AKT Inhibitor Hu7691 Induces Differentiation Of Neuroblastoma Cells And Its Mechanism Study

Objective:Neuroblastoma（NB）is one of the most common extracranial solid tumors in children,nearly half of which occur in infants and young children within 2 years of age.Neuroblastoma accounts for 25～50 cases per million people and 15%of childhood tumor related deaths.At present,clinical treatments against neuroblastoma are scarce,mainly based on cytotoxic chemotherapeutic drugs.Whereas for recurrent and metastatic neuroblastoma,existing treatment modalities are more unmet for patients.Clinically,induction of differentiation by maintenance therapy using 13-cis retinoic acid（Isotretinoin）is the standard of care for neuroblastoma.However,differentiation therapy has not been used as a first-line treatment for neuroblastoma due to the existing problems of differentiation therapy with low efficacy,unclear mechanism,and few in drug options.The phosphatidylinositol kinase/protein kinase B（PI3K/AKT）pathway is an important signaling pathway for regulating tumorigenesis and development.Studies have reported that the PI3K/AKT pathway is involved in regulating neural differentiation,but the relationship between the PI3K/AKT pathway and neuroblastoma differentiation remains unclear and needs to be studied.Hu7691 is a novel AKT inhibitor developed independently in our laboratory and currently under clinical trials.Therefore,the aim of this study was to investigate the therapeutic and differentiation effects of Hu7691 on neuroblastoma in vitro and in vivo,while elucidating the role of AKT in the differentiation process of neuroblastoma and the underlying mechanisms.Our study may provide a theoretical basis and a novel candidate molecule for the design and discovery of differentiation therapies.Methods:Several neuroblastoma cell lines（Neuro2a,IMR-32,SK-N-BE（2）,SK-N-DZ,CHP-126 and SK-N-SH）and PDC cells（NB-2,NB-7p,NB-18,NB-22,NB-50,NB-51,NB-58 and NB-61）were selected to study the effects of three AKT inhibitors（Hu7691,AZD5363 and GSK2141795）on proliferation inhibition and induction of differentiation.SRB was used to detect the proliferation of different NB cells after the treatment of AKT inhibitors.PI-Annexin V double staining was used to exam the apoptosis cells after the treatment of AKT inhibitors.Photographed and analyzing by Neuron J to determind the growth of neurites after the treatment of AKT inhibitors.β-III tubulin was examined by immunofluorescence after the treatment of AKT inhibitors.The cell cycle after AKT inhibitor treatment was assessed by PI single staining.The m RNA levels of three neural differentiation assoiated markers（Tubb3,Eno2 and Rbfox3）were assessed by q RT-PCR after AKT inhibitor treatment.The protein expression of N-Myc and AKT pathway inhibition were detected by Western blot.The correlation between genomic changes and neuroblastoma cell differentiation after AKT inhibitor treatment was analyzed by RNAseq and KEGG pathway enrichment tools.Neuro2a was selected to examine the contribution of the three subtypes of AKT（AKT1,AKT2,and AKT3）to the induction of neuroblastoma cell differentiation.q RT-PCR and Western blot were used to determine the silencing efficiency of different sh RNA sequences.Neurites outgrowth after AKT silencing was photographed under constant light and analyzed by Neuron J.m RNA level changes of neural differentiation related markers（Eno2 and Gap43）after silencing AKT were measured by q RT-PCR.Neuro2a xenografts model was established by inoculating Neuro2a cells into the armpits of nude mice,and Hu7691 was administered to investigate its therapeutic effects in vivo.Hu7691 was administrated by gavage,and the tumor volume and body weight of nude mice were measured daily.After 17 days of administration,the mice were dissected and the tumors were photographed and weighed.The relative tumor volume and relative tumor proliferation rate were calculated.Western blot was used to examine the inhibition of AKT pathway in the Hu7691 treatment group in vivo.Results:1.Hu7691 could inhibit cell proliferation and induce morphological changes in multiple neuroblastoma cell lines（1）Hu7691 could inhibit the proliferation of multiple neuroblastoma cell lines,with an average IC₅₀=12.49±6.00μM.On the other hand,several primary PDC cells also showed a certain sensitivity to Hu7691.The average three-day proliferation inhibition rate of 20μM was 57.2±9.43%.（2）The apoptosis of Neuro2a and chp-126was not induced by Hu7691,which was given the concentration over IC₅₀.Suggesting that the mechanism of Hu7691 was not achieved by inducing apoptosis.（3）Microscopically,we found that Hu7691 could cause differentiation like morphological changes in several neuroblastoma cell lines and multiple primary PDC cell lines,suggesting that Hu7691 may have the ability to induce neuroblastoma cell differentiation.2.Hu7691 could induce cell differentiation of Neuro2a cellsWe chose Neuro2a cells(IC₅₀=2.73μM)which processed the strongest anti-proliferation effect and introduced the differentiation positive drug ATRA for comparison.（1）By quantifying and analyzing neurites length in Neuro2a cells,it was clear that Hu7691 induced rapid and significant morphological changes in Neuro2a cells.（2）Immunofluorescence staining forβ-III tubulin,clarified that Hu7691 induced morphological changes in Neuro2a cells is correlated with differentiation and was dependent on Hu7691 concentration.（3）Examination of cell cycle by PI staining revealed that Hu7691 could concentration dependently induce cycle arrest in G₀/G₁phase of Neuro2a cells.（4）The m RNA levels of neural differentiation related markers Eno2,Tubb3 and Rbfox3 were up-regulated after Hu7691 treatment by q RT-PCR.（5）Hu7691 could concentration dependently downregulate N-Myc protein levels by Western blot.Combined with the above results,we found that in contrast to ATRA,Hu7691 could induce the differentiation of Neuro2a cells,while its differentiation effect is likely through regulation of N-Myc protein levels.3.AKT inhibitors AZD5363 and GSK2141795 induce cell differentiation of Neuro2a cells,similarlyTo examine the specificity of Hu7691 in inducing differentiation of Neuro2a cells,we introduced additional AKT inhibitors,AZD5363 and GSK2141795.（1）AZD5363and GSK2141795 could inhibit the proliferation of several neuroblastoma cell lines by SRB assay.（2）By quantifying and analyzing Neuro2a cell neurites length,it was clear that AZD5363 and GSK2141795 could rapidly and significantly induce morphological changes in Neuro2a cells.（3）AZD5363 and GSK2141795 could concentration dependently upregulate the m RNA levels of neural differentiation related markers Eno2and Rbfox3 by q RT-PCR.（4）Gene expression changes after AZD5363,GSK2141795and Hu7691 were examined by RNAseq.By Analyzing of the RNAseq results,we enriched multiple pathways involved in neural differentiation,suggesting that these AKT inhibitor may be involved in the possible signaling pathways related to differentiation process.4.The AKT regulation of neuroblastoma differentiation is dependent on its enzymatic activity（1）To clarify the correlation between AKT and differentiation,we examined AKT downstream by western blot and found that Hu7691,AZD5363 and GSK2141795 could significantly inhibit AKT pathway,suggesting the importance of AKT in neuroblastoma differentiation.（2）We knockdown different isoforms of AKT individually or co-knockdown different isoforms of AKT（pan AKT）by lentiviral infection.By q RT-PCR and Western blot,we found that the expression of different isoforms of AKT could be effectively inhibited in Neuro2a.（3）By quantifying and analyzing Neuro2a cell neurites length,we found that silencing different isoforms of AKT alone failed to cause obvious morphological changes.Whereas pan AKT knockdown resulted in significant Neuro2a changes and no obvious differences compared to the effects of Hu7691treatment was observed.（3）By q RT-PCR,we found that AKT single isoform knochdown could only upregulate the m RNA levels of Gap43,a primary differentiation related marker,while pan AKT knockdown could upregulate the m RNA levels of Gap43and Eno2.Taken together the above results,we found that pan AKT knockdown induced Neuro2a differentiation with similar effects to those exerted by Hu7691,suggesting a key role of AKT in the differentiation process.5.Hu7691 is therapeutic in Neuro2a transplanted tumor nude miceHu7691 showed excellent therapeutic effects on Neuro2a xenografts.On the last day of Hu7691 treatment,with tumor volume inhibition of 39.90±4.67%and 55.35±4.67%and tumor weight inhibition of 31.21±23.69%and 47.65±15.47%in Hu769140 mg/kg group and Hu7691 80 mg/kg groups,respectively.However,the ATRA 80mg/kg group did not show any tumor suppressive effect.By western bot,we found that Hu7691 could significantly inhibit the AKT pathway in vivo.Conclusion:Here,we demonstrated a novel AKT inhibitor Hu7691 exerts an anti-proliferation effect and differentiation inducing ability on multiple neuroblastoma cell lines and further confirmation of the in vivo therapeutic effects of Hu7691,suggesting that Hu7691 is a potential molecule against neuroblastoma.With the introduction of other AKT inhibitors,it is now clear that multiple AKT inhibitors also have the effect of inducing neuroblastoma differentiation.Further more,silencing AKT was found to have the effect of inducing neuroblastoma differentiation.Through this study,we not only define the key role of AKT in the progression of neuroblastoma differentiation,but also provide potential drugs and key targets for the application of differentiation therapies for neuroblastoma clinically.