RESEARCH Open Access

Reprogramming glioblastoma multiformecells into neurons by protein kinaseinhibitorsJie Yuan1,2,3, Fan Zhang1, Dennis Hallahan1, Zhen Zhang4, Liming He4, Ling-Gang Wu4, Meng You1 and Qin Yang1*

Abstract

Background: Reprogramming of cancers into normal-like tissues is an innovative strategy for cancer treatment.Recent reports demonstrate that defined factors can reprogram cancer cells into pluripotent stem cells.Glioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumor in humans. Despitemultimodal therapy, the outcome for patients with GBM is still poor. Therefore, developing novel therapeuticstrategy is a critical requirement.

Methods: We have developed a novel reprogramming method that uses a conceptually unique strategy for GBMtreatment. We screened a kinase inhibitor library to find which candidate inhibitors under reprogrammingcondition can reprogram GBM cells into neurons. The induced neurons are identified whether functional and lossof tumorigenicity.

Results: We have found that mTOR and ROCK kinase inhibitors are sufficient to reprogram GBM cells into neural-like cells and “normal” neurons. The induced neurons expressed neuron-specific proteins, generated actionpotentials and neurotransmitter receptor-mediated currents. Genome-wide transcriptional analysis showed that theinduced neurons had a profile different from GBM cells and were similar to that of control neurons induced byestablished methods. In vitro and in vivo tumorigenesis assays showed that induced neurons lost their proliferationability and tumorigenicity. Moreover, reprogramming treatment with ROCK-mTOR inhibitors prevented GBM localrecurrence in mice.

Conclusion: This study indicates that ROCK and mTOR inhibitors-based reprogramming treatment prevents GBMlocal recurrence. Currently ROCK-mTOR inhibitors are used as anti-tumor drugs in patients, so this reprogrammingstrategy has significant potential to move rapidly toward clinical trials.

Keywords: GBM, mTOR, Neuron, Protein kinase inhibitors, Reprogramming, ROCK

BackgroundThe ability to convert somatic cells into disease-relevantcell types through cellular reprogramming has openednew doors for basic research and cell replacement therapy[1]. Takahashi et al. demonstrated that defined factorscould drive skin-derived fibroblasts to induced pluripotentstem (iPS) cells that could be further differentiated intothe desired cell type. Direct somatic lineage reprogram-ming has attracted much attention as it bypasses

conversion into iPS cells. Recently studies have demon-strated that a set of neural factors can directly convert fi-broblasts into neurons [2–11]. Direct fibroblast-neuronreprogramming provides an alternative, potentially com-plementary tool to many of the proposed applications ofiPS technology for both disease modeling and develop-ment of cell-based therapies.Reprogramming healthy somatic cells with defined fac-

tors has been extensively investigated. However, repro-gramming cancer cells has comparatively lagged behind[12–14]. Differentiation of tumors into post-mitotic cellsis an ancient idea. For example, tretinoin (all-trans-reti-noic acid) was used as differentiation therapy of acute

* Correspondence: qyang@wustl.edu1Cancer Biology Division, Department of Radiation Oncology, WashingtonUniversity School of Medicine, 4511 Forest Park, St. Louis, MO 63108, USAFull list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Yuan et al. Journal of Experimental & Clinical Cancer Research (2018) 37:181 https://doi.org/10.1186/s13046-018-0857-5

promyelocytic leukemia [15]. Recent established repro-gramming method should significantly improve differenti-ation therapy. Reprogramming and oncogenictransformation are stepwise processes that share manysimilarities. Overexpression of a set of oncogenes inducesreprogramming and oncogenic transformation, whiletumor suppressor gene such as p53 suppresses both pro-cesses. Although unidentified biological barriers may exist[14, 16, 17], reprogramming of both solid and liquid tumorsto iPS cells has been reported by different groups [16, 18–27]. Loss of tumorigenicity by unknown mechanisms andinduced dedifferentiation to pluriopotency seem to be com-mon features of reprogrammed cells from different cancers.However, robust differentiation into specific lineages re-mains a stumbling block [28–32]. To overcome these road-blocks, therefore, we will detect whether cancer cells can bereprogrammed into normal-like cells under defined repro-gramming conditions.Glioblastoma multiforme (GBM) is the most common

and most aggressive malignant primary brain tumor inhumans. Despite multimodal therapy [surgery with ioniz-ing radiation and chemotherapy (temozolomide, TMZ)],the outcome for patients with GBM is still poor, with amedian survival of only 14 months [33–35]. Therefore, itis imperative to develop novel therapeutic strategies forGBM treatment. Integration-free reprogramming of can-cer cells is a safe and preferable strategy for clinical use.We screened a kinase inhibitor library and found that acombination of two inhibitors, Rho-associated proteinkinase (ROCK) and mammalian target of rapamycin(mTOR), could substitute for transcription factors andconvert human GBM cells into neural-like cells. The in-duced neural cells were subsequently trans-differentiatedinto functional neurons. The induced neurons lost theirtumorigenicity and reverted to the ‘normal state’. Import-antly, ROCK-mTOR inhibitors coupled with reprogram-ming treatment prevented GBM local recurrence in mice,suggesting that our reprogramming treatment had a keyrole in preventing GBM recurrence.

MethodsCell cultureHuman GBM cells, U118, GM97 and PDC1 were rou-tinely cultured in 60mm dish (MidSci) in Dulbecco’smodified Eagle medium (Hyclone) containing 10% fetalbovine serum (Millipore-Sigma), non-essential aminoacids (Invitrogen) and penicillin/streptomycin (Invitro-gen) at 37 °C in 5% CO2. U118 and GM97 have p53 mu-tation or inactivation (p53 data base, p53.fr), while U118also carries pten mutation [36]. Human fibroblastsIMR90 from ATCC are lung-derived fibroblasts from a16-week fetus. All cell lines have been tested for myco-plasma infection and were authenticated by short tan-dem repeat DNA profiling analysis.

Neural cell conversionFor neuronal conversion, GBM cells were plated at adensity of 3.0 × 104 cells cm− 2 on microscope glass cov-erslips coated with matrigel (BD) in 35mm dishes. Forneural induction, the media was changed to defined in-duction medium including DMEM/F12 (2% FBS) plus1 μM dexamethasone (Millipore-Sigma), 0.5 mM isobu-tylmethylxanthine (Millipore-Sigma), 200 μM indometh-acin (Millipore-Sigma), 2 μM Y-27632 (Enzo LifeSciences) and 2 μM P529 (Millipore-Sigma). For kinaseinhibitor screening experiments, we used 2 μM proteinkinase inhibitor from a library (Calbiochem, 355 inhibi-tors). For neuronal differentiation, we used neuronal ma-ture medium including Neural medium (ScienCell) with50 μM dbcAMP (Millipore-Sigma), 10 ng/ml NT3(PROSPEC), 10 ng/ml BDNF (PROSPEC), 0.5 μM Retin-oic acid (Millipore-sigma), 2 μM Y-27632 and 2 μMP529. Quantitative of mean % induced neuron (iN) pur-ity is counted by morphology of MAP2-positive staining,and numbers represent the percentage of iN cells at thetime point of quantification.

Cortical neuron culture and co-culture with iNsPrimary cortical neurons were isolated from P0 rats.Cortices were dissected and dissociated by trypsin diges-tion (0.25% Trypsin, 137mM NaCl, 5 mM KCl, 7 mMNa2HPO4, 25 mM HEPES) and plated on poly-D-lysinecoated glass coverslips. The neurons were maintained ingrowth medium consisting of MEM supplemented withB27, glutamine (all from Invitrogen), glucose, transferrin(Calbiochem), fetal bovine serum and Ara-C (both fromMillipore-Sigma) for a week before co-culture with iNs.iNs were induced for 7 days by induction medium withP + Y and dissociated by trypsin (0.05% Trypsin). iNswere seeded onto a cortical neuron bed and maintainedin neuronal mature medium.

Viral preparation, western blot and immunofluorescenceROCK1/2 and mTORC1 (Raptor) /C2 (Rictor) shRNAswere obtained from Millipore-Sigma. Western blottinganalyses were performed to check the knockdown effi-ciency. Immunofluorescence staining was performed asfollows: 5 × 104 modified human fibroblasts were plantedon Matrigel-coated glass coverslips the day before induc-tion. Cells were fixed for 20 min at room temperature in4% paraformaldehyde in PBS, permeabilized for 30 minin PBS containing 0.2% Triton X-100 and 10% normalgoat serum (NGS) and incubated overnight at 4 °C inPBS containing 10% NGS and primary antibodies. Cellswere washed three times with PBS and incubated for 2 hat room temperature with anti-rabbit or anti-mouse sec-ondary antibodies conjugated to Alexa Fluor-488 orAlexa Fluor-594 (1:500, Invitrogen). Images were ac-quired on immunofluorescence microscope or Zeiss

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LSM 510 META confocal microscope at 40× magnifica-tion and 1.3 numerical aperture oil-immersion objective.The following antibodies were used for the immuno-

fluorescence studies: rabbit anti-MAP2 (Millipore-Sigma,1:200), mouse anti-Tuj1 (R&D Systems, 1:100), rabbitanti-synapsin 1 (Cell Signaling, 1:200), mouse anti-TUJ1(1:1000, Covance) and rabbit anti-Tuj1 (1:2000, Covance).

Trypan blue dye exclusion assays, qRT-PCR and TUNELassaysGBM, iP and iN cells were seeded at a density of 10,000cells/well in a 12-well plate. Cells were counted with aViCell cell viability analyzer (Beckman Coulter). Cellproliferation was normalized to the percentage of con-trol cells. Three independent experiments were per-formed with triplicates for each cell line. For qRT-PCRassay, total RNA was extracted using Trizol isolationsystem (Invitrogen) according to the manufacturer’s in-structions. Total RNA was treated with DNase I (Qia-gen) to prevent DNA contamination. 2 μg RNA wasreverse transcribed using Transcriptor High FidelitycDNA Synthesis Kit (Roche). Reverse transcription andreal-time PCR were performed as a standard procedure.The terminal deoxynucleotidyltransferase-mediateddUTP-biotin nick end labelling (TUNEL) assay was per-formed with TUNEL assay kit (Roche).

ElectrophysiologyCoverslips plated with iNs were placed in the recordingchamber mounted on an Olympus 1 × 81 microscope.All electrophysiology recordings were recorded at roomtemperature (22–25 °C) via whole-cell recording with apatch electrode. Signals were amplified using HekaEPC-10 amplifier and filtered at 2 KHz via a Bessellow-pass filter. Data were sampled using Puls8.8 soft-ware, and analysed using Igor6.04.For recording voltage-gated currents and action poten-

tials, patch electrodes were filled with solution (in mM):130 K-gluconate, 10 KCl, 10 HEPES, 0.5 EGTA, 4MgATP, 0.3 Na2GTP, 10 P-Creatin, pH adjusted byKOH to 7.2, osmolarity measured at 310mOsm. Thecomposition of the external solution used for recordingwas as follows (in mM): 105 NaCl, 25 NaHCO3, 3Myo-Inositol, 2 Na-Pyruvate, 2.5 KCl, 1.25 NaH2PO4, 25Glucose, 2 CaCl2, 1.5 MgCl2, pH adjusted by NaOH to7.4, osmolarity measured at 300 mOsm. To isolatevoltage-dependent sodium currents, 1 μM tetrodotoxinwas added to the external solution. To monitorvoltage-gated currents, we applied step potentials ran-ging from − 60 mV to + 60mV for 20 ms. To monitoraction potentials, we applied step currents ranging from10 to 100 pA for 200 ms. External solution changes wereachieved rapidly, within 100 ms.

Microarray analysisMicroarray analysis was performed at the Washington Uni-versity Genome Center. Briefly, Illumina HumanHT-12 v4Expression BeadChip was used and samples were labeledby biotin. The Direct Hybridization Assay was performedand the data were scanned on the BeadArray Reader.Scanned images were quantitated by Illumina Beadscan, v3.Quantitative data was imported into Illumina GenomeStu-dio software and normalized by Illumina’s quantile method.The quantile-normalized background-subtracted data werecalculated in excel. Data were filtered based on the averagesignal with baseline set as 50. Only the genes with divisionvalue > 3 were selected and considered as differentiallyexpressed. All differentially expressed genes were clusteredusing MeV software.Gene expression for each sample was analyzed to de-

termine the relationship among GBM cells and iNs. Datawere filtered based on average signal and baseline wasset as 10 to increase the sensitivity. Differentiallyexpressed genes were identified with a 3-fold change asthe threshold. To examine the potential neuron differen-tiation pathways relevant to immortalization, all neurondifferentiation genes from Gene Ontology Website(http://www.geneontology.org/, GO: 0030182) werecompared with the microarray data. For gene enrich-ment analysis, we used web-based Gorilla program(http://cbl-gorilla.cs.technion.ac.il/). Thus, these resultswill greatly help for understanding mechanisms of GBMcell reprogramming.

GBM and iN cell transplantation and GBM recurrencetreatment in miceWe implanted GBM cells and iNs in the corpus callosumof nude mice. On the day of transplantation, cells wereharvested with accutase, counted, and re-suspended atconcentration of 25,000 cells/μL. Four microliter cell sus-pension was injected into the corpus callosum of miceusing the following stereotactic co-ordinates (in referenceto Bregma point): 10.98 mm (anterio-posterior axis),21.75 mm (latero-medial axis) and 22.25 mm (verticalaxis). Control GBM cells were injected into left side andiNs were injected into right side of the same mouse. Micewith cell transplants were perfusion fixated at 2 weeks,1 month and 4 months. Brains were excised and sectionedon a cryostat for IHC analysis of the cell implants. Theprimary antibodies used are described above. Subse-quently, various secondary antibodies were used tovisualize the specific primary immunoreaction product insingle and double IHC staining. For GBM local recurrenceexperiments, we used a mouse model of local recurrencefollowing surgical resection of orthotopic GBM tumors[37–40]. GBM tumors generated by GM97 cells in nudemice were surgically removed after 4 weeks when the pri-mary tumors had reached ~ 50 mm3. 95% of each tumor

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was excised and a small fragment of tumor, the largest be-ing 2 mm3, was not removed to model postoperative re-sidual GBM. Matrix-Driven Delivery (MDD) Pellet ofROCK-mTOR inhibitors with dried induction mediumwas prepared by Innovative Research of America. We im-planted the MDD pellet including mTOR-ROCK inhibi-tors (2 μM P+Y, 5 μl per day for 21 days) with or withoutdried induction medium into the resection cavity ofGBMs. MDD pellet with dried induction medium, pelletonly and no treatment were controls. Induction media ineach MDD pellet include 1 μM dexamethasone, 0.5 mMisobutylmethylxanthine and 200 μM indomethacin with105 μl DMEM/F12 (2% FBS). Mouse brains were excisedand sectioned on a cryostat for IHC and TUNEL assayanalyses.

ResultsScreening a protein kinase inhibitor library to reprogramGBM cellsReprogramming somatic cells are into iPS cells by tran-scription factors may cause genomic instability, which in-creases the risk of cancer-cell induction [41–45]. Therefore,we tried to develop a transgene-free method to efficientlyreprogram GBM cells into normal-like cells. Cellular senes-cence has been shown to regulate reprogramming fibro-blasts to iPS cells and fibroblast-neuron conversion [46–49]. Since many protein kinases are involved in senescenceand proliferation processes, we screened a protein kinaseinhibitor library (355 inhibitors, Calbiochem). We prepareda GBM cell line (U118) with expression of MAP2promoter-RFP. Through phenotypic change screening, wefound that candidate kinase inhibitors reprogrammed GBMcells into induced progenitor-like cells in inductionmedium (Fig. 1a, b). We observed that a subpopulation ofcells was MAP2-RFP positive with marked morphologicalchanges after 3–7 days in induction medium with candidatekinase inhibitor treatment. Cell morphology ranged fromlarge nuclear and flat shaped cells (cancer cells) to small, bi-polar cells, termed induced neural-like cells (Fig. 1a,b).Eight candidate small molecules/compounds were selectedfor further confirmation (Fig. 1c). Secondary screening con-firmed that three compounds induced morphologicalchanges with higher efficiency (Fig. 1c). Two candidatesmall molecules, namely rapamycin and palomid 529(P529), are mTOR inhibitors and the third one (Y27632) isa ROCK inhibitor. To determine the combinatorial effectsof these inhibitors on GBM-neural cell conversion, wefound that using mTOR-ROCK inhibitors (P529/Y27632)together converted GBM cells into neural-like cells withthe highest efficiency (~ 90% induced efficacy after 7 daysinduction) (Fig. 1c).To investigate whether ROCK-mTOR inhibitors have

a general effect on human GBM cells, another GBM cellline (GM97) and one patient-derived primary GBM cell

(PDC1) were treated with ROCK-mTOR inhibitors.After induction for 3–7 days, about 70–90% of theseGBM cells converted into neural-like cells (Additionalfile 1: Figure S1). Thus, reprogramming treatment ofROCK-mTOR inhibitors is able to convert multipleGBM cells into neural-like cells.

Reprogramming GBM cells into induced neuronsNext, we examined whether the GBM cells could be re-programmed into another lineage (neuron, a lineageswitch) in a defined neuronal medium. After 3 days inneuronal medium with mTOR-ROCK inhibitors, in-duced neural-like cells further differentiated into bi- ormulti-polar neuron-like cells. In 2–3 weeks, 70–90% ofGBM U118 cells converted to neurons with matureneuronal morphology that expressed the neuronalmarkers, Tuj1 and MAP2 (Fig. 2a & c). We calculatedthe iN yield as the percentage of MAP2+ cells in relationto the initial number of plated GBM cells. At 3 weeksafter induction, we obtained yields of 90 ± 15% (Fig. 2a).In contrast, after 4 weeks in neuronal medium withoutkinase inhibitors, control GBM cells maintained GBMcell morphology with negative staining of MAP2 (Fig. 2b).Furthermore, we verify the neuron induction in two otherGMB cells, GM97 and PDC1. Three weeks afterinduction, ~ 75–90% of GBM cells converted into iNs(Additional file 1: Figures. S1-S2). Moreover, iNs did notexpress astrocyte (GFAP) and oligodendrocyte marker(O4) (Additional file 1: Figure S3). These results suggestthat ROCK-mTOR inhibitor treatment led to conversionof GBM cells to neurons.So far, we identified iNs based on their morphology and

positive staining of several neuronal markers. Next, we fur-ther characterized the functional aspects of iNs induced byROCK-mTOR inhibitor treatment from GBM U118 cells.We found that ~ 80% of iNs showed Synapsin-positivepuncta that label the vesicles in the presynaptic nerve ter-minal (Fig. 2c). Furthermore, iNs showed positive immu-nostainings with TBR1 (60%) or LHX6 (55%), indicatingthat iNs have properties of different neuronal populationspresent in the forebrain (Fig. 2c).To determine whether iNs generate action potentials

and neurotransmitter-induced currents, the basic func-tions of neurons, we cultured U118 cells (7 dayspost-induction) with a monolayer culture of primary ratneurons, which might provide a better environment formaturation [2, 3]. Three weeks after co-culture,whole-cell voltage-clamp recordings of iNs showed that70% of iNs exhibited inward fast activating and inactivat-ing sodium currents and outward potassium currentsduring a ramp depolarization. This was also confirmedby applying a series of voltage steps to evoke inward so-dium currents and outward potassium currents (Fig. 2d,e, 70% positive efficacy, n = 30). Miniature excitatory

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Fig. 1 (See legend on next page.)

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postsynaptic currents were recorded from inducedneurons without co-culture, suggesting that iNs canform functional synapses by themselves (Fig. 2f, 65%positive efficacy, n = 30). These data indicate that iNsare functional neurons.We also tested the effects of reprogramming treatment

on normal primary neurons and astrocytes. We preparedprimary mouse neurons/astrocytes and found that the re-programming treatment of mTOR-ROCK inhibitors didnot affect neuron/astrocyte properties under drug concen-tration for reprogramming (Additional file 1: Figure S4).These data indicate that normal neurons/astrocytes areonly marginally responsive to our treatment strategy.

Genome-wide transcriptional profiling of inducedneuronsTo analyze the similarities between iNs and parentalGBM cells, we performed microarray-based compara-tive global gene expression profiling. Microarray datawas quantile normalized and filtered based on theaverage signal, and differently expressed genes wereselected for further analysis. Hierarchical cluster ana-lysis revealed a significant difference between iN cellsand their parental GBM U118 cells (Fig. 3a, b). Theclustering analysis of global gene expression revealedthat iNs from GBM cells by the reprogramming treat-ment of ROCK-mTOR inhibitors are particularly simi-lar to control neurons, which are induced fromfibroblasts by p53 knockdown (Fig. 3b) [49]. This in-dicates that iNs generated by different methods showsimilarity of gene expression profiling. Using theOntology Data-base for enrichment analysis, we foundthat the key factors involved in neuron differentiationwere significantly changed in iNs, compared withthose in parental GBM cells. Moreover, we examinedmRNA levels of key factors reported to be involvedin neuron conversion. We found that, in iNs inducedby the reprogramming treatment of ROCK-mTOR in-hibitors, expressions of a set of neurogenic transcrip-tion factors were increased by > 10-fold (Fig. 3c),suggesting that these factors may be involved inGBM-neuron conversion. These findings indicate thatgenetic trans-differentiation erases majority of the ex-pression hallmarks of the cell of origin, while specific-ally inducing the neuronal phenotype.

ROCK1/2 and mTORC1 shRNAs generate iNsBoth ROCK and mTOR are serine/threonine protein ki-nases. ROCK is a kinase belonging to the AGC (PKA/PKG/PKC) family, including ROCK1 and ROCK2 [50–53]. mTOR interacts with other proteins and serves as acore component of two protein complexes, mTOR com-plex 1 and mTOR complex 2 [54–60]. To determine ifROCK and mTOR pathways are involved inGBM-neuron conversion, we examined the effects ofROCK and mTOR knockdown on GBM-neuron conver-sion (Fig. 4a). Lentiviral constructs expressing short hair-pin RNA (shRNA) against ROCK1/2 and mTORC1(Raptor)/C2 (Rictor) were transfected into GBM U118cells. We found that knockdown of ROCK1, ROCK2 ormTORC1 (Raptor) could reprogram GBM cells intoneural-like cells, while knockdown of mTORC2 did not(Fig. 4b). Combination treatment of ROCK1/2 andmTORC1 shRNAs converted GBM cells to neural-likecells with kinetics and conversion efficiency similar toROCK-mTOR inhibitors. Moreover, the iNs showed typ-ical neuronal morphology and positive immunostainingfor neuronal markers TUJ1, MAP2 and Synapsin. Effi-ciency of neuronal conversion by shRNA treatment wassimilar to ROCK-mTOR inhibitors (Fig. 4c & d). Thus,we conclude that ROCK1/2 and mTORC1 kinases areinvolved in conversion of GBM to neurons.

Loss of proliferation and tumorigenicity in iNsWe performed proliferation assays to determine iN cellproliferation. Trypan blue exclusion assays were per-formed after treating cells with ROCK-mTOR inhibitors.We observed that ROCK-mTOR inhibitor treatment re-duced cell proliferation in time-dependent manner inGBM cells, but not in iNs (Fig. 5a and Additional file 1:Figures S5A & S6A). In addition, apoptosis increased inGBM cells upon ROCK-mTOR inhibitor treatment, butnot in iNs (Fig. 5b and Additional file 1: Figures S5B &S6B). These data suggest that ROCK-mTOR inhibitortreatment does not affect iN cell proliferation and sur-vival, but may mediate GBM cell proliferation by regu-lating apoptosis.We further evaluated cell proliferation and invasion.

The mature phenotype of iNs was accompanied by ces-sation of proliferation and invasion as measured bysoft-agar proliferation and invasion assays (Figs. 5c-e

(See figure on previous page.)Fig. 1 Protein kinase inhibitor screen for reprogramming GBM cells. a & b Screening design and primary screening results. Human GBM cells(U118) expressing MAP2-promoter-RFP were seeded in 96-well plates. Kinase inhibitors were added at a final concentration of 2 μM in theinduction medium. The medium was changed every other day until day 7 when cells converted to RFP-positive cells. Images were taken on day7 after inhibitor treatment. Positive induced neural-like cells were counted by RFP-positive staining and quantified on day 7. The histogramrepresents the ratio of normalized data for kinase inhibitor treatment as compared with DMSO. c Secondary screening results. U118 cells weretreated with candidate kinase inhibitors. R + Y: Rapamycin+Y27632; P + Y: P529 + Y27632. Quantitative data are the mean ± SEM from threeindependent experiments

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and Additional file 1: Figure S6C & S6D). To determinetumorigenic properties in vivo, we injected parentalGBM cells and iNs (1.5 × 105) into the brain frontal lobe,2 mm lateral to the bregma of nude mice (Fig. 6a) [49].

No tumors were formed following iN transplantation upto 16 weeks (Fig. 6b). Transplantation of GBM cellstreated with induction medium without ROCK-mTORinhibitors resulted in tumor formation in 10 of 10 mice

Fig. 2 ROCK-mTOR inhibitors induce GBM-Neuron cell conversion. a Kinetic analysis of iNs from GBM U118 cells was performed after inductionwith ROCK-mTOR inhibitors in neuronal medium. The bar graph shows quantity of iN cells with MAP2 positive staining. Quantitative data arepresented as mean ± SEM from three independent experiments. b No expression of MAP2 and Synapsin in U118 control cells withoutreprogramming treatment. c Expression of TUJ1, MAP2, Synapsin, LHX6 and TBR1 was examined in iNs 3 weeks after induction. B & C: Nucleiwere counterstained with DAPI (blue). Scale bar, 10 μm. d-f Electrophysiological characterization of U118 cells was performed 3 weeks afterinduction. d Representative traces of membrane currents were recorded with a ramp protocol (lower panel, a voltage ramp from -80 mV to +60 mV over 500 ms). Fast activating Na + currents were prominent. e Representative current traces (upper panel) were recorded in voltage-clampmode. Cells were depolarized by voltage steps from − 60 to + 60 mV in 10-mV increments (Δ10mV, upper panel). The lower panel shows thecurrent-voltage (I-V) relationship for sodium current. f Sample traces of spontaneous synaptic currents (without pharmacological blockers) wererecorded at a holding potential of − 80 mV

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after 4 weeks. Thus, we conclude that reprogramingGBM cells to neurons can abrogate the parental tumorcell tumorigenicity.

Reprogramming orthotopic intracranial GBM preventslocal recurrenceDespite advances in varieties of treatment modalities forGBM, local relapse remains a clinically significant problem.We examined whether our reprogramming treatment couldprevent GBM local recurrence using a mouse model [37–40]. GBM tumors were generated by transplantingGFP-GM97 cells in nude mice. After four weeks, when pri-mary tumors had reached ~ 50 mm3, ~ 95% of each tumorwas excised except for a small fragment (2 mm3). This frag-ment was not removed to model residual tumor cells aftersurgery. Matrix-Driven Delivery (MDD) Pellet ofROCK-mTOR inhibitors with dried induction medium wasprepared by Innovative Research of America. The finishedpellet had a biodegradable matrix that effectively and con-tinuously releases the active product in the animal for threeweeks. We implanted the mTOR-ROCK inhibitors with orwithout induction medium in the MDD pellet into the re-section cavity of xenografts. Controls included no treatment,induction medium only and ROCK-mTOR inhibitors with-out induction medium in the MDD pellet. Tumor recur-rence and mouse survival were measured (Fig. 6c, d, n= 10).

Tumor recurrence was not observed up to 12 weeksin mice treated with mTOR-ROCK inhibitors withinduction medium treatment (0/10 mice, Fig. 6c).Local recurrence was observed within 3–4 weeks in10 of 10 mice without treatment or with inductionmedium only (Fig. 6c). 8/10 mice displayed tumorrecurrence in mTOR-ROCK inhibitors without in-duction medium, suggesting that there is only aweak effect on GBM when using mTOR-ROCK in-hibitors alone (direct chemotherapy). Immunohisto-chemistry (IHC) analysis revealed that 90% ± 5 ofGFP/MAPs2 and 70% ± 8 of GFP/synapsin positivecells were detected 2 weeks after mTOR-ROCK in-hibitor treatment with induction medium (Fig. 6e).These results suggest that GFP-GBM cells convertedinto mature neurons in vivo. Recurred tumors wereexamined from mTOR-ROCK inhibitors without in-duction medium, medium only or no treatment. Itwas observed that GFP-positive cells did not expressMAP2 (Additional file 1: Figure S7). Tumors frommTOR-ROCK inhibitor treatment without inductionmedium had fewer Ki67 positive cells and moreapoptotic cells as compared with tumors frommedium only or no treatment control (Fig. 6f-g).Taken together, these results indicate that thatmTOR-ROCK inhibitors under the reprogramming

Fig. 3 Global gene expression profiling of GBM and iNs. a Heat-map of differentially genes expressed was generated for GBM U118 (WT) and iNs.Positive control neurons are induced from IMR90 fibroblasts by p53 knockdown [49]. b Hierarchical clustering was performed. c qRT-PCR resultsshow relative mRNA levels of defined transcription factors in GBM U118 control and iNs (n = 3)

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condition play a key role in preventing GBM localrecurrence.

DiscussionCollectively, we screened a kinase inhibitor library andfound that a combination of the ROCK-mTOR kinase

inhibitors can substitute for all transcription factors toreprogram GBM cells. This conversion is highly efficient,fast, and yields mature neurons. Induced neurons losetheir tumorigenicity and reprogramming treatment pre-vents GBM local recurrence in mice. ReprogrammingGBM by kinase inhibitors is a transgene-free method

Fig. 4 Effects of ROCK1/2 and mTORC1 shRNAs on GBM cell conversion. a Western blot analysis from U118 cells showed that shRNA1 andshRNA2 of ROCK1/2 and mTORC1 (Raptor) /C2 (Rictor) are sufficient to inhibit ROCK1/2, Raptor or Rictor expression, respectively. Expressions ofdownstream targets pS6 for Raptor and pAKT1-S473 for Rictor were reduced by Raptor or Rictor shRNAs. R1: ROCK1, R2: ROCK2. b GBM U118 cellswere transduced with ROCK1/2 and mTOR1/2 shRNAs and then cultured in induction medium. Positive induce neural cells were counted usingRFP-positive staining and quantified on day 7. The histogram represents the ratio of normalized data between treatment with shRNAs or DMSO.R1: ROCK1, R2: ROCK2, m1: mTORC1. c Kinetic analysis was performed for iNs after induction with ROCK1/2-mTORC1 shRNAs in induction medium(induction for the first week) and neuronal mature medium (induction after 1 week). iN cells was quantified as MAP2 positive cells. Quantitativedata are the mean ± SEM from three independent experiments. d Expression of TUJ1, MAP2 and Synapsin in iNs was examined 3 weeks afterinduction with ROCK1/2-mTORC1 shRNAs. Scale bar, 10 μm

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and ROCK-mTOR inhibitors have been used in patients.We believe that our findings will assist in the develop-ment of GBM therapy.ROCK1 and ROCK2 have tissue-specific expression

patterns. ROCK1 is mainly expressed in lung, liver,

spleen, kidney and testis, while ROCK2 is distributedmostly in brain and heart. By acting on the cytoskeleton,ROCK regulates cell shape and movement, cellularimmortalization and differentiation [50–53]. mTOR reg-ulates cell growth, proliferation, protein synthesis, and

Fig. 5 Proliferation and invasion ability of iNs. a GBM GM97 and iN (3 weeks induction) cells were treated with 2 μM P + Y. Proliferating cells (viable)were evaluated by trypan blue dye exclusion assay at day 4 after treatment. Shown are the mean fold change in cell number relative to the 24-h timepoint and SEM from three treatments. b TUNEL assay was performed for GBM GM97 and iN cells treated with 2 μM P + Y. Apoptotic cells wereevaluated at day 4 after treatment from three treatments. Etoposide treatment (5 μM) was used as a positive control. c-e GBM GM97 and iN cells wereseeded for soft agar assays (c & d) and invasion assays (e). Quantitative data are the mean ± SEM from three independent experiments

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transcription [54–60]. ROCK inhibitors are already usedas anti-tumor drugs in patients, while mTOR inhibitorsare used to prevent transplant rejection, promote lon-gevity, and treat cancer in clinical trials. Thus, ofROCK-mTOR inhibitors-based reprogramming strategyis expected to have low risk in vivo and may rapidlymove towards clinical trials. GBM cell lines used in thisstudy also have p53 mutation or inactivation. In futurestudies, we will examine the effects of p53 on GBM

reprogramming. Among the various proteins involved inthe mTOR pathway, much interest in GBM has coatedSGK1 kinase. Recently, a new SGK1 kinase inhibitor hasshown good antiproliferative, proapoptotic capacity andsynergistic potential with radiotherapy in GBM in vitroand in vivo. [61, 62] It is interesting to determinewhether SGK1 regulates GBM reprogramming.One key issue with reprogramming therapy is that re-

programmed cells may be reverted back to GBM cells

Fig. 6 Reprogramming orthotopic GBMs in mice. a Experimental design for tumor formation in mice. b Parental GBM cells (Control) and iNs wereimplanted into the same nude mice (left and right side each mouse, respectively) and the tumor formation was measured. c & d GBM tumorsgenerated by GM97 cells in nude mice with postoperative recurrence model. ROCK-mTOR inhibitors (P529 and Y27632, P + Y) with inductionmedia in the MDD pellet were administered directly into the resection cavity of GBMs. P + Y without induction medium, medium only (both inthe MDD pellets) or no treatment were used as controls. Tumor recurrence (c) and mouse survival (d) were measured (n = 10). e Postoperativeresidual GFP-GBM were treated with ROCK-mTOR inhibitors (P + Y) with induction medium. GFP-positive cells expressed the mature neuronalmarkers MAP2 and Synapsin 2 weeks after transplantation. Data are presented as mean ± SEM from 5 mice (300 cells/section and 2 sections permouse were counted). The scale bar represents 10 μm. f-g Ki67 (IHC) and apoptotic (TUNEL assay) cells were evaluated in postoperative braintissues treated with P + Y and induction media. Normal brain tissue was as the control. Ki67 and apoptotic cells from recurred tumors treatedwith P + Y without induction media, media only or no treatment were also examined. Scale bar 10 μm

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and form a tumor in vivo. We found that induced neu-rons do not show signs of further proliferation following4 weeks in replacement of differentiation media withmaintenance media. Although we did not observe thatthe reprogrammed cells revert back, we could not ruleout reversal of reprogrammed cells into tumors. Add-itional experiments with genetically different samplesand long-term in vivo observation are needed to addressthis issue. It is known that GBM cell lines are highlypassaged cells that may not recapitulate the properties ofprimary GBM. We reprogrammed patient-derived pri-mary GBM cells in vitro and in vivo. In the future stud-ies, we will work on designing reprogramming strategiesfor patient-derived primary GBMs with different geneticbackground.Delineating the molecular mechanism behind neural

conversion will aid further development of the method.Gene expression analysis suggests that ROCK-mTORmay act as the “master regulator” to coordinate a set ofdefined factors in blocking cell reprogramming inphysiological conditions. Consistent with this view,ROCK-mTOR controls both iPS reprogramming andneural conversion, implicating a general mechanism ofreprogramming, where loss of ROCK-mTOR may gener-ate lineage progenitors that develop complete and func-tional lineages in various induction media. In previousstudies, we found that cellular senescence is a key stepin the conversion of human fibroblasts into functionalneural cells, including three neural lineages (astrocytes,neurons, and oligodendrocytes) [48, 49]. By geneticallymanipulating the senescence process, we have provideda new method for converting most human fibroblastsinto neural cells in only 1–2 weeks. Both ROCK andmTOR are involved in senescence and proliferation pro-cesses, implicating that the reprogramming process mayalso be regulated through the kinase-senescence signal-ing pathways. We found that expressions of a set ofneurogenic transcription factors, such as Neurod2, Ascl1and Myt1l, were increased during GBM-neuron cell con-version. Neurod2 is significantly downregulated in GBMpatients, which is involved in the p53 pathway [63].Ascl1 and Myt1l suppress GBM tumorigenicity and arerequired for glioblastoma stem cells to undergo neuronallineage differentiation [64–66]. Correlations of repro-gramming factors and GBM tumorigenesis need furtherinvestigation using patient tissues.The reprogramming-based therapy presents new ave-

nues for the development of GBM therapeutics.ROCK-mTOR inhibitors are already used as anti-tumordrugs in patients with few or no side effects. We expectthat neurons generated by ROCK-mTOR inhibitors are“safe” in vivo. However, it is been shown that aberrantsynapse formation between implanted neurons of thewrong subtype and endogenous cells can lead to

significant motor side effects in human clinical trials forParkinson’s disease. We will identify the subtypes of ourinduced neurons and evaluate their functional propertiesin vivo. Our results also show that the reprogrammingtreatment with kinase inhibitors does not affect normalneurons and astrocytes, supporting the safety of our re-programming strategy. We believe that our strategy is anovel, low risk, and effective method that can be com-bined with conventional therapy for clinical use.

ConclusionsIn this study, we have developed a novel cellreprogramming-based method for GBM treatment. Wescreened a kinase inhibitor library, and found mTOR andROCK inhibitors are sufficient to reprogram GBM cellsinto “normal” neurons. In vitro and in vivo tumorigenesisassays showed that induced neurons lose tumorigenicityand this reprogramming treatment prevented GBM localrecurrence in mice. The role of the ROCK-mTOR path-ways in GBM reprogramming is entirely novel.ROCK-mTOR inhibitors are already used as anti-tumordrugs in patients and promise few or no side effects in re-programming. Therefore, the reprogramming-based ther-apy will provide new avenues for development oftherapeutics for GBM in patients.

Additional file

Additional file 1: Figure S1. ROCK-mTOR kinase inhibitors reprogramGBM cells into iNs. Figure S2. ROCK-mTOR kinase inhibitors reprogramGBM cells into iNs. Figure S3. iNs did not express astrocyte and oligo-dendrocyte markers. Figure S4. Effects of mTOR-ROCK inhibitor treat-ment on normal primary neurons/astrocytes. Figure S5. iNs lose abilities ofproliferation. Figure S6. Proliferation and invasion ability of iNs. Figure S7.Recurred tumors did not express MAP2. (DOC 6831 kb)

AbbreviationsGBM: Glioblastoma multiforme; iN: induced neurons; Inhibitor P: palomid529; Inhibitor Y: Y27632; iPS: Induced pluripotent stem

AcknowledgementsWe thank the Animal Surgery Core at the Hope Center of WashingtonUniversity to help performing animal experiments. We thank Buck Rogersand Xiaowei Wang for proofreading this manuscript.

FundingThis work is supported in part by grants from NIH (1R01NS100950, QY),Children’s Discovery Institute (MC-II-2016-511, QY), Institute of Clinical andTranslational Sciences (1UL1TR00234501, QY) and Medical Scientific ResearchFoundation of Guangdong Province in China (A2017558, JY).

Availability of data and materialsNot applicable.

Authors’ contributionsQY generated the hypotheses, designed experiments and analyzed data. JY,FZ, ZZ, LMH and MY performed experiments and generated data. LGW & DHdesigned experiments and analyzed data. QY and DH wrote the manuscript.All authors read and approved the final manuscript.

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Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Author details1Cancer Biology Division, Department of Radiation Oncology, WashingtonUniversity School of Medicine, 4511 Forest Park, St. Louis, MO 63108, USA.2Medical Center of Stomatology, the First Affiliated Hospital of JinanUniversity, Guangzhou 510630, China. 3School of Stomatology, JinanUniversity, Guangzhou 510630, China. 4Synaptic Transmission Section,National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892,USA.

Received: 23 May 2018 Accepted: 19 July 2018

References1. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells

from adult human fibroblasts by defined factors. Cell. 2007;131:861–72.2. Pang ZP, Yang N, Vierbuchen T, et al. Induction of human neuronal cells by

defined transcription factors. Nature. 2011;476:220–3.3. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M.

Direct conversion of fibroblasts to functional neurons by defined factors.Nature. 2010;463:1035–41.

4. Qiang L, Fujita R, Yamashita T, et al. Directed conversion of Alzheimer's diseasepatient skin fibroblasts into functional neurons. Cell. 2011;146:359–71.

5. Caiazzo M, Dell'Anno MT, Dvoretskova E, et al. Direct generation offunctional dopaminergic neurons from mouse and human fibroblasts.Nature. 2011;476:224–7.

6. Pfisterer U, Kirkeby A, Torper O, et al. Direct conversion of human fibroblaststo dopaminergic neurons. Proc Natl Acad Sci U S A. 2011;108:10343–8.

7. Yoo AS, Sun AX, Li L, et al. MicroRNA-mediated conversion of humanfibroblasts to neurons. Nature. 2011;476:228–31.

8. Ambasudhan R, Talantova M, Coleman R, et al. Direct reprogramming ofadult human fibroblasts to functional neurons under defined conditions.Cell Stem Cell. 2011;9:113–8.

9. Son EY, Ichida JK, Wainger BJ, et al. Conversion of mouse and humanfibroblasts into functional spinal motor neurons. Cell Stem Cell. 2011;9:205–18.

10. Ring KL, Tong LM, Balestra ME, et al. Direct reprogramming of mouse andhuman fibroblasts into multipotent neural stem cells with a single factor.Cell Stem Cell. 2012;11:100–9.

11. Kim J, Su SC, Wang H, et al. Functional integration of dopaminergic neuronsdirectly converted from mouse fibroblasts. Cell Stem Cell. 2011;9:413–9.

12. Izgi K, Canatan H, Iskender B. Current status in cancer cell reprogrammingand its clinical implications. J Cancer Res Clin Oncol. 2017;143:371–83.

13. Powers S, Pollack RE. Inducing stable reversion to achieve cancer control.Nat Rev Cancer. 2016;16:266–70.

14. Lang JY, Shi Y, Chin YE. Reprogramming cancer cells: back to the future.Oncogene. 2013;32:2247–8.

15. Warrell RP Jr, Frankel SR, Miller WH Jr, et al. Differentiation therapy of acutepromyelocytic leukemia with tretinoin (all-trans-retinoic acid). N. Engl. J.Med. 1991;324:1385–93.

16. Hochedlinger K, Blelloch R, Brennan C, et al. Reprogramming of a melanomagenome by nuclear transplantation. Genes Dev. 2004;18:1875–85.

17. Ramos-Mejia V, Fraga MF, Menendez P. iPSCs from cancer cells: challengesand opportunities. Trends Mol Med. 2012;18:245–7.

18. Zhang X, Cruz FD, Terry M, Remotti F, Matushansky I. Terminaldifferentiation and loss of tumorigenicity of human cancers viapluripotency-based reprogramming. Oncogene. 2013;32:2249–60. 2260

19. Carette JE, Pruszak J, Varadarajan M, et al. Generation of iPSCs from culturedhuman malignant cells. Blood. 2010;115:4039–42.

20. Utikal J, Maherali N, Kulalert W, Hochedlinger K. Sox2 is dispensable for thereprogramming of melanocytes and melanoma cells into inducedpluripotent stem cells. J Cell Sci. 2009;122:3502–10.

21. Miyoshi N, Ishii H, Nagai K, et al. Defined factors induce reprogramming ofgastrointestinal cancer cells. Proc.Natl.Acad.Sci.U.S.A. 2010;107:40–5.

22. Chang G, Miao YL, Zhang Y, et al. Linking incomplete reprogramming tothe improved pluripotency of murine embryonal carcinoma cell-derivedpluripotent stem cells. PLoS.One. 2010;5:e10320.

23. Choi SM, Liu H, Chaudhari P, et al. Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells. Blood. 2011;118:1801–5.

24. Hu K, Yu J, Suknuntha K, et al. Efficient generation of transgene-freeinduced pluripotent stem cells from normal and neoplastic bone marrowand cord blood mononuclear cells. Blood. 2011;117:e109–19.

25. Lin SL, Chang DC, Chang-Lin S, et al. Mir-302 reprograms human skincancer cells into a pluripotent ES-cell-like state. RNA. 2008;14:2115–24.

26. Amson R, Pece S, Lespagnol A, et al. Reciprocal repression between P53and TCTP. Nat Med. 2011;18:91–9.

27. Amson R, Karp JE, Telerman A. Lessons from tumor reversion for cancertreatment. Curr Opin Oncol. 2013;25:59–65.

28. Papp B, Plath K. Reprogramming to pluripotency: stepwise resetting of theepigenetic landscape. Cell Res. 2011;21:486–501.

29. Ho R, Chronis C, Plath K. Mechanistic insights into reprogramming toinduced pluripotency. J Cell Physiol. 2011;226:868–78.

30. Han SS, Williams LA, Eggan KC. Constructing and deconstructing stem cellmodels of neurological disease. Neuron. 2011;70:626–44.

31. Cherry AB, Daley GQ. Reprogramming cellular identity for regenerativemedicine. Cell. 2012;148:1110–22.

32. Robinton DA, Daley GQ. The promise of induced pluripotent stem cells inresearch and therapy. Nature. 2012;481:295–305.

33. Bleeker FE, Molenaar RJ, Leenstra S. Recent advances in the molecularunderstanding of glioblastoma. J Neurooncol. 2012;108:11–27.

34. Tamimi AF, Juweid M, Thakkar JP, et al. Epidemiology and Outcome ofGlioblastoma. Epidemiologic and molecular prognostic review ofglioblastoma. Cancer Epidemiol Biomarkers Prev. 2014;23:1985–96.

35. Young RM, Jamshidi A, Davis G, Sherman JH. Current trends in the surgicalmanagement and treatment of adult glioblastoma. Ann Transl Med. 2015;3:121–5839.

36. Qin Y, Takahashi M, Sheets K, et al. Epithelial membrane protein-2 (EMP2)promotes angiogenesis in glioblastoma multiforme. J Neurooncol. 2017;134:29–40.

37. Hingtgen S, Figueiredo JL, Farrar C, et al. Real-time multi-modality imaging ofglioblastoma tumor resection and recurrence. J Neurooncol. 2013;111:153–61.

38. Kauer TM, Figueiredo JL, Hingtgen S, Shah K. Encapsulated therapeutic stemcells implanted in the tumor resection cavity induce cell death in gliomas.Nat Neurosci. 2011;15:197–204.

39. Fomchenko EI, Holland EC. Mouse models of brain tumors and theirapplications in preclinical trials. Clin Cancer Res. 2006;12:5288–97.

40. Jacobs VL, Valdes PA, Hickey WF, De Leo JA. Current review of in vivo GBMrodent models: emphasis on the CNS-1 tumour model. ASN.Neuro. 2011;3:e00063.

41. Collado M, Blasco MA, Serrano M. Cellular senescence in cancer and aging.Cell. 2007;130:223–33.

42. Yang Q. Cellular senescence, telomere recombination and maintenance.Cytogenet Genome Res. 2008;122:211–8.

43. Yang Q, Zhang R, Horikawa I, et al. Functional diversity of human protectionof telomeres 1 isoforms in telomere protection and cellular senescence.Cancer Res. 2007;67:11677–86.

44. Beausejour CM, Krtolica A, Galimi F, et al. Reversal of human cellularsenescence: roles of the p53 and p16 pathways. EMBO J. 2003;22:4212–22.

45. Campisi J. Senescent cells, tumor suppression, and organismal aging: goodcitizens, bad neighbors. Cell. 2005;120:513–22.

46. Utikal J, Polo JM, Stadtfeld M, et al. Immortalization eliminates a roadblockduring cellular reprogramming into iPS cells. Nature. 2009;460:1145–8.

47. Li H, Collado M, Villasante A, et al. The Ink4/Arf locus is a barrier for iPS cellreprogramming. Nature. 2009;460:1136–9.

48. Sun CK, Zhou D, Zhang Z, et al. Senescence impairs direct conversion ofhuman somatic cells to neurons. Nat Commun. 2014;5:4112.

49. Zhou D, Zhang Z, He LM, et al. Conversion of fibroblasts to neural cells byp53 depletion. Cell Rep. 2014;9:2034–42.

50. Wong CC, Wong CM, Au SL, Ng IO. RhoGTPases and rho-effectors inhepatocellular carcinoma metastasis: ROCK N'Rho move it. Liver Int.2010;30:642–56.

Yuan et al. Journal of Experimental & Clinical Cancer Research (2018) 37:181 Page 13 of 14

51. Micuda S, Rosel D, Ryska A, Brabek J. ROCK inhibitors as emerging therapeuticcandidates for sarcomas. CurrCancer Drug Targets. 2010;10:127–34.

52. Narumiya S, Tanji M, Ishizaki T. Rho signaling, ROCK and mDia1, in transformation,metastasis and invasion. Cancer Metastasis Rev. 2009;28:65–76.

53. Olson MF. Applications for ROCK kinase inhibition. Curr Opin Cell Biol. 2008;20:242–8.

54. Chen T, Shen L, Yu J, et al. Rapamycin and other longevity-promotingcompounds enhance the generation of mouse induced pluripotent stemcells. Aging Cell. 2011;10:908–11.

55. Menendez JA, Vellon L, Oliveras-Ferraros C, Cufi S, Vazquez-Martin A. mTOR-regulated senescence and autophagy during reprogramming of somaticcells to pluripotency: a roadmap from energy metabolism to stem cellrenewal and aging. Cell Cycle. 2011;10:3658–77.

56. Cornu M, Albert V, Hall MN. mTOR in aging, metabolism, and cancer. CurrOpin Genet Dev; 2013;23:53–62.

57. Kaeberlein M. Lessons on longevity from budding yeast. Nature. 2010;464:513–9.

58. Weichhart T. Mammalian target of rapamycin: a signaling kinase for everyaspect of cellular life. Methods Mol Biol. 2012;821:1–14.

59. McCormick MA, Tsai SY, Kennedy BK. TOR and ageing: a complex pathwayfor a complex process. Philos.Trans.R.Soc.Lond B Biol Sci. 2011;366:17–27.

60. Harrison DE, Strong R, Sharp ZD, et al. Rapamycin fed late in life extendslifespan in genetically heterogeneous mice. Nature. 2009;460:392–5.

61. Abbruzzese C, Catalogna G, Gallo E, et al. The small molecule SI113synergizes with mitotic spindle poisons in arresting the growth of humanglioblastoma multiforme. Oncotarget. 2017;8:110743–55.

62. Talarico C, Dattilo V, D'Antona L, et al. SI113, a SGK1 inhibitor, potentiatesthe effects of radiotherapy, modulates the response to oxidative stress andinduces cytotoxic autophagy in human glioblastoma multiforme cells.Oncotarget. 2016;7:15868–84.

63. Agrawal R, Garg A, Benny MP, Sharma V, Sarkar C, Kulshreshtha R. p53 andmiR-210 regulated NeuroD2, a neuronal basic helix-loop-helix transcriptionfactor, is downregulated in glioblastoma patients and functions as a tumorsuppressor under hypoxic microenvironment. Int J Cancer. 2018;142:1817–28.

64. Park NI, Guilhamon P, Desai K, et al. ASCL1 reorganizes chromatin to directneuronal fate and suppress Tumorigenicity of glioblastoma stem cells. CellStem Cell. 2017;21:209–24.

65. Guichet PO, Guelfi S, Teigell M, et al. Notch1 stimulation induces avascularization switch with pericyte-like cell differentiation of glioblastomastem cells. Stem Cells. 2015;33:21–34.

66. Hu J, Ho AL, Yuan L, et al. From the cover: neutralization of terminaldifferentiation in gliomagenesis. Proc Natl Acad Sci USA. 2013;110:14520–7.

Yuan et al. Journal of Experimental & Clinical Cancer Research (2018) 37:181 Page 14 of 14