Mechanistic And Functional Insight Into The Glutamine Transporter ASCT2in MYCN-amplified Neuroblastomas

Part I Functional Insight into the Glutamine Transporter ASCT2in MYCN-amplified neuroblastomasBackgroud and objective:Neuroblastoma (NB) is one of the most frequent solid tumors detected in childhood. MYCN amplification occurs in20-25%of neuroblastomas. MYCN-amplified neuroblastomas is frequently resistant to conventional therapeutic drugs and is correlated with poor prognosis. At present there is no ideal drug to treat MYCN-amplified neuroblastomas. The study of the molecular mechanisms of tumor development to find a new drug targets is significant. We have previously found a series of MYCN-amplified neuroblastoma cells are consistently "addicted" to exogenous glutamine (Gln) and uptake the abundant Gln which dependend on glutamine transport. However, the identity and regulation of the transporter (s) that how to capture glutamine in MYCN-amplified neuroblastoma remain largely unknown. To prove our hypothesis, we first analyzed ASCT2’s biologic function in MYCN-amplified neuroblastomas in order to provide a new potential target for anticancer drug development.Methods:In the present study, we used Kelly, BE-2C and NLF cell, as the cell model of MYCN-amplified neuroblastomas. The growth of cells was measured by cell counting; the clone formation was detected by crystal violet staining; Trypan blue staining was used for detection of cell viability; the apoptosis rate was measuerd by Annexin V-FITC/PI apoptosis detection Kit, the cell cycle was detected by BrdU assay; the changes of Gln uptake was determined by the experiment of [3H]-L-glutamine uptake; metabolism analysis kit was use to detect the level of glutamate, succinate, fumarate in cells. The efficiency of inhibition of ASCT2gene expression was measured by fluorescence quantitative PCR and the Western blot method, when the RNAi technology was used. Then, Western blot method was used to detect the changes of expression levels of p70S6K, p70S6K, p4E-BP1; the tumor weight changes was measured and the expression of PCNA and c-Caspase-3was detected by immunohistochemistry (IHC) after the nude mouse model of xenograft tumor was established for four weeks.Results:1. The cell growth and survival were significantly inhibited and colony formation decreases in the absence of Gln; Alpha ketoglutarate (α-KG) or non essential amino acid (NEAA) could significantly save the cell death induced by glutamine deprivation, but could not restore cell proliferation ability, while α-KG and NEAA cooperate to restore the proliferation of Kelly cells;2. Asparagine, serine, alanine, glutamine significantly inhibited glutamine uptake in Kelly cell, while BCH, MeAIB, histidine did not interfere with glutamine transport. The Gln uptake was inhibited in dose-dependent manner by incubating0.5,1, and2mM GPNA;3. Glutamine uptake rate decreased more than50%and glutamic acid, succinic acid, fumaric acid levels decreased significantly in these cell lines by silencing ASCT2gene;4. The three stable ASCT2knockdown cell lines were established. The ASCT2gene expression in both mRNA and protein levels was efficiently suppressed by PLKO.1-shASCT2with the inhibition rate of80%or so. These cells growth were inhibited, the apoptosis rate of Kelly cells increased by29.71%, while the proportion of cells in Gl phase increased obviously, the percentage of S phase cells decreased. The phosphorylation levels of p70S6K,70S6K,4E-BP1decreased significantly, which show that ASCT2plays an important role in the activation process of the mTORCl signaling pathway;5. The speed of tumorigenesis and weight of tumors in vivo by silencing ASCT2gene significantly decrease. The expression of PCNA decreased and the expression of c-Caspase-3increased in the tumor tissue, which indicated that the silencing of ASCT2gene inhibit tumorigenicity by inhibiting tumor cell proliferation and induce apoptosis.Conclusion:1. MYCN-amplification neuroblastoma cells in global strictly rely on elevated glutamine metabolism for both survival and growth;2. The majority of the cellular Gln uptake in MYCN-amplified neuroblastomas is mediated by ASCT2;3. The expression of to maintain sufficient levels of glutamine essential for mTORC1signaling and TCA scycle anapleurosis;4. ASCT2silencing significantly inhibits the proliferation and survival in MYCN-amplified neuroblastoma cells and tumorigenesis in vivo which suggest ASCT2play an important role in the occurrence and development of tumor. We proposed that ASCT2may be a therapeutic target for MYCN-amplified neuroblastoma treatment; Part II Mechanistic Insight into the Glutamine Transporter ASCT2in MYCN-amplified neuroblastomas and its Clinical SignificanceBackgroud and objective:We have confirmed that ASCT2is a key factor to maintain glutamine transport in MYCN-amplified neuroblastomas cells. What is the regulation mechanism of ASCT2? We found that MYC overexpression induced metabolic reprogramming, which triggers cellular dependency on exogenous glutamine to sustain viability. We have previously found that ASCT2mRNAs were significantly elevated in MYCN-amplified tumors when compared with nonamplified tumors, which indicate that there is correlation between the expression of N-Myc and ASCT2in some extent. But the MYC oncogene is the only key factor regulating overexpressing of ASCT2? We found that activation of transcription factor-4(ATF4) is another key factor involved regulating the expression of ASCT2by information biology analysis. Is there a correlation between these results and clinical stage and prognosis? So in this part, we will study the mechanism of ASCT2transcriptional regulation by N-Myc and ATF4and its clinical significance.Methods:The effect of N-Myc and ATF4on the expression of ASCT2and the changes of ASCT2mRNA expression in stress conditions were measured by quantitative PCR and Western-blot method. To identify ASCT2regulatory factor(s), we performed bioinformatic analyses and identified a conserved N-Myc-binding site and ATF4-binding site within the ASCT2promoter region. We created a luciferase reporter construct using a pGL3plasmid containing the putative (in triplicate) N-Myc-binding or ATF4-binding site. As a control, we constructed an additional construct harboring mutation in the ASCT2-binding motif and detect the change of relative luciferase activity to study the effect N-Myc and ATF4on the transcriptional activity of ASCT2. The dynamic interaction of ATF4and N-Myc with ASCT2was alalyzed by chromatin immunoprecipitation experiments (CHIP). The relative expression of ASCT2, N-Myc, ATF4in primary neuroblastoma tumors was analyzed by gene chip (microarray) analysis. The relationship between ASCT2and the malignant degree, prognosis was analyzed by Meta-Analysis results on the basis of microarraydata accessible at the oncogenomics neuroblastoma prognsis database (website:http://pob.abcc.ncifcrf. gov/cgi-bin/JK). The protein expression of ATF4, ASCT2, PCNA in primary neuroblastoma tumors was measured by immunohistochemical method (IHC).Results: 1. Downregulated N-Myc significantly decreased the mRNA and protein expression of ASCT2. N-Myc could up-regulate the expression of ASCT2at transcriptional level through directly binding to the promoter region of ASCT2;2. A significant ASCT2activation upon glucose or glutamine deprivation and tunicamycin treatment, whereas hypoxia less affected its expression;3. ATF4knock-down potently inhibited ASCT2activation in Gln-starved Kelly. Both ChIP and luciferase assays confirmed that ATF4directly activate ASCT2expression;4. ASCT2mRNA expression was found to correlate with both MYCN amplification and poor prognosis;5. The protein expression of N-Myc, ATF4, ASCT2in three favorable neuroblastma cells are very low, in contrast, the expression was higher in thirteen favorable neuroblastma.Conclusion:ATF4as a novel regulator which cooperates with N-Myc to directly activate ASCT2expression. ASCT2expression which correlates with that of N-Myc and ATF4, is markedly elevated in high-grade serious neuroblastoma tumor samples compared with low-grade ones. High ASCT2expression is significantly associated with poor prognosis and survival of neuroblastoma patients. These dates suggest ASCT2may represent a novel molecular marker of clinical diagnosis and prognosis.