The Potential Functional Roles of NME1 Histidine Kinase Activity in Neuroblastoma Pathogenesis

Abstract

Neuroblastoma is the most common extracranial solid tumor in childhood. Gain of chromosome 17q material is found in >60% of neuroblastoma tumors and is associated with poor patient prognosis. The NME1 gene is located in the 17q21.3 region, and high NME1 expression is correlated with poor neuroblastoma patient outcomes. However, the functional roles and signaling activity of NME1 in neuroblastoma cells and tumors are unknown. NME1 and NME2 have been shown to possess histidine (His) kinase activity. Using anti-1- and 3-pHis specific monoclonal antibodies and polyclonal anti-pH118 NME1/2 antibodies, we demonstrated the presence of pH118-NME1/2 and multiple additional pHis-containing proteins in all tested neuroblastoma cell lines and in xenograft neuroblastoma tumors, supporting the presence of histidine kinase activity in neuroblastoma cells and demonstrating the potential significance of histidine kinase signaling in neuroblastoma pathogenesis. We have also demonstrated associations between NME1 expression and neuroblastoma cell migration and differentiation. Our demonstration of NME1 histidine phosphorylation in neuroblastoma and of the potential role of NME1 in neuroblastoma cell migration and differentiation suggest a functional role for NME1 in neuroblastoma pathogenesis and open the possibility of identifying new therapeutic targets and developing novel approaches to neuroblastoma therapy.

Keywords: NME/NM23/NDPK; histidine; kinase; neuroblastoma; phosphorylation.

Figure 1

NME1 in neuroblastoma: (A) The chromosome 17q21 region amplified in neuroblastoma tumors is shown, with the NME1 gene located within the amplified region (top) [29]. Relative NME1 expression levels were plotted in patients with tumors with and without chromosome 17q amplification (bottom) using the neuroblastoma Lastowska patient dataset (p value = 9.56e-10) in the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl) [30]. (B) Using the SEQC patient dataset in the R2 Genomics Analysis and Visualization Platform, patients were divided into high (blue) and low (red) NME1 gene expression groups and survival curves were generated. Overall survival (OS; left) and event-free survival (EFS; right) are shown with respective p values of 2.1e-14 and 6.0e-11 and patient numbers in parentheses. (C) Relative NME1 expression levels from the SEQC patient dataset were plotted in patients with MYCN amplified and non-amplified tumors (p value = 8.12e-32) and in patients with stage 1, 2, 3, and 4 tumors (p value = 1.35e-18), respectively, with patient numbers shown in parentheses. The clinical characteristics of the 498 neuroblastoma patients included in Figure 1B,C are the following: Age (<18 months: 300 patients, >18 months: 198 patients); Sex (278 males, 205 females and 15 N.A). For more information, the full details of this cohort have been previously published and are available through the R2 platform [31].

Figure 2

Histidine phosphorylation in neuroblastoma cell lines: (A) Immunoblots of neuroblastoma cell lysates were performed with a mixture of anti-1-pHis (phosphorylated histidine) and anti-3-pHis antibodies [28] and with antibodies to phosphorylated His118 (pNME1/2 95-8) (upper panels), comparing conditions where pHis is preserved (pH 10, 4 °C) and where it is lost (pH 3 (0.01% acetic acid), 90 °C). The levels of total NME1/2, lysine-histidine-pyrophosphate phosphatase (LHPP), and actin in each sample were evaluated by immunoblotting with specific antibodies (lower panels). (B) Immunoblots of neuroblastoma cell lysates were performed with anti-1-pHis and anti-3-pHis antibodies separately, with immunoblots for actin used as a control. Arrows indicate 1- phosphorylated histidine in NME1/NME2.

Figure 3

Histidine phosphorylation in neuroblastoma cell lines and tumor samples. (A) Immunoblots of neuroblastoma cell lysates and xenograft tumor lysates generated immediately after harvesting or after freezing in liquid N₂ for 1/3-pHis and actin are shown, comparing conditions where pHis is preserved (pH 10, 4 °C) and where it is lost (pH 3 (0.01% acetic acid), 90 °C). (B) Immunoblots of neuroblastoma cell lines lysed immediately after harvesting or after a 5-min delay (+5) and of orthotopic neuroblastoma tumor samples lysed immediately after harvesting or after 5 (+5) and 30 (+30) minute delays were performed with antibodies to 1/3-pHis and actin. (C) Immunofluorescence images using antibodies to 1/3-pHis and total NME1/2 labeled with Alexa Fluor 568 (red) in SK-N-SH neuroblastoma cells are shown. (D) Immunofluorescence images using antibodies to 1/3-pHis and NME1/2 in sections of SK-N-SH neuroblastoma cells from the optimal cutting temperature (OCT) block. Combined 1- and 3-pHis, NME1/2 and rabbit IgG (Rb IgG Ctrl) were tested, comparing conditions where pHis is preserved (pH 10, 4 °C) and where it is lost (pH 4, 90 °C).

Figure 4

NME1 depletion in neuroblastoma cells. (A) SK-N-BE(2) neuroblastoma cells were transfected with shRNA’s directed against NME1 (shNME1-99, shNME1-183) or with control shRNA (mock) and analyzed by Western blot (A) and Q-PCR (B) for NME1 and NME2 protein and gene expression, respectively. (C) Control SK-N-BE(2) neuroblastoma cells and cells with depleted NME1 (shNME1-99, shNME1-183) were plated and analyzed for cell confluence over time using continuous live cell imaging.

Figure 5

NME1 depletion affects neuroblastoma cell migration. (A) Control SK-N-BE(2) neuroblastoma cells and cells with depleted NME1 (shNME1-99, shNME1-183) were plated and analyzed for migration into a scratch wound using continuous live cell imaging. Images were taken every six hours and wound width was measured at each time point. Representative images (A) and a graph of relative wound width over time (B) are shown. Respective p values of depleted NME1 with shNME1-99 (black) and shNME1-183 (red) to control SK-N-BE(2) (blue) were 1.3e-2 and 2.6e-2.

Figure 6

Association of NME1 with neuroblastoma differentiation. Control SK-N-BE(2) cells and cells with depleted NME1 (shNME1-99, shNME1-183) were treated with 5 µM 13-cis-retinoic acid (CRA) or vehicle alone and analyzed using continuous live cell imaging with NeuroTrack^TM software. (A) Imaging obtained of cells after 5 days of vehicle or CRA treatment, with cell bodies in yellow and neurite extensions mapped in blue. (B) Total neurite length in parental neuroblastoma cells and in those with depleted NME1 per high-power field were plotted over time. The SK-N-BE(2), the depleted SK-N-BE(2) shNME1-99, and the depleted SK-N-BE(2) shNME1-183 present respective values of p = 1.73e-12, p = 2.5e-06, and p = 6.5e-09 between vehicle and CRA treatment.

Figure 7

Intracellular pathways associated with NME1 in neuroblastoma. (A) Numbers of proteins identified by mass spectrometry from NME1/2 or 1/3-pHis pull-down experiments using a pool of SK-N-BE(2) and SK-N-AS neuroblastoma cell lines or orthotopic xenograft neuroblastoma tumors. (B) Neuroblastoma cell lysates were immunoprecipitated using antibodies to NME1 or 1/3-pHis, and isolated proteins were analyzed by mass spectrometry. Proteins identified in all three enrichments with an enrichment score derived from the interaction with NME1 higher than 20 are listed (a complete list and matching comparison of the proteins identified among the three different pull-down experiments are provided as a Supplementary file). (C) 59 identified potential NME1 substrates were subjected to pathway analysis using EnrichR (https://amp.pharm.mssm.edu/Enrichr/) [36]. The top four identified pathways (using the Elsevier pathway collection) and gene ontology (using GO_Cellular_Component_2018 section) annotation clusters are listed.