Extracellular vesicle proteomic analysis leads to the discovery of HDGF as a new factor in multiple myeloma biology.

Identifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study, hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration, indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed, HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters naïve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary, HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME.

Stage-Specific Non-Coding RNA Expression Patterns during In Vitro Human B Cell Differentiation into Antibody Secreting Plasma Cells.

The differentiation of B cells into antibody secreting plasma cells (PCs) is governed by a strict regulatory network that results in expression of specific transcriptomes along the activation continuum. In vitro models yielding significant numbers of PCs phenotypically identical to the in vivo state enable investigation of pathways, metabolomes, and non-coding (ncRNAs) not previously identified. The objective of our study was to characterize ncRNA expression during human B cell activation and differentiation. To achieve this, we used an in vitro system and performed RNA-seq on resting and activated B cells and PCs. Characterization of coding gene transcripts, including immunoglobulin (Ig), validated our system and also demonstrated that memory B cells preferentially differentiated into PCs. Importantly, we identified more than 980 ncRNA transcripts that are differentially expressed across the stages of activation and differentiation, some of which are known to target transcription, proliferation, cytoskeletal, autophagy and proteasome pathways. Interestingly, ncRNAs located within Ig loci may be targeting both Ig and non-Ig-related transcripts. ncRNAs associated with B cell malignancies were also identified. Taken together, this system provides a platform to study the role of specific ncRNAs in B cell differentiation and altered expression of those ncRNAs involved in B cell malignancies.

Chemotherapy can induce weight normalization of morbidly obese mice despite undiminished ingestion of high fat diet.

Morbidly obese patients who accomplish substantial weight loss often display a long-term decline in their resting metabolism, causing even relatively restrained caloric intake to trigger a relapse to the obese state. Paradoxically, we observed that morbidly obese mice receiving chemotherapy for cancer experienced spontaneous weight reduction despite unabated ingestion of their high fat diet (HFD). This response to chemotherapy could also be achieved in morbidly obese mice without cancer. Optimally dosed methotrexate (MTX) or cyclophosphamide (CY) enabled the mice to completely and safely normalize their body weight despite continued consumption of obesogenic quantities of HFD. Weight reduction was not attributable to decreased HFD intake, enhanced energy expenditure or malabsorption. MTX or CY dosing significantly depleted both adipose tissue and preadipocyte progenitors. Remarkably, however, despite continued high fat feeding, a compensatory increase in hepatocyte lipid storage was not observed, but rather the opposite. Gene microarray liver analyses demonstrated that HFD mice receiving MTX or CY experienced significantly inhibited lipogenesis and lipid storage, whereas Enho (energy homeostasis) gene expression was significantly upregulated. Further metabolic studies employing a human hepatocellular line revealed that MTX treatment preserved robust oxidative phosphorylation, but also promoted mitochondrial uncoupling with a surge in proton leak. This is the first report that certain optimally dosed chemotherapeutic agents can induce weight loss in morbidly obese mice without reduced dietary intake, apparently by depleting stores of adipocytes and their progenitors, curtailment of lipogenesis, and inconspicuous disposal of incoming dietary lipid via a steady state partial uncoupling of mitochondrial oxidative phosphorylation.

MUC1 Vaccines, Comprised of Glycosylated or Non-Glycosylated Peptides or Tumor-Derived MUC1, Can Circumvent Immunoediting to Control Tumor Growth in MUC1 Transgenic Mice.

It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4(+) and CD8(+) T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape.

Inhibition of adaptive immunity by IL9 can be disrupted to achieve rapid T-cell sensitization and rejection of progressive tumor challenges.

The tolerogenic cytokine IL9 promotes T regulatory cell function and allergic airway inflammation, but it has not been extensively studied in cancer. In this report, we used IL9-deficient mice to investigate the effects of IL9 in multiple models of breast and colon cancer development. Eliminating endogenous IL9 enabled sensitization of host T cells to tumors, leading to their early rejection without the requirement of vaccines or immunomodulatory therapies. Notably, IL9-deficient mice acquired immunologic memory, which actively protected from residual disease and tumor rechallenge, an effect linked to activation of CD8(+) T cells. Depletion of either CD8(+) or CD4(+) T cells abolished the benefits of IL9 loss to tumor control. Adoptive transfer experiments showed that T cells from tumor-rejecting IL9-deficient mice retained their effector competency in wild-type animals. Moreover, neutralizing IL9 antibody phenocopied the effects of IL9 gene deletion by slowing tumor progression in wild-type animals. Our results show the ability of IL9 to function as an inhibitor of adaptive immunity that prevents the formation of immunologic memory to a growing tumor, highlighting the potential for IL9 neutralization as a unique tool for cancer immunotherapy.

Signals through 4-1BB inhibit T regulatory cells by blocking IL-9 production enhancing antitumor responses.

Previous studies from our laboratory indicate that intratumoral (i.t.) injections of CpG-ODN are the most effective adjuvant strategy to induce an antitumor immune response in tolerant BALB-neuT mice but insufficient for tumor eradication. We evaluated whether this treatment strategy could be enhanced by the presence of anti-OX40 and anti-4-1BB antibodies. Treatment with anti-4-1BB resulted in a greater antitumor response than anti-OX40. The results indicate that anti-4-1BB but not anti-OX40 inhibited the suppressive function of T regulatory cells (Tregs). Through microarray analysis we evaluated the mechanism by which anti-4-1BB inhibits iTregs using the Foxp3-GFP mice. We observed specific transcriptional differences in over 100 genes in iTregs treated with anti-4-1BB, and selected those genes that remained unaffected by exposure to anti-OX40. Interleukin 9 was transcriptionally down-regulated 28-fold by anti-4-1BB treatment, and this was matched by a significant reduction of IL-9 secretion by iTregs. Furthermore, blockade of the common γ-chain receptor resulted in the inhibition of iTreg-suppressive function. More importantly, neutralization of IL-9 plus i.t. injections of CpG-ODN induces tumor rejection in BALB-neuT and MUC-1 tolerant transgenic mice. These results indicate that IL-9 plays a role in iTreg biology during the tumor inflammatory process enhancing/promoting the suppressive function of these cells and that the blockade of IL-9 could serve as a novel strategy to modulate the function of Tregs to enhance the antitumor effect of tumor vaccines.

Foxp3-positive macrophages display immunosuppressive properties and promote tumor growth.

Regulatory T cells (T reg cells) are characterized by the expression of the forkhead lineage-specific transcription factor Foxp3, and their main function is to suppress T cells. While evaluating T reg cells, we identified a population of Foxp3-positive cells that were CD11b(+)F4/80(+)CD68(+), indicating macrophage origin. These cells were observed in spleen, lymph nodes, bone marrow, thymus, liver, and other tissues of naive animals. To characterize this subpopulation of macrophages, we devised a strategy to purify CD11b(+)F4/80(+)Foxp3(+) macrophages using Foxp3-GFP mice. Analysis of CD11b(+)F4/80(+)Foxp3(+) macrophage function indicated that these cells inhibited the proliferation of T cells, whereas Foxp3(-) macrophages did not. Suppression of T cell proliferation was mediated through soluble factors. Foxp3(-) macrophages acquired Foxp3 expression after activation, which conferred inhibitory properties that were indistinguishable from natural Foxp3(+) macrophages. The cytokine and transcriptional profiles of Foxp3(+) macrophages were distinct from those of Foxp3(-) macrophages, indicating that these cells have different biological functions. Functional in vivo analyses indicated that CD11b(+)F4/80(+)Foxp3(+) macrophages are important in tumor promotion and the induction of T reg cell conversion. For the first time, these studies demonstrate the existence of a distinct subpopulation of naturally occurring macrophage regulatory cells in which expression of Foxp3 correlates with suppressive function.

Comparative kinetic analyses of gene profiles of naïve CD4+ and CD8+ T cells from young and old animals reveal novel age-related alterations.

It is well established that immune responses are diminished in the old. However, we still do not have a clear understanding of what dictates the dysfunction of old T cells at the molecular level. Although microarray analysis has been used to compare young and old T cells, identifying hundreds of genes that are differentially expressed among these populations, it has been difficult to utilize this information to pinpoint which biological pathways truly affect the function of aged T cells. To better define differences between young and old naïve CD4+ and CD8+ T cells, microarray analysis was performed pre- and post-TCR stimulation for 4, 12, 24 and 72 h. Our data indicate that many genes are differentially expressed in the old compared to the young at all five time points. These genes encode proteins involved in multiple cellular functions such as cell growth, cell cycle, cell death, inflammatory response, cell trafficking, etc. Additionally, the information from this microarray analysis allowed us to underline both intrinsic deficiencies and defects in signaling only seen after activation, such as pathways involving T-cell signaling, cytokine production, and Th2 differentiation in old T cells. With the knowledge gained, we can proceed to design strategies to restore the function of old T cells. Therefore, this microarray analysis approach is a powerful and sensitive tool that reveals the extensive changes seen between young and old CD4+ and CD8+ naïve T cells. Evaluation of these differences provides in-depth insight into potential functional and phenotypical differences among these populations.

Blockade of CCL1 inhibits T regulatory cell suppressive function enhancing tumor immunity without affecting T effector responses.

Intratumoral accumulation of T regulatory cells (Tregs) creates an immunosuppressive environment that reduces the efficacy of antitumor immunotherapy. The immunosuppressive milieu within tumors is largely brought about by the presence of Tregs, which maintain self-tolerance by directly inhibiting T cells, NK cells, and dendritic cells. Depletion of Tregs enhances antitumor immune responses; however, current depletion therapies also affect the function of CD4 and CD8 T effector cells. Previous studies from our laboratory indicate that intratumoral delivery of CpG-ODN strongly reduces the levels of Tregs within the tumor, which is mainly mediated by IL-6. Because IL-6 promotes growth of some human cancers, alternate pathways to inactivate Tregs were sought through microarray analysis, resulting in gene candidates that can be exploited to modulate the function of Tregs. Analysis of these candidates indicates that neutralization of chemokine (C-C motif) ligand 1 (CCL1) prevented de novo conversion and suppressive function of Tregs without affecting the function of T effector cells. The combination of CpG-ODN and anti-CCL1 treatments induced complete rejection of tumors in BALB-neuT tolerant mice, and result in the generation of long-term protective memory responses. Tumor rejection correlated with changes in the lymphocyte composition within the tumor; we observed decreased Treg numbers and a concomitant accumulation of tumoricidal cells such as CD8+NKG2D+ and NK cells. These studies demonstrate that neutralization of CCL1 can be used as an adjuvant to antitumor immunotherapy, as a means of reversing the immunosuppressive function of Tregs without compromising T cell effector function.

The phosphorylation of ephrin-B2 ligand promotes glioma cell migration and invasion.

To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. Pathway enrichment analysis highlighted the bidirectional receptor/ligand tyrosine kinase system, EphB/ephrin-B, as the most tightly linked system to the invading cell phenotype. Clinical relevance of ephrin-B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of ephrin-B1 and -B2 mRNA were significantly higher in GBM (n = 82) than in normal brain (n = 24). Kaplan-Meier analysis demonstrated ephrin-B2, but not ephrin-B1, expression levels were significantly associated with short term survival in malignant astrocytomas (n = 97, p = 0.016). In human brain tumor specimens, the production and phosphorylation of ephrin-B2 were high in GBM. Immunohistochemistry demonstrated ephrin-B2 localization primarily in GBM cells but not in normal brain. A highly invasive glioma cell line, U87, expressed high levels of ephrin-B2 compared with relatively less invasive cell lines. Treatment with EphB2/Fc chimera further enhanced migration and invasion of U87 cells, whereas treatment with an ephrin-B2 blocking antibody significantly slowed migration and invasion. Forced expression of ephrin-B2 in the U251 cell line stimulated migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B2. These results demonstrate that high expression of ephrin-B2 is a strong predictor of short-term survival and that ephrin-B2 plays a critical role in glioma invasion rendering this signaling pathway as a potential therapeutic target.

Glioma cells on the run - the migratory transcriptome of 10 human glioma cell lines.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. RESULTS: Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. CONCLUSION: Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm.

Autotaxin: a secreted autocrine/paracrine factor that promotes glioma invasion.

Glioblastoma multiforme (GBM) is inherently invasive, and it is from the invasive cell population that the tumor recurs. The GBM invasion transcriptome reveals over-expression of various autocrine factors that could act as motility drivers, such as autotaxin (ATX). Some of these factors could also have paracrine roles, modulating the behavior of cells in the peri-tumoral brain parenchyma. ATX generates lysophosphatidic acid (LPA), which signals through LPA receptors expressed by GBM as well as in astrocytes, oligodendrocytes (ODC) and microglia; their activation manifest cell specific effects. ATX stimulates invasion of GBM cells in vitro and ex vivo invasion assays. ATX activity enhances GBM adhesion in cells expressing the LPA1 receptor, as well as stimulating rac activation. GBM secreted ATX can also have paracrine effects: ATX activity results in reduced ODC adhesion. ODC monolayer invasion showed that U87 and U251 GBM cells expressing ATX invaded through an ODC monolayer significantly more than cells depleted of ATX or cells expressing inactive ATX, suggesting that GBM cells secreting ATX find ODCs less of a barrier than cells that do not express ATX. Secreted factors that drive GBM invasion can have autocrine and paracrine roles; one stimulates GBM motility and the other results in ODC dis-adhesion.

CpG-ODN but not other TLR-ligands restore the antitumor responses in old mice: the implications for vaccinations in the aged.

AIM: There is accumulative evidence indicating that targeting antigen presenting cells (APCs) with different types of adjuvants could result in the induction of antitumor immune responses. It has been hypothesized that APCs function may be altered in the elderly contributing to a decline in the immune function. We evaluated whether targeting APCs following injection with Poly I:C, LPS, flagellin, imiquimod and CpG-ODN would induce an antitumor response in the old. MATERIALS AND METHODS: The immune and antitumor responses induce Poly I:C, LPS, flagellin, imiquimod and CpG-ODN were compared in young (2 month old) and old (18 months) mice. RESULTS: Our results indicated that only intratumoral (i.t.) injections of CpG-ODN completely rejected the tumor in both young and old mice. Injections of Poly I:C also induced the rejection of tumors in the young but not in the old. Furthermore, i.t. injections of CpG-ODN promoted the development of protective memory responses in the young and the old. Analysis of the immune responses in the old indicated that CpG-ODN but not Poly-I:C induces: a pro-inflammatory Th1 type response; accumulation and activation of CD4+, CD8+ T and, NK cell responses; activation of APCs; and reduction in the number of Tregs. The activation of these immune-parameters positively correlates with the induction of an antitumor response. CONCLUSIONS: These studies indicate that there are differences in the level of stimulation with TLR-ligands between young and old APCs and that the aged immune responses can be rescued and exploited for the induction of tumor immunity by targeting APCs with specific TLR-ligands. These results have important clinical implications for developing immunization strategies containing TLR-ligands that will be effective in both the young and old.

Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.

Invasion is a defining hallmark of glioblastoma multiforme, just as metastasis characterizes other high-grade tumors. Glial tumors invariably recur due to the regrowth of invasive cells, which are unaffected by standard treatment modalities. Drivers of glioma invasion include autocrine signals propagated by secreted factors that signal through receptors on the tumor. These secreted factors are able to diffuse through the peritumoral stroma, thereby influencing parenchymal cells that surround the tumor mass. Here we describe various autocrine motility factors that are expressed by invasive glioma cells and explore the effects that they may have on normal cells present in the path of invasion. Conversely, normal brain parenchymal cells secrete ligands that can stimulate receptors on invasive glioma cells and potentially facilitate glioma invasion or create a permissive microenvironment for malignant progression. Parallel observations have been made for solid tumors of epithelial origin, in which parenchymal and stromal cells either support or suppress tumor invasion. Most autocrine and paracrine interactions involved in glioma invasion constitute known signaling systems in stages of central nervous system development that involve the migration of precursor cells that populate the developing brain. Key paracrine interactions between glioma cells and the brain microenvironment can influence glioma pathobiology and therefore contribute to its poor prognosis. Current therapies for glioma that could have an impact on paracrine communication between tumors and normal cells are discussed. We suggest that cells in the normal brain parenchyma be considered as potential targets for adjuvant therapies to control glioma growth because such cells are less likely to develop resistance than glioma cells.

MAP-ing glioma invasion: mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival.

Although astrocytic brain tumors do not metastasize systemically, during tumorigenesis glioma cells adopt an invasive phenotype that is poorly targeted by conventional therapies; hence, glioma patients die of recurrence from the locally invasive tumor population. Our work is aimed at identifying and validating novel therapeutic targets and biomarkers in invasive human gliomas. Transcriptomes of invasive glioma cells relative to stationary cognates were produced from a three-dimensional spheroid in vitro invasion assay by laser capture microdissection and whole human genome expression microarrays. Qualitative differential expression of candidate invasion genes was confirmed by quantitative reverse transcription-PCR, clinically by immunohistochemistry on tissue microarray, by immunoblotting on surgical specimens, and on two independent gene expression data sets of glial tumors. Cell-based assays and ex vivo brain slice invasion studies were used for functional validation. We identify mitogen-activated protein kinase (MAPK) kinase 3 (MKK3) as a key activator of p38 MAPK in glioma; MKK3 activation is strongly correlated with p38 activation in vitro and in vivo. We further report that these members of the MAPK family are strong promoters of tumor invasion, progression, and poor patient survival. Inhibition of either candidate leads to significantly reduced glioma invasiveness in vitro. Consistent with the concept of synthetic lethality, we show that inhibition of invasion by interference with these genes greatly sensitizes arrested glioma cells to cytotoxic therapies. Our findings therefore argue that interference with MKK3 signaling through a novel treatment combination of p38 inhibitor plus temozolomide heightens the vulnerability of glioma to chemotherapy.

Increased fibroblast growth factor-inducible 14 expression levels promote glioma cell invasion via Rac1 and nuclear factor-kappaB and correlate with poor patient outcome.

Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion throughout the central nervous system. Understanding genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. Our current report shows that the fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 overexpression stimulates glioma cell migration and invasion, and depletion of Rac1 by small interfering RNA inhibits this cellular response. Activation of Fn14 signaling by the ligand TNF-like weak inducer of apoptosis (TWEAK) stimulates migration and up-regulates expression of Fn14; this TWEAK effect requires Rac1 and nuclear factor-kappaB (NF-kappaB) activity. The Fn14 promoter region contains NF-kappaB binding sites, which mediate positive feedback causing sustained overexpression of Fn14 and enduring glioma cell invasion. Furthermore, Fn14 gene expression levels increase with glioma grade and inversely correlate with patient survival. These results show that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Such a feedback loop argues for aggressive targeting of the Fn14 axis as a unique and specific driver of glioma malignant behavior.

Gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets.

The invasive phenotype of glioblastoma multiforme (GBM) is a hallmark of malignant process, yet molecular mechanisms that dictate this locally invasive behavior remain poorly understood. Gene expression profiles of human glioma cells were assessed from laser capture-microdissected GBM cells collected from paired patient tumor cores and white matter-invading cell populations. Changes in gene expression in invading GBM cells were validated by quantitative reverse transcription polymerase chain reaction (QRT-PCR) and immunohistochemistry in an independent sample set. QRT-PCR confirmed the differential expression in 19 of 21 genes tested. Immunohistochemical analyses of autotaxin (ATX), ephrin B3, B-cell lymphoma-w (BCLW), and protein tyrosine kinase 2 beta showed them to be expressed in invasive glioma cells. The known GBM markers, insulin-like growth factor binding protein 2 and vimentin, were robustly expressed in the tumor core. A glioma invasion tissue microarray confirmed the expression of ATX and BCLW in invasive cells of tumors of various grades. GBM phenotypic and genotypic heterogeneity is well documented. In this study, we show an additional layer of complexity: transcriptional differences between cells of tumor core and invasive cells located in the brain parenchyma. Gene products supporting invasion may be novel targets for manipulation of brain tumor behavior with consequences on treatment outcome.

Differential gene expression in auristatin PHE-treated Cryptococcus neoformans.

The antifungal pentapeptide auristatin PHE was recently shown to interfere with microtubule dynamics and nuclear and cellular division in the opportunistic pathogen Cryptococcus neoformans. To gain a broader understanding of the cellular response of C. neoformans to auristatin PHE, mRNA differential display (DD) and reverse transcriptase PCR (RT-PCR) were applied. Examination of approximately 60% of the cell transcriptome from cells treated with 1.5 times the MIC (7.89 micro M) of auristatin PHE for 90 min revealed 29 transcript expression differences between control and drug-treated populations. Differential expression of seven of the transcripts was confirmed by RT-PCR, as was drug-dependent modulation of an additional seven transcripts by RT-PCR only. Among genes found to be differentially expressed were those encoding proteins involved in transport, cell cycle regulation, signal transduction, cell stress, DNA repair, nucleotide metabolism, and capsule production. For example, RHO1 and an open reading frame (ORF) encoding a protein with 91% similarity to the Schizophyllum commune 14-3-3 protein, both involved in cell cycle regulation, were down-regulated, as was the gene encoding the multidrug efflux pump Afr1p. An ORF encoding a protein with 57% identity to the heat shock protein HSP104 in Pleurotus sajor-caju was up-regulated. Also, three transcripts of unknown function were responsive to auristatin PHE, which may eventually contribute to the elucidation of the function of their gene products. Further study of these differentially expressed genes and expression of their corresponding proteins are warranted to evaluate how they may be involved in the mechanism of action of auristatin PHE. This information may also contribute to an explanation of the selectivity of auristatin PHE for C. neoformans. This is the first report of drug action using DD in C. neoformans.

The human Fn14 receptor gene is up-regulated in migrating glioma cells in vitro and overexpressed in advanced glial tumors.

Glioblastoma multiforme comprises the majority of human brain tumors. Patients with glioblastoma multiforme have poor survival rates, with an average life expectancy of <1 year. To assess possible mechanisms and to potentially target invasive glioma cells, we previously measured the gene expression profiles of glioma cells under migration-activated or passive states. One of the genes identified was Fn14, which encodes a cell surface receptor for the tumor necrosis factor superfamily member named TWEAK. In this study, we show that Fn14 gene expression is induced in migration-activated glioma cells in vitro and significantly increases according to tumor grade in vivo (P < 0.01), with highest levels in glioblastoma tissue specimens. The in situ expression pattern of Fn14 mRNA and protein was confined to primary glioma cells and the vascular endothelium, with no detection in adjacent normal brain. Conversely, TWEAK mRNA levels are low in glioblastoma samples relative to normal brain tissue. In addition, activation of the Fn14 receptor by addition of recombinant TWEAK resulted in increased glioma cell migration in vitro. These results suggest a positive role for TWEAK and Fn14 in glioma progression and indicate that Fn14 gene expression may serve as a marker for invasive glioma cells.