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.
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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
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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.).
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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.
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Work was performed with Institutional Biosafety Committee approval from the University of Texas Rio Grande Valley: Registration number: 2016–003-IBC.
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432_2020_3133_MOESM1_ESM.tif
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)
432_2020_3133_MOESM2_ESM.tif
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)
432_2020_3133_MOESM3_ESM.tif
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)
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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 146, 593–604 (2020). https://doi.org/10.1007/s00432-020-03133-w
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DOI: https://doi.org/10.1007/s00432-020-03133-w