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Intratumoral drug delivery is a promising approach for the treatment of glioblastoma multiforme (GBM)., Intratumoral drug delivery is a promising approach for the treatment of glioblastoma multiforme (GBM). However, drug washout remains a major challenge in GBM therapy. Our strategy, aimed at reducing drug clearance and enhancing site-specific residence time, involves the local administration of a multi-component system comprised of nanoparticles (NPs) embedded within a thermosensitive hydrogel (HG). Herein, our objective was to examine the distribution of NPs and their cargo following intratumoral administration of this system in GBM. We hypothesized that the HG matrix, which undergoes rapid gelation upon increases in temperature, would contribute towards heightened site-specific retention and permanence of NPs in tumors. BODIPY-containing, infrared dye-labeled polymeric NPs embedded in a thermosensitive HG (HG–NPs) were fabricated and characterized. Retention and distribution dynamics were subsequently examined over time in orthotopic GBM-bearing mice. Results demonstrate that the HG–NPs system significantly improved site-specific, long-term retention of both NPs and BODIPY, with co-localization analyses showing that HG–NPs covered larger areas of the tumor and the peri-tumor region at later time points. Moreover, NPs released from the HG were shown to undergo uptake by surrounding GBM cells. Findings suggest that intratumoral delivery with HG–NPs has immense potential for GBM treatment, as well as other strategies where site-specific, long-term retention of therapeutic agents is warranted.

TWIST1 (TW) is a bHLH transcription factor (TF) and master regulator of the epithelial-to-mesenchymal transition (EMT). In vitro, TW promotes mesenchymal change, invasion, and self-renewal in glioblastoma (GBM) cells. However, the potential therapeutic relevance of TW has not been established through loss-of-function studies in human GBM cell xenograft models. The effects of TW loss of function (gene editing and knockdown) on inhibition of tumorigenicity of U87MG and GBM4 glioma stem cells were tested in orthotopic xenograft models and conditional knockdown in established flank xenograft tumors. RNAseq and the analysis of tumors investigated putative TW-associated mechanisms. Multiple bioinformatic tools revealed significant alteration of ECM, membrane receptors, signaling transduction kinases, and cytoskeleton dynamics leading to identification of PI3K/AKT signaling. We experimentally show alteration of AKT activity and periostin (POSTN) expression in vivo and/or in vitro. For the first time, we show that effect of TW knockout inhibits AKT activity in U87MG cells in vivo independent of PTEN mutation. The clinical relevance of TW and candidate mechanisms was established by analysis of the TCGA and ENCODE databases. TW expression was associated with decreased patient survival and LASSO regression analysis identified POSTN as one of top targets of TW in human GBM. While we previously demonstrated the role of TW in promoting EMT and invasion of glioma cells, these studies provide direct experimental evidence supporting protumorigenic role of TW independent of invasion in vivo and the therapeutic relevance of targeting TW in human GBM. Further, the role of TW driving POSTN expression and AKT signaling suggests actionable targets, which could be leveraged to mitigate the oncogenic effects of TW in GBM.

Twist1 is a master regulator of epithelial mesenchymal transition and carcinoma metastasis. Twist1 has also been associated with increased malignancy of human glioma. However, the impact of inhibiting Twist1 on tumorigenicity has not been characterized in glioma models in the context of different oncogenic transformation paradigms. Here we used an orthotopic mouse glioma model of transplanted transformed neural progenitor cells (NPCs) to demonstrate the effects of Twist1 loss of function on tumorigenicity. Decreased tumorigenicity was observed after shRNA mediated Twist knockdown in HPV E6/7 Ha-RasV12 transformed NPCs and Cre mediated Twist1 deletion in Twist1 fl/fl NPCs transformed by p53 knockdown and Ha-RasV12 expression. By contrast, Twist1 deletion had no effect on tumorigenicity of NPCs transformed by co-expression of Akt and Ha-RasV12. We demonstrated a dramatic off-target effect of Twist1 deletion with constitutive Cre expression, which was completely reversed when Twist1 deletion was achieved by transient administration of recombinant Cre protein. Together these findings demonstrate that the function of Twist1 in these models is highly dependent on specific oncogenic contexts of NPC transformation. Therefore, the driver mutational context in which Twist1 functions may need to be taken into account when evaluating mechanisms of action and developing therapeutic approaches to target Twist1 in human gliomas.

Current methods to assess the drug response of individual human cancers are often inaccurate, costly, or slow. Functional approaches that rapidly and directly assess the response of patient cancer tissue to drugs or small molecules offer a promising way to improve drug testing, and have the potential to identify the best therapy for individual patients. We developed a digitally-manufactured microfluidic platform for multiplexed drug testing of intact cancer slice cultures, and demonstrate the use of this platform to evaluate drug responses in slice cultures from human glioma xenografts and patient tumor biopsies. This approach retains much of the tissue microenvironment and can provide results rapidly enough, within days of surgery, to guide the choice of effective initial therapies. Our results establish a useful preclinical platform for cancer drug testing and development with the potential to improve cancer personalized medicine.

The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy.

Periostin is a matricellular fascilin-like protein that was first described as a component of periodontal ligaments.1 Subsequently, periostin has been shown to have critical functions in development, pathologic fibrosis, and cancer biology (reviewed in2–5). In carcinomas, periostin, secreted by cancer and stromal cells, promotes malignancy by promoting invasion, cell survival, treatment resistance, and metastasis. Secreted periostin functions in part through integrin interactions (most commonly with αvβ3 and αvβ5), which in turn activates proinvasive and survival-signaling pathways including focal adhesion kinase (FAK) and PI3K/Akt.5 Periostin promotes epithelial-mesenchymal transition (EMT)4 and is accordingly implicated in metastatic cell colonization and survival.6,7 Unlike carcinomas, relatively little is known about the prognostic or functional significance of periostin in human gliomas, this despite the early recognition that the periostin gene (POSTN) is among the most upregulated genes in GBM compared with normal brain.8,9 The few studies of periostin in glioma have reported direct correlations between expression levels and increased tumor edema, tumor grade, and survival.10,11 Functionally, periostin is reported to promote glioma cell invasion in vitro.11 In addition to expression in glioma cells, periostin is also reported to be a tumor-specific marker of GBM-associated angiogenesis.12 These associations support the potential importance of periostin in glioma malignancy, but the mechanisms by which periostin may contribute to glioma malignancy require further investigation. This study broadly examined periostin expression and function to extend current understanding of its clinical relevance and therapeutic potential. Expression in clinical samples and public databases have confirmed robust prognostic significance across all glioma grades. Periostin gene signatures also predicted outcome and demonstrated relationships of periostin with biological hallmarks of malignancy. In functional analysis, periostin regulated glioma cell invasion, adhesion, migration, cell morphology, and glioma stem cell survival in vivo. Interactions with αvβ3 and αvβ5 integrins and alteration of FAK localization suggested putative mechanisms of action for secreted periostin. Together, these data strongly support the potential of periostin as a biomarker and therapeutic target in human gliomas and provide direction for future investigations critical to realizing this potential.

Diffuse invasion into adjacent brain matter by glioblastoma (GBM) is largely responsible for their dismal prognosis. Previously, we showed that the TWIST1 (TW) bHLH transcription factor and its regulated gene periostin (POSTN) promote invasive phenotypes of GBM cells. Since TW functional effects are regulated by phosphorylation and dimerization, we investigated how phosphorylation of serine 68 in TW regulates TW dimerization, POSTN expression, and invasion in glioma cells. Compared with wild-type TW, the hypophosphorylation mutant, TW(S68A), impaired TW heterodimerization with the E12 bHLH transcription factor and cell invasion in vitro but had no effect on TW homodimerization. Overexpression of TW:E12 forced dimerization constructs (FDCs) increased glioma cell invasion and upregulated pro-invasive proteins, including POSTN, in concert with cytoskeletal reorganization. By contrast, TW:TW homodimer FDCs inhibited POSTN expression and cell invasion in vitro. Further, phosphorylation of analogous PXSP phosphorylation sites in TW:E12 FDCs (TW S68 and E12 S139) coordinately regulated POSTN and PDGFRa mRNA expression. These results suggested that TW regulates pro-invasive phenotypes in part through coordinated phosphorylation events in TW and E12 that promote heterodimer formation and regulate downstream targets. This new mechanistic understanding provides potential therapeutic strategies to inhibit TW-POSTN signaling in GBM and other cancers.

Although new neurons are produced in the subventricular zone (SVZ) of the adult mammalian brain, fewer functional neurons are produced with increasing age. The age-related decline in neurogenesis has been attributed to a decreased pool of neural progenitor cells (NPCs), an increased rate of cell death, and an inability to undergo neuronal differentiation and develop functional synapses. The time between mitotic events has also been hypothesized to increase with age, but this has not been directly investigated. Studying primary-cultured NPCs from the young adult and aged mouse forebrain, we observe that fewer aged cells are dividing at a given time; however, the mitotic cells in aged cultures divide more frequently than mitotic cells in young cultures during a 48-hour period of live-cell time-lapse imaging. Double-thymidine-analog labeling also demonstrates that fewer aged cells are dividing at a given time, but those that do divide are significantly more likely to re-enter the cell cycle within a day, both in vitro and in vivo. Meanwhile, we observed that cellular survival is impaired in aged cultures. Using our live-cell imaging data, we developed a mathematical model describing cell cycle kinetics to predict the growth curves of cells over time in vitro and the labeling index over time in vivo. Together, these data surprisingly suggest that progenitor cells remaining in the aged SVZ are highly proliferative.

Approximately 50-80% of oligodendrogliomas demonstrate a combined loss of chromosome 1p and 19q. Chromosome 1p/19q deletion, appearing early in tumorigenesis, is associated with improved clinical outcomes, including response to chemotherapy and radiation. Although many hypotheses have been proposed, the molecular mechanisms underlying improved clinical outcomes with 1p/19q deletion in oligodendrogliomas have not been characterized fully. To investigate the molecular differences between oligodendrogliomas, we employed an unbiased proteomic approach using microcapillary liquid chromatography mass spectrometry, along with a quantitative technique called isotope-coded affinity tags, on patient samples of grade II oligodendrogliomas. Following conventional biochemical separation of pooled tumor tissue from five samples of undeleted and 1p/19q deleted grade II oligodendrogliomas into nuclei-, mitochondria-, and cytosol-enriched fractions, relative changes in protein abundance were quantified. Among the 442 total proteins identified, 163 nonredundant proteins displayed significant changes in relative abundance in at least one of the three fractions between oligodendroglioma with and without 1p/19q deletion. Bioinformatic analyses of differentially regulated proteins supported the potential importance of metabolism and invasion/migration to the codeleted phenotype. A subset of altered proteins, including the pro-invasive extracellular matrix protein BCAN, was further validated by Western blotting as candidate markers for the more aggressive undeleted phenotype. These studies demonstrate the utility of proteomic analysis to identify candidate biological motifs and molecular mechanisms that drive differential malignancy related to 1p19q phenotypes. Future analysis of larger patient samples are warranted to further refine biomarker panels to predict biological behavior and assist in the identification of deleted gene products that define the 1p/19q phenotype.

The capacity of adult human glial progenitor cells (AGPs), to proliferate and undergo multipotent differentiation, positions them as ideal candidate cells of origin for human gliomas. To investigate this potential role we identified AGPs as mitotically active Olig2 cells in nonneoplastic adult human brain and gliomas. We conservatively estimated that one in 5,000 human temporal lobe neocortical gray or subcortical white matter cells is mitotic. Extrapolating from a mean Olig2/Mib-1 labeling index (LI) of 52% and total cell number of 100 billion, we estimated the overall prevalence of mitotic Olig2 AGPs in nonneoplastic human brain parenchyma at 10 million. These data identify a large reservoir of Olig2 AGPs which could be potential targets for human gliomagenesis. The vast majority of mitotic cells in Grade II and Grade III gliomas of all histologic subtypes expressed Olig2 (mean LI 75%) but rarely S100B (LI 0.6%), identifying the Olig2 cell as a distinct contributor to the proliferating cell population of human gliomas of both oligodendroglial and astrocytic lineages. In the most malignant Grade IV glioma, or glioblastoma multiforme (GBM), the prevalence of Olig2/Mib-1 cells was significantly decreased (24.5%). The significantly lower Olig2/Mib-1 LI in GBMs suggests that a decrease in the prevalence of Olig2 cells to the total mitotic cell pool accompanies increasing malignancy. The novel framework provided by this quantitative and comparative analysis supports future studies to examine the histogenetic role of Olig2 AGPs in adult gliomas, their potential contribution to the tumor stroma and the molecular role of Olig2 in glioma pathogenesis.

Dickkopf-1 (DKK-1) is known inhibitor of the canonical Wnt pathway. Recent studies strongly suggested that activation of DKK-1 expression results in inhibition of cell tumorigenicity. Reduced levels of DKK-1 in melanomas were recently shown. However, it is not known if DKK-1 activation in melanoma cells will inhibit cell tumorigenicity. In the present study, we overexpressed DKK-1 in melanoma cell line MDA-MB435. We show that while DKK-1 did not affect cell growth in soft agar, weak but significant inhibition of tumorigenicity in nude mice in vivo was observed. Analysis of resulting tumors revealed activation of cell death. In tumors originating from cells transduced with DKK-1, tumor mass was permeated with areas of necrosis. In tumors, originated from control cells, areas of necrosis were limited to the central region, a common feature of large tumors growing in nude mice. TUNEL assay revealed that in tumors originating from cells transduced with DKK-1 apoptotic cells were detected along the border of necrotic and viable areas of the tumors indicating significant increase in apoptotic process. Thus, our results indicate that activation of DKK-1 in melanoma cells leads to activation of apoptosis in vivo and, thus, is incompatible with tumor growth in nude mice.

Background The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors. Results We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations. Conclusions Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users.

There is a critical unmet need to tailor chemotherapies to individual patients. Personalized approaches could lower treatment toxicity, improve the patient’s quality of life, and ultimately reduce mortality. However, existing models of drug activity (based on tumor cells in culture or animal models) cannot accurately predict how drugs act in patients in time to inform the best possible treatment. Here we demonstrate a microfluidic device that integrates live slice cultures with an intuitive multi well platform that allows for exposing the slices to multiple compounds at once or in sequence. We demonstrate the response of live mouse brain slices to a range of drug doses in parallel. Drug response is measured by imaging of markers for cell apoptosis and for cell death. The platform has the potential to allow for identifying the subset of therapies of greatest potential value to individual patients, on a timescale rapid enough to guide therapeutic decision-making.

Summary Increasing age is the most robust predictor of greater malignancy and treatment resistance in human gliomas. However, the adverse association of clinical course with aging is rarely considered in animal glioma models, impeding delineation of the relative importance of organismal versus progenitor cell aging in the genesis of glioma malignancy. To address this limitation, we implanted transformed neural stem/progenitor cells (NSPCs), the presumed cells of glioma origin, from 3- and 18-month-old mice into 3- and 20-month host animals. Transplantation with progenitors from older animals resulted in significantly shorter (P ≤ 0.0001) median survival in both 3-month (37.5 vs. 83 days) and 20-month (38 vs. 67 days) hosts, indicating that age-dependent changes intrinsic to NSPCs rather than host animal age accounted for greater malignancy. Subsequent analyses revealed that increased invasiveness, genomic instability, resistance to therapeutic agents, and tolerance to hypoxic stress accompanied aging in transformed NSPCs. Greater tolerance to hypoxia in older progenitor cells, as evidenced by elevated HIF-1 promoter reporter activity and hypoxia response gene (HRG) expression, mirrors the upregulation of HRGs in cohorts of older vs. younger glioma patients revealed by analysis of gene expression databases, suggesting that differential response to hypoxic stress may underlie age-dependent differences in invasion, genomic instability, and treatment resistance. Our study provides strong evidence that progenitor cell aging is responsible for promoting the hallmarks of age-dependent glioma malignancy and that consideration of progenitor aging will facilitate development of physiologically and clinically relevant animal models of human gliomas.

Cross-relaxation imaging (CRI) is a quantitative magnetic resonance technique that measures the kinetic parameters of magnetization transfer between protons bound to water and protons bound to macromolecules. In this study, in vivo, four-parameter CRI of normal rat brains (N = 5) at 3.0 T was first directly compared to histology. The bound pool fraction, f, was strongly associated with myelin density (Pearson's r = 0.99, p

18 páginas, 9 figuras, 1 tabla.-- et al., [Background]: Tumor cell invasion into adjacent normal brain is a mesenchymal feature of GBM and a major factor contributing to their dismal outcomes. Therefore, better understandings of mechanisms that promote mesenchymal change in GBM are of great clinical importance to address invasion. We previously showed that the bHLH transcription factor TWIST1 which orchestrates carcinoma metastasis through an epithelial mesenchymal transition (EMT) is upregulated in GBM and promotes invasion of the SF767 GBM cell line in vitro. [Results]: To further define TWIST1 functions in GBM we tested the impact of TWIST1 over-expression on invasion in vivo and its impact on gene expression. We found that TWIST1 significantly increased SNB19 and T98G cell line invasion in orthotopic xenotransplants and increased expression of genes in functional categories associated with adhesion, extracellular matrix proteins, cell motility and locomotion, cell migration and actin cytoskeleton organization. Consistent with this TWIST1 reduced cell aggregation, promoted actin cytoskeletal re-organization and enhanced migration and adhesion to fibronectin substrates. Individual genes upregulated by TWIST1 known to promote EMT and/or GBM invasion included SNAI2, MMP2, HGF, FAP and FN1. Distinct from carcinoma EMT, TWIST1 did not generate an E- to N-cadherin "switch" in GBM cell lines. The clinical relevance of putative TWIST target genes SNAI2 and fibroblast activation protein alpha (FAP) identified in vitro was confirmed by their highly correlated expression with TWIST1 in 39 human tumors. The potential therapeutic importance of inhibiting TWIST1 was also shown through a decrease in cell invasion in vitro and growth of GBM stem cells. [Conclusions]: Together these studies demonstrated that TWIST1 enhances GBM invasion in concert with mesenchymal change not involving the canonical cadherin switch of carcinoma EMT. Given the recent recognition that mesenchymal change in GBMs is associated with increased malignancy, these findings support the potential therapeutic importance of strategies to subvert TWIST1-mediated mesenchymal change., This research was funded in part through an NIH/NINDS T32-NS-0007144 Clinical Neuroscience Training Grant (RO, LK, JM) and a University of Washington Institutional Bridge Funding Grant (RCR, AKM). Research in ISG group is supported partially by FEDER and by MICINN (SAF2009-08803), Junta de Castilla y León (CSI13A08 and proyecto Biomedicina 2009-2010), MEC OncoBIO Consolider-Ingenio 2010 (Ref. CSD2007-0017), Sandra Ibarra Foundation, NIH grant (R01 CA109335-04A1) and by Group of Excellence Grant (GR15) from Junta de Castilla y Leon.

Human glioma incidence, malignancy and treatment resistance are directly proportional to patient age. Cell intrinsic factors are reported to contribute to human age-dependent glioma malignancy but suitable animal models to examine the role of aging are lacking. Here we developed an orthotopic syngeneic glioma model to test the hypothesis that the age of neural progenitor cells (NPCs), presumed cells of glioma origin, influences glioma malignancy. Gliomas generated from transformed donor 3-, 12-, and 18-month-old NPCs in same-aged adult hosts all formed highly invasive glial tumors that phenocopied the human disease. Survival analysis indicated increased malignancy of gliomas generated from older 12- and 18-month-old transformed NPCs compared with their 3-month counterparts (median survival of 38.5 and 42.5 vs. 77 days, respectively). This study showed for the first time that age of target cells at the time of transformation can affect malignancy and demonstrated the feasibility of a syngeneic model using transformed NPCs for future examination of the relative impacts of age-related cell intrinsic and cell-extrinsic factors in glioma malignancy.

We used the whole-genome approach to identify major functional categories of genes whose expression depends on gestational age. Using microarray analysis, we compared gene expression profiles in the villous tissues of first (45-59 days) and second trimester (109-115 days) placentae with C-section term placentae. We found that in first trimester placentae, genes related to cell cycle, DNA, amino acids, and carbohydrate metabolism were significantly overrepresented, while genes related to signal transduction were underrepresented. Among genes involved in organism defense, we identified genes involved in chemical response, metabolism, and transport. Analysis of signal transduction pathways suggested, and subsequently confirmed independently, that the Wnt pathway was changed with gestational age leading to inhibition of beta-catenin protein expression. Our study will serve as a reference database to gain insight into the regulation of gene expression in the developing placentae and to compare with gene expression in placentae from complicated pregnancies.

The plasma concentrations of orally administered anti-human immunodeficiency virus protease inhibitors are significantly reduced during human and mouse pregnancy. We have shown that in the mouse, at gestational day 19, this reduction is due to increased hepatic cytochrome P450 3a (Cyp3a) protein expression and activity. In the current study, we investigated the mechanisms by which Cyp3a activity is increased by pregnancy and the time course of change in expression of Cyp3a and P-glycoprotein (P-gp) in various tissues. We found that hepatic transcripts of Cyp3a16, Cyp3a41, and Cyp3a44 were significantly increased during pregnancy, whereas those of Cyp3a11 and Cyp3a25 were significantly decreased. This resulted in a net increase in Cyp3a protein expression and activity in the liver during pregnancy. The increase in Cyp3a41 and Cyp3a44 transcripts was positively correlated (p0.05) with hepatocyte nuclear factor 6 and estrogen receptor-alpha transcripts. The pregnancy-related factors that transcriptionally activated mouse Cyp3a isoforms also activated the human CYP3A4 promoter in pregnant CYP3A4-promoter-luciferase transgenic (CYP3A4-tg) mice. In contrast, intestinal Cyp3a protein expression was not significantly affected by pregnancy. No change in P-gp protein expression was observed in the liver or kidney during pregnancy, although a significant decrease was observed in the placenta. Because hepatic CYP3A activity also seems to be induced during human pregnancy, the mouse (including CYP3A4-tg mouse) seems to be an excellent animal model to determine the molecular mechanisms for such an induction.

Dickkopf-1 (DKK-1) is a secreted inhibitor of the Wnt signaling pathway. We previously identified DKK-1 as a candidate tumor suppressor and demonstrated that ectopic expression of the DKK-1 suppressed the tumorigenicity of HeLa cells in vitro and in vivo. Since suppression of tumorigenicity of HeLa cells by DKK-1 overexpression was not mediated by effects on beta-catenin dependent transcription, we hypothesized that DKK-1 might also inhibit tumorigenicity of breast carcinoma cell lines lacking an activated canonical Wnt pathway. In the present study we show that ectopic expression of DKK-1 in various breast cancer cell lines resulted in a change in the cell phenotype, increased sensitivity to apoptosis, inhibition of anchorage independent growth in vitro, and suppression of tumorigenicity in vivo. Consistent with known effects of DKK-1 on the canonical Wnt signaling pathway, ectopic expression of DKK-1 in breast carcinoma cells was associated with increased phosphorylation and degradation of beta-catenin. However, none of the breast tumor cells used in this study showed detectable levels of beta-catenin dependent activation of TCF/Lef promoter activity measured by reporter constructs. Consistent with the results of these transient transfection assays, we were unable to demonstrate the expected beta-catenin dependent, TCF/Lef mediated inhibition of cyclin D1 and c-myc gene transcription in breast cells overexpressing DKK-1. However, we found that cells with DKK-1 overexpression have increased activity of CamKII pathway. Overexpression of the constitutively active form of CamKII (T286D) resulted in inhibition of breast cancer cell tumorigenicity. Thus, our study supports the hypothesis that DKK-1 mediated tumor suppressor effect is independent of beta-catenin dependent transcription and identified the CamKII pathway that contributes into DKK-1 signaling.

To elucidate the molecular basis for differential susceptibilities to mammary carcinogenesis, we compared the transcriptomes of normal mammary glands from pubescent female rats of the resistant Copenhagen (Cop) strain with those of the susceptible Fischer 344 (F344), August x Copenhagen Irish (ACI), Buffalo/N (Buf/N), Wistar-Furth (WF) strains and F1 (Cop x F344) progeny (F1). Gene expression profiles in mammary tissue within each rat strain were remarkably similar, indicating that gene expression was determined by genetic background. We next identified the subset of genes that were differentially expressed in all susceptible strains relative to the resistant Cop strain. Among these, the messenger RNAs encoding prolactin (Prl) and its cell surface receptor were significantly elevated in all susceptible strains. The expression levels of several Prl-regulated genes were also significantly elevated, indicating the presence of increased Prl signaling in mammary glands of all susceptible strains. Pathway analysis of gene expression profiles further identified the Prl-activated Jak/STAT-signaling pathway among the pathways that most distinguished sensitive rat strains from the resistant Cop rat. To test the hypothesis that reduced levels of the Prl signaling in mammary tissue partially contributed to the genetic resistance to mammary carcinogenesis, we used the neuroleptic drug, perphenazine, to transiently elevate serum Prl levels in the Cop strain. Whereas Cop rats are resistant to N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis, approximately 5% of pubescent Cop females treated with perphenazine and NMU exposure developed mammary adenocarcinomas with latencies comparable with those of sensitive strains. Together, these finding indicated that in the rat, the molecular mechanisms underlying genetic susceptibility to mammary carcinogenesis include de-regulation of Prl signaling.

The breast cancer resistance protein (BCRP) plays an important role in drug disposition, including limiting drug penetration across the placental barrier. Our goal was to investigate the effects of pregnancy on Bcrp1 expression in pregnant mice. We examined Bcrp1 expression in placenta, kidney, liver, and small intestine at various gestational ages. Bcrp1 protein levels peaked at gestation day (gd) 15 in placenta, at gd 10 and 15 in kidney, and at gd 15 in liver; however, Bcrp1 protein levels in small intestine did not change significantly with gestational ages. Immunohistochemistry analysis revealed that the cellular localization of Bcrp1 in placenta, kidney, liver, and small intestine was not influenced by pregnancy. Bcrp1 mRNA levels were analyzed by quantitative real-time RT-PCR. In general, the effects of pregnancy on Bcrp1 protein somewhat lagged behind the effects on Bcrp1 mRNA. To further investigate the possible roles of nuclear receptors in the regulation of the Bcrp1 gene during pregnancy, we examined mRNA levels of aryl hydrocarbon receptor (AhR), hypoxia-inducible factor 1alpha (HIF1alpha), estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), or progesterone receptor and compared them with those of Bcrp1. Bcrp1 mRNA was significantly correlated with mRNA of AhR, HIF1alpha, and ERbeta in placenta, with mRNA of HIF1alpha in kidney, and with mRNA of AhR and ERalpha in liver. These data suggest that Bcrp1 expression in mouse tissues can be altered by pregnancy in a gestational age-dependent manner. Such effects are likely mediated by certain nuclear receptors through a transcriptional mechanism.

We previously identified a positive transcriptional element identical to human Ha-ras response element (HRE) within the promoter of the rat Ha-ras gene. We further identified CArG binding factor A (CBF-A), a member of heterogeneous nuclear ribonuclear protein (hnRNP) gene family, as a trans-acting factor that binds the HRE sequence with high affinity in rat mammary carcinoma cells. To determine if activation of CBF-A plays a role in tumor development in vivo , we investigated CBF-A expression and binding activity in rat mammary tumors induced by N-methyl-N-nitrosourea. We found that approximately 82% of tumors expressed CBF-A at levels that were 3-20 fold higher than detected in normal mammary gland. Moreover, elevated CBF-A protein levels were invariably associated with increased binding activity to the HRE. CBF-A mRNA levels in tumors were on average elevated only two fold as compared to normal mammary gland, indicating that increased CBF-A protein levels in tumors resulted from both translational and/or post-translational regulation. The level of CBF-A expression in mammary tumors was independent of Ha-ras mutational status. Together, these findings indicated that deregulation of CBF-A contributes to mammary carcinogenesis via a mechanism that is distinct from its hnRNP functions in binding and post-transcriptional regulation of RNA.

Teratogen-induced alterations in gene expression play an important role in the genesis of malformations in animals. The recent development of DNA microarrays now offers the opportunity to monitor global changes in gene expression and therefore the potential to obtain significant new information concerning both normal and abnormal development. RNA was isolated from day-9 mouse embryos at 1 and 5 h after exposure to hyperthermia (HS) or 4-hydroperoxycyclophosphamide (4CP) and compared to RNA isolated from concurrent controls using mouse cDNA microarrays. Cy5/Cy3 intensity data were extracted using Spot-on Image software and then normalized using the statistical software program R/maanova. Differentially expressed genes were identified using a linear mixed-effects model and p values derived from t-test statistics. Approximately 9000 genes show statistically significant alterations in expression in day-9 mouse embryos exposed to HS or 4CP. HS and 4CP also induce alterations in the expression of distinct sets of genes, e.g., DNA replication/repair, cell cycle, signal transduction, and transcription-related genes. As expected, a variety of heat shock genes are upregulated by HS but not 4CP. Among genes whose expression is altered by both HS and 4CP, cluster analysis identified three p53 target genes (Cyclin G1, Gtse1, and Mdm2), and follow up studies confirmed that p53 is activated in embryos exposed to these two teratogens. In addition, cluster analyses also revealed that HS but not 4CP induces the downregulation of genes encoding key enzymes in the cholesterol biosynthesis pathway. Thus, our microarray data have identified one potentially important pathway (p53) common to both HS- and 4CP-induced teratogenesis and another pathway (cholesterol biosynthesis) potentially important, but specific to HS-induced teratogenesis.

We described previously the isolation and characterization of two non-tumorigenic revertants from the HeLa cervical carcinoma cell line, and demonstrated that loss of the transformed phenotype in these cells was the result of dominant somatic mutations. The goal of the present study was to use cDNA microarrays to identify candidate tumor suppressors among the set of genes whose increased expression correlated with loss of tumorigenicity in both revertants. Among the genes with significantly increased expression levels in both HA and HF revertants we identified Insulin Growth Factor Binding Protein-3 (IGFBP-3) and the Dickkopf-1 (DKK-1) genes. Both of these genes encode secreted proteins implicated in the modulation cell growth and differentiation, and IGFBP-3 was shown previously to have tumor suppressing activity. To test the hypothesis that increased expression of IGFBP-3 or the DKK-1 genes could have contributed to the suppression of tumorigenicity in the revertants, we expressed IGFBP-3 or DKK-1 in HeLa cells, and assessed their effects on anchorage dependent and independent growth, and tumor formation in athymic nude mice. Ectopic expression of IGFBP-3 or DKK-1 resulted in significantly decreased growth in soft agar. HeLa cells expressing ectopic IGFBP-3 or DKK-1 showed statistically significant differences in the kinetics of tumor formation. In any tumors that arose in animals injected with the IGFBP-3 expressing cells, there was a complete loss of IGFBP-3 activity, as measured by binding to IGF-1 and IGF-2 proteins. All tumors that arose after injection of cells expressing DKK-1, invariably showed almost a complete loss of ectopic DKK-1 expression. The observations that loss of DKK-1 expression or IGFBP-3 activity was required for tumorigenicity suggested that both proteins encode putative tumor suppressor genes. We also show that while DKK-1 expression does not affect cell growth in vitro, the protein does sensitize cells to apoptosis. We also demonstrated that effect of DKK-1 was not due to inhibition of beta-catenin/TCF4-regulated transcription. Taken together, our results indicate that somatic cell genetics combining with gene expression profiling may be a useful approach for the identification of functional suppressors of malignant cell growth.

In human adult gliomas increased patient age is a robust predictor of malignant clinical behavior evidenced by increased incidence, histologic grade, resistance to therapy and shortened patient survival [1]. Despite these associations, the mechanisms responsible for age-related increases in glioma malignancy are poorly understood; a deficiency compounded by the lack of informative animal models. Neural stem/progenitor (NSPCs) cells are the presumed cells of glioma origin. Using a syngeneic mouse glioma model we showed that the “age” of neural stem and progenitor cells (i.e., the age of donors from which NSPCs were isolated prior to transformation) dictated survival in same-aged hosts and differently-aged (3mo and 20mo) hosts [1]. Surprisingly, host age had no significant effect on survival [1]. After malignant transformation, aging in NSPCs translated to increased invasive potential, genomic instability, resistance to genotoxic stress (radiation and alkylating chemotherapy) and tolerance of hypoxia. These observations suggest that aging related changes in putative glioma cells of origin contribute to increased malignant potential. However, many aging associated changes observed in normal NSPCs would seem more likely to inhibit malignant potential. For example, with aging there is an overall attrition of NSPCs associated with acquisition of a senescent phenotype with decreased overall proliferation and self-renewal [2]. How then does a senescing cell population generate a more robust cancer cell? Emerging understanding of aging NSPCs suggests that mechanisms associated with aging may actually prime NSPCs for enhanced malignant potential. Conceivably, the increased genomic instability and tolerance of hypoxic stress with aging could enhance NSPC “malignant fitness” or potential [1]. In addition, although less proliferative overall, a small sub-population of aging NSPCs display an increased propensity to re-enter the cell cycle and by conferring a selection advantage could also enhance their malignant potential [3]. Presumably, after malignant transformation the age-dependent state of malignant “pre-conditioning” present in normal NSPCs translates into differential growth advantages within the brain and glioma micro-environment. Genomic instability and hypoxia are speculated to be among the mechanisms fundamental for defining malignant potential and its manifestations of increased invasion, proliferation and treatment resistance. Genomic instability could drive emergence and selection of hypoxia tolerant and responsive cells in the aging brain microenvironment while activation of hypoxic responses in turn could foster acquisition of or enhancement of genomic instability. Finally, senescent and malignant states share common features of altered chromatin structure, epigenetic changes and DNA damage. Therefore, age-dependent mechanisms that regulate DNA integrity and DNA damage responses may provide clues to reconciling the co-existence of senescence and enhanced malignant potential. One candidate mechanism could be inverse changes observed in p53 and p16 tumor suppressor (TS) proteins in normal NSPCs with aging [1, 2]. In young (3mo) NSPCs p16 is not detectable while robust levels of p53 basal and inducible expression and target gene activation (p21) are present. Conversely, in aged (18mo) NSPCs, p16 expression is dramatically increased concurrent with a dramatic abrogation of p53 activity [4]. Increased p16 contributes to age-related attrition of NSPCs in the SVZ through effects on proliferation and self-renewal [2] but its functional relevance for age-acquired malignant potential is not known. Conversely, abrogation of p53 activity with age could promote increased malignant potential through its known pleiotropic functions to regulate cell cycle progression, apoptosis/senescence, genomic integrity, glycolysis, HIF1 mediated hypoxic responses and invasion. Therefore, differential expression and function of the p16/p53 tumor suppressors, possibly in response to age-related accumulation of DNA damage, represents a candidate mechanism underlying the concurrent acquisition of increased senescent and malignant potential in aging NSPCs. An additional mechanism related to p53 that may also contribute to age-dependent NSPC malignant potential is activation of the mTOR pathway in aging NSPCs (unpublished observation). Decreased p53 may contribute to mTOR activation [5] which in turn may prime aged NSPCs for increased malignant potential. Of interest, mTOR also has been linked to epithelial stem cell senescence [6,7]; therefore, persistence of increased mTOR activity in a sub-set of aged NSPCs that evade senescence may ultimately provide a growth advantage upon malignant transformation. The incorporation of cell-intrinsic and micro-environmental influences of aging into a glioma model was anticipated to reveal age-related mechanisms relevant to human glioma malignancy. In two respects- increased resistance to genotoxic agents and increased activation of HIF-1 hypoxia response genes- this model recapitulated features associated with age-dependent malignancy of human gliomas. Aging human glioma cells/tumors are more resistant to genotoxic stresses (radiation and alkylating chemotherapy) [1, 8] but in line with age-related increases in VEGF expression, they are more responsive to the VEGF inhibitor bevacizumab [9]. This latter observation echoes the increased activation of hypoxia response genes (HRGs) including VEGF seen in aged transformed NSPCs as well as a general increase in HRG expression in aged human glioma patients [1]. Incorporating aging in an animal glioma model therefore served to identify shared mechanisms that contribute to normal aging and acquisition of malignant potential in NSPCs (Figure ​(Figure1).1). Further, the preliminary results provide proof of principle that accounting for aging has relevance in the pre-clinical setting. Figure 1 A model whereby mechanisms of aging concurrently contribute to reduced physiologic “fitness” and increased malignant potential of neural stem/progenitor cells. Reduced NSPC “fitness” regulated in part by increased p16 manifests ...

In the present study we identified a positive transcriptional element within the rat Ha-ras promoter previously known as Ha-ras response element (HRE) and identified a trans-acting factor that binds HRE sequences in rat mammary cells. To identify the binding protein we employed sequence specific DNA affinity chromatography. Amino acid sequence analysis of the affinity-purified proteins was performed by tandem mass spectroscopy. The results unexpectedly demonstrated that in rat mammary cells CArG box-binding factor A (CBF-A) is the major protein species that bind specifically to the rat and human HRE sequences with high affinity. The affinity of CBF-A binding to HRE was significantly higher than to the CArG box described as a recognition sequence for CBF-A protein. Transient transfection assays using reporter plasmids verified that mutations within the HRE that disrupt binding of CBF-A also reduced the activity of the rat Ha-ras promoter. Despite the fact that the HRE within the Ha-ras promoter resembles a binding site for Ets transcription factors, we did not detect the binding of Ets-related proteins to the rat HRE in BICR-M1Rk cells. We further demonstrated a correlation between the presence of HRE binding activity and induction of Ha-ras mRNA expression following serum stimulation in the mammary carcinoma cell line. Taken together, our results suggest that CBF-A may play an important role in transcriptional regulation of Ha-ras promoter activity during normal mammary cell growth and carcinogenesis.

Rat strains differ dramatically in their susceptibility to mammary carcinogenesis. On the assumption that susceptibility genes are conserved across mammalian species and hence inform human carcinogenesis, numerous investigators have used genetic linkage studies in rats to identify genes responsible for differential susceptibility to carcinogenesis. Using a genetic backcross between the resistant Copenhagen (Cop) and susceptible Fischer 344 (F344) strains, we mapped a novel mammary carcinoma susceptibility (Mcs30) locus to the centromeric region on chromosome 12 (LOD score of ∼8.6 at the D12Rat59 marker). The Mcs30 locus comprises approximately 12 Mbp on the long arm of rat RNO12 whose synteny is conserved on human chromosome 13q12 to 13q13. After analyzing numerous genes comprising this locus, we identified Fry, the rat ortholog of the furry gene of Drosophila melanogaster, as a candidate Mcs gene. We cloned and determined the complete nucleotide sequence of the 13 kbp Fry mRNA. Sequence analysis indicated that the Fry gene was highly conserved across evolution, with 90% similarity of the predicted amino acid sequence among eutherian mammals. Comparison of the Fry sequence in the Cop and F344 strains identified two non-synonymous single nucleotide polymorphisms (SNPs), one of which creates a putative, de novo phosphorylation site. Further analysis showed that the expression of the Fry gene is reduced in a majority of rat mammary tumors. Our results also suggested that FRY activity was reduced in human breast carcinoma cell lines as a result of reduced levels or mutation. This study is the first to identify the Fry gene as a candidate Mcs gene. Our data suggest that the SNPs within the Fry gene contribute to the genetic susceptibility of the F344 rat strain to mammary carcinogenesis. These results provide the foundation for analyzing the role of the human FRY gene in cancer susceptibility and progression.

Background: Gliomas are the most common primary brain tumors and their prognosis is related to WHO histopathological grade. Glioblastoma (grade IV astrocytoma or GBM) is characterized by microscopic invasion into surrounding brain and universally poor prognosis despite treatment with surgery, radiation and chemotherapy . Recently, the matricellular protein periostin (POSTN) has been shown to be associated with increased parenchymal invasion (measured by edema on MRI) and poor prognosis in GBM. The specific aim of the present analysis was to determine the mechanistic impact of POSTN on glioma outcome. Methods: We used the gene expression of POSTN from 559 GBM patients (pts) included in the Cancer Genome Atlas (TCGA) to build a gene expression model of the expression of 12184 genes to predict POSTN expression using elastic net. This model was used to predict the POSTN expression in a testing set of 419 pts with gliomas included in Repository for Brain Neoplasia Data (REMBRANDT) (99 grade II, 71 grade III and 125 grade IV and 124 with no grade). Receiver-operating characteristic (ROC) and survival analysis were performed to measure the performance of the model, and gene-set enrichment analysis (GSEA) was used to reveal network topology perturbations. Results: The gene expression model discovered 721 genes highly correlated to POSTN expression that predict POSTN in the testing set with an area under the curve (AUC) of 0.96 on ROC curve. GSEA reveals genes involved in “mesenchymal transition,” transition of invasive ductal carcinoma (IDC) from ductal carcinoma in situ (DCIS) in breast cancer and stem cell signatures. Survival analysis of the TCGA GBM pts showed that the cohort with “high POSTN” had worse survival (median 12 months versus 15 months, log-rank p=0.0002). When applied to all pts in REMBRANDT, the model predicted classes also had significantly different survival (median 13.4 versus 38 months, log-rank p Conclusions: We have developed a gene expression model related to POSTN, a gene linked to poor prognosis in GBM, to investigate the role correlated genes may play in the aggressive phenotype. Some of the genes found to be highly correlated to POSTN are related to mesenchymal transition, invasive behavior in breast cancer and stemness. We verify that high POSTN expression is a strong prognostic indicator for poor outcome in GBM, and reveal for the first time that pts with grade II and III glioma with a “high” POSTN signature have significantly worse survival. Given that low grade glioma pts often have less aggressive treatment at time of diagnosis, we propose studying the role of early chemoradiation in the subset of pts with poor POSTN signature to potentially improve their outcome. Citation Format: Andrew D. Trister, Andrei M. Mikheev, Jason K. Rockhill, Stephen H. Friend, Robert C. Rostomily. Periostin expression in glioma correlates with genes related to mesenchymal transition and survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1210. doi:10.1158/1538-7445.AM2013-1210

Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) are structurally related survival and differentiation factors for distinct sets of peripheral and central neurons. We previously reported that BDNF and NGF gene expression are differentially regulated in mouse L929 fibroblasts. Here we examine expression of these three neurotrophins in human fibroblasts. Northern blots detected BDNF and NT-3 mRNAs in fibroblasts derived from lung (WI-38), calvarium and foreskin. WI-38 cells and foreskin fibroblasts expressed 1.6 kb as well as 4 kb BDNF mRNAs whereas only the smaller BDNF mRNA was detected in calvarium fibroblasts. NGF mRNA was present in foreskin and calvarium but not lung fibroblasts. In WI-38 cells serum treatment increased levels of BDNF mRNA within 2 hr. Cycloheximide did not inhibit the increase. Treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) transiently suppressed BDNF mRNA. Treatment with both serum and TPA first stimulated and then transiently suppressed BDNF mRNA. TPA and/or serum did not significantly affect BDNF mRNA in calvarium fibroblasts. These results show that human fibroblasts derived from different tissues express and regulate neurotrophin genes differentially.

Using Quantitative Trait Locus (QTL) mapping in a backcross between susceptible Fischer 344 and resistant Copenhagen rat strains, we identified Fry, the rat ortholog of the Drosophila melanoganster furry gene, as a mammary carcinoma susceptibility (Mcs) gene. The Fischer 344 Fry allele harbors two single nucleotide polymorphisms that alter amino acid residues that are highly conserved from yeast to humans. One of these SNPs replaces an Alanine with a Serine residue, creating a de novo concensus sequence for several cancer-associated protein kinases. In the lower eucaryoyes, the furry orthologs are implicated in diverse cell processes including epithelial cell polarization, morphogenesis, proliferation, cytokinesis, and regulation of the NDR family of kinases, some of which are putative human tumor suppressor genes. Consistent with its function as a tumor suppressor gene, ectopic expression of the FRY gene was reduced in 3 of 4 human breast cancer cell lines. Ectopic expression of the wildtype allele significantly reduced the tumorigenicity of the human MDA-MB-231 mammary carcinoma cell line. In contrast to the parental MDA-MB-231 cells, which grew randomly in Matrigel® matrix, those expressing Fry (231CFry) formed spherical colonies of differentiated cells, closely resembling those formed by MCF-10A normal mammary epithelial cell line. When xenographed into nude mice, the 231CFry cells demonstrated a dramatic reduction in tumorigenicity (8x reduction in tumor volume at 15 days) and diminished invasiveness into surrounding tissues. Gene expression profiling suggested that in MDA-MB-231 cells, ectopic Fry induced the expression of gene networks involved in cell differentiation, proliferation, and polarization, consistent with a role for FRY in suppressing epithelial-mesenchymal transition (EMT). To investigate the role of human FRY in breast cancer, we examined FRY mRNA expression in >1,500 human breast cancers, representing distinct eight cohorts entered into the Oncomine 3.0 Cancer Profiling Database. Our analysis revealed that decreased FRY mRNA was highly correlated with poorly differentiated breast cancer phenotypes (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-131. doi:10.1158/1538-7445.AM2011-LB-131

Summary Monoclonal and polyclonal antibodies specific for methylation adducts have been applied in an immunohistochemical study of DNA damage in rat and hamster liver following exposure to dimethylnitrosamine. The approach was validated, for frozen and paraffin-embedded sections, by comparison with biochemical data on adduct levels in whole tissues or specific cell populations in the same experimental systems. The potential application of this method to human exposure assessment is discussed.

Patterns of gene expression in mammary cells following carcinogen exposure of strains of rat sensitive or resistant to carcinogenesis