Inhibition of WNT signaling attenuates self-renewal of SHH-subgroup medulloblastoma.

The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.

Alternative splicing in cancer: implications for biology and therapy.

Alternative splicing has critical roles in normal development and can promote growth and survival in cancer. Aberrant splicing, the production of noncanonical and cancer-specific mRNA transcripts, can lead to loss-of-function in tumor suppressors or activation of oncogenes and cancer pathways. Emerging data suggest that aberrant splicing products and loss of canonically spliced variants correlate with stage and progression in malignancy. Here, we review the splicing landscape of TP53, BARD1 and AR to illuminate roles for alternative splicing in cancer. We also examine the intersection between alternative splicing pathways and novel therapeutic approaches.

Myc proteins as therapeutic targets.

Myc proteins (c-myc, Mycn and Mycl) target proliferative and apoptotic pathways vital for progression in cancer. Amplification of the MYCN gene has emerged as one of the clearest indicators of aggressive and chemotherapy-refractory disease in children with neuroblastoma, the most common extracranial solid tumor of childhood. Phosphorylation and ubiquitin-mediated modulation of Myc protein influence stability and represent potential targets for therapeutic intervention. Phosphorylation of Myc proteins is controlled in-part by the receptor tyrosine kinase/phosphatidylinositol 3-kinase/Akt/mTOR signaling, with additional contributions from Aurora A kinase. Myc proteins regulate apoptosis in part through interactions with the p53/Mdm2/Arf signaling pathway. Mutation in p53 is commonly observed in patients with relapsed neuroblastoma, contributing to both biology and therapeutic resistance. This review examines Myc function and regulation in neuroblastoma, and discusses emerging therapies that target Mycn.

Cyclic GMP-dependent protein kinase II inhibits cell proliferation, Sox9 expression and Akt phosphorylation in human glioma cell lines.

Earlier we used a glioma model to identify loci in the mouse genome, which were repeatedly targeted by platelet-derived growth factor (PDGF)-containing Moloney murine leukemia viruses. The gene Prkg2, encoding cyclic guanosine monophosphate (cGMP)-dependent protein kinase II, cGKII, was tagged by retroviral insertions in two brain tumors. The insertions were both situated upstream of the kinase domain and suggested creating a truncated form of the cGKII protein. We transfected different human glioma cell lines with Prkg2 and found an overall reduction in colony formation and cell proliferation compared with controls transfected with truncated Prkg2 (lacking the kinase domain) or empty vector. All glioma cells transfected with the cGKII phosphorylate vasodilator-stimulated phosphoprotein, VASP, after cGMP analog treatment. Glioma cell lines positive for the Sox9 transcription factor showed reduced Sox9 expression when Prkg2 was stably transfected. When cGKII was activated by cGMP analog treatment, Sox9 was phosphorylated, Sox9 protein expression was suppressed and the glioma cell lines displayed loss of cell adhesion, inhibition of Akt phosphorylation and G1 arrest. Sox9 repression by siRNA was similarly shown to reduce glioma cell proliferation. Expression analysis of stem and glial lineage cell markers also suggests that cGKII induces differentiation of glioma cell lines. These findings describe an anti-proliferative role of cGKII in human glioma biology and would further explain the retroviral tagging of the cGKII gene during brain tumor formation in PDGF-induced tumors.

Genetics of brain tumors.

Brain tumors are among the most common forms of cancer in children and account for most cancer-related deaths in this age group. The incidence of brain tumors appears to be increasing in children, while therapeutic advances have been modest. Few genetic studies exist on pediatric brain tumors, in part because tissue from low-grade and brain stem tumors is not readily available, and also because individual centers have relatively few cases. Genetic changes in infiltrating astrocytomas involve genes in the p53 and RB pathways, and show alterations that are similar to infiltrating astrocytomas in adults. The PTC gene is mutated in a subgroup of medulloblastomas, and may lead to increased proliferation in granule cells that normally express this receptor. Further studies are needed to identify genetic alterations in pilocytic and low-grade astrocytomas, which account for 40% of brain tumors in children.

Expression of N-myc and MRP genes and their relationship to N-myc gene dosage and tumor formation in a murine neuroblastoma model.

BACKGROUND: Although the association between N-myc gene amplification and poor clinical outcome in neuroblastoma is well established, the mechanism by which amplification influences prognosis is not well defined. PROCEDURE: We used a human N-myc transgenic mouse model to investigate the role of N-myc in neuroblastoma, including its relationship to the multidrug-resistance-associated protein (MRP) gene. We developed a rapid real-time PCR method to distinguish homozygous and hemizygous N-myc mice that is comparable to Southern analysis. RESULTS: A highly significant correlation (P < 0.0001) between N-myc and MRP expression was demonstrated in murine tumors. Amplification of the transgene was observed in the majority of tumors, highlighting the clinical relevance of this model. However, no correlation between N-myc expression and transgene dosage or tumor latency was observed. CONCLUSIONS: The data suggest that increased N-myc dosage contributes to increased tumor incidence and decreased latency by mechanisms independent of N-myc expression.

Genome-wide screen for allelic imbalance in a mouse model for neuroblastoma.

We have used the rat tyrosine hydroxylase promotor to overexpress MYCN in the neural crest of transgenic mice, resulting in a mouse model for neuroblastoma. Using PCR analysis of microsatellite markers, we conducted a genome-wide analysis in tumors from these animals. Regions of chromosomes 1, 3, 10, 11, 14, and 18 were affected in 20-50% of tumors. Analysis of a subset of these tumors by comparative genomic hybridization was consistent with the microsatellite data. The changes on mouse chromosomes 1, 11, 14, and 18 were syntenic with corresponding regions of loss of heterozygosity in human neuroblastoma, suggesting that genes implicated in the mouse tumors may also play a role in the pathogenesis of the human disease. One-third of the mouse tumors shared abnormalities on chromosomes 1, 3, and 10, whereas the remainder of tumors did not show this combination. These data suggest that genetic mutations on chromosomes 1, 3, and 10 cooperate in the pathogenesis of neuroblastoma and that neuroblastoma in the mouse arises from at least two distinct genetic pathways, one of which is dependent on lesions in chromosomes 1, 3, and 10, the other of which is not.

Targeted expression of MYCN causes neuroblastoma in transgenic mice.

The proto-oncogene MYCN is often amplified in human neuroblastomas. The assumption that the amplification contributes to tumorigenesis has never been tested directly. We have created transgenic mice that overexpress MYCN in neuroectodermal cells and develop neuroblastoma. Analysis of tumors by comparative genomic hybridization revealed gains and losses of at least seven chromosomal regions, all of which are syntenic with comparable abnormalities detected in human neuroblastomas. In addition, we have shown that increases in MYCN dosage or deficiencies in either of the tumor suppressor genes NF1 or RB1 can augment tumorigenesis by the transgene. Our results provide direct evidence that MYCN can contribute to the genesis of neuroblastoma, suggest that the genetic events involved in the genesis of neuroblastoma can be tumorigenic in more than one chronological sequence, and offer a model for further study of the pathogenesis and therapy of neuroblastoma.

Characterization of the RAD10 gene of Saccharomyces cerevisiae and purification of Rad10 protein.

The RAD10 gene of Saccharomyces cerevisiae is one of at least five genes required for damage-specific incision of DNA during nucleotide excision repair. This gene was previously cloned and sequenced [Weiss, W. A., & Friedberg, E. C. (1985) EMBO J. 4, 1575-1582; Reynolds et al. (1985) EMBO J. 4, 3549-3552]. In the present studies, we have mapped one major and three minor transcriptional start sites in the RAD10 gene. The locations of these sites relative to the translational start codon are remarkably similar to those previously identified in the yeast RAD2 gene [Nicolet et al. (1985) Gene 36, 225-234]. The two genes also share common sequences in these regions. However, in contrast to RAD2 [Robinson et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1842-1846], RAD10 is not induced following exposure of cells to the DNA-damaging agent 4-nitroquinoline 1-oxide. Native RAD10 protein and also two different Rad10 fusion proteins are rapidly degraded in most Escherichia coli strains. However, following overexpression of the cloned RAD10 gene in yeast, native Rad10 protein was purified to greater than 90% homogeneity. A catalytic function has not been identified for the purified protein. RAD10 cells (untransformed with the cloned gene) contain fewer than 500 molecules per cell. This is similar to the levels of the UvrA, UvrB, and UvrC nucleotide excision repair proteins in E. coli.

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.

Physiological levels of normal tRNA(CAGGln) can effect partial suppression of amber mutations in the yeast Saccharomyces cerevisiae.

A number of ciliated protozoa are known to read the stop codons UAA and UAG as sense codons that specify glutamine during protein synthesis. In considering evolutionary mechanisms for this curious divergence from the standard genetic code, we propose the existence of progenitor tRNAs for glutamine that can weakly suppress UAA and UAG codons. It has been previously shown that multicopy plasmids that overexpress normal tRNA(CAAGln) and tRNA(CAGGln) genes from the yeast Saccharomyces cerevisiae can partially suppress a number of yeast ochre and amber mutations, respectively. In the present study we show that the tRNA(CAGGln) gene can also function as a weak amber suppressor when expressed in cells at physiological levels. This observation is consistent with a role of tRNA(CAGGln) as an evolutionary progenitor of tRNAs that strongly decode UAG codons.

Normal yeast tRNA(CAGGln) can suppress amber codons and is encoded by an essential gene.

We have isolated a gene that can encode yeast tRNA(CAGGln). When present on a multicopy plasmid, this gene suppresses the phenotype of a number of amber mutants, but has no effect on the ocher mutants tested. We therefore conclude that the anticodon CUG in tRNA(CAGGln) can decode the amber codon UAG by G-U mispairing, possibly by wobble base-pairing in the first codon position. This represents the second example we have observed in this laboratory of nonsense suppression in yeast by natural tRNA(Gln), involving G-U mispairing in the first codon position. Replacing the genomic copy of the cloned gene with a disrupted tRNA gene results in recessive lethality in heterozygous diploids and is lethal to haploid cells. This lethality can be rescued by transformation of cells with a single copy plasmid containing the tRNA(CAGGln) gene. Thus, the gene encoding tRNA(CAGGln) is apparently essential for viability in yeast, suggesting that it is normally present as a single copy gene.

Molecular approaches to the study of nucleotide excision repair in eukaryotes.

Very little is known about the molecular mechanism of nucleotide excision repair in eukaryotes. Studies on human cells have been stimulated by the availability of excision repair-defective cell lines from patients suffering from the autosomal recessive disease xeroderma pigmentosum (XP). Such studies have contributed significantly to an understanding of the genetic complexity of excision repair in human cells. However, to date, no human excision repair genes or gene products known to complement the repair defect in XP cells have been isolated. The yeast Saccharomyces cerevisiae is an interesting model for exploring the molecular mechanism of nucleotide excision repair in eukaryotic cells. As is true in human cells, multiple yeast genes are involved and at least five genes are required for the specific incision of UV-irradiated DNA in vivo. These five genes have been isolated by molecular cloning and the nucleotide sequences of four of them have been determined. Each of these cloned genes is being used for overexpression of protein.

Nucleotide excision repair genes from the yeast Saccharomyces cerevisiae.

The genetics of nucleotide excision repair in the yeast Saccharomyces cerevisiae is complex, apparently requiring at least 10 genes. We have isolated 5 of these genes (designated RAD1, RAD2, RAD3, RAD4, and RAD10) by molecular cloning and plan to overexpress them in order to generate proteins for biochemical study. We have sequenced four of these five genes and have noted regions of homology with other proteins in the predicted amino acid sequence of some of them. In particular, there is striking homology between Rad3 protein and a number of prokaryotic and eukaryotic proteins that bind nucleotides and hydrolyze ATP or GTP. Mutations in this region of the RAD3 gene render cells defective in the nucleotide excision repair function. In addition to its role in nucleotide excision repair, the RAD3 gene is essential for the viability of haploid cells in the absence of DNA damage. The nature of the essential function is unknown. The RAD1 and RAD3 genes are not inducible by DNA damaging agents. However, exposure of cells to UV radiation, 4-nitroquinoline 1-oxide, or gamma radiation results in 4- to 6-fold enhanced expression of the RAD2 gene.

Molecular cloning and characterization of the yeast RAD10 gene and expression of RAD10 protein in E. coli.

A plasmid designated pNF101 was isolated by transforming rad10 mutants with a yeast genomic library and screening transformed cells for enhanced resistance to killing by u.v. radiation. Plasmid pNF101 fully complements the u.v. sensitivity of rad10 mutant strains and was shown to contain the RAD10 gene by genetic analysis of integrant strains. The nucleotide sequence of the RAD10 gene was determined. The coding region consists of 195 codons and could encode a polypeptide of calculated mol. wt. 22 616 daltons. RAD10 protein expressed in Escherichia coli maxicells has a mol. wt of approximately 30 kd measured by gel electrophoresis. The RAD10 gene was localized to chromosome XIII of Saccharomyces cerevisiae by hybridization of the cloned gene to yeast chromosomes resolved by electrophoresis, and by genetic analysis.

Lung cancer type in relation to cigarette dosage.

In a retrospective study of 1,228 white males with histologically confirmed bronchogenic carcinoma, the proportion of squamous cell carcinoma increased with increasing age at diagnosis. Since the distribution of cell types was much the same in the 73% of cases aged 50-69, the relationship of cancer type to daily cigarette dosage was studied in this group. Squamous cell carcinoma increased from 48% of those men who smoked less than 20 cigarettes per day to 61% of those who smoked 40 or more cigarettes per day. Comparison with other studies showed conflicting results.