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Molecular Biology, Pathobiology, and Genetics |
Departments of 1 Pathology and 2 Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland and 3 Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York
Requests for reprints: Charles G. Eberhart, Department of Pathology, Johns Hopkins University School of Medicine, Ross Building 558, 720 Rutland Avenue, Baltimore, MD 21205. Phone: 410-502-5185; Fax: 410-955-9777; E-mail: ceberha{at}jhmi.edu.
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Abstract |
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-secretase. Notch blockade suppressed expression of the pathway target Hes1 and caused cell cycle exit, apoptosis, and differentiation in medulloblastoma cell lines. Interestingly, viable populations of better-differentiated cells continued to grow when Notch activation was inhibited but were unable to efficiently form soft-agar colonies or tumor xenografts, suggesting that a cell fraction required for tumor propagation had been depleted. It has recently been hypothesized that a small population of stem-like cells within brain tumors is required for the long-term propagation of neoplastic growth and that CD133 expression and Hoechst dye exclusion (side population) can be used to prospectively identify such tumor-forming cells. We found that Notch blockade reduced the CD133-positive cell fraction almost 5-fold and totally abolished the side population, suggesting that the loss of tumor-forming capacity could be due to the depletion of stem-like cells. Notch signaling levels were higher in the stem-like cell fraction, providing a potential mechanism for their increased sensitivity to inhibition of this pathway. We also observed that apoptotic rates following Notch blockade were almost 10-fold higher in primitive nestin-positive cells as compared with nestin-negative ones. Stem-like cells in brain tumors thus seem to be selectively vulnerable to agents inhibiting the Notch pathway. (Cancer Res 2006; 66(15): 7445-52) |
Introduction |
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In addition to arising from stem or precursor cells, medulloblastoma may also contain functionally important subsets of cells with stem-like properties. It has recently been hypothesized that in many types of cancer, including medulloblastoma, such stem-like cells are uniquely capable of propagating tumor growth (16–18). This "cancer stem cell" hypothesis is based, in part, on paradigms explaining the development and repair of normal organs, in which only stem cells are thought to have a long-term capacity for self-renewal and are therefore necessary for the generation and regeneration of tissues. Experimental support for the existence of a discrete cancer cell subpopulation required for tumor propagation initially came from clonogenic assays, which showed that only a small portion of tumor cells could form colonies in vitro or engraft in vivo (19–21). Only recently the prospective isolation of cells uniquely capable of propagating neoplasms has been achieved, providing firmer support for the presence of cancer stem cells (22, 23). This has been made possible by the characterization of markers that identify stem-like cells in tumor specimens. Two such markers, CD133 and side population, have been used to prospectively isolate a small percentage of cells in brain tumors uniquely able to generate tumor neurospheres and xenografts (24–27). Interestingly, both markers were initially identified in nonneoplastic stem cells (28–31), highlighting the similarities between normal and malignant stem cells.
To date, no agent has been shown to target the cancer stem cell subpopulation in solid tumors. However, similarities in the growth characteristics and gene expression patterns of benign neural stem cells and brain tumor stem cells suggest that the same signaling pathways may be required for survival and growth in both (18, 32–35). Notch is known to promote the survival and proliferation of nonneoplastic neural stem cells and to inhibit their differentiation (36, 37). Signaling is initiated by ligand binding, followed by intramembranous proteolytic cleavage of the Notch receptor by the -secretase complex. Inhibitors of this complex slow the growth of Notch-dependent tumors such as medulloblastoma and T-cell leukemia (8, 9, 38). We therefore used a potent small-molecule -secretase inhibitor to further investigate the role for Notch signaling in medulloblastoma, comparing the effects on overall tumor cell mass to those on cells expressing stem-like markers. Our data indicate that stem-like brain tumor cells may be especially vulnerable to attacks on molecular pathways, such as Notch, which are required in their nonneoplastic cognate cells.
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Materials and Methods |
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-secretase inhibitor dissolved in DMSO at the concentrations indicated. RNA and protein extractions and all cell-based assays were done 48 hours after drug application unless otherwise noted. All experiments with error bars were done in triplicate and shown as mean values with SE unless otherwise noted. Cell mass was measured using CellTiter assays according to the instructions of the manufacturer (Promega, Madison, WI). Cell number and viability were assessed using the Guava PCA and Viacount reagent according to instructions (Guava Technologies, Heyward, CA). Soft-agar assays were done as previously described, with colonies counted using an automated reader (8).
-Secretase inhibitor synthesis. The potent -secretase inhibitor [11-endo]-N-(5,6,7,8,9,10-hexahydro-6,9-methanobenzo[a][8]annulen-11-yl)-thiophene-2-sulfonamide was listed as compound 18 in the recent report by Lewis et al. (39) describing its synthesis and testing, and we refer to it as GSI-18. It was synthesized as previously described (39) and its identity and quality were confirmed by nuclear magnetic resonance and mass spectral analysis. The biological activity of this inhibitor against -secretase was confirmed using in vitro and cell-based assays as previously described (40).
Immunocytochemistry. The cell lines DAOY and MB2 were seeded in 12-well tissue culture plates and allowed to adhere overnight. After treating with 2 µmol/L GSI-18 for 48 hours, cells were washed with PBS and fixed using 4% paraformaldehyde solution in PBS at room temperature for 30 minutes. Cells were then permeabilized with 0.4% Triton X-100 in PBS for 5 minutes at room temperature, washed with PBS, and incubated in 5% bovine serum albumin (BSA)-PBS for 1 hour and then in 1:1,000 anti-nestin antibody (Chemicon, Temecula, CA) or 1:500 anti–cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) in 1% BSA-PBS for 2 hours. After washing with PBS, a final 60-minute incubation with a 1:300 dilution of Cy3-conjugated goat anti-mouse and Cy2-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in PBS containing 1% BSA was done. After washing with PBS, cells were then counterstained with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA), mounted, and visualized with fluorescence microscope (Zeiss, Jena, Germany). In the single-immunolabeling study (Fig. 6A and C), six high-power fields of DAOY cultures containing 147 to 333 (mean, 193) cells were photographed and the percentages of cells positive for nestin were scored in a blinded fashion. In the double-immunolabeling study (Fig. 6D-F), 10 high-power fields were photographed and counted.
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Real-time reverse transcription-PCR. Quantitative reverse transcription-PCR (RT-PCR) for Notch1, Notch2, and Hes1 was done as previously described (31), with all reactions normalized to actin (Applied Biosystems, Foster City, CA). Commercially available Assay on Demand TaqMan primers and probes were used to measure mRNA for Tuj1 and the 
6 subunit of the -aminobutyric acid type A receptor (GABRA6). Each quantitative RT-PCR reaction was done in triplicate and error bars represent SE.
Protein analysis. Western blots contained 20 µg of protein per lane on a 10% Tris-glycine SDS-PAGE gel (Invitrogen) and electrophoresing for several hours in 1x TG-SDS buffer (Amresco, Solon, OH). Proteins were transferred to 0.45-µm Optitran nitrocellulose (Schleicher & Schuell, Keene, NH) in 1x Tris-glycine buffer (Amresco). Blots were blocked in PBS containing 5% nonfat dry milk powder and incubated overnight at 4°C with antibodies directed against Hes1 (kind gift of Dr. Tetsuo Sudo, Toray Industries, Tebiro, Kamakura, Japan; 1:2,000) or glyceraldehyde-3-phosphate dehydrogenase (Research Diagnostics, Flanders, NJ; 1:20,000). Blots were then washed several times with PBS containing 0.1% Tween 20 and incubated in peroxidase-conjugated IgG diluted 1:2,000 in blocking solution. After washing several times in PBS with 0.1% Tween 20, blots were developed with enhanced chemiluminescence reagent (Pierce, Rockford, IL) and exposed to film.
Statistical analysis. Statistical analyses were done using GraphPad Prism 4 (GraphPad Software, San Diego, CA). Data graphed with error bars represent mean and SE from experiments done in triplicate unless otherwise noted. A two-sided Student's t test was used to determine the significance of any differences.
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Results |
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-secretase inhibitor with a sulfonamide core (39). We first sought to determine what concentration of GSI-18 effectively inhibited Notch activity in tumor cells by measuring expression of the pathway target Hes1. GSI-18 at 2 µmol/L reduced both mRNA and protein levels of Hes1 in DAOY cells by >70% (Fig. 1A and B
), suggesting that this concentration should be sufficient to cause antitumor effects mediated by Notch pathway inhibition. GSI-18 levels <0.4 µmol/L did not affect Hes1 mRNA expression (data not shown).
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-secretase inhibitor with a benzodiazepine core, also slowed but did not arrest growth in medulloblastoma cell mass when used at concentrations that effectively inhibited the Notch pathway, indicating the findings were not specific to one structural class of compound (data not shown). DAOY cells stably transfected with a NICD2 were used to further control for the specificity of the pharmacologic effects. This truncated Notch receptor does not require ligand binding or -secretase activity for nuclear translocation and signaling, and cells expressing it should be insensitive to effects of GSI-18 mediated by Notch2 inhibition. NICD2 expression in a stable subclone we have previously described (8) rescued the negative effects on DAOY cell mass (Fig. 1D). A second stable subclone, as well as a pool of cells into which NICD2 was introduced by transfection, was also resistant to the growth-inhibiting effects of GSI-18 (data not shown), suggesting that this compound acts through Notch and not other pathways regulated by -secretase. It has previously been reported that in pre-T cells, down-regulation of Notch activity reduces cellular metabolism and cell size (41). We also observed decreased cell size in medulloblastoma cultures following GSI-18 exposure (Fig. 1E). To rule out the possibility that our measurements of viable cell mass bioreductive capacity (CellTITER assay) were being altered by metabolic affects of Notch, we quantitated cell number using flow cytometry. The number of live cells in DAOY, D283Med, D425Med, and PFSK cultures exposed to 2 µmol/L GSI-18 for 48 hours was reduced as compared with vehicle-treated controls, but the reduction was less than that of cell mass (Fig. 1F). Thus, measures of both cell mass and cell number show a slowing, but not an arrest, of tumor growth under conditions of Notch blockade.
Decreased proliferation and increased neuronal differentiation following Notch inhibition. Flow cytometric analysis showed that 2 µmol/L GSI-18 increased the G1-G0 cell fraction and decreased the S-phase and G2-M fractions of DAOY and PFSK cell lines (Fig. 1G), suggesting that cell cycle exit likely plays a role in its antigrowth effects. Notch pathway down-regulation has also been linked to cellular differentiation in both normal development and in neoplasms (42, 43) and we therefore examined whether Notch inhibition caused cellular differentiation. -Secretase inhibition in DAOY cells increased RNA levels of two markers of cerebellar neuronal differentiation, Tuj1 and GABRA6, in a dose-dependent fashion (Fig. 1H). One of these neuronal markers, GABRA6, is specifically found in cerebellar granule cells in the brain, where its expression is induced as they mature (44). Its induction after Notch pathway inhibition suggests that the differentiation pathway being actuated resembles that in normal cerebellar granule neuron precursors, and highlights the similarities between the DAOY line and developing cerebellum.
Notch blockade by -secretase inhibitors suppresses tumor formation. We next examined the effects of Notch pathway blockade on tumor formation in vitro and in vivo. We first used a clonogenic assay to determine whether cells capable of forming anchorage-independent colonies were depleted by GSI-18. Vehicle-treated DAOY cultures seeded in soft agar formed 50 colonies per field on average (Fig. 2
). This number dropped to 4 when an equal number of viable cells was counted and seeded after 48 hours of treatment with GSI-18 and increased to 119 in the presence of constitutive Notch2 activation. Thus, whereas many tumor cells continued to grow in 2 µmol/L GSI-18, their clonogenicity in soft agar was suppressed by >90%.
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-secretase inhibition, the experiment was repeated with cells counted after a recovery period. After 2 days of treatment, followed by 24 hours of recovery in medium containing 10% FBS and no -secretase inhibitor, both GSI-18-treated and vehicle-treated cells proliferated at similar rates, suggesting that any overall effects on growth had normalized (Fig. 3D). When equal numbers of these "recovered" cells were injected into mice, GSI-18-treated cultures were still unable to form tumors (n = 4) whereas bulky tumor xenografts formed in all 12 controls. D425Med and D283Med cells also failed to engraft (n = 2 animals D425Med, n = 3 animals D283Med) when pretreated for 48 hours with GSI-18, whereas vehicle-treated cells injected on the contralateral flank formed large tumors at all but one site (Fig. 3E).
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Notch regulates the percentage of medulloblastoma cells expressing stem/precursor markers. The data presented above suggest that while viable and proliferative following Notch pathway blockade, medulloblastoma cultures are altered in some way, blocking their ability to form soft-agar colonies and tumor xenografts. It has been shown that stem-like "side-populations" in established brain tumor cell lines are uniquely able to form xenografts (25, 26). Thus, cancer stem cells seem to be able to persist in established brain tumor lines maintained for many years in culture. Because stem-like tumor cells are thought to be critical for xenograft formation, we sought to determine if this subpopulation might be especially sensitive to Notch pathway blockade.
We first examined the requirement for ongoing Notch activity in CD133-positive cells. Singh et al. (24, 33) have shown that only CD133-positive medulloblastoma and glioblastoma brain tumor cells are able to form multipotent neurosphere clones or tumor xenografts. Another type of brain tumor, ependymoma, also seems to contain a small fraction of CD133-positive cells uniquely capable of engrafting in immunocompromised mice (27). In the adherent embryonal brain tumor cell lines DAOY and PFSK, flow cytometric analysis revealed that 9.9% and 10.8% of cells, respectively, expressed CD133 on their surface (Fig. 4A and B
). These CD133-positive fractions fell within the 6.1% to 45.4% range (mean, 19.7%) reported in primary medulloblastoma (33). The D283Med and D425Med lines, which have elevated c-Myc levels and grow in suspension, contained higher percentages of CD133-positive cells. Sorting of DAOY and PFSK cultures generated populations enriched 
3-fold for cells expressing CD133 and negative fractions in which <1% of cells were CD133 positive. Interestingly, mRNA levels of Hes1, a marker of Notch pathway activity, were elevated 4-fold in the CD133-enriched fraction of DAOY and 5-fold in the CD133-enriched fraction of PFSK (Fig. 4C). This suggests that Notch signaling is especially active in stem-like cancer cells and supports the possibility that Notch pathway inhibition may target this population.
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Side population was analyzed to obtain a second measure of the effects of Notch on stem-like tumor cells. DAOY cultures contained a Hoechst 33342–excreting side population of 1.9% (Fig. 5A ). The multiple drug resistance pumps mediating dye efflux are verapamil sensitive (28), and adding verapamil to the medulloblastoma culture abolished Hoechst excretion, verifying that this was a bona fide side population. Consistent with our hypothesis that Notch activity regulates stem-like cancer cells, constitutive activation of Notch2 increased the side population almost 4-fold to 7.3%, whereas Notch inhibition essentially ablated it, with only 0.01% of remaining cells negative for dye (Fig. 5A and B). NICD2 expression protected the side population from GSI-18 exposure, but not from verapamil, indicating that the effect of GSI-18 was specifically mediated through its effects on Notch signaling. Small side populations were also present in the PFSK, D283Med, and D425Med cell lines and were ablated by both verapamil (data not shown) and 2 µmol/L GSI-18 (Fig. 5B). Thus, Notch pathway blockade depletes the stem-like subpopulation defined either by CD133 or by side population.
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-secretase inhibitor GSI-18 reduced this population 4-fold to 2.4% (P < 0.01; Fig. 6C). A significant reduction was also observed in MB2 cells following Notch blockade. We used double immunofluorescence to study cell death in medulloblastoma cells positive or negative for nestin. The basal apoptotic rate was measured using antibodies specific for cleaved caspase-3. In DAOY cultures to which vehicle was added for 48 hours, the apoptotic rate was low in both nestin-negative (26 of 1,841; 1.4%) and nestin-positive (4 of 160; 2.5%) cells (Fig. 6D-F). However, after 48 hours of treatment with 2 µmol/L GSI-18, the apoptotic rate increased to 37% in the nestin-positive population (23 of 62 cells) whereas apoptosis in the better-differentiated cells lacking nestin only increased to 3.9% (40 of 1,017 cells). Interestingly, nestin was less evenly distributed in the apoptotic cells and was sometimes observed surrounding the degenerating nuclei. Such cagelike perinuclear nestin staining has been previously described in mitotic cells (47). Our immunofluorescent assessment of cell death using cleaved caspase-3 corresponds relatively well to apoptotic induction measured by flow cytometric analysis of Annexin staining, suggesting that our manual counting procedure is accurate (Fig. 6G). Taken together, these data indicate that the survival of stem-like tumor cells is more sensitive to Notch pathway inhibition than the survival of better-differentiated cells.
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Discussion |
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-secretase inhibitor GSI-18 slowed the growth of tumor cells in vitro but had a much more dramatic effect on the formation of colonies in soft agar and tumor xenografts in nude mice. Indeed, our most striking finding was that large numbers of viable, rapidly proliferating cells were not able to generate bulky xenografts if they had previously been treated with GSI-18, whereas equal numbers of vehicle-treated cells always formed large tumors. Systemic (i.p.) treatment of animals with GSI-18 also blocked xenograft formation with no apparent side effects, indicating this agent may be effective therapeutically in preventing tumor metastasis or regrowth following debulking. We believe that these dramatic effects on tumor propagation are mediated by depletion of cancer stem cells, as subpopulations expressing the stem cell marker CD133, as well as the stem-like side population, were profoundly reduced by Notch blockade. These results must be extended to primary tumors or additional low-passage lines and might provide a first indication that stem-like cells can be successfully ablated from brain tumors. The targeted depletion of stem-like cells we observe following Notch pathway blockade contrasts sharply with prior reports of cancer stem cell survival following standard chemotherapies. For example, Wulf et al. (48) showed higher efflux of chemotherapeutic agents and better survival in leukemic side population cells as compared with non-side population cells. Based on this and on similar studies, it has been suggested that the efflux pumps which define side population also function to remove toxic chemotherapy drugs from cancer stem cells (reviewed in refs. 49, 50). In support of this concept, treatment of neuroblastoma cell lines with mitoxantrone actually increases the side population, suggesting that stem-like cancer cells are relatively resistant to this chemotherapeutic agent and accumulate as more differentiated cells are killed (51). Thus, conventional chemotherapies effectively remove the better-differentiated cells while leaving most stem-like cells alive. In contrast, Notch blockade depletes stem-like cells but leaves many better-differentiated cells capable of limited growth intact.
We examined only one type of malignant brain tumor in this study, but our findings may be applicable to other neoplasms as well. The Notch pathway is known to regulate stem cells in a wide variety of tissues and Notch blockade seems to affect survival and proliferation of multiple types of cancer (52, 53). For example, Notch is activated by translocation or mutation in >50% of T-cell acute lymphoblastic leukemia, and anti-Notch therapies have been shown to slow acute lymphoblastic leukemia growth in vitro (38). Aberrantly activated Notch signaling has also been documented in lung, breast, salivary gland, and pancreatic carcinoma (54–57). -Secretase inhibitors may therefore be useful in targeting stem-like cancer cells in a wide range of neoplasms.
In the future, multiagent chemotherapeutic regimens may target both stem-like and better-differentiated cells. Drugs blocking Notch signaling, or other pathways required in stem cells such as Wnt and Hedgehog, will be used to deplete the cancer stem cell population, and traditional chemotherapeutic agents can be used at the same time to debulk the larger mass of tumor cells. This will effect a rapid removal of both subpopulations and might circumvent the possibility that some differentiated tumor cells can dedifferentiate and repopulate the stem cell fraction (58). Another potential complication of cancer stem cell–directed therapies is that nonneoplastic stem cells may also be depleted by such strategies. This is of particular concern in children who will presumably require stem cells for the maintenance and repair of a host of tissues over the course of their lives. Of note, we found that i.p. injections of GSI-18 for 5 days blocked tumor formation with no obvious ill effects on the animal's health over the subsequent months. Indeed, it has recently been shown that inhibition of -secretase activity using small-molecule drugs can actually enhance long-term memory in rodents (59).
In summary, we show that in the malignant brain tumor medulloblastoma, Notch pathway blockade depletes a population of cells required for in vivo tumor formation by suppressing proliferation and inducing apoptosis or differentiation in stem-like cells. Our data suggest that Notch pathway blockers may be the first of a new class of chemotherapeutic agents—those targeting cancer stem cells.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Josh Copeland for assistance with Western blots, Dr. Tetsuo Sudo for his generous gift of Hes1 antiserum, and Dr. J.C. Zuniga-Pflucker for his suggestions on Notch inhibition and cell size.
Received 3/13/06. Revised 5/17/06. Accepted 5/25/06.
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References |
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