Final Report Summary - NOTCH PATHWAY IN GSC (Role of Notch signaling pathway in Glioma Stem Cells)
Abstract
Recently, a subpopulation of tumor cells with stem-like properties has been identified in gliomas, as well as in many other tumors. Expression of high levels of CD44 and Id1 has been described in a subpopulation of glioma stem cells (GSC). Notch signaling pathway has been shown to play a crucial role in normal neural stem cells and is deregulated in brain tumors, especially gliomas. Interactions in both directions between the TGF-β and Notch pathways have been described in several cell types. The stimulation of gliomasphere cultures with TGF-β induced transactivation of the Notch pathway, as observed by cleavage (activation) of Notch1 by western blot and by increase of Notch target genes (Hes1, Hey1). The induction of Notch targets was completely blocked by pretreatment with the γ-secretase inhibitor. TGF-β treatment also induced increased expression of the Notch ligand Jagged1. By blocking Jagged1 expression by RNA interference we were able to prevent TGF-β induced activation of Notch1. The treatment of gliomasphere cultures with γ-secretase inhibitor caused a strong decrease in the percentage of cells with high CD44 expression. The combination of the two inhibitors produced a further decrease of CD44high cells suggesting that combination of both treatments might be more effective than therapies based on either drug alone.
Introduction
Notch signaling pathway has been shown to play a role in the normal development and neoplastic progression in the central nervous system, having important roles in proliferation, differentiation, apoptosis and stem cell regulation [1]. Notch signaling is required for preventing neuronal differentiation and promoting neural stem cell maintenance, so a role of Notch in glioma stem cells (GSC) maintenance has been suggested [2-3].
Glioblastoma multiforme (GBM) is the most common malignant intracranial tumor in adults with a mean survival of 14 months [4]. Even though the advances in neurosurgery, radio- and chemo-therapy little improvement in the survival has been achieved, being the glioma the third cause of cancer related death in median age males and the fourth in women between 15-34 years old [5]. Notch pathway that has been demonstrated to be important in normal neural stem cells [6] and that is deregulated in GBM [7-8]. Over-expression of Notch signaling components has been observed in GBM harboring EGFR amplification [9]. Due to the aggressive nature of GBM and its incidence it is necessary to develop new therapeutic strategies in order to improve the patient outcome. Targeting Notch pathway therefore holds a promise of being a particularly appropriate therapeutic target in GBM and the role of Notch inhibition in GSC will provide new insights in GSC biology.
Results and discussion
It has been identified a cell population enriched for GSCs that expresses high levels of CD44 and Id1 and tend to be located in a perivascular niche [10]. We obtained patient-derived glioma cell cultures enriched in GSCs from patients diagnosed with GBM and analyzed the expression of Notch signaling pathway components and Notch pathway activity. We demonstrated that Notch pathway is active in patient-derived GSCs by western blot analysis using an antibody specific for the cleaved (active) form of Notch1 (Fig. 1a), and by transfection with a Notch reporter plasmid. By real-time PCR we detected expression of Jagged 1 and 2, Dll1 and 3, as well as Notch1, 2 and 3 (Fig. 1b). No expression of Dll4 and Notch4 was detected in any of the GSCs analyzed.
The inhibition of the TGFβ pathway decreases the CD44high/Id1high GSC population through the repression of Id1 and Id3 levels, therefore inhibiting the capacity of cells to initiate tumors [10]. We decided to analyze if TGFβ was able to induce Notch pathway. Western blot analysis of cleaved Notch1 in patient-derived GSCs treated with TGFβ demonstrated that TGFβ was able to activate Notch pathway in 9/9 patient-derived tumor cultures without affecting Notch total levels (Fig.2). The activation of Notch pathway was accompanied by an induction in the expression of Notch ligand Jagged1, that was not prevented by treatment with the γ-secretase inhibitor, a well known Notch inhibitor.
To further analyze the activation of Notch pathway by TGFβ, we treated patient-derived cultures with TGFβ and analyzed the expression of Notch targets Hes1 and Hey1 by real-time PCR. TGFβ treatment was able to transactivate Notch pathway inducing the expression of both, Hes1 and Hey1. Treatment with γ-secretase inhibitor completely blocked Notch targets induction by TGFβ (Fig.3).
We then decided to analyze the role Notch activation in the maintenance of the GSCs in patient-derived cultures. Ectopical expression of the constitutively active Notch intracellular domain (Nicd) in gliomasphere cultures resulted in an increase in CD44 GSC marker expression, by flow cytometry analysis (Fig.4). Similarly, expression of Notch target Hes1 also resulted in the induction of CD44 expression (Fig.4). Furthermore, cells over-expressing Notch ligands Jagged1 or Dll1 presented activation of Notch pathway and increased expression of CD44. Blockade of Notch pathway through treatment with γ-secretase inhibitor reduced Notch targets Hes1 and Hey expression as well as GSC markers CD44 and Id1.
Our previous studies described that CD44 expression increased upon TGFβ treatment in GBM patient-derived cultures [10]. Similarly, CD44 expression detected by flow cytometry and real-time PCR was upregulated in all GBM patient-derived cell cultures. In 6 out of 9 patient-derived cell cultures we observed that the treatment with the γ-secretase inhibitor significantly reduced the percentage of CD44high population. Furthermore, combination of Notch and TGFβ receptor inhibitors presented and additive effect decreasing CD44high population (Fig.3). Knock-down of Jagged1 partially prevented TGFβ induction of Notch target expression and blocked Notch cleavage.
All together, we described a transactivation of Notch pathway by TGFβ. Notch activation induced CD44 stem cell marker expression in patient-derived glioma tumor cells. The treatment of gliomasphere cultures with γ-secretase inhibitor caused a strong decrease in the percentage of cells with high CD44 expression, which are enriched in GSCs, similarly to what observed with the TGF-β receptor inhibitor. The combination of both inhibitors produced a further decrease of CD44high cells. We propose that a combinatorial treatment to target both TGF-β and Notch might be more effective in cases of partial or no response to therapies based on either drug alone.
Bibliography
1. Lino, M.M.; Merlo, A.; Boulay, J.L. Notch signaling in glioblastoma: A developmental drug target? BMC Med 2010, 8, 72.
2. Hitoshi, S.; Alexson, T.; Tropepe, V.; Donoviel, D.; Elia, A.J.; Nye, J.S.; Conlon, R.A.; Mak, T.W.; Bernstein, A.; van der Kooy, D., Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 2002, 16, 846-858.
3. Morrison, S.J.; Perez, S.E.; Qiao, Z.; Verdi, J.M.; Hicks, C.; Weinmaster, G.; Anderson, D.J. Transient notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 2000, 101, 499-510.
4. Kleihues, P.; Cavenee, W.K.; International Agency for Research on Cancer., Pathology and genetics of tumours of the nervous system. IARC Press: Lyon, 2000; p 314 p.
5. Stiles, C.D.; Rowitch, D.H. Glioma stem cells: A midterm exam. Neuron 2008, 58, 832-846.
6. Beatus, P.; Lendahl, U., Notch and neurogenesis. J Neurosci Res 1998, 54, 125-136.
7. Schreck, K.C.; Taylor, P.; Marchionni, L.; Gopalakrishnan, V.; Bar, E.E.; Gaiano, N.; Eberhart, C.G. The notch target hes1 directly modulates gli1 expression and hedgehog signaling: A potential mechanism of therapeutic resistance. Clin Cancer Res 2010, 16, 6060-6070.
8. Somasundaram, K.; Reddy, S.P.; Vinnakota, K.; Britto, R.; Subbarayan, M.; Nambiar, S.; Hebbar, A.; Samuel, C.; Shetty, M.; Sreepathi, H.K. et al., Upregulation of ascl1 and inhibition of notch signaling pathway characterize progressive astrocytoma. Oncogene 2005, 24, 7073-7083.
9. Brennan, C.; Momota, H.; Hambardzumyan, D.; Ozawa, T.; Tandon, A.; Pedraza, A.; Holland, E., Glioblastoma subclasses can be defined by activity among signal transduction pathways and associated genomic alterations. PLoS One 2009, 4, e7752.
10. Anido, J.; Saez-Borderias, A.; Gonzalez-Junca, A.; Rodon, L.; Folch, G.; Carmona, M.A.; Prieto-Sanchez, R.M.; Barba, I.; Martinez-Saez, E.; Prudkin, L., et al., Tgf-beta receptor inhibitors target the cd44(high)/id1(high) glioma-initiating cell population in human glioblastoma. Cancer Cell 2010, 18, 655-668.
Recently, a subpopulation of tumor cells with stem-like properties has been identified in gliomas, as well as in many other tumors. Expression of high levels of CD44 and Id1 has been described in a subpopulation of glioma stem cells (GSC). Notch signaling pathway has been shown to play a crucial role in normal neural stem cells and is deregulated in brain tumors, especially gliomas. Interactions in both directions between the TGF-β and Notch pathways have been described in several cell types. The stimulation of gliomasphere cultures with TGF-β induced transactivation of the Notch pathway, as observed by cleavage (activation) of Notch1 by western blot and by increase of Notch target genes (Hes1, Hey1). The induction of Notch targets was completely blocked by pretreatment with the γ-secretase inhibitor. TGF-β treatment also induced increased expression of the Notch ligand Jagged1. By blocking Jagged1 expression by RNA interference we were able to prevent TGF-β induced activation of Notch1. The treatment of gliomasphere cultures with γ-secretase inhibitor caused a strong decrease in the percentage of cells with high CD44 expression. The combination of the two inhibitors produced a further decrease of CD44high cells suggesting that combination of both treatments might be more effective than therapies based on either drug alone.
Introduction
Notch signaling pathway has been shown to play a role in the normal development and neoplastic progression in the central nervous system, having important roles in proliferation, differentiation, apoptosis and stem cell regulation [1]. Notch signaling is required for preventing neuronal differentiation and promoting neural stem cell maintenance, so a role of Notch in glioma stem cells (GSC) maintenance has been suggested [2-3].
Glioblastoma multiforme (GBM) is the most common malignant intracranial tumor in adults with a mean survival of 14 months [4]. Even though the advances in neurosurgery, radio- and chemo-therapy little improvement in the survival has been achieved, being the glioma the third cause of cancer related death in median age males and the fourth in women between 15-34 years old [5]. Notch pathway that has been demonstrated to be important in normal neural stem cells [6] and that is deregulated in GBM [7-8]. Over-expression of Notch signaling components has been observed in GBM harboring EGFR amplification [9]. Due to the aggressive nature of GBM and its incidence it is necessary to develop new therapeutic strategies in order to improve the patient outcome. Targeting Notch pathway therefore holds a promise of being a particularly appropriate therapeutic target in GBM and the role of Notch inhibition in GSC will provide new insights in GSC biology.
Results and discussion
It has been identified a cell population enriched for GSCs that expresses high levels of CD44 and Id1 and tend to be located in a perivascular niche [10]. We obtained patient-derived glioma cell cultures enriched in GSCs from patients diagnosed with GBM and analyzed the expression of Notch signaling pathway components and Notch pathway activity. We demonstrated that Notch pathway is active in patient-derived GSCs by western blot analysis using an antibody specific for the cleaved (active) form of Notch1 (Fig. 1a), and by transfection with a Notch reporter plasmid. By real-time PCR we detected expression of Jagged 1 and 2, Dll1 and 3, as well as Notch1, 2 and 3 (Fig. 1b). No expression of Dll4 and Notch4 was detected in any of the GSCs analyzed.
The inhibition of the TGFβ pathway decreases the CD44high/Id1high GSC population through the repression of Id1 and Id3 levels, therefore inhibiting the capacity of cells to initiate tumors [10]. We decided to analyze if TGFβ was able to induce Notch pathway. Western blot analysis of cleaved Notch1 in patient-derived GSCs treated with TGFβ demonstrated that TGFβ was able to activate Notch pathway in 9/9 patient-derived tumor cultures without affecting Notch total levels (Fig.2). The activation of Notch pathway was accompanied by an induction in the expression of Notch ligand Jagged1, that was not prevented by treatment with the γ-secretase inhibitor, a well known Notch inhibitor.
To further analyze the activation of Notch pathway by TGFβ, we treated patient-derived cultures with TGFβ and analyzed the expression of Notch targets Hes1 and Hey1 by real-time PCR. TGFβ treatment was able to transactivate Notch pathway inducing the expression of both, Hes1 and Hey1. Treatment with γ-secretase inhibitor completely blocked Notch targets induction by TGFβ (Fig.3).
We then decided to analyze the role Notch activation in the maintenance of the GSCs in patient-derived cultures. Ectopical expression of the constitutively active Notch intracellular domain (Nicd) in gliomasphere cultures resulted in an increase in CD44 GSC marker expression, by flow cytometry analysis (Fig.4). Similarly, expression of Notch target Hes1 also resulted in the induction of CD44 expression (Fig.4). Furthermore, cells over-expressing Notch ligands Jagged1 or Dll1 presented activation of Notch pathway and increased expression of CD44. Blockade of Notch pathway through treatment with γ-secretase inhibitor reduced Notch targets Hes1 and Hey expression as well as GSC markers CD44 and Id1.
Our previous studies described that CD44 expression increased upon TGFβ treatment in GBM patient-derived cultures [10]. Similarly, CD44 expression detected by flow cytometry and real-time PCR was upregulated in all GBM patient-derived cell cultures. In 6 out of 9 patient-derived cell cultures we observed that the treatment with the γ-secretase inhibitor significantly reduced the percentage of CD44high population. Furthermore, combination of Notch and TGFβ receptor inhibitors presented and additive effect decreasing CD44high population (Fig.3). Knock-down of Jagged1 partially prevented TGFβ induction of Notch target expression and blocked Notch cleavage.
All together, we described a transactivation of Notch pathway by TGFβ. Notch activation induced CD44 stem cell marker expression in patient-derived glioma tumor cells. The treatment of gliomasphere cultures with γ-secretase inhibitor caused a strong decrease in the percentage of cells with high CD44 expression, which are enriched in GSCs, similarly to what observed with the TGF-β receptor inhibitor. The combination of both inhibitors produced a further decrease of CD44high cells. We propose that a combinatorial treatment to target both TGF-β and Notch might be more effective in cases of partial or no response to therapies based on either drug alone.
Bibliography
1. Lino, M.M.; Merlo, A.; Boulay, J.L. Notch signaling in glioblastoma: A developmental drug target? BMC Med 2010, 8, 72.
2. Hitoshi, S.; Alexson, T.; Tropepe, V.; Donoviel, D.; Elia, A.J.; Nye, J.S.; Conlon, R.A.; Mak, T.W.; Bernstein, A.; van der Kooy, D., Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 2002, 16, 846-858.
3. Morrison, S.J.; Perez, S.E.; Qiao, Z.; Verdi, J.M.; Hicks, C.; Weinmaster, G.; Anderson, D.J. Transient notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 2000, 101, 499-510.
4. Kleihues, P.; Cavenee, W.K.; International Agency for Research on Cancer., Pathology and genetics of tumours of the nervous system. IARC Press: Lyon, 2000; p 314 p.
5. Stiles, C.D.; Rowitch, D.H. Glioma stem cells: A midterm exam. Neuron 2008, 58, 832-846.
6. Beatus, P.; Lendahl, U., Notch and neurogenesis. J Neurosci Res 1998, 54, 125-136.
7. Schreck, K.C.; Taylor, P.; Marchionni, L.; Gopalakrishnan, V.; Bar, E.E.; Gaiano, N.; Eberhart, C.G. The notch target hes1 directly modulates gli1 expression and hedgehog signaling: A potential mechanism of therapeutic resistance. Clin Cancer Res 2010, 16, 6060-6070.
8. Somasundaram, K.; Reddy, S.P.; Vinnakota, K.; Britto, R.; Subbarayan, M.; Nambiar, S.; Hebbar, A.; Samuel, C.; Shetty, M.; Sreepathi, H.K. et al., Upregulation of ascl1 and inhibition of notch signaling pathway characterize progressive astrocytoma. Oncogene 2005, 24, 7073-7083.
9. Brennan, C.; Momota, H.; Hambardzumyan, D.; Ozawa, T.; Tandon, A.; Pedraza, A.; Holland, E., Glioblastoma subclasses can be defined by activity among signal transduction pathways and associated genomic alterations. PLoS One 2009, 4, e7752.
10. Anido, J.; Saez-Borderias, A.; Gonzalez-Junca, A.; Rodon, L.; Folch, G.; Carmona, M.A.; Prieto-Sanchez, R.M.; Barba, I.; Martinez-Saez, E.; Prudkin, L., et al., Tgf-beta receptor inhibitors target the cd44(high)/id1(high) glioma-initiating cell population in human glioblastoma. Cancer Cell 2010, 18, 655-668.
