A novel preclinical model of craniospinal irradiation in pediatric diffuse midline glioma demonstrates decreased metastatic disease.

Background: Diffuse midline glioma (DMG) is an aggressive pediatric central nervous system tumor with strong metastatic potential. As localized treatment of the primary tumor improves, metastatic disease is becoming a more important factor in treatment. We hypothesized that we could model craniospinal irradiation (CSI) through a DMG patient-derived xenograft (PDX) model and that CSI would limit metastatic tumor. Methods: We used a BT245 murine orthotopic DMG PDX model for this work. We developed a protocol and specialized platform to deliver craniospinal irradiation (CSI) (4 Gy x2 days) with a pontine boost (4 Gy x2 days) and compared metastatic disease by pathology, bioluminescence, and MRI to mice treated with focal radiation only (4 Gy x4 days) or no radiation. Results: Mice receiving CSI plus boost showed minimal spinal and brain leptomeningeal metastatic disease by bioluminescence, MRI, and pathology compared to mice receiving radiation to the pons only or no radiation. Conclusion: In a DMG PDX model, CSI+boost minimizes tumor dissemination compared to focal radiation. By expanding effective DMG treatment to the entire neuraxis, CSI has potential as a key component to combination, multimodality treatment for DMG designed to achieve long-term survival once novel therapies definitively demonstrate improved local control.

Novel RAF Fusions in Pediatric Low-Grade Gliomas Demonstrate MAPK Pathway Activation.

Brain tumors are the most common solid tumor in children, and low-grade gliomas (LGGs) are the most common childhood brain tumor. Here, we report on 3 patients with LGG harboring previously unreported or rarely reported RAF fusions: FYCO1-RAF1, CTTNBP2-BRAF, and SLC44A1-BRAF. We hypothesized that these tumors would show molecular similarity to the canonical KIAA1549-BRAF fusion that is the most widely seen alteration in pilocytic astrocytoma (PA), the most common pediatric LGG variant, and that this similarity would include mitogen-activated protein kinase (MAPK) pathway activation. To test our hypothesis, we utilized immunofluorescent imaging and RNA-sequencing in normal brain, KIAA1549-BRAF-harboring tumors, and our 3 tumors with novel fusions. We performed immunofluorescent staining of ERK and phosphorylated ERK (p-ERK), identifying increased p-ERK expression in KIAA1549-BRAF fused PA and the novel fusion samples, indicative of MAPK pathway activation. Geneset enrichment analysis further confirmed upregulated downstream MAPK activation. These results suggest that MAPK activation is the oncogenic mechanism in noncanonical RAF fusion-driven LGG. Similarity in the oncogenic mechanism suggests that LGGs with noncanonical RAF fusions are likely to respond to MEK inhibitors.

Mammalian Ste20-like kinase 4 promotes pituitary cell proliferation and survival under hypoxia.

The genetic and molecular mechanisms that initiate and maintain pituitary tumorigenesis are poorly understood. Nonfunctioning tumors of the gonadotrope lineage represent 35% of all tumors; are usually macroadenomas, often resulting in hypopituitarism; and have no medical treatments. Using expression microarrays combined with whole-genome copy number screens on individual human tumors, we identified the mammalian sterile-20-like kinase (MST4) transcript, which was amplified within chromosome Xq26.2 in one tumor and up-regulated in all gonadotrope tumor samples. MST4 mRNA and protein were consistently overexpressed in human tumors compared with normal pituitaries. To mimic the pituitary tumor microenvironment, a hypoxia model using LßT2 murine gonadotrope cells was created to examine the functional role of the kinase. During long-term hypoxia, MST4 expression increased colony formation in a soft agar assay and rates of cell proliferation by activating p38 MAPK and AKT. Under short-term severe hypoxic stress, MST4 decreased the rates of apoptosis via p38 MAPK, AKT, hypoxia-inducible factor-1, and its cell-specific downstream targets. Analysis of MST4 mutants confirmed the importance of the kinase sequence but not the regulatory C terminus for its functional effects. Together these data identify the MST4 kinase as a novel candidate to mediate human pituitary tumorigenesis in a hypoxic environment and position it as a potential therapeutic target.

Family with sequence similarity 5, member C (FAM5C) increases leukocyte adhesion molecules in vascular endothelial cells: implication in vascular inflammation.

Identification of the regulators of vascular inflammation is important if we are to understand the molecular mechanisms leading to atherosclerosis and consequent ischemic heart disease, including acute myocardial infarction. Gene polymorphisms in family with sequence similarity 5, member C (FAM5C) are associated with an increased risk of acute myocardial infarction, but little is known about the function of this gene product in blood vessels. Here, we report that the regulation of the expression and function of FAM5C in endothelial cells. We show here that FAM5C is expressed in endothelial cells in vitro and in vivo. Immunofluorescence microscopy showed localization of FAM5C in the Golgi in cultured human endothelial cells. Immunohistochemistry on serial sections of human coronary artery showed that FAM5C-positive endothelium expressed intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). In cultured human endothelial cells, the overexpression of FAM5C increased the reactive oxygen species (ROS) production, nuclear factor-κB (NF-κB) activity and the expression of ICAM-1, VCAM-1 and E-selectin mRNAs, resulting in enhanced monocyte adhesion. FAM5C was upregulated in response to inflammatory stimuli, such as TNF-α, in an NF-κB- and JNK-dependent manner. Knockdown of FAM5C by small interfering RNA inhibited the increase in the TNF-α-induced production of ROS, NF-κB activity and expression of these leukocyte adhesion molecule mRNAs, resulting in reduced monocyte adhesion. These results suggest that in endothelial cells, when FAM5C is upregulated in response to inflammatory stimuli, it increases the expression of leukocyte adhesion molecules by increasing ROS production and NF-κB activity.

Modeling luminal breast cancer heterogeneity: combination therapy to suppress a hormone receptor-negative, cytokeratin 5-positive subpopulation in luminal disease.

INTRODUCTION: Many Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+ PR+ ones. One such subpopulation we call "Luminobasal" is ER-, PR- and cytokeratin 5 (CK5)-positive. It is not targeted for treatment. METHODS: To address the relationships between ER+PR+CK5- and ER-PR-CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from the same parental Luminal human breast cancer cell line. We used high-throughput screening to identify pLB-specific drugs and examined their efficacy alone and in combination with hormone therapy in mixed-cell tumor models. RESULTS: We show that pLUM and MCF7 cells suppress proliferation of pLB cells in mixed-cell 3D colonies in vitro and that pLUM cells suppress growth of pLB cells in mixed-cell xenografts in vivo. High-throughput screening of 89 FDA-approved oncology drugs shows that pLB cells are sensitive to monotherapy with the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib. By exploiting mixed-cell 3D colonies and mixed-cell solid mouse tumors models we demonstrate that combination therapy with gefitinib plus the anti-estrogen fulvestrant constitutes a robust treatment strategy. CONCLUSIONS: We propose that response to combination endocrine/EGFR inhibitor therapies in heterogeneous Luminal cancers may improve long-term survival in patients whose primary tumors have been preselected for appropriate biomarkers, including ER, PR, CK5 and EGFR.

Reprimo (RPRM) is a novel tumor suppressor in pituitary tumors and regulates survival, proliferation, and tumorigenicity.

Reprimo (RPRM), initially identified as a downstream effector of p53-induced cell cycle arrest at G(2)/M, is a putative tumor suppressor silenced in some types of cancer. In microarrays, the RPRM transcript was repressed 26-fold in gonadotrope (null cell) human pituitary tumors compared with normal pituitary but in the absence of changes in p53. Inhibition of RPRM mRNA was confirmed by RT-PCR in all gonadotrope tumors, most GH samples, and variably in other tumor types. Human pituitary tumors showed no evidence of abnormal promoter hypermethylation as a mechanism of RPRM repression. RPRM stable expression in gonadotrope (LßT2) and GH (GH3) pituitary cells resulted in decreased rates of cell proliferation by 55 and 30%, respectively; however, RPRM reexpression did not alter G(2)/M transition. In addition, RPRM increased rates of apoptosis in response to growth factor deprivation as assessed by caspase-3 cleavage and nuclear condensation. Clonagenic assays showed a 5.3- and 3.7-fold suppression of colony growth in RPRM-overexpressing LßT2 and GH3 cells, respectively, supporting its role as a tumor suppressor. In cells stably expressing RPRM mRNA, protein levels were actively suppressed due to rapid degradation through ubiquitination and proteasomal targeting. Growth factor withdrawal, as a model of cellular stress, stabilized RPRM protein levels. Together these data suggest that RPRM is transiently up-regulated at a posttranscriptional level in times of cellular stress to restrict cell survival, proliferation, and tumor formation. When RPRM is silenced as in human pituitary tumors, unrestrained growth and tumor progression may occur.

Identification of growth arrest and DNA-damage-inducible gene beta (GADD45beta) as a novel tumor suppressor in pituitary gonadotrope tumors.

Gonadotrope and null cell pituitary tumors cause significant morbidity, often presenting with signs of hypogonadism together with visual disturbances due to mass effects. Surgery and radiation are the only therapeutic options to date. To identify dysregulated genes and pathways that may play a role in tumorigenesis and/or progression, molecular profiling was performed on 14 gonadotrope tumors, with nine normal human pituitaries obtained at autopsy serving as controls. Bioinformatic analysis identified putative downstream effectors of tumor protein 53 (p53) that were consistently repressed in gonadotrope pituitary tumors, including RPRM, P21, and PMAIP1, with concomitant inhibition of the upstream p53 regulator, PLAGL1(Zac1). Further analysis of the growth arrest and DNA damage-inducible (GADD45) family revealed no change in the p53 target, GADD45α, but identified repression of GADD45ß in pituitary tumors in addition to the previously reported inhibition of GADD45γ. Overexpression of GADD45ß in LßT2 mouse gonadotrope cells blocked tumor cell proliferation and increased rates of apoptosis in response to growth factor withdrawal. Stable gonadotrope cell transfectants expressing increased GADD45ß showed decreased colony formation in soft agar, confirming its normal role as a tumor suppressor. Unlike previous studies of GADD45γ in pituitary tumors and α and ß in other tumors, bisulfite sequencing showed no evidence of hypermethylation of the GADD45ß promoter in human pituitary tumor samples to explain the repression of its expression. Thus, GADD45ß is a novel pituitary tumor suppressor whose reexpression blocks proliferation, survival, and tumorigenesis. Together these studies identify new targets and mechanisms to explore in pituitary tumor initiation and progression.

Giant pituitary adenomas: pathologic-radiographic correlations and lack of role for p53 and MIB-1 labeling.

Giant pituitary adenomas, with diameter ≥4 cm, were formerly considered rare and not surgically approachable. Few United States-based series exist. We reviewed our 10-year experience with these tumors and identified 17 patients, 11 male and 6 female, aged 27 to 65 years. Twelve of 17 cases were either gonadotroph or null cell adenomas and 5 were giant prolactinomas. By neuroimaging, all invaded the cavernous sinus(es) and tumors in 13 patients invaded the skull base. Despite massive size, only 5 showed apoplectic clinical and neuroimaging features. When present, this feature occasionally prompted preoperative consideration of craniopharyngioma. Transsphenoidal surgical excision was possible in all patients, with 3 undergoing planned second-stage reoperations and 2 requiring a second surgery for recurrence (both at 6-year intervals). Despite the aggressive features of massive size and cavernous sinus invasion, mitotic rates and immunohistochemistry (IHC) labeling for p53 and MIB-1, features alleged to be associated with atypical adenomas, were minimally increased. Absence of a role for TP53 and cell cycle markers was further verified on a subset of our cases by microarray and quantitative reverse transcription polymerase chain reaction analyses. Five giant gonadotroph adenomas were compared with 7 nonaggressive, nongiant gonadotroph cell adenomas, and no statistically significant changes in transcript levels of MIB-1 (MKI67) or TP53 were observed. A number of other genes, however, did show differential gene expression. In conclusion, most giant pituitary adenomas are gonadotroph cell adenomas or giant prolactinomas in men. Microarray profiling validates the IHC impression that MIB-1 and p53 IHC do not correlate with aggressive features in the most common type of giant adenoma.

Epidermal growth factor receptor pathway substrate 8 is overexpressed in human pituitary tumors: role in proliferation and survival.

Based on prior work showing that human pituitary tumors overexpress epidermal and fibroblast growth factor receptors, we hypothesized that downstream components of growth factor signaling pathways may also be dysregulated. Epidermal growth factor pathway substrate number 8 (Eps8) was identified as a transcript overexpressed (5.9-fold) in human pituitary tumors compared with normal pituitary by DNA microarrays. Eps8 mRNA up-regulation was confirmed by semiquantitative RT-PCR. Immunoblot analysis showed that Eps8 protein levels and its downstream target phosphorylated ERK were also up-regulated in human pituitary tumors. Stable overexpression of Eps8 in LbetaT2 gonadotrope pituitary cells augmented colony formation in soft agar at d 21. Eps8 cells proliferated more robustly compared with controls in growth factor replete as well as growth-restricted conditions. In addition, the Eps8 overexpressing cells were protected from serum withdrawal-induced apoptosis compared with controls as assessed by caspase-3 cleavage. Epidermal growth factor activated a robust amplification of ERK and modest up-regulation of Akt in Eps8-overexpressing pituitary cells compared with vector controls. MAPK kinase inhibition or silencing of Eps8 blunted the proliferation of the cells in response to growth factor stimulation. Blockade of the phosphatidylinositol 3-kinase pathway or silencing of Eps8 resulted in a loss of the Eps8 protection from growth factor withdrawal-induced apoptosis. Together these data support a role of Eps8 in amplifying growth factor receptor signaling in human pituitary tumors to promote proliferation and cell survival.

Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis.

Mutations in several steps of de novo purine synthesis lead to human inborn errors of metabolism often characterized by mental retardation, hypotonia, sensorineural hearing loss, optic atrophy, and other features. In animals, the phosphoribosylglycinamide transformylase (GART) gene encodes a trifunctional protein carrying out 3 steps of de novo purine synthesis, phosphoribosylglycinamide synthase (GARS), phosphoribosylglycinamide transformylase (also abbreviated as GART), and phosphoribosylaminoimidazole synthetase (AIRS) and a smaller protein that contains only the GARS domain of GART as a functional protein. The GART gene is located on human chromosome 21 and is aberrantly regulated and overexpressed in individuals with Down syndrome (DS), and may be involved in the phenotype of DS. The GART activity of GART requires 10-formyltetrahydrofolate and has been a target for anti-cancer drugs. Thus, a considerable amount of information is available about GART, while less is known about the GARS and AIRS domains. Here we demonstrate that the amino acid residue glu75 is essential for the activity of the GARS enzyme and that the gly684 residue is essential for the activity of the AIRS enzyme by analysis of mutations in the Chinese hamster ovary (CHO-K1) cell that require purines for growth. We report the effects of these mutations on mRNA and protein content for GART and GARS. Further, we discuss the likely mechanisms by which mutations inactivating the GART protein might arise in CHO-K1 cells.

The production of transgenic mice expressing human cystathionine beta-synthase to study Down syndrome.

Down syndrome (DS) is the most common genetic cause of significant cognitive disability. We hypothesize that by identifying metabolic alterations associated with cognitive impairment, it may be possible to develop medical or dietary interventions to ameliorate cognitive disabilities in persons with DS. Evidence suggests that one-carbon/transsulfuration (1C-TS) metabolism is abnormal in persons with DS. Cystathionine beta-synthase (CBS) plays a critical role in this metabolic system. The gene for CBS is on human chromosome 21, and there is evidence of elevated CBS enzyme activity in tissues and cells from individuals with DS. To analyze the possible role of CBS in Down syndrome, we have produced several lines of transgenic mice expressing the human CBS gene. We describe the use of Florescence Situ Hybridization (FISH) analysis to characterize the transgene insertion site for each line. Our initial expression analysis of each transgenic line by RT-PCR shows that the tissue specificity of human CBS mRNA levels in these mice may differ from the tissue specificity of mouse CBS mRNA levels in the same animals. These mice will be invaluable for assessing the regulation of the CBS gene and the role of CBS in cognition. They can also be used to develop therapies that target abnormalities in 1C-TS metabolism to improve cognition in persons with DS.