21st Century Science Initiative Grant: Researching Brain Cancer
Glioblastoma multiforme (GBM or astrocytoma WHO grade IV) is a devastating and highly invasive brain tumor. GBMs have an incidence of ~3/100,000 per year and often occur in patients of >50 years of age. However, they are an important cause of cancer-related deaths in younger adults.
Cure rates for GBMs are below 5%. These tumors cannot be routinely removed entirely by surgery as their invasive nature precludes the extirpation of all cells without massive brain damage. Since GBMs harbor cancer stem cells with enhanced DNA repair programs, that render them more resistant to radiation and conventional chemotherapy, leaving behind even a few of them after surgery poses the problem of recurrence and eventual patient death (Singh et al., 2003; Bao et al., 2006; Furnari et al., 2007). Developing an effective GBM therapy represents a great challenge. For instance, targeting mutant deltaEGFR, one of the major GBM oncogenes, leads to tumor escape through gain of an alternative mechanism (e.g. Mukasa et al., 2010).
New possibilities for GBM therapies derive from the study of stem cells. Cancer stem cells are defined as cells that can self-renew and give rise to more differentiated progeny, much like normal stem cells, but unlike these, cancer stem cells can induce tumor growth as assayed in immunodeficient mice (reviewed Reya et al., 2001; Ruiz i Altaba and Brand, 2009). Understanding the factors that regulate cancer stem cell self-renewal will likely shed light on novel ways to attack and kill these cells that maintain the growing GBMs and may induce recurrence. Our goal is to induce GBM stem cell death or differentiation and thus tumor regression and disappearance.
The state of a cell is plastic. Many types of normal differentiated cells have been shown to be directly reprogrammable to embryonic stem cell (ESC)-like (iPS) cells through the transient expression of a limited cohort of genes (Takahashi and Yamanaka, 2006; Yu et al., 2007; Wernig et al., 2007). The original reprogramming gene cohort comprises Oct4, Sox2, cMyc, Klf4 (Takahashi and Yamanaka, 2006) but variants include, directly (Yu et al., 2007) or indirectly, the action of the ESC transcription factor Nanog (Chambers et al., 2003; Mitsui et al. 2003). Reprogramming functions act in a network so that activation of a few nodes (e.g. Oct4, Nanog) leads to the activation of the network that drives a drastic change in fate. Depending on the functions already present in the target cells, reprogramming may occur with very few alterations. For example, neural stem cells have been reported to be reprogrammable to iPS cells with only 1 factor (Oct4; Kim et al., 2009).
Cancer stem cells appear not to be an exception; for example, malignant human melanoma cells can be induced to become normal neural crest cells when introduced into the dorsal mid/hindbrain of chick embryos, a place where neural crest cells normally develop (reviewed in Hendrix et al., 2007). These findings suggest that the state of cancer cells is not fixed and that once we identify and manipulate the right regulators we will be able to induce their death, permanent arrest, or loss of malignancy through differentiation.
One key question is thus the nature and function of such genes that characterize and maintain GBM stem cells. One possibility is that reprogramming genes are involved in cancer stemness. This is an exciting idea and different recent lines of evidence from our and other laboratories support this possibility in GBMs.
Our interest in GBMs allowed us to show for the first time that GBM cancer stem cells require SONIC HEDGEHOG (SHH)-GLI signaling for growth, proliferation and survival (Dahmane et al., 2001; Clement et al., 2007; Zbinden et al., 2010). Additional studies provided further support for our findings (e.g. Bar et al., 2007; Ehtesham et al., 2007) and suggest that GLI1 may be a prognostic marker for GBM (Rossi et al., 2011). SHH-GLI signaling is a key intercellular communication pathway that we and others have shown also participates in the control of normal neural stem cell and brain precursor behavior (e.g. Dahmane and Ruiz i Altaba, 1999; Dahmane et al., 2001; Lai et al., 2003; Palma and Ruiz i Altaba, 2004). Thus these data raised the possibility that SHH-GLI signaling is an essential signaling pathway involved in the maintenance of both normal brain stem cells and of GBM stem cells.
We also originally found that GBMs and lower grade gliomas express a core ESC-like signature that includes the expression of a number of genes previously involved in regulating ESCs and reprogramming that include NANOG, OCT4 and SOX2 (Clement et al., 2007). Later on such a signature was also detected in other cancer types (Ben-Porath et al., 2008). Upon blockade of SHH-GLI signaling, the levels of this ESC-like signature (including those of the key stemness gene NANOG), GBM growth, and the number of GBM stem cells drastically decreased (Clement et al., 2007), suggesting a connection between GBM stem cells and SHH-GLI signaling, and thus beginning to highlight the molecular mechanisms involved in GBM stemness.
Other evidence for a link derives from parallel studies we have carried out on the role of GLI1, the downstream effector of SHH signaling and a key node in signaling integration (e.g. Lee et al., 1997; Stecca and Ruiz i Altaba, 2010). Enhanced GLI1 function in the brain of transgenic mice drives increased neural stem cell self-renewal, brain overgrowth and much increased levels of Nanog (Stecca and Ruiz i Altaba, 2009). GLI1 not only imparted normal stem cells/early precursors with enhanced stem cell properties but also with increased invasiveness of the brain parenchyma (Stecca and Ruiz i Altaba, 2009). GLI1 thus appeared as a bona fide GBM stem cell regulator that we ought to target.
However, it remained unclear if the genes - composing the ESC-like signature in gliomas that is regulated by SHH-GLI - are functional and drive GBM stemness and thus tumor persistence and growth. To address this question we decided to directly test the function of the key pluripotency homeodomain protein NANOG in human GBM, using primary tumors implanted into the brain of immunodeficient mice (orthotopic xenografts). Our very recent work shows for the first time that NANOG is essential for GBM growth in vivo. Its knockdown with lentivector-encoded shRNAs (against NANOG and NANOGP8 thus targeting the two NANOG-producing genes) abolished the growth of cell line and primary GBMs (Zbinden et al., 2010). In other words, NANOG function is absolutely essential for GBM growth in mice, suggesting a similar requirement in patients.
Mechanistically, we showed that NANOG is an essential downstream mediator of HH-GLI signaling in gliomas, forming a positive regulatory loop with GLI1. In contrast, and like GLI1 (Stecca and Ruiz i Altaba, 2009; see also Abe et al., 2008), it acts in a negative loop with p53, which is often lost in GBMs (Furnari et al., 2007). A GLI1-NANOG module is thus an essential node in the control human GBM stemness and tumor growth (Zbinden et al., 2010).
Our data open exciting possibilities for therapeutic intervention against human GBMs. For the first time we have provided the proof of principle that blocking the function of NANOG, a single key stemness factor in primary human GBMs, is sufficient to repress GLI1 and to prevent tumor growth in vivo (Zbinden et al., 2010). Since endogenous normal NANOG function may be restricted to the embryonic brain and adult stem cell niches (e.g. Po et al., 2010), we reasoned that inhibiting its function over a defined period of time should obliterate GBMs and prevent recurrence while allowing normal recovery of endogenous stem cell lineages, possibly recapitulating our previous findings in colon cancers and melanomas with time-limited SHH-GLI inhibition (Stecca et al., 2007; Varnat et al., 2009). In this project we propose to develop innovative NANOG repressors able to penetrate tumor cells in vivo, with the aim to kill GBM stem cells and thus the tumor itself and any and all recurrences. Our strategy should also allow for the destruction of invasive stem cells that are impossible to target otherwise.
- Abe, Y., Oda-Sato, E., Tobiume, K., Kawauchi, K., Taya, Y., Okamoto, K., Oren, M., Tanaka, N. (2008). Hedgehog signaling overrides p53-mediated tumor suppression by activating Mdm2. Proc Natl Acad Sci U S A. 105,4838-43.
- Bao, S., Wu, Q., McLendon, R.E., Hao, Y., Shi, Q., Hjelmeland, A.B., Dewhirst, M.W., Bigner, D.D., Rich, J.N. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756-60.
- Bar, E.E., Chaudhry, A., Lin, A., Fan, X., Schreck, K., Matsui, W., Piccirillo, S., Vescovi, A.L., DiMeco, F., Olivi, A., Eberhart, C.G. (2007) Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma. Stem Cells 25,2524-33.
- Ben-Porath, I., Thomson, M.W., Carey, V.J., Ge, R., Bell, G.W., Regev, A., Weinberg, R.A. (2008) An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 40, 499-507.
- Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., Smith, A. (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.Cell 113,643-55.
- Clement, V., Sanchez, P., de Tribolet, N., Radovanovic, I., Ruiz i Altaba, A. (2007) HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr Biol. 17,165-72.
- Dahmane, N. and Ruiz i Altaba, A. (1999) Sonic hedgehog regulates the growth and patterning of the cerebellum. Development 126,3089-100.
- Dahmane, N., Sánchez, P., Gitton, Y., Palma, V., Sun, T., Beyna, M., Weiner, H., Ruiz i Altaba, A. (2001) The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development 128,5201-12.
- Ehtesham, M., Sarangi, A., Valadez, J.G., Chanthaphaychith, S., Becher, M.W., Abel, T.W., Thompson, R.C., Cooper, M.K. (2007) Ligand-dependent activation of the hedgehog pathway in glioma progenitor cells. Oncogene 26,5752-61.
- Furnari, F.B., Fenton, T., Bachoo, R.M., Mukasa, A., Stommel, J.M., Stegh, A., Hahn, W.C., Ligon, K.L., Louis, D.N., Brennan, C., Chin, L., DePinho, R.A., Cavenee, W.K. (2007) Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev. 21, 2683-710.
- Hendrix, M.J., Seftor, E.A., Seftor, R.E., Kasemeier-Kulesa, J., Kulesa, P.M., Postovit, L.M. (2007) Re-programming metastatic tumour cells with embryonic microenvironments. Nat Rev Cancer. 7, 246-55.
- Kim, J.B., Greber, B., Araúzo-Bravo, M.J., Meyer, J., Park, K.I., Zaehres, H., Schöler, H.R. (2009) Direct reprogramming of human neural stem cells by OCT4. Nature 461, 649-3.
- Lai, K., Kaspar, B.K., Gage, F.H., Schaffer, D.V. (2003) Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat Neurosci 6, 21-7.
- Lee, J., Platt, K.A., Censullo, P., Ruiz i Altaba, A. (1997) Gli1 is a target of Sonic hedgehog that induces ventral neural tube development. Development 124, 2537¬ 52.