Pathway dissection and model development
Although Akt activity is elevated in the majority of GBMs, mutations in the gene for Akt have not been found. Rather, deletion of the pten tumor suppressor is frequently found and correlates with elevated Akt activity in these tumors. However, our original modeling system uses a mutant, activated form of Akt (1, 2, 3, 4, 5). Therefore, we determined if loss of PTEN and activation of Akt were the same in the context of glioma formation. Towards this aim, we have collaborated with Pier Paolo Pandolfi and used his floxed pten genetic background. Deletion of pten with an RCAS vector that expresses Cre cooperates with Ras to form tumors very similar to those generated by Ras+Akt (6). We used Ras+Akt-induced glioma-bearing mice in preclinical trials to determine the requirement for mTOR signaling in tumor maintenance. We demonstrated that as little as 48 hours of inhibition of mTOR in vivo results in the onset of apoptotic cell death within the Ras+Akt-induced GBMs, and 7 days of treatment results in substantial killing of the tumor (6, 7). We have also recently shown that the oncogenic action of Ras in this modeling system is through Raf (39).
Alterations in translational efficiencies of specific mRNAs by combined Ras and Akt signaling
We are testing blockade of the PI3K/Akt/mTOR pathway that modulates translational efficiencies of mRNAs. We investigated the effect of small molecule signal transduction blockade on the conversion of the transcriptome to the proteome in glial cells. The Ras and Akt pathways intersect at the interaction between 4EBP and eIF4E to regulate translation. Oncogenic signal transduction pathways, such as those involving Ras and Akt, have been shown to affect initiation of mRNA translation by this mechanism. We used our GBM model system because of its dependence on combined Ras and Akt signaling. We employed microarray gene expression profiling of total cellular mRNA and found that the immediate effect of Ras and Akt signaling blockade on transcription was relatively modest. By contrast, array analysis of mRNA associated with polysomes indicated that these pathways substantially altered the composition of the polysome-associated mRNA. These observations indicate that Ras and Akt signaling regulate the recruitment of specific mRNAs to ribosomes to a far greater extent than they regulate the production of mRNAs by transcriptional effects. The mRNAs most affected by polysome recruitment are those encoding growth-regulating and oncogenic proteins as well as proteins with a wide range of functions including transcription regulation as well as cell to cell interactions and morphology (8, 9, 10).
Using the RCAS/tv-a system, we investigated the functions of PDGF autocrine signaling in gliomagenesis by transferring the over-expression of PDGF-B into either nestin-expressing neural progenitors or GFAP-expressing astrocytes (11). There is dose-dependent effects of PDGF-B on the histological grade of gliomas induced in our system. Using vectors that produce elevated amounts of PDGF, we generated tumors with shortened latency, increased cellularity, regions of necrosis, and general high-grade character. In addition, elevated PDGF-B in these tumors also mediates vascular smooth muscle cell recruitment that supports tumor angiogenesis. PDGFR signaling appears to be required for the maintenance of these high-grade characteristics since treatment of high-grade PDGF-B induced tumors with a small molecule inhibitor of PDGFR (PTK787) results in reversion to a lower grade tumor histology. This data indicates that PDGFR signaling quantitatively regulates glioma grade and is required to sustain these high-grade oligodendrogliomas in vivo (12, 28, 32).
We measured the activity of Akt and the Ras pathway (by Erk phosphorylation) in these tumors using immunohistochemical staining and found that although the phosphorylated forms of these proteins are easily detectable in Ras+Akt driven GBMs, they are undetectable in PDGF induced oligodendrogliomas. We looked in culture for the effects of PDGF signaling and found that although both Ras and Akt activities are activated transiently, this effect is rapidly lost and replaced with elevation of p21 and PCNA (34). Furthermore, cultured astrocytes infected with RCAS-PDGF and chronically stimulated with PDGF have similar signaling characteristics (low Ras and Akt signaling), and are reversible by blockade of the PDGF receptor. We do not fully understand the signaling downstream of PDGF that leads to glioma formation but it appears not to be combined Ras and Akt (13).
Modeling of Medulloblastomas
Medulloblastomas are primary tumors arising in the cerebellum and the most common solid tumor in children. In these tumors Myc amplification is a poor prognostic factor. One of the critical signaling abnormalities is the activation of Akt via the IGF receptor and activation of the sonic hedgehog pathway by loss of the PTC gene. In mice, PTC heterozygous knockouts develop medulloblastomas (Matt Scott lab) at low frequency. The incidence of these tumors is dramatically enhanced by loss of p53 (Tom Curran lab). Human medulloblastomas do not show p53 loss making interpretation of the result relative to human tumors difficult. We showed that transfer of c-Myc to nestin expressing cells in the cerebellum generates clusters of CNS progenitor cells but no tumors (14, 27). In collaboration with Dan Fults now back at the University of Utah we showed that SHH gene transfer to the same cells generates medulloblastomas in 15 percent of mice by 12 weeks, the addition of Myc to SHH elevates the incidence to 25 percent (14, 15) the addition of IGF2 elevates the incidence to 40 percent while the addition of activated Akt elevates the incidence to 50 percent. Just recently we have shown that these medulloblastomas models can be used in preclinical trials to determine the biology of therapeutic response in stem cells of these tumors (29, 35). We have also generated a BLI reporter mouse that images the activity of SHH signaling in vivo in medulloblastomas and gliomas (37). In addition, we have created a model of PNETs that include a subpopulation of medulloblastomas (38).
Imaging strategies and preclinical trials
We have utilized MRI and PET strategies as a method for screening and detecting brain tumors in mice based on their pattern of enhancement. The imaging characteristics of these tumors show many similarities to those seen in humans with comparable tumor types (16). We are using this technology in a medium throughput fashion to identify mice with large gliomas as inclusion criteria in preclinical trials (17). We have used MRI in two preclinical including PDGF-induced gliomas treated with the PDGF receptor inhibitor PTK787 (12, 30, 33) and Ras+Akt-induced GBMs treated with the mTOR inhibitor CCI779 (6). In both cases, tumors were identified by MRI scanning and contrast enhancement was found to be decreased post treatment correlating with histological changes in the tumors on analysis. Diffusion imaging by MRI was a good predictor of therapeutic response in PDGF-driven gliomas and that in order to obtain reproducible data the mice needed to be stratified by tumor grade using MRI characteristics (18). In addition, FLT PET scanning can be used as a readout of therapeutic response in our glioma models (36).
Bioluminescence imaging in mice
The Efluc mouse has been designed to allow the use of bioluminescence as a readout for the proliferation in tumors noninvasively in vivo. The Efluc transgene utilizes the E2F1 promoter that is activated when cells leave G1 and enter S phase to drive the expression of luciferase. Because of the high proliferative rate of tumors due to deregulation of the Rb pathway in these cells, tumor cells arising in the Efuc transgenic mouse are detectable by this technique. We crossed the Efluc mouse with our Ntv-a transgenic mice that allow transfer to glial progenitors using the RCAS vectors. As proof of principal for the use of this technology in preclinical trials, we have demonstrated that treatment with the PDGF receptor inhibitor PTK787 described above will substantially reduce light emission from these tumors in an Efluc background. We then tested the effect of mTOR blockade on these tumors using CCI779. We found that blockade of mTOR gave a similar result as PDGF receptor blockade indicating that proliferation but not survival of these tumors was dependent on mTOR activity (19). As such imaging techniques allow each mouse to be its own control, this technique provides statistically significant results with far fewer mice than standard comparisons between populations of mice (20, 21, 22, 23). We have also generated another BLI mouse reporter line that reports on Gli singling downstream of SHH signaling. This mouse demonstrates SHH signaling not only in SHH-driven medulloblastomas but in PDGF driven gliomas as well (37).
We have proceeded to test other small molecules targeting members of the Akt signaling pathway in this tumor system. We have obtained an Akt inhibitor perifosine from Kyrex Pharmaceuticals and are working out the signaling effects of these drugs in cultured glia and in our mouse models of gliomas (24). In addition to understanding the effect of single agents in detail, we are now combining these drugs with each other and with the standard cytotoxic drugs and radiation used to treat gliomas in humans (25, 26, 31). Our work has resulted in an ongoing clinical trial of perifosine in glioma patients at Memorial Sloan-Kettering Cancer Center.
The glioma cell of origin and stem like cells in brain tumors
The cell of origin for gliomas has been debated extensively. The glial characteristics of these tumors could imply that they arise from the differentiated glia they resemble or their precursors. Mutations found in gliomas destabilize the differentiation status of these cells, rendering it difficult to determine which cell type gives rise to a given tumor histology. Our results from the RCAS/tv-a system, (essentially lineage tracing studies) correlate specific cell characteristics with the histology of gliomas that arise from these cells. From these experiments it appears that undifferentiated, nestin-expressing cells are more sensitive to the oncogenic effects of certain signaling abnormalities than are differentiated astrocytes. However, specific genetic alterations such as loss of ink4a-arf (1), inappropriate myc expression (3), or chronically elevated PDGF signaling (12) can allow differentiated astrocytes to act as the cell-of-origin for gliomas. These issues have been discussed at length in several opinion papers (11, 21, 28). Our original paper on PDGF-induced gliomas discussed the role of differentiation blockade in the formation of gliomas (40).
Uhrbom L. DC, Celestino J.C., Rosenblum M.K., Fuller G.N. and Holland E.C. Ink4a-Arf loss cooperates with KRas activation in astrocytes and neural progenitors to generate glioblastomas of various morphologies adepending on amount of Akt activity. Cancer Res 2002;1;62(19):5551-5558. [PubMed Abstract]
Weiss WA, Israel M, Cobbs C, Holland E.C, James CD, Louis DN, Marks C, McClatchey AI, Roberts T, Van Dyke T and others. Neuropathology of genetically engineered mice: consensus report and recommendations from an international forum. Oncogene 2002;21(49):7453-63. [PubMed Abstract]
Lassman A. DC, Fuller G.N. and Holland E.C. Overexpression of c-Myc promotes an undifferentiated phenotype in cultured astrocytes and allows elevated Ras and Akt signaling to induce gliomas from GFAP-expressing cells in mice. Neuron Glia Biology 2004;1:157-163. [PubMed Abstract]
Uhrbom L, Kastemar M, Johansson FK, Westermark B, Holland EC. Cell type-specific tumor suppression by Ink4a and Arf in Kras-induced mouse gliomagenesis. Cancer Res 2005;65(6):2065-9. [PubMed Abstract]
Dunlap SM, Celestino J, Wang H, Jiang R, Holland EC, Fuller GN, Zhang W. Insulin-like growth factor binding protein 2 promotes glioma development and progression. Proc Natl Acad Sci U S A 2007;104(28):11736-41. [PubMed Abstract]
Hu X, Pandolfi PP, Li Y, Koutcher JA, Rosenblum M, Holland EC. mTOR promotes survival and astrocytic characteristics induced by Pten/AKT signaling in glioblastoma. Neoplasia 2005;7(4):356-68. [PubMed Abstract]
Hu X, Holland EC. Applications of mouse glioma models in preclinical trials. Mutat Res 2005;576(1-2):54-65. [PubMed Abstract]
Rajasekhar VK, Viale A, Socci ND, Wiedmann M, Hu X, Holland EC. Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol Cell 2003;12(4):889-901. [PubMed Abstract]
Rajasekhar VK, Holland EC. Postgenomic global analysis of translational control induced by oncogenic signaling. Oncogene 2004;23(18):3248-64. [PubMed Abstract]
Holland EC, Sonenberg N, Pandolfi PP, Thomas G. Signaling control of mRNA translation in cancer pathogenesis. Oncogene 2004;23(18):3138-44. [PubMed Abstract]
Dai C, Holland EC. Astrocyte differentiation states and glioma formation. Cancer J 2003;9(2):72-81. [PubMed Abstract]
Shih AH, Dai C, Hu X, Rosenblum MK, Koutcher JA, Holland EC. Dose-dependent effects of platelet-derived growth factor-B on glial tumorigenesis. Cancer Res 2004;64(14):4783-9. [PubMed Abstract]
Dai C, Lyustikman Y, Shih A, Hu X, Fuller GN, Rosenblum M, Holland EC. The characteristics of astrocytomas and oligodendrogliomas are caused by two distinct and interchangeable signaling formats. Neoplasia 2005;7(4):397-406. [PubMed Abstract]
Rao G, Pedone CA, Valle LD, Reiss K, Holland EC, Fults DW. Sonic hedgehog and insulin-like growth factor signaling synergize to induce medulloblastoma formation from nestin-expressing neural progenitors in mice. Oncogene 2004;23(36):6156-62. [PubMed Abstract]
Rao G, Pedone CA, Coffin CM, Holland EC, Fults DW. c-Myc enhances sonic hedgehog-induced medulloblastoma formation from nestin-expressing neural progenitors in mice. Neoplasia 2003;5(3):198-204. [PubMed Abstract]
Xu S, Gade TP, Matei C, Zakian K, Alfieri AA, Hu X, Holland EC, Soghomonian S, Tjuvajev J, Ballon D and others. In vivo multiple-mouse imaging at 1.5 T. Magn Reson Med 2003;49(3):551-7. [PubMed Abstract]
Koutcher JA, Hu X, Xu S, Gade TP, Leeds N, Zhou XJ, Zagzag D, Holland EC. MRI of mouse models for gliomas shows similarities to humans and can be used to identify mice for preclinical trials. Neoplasia 2002;4(6):480-5. [PubMed Abstract]
McConville P HD, Moody JB, Leopold WR, Kreger AR, Woolliscroft MJ, Rehemtulla A, Ross BD and Holland EC. Magnetic resonance imaging determination of tumor grade and early response to temozolomide in a genetically engineered mouse model of glioma. Clin Cancer Res. 2007;15;13(10):2897-904. [PubMed Abstract]
Uhrbom L, Nerio E, Holland EC. Dissecting tumor maintenance requirements using bioluminescence imaging of cell proliferation in a mouse glioma model. Nat Med 2004;10(11):1257-60. [PubMed Abstract]
Zhang L, Lee KC, Bhojani MS, Khan AP, Shilman A, Holland EC, Ross BD, Rehemtulla A. Molecular imaging of Akt kinase activity. Nat Med 2007;13(9):1114-9. [PubMed Abstract]
Fomchenko EI, Holland EC. Mouse models of brain tumors and their applications in preclinical trials. Clin Cancer Res 2006;12(18):5288-97. [PubMed Abstract]
Becher OJ, Holland EC. Genetically engineered models have advantages over xenografts for preclinical studies. Cancer Res 2006;66(7):3355-8, discussion 3358-9. [PubMed Abstract]
Momota H, Holland EC. Bioluminescence technology for imaging cell proliferation. Curr Opin Biotechnol 2005;16(6):681-6. [PubMed Abstract]
Momota H, Nerio E, Holland EC. Perifosine inhibits multiple signaling pathways in glial progenitors and cooperates with temozolomide to arrest cell proliferation in gliomas in vivo. Cancer Res 2005;65(16):7429-35. [PubMed Abstract]
Holland EC. Mouse models of human cancer as tools in drug development. Cancer Cell 2004;6(3):197-8. [PubMed Abstract]
Hambardzumyan D, Squatrito M, Holland EC. Radiation resistance and stem-like cells in brain tumors. Cancer Cell 2006;10(6):454-6. [PubMed Abstract]
Fults D, Pedone C, Dai C, Holland EC. MYC expression promotes the proliferation of Neural progenitors cells in culture and in vivo. Neoplasia. 2002;4:32-39. [PubMed Abstract]
Shih A, Holland EC. Developmental neurobiology and the origin of brain tumors. J. Neuroonc. 2004 70:125-136. [PubMed Abstract]
Fomchenko El, Holland EC. Stem cells and brain cancer. Exp Cell Res 2005;306(2):323-9. [PubMed Abstract]
Lassman AB, Rossi MR, Raizier JJ, Abrey LE, Lieberman FS, Grefe CN, Lamborn K, Pao W, Shih AH, Kuhn JG, Wilson R, Novak NJ, Cowell JK, Deangelis LM, Wen P, Gilbert MR, Chang S, Yung WA, Prados M, Holland EC. Molecular study of malignant gliomas treated with epidermal growth factor receptor inhibitors: tissue analysis from north american brain tumor consortium trials 01-03 and 00-01. Clin Cancer Res 2005; 11 (21):7841-50. [PubMed Abstract]
Shih AH, Holland EC. Notch signaling enhances nestin expression in gliomas. Neoplasia. 2006;8(12):1072-82. [PubMed Abstract]
Tchougounova E, Kastemar M, Brasater D, Holland EC, Westermark B, Uhrbom L. Loss of Arf causes tumor progression of PDGFB-induced oligodendroglioma. Oncogene. 2007 Sep 20;26(43):6289-96. [PubMed Abstract]
Bradley SV, Holland EC, Liu GY, Thomas D, Hyun TS, Ross TS. Huntingtin Interacting Protein 1 Is a Novel Brain Tumor Marker that Associates with Epidermal Growth Factor Receptor. Cancer Res. 2007 Apr 15;67(8):3609-15. [PubMed Abstract]
Liu Y, Yeh N, Zhu XH, Leversha M, Cordon-Cardo C, Ghossein R, Singh B, Holland E, Koff A.Somatic cell type specific gene transfer reveals a tumor-promoting function for p21(Waf1/Cip1). EMBO J. 2007 Nov 14;26(22):4683-93. [PubMed Abstract]
Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland, EC. PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo. Genes Dev. 2008 Feb 15;22(4):436-48. [PubMed Abstract]
Bradbury MS, Hambardzumyan D, Zanzonico PB, Schwartz J, Cai S, Burnazi EM, Longo V, Larson SM, Holland EC. Abstract Dynamic Small-Animal PET Imaging of Tumor Proliferation with 3'-Deoxy-3'-18F-Fluorothymidine in a Genetically Engineered Mouse Model of High-Grade Gliomas. J Nucl Med. 2008 Mar;49(3):422-429. [PubMed Abstract]
Becher OJ, Hambardzumyan D, Fomchenko EI, Momota H, Mainwaring L, Bleau AM, Katz AM, Edgar M, Kenney AM, Cordon-Cardo C, Blasberg RG, Holland EC. Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor-Induced Gliomas. Cancer Res. 2008 Apr 1;68(7):2241-9. [PubMed Abstract]
Momota H, Shih AH, Edgar MA, Holland EC. c-Myc and β-catenin Cooperate with Loss of p53 to Generate Multiple Members of the Primitive Neuroectodermal Tumor (PTEN) Family in Mice. Oncogene. 2008 Mar 31; [Epub ahead of print] [PubMed Abstract]
Lyustikman Y, Momota H, Pao W, Holland EC. Constitutive activation of Raf-1 induces glioma formation in mice. Neoplasia (In Press).
Dai C, Celestino JC, Okada Y, Louis DN, Fuller GN, Holland EC. PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev. 2001 Aug 1;15(15):1913-25. [PubMed Abstract]