Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition

Abstract

SUMMARYNeuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. Here, we focus on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivo inhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression. Activation of Alk signaling, in particular overexpression of ALK wt and NB ALK mutants resulted in the upregulation of NMyc and trkB, which may contribute to continued proliferation and NB predisposition. MATERIALS AND METHODS Expression plasmidsPcDNA3.1 expression plasmids for human ALK wt , ALK F1174L and ALK R1275Q, and pCAGGS expression plasmids for Hand2, Phox2b wt and Phox2b K155X have been described previously (Janoueix-Lerosey et al., 2008;Reiff et al., 2010). shRNA constructsThe shRNA against Gallus gallus Alk and Mk were designed using BLOCK-iT RNAi Designer (Invitrogen, Karlsruhe, Germany) and target the following sequences: shAlk, 5Ј-AAU GGU UUC UCU CUA UGU CCA ACU C-3Ј; shMk, 5Ј-GAG CUG ACU GCA AGU ACA AGU UUG A-3Ј. Scrambled shRNAs (sc-shRNA, Invitrogen, Karlsruhe, Germany) served as controls. In situ hybridizationNon-radioactive in situ hybridization on cryosections and preparation of digoxigenin labeled probes for chick Phox2b was carried out as described previously (Ernsberger et al., 1997;Stanke et al., 1999). qPCR analysisEqual amounts of RNA were used to synthesize cDNA with Oligo(dT)-primers and Superscript-III-reverse-transcriptase according to manufacturer's instructions (Invitrogen, Karlsruhe, Germany). The PCR was carried out using Abgene's Absolute Blue SYBR-Green qPCR Mix (Abgene, Epsom, UK) in a Stratagene Mx3000p Light Cycler (Stratagene, Waldbronn, Germany). All primers (MWG Biotech AG, Ebersberg, Germany) were designed to anneal optimally at 58°C with PerlPrimer (Marshall, 2004) (95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, repeated for 50 cycles and a subsequent dissociation curve). The primer pairs (see Table S1 in the supplementary material) were ...

Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma

“…Interestingly, the ALK gene itself is upregulated in human NB presenting with ALK mutations [29]. Strikingly, this upregulation is also observed in murine MYCN / Alk tumors compared with MYCN tumors and in the ganglia of mutated animals, in agreement with the reported ALK autoregulation [19,33]. Altogether, these data strongly indicate that tumors obtained in our models are fully relevant to human NB.…”

“…In vitro , sympathetic neuroblast proliferation has been shown to be increased upon overexpression of wild-type (Wt) ALK as well as R1275Q and F1174L mutant forms in a culture model of chick embryonic neuroblasts [19]. Moreover, it has been recently reported that F1174L ALK may transform murine neural crest progenitor cells [20].…”

The prenatal origins of cancer

“…ALK has a key role in early sympathoadrenal development to protect neuroblast growth in utero against nutrient deprivation 37, 38 . Germline and somatic mutations in ALK causing constitutive kinase activation are present in 8–10% of neuroblastoma cases and correlate with poor prognosis 39–42 .…”

Wild-type ALK and activating ALK-R1275Q and ALK-F1174L mutations upregulate Myc and initiate tumor formation in murine neural crest progenitor cells

“…ALK has been recently extensively studied as a candidate in the development of new targeted treatments for progressive and resistant NB. ALK is expressed in the developing central and peripheral nervous system during embryogenesis [5], and in the developing sympathoadrenal lineage of the NC, where its signaling may regulate the balance between cell proliferation and differentiation [1, 6, 7]. ALK physiological role in the normal development of the nervous system is not yet fully understood, but ALK having a role in neurogenesis control was demonstrated in Drosophila , zebrafish, and chicken [6, 8, 9].…”