Increased expression of the glioma-associated antigen ARF4L after loss of the tumor suppressor PTEN

Laboratory investigation

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Object

Despite recent advances in cancer immunotherapy, cellular mechanisms controlling expression of tumor-associated antigens are poorly understood. Mutations in cancer cells, such as loss of PTEN, may increase expression of tumor-associated antigens. The authors investigated the relationship between PTEN status and the expression of a glioma-associated antigen, adenosine diphosphate–ribosylation factor 4–like (ARF4L) protein.

Methods

Human glioma cell lines with confirmed PTEN status were examined by Northern blot analysis and quantitative polymerase chain reaction. Western blot analysis was used to measure ARF4L protein levels across multiple cell lines.

Results

The loss of PTEN was shown to lead to increased levels of ARF4L protein but no change in transcript levels. Cell lines with serial mutations, including activation of Ras and Akt pathways, also demonstrated increased levels of ARF4L protein, which decreased after treatment with rapamycin. The ARF4L transcript preferentially localized to the polysomal compartment after PTEN loss in glioma or activation of Akt in human astrocytes.

Conclusions

Expression of ARF4L is controlled by the activated Akt/mTOR pathway, which is a downstream effect of the loss of PTEN function. Mutations leading to oncogenesis may impact the regulation and expression of tumor specific antigens. Screening of mutation status in glioma may be helpful in selecting patients for immunotherapy trials in the future.

Abbreviations used in this paper:ARF4L = adenosine diphosphate–ribosylation factor 4–like; EDTA = ethylenediaminetetraacetic acid; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GBM = glioblastoma multiforme; hTERT = human telomerase reverse transcriptase; mTOR = mammalian target of rapamycin; NHA = normal human astrocyte; PCR = polymerase chain reaction; PI3-K = phosphatidylinositol 3-kinase; PTEN = phosphatase and tensin homolog; TAA = tumor-associated antigen; UCSF = University of California, San Francisco; 4-HT = 4-hydroxytamoxifen.

Article Information

Address correspondence to: Andrew T. Parsa, M.D., Ph.D., 505 Parnassus Avenue, M779, Box 0112, San Francisco, California 94143. email: parsaa@neurosurg.ucsf.edu.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Western blot showing that the PTEN status correlates with ARF4L levels in glioma tissue. Fresh-frozen human Grade IV glioma tissue (4-digit sample numbers) and human Grade IV glioma xenograft tissue (samples 5, 6, 8, 10, 12, 14) were previously tested for PTEN status and confirmed with S6 kinase activation and phospho-S6 activity. Protein from specimens with loss of PTEN are denoted with a minus sign, whereas specimens with wild-type PTEN are denoted with a plus sign. Western blot analysis demonstrates that the loss of PTEN correlates with higher levels of ARF4L in tissue and cell line samples, including U87 cells, compared with samples from tissue and cells lines with wild-type PTEN.

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    Western blot showing that activated Akt and Ras pathways correlate with elevated ARF4L levels. Normal human astrocytes were serially altered with mutations conferring activated E6, E7, hTERT, Ras, and Akt function. Western blot analysis shows ARF4L levels are higher in cells with activated Ras and Akt function compared with NHAs and E6/E7/hTERT activation. The Ras effector mutant cell lines, C40, S35, and G37, also demonstrate elevated ARF4L levels, and correspond to constitutively active PI3-K, Ras and Ral, respectively.

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    Western blot showing that rapamycin reduces ARF4L protein levels in cells with activated Akt/Ras pathways. Rapamycin treatment of NHAs and cells with E6/E7/hTERT and E6/E7/hTERT+Ras mutations did not change protein expression levels of ARF4L. However, rapamycin treatment did reduce levels of ARF4L in cells with activated Akt mutation.

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    Blot showing that inducing Akt function increases ARF4L expression and can be blocked by rapamycin treatment. The U87 cells stably transfected with an Akt construct induced by estrogen show significant increase in ARF4L with 4HT. This inducible ARF4L expression is then reduced by treatment with rapamycin.

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    Bar graphs showing that ARF4L mRNA differentially localizes to polysomes according to Akt activation and PTEN status. Upper: The ARF4L is preferentially found in monosomes in NHA and PTEN+/+ cell lines, and they preferentially localize to polysomes in Akt-activated and PTEN/ cell lines. Asterisks denote a statistically significant difference between comparison groups. Lower: With rapamycin treatment, ARF4L transcripts shift from polysomes to monosomes in Akt-activated and PTEN/ cell lines. Data shown are results from quantitative real-time PCR experiments using percentage of fluorescence intensity standardized to GAPDH. The ARF4L in total mRNA did not change significantly between rapamycin treated and untreated groups.

References

  • 1

    Brossart PStuhler GFlad TStevanovic SRammensee HKanz L: Her-2/neu-derived peptides are tumor-associated antigens expressed by human renal cell and colon carcinoma lines and are recognized by in vitro induced specific cytotoxic T lymphocytes. Cancer Res 58:7327361998

    • Search Google Scholar
    • Export Citation
  • 2

    Hirose YKatayama MStokoe DHaas-Kogan DABerger MSPeiper RO: The p38 mitogen-activated protein kinase pathway links the DNA mismatch repair system to the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents. Mol Cell Biol 23:830683152003

    • Search Google Scholar
    • Export Citation
  • 3

    Johannes GSarnow P: Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites. RNA 4:150015131998

    • Search Google Scholar
    • Export Citation
  • 4

    Kahn RAKern FGClark JGelmann EPRulka C: Human ADP-ribosylation factors. A functionally conserved family of GTP-binding proteins. J Biol Chem 266:260626141991

    • Search Google Scholar
    • Export Citation
  • 5

    Katayama TImaizumi KYoneda TTaniguchi MHonda AManabe T: Role of ARF4L in recycling between endosomes and the plasma membrane. Cell Mol Neurobiol 24:1371472004

    • Search Google Scholar
    • Export Citation
  • 6

    Kleihues POhgaki H: Primary and secondary glioblastomas: from concept to clinical diagnosis. Neurooncol 1:44511999

  • 7

    Kohn ADTakeuchi FRoth RA: Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. J Biol Chem 271:21920219261996

    • Search Google Scholar
    • Export Citation
  • 8

    Konopka GBonni A: Signaling pathways regulating gliomagenesis. Curr Mol Med 3:73842003

  • 9

    Liau LMBlack KLMartin NASykes SNBronstein JMJouben-Steele L: Treatment of a patient by vaccination with autologous dendritic cells pulsed with allogeneic major histocompatibility complex class I-matched tumor peptides. Case report. Neurosurg Focus 9:6E82000

    • Search Google Scholar
    • Export Citation
  • 10

    Mao CKoutsky LAAult KAWheeler CMBrown DRWiley DJ: Efficacy of human papillomavirus-16 vaccine to prevent cervical intraepithelial neoplasia: a randomized controlled trial. Obstet Gynecol 107:18272006

    • Search Google Scholar
    • Export Citation
  • 11

    Nair SKHeiser ABoczkowski DMajumdar ANaoe MLebkowski JS: Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. Nat Med 6:101110172000

    • Search Google Scholar
    • Export Citation
  • 12

    Neshat MSMellinghoff IKTran CStiles BThomas GPetersen R: Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci U S A 98:10314103192001

    • Search Google Scholar
    • Export Citation
  • 13

    Nestle FOAlijagic SGilliet MSun YGrabbe SDummer R: Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4:3283321998

    • Search Google Scholar
    • Export Citation
  • 14

    Nonaka YTsuda NShichijo SIto MMaeda YHarada M: Recognition of ADP-ribosylation factor 4-like by HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes from patients with brain tumors. Tissue Antigens 60:3193272002

    • Search Google Scholar
    • Export Citation
  • 15

    Panner AParsa ATPieper RO: Translational regulation of TRAIL sensitivity. Cell Cycle 5:1471502006

  • 16

    Parsa ATHolland EC: Cooperative translational control of gene expression by Ras and Akt in cancer. Trends Mol Med 10:6076132004

  • 17

    Rajasekhar VKViale ASocci NDWiedmann MHu XHolland EC: Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol Cell 12:8899012003

    • Search Google Scholar
    • Export Citation
  • 18

    Sonoda YOzawa TAldape KDDeen DFBerger MSPieper RO: Akt pathway activation converts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma. Cancer Res 61:667466782001

    • Search Google Scholar
    • Export Citation
  • 19

    Sonoda YOzawa THirose YAldape KDMcMahon MBerger MS: Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma. Cancer Res 61:495649602001

    • Search Google Scholar
    • Export Citation
  • 20

    Wang JYDel Valle LGordon JRubini MRomano GCroul S: Activation of the IGF-IR system contributes to malignant growth of human and mouse medulloblastomas. Oncogene 20:385738682001

    • Search Google Scholar
    • Export Citation
  • 21

    Wei QClarke LScheidenhelm DKQian BTong ASabha N: High-grade glioma formation results from postnatal pten loss or mutant epidermal growth factor receptor expression in a transgenic mouse glioma model. Cancer Res 66:742974372006

    • Search Google Scholar
    • Export Citation
  • 22

    Yannelli JRHyatt CJohnson SHwu PRosenberg SA: Characterization of human tumor cell lines transduced with the cDNA encoding either tumor necrosis factor alpha (TNF-a) or interleu-kin-2 (IL-2). J Immunol Methods 161:77901993

    • Search Google Scholar
    • Export Citation

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