Advertisement

Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

The PI3K pathway impacts stem gene expression in a set of glioblastoma cell lines

  • 56 Accesses

Abstract

Background

The PI3K pathway controls diverse cellular processes including growth, survival, metabolism, and apoptosis. Nuclear FOXO factors were observed in cancers that harbor constitutively active PI3K pathway output and stem signatures. FOXO1 and FOXO3 were previously published to induce stem genes such as OCT4 in embryonic stem cells. Here, we investigated FOXO-driven stem gene expression in U87MG glioblastoma cells.

Methods

PI3K-activated cancer cell lines were investigated for changes in gene expression, signal transduction, and clonogenicity under conditions with FOXO3 disruption or exogenous expression. The impact of PI3K pathway inhibition on stem gene expression was examined in a set of glioblastoma cell lines.

Results

We found that CRISPR-Cas9-mediated FOXO3 disruption in U87MG cells caused decreased OCT4 and SOX2 gene expression, STAT3 phosphorylation on tyrosine 705 and clonogenicity. FOXO3 over expression led to increased OCT4 in numerous glioblastoma cancer cell lines. Strikingly, treatment of glioblastoma cells with NVP-BEZ235 (a dual inhibitor of PI3K and mTOR), which activates FOXO factors, led to robust increases OCT4 gene expression. Direct FOXO factor recruitment to the OCT4 promoter was detected by chromatin immunoprecipitation analyses using U87MG extracts.

Discussion

We show for the first time that FOXO transcription factors promote stem gene expression glioblastoma cells. Treatment with PI3K inhibitor NVP-BEZ235 led to dramatic increases in stem genes in a set of glioblastoma cell lines.

Conclusion

Given that, PI3K inhibitors are actively investigated as targeted cancer therapies, the FOXO-mediated induction of stem genes observed in this study highlights a potential hazard to PI3K inhibition. Understanding the molecular underpinnings of stem signatures in cancer will allow refinements to therapeutic strategies. Targeting FOXO factors to reduce stem cell characteristics in concert with PI3K inhibition may prove therapeutically efficacious.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Data availability

All cell lines and additional data prepared from this work are available upon request.

Abbreviations

PI3K:

Phosphatidylinositol 3 Kinase

PIP2:

Phosphatidylinositol 4,5-bisphosphate

PIP3:

Phosphatidylinositol 3,4,5-trisphosphate

AKT:

Protein Kinase B

PTEN :

Phosphatase and Tensin homolog deleted on chromosome ten

FOXO:

Forkhead box subfamily O

ES:

Embryonic stem

GBM:

Glioblastoma multiforme

BBC:

Basal breast cancer

OCT4 :

Octamer-binding Transcription factor 4

SOX2 :

Sex determining region Y-box 2

CRISPR:

Clustered Regularly Interspaced Short Palindromic Repeats

Cas9:

CRISPR-associated sequence 9

NPTII :

Neomycin resistance cassette (Neomycin phosphotransferase)

ATCC:

American Type Culture Collection

MEM:

Minimal essential media

DMEM:

Dulbecco's Modified Eagle Medium

RPMI:

Roswell Park Memorial Institute (RPMI) 1640 Medium

FBS:

Fetal bovine serum

PBS:

Phosphate-buffered saline

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene fluoride

TBST:

1X Tris-buffered saline with Tween 20

kDa:

Kilo Dalton

ACTB:

Beta actin

LIF :

Leukemia Inhibitory Factor

IL6 :

Interleukin 6

NANOG :

Nanog homeobox

SHH :

Sonic Hedge Hog

TGFB1 :

Transforming Growth Factor beta1

EGF :

Epidermal Growth Factor

ALPP :

Alkaline Phosphatase Placental

TUBB3 :

Tubulin Beta 3 class III

STAT3:

Signal transducer and activator of transcription 3

JAK2:

Janus Kinase 2

IBC:

Institutional Biosafety Committee

Cyt:

Cytoplasm

Nuc:

Nucleus

References

  1. Bellacosa A et al (1998) Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene 17:313–325. https://doi.org/10.1038/sj.onc.1201947

  2. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, Weinberg RA (2008) An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 40:499–507. https://doi.org/10.1038/ng.127

  3. Bigarella CL, Li J, Rimmele P, Liang R, Sobol RW, Ghaffari S (2017) FOXO3 transcription factor is essential for protecting hematopoietic stem and progenitor cells from oxidative DNA damage. J Biol Chem 292:3005–3015. https://doi.org/10.1074/jbc.M116.769455

  4. Brunet A et al (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868

  5. Caino MC et al (2015) PI3K therapy reprograms mitochondrial trafficking to fuel tumor cell invasion. Proc Natl Acad Sci USA 112:8638–8643. https://doi.org/10.1073/pnas.1500722112

  6. Calnan DR, Brunet A (2008) The FoxO code Oncogene 27:2276–2288. https://doi.org/10.1038/onc.2008.21

  7. Daniele S et al (2015) Combined inhibition of AKT/mTOR and MDM2 enhances Glioblastoma Multiforme cell apoptosis and differentiation of cancer stem cells. Sci Rep 5:9956. https://doi.org/10.1038/srep09956

  8. Galoczova M, Coates P, Vojtesek B (2018) STAT3, stem cells, cancer stem cells and p63. Cell Mol Biol Lett 23:12. https://doi.org/10.1186/s11658-018-0078-0

  9. Ghaffari SG et al (2010) Foxo1 is essential for the regulation of pluripotency in embryonic stem cells. Exp Hematol 38:S120–S120

  10. Hagenbuchner J et al (2016) Nuclear FOXO3 predicts adverse clinical outcome and promotes tumor angiogenesis in neuroblastoma. Oncotarget 7:77591–77606. https://doi.org/10.18632/oncotarget.12728

  11. Hopkins BD et al (2018) Suppression of insulin feedback enhances the efficacy of PI3K inhibitors. Nature 560:499–503. https://doi.org/10.1038/s41586-018-0343-4

  12. Ikeda M, Toyoshima F (2017) Dormant pluripotent cells emerge during neural differentiation of embryonic stem cells in a FoxO3-dependent manner. Mol Cell Biol. https://doi.org/10.1128/MCB.00417-16

  13. Iwadate Y et al (2017) The pluripotent stem-cell marker alkaline phosphatase is highly expressed in refractory glioblastoma with DNA hypomethylation. Neurosurgery 80:248–256. https://doi.org/10.1093/neuros/nyw026

  14. Jacobs FM, van der Heide LP, Wijchers PJ, Burbach JP, Hoekman MF, Smidt MP (2003) FoxO6, a novel member of the FoxO class of transcription factors with distinct shuttling dynamics. J Biol Chem 278:35959–35967. https://doi.org/10.1074/jbc.M302804200

  15. Jones NM, Rowe MR, Shepherd PR, McConnell MJ (2016) Targeted inhibition of dominant PI3-kinase catalytic isoforms increase expression of stem cell genes in glioblastoma cancer stem cell models. Int J Oncol 49:207–216. https://doi.org/10.3892/ijo.2016.3510

  16. Keniry M et al (2013) Survival factor NFIL3 restricts FOXO-induced gene expression in cancer. Genes Dev 27:916–927. https://doi.org/10.1101/gad.214049.113

  17. Keniry M, Parsons R (2008) The role of PTEN signaling perturbations in cancer and in targeted therapy. Oncogene 27:5477–5485. https://doi.org/10.1038/onc.2008.248

  18. Kumazoe M et al (2017) The FOXO3/PGC-1beta signaling axis is essential for cancer stem cell properties of pancreatic ductal adenocarcinoma. J Biol Chem 292:10813–10823. https://doi.org/10.1074/jbc.M116.772111

  19. Li J et al (1997) PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275:1943–1947

  20. Li Y et al (2011) Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Res 21:196–204. https://doi.org/10.1038/cr.2010.142

  21. Li X, Dai D, Chen B, Tang H, Xie X, Wei W (2018) Efficacy of PI3K/AKT/mTOR pathway inhibitors for the treatment of advanced solid cancers: a literature-based meta-analysis of 46 randomised control trials. PLoS ONE 13:e0192464. https://doi.org/10.1371/journal.pone.0192464

  22. Liang R, Rimmele P, Bigarella CL, Yalcin S, Ghaffari S (2016) Evidence for AKT-independent regulation of FOXO1 and FOXO3 in haematopoietic stem and progenitor cells. Cell Cycle 15:861–867. https://doi.org/10.1080/15384101.2015.1123355

  23. Lin SF, Huang YY, Lin JD, Chou TC, Hsueh C, Wong RJ (2012) Utility of a PI3K/mTOR inhibitor (NVP-BEZ235) for thyroid cancer therapy. PLoS ONE 7:e46726. https://doi.org/10.1371/journal.pone.0046726

  24. Lin A, Piao HL, Zhuang L, dos Sarbassov D, Ma L, Gan B (2014) FoxO transcription factors promote AKT Ser473 phosphorylation and renal tumor growth in response to pharmacologic inhibition of the PI3K-AKT pathway. Cancer Res 74:1682–1693. https://doi.org/10.1158/0008-5472.CAN-13-1729

  25. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Method Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262

  26. Loh YH et al (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38:431–440. https://doi.org/10.1038/ng1760

  27. Luo J, Manning BD, Cantley LC (2003) Targeting the PI3K-Akt pathway in human cancer: rationale and promise. Cancer Cell 4:257–262

  28. Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream. Cell 129:1261–1274. https://doi.org/10.1016/j.cell.2007.06.009

  29. Marotta LL et al (2011) The JAK2/STAT3 signaling pathway is required for growth of CD44(+)CD24(-) stem cell-like breast cancer cells in human tumors. J Clin Invest 121:2723–2735. https://doi.org/10.1172/JCI44745

  30. Matsushima M et al (2015) Intravesical dual PI3K/mTOR complex 1/2 inhibitor NVP-BEZ235 therapy in an orthotopic bladder cancer model. Int J Oncol 47:377–383. https://doi.org/10.3892/ijo.2015.2995

  31. Miyamoto K et al (2007) Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell Stem Cell 1:101–112. https://doi.org/10.1016/j.stem.2007.02.001

  32. Molyneaux KA, Schaible K, Wylie C (2003) GP130, the shared receptor for the LIF/IL6 cytokine family in the mouse, is not required for early germ cell differentiation, but is required cell-autonomously in oocytes for ovulation. Development 130:4287–4294

  33. Nakae J, Kitamura T, Silver DL, Accili D (2001) The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. J Clin Invest 108:1359–1367. https://doi.org/10.1172/JCI12876

  34. Niu H, Cattoretti G, Dalla-Favera R (2003) BCL6 controls the expression of the B7–1/CD80 costimulatory receptor in germinal center B cells. J Exp Med 198:211–221. https://doi.org/10.1084/jem.20021395

  35. O'Connor MD et al (2008) Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem Cells 26:1109–1116. https://doi.org/10.1634/stemcells.2007-0801

  36. Oh HM, Yu CR, Dambuza I, Marrero B, Egwuagu CE (2012) STAT3 protein interacts with Class O Forkhead transcription factors in the cytoplasm and regulates nuclear/cytoplasmic localization of FoxO1 and FoxO3a proteins in CD4(+) T cells. J Biol Chem 287:30436–30443. https://doi.org/10.1074/jbc.M112.359661

  37. Okkenhaug K, Vanhaesebroeck B (2003) PI3K in lymphocyte development, differentiation and activation. Nat Rev Immunol 3:317–330. https://doi.org/10.1038/nri1056

  38. Paik JH et al (2007) FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 128:309–323. https://doi.org/10.1016/j.cell.2006.12.029

  39. Poirier K et al (2010) Mutations in the neuronal ss-tubulin subunit TUBB3 result in malformation of cortical development and neuronal migration defects. Hum Mol Genet 19:4462–4473. https://doi.org/10.1093/hmg/ddq377

  40. Raz R, Lee CK, Cannizzaro LA, d'Eustachio P, Levy DE (1999) Essential role of STAT3 for embryonic stem cell pluripotency. Proc Natl Acad Sci USA 96:2846–2851

  41. Renault VM et al (2009) FoxO3 regulates neural stem cell homeostasis. Cell Stem Cell 5:527–539. https://doi.org/10.1016/j.stem.2009.09.014

  42. Rivas S, Gomez-Oro C, Anton IM, Wandosell F (2018) Role of Akt isoforms controlling cancer stem cell survival. Phenotype Self Renew Biomed. https://doi.org/10.3390/biomedicines6010029

  43. Saal LH et al (2005) PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma. Cancer Res 65:2554–2559. https://doi.org/10.1158/0008-5472-CAN-04-3913

  44. Saal LH et al (2007) Poor prognosis in carcinoma is associated with a gene expression signature of aberrant PTEN tumor suppressor pathway activity. Proc Natl Acad Sci USA 104:7564–7569. https://doi.org/10.1073/pnas.0702507104

  45. Saal LH et al (2008) Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair. Nat Genet 40:102–107. https://doi.org/10.1038/ng.2007.39

  46. Sunayama J et al (2011) FoxO3a functions as a key integrator of cellular signals that control glioblastoma stem-like cell differentiation and tumorigenicity. Stem Cells 29:1327–1337. https://doi.org/10.1002/stem.696

  47. Sykes SM et al (2011) AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias. Cell 146:697–708. https://doi.org/10.1016/j.cell.2011.07.032

  48. Tothova Z, Gilliland DG (2007) FoxO transcription factors and stem cell homeostasis: insights from the hematopoietic system. Cell Stem Cell 1:140–152. https://doi.org/10.1016/j.stem.2007.07.017

  49. Tothova Z et al (2007) FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell 128:325–339. https://doi.org/10.1016/j.cell.2007.01.003

  50. Trinh DL et al (2013) Analysis of FOXO1 mutations in diffuse large B-cell lymphoma. Blood 121:3666–3674. https://doi.org/10.1182/blood-2013-01-479865

  51. van der Heide LP, Jacobs FM, Burbach JP, Hoekman MF, Smidt MP (2005) FoxO6 transcriptional activity is regulated by Thr26 and Ser184, independent of nucleo-cytoplasmic shuttling. Biochem J 391:623–629. https://doi.org/10.1042/BJ20050525

  52. Vazquez N et al (2018) A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone. BMC Mol Biol 19:3. https://doi.org/10.1186/s12867-018-0105-8

  53. Xu K, Zhang Z, Pei H, Wang H, Li L, Xia Q (2017) FoxO3a induces temozolomide resistance in glioblastoma cells via the regulation of beta-catenin nuclear accumulation. Oncol Rep 37:2391–2397. https://doi.org/10.3892/or.2017.5459

  54. Yu Z et al (2015) Differential properties of human ALP(+) periodontal ligament stem cells vs their ALP(-) counterparts. J Stem Cell Res Ther. https://doi.org/10.4172/2157-7633.1000292

  55. Yu F et al (2018) FoxO1 inhibition promotes differentiation of human embryonic stem cells into insulin producing cells. Exp Cell Res 362:227–234. https://doi.org/10.1016/j.yexcr.2017.11.022

  56. Zhang X et al (2011) FOXO1 is an essential regulator of pluripotency in human embryonic stem cells. Nat Cell Biol 13:1092–1099. https://doi.org/10.1038/ncb2293

Download references

Acknowledgements

The authors would like to thank the UTRGV Department of Biology and COS for their support, reagents, and expertise. This work was supported by National Institute of Health: 1SC3GM132053-01 (M.K.), Howard Hughes Medical Association: 52007568 (N.V. and R.M.), United States Department of Agriculture: Step 2 2015–38422-24061(A.L.), United States Department of Agriculture: H.S.I. 2016–38422-25760 (M.K. and E.M), National Institute of Health 5R25GM10086606 (A.S.), UTRGV College of Sciences (COS) Seed Grant (M.K.), National Science Foundation: Advance 1209210 (M.K.), and National Science Foundation: 1463991 (E.S and M.K.).

Funding

This work was supported by NIH 1SC3GM132053-01 (M. K.), HHMI 52007568 (N. V. and R. M.), USDA Step 2 2015–38422-24061(A. L.), USDA H. S. I. 2016–38422-25760 (M. K. and E. M.), NIH 5R25GM10086606 (A. S.), UTRGV College of Sciences (COS) Seed Grant (M. K.), NSF Advance 1209210 (M. K.), and NSF 1463991 (E. S. and M. K.).

Author information

EM, NV, WI, ES, MP, and MK formulated the hypothesis, organized the study, designed the protocol, analyzed the data, and wrote the manuscript. NV, RM. VF, LS, AL, AS, LH, VC, AR, CT, MC, MA, MH, NG, AA, KA, LM, VR, AB, and MK performed the experiments.

Correspondence to Megan Keniry.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval

Work was performed with Institutional Biosafety Committee approval from the University of Texas Rio Grande Valley: Registration number: 2016–003-IBC.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Exogenous FOXO3 and Dual PI3K Inhibitor NVP-BEZ235 Induced OCT4 in HEK 293 and BT549 Cells. A. OCT4 gene expression (determined by qRT-PCR) in indicated cell line with exogenous FOXO3. OCT4 gene expression was increased in FOXO3 transfected cells. B. HEK 293 and BT549 cells were treated with 50nM NVP-BEZ235 for five days and analyzed by q-RT-PCR. NVP-BEZ235 treated cells had increased OCT4 gene expression. C. Alkaline Phosphatase ALPP gene expression was assessed by q-RT-PCT in indicted cell lines treated with NVP-BEZ235 for five days. ALPP was induced in NVP-BEZ235 treated cell lines. *Significant difference indicated by Students T-test (TIF 93 kb)

Figure S2. FOXO3 Nuclear Localization was not impacted by NVP-BEZ235 Treatment. A. U87MG cells that harbor wild-type or mutant FOXO3 were treated with 1mM NVP-BEZ235 for 5 days and fractionated. GAPDH and Histone H3 served as cytoplasmic (cyt.) and nuclear (nuc.) controls respectively. NVP-BEZ235 treated samples had overall less protein. The proportion of nuclear FOXO3 (wild-type as well as mutant) was unchanged with NVP-BEZ235 treatment compared to DMSO controls (TIF 135 kb)

Figure S3. NVP-BEZ235 Treatment of U87MG Cells induced both OCT4 and TUBB3 gene expression. A. U87MG cells were treated with DMSO, 1mM NVP-BEZ235, 10mM UO126 or both drugs as indicated for three days and analyzed for q-RT-PCR. NVP-BEZ235 treated cells had increased OCT4 and TUBB3 gene expression compared to the DMSO control cells. Tukey Test was performed * and ** denote P<0.05 in comparison to cognate DMSO control for OCT4 and TUBB3 gene expression respectively. Induction of OCT4 and TUBB3 were also significantly higher in double treatment samples (with both NVP-BEZ235 and UO126) compared to single treatment with NVP-BEZ235 based on Tukey Test (P<0.05). B. Western blot analysis was performed with lysates from U87MG cells with indicated drug treatments for three days (same drug treatments were utilized as in panel A) (TIF 128 kb)

Supplementary file4 (DOCX 17 kb)

Supplementary file5 (DOCX 14 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Martinez, E., Vazquez, N., Lopez, A. et al. The PI3K pathway impacts stem gene expression in a set of glioblastoma cell lines. J Cancer Res Clin Oncol (2020). https://doi.org/10.1007/s00432-020-03133-w

Download citation

Keywords

  • FOXO transcription factors
  • Stem genes
  • OCT4
  • PI3K inhibition
  • Glioblastoma