Michael B. Armstrong

The PICALM Protein Plays a Key Role in Iron Homeostasis and Cell Proliferation

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimers disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM’s function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.

Effect of pentoxifylline on the flow of polymorphonuclear leukocytes through a model capillary.

Pentoxifylline is a methylxanthine derivative used to increase blood flow in peripheral atherosclerosis. Pentoxifylline is known to increase whole blood filtration rate, and recent evidence suggests that pentoxifylline increases the filtration rate of polymorphonuclear leukocytes (PMNs). The purpose of this study was to directly observe and quantitate the effect of pentoxifylline on the flow of individual PMNs into a model capillary. Short-term incubation of human PMNs with 10 mM pentoxifylline inhibited cell activation, as judged by a significant reduction in the number of neutrophils forming pseudopods. Furthermore, incubation of PMNs from 6 healthy men with 0.1, 1.0 and 10 mM pentoxifylline significantly decreased the time required for individual cells to be aspirated into a 4 μm pipet under constant pressure by 16 ± 5%, 21 ± 7%, and 41 ± 8%, respectively (mean ± SEM, p≤0.05), compared with control. These experiments are the first direct demonstration of increased deformability in neutrophils treated with pentoxifylline. The results are consistent with the hypothesis that the beneficial effect of pentoxifylline on microvascular perfusion is partly due to an inhibition of PMN stiffness and activation.

Type III TGF-β receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the pediatric tumor neuroblastoma remain unclear. Comparison of mRNA from benign neuroblastic tumors and neuroblastomas revealed that expression of the type III TGF-β receptor (TGFBR3) decreases with advancing stage of neuroblastoma and this loss correlates with a poorer prognosis. Patients with MYCN oncogene amplification and low TGFBR3 expression were more likely to have an adverse outcome. In vitro, TβRIII expression was epigenetically suppressed by MYCN-mediated recruitment of histone deacetylases to regions of the TGFBR3 promoter. TβRIII bound FGF2 and exogenous FGFR1, which promoted neuronal differentiation of neuroblastoma cells. TβRIII and FGF2 cooperated to induce expression of the transcription factor inhibitor of DNA binding 1 via Erk MAPK. TβRIII-mediated neuronal differentiation suppressed cell proliferation in vitro as well as tumor growth and metastasis in vivo. These studies characterize a coreceptor function for TβRIII in FGF2-mediated neuronal differentiation, while identifying potential therapeutic targets and clinical biomarkers for neuroblastoma.

Neuroblastoma in a pediatric patient with a microduplication of 2p involving the MYCN locus

Neuroblastoma is the most common solid tumor of infancy, and mutations in several genes have been implicated as playing a role in tumor development. Here, we describe a pediatric patient with a constitutional microduplication of 2p24.3 who developed Stage 4 neuroblastoma at age 11 months. He represents the sixth patient described in the literature with partial trisomy 2p and neuroblastoma. All previous cases had duplication events spanning two genes implicated in neuroblastoma, MYCN and ALK. Our patient is unique because his duplicated region includes the MYCN gene only; the ALK gene is unaffected. These data, combined with the relatively high incidence of neuroblastoma reported in partial trisomy 2p patients, support the notion that MYCN duplication should be added to the growing list of genetic factors associated with an increased risk of neuroblastoma. The mechanism of increased risk is unclear, but the fact that our patient had dramatic amplification of MYCN in his tumor suggests that a germline duplication might predispose to further amplification. Additionally, our patient has several morphologic features common to patients with partial trisomy 2p including high forehead, hypertelorism, postaxial polydactyly, and developmental delay despite having a microduplication spanning approximately 1 Mb and including just three intact genes. This case may therefore help further delineate the genotype–phenotype correlations associated with partial trisomy 2p.

Hereditary Intrinsic Factor Deficiency in Chaldeans

Juvenile vitamin B(12) or cobalamin (Cbl) deficiency is notoriously difficult to explain due to numerous acquired and inherited causes. The consequences of insufficient Cbl are megaloblastic anemia, nutrient malabsorption, and neurological problems. The treatment is straightforward with parenteral Cbl supplementation that resolves most health issues without an urgent need to clarify their cause. Aside from being clinically unsatisfying, failing to elucidate the basis of Cbl deficiency means important information regarding recurrence risk is not available to the individual if the cause is contagious or inherited. Acquired causes have largely disappeared in the Modern World because they were mostly due to parasites or malnutrition. Today, perhaps the most common causes of juvenile Cbl deficiency are Imerslund-Gräsbeck syndrome and inherited intrinsic factor deficiency (IFD). Three genes are involved and genetic testing is complicated and not widely available. We used self-identified ancestry to accelerate and confirm the genetic diagnosis of IFD in three families of Chaldean origin. A founder mutation limited to Chaldeans from Iraq in the intrinsic factor gene GIF was identified as the cause. World events reshape the genetic structure of populations and inherited diseases in many ways. In this case, all the patients were diagnosed in the USA among recent immigrants from a single region. While IFD itself is not restricted to one kind of people, certain mutations are limited in their range but migrations relocate them along with their host population. As a result, self-identified ancestry as a stratifying characteristic should perhaps be considered in diagnostic strategies for rare genetic disorders.

Mxi1 and Mxi1-0 Antagonize N-Myc Function and Independently Mediate Apoptosis in Neuroblastoma

Neuroblastoma (NB) is the third most common malignancy of childhood, and outcomes for children with advanced disease remain poor; amplification of the MYCN gene portends a particularly poor prognosis. Mxi1 antagonizes N-Myc by competing for binding to Max and E-boxes. Unlike N-Myc, Mxi1 mediates transcriptional repression and suppresses cell proliferation. Mxi1 and Mxi1-0 (an alternatively transcribed Mxi1 isoform) share identical Max and DNA binding domains but differ in amino-terminal sequences. Because of the conservation of these critical binding domains, we hypothesized that Mxi1-0 antagonizes N-Myc activity similar to Mxi1. SHEP NB cells and SHEP cells stably transfected with MYCN (SHEP/MYCN) were transiently transfected with vectors containing full-length Mxi1, full-length Mxi1-0, or the common Mxi domain encoded by exons 2 to 6 (ex2-6). After incubation in low serum, parental SHEP/MYCN cell numbers were reduced compared with SHEP cells. Activated caspase-3 staining and DNA fragmentation ELISA confirmed that SHEP/MYCN cells undergo apoptosis in low serum, while SHEP/MYCN cells transfected with Mxi1 or Mxi1-0 do not. However, SHEP/MYCN cells transfected with Mxi1 or Mxi1-0 and grown in normal serum showed proliferation rates similar to SHEP cells. Mxi ex2-6 did not affect cell number in low or normal serum, suggesting that amino terminal domains of Mxi1 and Mxi1-0 are critical for antagonism. In the absence of N-Myc, Mxi1 and Mxi1-0 induce apoptosis independently through the caspase-8–dependent extrinsic pathway, while N-Myc activates the caspase-9–dependent intrinsic pathway. Together, these data indicate that Mxi1 and Mxi1-0 antagonize N-Myc but also independently impact NB cell survival.

Abstract B32: The role of Exon 0 in mediating Mxi0 activity in neuroblastoma

Background: Neuroblastoma is the most common extracranial malignancy of childhood. The Myc family of proteins regulates cell growth and proliferation and has been implicated in the etiology of many cancers. MYCN amplified neuroblastoma is associated with a poor prognosis. Investigating specific tumor pathways will further our understanding of neuroblastoma pathogenesis and lead to future therapeutic options. Mxi1 is a member of the MAD family that inhibits N-Myc activity. Mxi0 is an alternatively spliced variant of Mxi1 with a different first exon (Exon 0) whose function has not been determined. These proteins appear to have differential functions in neuroblastoma pathogenesis. Objective: Elucidate the impact of Mxi1 and Mxi0 expression on neuroblastoma physiology and determine the role of Exon 0 in the function of Mxi0. Design: We created neuroblastoma cell lines with inducible expression of Mxi1 and Mxi0 to tightly control expression. Cell proliferation and survival were quantified using BrdU and MTT assays. Chemosensitivity was assessed by treating cells with doxorubicin or etoposide after induction of Mxi1 or Mxi0 expression. Cell viability was then measured by by MTT assay. To help examine the role of Exon 0, we utilized GFP tagged constructs of Mxi1, Mxi0, and Exon 0. These proteins were expressed in 293T cells and subcellular localization of Mxi1, Mxi0, and Exon 0 proteins was then detected by immunofluorescence. To assess the role of nuclear export in cell localization, cells were treated with leptomycin B. Results: Overexpression of Mxi1 inhibits N-Myc mediated cell proliferation. Conversely, overexpression of Mxi0 in neuroblastoma cell lines leads to enhanced proliferation, suggesting that Mxi0 has a counter-regulatory role to that of Mxi1. Compared with Mxi1, expression of Mxi0 results in cells becoming more chemoresistant. Examination of Mxi1 and Mxi0 subcellular location reveals that Mxi1 resides in the nucleus while Mxi0 is found primarily in the cytoplasm. Exon 0 alone also is found in the cytoplasm, indicating that this differential localization is determined by the presence of Exon 0. Finally, treatment with leptomycin B resulted in accumulation of Mxi0 in the nucleus, suggesting that it may cycle in and out of the nucleus in response to appropriate signals. Conclusions: Overexpression of Mxi1 in neuroblastoma cell lines leads to inhibition of N-Myc-mediated cell proliferation while Mxi0 appears to promote cell growth. Mxi1 expression enhances chemosensitivity of neuroblastoma cells, while Mxi0 has the opposite effect. Exon 0 directs the cytoplasmic localization of Mxi0 and may play an important role in its differential function. A better understanding of how the interaction between Mxi1 and Mxi0 affects neuroblastoma physiology and how Exon 0 imparts the differential function of Mxi0 may aid in developing more effective targeted therapies to improve outcomes in children with neuroblastoma. Citation Format: Stephen J. Kirchner, James T. Bartram, D. Christian Ellis, Daniel S. Wechsler, Michael B. Armstrong. The role of Exon 0 in mediating Mxi0 activity in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B32.

Abstract B37: The impact of modulating Mxi1 and Mxi0 expression on N-Myc-mediated neuroblastoma tumor pathogenesis and chemosensitivity

Background: Neuroblastoma is the most common extracranial malignancy of childhood. Myc family proteins regulate cell growth and proliferation in response to mitogenic stimulation, and Myc proteins are implicated in the etiology of many cancers. MYCN amplified neuroblastoma carries a poor overall survival with limited therapeutic options. Investigating specific tumor pathways will further our understanding of neuroblastoma pathogenesis and lead to future therapeutic options for children with this disease. Mxi1 is a member of the MAD family of proteins that inhibit N-Myc function. Mxi0 is an alternatively-spliced variant of Mxi1 whose function has not been determined. We hypothesize that Mxi1 and Mxi0 expression levels affect N-Myc-dependent neuroblastoma cell growth. Objective: To determine the impact of modulating Mxi1 and Mxi0 expression on N-Myc-dependent neuroblastoma cell proliferation and survival. Design: We expressed Mxi1 and Mxi0 in SHEP neuroblastoma cells and SHEP cells stably transfected to express high levels of MYCN (SHEP/ MYCN ). We also utilized native neuroblastoma cell lines with inducible expression of Mxi1 and Mxi0. Cell proliferation and survival were quantified using BrdU and MTT assays, respectively. Apoptosis was measured by propidium iodide staining and caspase-3 immunohistochemistry. Cellular localization of Mxi1 and Mxi0 proteins was detected by immunofluorescence. Results: Overexpression of Mxi1 inhibits N-Myc mediated cell proliferation. Additionally, in the absence of N-Myc, Mxi1 overexpression independently inhibits cell proliferation and induces cell apoptosis. Conversely, overexpression of Mxi0 in neuroblastoma cell lines leads to enhanced proliferation, suggesting that Mxi0 has a counter-regulatory role to that of Mxi1. Expression of Mxi1 increased sensitivity of the neuroblastoma cells to doxorubicin, while higher levels of Mxi0 made the cells more chemoresistant. Finally, examination of Mxi1 and Mxi0 cellular location reveals that Mxi1 resides in the nucleus while Mxi0 is found primarily in the cytoplasm. Conclusions: Overexpression of Mxi1 in neuroblastoma cell lines leads to inhibition of N-Myc-mediated cell proliferation while Mxi0 appears to promote cell growth. Mxi1 expression enhanced chemosensitivity of neuroblastoma cells, while Mxi0 had the converse effect. A better understanding of the interaction between Mxi1 and Mxi0 and how the balance of these proteins affect neuroblastoma physiology may aid in developing more effective targeted therapies to improve outcomes in children with neuroblastoma. Citation Format: D. Christian Ellis, R. Scott Widemon, Daniel S. Wechsler, Michael B. Armstrong. The impact of modulating Mxi1 and Mxi0 expression on N-Myc-mediated neuroblastoma tumor pathogenesis and chemosensitivity. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B37.

Abstract 5041: The type III TGF-beta receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma.

Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the pediatric tumor neuroblastoma remain unclear. Here we report that expression of type III TGF-beta receptor (TβRIII) mRNA and protein decreases with advancing stage of neuroblastoma and positively correlates with prognosis. TβRIII expression is epigenetically suppressed by MYCN oncogene amplification and TβRIII expression can be used as a prognostic marker in neuroblastoma patients with MYCN amplification. TβRIII expression in neuroblastoma cells promotes neuronal differentiation and enhances the differentiating effects of FGF2 treatment. Mechanistically, glycosaminoglycan modifications on TβRIII bind FGF2 and FGFR1 to promote neuronal differentiation via Erk MAPK and the transcription factor ID1. TβRIII-mediated differentiation suppresses tumor cell proliferation in vitro and in vivo. These studies characterize a novel co-receptor function for TβRIII in FGF2-mediated neuronal differentiation of neuroblastoma cells, while identifying potential therapeutic targets and clinical biomarkers for advanced-stage disease. More generally, our results suggest that the targeting of growth factor receptors and downstream signaling pathways may prove useful in promoting neuronal differentiation to suppress neuroblastoma tumor growth. Citation Format: Erik H. Knelson, Angela L. Gaviglio, Alok K. Tewari, Michael B. Armstrong, Andrew B. Nixon, Mark D. Starr, Karthikeyan Mythreye, Gerard C. Blobe. The type III TGF-beta receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma. [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 5041. doi:10.1158/1538-7445.AM2013-5041