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Cancer Research 67, 3871, April 15, 2007. Published Online First April 5, 2007;
doi: 10.1158/0008-5472.CAN-07-0493
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Medulloblastomas Derived from Cxcr6 Mutant Mice Respond to Treatment with a Smoothened Inhibitor

Ken Sasai1,2, Justyna T. Romer1, Hiromichi Kimura1, Derek E. Eberhart3, Dennis S. Rice3 and Tom Curran4

1 Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee; 2 Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo, Japan; 3 Lexicon Genetics, Inc., The Woodlands, Texas; and 4 Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

Requests for reprints: Tom Curran, Children's Hospital of Philadelphia, 517 Abramson Research Center, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318. Phone: 267-426-2819; Fax: 215-590-3709; E-mail: currant{at}chop.edu.


   Abstract
Top
 Abstract
Introduction
 Experimental Procedures
 Results
 Discussion
 References
 
The sonic hedgehog (Shh) pathway is activated in ~30% of human medulloblastoma resulting in increased expression of downstream target genes. In about half of these cases, this has been shown to be a consequence of mutations in regulatory genes within the pathway, including Ptc1, Smo, and Sufu. However, for some tumors, no mutations have been detected in known pathway genes. This suggests that either mutations in other genes promote tumorigenesis or that epigenetic alterations increase pathway activity in these tumors. Here, we report that 3% to 4% of mice lacking either one or both functional copies of Cxcr6 develop medulloblastoma. Although CXCR6 is not known to be involved in Shh signaling, tumors derived from Cxcr6 mutant mice expressed Shh pathway target genes including Gli1, Gli2, Ptc2, and Sfrp1, indicating elevated pathway activity. Interestingly, the level of Ptc1 expression was decreased in tumor cells although two normal copies of Ptc1 were retained. This implies that reduced CXCR6 function leads to suppression of Ptc1 thereby increasing Smoothened function and promoting tumorigenesis. We used a direct transplant model to test the sensitivity of medulloblastoma arising in Cxcr6 mutant mice to a small-molecule inhibitor of Smoothened (HhAntag). We found that transplanted tumors were dramatically inhibited in mice treated for only 4 days with HhAntag. These findings suggest that HhAntag may be effective against tumors lacking mutations in known Shh pathway genes. [Cancer Res 2007;67(8):3871–7]


   Introduction
Top
 Abstract
 Introduction
Experimental Procedures
 Results
 Discussion
 References
 
Medulloblastoma, a primitive neuroectodermal tumor that arises in the cerebellum, is the most common malignant brain tumor of childhood. Current therapy is associated with long-term deleterious side effects particularly in young children (1, 2), and therefore it is important to develop alternative therapeutic strategies. Small-molecule inhibitors that target cell signaling pathways aberrantly activated in medulloblastoma could be very useful in the future management of this disease. The sonic hedgehog (Shh) pathway provides a unique opportunity for intervention because it is activated in ~30% of human medulloblastoma, based on gene expression signatures (3, 4). Using the Ptc1+/–p53–/– mouse model of medulloblastoma (5), we conducted a proof-of-principle preclinical study of a small molecule (HhAntag) that binds to and inhibits Smoothened (6–8) thereby down-regulating the Shh pathway (9). Remarkably, HhAntag eliminated spontaneous medulloblastoma in these mice after only 2 weeks of oral dosing (9). Similarly, HhAntag was very effective at reversing the growth of transplantable mouse medulloblastoma (10). In contrast, we found that it did not affect growth of medulloblastoma cell lines because the Shh pathway is down-regulated when tumor cells are placed in culture (9, 10).

Mutations in patched-1 (PTCH1) occur in ~10% of sporadic human medulloblastoma (11–14). Other components of the Shh pathway, including suppressor of fused (SUFU; ref. 15) and SMO (16), which also result in pathway activation, have been described. However, half of the tumors in which the Shh pathway is activated fail to show any mutations in the known pathway members (4). This indicates that there are either mutations in unknown genes that regulate Shh signaling, directly or indirectly, or that epigenetic mechanisms contribute to tumorigenesis. In addition, mutation of several genes involved in cell cycle control and DNA repair in mice promotes medulloblastoma formation, and in some of these cases, it seems that tumors lose one or both copies of Ptc1, resulting in increased Shh pathway activity (3, 17–23). Thus, the Shh pathway can be activated in tumors in a number of ways.

Here, we characterize Cxcr6 mutant mice, which were generated as a part of the Lexicon Genome5000 program using the pKOS gene-targeting system (24), and we report that heterozygous or homozygous mutation of Cxcr6 causes a low (3–4%) incidence of medulloblastoma. CXCR6 (also named BONZO, STRL33, or TYMSTR) is a chemokine receptor that was identified as the principal coreceptor for several strains of human and simian immunodeficiency viruses (25–27). Its normal biological function is unclear and gene ablation studies indicated no obvious phenotypes (28). Although the tumors arising in Cxcr6 mutant mice express several Shh pathway signature genes, both normal copies of Ptc1 are present. However, the expression level of Ptc1 is low, indicating the possibility of epigenetic suppression in these tumors. We found that directly transplanted medulloblastoma cells derived from Cxcr6 mutant mice responded to treatment with HhAntag. This implies that altered CXCR6 function led to suppression of Ptc1 expression, which in turn increased Shh pathway activity, thus promoting medulloblastoma formation.


   Experimental Procedures
Top
 Abstract
 Introduction
 Experimental Procedures
Results
 Discussion
 References
 
Generation of Cxcr6 mutant mice. Mutant mice were generated by Lexicon Genetics, Inc. (The Woodlands, TX). A targeting vector, designed to delete exon 2, was constructed using the Lambda KOS system previously described by Wattler et. al. (24). Briefly, the targeted embryonic stem cell clone (derived from the 129/SvEvBrd strain), containing a LacZ-Neo cassette in place of exon 2 of the Cxcr6 gene (Fig. 1A ), was microinjected into host blastocysts. The resulting chimeric mice were bred with C57BL/6J albino mice to achieve germ line transmission of the Cxcr6 mutation. The mice used in this study were of mixed genetic background (129/SvEvBrd and C57BL/6J). All mouse studies were carried out according to protocols approved by the institutional Animal Care and Use Committees at Lexicon Genetics and St. Jude Children's Research Hospital.


Figure 1
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  		Figure 1. Targeted disruption of the Cxcr6 locus and the establishment of a mutant mouse strain. A, top, wild-type allele of the Cxcr6 gene; middle, targeting vector; bottom, targeted allele. The filled and open boxes in the wild-type allele represent the protein coding and noncoding regions of Cxcr6 mRNA, respectively. A LacZ-Neo cassette (gray box) replaced exon 2 of the Cxcr6 gene. The expected sizes of the HindIII (H) and EcoRV (E) digestion products of the gene, hybridized with the indicated probes, are shown. B, genomic DNA isolated from embryonic stem cells (wild-type and a positive clone) was digested with either HindIII (left) or EcoRV (right) and analyzed by Southern blotting with the probes shown in (A). C, PCR genotype analysis with primer pair F1/R1 (lane 1 and 2), which results in a 765-bp product in the wild-type allele, and with primer pair F2/R1 (lane 2 and 3), which results in a 389-bp product in the targeted allele. D, gene expression of Cxcr6 (top) and lacZ (middle) was analyzed by RT-PCR using total RNA isolated from mouse spleen. Gapdh was used as an internal control (bottom). Genotype of each sample is indicated at the top of each lane.

 
Genotyping of mice. Genomic DNA samples (10 µg) isolated from targeted embryonic stem cells were digested with HindIII or EcoRV and analyzed by Southern blotting. Probes for Southern blotting were generated by genomic PCR. Primer sequences used in this experiment included 5'-CCTCTGCTGCAAACCTATTC-3' and 5'-AAACCGTGTGCCCAGAACG-3' for probe-I and 5'-TATAGGCAGCTTGGTACTATC-3' and 5'-ACTGTGGTCAGCAGCCGAG-3' for probe-II. Routine genotyping of mice was done by PCR using three primers (F1, 5'-CTGTCAGTACCACAGTGAGG-3'; R1, 5'-AACCGTCATGACCTTGATTTC-3'; and F2, 5'-GCAGCGCATCGCCTTCTATC-3'), which resulted in 765-bp (F1/R1) and 389-bp (F2/R1) products from the wild-type and targeted alleles, respectively.

Histologic analysis and immunohistochemistry. Mice were perfused with 4% paraformaldehyde and then the brains were removed, postfixed, embedded in paraffin, and sectioned in the sagittal plane, as previously described (9). Paraffin sections were stained with H&E using standard histologic protocols and processed for immunohistochemistry with antibodies specific for glial fibrillary acidic protein (Shandon Immunon, Waltham, MA) and synaptophysin (Dako, Copenhagen, Denmark).

Allograft propagation and HhAntag treatment. Six-week-old female athymic nude mice (CD1 nu/nu; Charles River Breeding Laboratory, Wilmington, MA) were injected s.c. in the flank with suspended tumor cells (3 x 106) derived from a Cxcr6+/– mouse. Tumor sizes were measured and tumor volumes were calculated as previously described (10). Cohorts of mice bearing allografts with volumes in the 200 to 400 mm3 range were treated with vehicle alone (n = 2) or with 100 mg kg–1 HhAntag (n = 4) twice a day for 4 days by oral gavage. Four hours after the last dose, mice were euthanized and allografts were harvested. HhAntag was obtained from Curis (Boston, MA) and Genentech (South San Francisco, CA).

Cell culture. Medulloblastoma cells derived from a Cxcr6+/– mouse were cultured as previously described (29), and total RNA was isolated after three passages.

Northern blotting and in situ hybridization. Gene expression was evaluated by Northern blotting (30) and in situ hybridization (9, 31) as previously described. Antisense RNA probes for Cxcr4 (corresponding to Ala102-Leu273 of mouse CXCR4) and Fyco1 (corresponding to Met1211-Pro1437 of mouse Fyco1) were synthesized with the Strip-EZ RNA kit (Ambion, Austin, TX). Other probes were previously described (9, 10, 30).

Reverse transcription-PCR. Expression of Cxcr6 and lacZ mRNA was analyzed by reverse transcription-PCR (RT-PCR) using a Superscript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA). Primer sequences used in this experiment included 5'-ATGTTTGCCCCAACAGATG-3' and 5'-CTAGAGTTGGAACATACTGGTGG-3' for mouse Cxcr6 and 5'-ATGGAAGATCCCGTCGTTTTAC-3' and 5'-ATGGGATAGGTCACGTTGGTG-3' for lacZ. PCR primers for glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were previously described (32).

Fluorescence in situ hybridization analyses. A probe specific for the Patched-1 gene was prepared from a bacterial artificial chromosome (ResGen Mouse BAC clone, 241013; Invitrogen) by labeling with digoxigenin-11-dUTP (Roche Applied Science, Indianapolis, IN) using nick translation. A biotinylated probe specific for mouse chromosome 13 (386H1O; BACPAC Resources, Oakland, CA) was used as an internal control for the fluorescence in situ hybridization (FISH) assays. Both labeled clones were combined with sheared mouse DNA and hybridized to 5-µm-thick paraffin sections. Specific hybridization signals were detected by incubating the slides with fluorescein-labeled anti-digoxigenin antibody (Roche Applied Science) and Texas red-avidin (Vector Laboratories, Burlingame, CA). The paraffin sections were then stained with 4,6-diamidino-2-phenylindole and analyzed.

Microarray gene expression analyses. Total RNA was prepared from developing cerebella (postnatal day 5) from wild-type mice and from medulloblastomas derived from Cxcr6+/– (n = 2) and Ptc1+/– mice (n = 2). The samples were then hybridized with the Mouse Genome 430 2.0 Array (45101 probe sets; Affymetrix, Santa Clara, CA) and analyzed using the Affymetrix Microarray Suite version 5.0 (MAS v5) and Spotfire 8.1 software, as previously described (10, 33). To identify the changes in gene expression, the numbers of probe sets scored as up-regulated (>2-fold increase, log ratio >1), down-regulated (>2-fold decrease, log ratio <1), and unchanged (–0.5 < log ratio < 0.5) were counted for each comparison (medulloblastoma versus developing cerebellum). Log ratios of signal are log 2 ratios of geometric means of the stabilized signals, which were generated using STATA/SE 8.2 for Linux. The data discussed in this publication have been deposited in National Center for Biotechnology Information Gene Expression Omnibus5 and are accessible through Gene Expression Omnibus Series accession no. GSE7212.


   Results
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
Discussion
 References
 
Cxcr6 mutant mice were generated as a part of the Lexicon Genome5000 program, which uses gene knockout technology to investigate the physiologic and behavioral functions of 5,000 human genes through analysis of the corresponding knockout mouse models. A targeting vector containing a LacZ-Neo cassette was used to replace exon 2 of the Cxcr6 gene via homologous recombination, eliminating >90% of the protein coding region (Fig. 1A). Southern blot (Fig. 1B) and PCR (Fig. 1C) analyses were used to confirm correct targeting of the Cxcr6 gene. Cxcr6+/– and Cxcr6–/– mice, born at the expected Mendelian ratios, were viable and fertile and did not immediately exhibit an evident phenotype. Targeting of the Cxcr6 gene was confirmed by RT-PCR using total RNA prepared from mouse spleen, which expresses high levels of Cxcr6 (25, 27). RT-PCR analyses showed that spleens from wild-type (Fig. 1D, lanes 2 and 3) and heterozygous (Fig. 1D, lanes 4 and 5) mice expressed Cxcr6 mRNA, but spleens from homozygous mice did not (Fig. 1D, lanes 6 and 7). As the targeted alleles contain a lacZ gene, we also did RT-PCR using specific primers for lacZ. Spleens from both homozygous and heterozygous mice expressed lacZ (Fig. 1D, lanes 4–7). Heterozygous mice seemed to make less Cxcr6 RNA than wild-type mice (although the assay is semiquantitative at best).

Exon 2 of Cxcr6 is located within intron 14 of Fyco1 in the mouse and human genomes in an antiparallel transcriptional orientation (34, 35). We found that wild-type Fyco1 is expressed at normal levels in the adult cerebellum of Cxcr6–/– mice using Northern blotting (Supplementary Fig. S1), RT-PCR, and DNA sequencing (data not shown) approaches. Thus, we conclude that the phenotype observed in Cxcr6 mutant mice is likely a consequence of the reduced level of Cxcr6 expression because Fyco1 is unchanged.

Previously, Unutmaz et al. (28) described mice in which Cxcr6 (Bonzo) was replaced with a gene encoding green fluorescent protein. These mice did not exhibit any obvious pathologies and, subsequently, they were used to investigate the function of CXCR6 in T-cell subsets. These studies led to the proposal that CXCR6 and its ligand CXCL16 play important roles in lung lymphocyte homing (36, 37) and liver-based immune responses (38) although the precise mechanism of action is unclear. We observed our Cxcr6 mutant mice for a period of 12 months and made the surprising observation that a small number of both heterozygous and homozygous mice develop brain tumors. This phenotype may have been missed by other investigators because the penetrance is low, the age of onset varies, and the cause of death can only be ascertained by careful examination of the brain. The tumors arose consistently in cerebellum (Fig. 2A ) and, histologically, they resembled Ptc1+/– tumors, composed of small round cells with hyperchromatic nuclei and scant cytoplasm. Both Cxcr6+/– and Cxcr6–/– mice developed brain tumors between the ages of 12 and 39 weeks, with a frequency of 4.2% and 2.9%, respectively (Table 1 ). The peak incidence occurred between 16 and 20 weeks and there was no significant difference in tumor incidence or time of onset between homozygous and heterozygous mice. We also found that all tumors examined (Cxcr6+/–, n = 1; Cxcr6–/–, n = 4) stained positive for both glial fibrillary acidic protein and synaptophysin (Fig. 2B) and they expressed Gli1 and Sfrp1 mRNA (Fig. 2C) similar to Ptc1+/– tumors (14, 39, 40). Based on the histologic appearance, location, and expression of markers, we concluded that tumors arising in Cxcr6 mutant mice were medulloblastomas.


Figure 2
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  		Figure 2. Cxcr6 mutant mice develop medulloblastoma. A, left, an example of medulloblastoma compared with normal cerebellum from Cxcr6–/– mouse. Right, high magnification of the boxed region. B, immunohistochemical studies showing that Cxcr6 mutant medulloblastoma was immunoreactive for glial fibrillary acidic protein (GFAP; left) and synaptophysin (right). C, in situ hybridization analysis showing Gli1 (left) and Sfrp1 expression (right) in Cxcr6–/– medulloblastoma.

 

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  		Table 1. Incidence of medulloblastoma in Cxcr6 mutant mice

 
To investigate the mechanisms responsible for medulloblastoma formation in the Cxcr6 mutant mice, we compared the expression levels of Shh pathway genes with those in developing (Fig. 3A, lanes 1–3 ) and adult (Fig. 3A, lanes 4–6) cerebella. The genotype of each sample was confirmed by RT-PCR analyses of both Cxcr6 and lacZ (Fig. 3B). Northern blot analysis showed that medulloblastomas in Cxcr6–/– (Fig. 3A, lane 7) and Cxcr6+/– (Fig. 3A, lanes 8 and 9) mice expressed high levels of Gli1 and Sfrp1, consistent with the results of in situ hybridization studies (Fig. 2C). The mRNA levels of Gli2, Ptc2, and Cxcr4, whose up-regulation has been reported in other mouse medulloblastomas and in a subset of human medulloblastomas (3, 4), were also increased (Fig. 3A). The levels of these genes (Gli1, Gli2, Ptc2, Sfrp1, and Cxcr4) were very similar to those observed in Ptc1+/– medulloblastoma (Fig. 3A, lane 10), indicating that the Shh pathway was activated in medulloblastomas arising in Cxcr6 mutant mice. Medulloblastomas observed in other mouse models have been reported to arise as a consequence of either direct or indirect activation of the Shh pathway leading to increased expression of downstream target genes, and one or both copies of Ptc1 were lost in some of these cases (3, 17–23, 40, 41).


Figure 3
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  		Figure 3. Expression of Shh pathway genes in developing (p7-CB) and adult cerebella (Ad-CB) and medulloblastomas (MB). A, total RNA (10 µg) was analyzed by Northern blotting using radiolabeled probes for Gli1, Gli2, Ptc1, Ptc2, Sfrp1, and Cxcr4 as indicated. Ethidium bromide staining of the 28S and 18S rRNA is shown as a loading control. The Cxcr6 genotype of each sample is indicated at the top of each lane. B, expression of Cxcr6 (top) and lacZ (middle) was analyzed by RT-PCR using the same RNA samples as in (A). Gapdh was used as an internal control (bottom). C, paraffin sections of medulloblastomas were subjected to FISH analyses using probes for Ptc1 (green) and mouse chromosome 13 (red). A representative example of a FISH image in which both Ptc1 alleles are present in Cxcr6+/– medulloblastoma.

 
We also found that the mRNA levels of Ptc1 were decreased in all medulloblastomas tested from Cxcr6 mutant mice (Fig. 3A, lanes 7–9). In fact, the level of Ptc1 expression in these tumors was comparable to that previously seen in tumors from Ptc1+/– mice (Fig. 3A, lane 10; ref. 30). This finding raised the possibility that one of the Ptc1 alleles may be lost or mutated in Cxcr6 mutant medulloblastomas. However, FISH analysis showed that both Ptc1 alleles were present in medulloblastomas from Cxcr6 mutant mice (Fig. 3C). Furthermore, RT-PCR and cDNA sequencing analyses failed to reveal any mutations in Ptc1 or Sufu in the four tumors examined (Cxcr6+/–, n = 3; Cxcr6–/–, n = 1), suggesting that, in Cxcr6 mutant mice, aberrant activation of the Shh pathway occurs indirectly by down-regulation of Ptc1 expression.

Gene expression profiles of medulloblastomas arising in Cxcr6+/– mice (n = 2) were compared with those of Ptc1+/– medulloblastomas (n = 2). We tabulated the number of probe sets scored as up-regulated, down-regulated, and unchanged, comparing each type of medulloblastoma to genes expressed in developing cerebellum (see Experimental Procedures for details). To exclude potential chip errors, we eliminated probes whose expression levels were not consistent between the two independent samples of each tumor. Specifically, 10,244 probes met the criteria described above. We found that the gene expression profiles of medulloblastomas derived from these two mouse models were very similar, with expression levels of 1,080 probes up-regulated, 1,453 down-regulated, and 7,006 unchanged, comparing both types of medulloblastomas to developing cerebellum (Table 2 ). Furthermore, expression levels of the Shh pathway–related genes were essentially the same between Cxcr6+/– and Ptc1+/– medulloblastomas (Supplementary Table S1), which is consistent with Northern blot analyses (Fig. 3A). On the other hand, 6.9% (705 of 10,244) of probes were differentially expressed (Table 2), and Supplementary Table S2 shows the probes whose expression levels were different between two types of medulloblastomas (log ratio >3). It is possible that some of the listed genes may contribute to the aberrant activation of the Shh pathway and development of medulloblastomas in Cxcr6 mutant mice.


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  		Table 2. Comparison of gene expression profiles between Ptc1+/– and Cxcr6+/– medulloblastomas

 
Microarray gene expression analysis provides a useful means to identify tumors containing specific gene mutations that are amendable to molecular targeted therapies. Because gene expression profiles, including the activation of the Shh pathway, are very similar between Cxcr6+/– and Ptc1+/– medulloblastomas, it is a reasonable assumption that the medulloblastomas derived from Cxcr6 mutant mice might respond to treatment with Shh pathway inhibitors. Previously, we showed that medulloblastomas arising in Ptcl+/–p53–/– mice responded to treatment with the Shh pathway inhibitor (HhAntag; ref. 9). However, as the tumor incidence in Cxcr6 mutant mice is very low and loss of p53 did not accelerate medulloblastoma formation (data not shown), it is not feasible to treat spontaneous tumors arising in Cxcr6 mice with the inhibitor. Therefore, we propagated the Cxcr6+/– medulloblastoma cells in the flank of nude mice. Previously, we reported that directly transplanted medulloblastomas from Ptc1+/–p53–/– mice closely resemble the parental tumors, both histologically and in terms of gene expression profiles (10). As a first step, we confirmed that the Shh pathway activity was maintained in the flank allograft (directly transplanted from fresh medulloblastoma) by testing the expression levels of Gli1 and Sfrp1 (Fig. 4B, lanes 4 and 5 ). In contrast, the Shh pathway activity could not be maintained in medulloblastoma culture (Fig. 4B, lane 3), indicating that cultured medulloblastoma cells are not suitable for testing the HhAntag, consistent with our previous studies (9, 10). To evaluate the effect of HhAntag on medulloblastomas derived from Cxcr6 mutant mice, cohorts of mice bearing tumor allografts were treated with 100 mg kg–1 HhAntag twice daily. We found that the mRNA levels of Gli1 and Sfrp1 in allografts were clearly reduced after treatment with eight doses of 100 mg kg–1 HhAntag (Fig. 4A, lanes 6 and 7). After treatment, the transplanted tumors rapidly lost volume (Fig. 4A), indicating that the Shh pathway inhibitor was very effective in this model of medulloblastoma.


Figure 4
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  		Figure 4. Effect of Smoothened antagonist (HhAntag) on the allografts derived from Cxcr6+/– medulloblastoma. A, cohorts of mice bearing flank allografts were treated with vehicle alone (n = 2) or with 100 mg kg–1 HhAntag (n = 4) twice a day for 4 d by oral gavage. The tumor volume was measured every other day. B, total RNA (10 µg) isolated from different sources, as indicated above the lanes, was analyzed by Northern blotting with antisense probes specific for Gli1, Sfrp1, and Gapdh as indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
 Discussion
References
 
Over the past several years, there has been increasing hope that molecular targeted therapies will prove efficacious in the treatment of specific cancers. However, one of the greatest challenges facing the field is to identify the specific tumor subsets that are capable of responding to the treatment. Previously, we tested a small-molecule inhibitor of the Shh pathway (HhAntag) in a spontaneous mouse model of medulloblastoma (9). These mice carried germ line mutations in the Ptc1 and p53 genes and they responded dramatically to treatment with HhAntag. In addition, we found that allograft tumors, derived directly from Ptc1+/–p53–/– mice but not tumor cell lines, were completely eliminated after a few oral doses of the drug (10). Although promising, these studies may only be specifically relevant for the 10% of human tumors that carry PTCH1 mutations. However, ~30% of human medulloblastomas exhibit a gene expression signature consistent with Shh pathway activation (3, 4). This raises the possibility that HhAntag may have broader application than just the tumor subset associated with heterozygous loss of Ptc1 or other known mutations in the Shh pathway. Here, we show that mice with heterozygous or homozygous mutations in Cxcr6 develop a low frequency of medulloblastomas. Remarkably, these tumors show high levels of expression of Shh pathway target genes but they do not harbor mutations in Ptc1 or Sufu. Although the Ptc1 locus is intact in the tumors, the levels of Ptc1 mRNA are quite low. Thus, we speculate that impartial or complete loss of Cxcr6 function leads indirectly to suppression of Ptc1 expression thereby activating the Shh pathway and promoting medulloblastoma formation. Tumors from these mice provide a model for testing the usefulness of HhAntag against medulloblastomas lacking known mutations in the Shh pathway.

The role of CXCR6 in human medulloblastoma is unclear at present. Microarray and semiquantitative RT-PCR analyses revealed that mRNA levels of Cxcl16, which encodes a ligand for CXCR6 (42), are elevated in medulloblastoma arising in both Ptc1+/– and Cxcr6 mutant mice, compared with normal developing cerebellum (data not shown). Because chemokines and their receptors mediate signals critical for the recruitment of effector immune cells to the site of inflammation, it is possible that loss of such receptors provides a selective advantage to the tumor cells. Indeed, the deletion of chromosome 3p21.3, which contains a large cluster of chemokine receptors including the CXCR6 gene (35), has been observed in several human cancers (43, 44). Thus, it is possible that some human medulloblastomas might harbor deletions in the chromosome 3p21.3 region.

Surprisingly, we were unable to accelerate development of medulloblastoma in Cxcr6 mutant mice by crossing onto a p53 null background. This was unexpected as loss of p53 dramatically accelerated medulloblastoma formation in Ptc1+/– mice (5). In addition, several other mouse models with homozygous loss of p53 have been reported, including p53–/–Rb–/– (17), Lig4–/–p53–/– (18), Ink4c–/–p53–/– (19), and Parp1–/–p53–/– mice (20). Although genetic instability resulting from inactivation of the p53 pathway contributes to neoplastic transformation of granule cell precursors (5, 41), the precise molecular mechanism is unclear. It is possible that loss of Cxcr6 also promotes medulloblastoma formation in a similar manner to loss of p53 and, therefore, tumorigenesis is not further accelerated by loss of p53.

The low tumor frequency in Cxcr6 mutant mice precluded drug studies in the spontaneous tumor background. However, using directly transplanted medulloblastoma cells, we were able to show that medulloblastoma cells harboring Cxcr6 mutations responded well to treatment with HhAntag. The Shh pathway remained active and gene expression profiles were maintained in medulloblastoma cells directly transplanted from tumors. In contrast, cultured medulloblastoma cells from Cxcr6 mutant mice did not maintain Shh pathway activity, so they could not be used for HhAntag studies. This observation is in agreement with our previous reports indicating that medulloblastoma cells, grown under standard serum-containing culture conditions, lose Shh pathway activity (9, 10). In the case of glioblastoma, Lee et al. (45) recently reported that cultured tumor cells in serum-containing media are very different, both genetically and biologically, from the parental tumors. These studies question the usefulness of cultured tumor cells for testing antitumor agents. We propose that directly transplanted tumors, derived from genetically engineered mouse models, offer a convenient system for target validation as well as a convenient model system for testing new therapies targeted against the molecular pathways that contribute to tumor formation and progression.


   Acknowledgments
 
Grant support: Accelerate Brain Cancer Cure, the Pediatric Brain Tumor Foundation, NIH grants PO1-CA096832 and P30-CA21765, and the American Lebanese and Syrian Association Charities.

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 Peter J. McKinnon and Richard J. Gilbertson for helpful discussions; David Finkelstein for cDNA microarray analysis; Marc Valentine and Virginia Valentine for FISH analysis; and Curis and Genentech for providing HhAntag.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

5 http://www.ncbi.nlm.nih.gov/geo/ Back

Received 2/ 6/07. Revised 3/ 8/07. Accepted 3/ 9/07.


   References
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
 Discussion
 References
 

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X. Fan and C. G. Eberhart
Medulloblastoma Stem Cells
J. Clin. Oncol., June 10, 2008; 26(17): 2821 - 2827.
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