identified and analyzed several potential downstream gene targets of MDA-9/Syntenin. One gene implicated in induction of vasculogenesis is insulin growth factor binding protein-2 (IGFBP-2), which is transcriptionally regulated by hypoxia inducible factor-1 as a consequence of MDA-9/Syntenin and Src interaction. IGFBP-2 transcriptionally regulates VEGF-A in an AKT-dependent manner through interaction with V3 integrin resulting in angiogenesis. Our studies delineate an unanticipated cell non-autonomous function of MDA-9/Syntenin in the context of angiogenesis by augmenting expression of several pro-angiogenic factors including IGFBP-2, which may provide a complementary way to promote metastasis. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (non-autonomous). Citation Format: Swadesh K. Das, Timothy P. Kegelman, Prasanna K. Santhekadur, Santanu Dasgupta, Paul Dent, Steven Grant, Luni Emdad, Devanand Sarkar, Paul B. Fisher, Maurizio Pellecchia. MDA-9/syntenin and IGFBP-2 promote angiogenesis in human melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3902. doi:10.1158/1538-7445.AM2013-3902

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

MDA-9/Syntenin and IGFBP-2 Promote Angiogenesis in Human Melanoma

Melanoma differentiation-associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacologic approaches were used to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma, and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, chorioallantoic membrane (CAM) assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several proangiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the extracellular matrix (ECM), activating Src and FAK resulting in activation by phosphorylation of Akt, which induces hypoxia inducible factor 1-α (HIF-1α). The HIF-1α activates transcription of insulin growth factor-binding protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell nonautonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (nonautonomous).

Fat Body dSir2 Regulates Muscle Mitochondrial Physiology and Energy Homeostasis Nonautonomously and Mimics the Autonomous Functions of dSir2 in Muscles

Sir2 is an evolutionarily conserved NAD(+)-dependent deacetylase which has been shown to play a critical role in glucose and fat metabolism. In this study, we have perturbed Drosophila Sir2 (dSir2) expression, bidirectionally, in muscles and the fat body. We report that dSir2 plays a critical role in insulin signaling, glucose homeostasis, and mitochondrial functions. Importantly, we establish the nonautonomous functions of fat body dSir2 in regulating mitochondrial physiology and insulin signaling in muscles. We have identified a novel interplay between dSir2 and dFOXO at an organismal level, which involves Drosophila insulin-like peptide (dILP)-dependent insulin signaling. By genetic perturbations and metabolic rescue, we provide evidence to illustrate that fat body dSir2 mediates its effects on the muscles via free fatty acids (FFA) and dILPs (from the insulin-producing cells [IPCs]). In summary, we show that fat body dSir2 is a master regulator of organismal energy homeostasis and is required for maintaining the metabolic regulatory network across tissues.

Tbx3 directs cell fate decision towards mesendoderm, pancreas and liver in mouse embryonic stem cells

Pluripotency depends on networks of key transcriptional regulators. Recent reports demonstrate additional functions of pluripotency-associated factors during early lineage commitment. The T-box transcription factor Tbx3 has been implicated in the regulation of embryonic stem cell (ESC) self-renewal and pluripotency. In this study, we demonstrate previously unappreciated roles of Tbx3 for the specification of mesendoderm. Tbx3 is dynamically expressed in murine embryos and in differentiating ESCs, concomitant with specification-onset of the mesendoderm lineages. Transcriptional profiling of differentiating ESCs during forced Tbx3 expression indicates enhanced mesendoderm specification and enrichment of mature endoderm derivatives namely pancreas and liver in prolonged differentiation cultures. Tbx3 directly activates key factors of lineage specification as assessed by ChIP and reporter assays and additionally promotes mesendoderm fate in a cell non-autonomous fashion by enhancing Nodal signaling. In summary, our data suggest cell autonomous and cell non-autonomous functions of Tbx3 for early lineage specification towards mesendoderm, thus, establishing a new path towards in vitro engineering of pancreas and liver for regenerative medicine.

Periodic orbits of the fourth-order non-autonomous differential equation [formula omitted

We provide sufficient conditions for the existence of periodic solutions of the fourth-order differential equationu⁗+qu″+pu=εF(t,u,u′,u″,u‴),where q,p and ε are real parameters, ε is small and F is a nonlinear non-autonomous periodic function with respect to t. Moreover we provide some applications.

Identification of NOG as a Specific Breast Cancer Bone Metastasis-supporting Gene

Metastasis requires numerous biological functions that jointly provide tumor cells from a primary site to seed and colonize a distant organ. Some of these activities are selected for in the primary site, whereas others are acquired at the metastatic niche. We provide molecular evidence showing that the BMP inhibitor, NOG, provides metastatic breast cancer cells with the ability to colonize the bone. NOG expression is acquired during the late events of metastasis, once cells have departed from the primary site, because it is not enriched in primary tumors with high risk of bone relapse. On the contrary, breast cancer bone metastatic lesions do select for high levels of NOG expression when compared with metastasis to the lung, liver, and brain. Pivotal to the bone colonization functions is the contribution of NOG to metastatic autonomous and nonautonomous cell functions. Using genetic approaches, we show that when NOG is expressed in human breast cancer cells, it facilitates bone colonization by fostering osteoclast differentiation and bone degradation and also contributes to metastatic lesions reinitiation. These findings reveal how aggressive cancer cell autonomous and nonautonomous functions can be mechanistically coupled to greater bone metastatic potential.

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions.

Limit cycles of the sixth-order non-autonomous differential equation

We provide sufficient conditions for the existence of periodic solutions of the sixth-order differential equationx(6)+(1+p2+q2)x....+(p2+q2+p2q2)x¨+p2q2x=εF(t,x,x˙,x¨,x...,x....,x.....),where p and q are rational numbers different from 1, 0, −1 and p≠q, ε is small and F is a nonlinear non-autonomous periodic function. Moreover we provide some applications.

Distinct Cell Guidance Pathways Controlled by the Rac and Rho GEF Domains of UNC-73/TRIO in Caenorhabditis elegans

The cytoskeleton regulator UNC-53/NAV2 is required for both the anterior and posterior outgrowth of several neurons as well as that of the excretory cell while the kinesin-like motor VAB-8 is essential for most posteriorly directed migrations in Caenorhabditis elegans. Null mutations in either unc-53 or vab-8 result in reduced posterior excretory canal outgrowth, while double null mutants display an enhanced canal extension defect, suggesting the genes act in separate pathways to control this posteriorly directed outgrowth. Genetic analysis of putative interactors of UNC-53 or VAB-8, and cell-specific rescue experiments suggest that VAB-8, SAX-3/ROBO, SLT-1/Slit, and EVA-1 are functioning together in the outgrowth of the excretory canals, while UNC-53 appears to function in a parallel pathway with UNC-71/ADAM. The known VAB-8 interactor, the Rac/Rho GEF UNC-73/TRIO operates in both pathways, as isoform specific alleles exhibit enhancement of the phenotype in double-mutant combination with either unc-53 or vab-8. On the basis of these results, we propose a bipartite model for UNC-73/TRIO activity in excretory canal extension: a cell autonomous function that is mediated by the Rho-specific GEF domain of the UNC-73E isoform in conjunction with UNC-53 and UNC-71 and a cell nonautonomous function that is mediated by the Rac-specific GEF domain of the UNC-73B isoform, through partnering with VAB-8 and the receptors SAX-3 and EVA-1.

MaizeRough Endosperm3Encodes an RNA Splicing Factor Required for Endosperm Cell Differentiation and Has a Nonautonomous Effect on Embryo Development

Endosperm and embryo development are coordinated via epigenetic regulation and signaling between these tissues. In maize (Zea mays), the endosperm-embryo signals are not known, but endosperm cellularization is a key event for embryos to form shoots and roots. We screened seed mutants for nonautonomous functions in endosperm and embryo development with genetically nonconcordant seeds and identified the recessive mutant rough endosperm3 (rgh3). The wild-type Rgh3 allele is required in the endosperm for embryos to develop and has an autonomous role in embryo and seedling development. Endosperm cell differentiation is defective in rgh3. Results from endosperm cell culture indicate that rgh3 mutants remain in a proliferative state through mid-seed development. Rgh3 encodes the maize U2AF(35) Related Protein (URP), an RNA splicing factor involved in both U2 and U12 splicing. The Rgh3 allele produces at least 19 alternative splice variants with only one isoform encoding a full-length ortholog to URP. The full-length RGH3α isoform localizes to the nucleolus and displays a speckled pattern within the nucleoplasm, and RGH3α colocalizes with U2AF(65). A survey of alternatively spliced transcripts found that, in the rgh3 mutant, a fraction of noncanonical splicing events are altered. Our findings suggest that differentiation of maize endosperm cell types is necessary for embryos to develop. The molecular cloning of Rgh3 suggests that alternative RNA splicing is needed for cell differentiation, development, and plant viability.

Planar polarity pathway and Nance-Horan syndrome-like 1b have essential cell-autonomous functions in neuronal migration

Components of the planar cell polarity (PCP) pathway are required for the caudal tangential migration of facial branchiomotor (FBM) neurons, but how PCP signaling regulates this migration is not understood. In a forward genetic screen, we identified a new gene, nhsl1b, required for FBM neuron migration. nhsl1b encodes a WAVE-homology domain-containing protein related to human Nance-Horan syndrome (NHS) protein and Drosophila GUK-holder (Gukh), which have been shown to interact with components of the WAVE regulatory complex that controls cytoskeletal dynamics and with the polarity protein Scribble, respectively. Nhsl1b localizes to FBM neuron membrane protrusions and interacts physically and genetically with Scrib to control FBM neuron migration. Using chimeric analysis, we show that FBM neurons have two modes of migration: one involving interactions between the neurons and their planar-polarized environment, and an alternative, collective mode involving interactions between the neurons themselves. We demonstrate that the first mode of migration requires the cell-autonomous functions of Nhsl1b and the PCP components Scrib and Vangl2 in addition to the non-autonomous functions of Scrib and Vangl2, which serve to polarize the epithelial cells in the environment of the migrating neurons. These results define a role for Nhsl1b as a neuronal effector of PCP signaling and indicate that proper FBM neuron migration is directly controlled by PCP signaling between the epithelium and the migrating neurons.