Chordoma Foundation


The PI3K/Akt/mTOR Pathway in Chordoma

Understanding the Pathway: Activation of Akt, either by growth factors or by RAS, leads to the phosphorylation of PI3k and PDK-1 and the subsequent inactivation of TSC1/TSC2 complex. When this complex is inactivated, Rheb is able to phosphorylate mTOR. This leads to the downstream phosphorylation of p70S6K and 4E-BP1. PTEN is a tumor suppressor gene whose expression negatively regulates the interactions of this pathway.

Activation of the PI3k/Akt/mTOR signaling pathway suppresses apoptosis, promotes cell growth, and drives cellular proliferation.1 Abnormalities involving the various components of this complicated pathway have been implicated in a number of human cancers.

Attention was first focused on this pathway after numerous reports of chordoma tumors in individuals with tuberous sclerosis syndrome. This disease involves the TSC1 and TSC2 genes, key players in the PI3K/Akt/mTOR pathway. Further investigation revealed that the PI3k/Akt/mTOR pathway is activated in the majority of chordomas and suggested that signaling through PI3k, Akt, and mTOR may be critical to chordoma pathogenesis.2  Cell lines and mouse models treated with substances that inhibit mTOR, PI3K, or Akt not only diminish the pathway’s activity but also reduce chordoma cellular proliferation and even trigger cell death. Though response has been mixed, a number of patients have been treated with drugs that inhibit mTOR have experienced positive results. Researchers continue to evaluate whether this pathway can be targeted to treat chordoma, and this page contains a summary of their published research.

Molecular Evidence

Copy Number Variation

Chromosomal Aberrations:

  • Loss of chromosomal regions harboring genes involved in the pathway have been reported in chordomas. Among the losses are PI3KCA at 3q26, mTOR at 1p36.2, and negative regulator PTEN at 10q23.3 4 5 6 7 8

Gene Copy Number Variation:

  • Gene copy number variations in mTOR, RPS6, and S6 have been reported.6 9 10
  • Copy number variation in TSC1 and TSC2 has not been observed, indicating that something else is responsible for inactivation of the mTOR-inhibiting TSC1/TSC2 complex.9

Germline Genetic Variant

  • In 11 cases of chordoma occurring in individuals with tuberous sclerosis (TSC), one individual was found to have a germline TSC2 mutation and another a germline TSC1 mutation. Biallelic inactivation was confirmed in both and served to inactivate the TSC1/TSC2 complex, abolishing its inhibitory effect on mTOR.11 Clinical features in TSC-associated pediatric chordomas differ from the general pediatric chordoma population.12
  • In a chordoma patient with family history of TSC, a nonsense germline mutation was detected in TSC1 and was shown to cause translation termination.13

Somatic Mutation

  • Somatic mutations are largely absent in mutation hotspots in genes Rheb, PI3KCA, or PTEN, indicating that mutation is not responsible for the abnormal signaling of these components of the pathway.6 9
  • In a 2014 study Choy et al. detected a mutation in PI3KCA in only 1 of 45 chordoma samples tested.14 Similarly, Tauziède-Espariat et al. found 2 mutations (p.H1047L and p.E542K) in the PIK3CA gene in 5% of cases analyzed (3 tumors from 2 patients).15
  • Analysis of 104 chordoma cases found mutations in PIK3CA in 9/104 samples, in PIK3R1 and MTOR in 1/104 samples and in PTEN in 6/104 samples along with a disruptive rearrangement in PTEN in an additional sample.16

Protein Expression and Activation


  • Total Akt and p-Akt are detected in the majority chordomas analyzed. Lack of mutations in the TSC1 and TSC2 genes, coupled with the presence of the TSC1 and TSC2 proteins and the phosphorylation of TSC2 in nearly all samples, indicate that phosphorylation of TSC2 by Akt, rather than genetic mutation, serves to inactivate the TSC1/TSC2 complex.1 6 9 10 17 18 19 
  • Strong staining for Akt has been observed in a significantly higher fraction of chordoma tissues than fetal nucleus pulposus tissues.20 
  • Kinase analysis of 10 chordoma samples revealed frequent and strong up regulation of the AKT signaling pathway.7 p-AKT expression varies among chordoma cell lines.21 22 
  • Poor survival rate has been directly associated with pAKT positivity.


  • PI3K is detected in nearly all chordomas.6


  • Total mTOR is present in nearly all chordomas evaluated, and p-mTOR has been detected in more than half.1 6 9 10 18 19 23 24


  • Chordomas show variable activation of p70S6K or RPS6, though most show expression of the unactivated proteins. Their activation is important for protein synthesis. Hypermethylation of the RPS6 promoter has not been observed, so the absence of activated p70S6K (p-p70S6K) and RPS6 (p-RPS6K) cannot be attributed to lack of RPS6 gene expression.1 6 9 10 15 18 25


  • Key downstream protein PDK-1 is present and phosphorylated in all samples analyzed, providing further evidence that the PI3K pathway is activated.


  • Though present, PTEN chromosomal alterations were not found to alter gene expression.6
  • Chordomas have shown variable immunoreactivity for the PTEN protein and absence of PTEN may lead to constitutive Akt phosphorylation and activation of the pathway.9 10 23 24 25 26 27
  • PTEN expression has been associated with degree of bone invasion.23
  • Loss of PTEN was observed in 17/23 pediatric tumors by immunohistochemistry.28


  • 4E-BP1 has an inhibitory effect on eIF-4E, but the inhibition is released when 4E-BP1 is phosphorylated so that mRNA translation continues. Total 4E-BP1 and p-4E-BP1 are detected in nearly all chordomas tested.6 9 10 15 18 25
  • Total eIF-4E is also detected, indicating that the protein is free in most chordomas to promote cellular proliferation and protein synthesis.

Pathway Activation

  • Serum-starved U-CH1 cells espress high levels of p-PRAS40, a negative regulator of MTORC1, p-TSC2, and p-AKT, indicating that Akt signaling is constitutively active independent of growth factors.25
  • p-mTOR has been detected in more than half of chordomas analyzed. p-p70S6K is thought to be a better biomarker for activation of the PI3K/Akt/mTOR pathway than p-mTOR, and it has also been detected in more than half of chordomas analyzed.9 10 18 Taking both these phosphorylated proteins into account, Presneau et. al. estimated that the pathway is activated in ~65% of chordomas.9 More recently, Tauziède-Espariat et al. found activation in 46% of of chordoma, based on expression of p4EBP1 and dually phosphorylated pS6 (ser 240/244 and ser 235/236).15
  • Gene loci that were found to be hypermethylated in cancer samples versus controls fell into a number of cancer-related pathways, including the PI3k/Akt/mTOR pathway.29
  • Transcriptome microarray analysis comparing 4 tumors with high brachyury expression versus 4 tumors with low brachyury expression found that the PI3K/AKT signaling pathway is significantly upregulated when brachyury is overexpressed.30
  • Microarray gene expression profiling found that the PI3K/ACT signaling pathway was significantly differentially expressed between clival and sacral chordoma.31
  • YBX1 is more highly expressed in chordoma tumors than normal control tissues and increased expression of YBX1 activated the EGFR/AKT pathway in chordoma cells.32

Preclinical Evidence

In-vitro Efficacy

  • Rapamycin: Treatment of chordoma cell lines with the mTOR inhibitor has been shown to have a variety of tumor suppressive effects, including inhibition of cell growth and proliferation, decreased cell number, and reduced phosphorylation of Akt and P70S6K.25 33
  • Wortmannin: Treatment of U-CH1 with the PI3K inhibitor significantly decreased phosphorylation of pathway activator Akt.25 Treatment of U-CH2 with wortmannin suppressed brachyury mRNA expression.30
  • PI-103 (+/-doxorubicin or cisplatin): Treatment of U-CH1 with the dual PI3K/mTOR inhibitor reduced cellular proliferation and increased apoptosis, and the addition of a chemotherapy drug increases the tumor-suppressive effects.1
  • Ly294002: Treatment of U-CH2 with BEZ235 suppressed brachyury mRNA expression. 30
  • BEZ235: Treatment of U-CH2 with BEZ235 suppressed cell growth and decreased brachyury mRNA and protein expression. 30
  • PDGFR Inhibition: PTEN-deficient primary culture cells showed greater resistance to a PDGFR inhibitor than those that were not PTEN-deficient. PTEN restoration in two cell lines decreased proliferation and established sensitivity to the PDGFR inhibitor in both cases.34
  • β-β-dimethylacrylshikonin: Treatment of 4 chordoma cell lines with β-β-dimethylacrylshikonin (DMAS) resulted in decreased Akt, p-Akt, and p-Erk protein expression in the majority of cell lines tested.35
  • Antagomirs inhibiting miR-140-3p and miR-155-5p directly increased PTEN protein expression in patient derived chordoma cells. Inhibition of miR-140-3p and miR-155-5p significantly reduced PI3K-Akt-mTOR signaling and sensitized cells to doxorubicin, paclitaxel and cisplatin treatment.36

In-vivo Efficacy

  • Rapamycin: Rapamycin treatment reduced tumor volume in mice grafted with tumors derived from chordoma cell line U-CH1.33
  • MLN0128: Treatment of chordoma mouse xenografts with this ATP-competitive dual MTORC1/2 inhibitor decreased activity of the PI3k/Akt/mTOR signaling pathway (p = 0.019).37
  • Rapamycin: Rapamycin delayed the onset of tumor formation in a newly developed zebrafish model of chordoma and improved the survival of tumor-bearing fish.38
  • LY294022: PI3K inhibitor LY294022 had no inhibitory/activating effects on tumor growth in a zebrafish model of chordoma, suggesting that mTOR can be activated by the Ras oncogene rather than by PI3K.38

Clinical Evidence

Case Reports

There have been a number of case reports publishing the results of treating chordoma patients with a therapeutic agent that targets the PI3k/Akt/mTOR signaling pathway.

  • Rapamycin: One patient experienced disease progression after two months treatment (after switching from PDGFR inhibitor Imatinib).39 Another patient with a recurrence experienced 6.1 times slower tumor growth during 10 months of treatment after surgery.33
  • Imatinib + Sirolimus: In a case series of 10 patients with progressive, advanced chordoma, 9 were evaluable for response. By RECIST criteria, 1/9 showed partial response, 7/9 reached stable disease, 1/9  remained progressive after 3 months. Clinical benefit rate was determined to by 89%, with 7 of 9 patients reporting improvement in the symptoms they had prior to treatment.40
  • Everolimus: A patient with sacral chordoma was treated with the PDGFR inhibitor imatinib for 4 months until slight progression occurred and then the mTOR inhibitor everolimus was added and stable disease was achieved for 16 months. After disease progression, the patient was treated with the EGFR inhibitor erlotinib which led to a brief clinical benefit.41
  • Temsirolimus: One patient treated with the mTOR-inhibitor temsirolimus was included in a retrospective review of 80 patients.42

Clinical Trials

  • Imatinib + Everolimus: A phase 2 clinical study (EUDRACT number: 2010-021755-34) of 43 adults with progressive advanced chordoma treated with imatinib plus everolimus found limited antitumor activity. However, a subgroup of patients with highly phosphorylated mTOR effectors were responsive, suggesting that activation of the mTOR pathway could be predictive of response to imatinib plus everolimus.43

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