Julianna T.D. Ross

MicroRNA-328 is associated with （non-small） cell lung cancer （NSCLC） brain metastasis and mediates NSCLC migration

Brain metastasis (BM) can affect ∼ 25% of nonsmall cell lung cancer (NSCLC) patients during their lifetime. Efforts to characterize patients that will develop BM have been disappointing. microRNAs (miRNAs) regulate the expression of target mRNAs. miRNAs play a role in regulating a variety of targets and, consequently, multiple pathways, which make them a powerful tool for early detection of disease, risk assessment, and prognosis. We investigated miRNAs that may serve as biomarkers to differentiate between NSCLC patients with and without BM. miRNA microarray profiling was performed on samples from clinically matched NSCLC from seven patients with BM (BM+) and six without BM (BM−). Using t‐test and further qRT‐PCR validation, eight miRNAs were confirmed to be significantly differentially expressed. Of these, expression of miR‐328 and miR‐330‐3p were able to correctly classify BM+ vs. BM− patients. This classifier was used on a validation cohort (n = 15), and it correctly classified 12/15 patients. Gene expression analysis comparing A549 parental and A549 cells stably transfected to over‐express miR‐328 (A549‐328) identified several significantly differentially expressed genes. PRKCA was one of the genes over‐expressed in A549‐328 cells. Additionally, A549‐328 cells had significantly increased cell migration compared to A549 cells, which was significantly reduced upon PRKCA knockdown. In summary, miR‐328 has a role in conferring migratory potential to NSCLC cells working in part through PRKCA and with further corroboration in additional independent cohorts, these miRNAs may be incorporated into clinical treatment decision making to stratify NSCLC patients at higher risk for developing BM.

Glycogene Expression Alterations Associated with Pancreatic Cancer Epithelial-Mesenchymal Transition in Complementary Model Systems

Background The ability to selectively detect and target cancer cells that have undergone an epithelial-mesenchymal transition (EMT) may lead to improved methods to treat cancers such as pancreatic cancer. The remodeling of cellular glycosylation previously has been associated with cell differentiation and may represent a valuable class of molecular targets for EMT. Methodology/Principal Findings As a first step toward investigating the nature of glycosylation alterations in EMT, we characterized the expression of glycan-related genes in three in-vitro model systems that each represented a complementary aspect of pancreatic cancer EMT. These models included: 1) TGFβ-induced EMT, which provided a look at the active transition between states; 2) a panel of 22 pancreatic cancer cell lines, which represented terminal differentiation states of either epithelial-like or mesenchymal-like; and 3) actively-migrating and stationary cells, which provided a look at the mechanism of migration. We analyzed expression data from a list of 587 genes involved in glycosylation (biosynthesis, sugar transport, glycan-binding, etc.) or EMT. Glycogenes were altered at a higher prevalence than all other genes in the first two models (p<0.05 and <0.005, respectively) but not in the migration model. Several functional themes were shared between the induced-EMT model and the cell line panel, including alterations to matrix components and proteoglycans, the sulfation of glycosaminoglycans; mannose receptor family members; initiation of O-glycosylation; and certain forms of sialylation. Protein-level changes were confirmed by Western blot for the mannose receptor MRC2 and the O-glycosylation enzyme GALNT3, and cell-surface sulfation changes were confirmed using Alcian Blue staining. Conclusions/Significance Alterations to glycogenes are a major component of cancer EMT and are characterized by changes to matrix components, the sulfation of GAGs, mannose receptors, O-glycosylation, and specific sialylated structures. These results provide leads for targeting aggressive and drug resistant forms of pancreatic cancer cells.

Integrated Genomic and Epigenomic Analysis of Breast Cancer Brain Metastasis

The brain is a common site of metastatic disease in patients with breast cancer, which has few therapeutic options and dismal outcomes. The purpose of our study was to identify common and rare events that underlie breast cancer brain metastasis. We performed deep genomic profiling, which integrated gene copy number, gene expression and DNA methylation datasets on a collection of breast brain metastases. We identified frequent large chromosomal gains in 1q, 5p, 8q, 11q, and 20q and frequent broad-level deletions involving 8p, 17p, 21p and Xq. Frequently amplified and overexpressed genes included ATAD2, BRAF, DERL1, DNMTRB and NEK2A. The ATM, CRYAB and HSPB2 genes were commonly deleted and underexpressed. Knowledge mining revealed enrichment in cell cycle and G2/M transition pathways, which contained AURKA, AURKB and FOXM1. Using the PAM50 breast cancer intrinsic classifier, Luminal B, Her2+/ER negative, and basal-like tumors were identified as the most commonly represented breast cancer subtypes in our brain metastasis cohort. While overall methylation levels were increased in breast cancer brain metastasis, basal-like brain metastases were associated with significantly lower levels of methylation. Integrating DNA methylation data with gene expression revealed defects in cell migration and adhesion due to hypermethylation and downregulation of PENK, EDN3, and ITGAM. Hypomethylation and upregulation of KRT8 likely affects adhesion and permeability. Genomic and epigenomic profiling of breast brain metastasis has provided insight into the somatic events underlying this disease, which have potential in forming the basis of future therapeutic strategies.

Cdc42 and the Guanine Nucleotide Exchange Factors Ect2 and Trio Mediate Fn14-Induced Migration and Invasion of Glioblastoma Cells

Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor–inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14–directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy. Mol Cancer Res; 10(7); 958–68. ©2012 AACR.

miRNA Expression Profiling in Migrating Glioblastoma Cells: Regulation of Cell Migration and Invasion by miR- 23b via Targeting of Pyk2

Background Glioblastoma (GB) is the most common and lethal type of primary brain tumor. Clinical outcome remains poor and is essentially palliative due to the highly invasive nature of the disease. A more thorough understanding of the molecular mechanisms that drive glioma invasion is required to limit dispersion of malignant glioma cells. Methodology/Principal Findings We investigated the potential role of differential expression of microRNAs (miRNA) in glioma invasion by comparing the matched large-scale, genome-wide miRNA expression profiles of migrating and migration-restricted human glioma cells. Migratory and migration-restricted cell populations from seven glioma cell lines were isolated and profiled for miRNA expression. Statistical analyses revealed a set of miRNAs common to all seven glioma cell lines that were significantly down regulated in the migrating cell population relative to cells in the migration-restricted population. Among the down-regulated miRNAs, miR-23b has been reported to target potential drivers of cell migration and invasion in other cell types. Over-expression of miR-23b significantly inhibited glioma cell migration and invasion. A bioinformatics search revealed a conserved target site within the 3′ untranslated region (UTR) of Pyk2, a non-receptor tyrosine kinase previously implicated in the regulation of glioma cell migration and invasion. Increased expression of miR-23b reduced the protein expression level of Pyk2 in glioma cells but did not significantly alter the protein expression level of the related focal adhesion kinase FAK. Expression of Pyk2 via a transcript variant missing the 3′UTR in miR-23b-expressing cells partially rescued cell migration, whereas expression of Pyk2 via a transcript containing an intact 3′UTR failed to rescue cell migration. Conclusions/Significance Reduced expression of miR-23b enhances glioma cell migration in vitro and invasion ex vivo via modulation of Pyk2 protein expression. The data suggest that specific miRNAs may regulate glioma migration and invasion to influence the progression of this disease.

Efficacy of Exercise in Reducing Depressive Symptoms across 5-HTTLPR Genotypes

INTRODUCTION Exercise is effective in the alleviation of depressive symptoms and may have physiological effects similar to those of selective serotonin reuptake inhibitors (SSRI). Recent research has identified the difference in treatment effects across genetic polymorphisms of the serotonin transporter polymorphic region (5-HTTLPR), in which the l allele has been associated with a better response to SSRI compared with the s allele. The purpose of the current research was to examine the antidepressant effects of exercise across 5-HTTLPR genotypes. METHODS Participants, ages 18–23 yr, were randomly assigned to a 5-wk exercise intervention or a no-treatment control group. Participants completed the Beck Depression Inventory before and after the intervention and provided a saliva sample for DNA analysis. RESULTS Exercise resulted in a significant reduction in depressive symptoms compared with the control group. In addition, individuals with at least one l allele demonstrated greater reductions in depressive symptoms compared with ss individuals. CONCLUSIONS The effects of exercise on depressive symptoms appear to be moderated by 5-HTTLPR genotype, suggesting that the mechanisms responsible for the alleviation of depressive symptoms are similar for exercise and SSRI treatment. Furthermore, these findings suggest that 5-HTTLPR genotype should be a factor in determining the proper line of treatment for depression.

TROY (TNFRSF19) Promotes Glioblastoma Survival Signaling and Therapeutic Resistance

Of the features that characterize glioblastoma, arguably none is more clinically relevant than the propensity of malignant glioma cells to aggressively invade into the surrounding normal brain tissue. These invasive cells render complete resection impossible, confer significant resistance to chemo- and radiation-therapy, and virtually assure tumor recurrence. Expression of TROY (TNFRSF19), a member of the TNF receptor superfamily, inversely correlates with patient survival and stimulates glioblastoma cell migration and invasion in vitro. In this study, we report that TROY is overexpressed in glioblastoma tumor specimens and TROY mRNA expression is increased in the invasive cell population in vivo. In addition, inappropriate expression of TROY in mouse astrocytes in vivo using glial-specific gene transfer in transgenic mice induces astrocyte migration within the brain, validating the importance of the TROY signaling cascade in glioblastoma cell migration and invasion. Knockdown of TROY expression in primary glioblastoma xenografts significantly prolonged survival in vivo. Moreover, TROY expression significantly increased resistance of glioblastoma cells to both IR- and TMZ-induced apoptosis via activation of Akt and NF-κB. Inhibition of either Akt or NF-κB activity suppressed the survival benefits of TROY signaling in response to TMZ treatment. These findings position aberrant expression and/or signaling by TROY as a contributor to the dispersion of glioblastoma cells and therapeutic resistance. Implications: Targeting of TROY may increase tumor vulnerability and improve therapeutic response in glioblastoma. Mol Cancer Res; 11(8); 865–74. ©2013 AACR.

The association of 5-HTTLPR genotype and depressive symptoms is moderated by physical activity

The s allele serotonin transporter polymorphic region (5-HTTLPR) is associated with a number of physiological mechanisms that may increase the risk of elevated depressive symptoms. However, reports of a relationship between serotonin transporter polymorphic region (5-HTTLPR) genotype and depressive symptoms have thus far been inconclusive. This heterogeneity of results suggests that other factors may be moderating the relationship between 5-HTTLPR and depressive symptoms. Higher levels of physical activity are associated with lower levels of depressive symptoms. Mechanisms responsible for this association include alterations of the serotonergic system and the hypothalamic-pituitary axis. The aim of the current study was to measure the moderating effect of physical activity on the relationship between 5-HTTLPR genotype and depressive symptoms. Participants, ages 18-23, provided a saliva sample for DNA analysis and completed questionnaires to assess depressive symptoms and physical activity. A hierarchical multiple regression analysis was conducted to examine the moderating effect of physical activity on the relationship between 5-HTTLPR genotype and depressive symptoms. Analysis revealed a significant interaction between 5-HTTLPR and physical activity (p = .010). At low levels of physical activity, individuals with at least one s allele had significantly higher levels of depressive symptoms compared to ll individuals (p = .011). This finding provides preliminary support for a moderating effect of physical activity on the relationship between 5-HTTLPR and depressive symptoms.

Abstract 3785: Elevated expression of Fn14 in non-small cell lung cancer correlates with c-MET and promotes tumor cell invasion and metastasis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor invasiveness and therapeutic resistance strongly contribute to the low (<10%) five-year survival rate for advanced stage lung cancer; thus, identification of novel interventions targeting metastasis and therapeutic resistance is a necessity. Hepatocyte growth factor receptor (c-Met) and fibroblast growth factor-inducible 14 (Fn14) are two cell surface receptors known to be associated with lung cancer invasiveness and cell survival. Our lab recently demonstrated that Fn14 was over-expressed in non-small cell lung cancer (NSCLC), correlated with activated epidermal growth factor receptor, and that expression of Fn14 modulated NSCLC cell invasiveness and metastasis. We hypothesize that elevated Fn14 expression can be maintained by c-Met activation, and that blockade of Fn14 can suppress NSCLC invasiveness driven by c-Met. Here we demonstrate that Fn14 protein expression significantly correlated with c-Met expression in primary NSCLC patient tumors. Fn14 and c-Met were more highly expressed in metastatic tumors compared to patient-matched primary NSCLC tumors, suggesting a role in the metastatic phenotype. To further elucidate the relationship between Fn14 and c-Met in NSCLC invasiveness, we employed two cell lines derived from a primary tumor (H2073) and metastatic lymph node (H1993) of the same patient. H1993 cells (c-Met receptor amplification) expressed significantly elevated protein levels of Fn14 compared to H2073 (low c-Met), which were significantly diminished in H1993 cells upon c-Met inhibition through SU11274 treatment. Moreover, exposure of H2073 cells to HGF induced the protein expression of Fn14. The elevated expression of Fn14 through HGF stimulation (H2073) or c-Met receptor amplification (H1993) was attenuated by pretreatment of the cells with U0126, a potent MEK inhibitor, suggesting that c-Met-induced Fn14 expression may depend on MAPK signaling. To further elucidate the effect of Fn14 on global c-Met-induced expression, genome-wide expression analysis was employed to determine genes both over-expressed in H1993 compared to H2073 and subsequently suppressed in Fn14 depleted H1993 cells. RALGPS2, a gene over-expressed in lung tumors that induces of c-fos and OPRL1, a gene elevated in metastatic melanoma, were elevated in the metastatic cell line and subsequently suppressed with Fn14 depletion. In addition, shRNA-mediated depletion of Fn14 in H1993 cells attenuated cell migration and invasion by 45% and 40%, respectively. This work demonstrates that c-Met and Fn14 may play a critical role in NSCLC metastasis, and suggests that targeting Fn14 may impact c-Met-driven NSCLC invasiveness. Successful targeting of NSCLC invasiveness would have significant clinical impact on a disease that is deadly in its advanced stages. Citation Format: Timothy G. Whitsett, Shannon P. Fortin, Paul A. Kurywchak, Julianna Ross, Janine LoBello, Christopher B. Kingsley, Jeffrey W. Allen, Glen J. Weiss, Nhan L. Tran. Elevated expression of Fn14 in non-small cell lung cancer correlates with c-MET and promotes tumor cell invasion and metastasis. [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 3785. doi:10.1158/1538-7445.AM2013-3785

Abstract 2621: TNFRSF19 (TROY) promotes glioma cell survival signaling and therapeutic resistance.

Glioblastoma multiforme (GBM) is the most common primary central nervous system tumor accounting for approximately 40% of all primary malignant brain tumors. The mechanism driving the development and recurrence of GBM is still largely unknown which greatly limits the successful treatment of this disease. The tumor necrosis factor receptor superfamily member TNFRSF19 (TROY) is a type I cell surface receptor protein containing the highly conserved TNFR cysteine-rich motifs in the extracellular domain and a tumor necrosis factor-receptor-associated factor (TRAF) - binding sequence in the cytoplasmic domain. We recently demonstrated that increased expression of TROY stimulated glioma cell migration in vitro and increased cell invasion in an organotypic brain slice model. Conversely, siRNA mediated knockdown of TROY expression inhibited glioma cell migration. In addition, profiling of TROY in brain tumor samples indicated that TROY mRNA expression was significantly increased in GBM samples, directly correlated with increasing glial tumor grade, and inversely correlated with patient outcome suggesting that TROY expression may play a role in GBM invasion and is a good indicator of survival outcome. In the current study, we investigated the role of TROY in therapeutic resistance and survival signaling. We report that TROY protein expression was significantly increased in patient GBM tumor samples with TROY mRNA exhibiting increased expression in the invasive cell population. Aberrant expression of TROY in mouse astrocytes in situ using glial-specific gene transfer in transgenic mice induced astrocyte migration within the brain supporting an important role for TROY in glioma cell migration. Notably, increased TROY expression did not increase cell proliferation but increased resistance of glioma cells to both radiation and temozolomide induced apoptosis while knockdown of TROY increased temozolomide sensitivity. TROY induced resistance to TMZ was dependent upon Akt and NF-κB activation. We also report that TROY induced NF-κB phosphorylation and stimulation of migration required the membrane proximal region of the TROY cytoplasmic domain and that knockdown of TROY expression increased survival in a xenograft model. The current results further support a role for TROY in GBM and suggest that targeting TROY and its signaling pathway represents a potential approach to increase tumor vulnerability and improve the therapeutic response of glioblastoma. Citation Format: Harshil D. Dhruv, Serdar Tuncali, Jean Kloss, Zhongbo Yang, Cassie Schumacher, Bart Williams, Julianna Ross, Nhan Tran, Joseph Loftus. TNFRSF19 (TROY) promotes glioma cell survival signaling and therapeutic resistance. [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 2621. doi:10.1158/1538-7445.AM2013-2621