Raelene Endersby

Cooperativity within and among Pten, p53, and Rb Pathways Induces High-Grade Astrocytoma in Adult Brain

Mutations in the PTEN, TP53, and RB1 pathways are obligate events in the pathogenesis of human glioblastomas. We induced various combinations of deletions in these tumor suppressors in astrocytes and neural precursors in mature mice, resulting in astrocytomas ranging from grade III to grade IV (glioblastoma). There was selection for mutation of multiple genes within a pathway, shown by somatic amplifications of genes in the PI3K or Rb pathway in tumors in which Pten or Rb deletion was an initiating event. Despite multiple mutations within PI3K and Rb pathways, elevated Mapk activation was not consistent. Gene expression profiling revealed striking similarities to subclasses of human diffuse astrocytoma. Astrocytomas were found within and outside of proliferative niches in the adult brain.

PTEN signaling in brain: neuropathology and tumorigenesis.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor that antagonizes the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway by functioning as a lipid phosphatase. This ubiquitous and evolutionarily conserved signaling cascade influences numerous functions including cell growth, survival, proliferation, migration and metabolism. Inherited mutations in PTEN cause pleiotropic effects including cancer predisposition as well as a range of neurological abnormalities revealing specialized roles for PTEN in nervous system development and maintenance. Somatic mutations in PTEN occur frequently as late events in sporadic brain tumors. Mouse models based on Pten deletion in the brain have provided insights into the normal functions of Pten in the nervous system as well as the initiation and progression of gliomas. Compromised PTEN function may contribute to gliomagenesis through disrupted regulation of proliferation, migration, invasion, angiogenesis, stem cell self-renewal and regulation of other tumor suppressor pathways such as p53. Clinical findings in high-grade glioma suggest that PTEN gene alterations are associated with poor prognosis and may influence response to specific therapies. Emerging research using specific pharmacological inhibitors of the PI3K pathway may provide novel therapeutic options for the treatment of PTEN-deficient tumors.

Novel Oncogenic PDGFRA Mutations in Pediatric High-Grade Gliomas

The outcome for children with high-grade gliomas (HGG) remains dismal, with a 2-year survival rate of only 10% to 30%. Diffuse intrinsic pontine glioma (DIPG) comprise a subset of HGG that arise in the brainstem almost exclusively in children. Genome-wide analyses of copy number imbalances previously showed that platelet-derived growth factor receptor α (PDGFRA) is the most frequent target of focal amplification in pediatric HGGs, including DIPGs. To determine whether PDGFRA is also targeted by more subtle mutations missed by copy number analysis, we sequenced all PDGFRA coding exons from a cohort of pediatric HGGs. Somatic-activating mutations were identified in 14.4% (13 of 90) of nonbrainstem pediatric HGGs and 4.7% (2 of 43) of DIPGs, including missense mutations and in-frame deletions and insertions not previously described. Forty percent of tumors with mutation showed concurrent amplification, whereas 60% carried heterozygous mutations. Six different mutations impacting different domains all resulted in ligand-independent receptor activation that was blocked by small molecule inhibitors of PDGFR. Expression of mutants in p53-null primary mouse astrocytes conferred a proliferative advantage in vitro and generated HGGs in vivo with complete penetrance when implanted into brain. The gene expression signatures of these murine HGGs reflected the spectrum of human diffuse HGGs. PDGFRA intragenic deletion of exons 8 and 9 were previously shown in adult HGG, but were not detected in 83 nonbrainstem pediatric HGG and 57 DIPGs. Thus, a distinct spectrum of mutations confers constitutive receptor activation and oncogenic activity to PDGFRα in childhood HGG.

The Tumor Suppressor PTEN Is Necessary for Human Sprouty 2-mediated Inhibition of Cell Proliferation

Sprouty family proteins are novel regulators of growth factor actions. Human Sprouty 2 (hSPRY2) inhibits the proliferation of a number of different cell types. However, the mechanisms involved in the anti-proliferative actions of hSPRY2 remain to be elucidated. Here we have demonstrated that hSPRY2 increases the amount of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and decreases its phosphorylation. The resultant increase in PTEN activity is reflected in decreased activation of Akt by epidermal growth factor and serum. Consistent with increased PTEN activity, in hSPRY2-expressing cells, the progression of cells from the G1 to S phase is decreased. By using PTEN null primary mouse embryonic fibroblasts and their isogenic controls as well as small interfering RNA against PTEN, we demonstrated that PTEN is necessary for hSPRY2 to inhibit Akt activation by epidermal growth factor as well as cell proliferation. Overall, we concluded that hSPRY2 mediates its anti-proliferative actions by altering PTEN content and activity.

Phase I and Pharmacokinetic Studies of Erlotinib Administered Concurrently with Radiotherapy for Children, Adolescents, and Young Adults with High-Grade Glioma

Purpose: To estimate the maximum-tolerated dose (MTD) of erlotinib administered during and after radiotherapy, and to describe the pharmacokinetics of erlotinib and its metabolite OSI-420 in patients between 3 and 25 years with newly diagnosed high-grade glioma who did not require enzyme-inducing anticonvulsants. Experimental Design: Five dosage levels (70, 90, 120, 160, and 200 mg/m2 per day) were planned in this phase I study. Dose-limiting toxicities (DLT) were evaluated during first 8 weeks of therapy. Local radiotherapy (dose between 54 and 59.4 Gy) and erlotinib started preferentially on the same day. Erlotinib was administered once daily for a maximum of 3 years. Pharmacokinetic studies were obtained after first dose and on day 8 of therapy. Mutational analysis of EGFR kinase domain, PIK3CA, and PTEN was done in tumor tissue. Results: Median age at diagnosis of 23 patients was 10.7 years (range, 3.7-22.5 years). MTD of erlotinib was 120 mg/m2 per day. Skin rash and diarrhea were generally well controlled with supportive care. Dose-limiting toxicities were diarrhea (n = 1), increase in serum lipase (n = 1), and rash with pruritus (n = 1). The pharmacokinetic variables of erlotinib and OSI-420 in children were similar to those described in adults. However, there was no relationship between erlotinib dosage and drug exposure. No EGFR kinase domain mutations were observed. Two patients with glioblastoma harbored mutations in PIK3CA (n = 1) or PTEN (n = 1). Conclusions: Although the MTD of erlotinib in children with newly diagnosed high-grade glioma was 120 mg/m2 per day, pharmacokinetic studies showed wide interpatient variability in drug exposure.

The PTEN/Akt Pathway Dictates the Direct αVβ3-Dependent Growth-Inhibitory Action of an Active Fragment of Tumstatin in Glioma Cells In vitro and In vivo

The collagen type IV cleavage fragment tumstatin and its active subfragments bind to integrin alpha(V)beta(3) and inhibit activation of focal adhesion kinase, phophoinositol-3 kinase, Akt, and mammalian target of rapamycin (mTOR) in what is thought to be an endothelial cell-specific manner. The resultant endothelial cell apoptosis accounts for the ability of tumstatin to function as an endogenous inhibitor of angiogenesis and an indirect suppressor of tumor growth. We hypothesized that the inability of tumstatin to directly suppress tumor cell growth might be the result of the constitutive activation of the Akt/mTOR pathway commonly seen in tumors. Consistent with this idea, several integrin alpha(V)beta(3)-expressing glioma cell lines with PTEN mutations and high levels of phospho-Akt (pAkt) were unaffected by exposure to an active fragment of tumstatin (T3), whereas alpha(V)beta(3)-expressing glioma cell lines with a functional PTEN/low levels of pAkt exhibited T3-induced growth suppression that could be bypassed by small interfering RNA-mediated suppression of PTEN, introduction of a constitutively expressed Akt, or introduction of the Akt and mTOR target eukaryotic translation initiation factor 4E. The direct tumor-suppressive actions of T3 were further shown in an alpha(V)beta(3)-deficient in vivo mouse model in which T3, while unable to alter the tumstatin-insensitive vasculature contributed by the alpha(V)beta(3)-deficient host, nonetheless suppressed the growth and proliferative index of i.c. implanted alpha(V)beta(3)-expressing PTEN-proficient glioma cells. These results show that tumstatin, previously considered to be only an endogenous inhibitor of angiogenesis, also directly inhibits the growth of tumors in a manner dependent on Akt/mTOR activation.

Nonredundant Functions for Akt Isoforms in Astrocyte Growth and Gliomagenesis in an Orthotopic Transplantation Model

The AKT family, comprising three highly homologous kinases, is an essential mediator of the PTEN/PI3K pathway, which is deregulated in many human cancers. A thorough understanding of the specific activities of each isoform in normal and disease tissues is lacking. We evaluated the role of each Akt isoform in gliomagenesis by using a model system driven by common glioma abnormalities, loss of function of p53 and Pten, and expression of EGFRvIII. Both Pten deletion and EGFRvIII expression accelerated the proliferation of p53-null primary murine astrocytes. All three Akt isoforms were expressed and phosphorylated in astrocytes, with significantly higher activation in Pten-null cells. Despite substantial compensation in many contexts when individual Akt isoforms were inhibited, isoform-specific effects were also identified. Specifically, loss of Akt1 or Akt2 decreased proliferation of Pten wild-type astrocytes, whereas combined loss of multiple isoforms was needed to inhibit proliferation of Pten-null astrocytes. In addition, Akt3 was required for anchorage-independent growth of transformed astrocytes and human glioma cells, and Akt3 loss inhibited invasion of transformed astrocytes. EGFRvIII expression transformed p53-null astrocytes with or without Pten deletion, causing rapid development of high-grade astrocytoma on intracranial transplantation. Furthermore, tumorigenesis of Pten;p53-null astrocytes expressing EGFRvIII was delayed by Akt1 loss and accelerated by Akt2 loss. Taken together, these results indicate context-dependent roles for individual Akt isoforms and suggest that there may be heterogeneous tumor response to isoform-specific inhibitors.

Ultraviolet Radiation Suppresses Obesity and Symptoms of Metabolic Syndrome Independently of Vitamin D in Mice Fed a High-Fat Diet

The role of vitamin D in curtailing the development of obesity and comorbidities such as the metabolic syndrome (MetS) and type 2 diabetes has received much attention recently. However, clinical trials have failed to conclusively demonstrate the benefits of vitamin D supplementation. In most studies, serum 25-hydroxyvitamin D [25(OH)D] decreases with increasing BMI above normal weight. These low 25(OH)D levels may also be a proxy for reduced exposure to sunlight-derived ultraviolet radiation (UVR). Here we investigate whether UVR and/or vitamin D supplementation modifies the development of obesity and type 2 diabetes in a murine model of obesity. Long-term suberythemal and erythemal UVR significantly suppressed weight gain, glucose intolerance, insulin resistance, nonalcoholic fatty liver disease measures; and serum levels of fasting insulin, glucose, and cholesterol in C57BL/6 male mice fed a high-fat diet. However, many of the benefits of UVR were not reproduced by vitamin D supplementation. In further mechanistic studies, skin induction of the UVR-induced mediator nitric oxide (NO) reproduced many of the effects of UVR. These studies suggest that UVR (sunlight exposure) may be an effective means of suppressing the development of obesity and MetS, through mechanisms that are independent of vitamin D but dependent on other UVR-induced mediators such as NO.

Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule

Myeloid leukemia factor 1 (MLF1) is an oncoprotein associated with hemopoietic lineage commitment and acute myeloid leukemia. Here we show that Mlf1 associated with a novel binding partner, Mlf1-associated nuclear protein (Manp), a new heterogenous nuclear ribonucleoprotein (hnRNP) family member, related to hnRNP-U. Manp localized exclusively in the nucleus and could redirect Mlf1 from the cytoplasm into the nucleus. The nuclear content of Mlf1 was also regulated by 14-3-3 binding to a canonical 14-3-3 binding motif within the N terminus of Mlf1. Significantly Mlf1 contains a functional nuclear export signal and localized primarily to the nuclei of hemopoietic cells. Mlf1 was capable of binding DNA, and microarray analysis revealed that it affected the expression of several genes, including transcription factors. In summary, this study reveals that Mlf1 translocates between nucleus and cytoplasm, associates with a novel hnRNP, and influences gene expression.

Comparative drug screening in NUT midline carcinoma

Background:The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed.Methods:On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts.Results:In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)–NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease.Conclusions:These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease.