|
|
|
 |
||
|
Vision for Clinical Research at the CCR
The desire to obtain maximal information from every clinical protocol offered at the Clinical Center drives our investment in genomic profiling. Analysis of every biospecimen donated by patients enrolled in our clinical trials is a major part of our information gathering. Our clinical researchers use a wealth of technologies and analysis platforms to obtain sophisticated data from each biospecimen collected. Aware that such molecular information may soon be critically relevant to establishing markers of drug response or patient stratification—information needed to usher new drugs through clinical trials more efficiently—we have established a centralized facility of human biospecimens for clinical research. We collect material using standardized methods and approaches to ensure that samples are of the highest quality. This care at the outset makes subsequent analysis both possible and reliable. Our newly established Clinical Molecular Profiling Core is at the heart of our commitment to biospecimen collection and our capacity to perform sophisticated analysis. This core, headed by Paul Meltzer, PhD, will coordinate a complex series of genetic and genomic analyses on human samples collected during a patient’s participation in clinical trials. The samples, with patient consent, will be procured under NCI’s new guidelines for biospecimens and will be subjected to the most advanced technologies to interrogate the underlying disease using as many approaches as are feasible, based on the amount and type of tissue available. We will analyze and mine our acquired data to advance our understanding of the underlying mechanisms and process of cancer, and we will correlate clinical results with molecular targets and pathways where possible. With much optimism, I welcome this addition to our clinical infrastructure. With this powerful technology, we will accelerate our ability to move discoveries made at the laboratory bench to the clinical setting and benefit many cancer patients.
Application of Integrative Functional Genomics To Decode Cancer Signatures
Orthologous human and mouse genes from both data sets were selected, and the gene expression data were integrated after standardizing the relative expression levels for both species. In hierarchical clustering analysis of integrated data, gene expression patterns of HCC from Myc, E2f1, and Myc/E2f1 mice were most similar to those of the better survival group of human HCC, whereas the expression patterns of Myc/Tgfa and diethylnitrosamine (DENA)–induced mouse HCC were most similar to those of the poorer survival group of human HCC. These results suggest that these two classes of mouse models might closely recapitulate the molecular patterns of the two subclasses of human HCC. The similarity of gene expression profiles between human and mouse models are in good agreement with the phenotypic characteristics of the tumors (Figure 1). The human tumors with increased proliferation, decreased apoptosis, and worse prognosis are paired with the mouse models that have the same characteristics. The gene expression–based prediction of mouse models is highly concordant with the phenotypes of mice. Myc/Tgfa mice have a typically poor prognosis phenotype, such as an earlier and higher incident rate of HCC development and higher mortality, genomic instability, and expression of poor prognostic marker. Myc and Myc/E2f1 mice have a relatively higher mutation frequency regarding β-catenin as well as a higher nuclear accumulation of the protein, which in human HCC are indicative of lower genomic instability and better prognosis. The fact that these findings were first uncovered by using unsupervised methods and validated later using supervised methods indicates that the underlying principles in gene expression changes are conserved between mouse and human HCC. Figure 1. Phenotypic similarities between hepatocellular carcinomas (HCCs) generated in transgenic mouse models and subclasses A and B of human HCC. The best-fit HCC mouse models can be used to test hypotheses on tumor progression that are generated by analysis of cross-species gene expression patterns or from other experimental data. These models should also be extremely valuable for testing both potential therapeutic targets identified in human studies and preclinical trials of drugs. H, high; L, low; M, medium. In our second study, we extended this comparative functional genomic approach to address the issue pertaining to the cell(s) of origin for tumors. It is axiomatic that cancer cells evolve from normal cells after accumulation of genetic and epigenetic alterations. Also, it has been shown that the gene expression patterns in cancer cells reflect these alterations. Nevertheless, a considerable fraction of the gene expression program of cancer cells is characteristic of the non-transformed cellular lineages from which they originated. Furthermore, analysis of gene expression profiles of cancer cell lines indicated that neither physiological adaptation in vivo nor experimental adaptation in vitro is sufficient to abolish the gene expression programs acquired during development. These data suggest that the global gene expression profiles of tumors might provide critical information on the cellular origin of tumors. Because HCC could originate from both adult hepatocytes and hepatic progenitor cells, we decided to test whether global gene expression analysis of human HCC could identify subtypes of HCC derived from these different cell types. The experimental strategy involved the generation of gene expression data from multiple species suitable for integration and cross-comparison. We integrated, using only orthologous genes, gene expression data from rat fetal hepatoblasts and adult rat hepatocytes with HCC gene expression data from human and mouse models. By applying hierarchical clustering analysis of gene expression patterns from human HCC, mouse HCC, rat fetal hepatoblasts, and adult rat hepatocytes, we identified a new prognostic subtype of HCC that shares gene expression patterns with fetal hepatoblasts. The hepatoblast (HB) subtype is distinguished from other types of HCC by the differential expression of hundreds of genes, and the robustness of this gene expression signature in the HB subtype was validated in an independent cohort of HCC patients. HCC patients who shared a gene expression pattern with fetal hepatoblasts had a poor prognosis. The gene expression program that distinguished this subtype from other types of HCC included markers of hepatic oval cells, suggesting that HCC of this subtype arises from hepatic progenitor cells. Application of network-based pathway analyses of gene expression provided important insights into the pathogenesis of the HB subtype of HCC (Figure 2). Enrichment of predicted JUN and FOS activity in the HB subtype led us to hypothesize that the activator protein 1 (AP-1) complex was the major driving force in tumorigenesis of the HB subtype. Previous studies have shown that Jun is essential for normal liver development, and it could also be crucial for the initiation of HCC development in mice. Furthermore, higher expression of JUN target genes involved in invasive phenotypes (e.g., MMP1, PLAUR, TIMP1, CD44, and VIL2) was observed in the HB subtype of HCC, indicating the cellular origin of these tumors and accounting for the poor prognosis of the affected individuals. Figure 2. Gene networks of activator protein 1 (AP-1) transcription factors in the HCC subtype with liver progenitor cell signature. Upregulated and downregulated genes in the HCC subtype, with progenitor signature, are indicated in red and green, respectively. Genes in gray color are not on the list but are associated with the regulated genes. Gray lines and arrows represent the direction of transcriptional regulation, and plus and minus signs indicate positive and negative regulation of gene expression. Purple lines represent known physical interactions between connected genes. The success of the new experimental and analytical approaches presented here strongly suggests that more integration of independent data sets will enhance our ability to identify key regulatory elements in cancer development. It is, therefore, reasonable to expect that the clinical inference from transcriptome analyses will be significantly strengthened when gene expression data are integrated with diverse genomics information obtained from DNA sequence, array-based comparative genomic hybridization (CGH), and noncoding gene (i.e., microRNA) expression analyses.
Nucleophosmin: A Ran/Crm1-associated Licensing Factor That Regulates Centrosome Duplication
The cellular components that regulate nucleocytoplasmic transport are also independently implicated in spindle assembly (Weis K. Cell 112: 441–51, 2003). This process involves import (importins α and β) and export (Crm1) receptors that bind to nuclear localization signals (NLS) or nuclear export signals (NES), respectively. Ran, a small GTPase, controls the interaction of these receptors with their substrates. A fraction of Ran, Crm1, and RanBP1 (a major regulator of Ran that promotes Crm1 dissociation from Ran) is found on centrosomes. Crm1 inactivation, either by a Crm1-specific inhibitor, leptomycin B (LMB), or hepatitis B virus (HBV) HBx protein interaction (via its NES motif), results in supernumerary centrosomes and multipolar spindles (Forgues M et al. Mol Cell Biol 23: 5282–92, 2003; Forgues M et al. J Biol Chem 276: 22797–803, 2001). This implies that Crm1 may function to prevent unscheduled centriole splitting during mitosis, thereby ensuring the formation of a bipolar spindle. Aneuploidy can be detected in livers chronically infected with HBV, a preneoplastic condition predisposing individuals to hepatocellular carcinoma. Multipolar spindles and mitotic abnormalities are also a consequence of Ran mutations or overexpression of RanBP1. Since Ran/Crm1 acts as a receptor to shuttle cellular proteins and interacts with many known cell cycle regulators, components of the Ran/Crm1 pathway likely function as licensing factors to ensure appropriate centrosome duplication during different stages of the cell cycle. Thus, one attractive model is that the Ran/Crm1 complex, through its NES binding activity, may ensure that proteins that regulate centrosome duplication are present at the correct location and time to safeguard the fidelity of this process. Nucleophosmin (NPM or B23) is a ubiquitously expressed phosphoprotein that mainly localizes in the nucleolus and shuttles between the nucleus and the cytoplasm during the cell cycle. NPM associates with unduplicated centrosomes and dissociates from centrosomes upon phosphorylation by CDK2/cyclin E, which coincides with the initiation of centrosome duplication and DNA replication. During mitosis, NPM reassociates with centrosomes. Since NPM has been implicated as a regulator of centrosome synthesis, we thought it plausible that NPM is a substrate for Ran/Crm1 to regulate centrosome duplication. This hypothesis was first tested by a heterokaryon assay to evaluate cytoplasmic-to-nuclear transport of NPM or an NPM NES mutant. In our study, the NES motif of NPM was shown to be functional and its local trafficking was mediated by a Ran/Crm1-dependent process. Since Crm1 and NPM could associate with centrosomes, we determined whether their localization required a functional NES motif. We demonstrated the association of NPM and Crm1 by immunofluorescence-based co-localization on centrosomes and co-fractionation from sucrose gradients to isolate centrosomes. Mutation of NPM NES or disruption of Crm1 function by LMB, RanBP1, or HBx led to NPM dissociation from centrosomes and initiation of premature centrosome duplication. Therefore, this process was mediated by the Ran/Crm1 pathway and required a functional NES motif. To examine the role of NPM in regulating centrosome duplication, we knocked down the expression of endogenous NPM with NPM siRNA. Consistently, NPM siRNA resulted in supernumerary centrosomes that could be nucleated in mitotic cells to form mitotic spindles, an activity that could be effectively inhibited by coexpression of NPM. Thus, loss of NPM is associated with centrosome reduplication. Furthermore, a novel proline-dependent kinase (PDK) phosphorylation site was identified at threonine residue (T95) within the NES motif of NPM. We tested whether phosphorylation at T95 could alter the NES property, thereby preventing NPM binding to Crm1 and docking on centrosomes. A mutant mimicking T95 phosphorylation displayed decreased centrosome binding and supernumerary centrosomes. Since this phosphorylation site is within the NPM NES, it may regulate the binding of NPM to Crm1. Taken together, these results suggest that proper centrosome duplication is mediated by NPM binding to centrosomes through the interaction of its NES motif with Crm1. These findings suggest that the Crm1/Ran complex may act as a “loading dock,” to spatially and temporally coordinate various checkpoint factors to regulate the fidelity of centrosome duplication during cell cycle progression. We have used genetic and biochemical approaches to demonstrate that NPM localization is mediated by a Ran/Crm1-dependent process and, through its NES-based interaction with Crm1, is a potential licensing factor for centrosome duplication (Figure 1). The disruption of such processes may lead to genomic instability and oncogenic acceleration. Figure 1. Ran/Crm1 functions as a loading dock to coordinate “licensing factors” that regulate centrosome duplication. The small GTPase, Ran, switches between an inactive GDP and an active GTP-bound state through interaction with RanBP1 and RCC1, respectively. During early G1 or mitosis, nucleophosmin (NPM) associates with centrosomes through its nuclear export signal (NES) interaction with the Ran/Crm1 complex, thus preventing centrosome reduplication. NPM is then phosphorylated and dissociates from the G1 centrosome upon activation of CDK2/cyclin E, or other kinases, to initiate centrosome duplication. NPM reassociates with mitotic centrosomes upon dephosphorylation. Thus, the Ran/Crm1 network serves as a loading lock to spatially and temporally coordinate NES-containing “licensing factors” that ensure the fidelity of the centrosome duplication process.
TNF Produced by Distinct Types of Leukocytes: The Good and the Bad
Mice with complete or partial TNF ablation may serve as useful models to evaluate the consequences of TNF blockade. In particular, studies in mice have suggested the possibility of deleterious side effects to anti-TNF therapy, which has held true for a fraction of patients who have indeed developed various bacterial infections, including tuberculosis. We used Cre-loxP technology to generate a panel of novel mice with conditional TNF ablation in distinct types of immune cells. One possibility we wanted to evaluate was whether beneficial TNF could predominantly be coming from one cell type and harmful TNF from another. In collaboration with Lino Tessarollo, PhD (Mouse Cancer Genetics Program, NCI, Frederick) and our sister lab at the Engelhardt Institute of Molecular Biology in Moscow, we generated mice with highly efficient and specific TNF ablation in cells of the innate immune system, such as macrophages and neutrophils (M-TNF mice), as well as in both major types of lymphocytes (T-TNF and B-TNF mice). All these mice have shown distinct phenotypes, indicating important and non-redundant functions in vivo for TNF produced by macrophages, T cells, and B cells (Figure 1). Figure 1. Tumor necrosis factor (TNF)–“floxed” mice were generated by homologous recombination in embryonic stem (ES) cells with subsequent removal of neo-cassette. Three different cell type–specific deleter mice were used to generate the experimental panel in the study. Beneficial (green) and detrimental (red) in vivo effects of TNF produced by various types of leukocytes are listed. LPS, lipopolysaccharide; SEB, staphylococcal enterotoxin B; MLys, macrophage lysozyme; loxP, target sequences for the site-specific Cre-recombinase. Mice with TNF ablation in macrophages and neutrophils produced almost no detectable systemic TNF in response to lipopolysaccharide (LPS) and were protected from LPS–D-galactosamine (Dgal) liver toxicity. Under these challenges, both B-TNF and T-TNF mice had the wild-type phenotype. However, in models of toxicity in which T cells were activated by staphylococcal enterotoxin B (SEB) or concanavalin A (ConA), T-TNF mice showed protection from TNF-mediated toxicities. Thus, different toxic agents induce either macrophage/neutrophil or T cell–derived TNF. Macrophage/neutrophil-derived TNF also turned out to be critical in resistance to the intracellular pathogen Listeria monocytogenes. Surprisingly, however, mice with TNF ablation only in T cells also showed defects in host defense against high doses of L. monocytogenes. Importantly, macrophages and neutrophils in T-TNF mice retained full ability to produce high levels of systemic TNF, as indicated by challenge experiments with LPS and other bacterial products. Why couldn’t this abundant TNF compensate for the lack of TNF produced by T cells? What is the intrinsic non-redundant role of T cell–derived TNF? These questions remain to be answered. We hypothesize that T cells produce TNF in such a way that it either remains membrane bound or is released within the space of cell-to-cell contacts. A possible alternative is that in different in vivo situations, macrophages are desensitized and TNF may be produced only by T cells. Although B-TNF mice had a wild-type phenotype in these challenge models, TNF produced by B cells is critically involved in providing maintenance signals for the organized lymphoid tissues, such as in the spleen (Endres R et al. J Exp Med 189: 159–68, 1999) or Peyer’s patches (Tumanov AV et al. J Immunol 173: 86–91, 2004). Thus, TNF produced by each type of immune cell analyzed in our study may be both good and bad, depending on the pathophysiological model. It is conceivable that the thresholds for protective and deleterious TNF functions may differ, and this could be exploited in future protocols of therapeutic TNF ablation.
CD4-CD8 Differentiation in the Thymus: The cKrox of the Matter
To examine cKrox function during T-cell development, we generated transgenic mice in which this protein is expressed in all developing and mature T cells. Remarkably, these mice had CD4 but not CD8 T cells. This raised the possibility that cKrox might impose CD4 choice to MHC-I–signaled precursors that are normally CD8-bound and, thus, might be one of the long sought-after effectors of CD4-CD8 differentiation. Although this was an appealing perspective, it was also possible that the cKrox transgene simply prevented the differentiation of CD8 T cells without affecting their lineage direction. To distinguish between these possibilities, we generated cKrox transgenic mice whose T cells all carry the same TCR specificity for a defined MHC-I–peptide complex. Normally, T-cell precursors in such mice fail to express cKrox and develop into CD8 cells. In the presence of the cKrox transgene, however, these precursors were redirected into CD4 cells, indicating that cKrox promoted CD4 choice at the expense of CD8 choice. Importantly, cKrox also imposed the functional helper differentiation characteristics of CD4 cells: Whereas MHC-I–specific CD8 T cells normally are cytotoxic, the MHC-I–specific CD4 T cells that developed in cKrox transgenic mice lacked cytotoxic properties (such as expression of the enzyme perforin) and had gained attributes of helper function. These findings indicate that cKrox is a master developmental regulator that imposes CD4 differentiation to developing thymocytes. In parallel to this work, the laboratory of Dietmar Kappes, PhD, Fox Chase Cancer Center, independently showed that a spontaneous point mutation in the gene encoding cKrox (which these authors called Thpok and that is now officially referred to as Zbtb7b) resulted in a phenotype mirroring the one observed in the cKrox transgene: Mice carrying this mutation lacked CD4 T cells and had MHC-II–specific cytotoxic CD8 T cells. The identification of cKrox as a master switch of CD4-CD8 lineage differentiation raises many questions. One key issue will be to investigate how cKrox is upregulated during the development of T cells recognizing MHC-II–peptide but not MHC-I–peptide complexes. The search for cKrox target genes should provide insight into the mechanism of lineage differentiation in the thymus. It is interesting that some cKrox homologs repress gene expression by recruiting enzymes (histone deacetylases) that promote the closure of chromatin to transcription. The possibility that cKrox affects lineage differentiation by altering chromatin is intriguing. Indeed, whereas many differentiation processes mediated by changes in chromatin organization are intimately associated with cell division (during which chromatin reorganization occurs), this is not the case with CD4-CD8 lineage differentiation.
Scientific Advisory CommitteeIf you have scientific news of interest to the CCR research community, please contact one of the scientific advisors (below) responsible for your areas of research.
CCR Frontiers in Science—StaffCenter for Cancer Research Robert H. Wiltrout, PhD, Director Deputy Directors Douglas R. Lowy, MD Editorial Staff Sue Fox, BA/BSW, Senior Editor * Palladian Partners, Inc. FOR INTERNAL USE ONLY |
||
 |
he
close association between basic and clinical research that exists within the
CCR enables our clinical program to benefit from a dynamic, collaborative environment
that drives our advances in translational research. Distinct from a comprehensive
cancer center, our clinical program is a low-volume, high-intensity clinical/translational
research enterprise focused intensely upon a small number of patients with cancer
or AIDS-related illnesses. Patient volunteers who participate in our clinical
trials receive state-of-the-art therapies and approaches to diagnosis and treatment.
Genomic profiling is becoming a very important addition to these cutting-edge
approaches.
n
mammalian cells, centrosome duplication is a critical event that must be completed
before mitosis, the culminating stage of the cell cycle, during which a bipolar
mother cell partitions into two genetically equivalent daughter cells. Centrosomes
are the major microtubule organizing centers that direct bipolar spindle assembly
and balance mitotic chromosome segregation, controlling microtubule number,
nucleation, polarity, and orientation. This process, which begins at G
D4
and CD8 T cells are essential components of the adaptive immune system and are
critical for defense against infection. CD4 T cells recognize peptides of extracellular
origin in complex with class II major histocompatibility complex (MHC-II) molecules,
and generally provide help to other immune-competent cells. In contrast, CD8
T cells recognize peptides of intracellular origin in complex with MHC-I molecules
and differentiate into cytotoxic effectors. Besides their role in infection
defense, CD8 T cells have attracted the attention of tumor immunologists, who
seek to harness their killing power to eliminate cancer cells. CD4- and CD8-lineage
T cells arise in the thymus from nonfunctional precursors upon interaction with
MHC-II or MHC-I peptide complexes expressed on the thymic stroma, respectively.
How this choice of lineage occurs has puzzled immunologists and others for years
and is the main focus of our laboratory. Although signals initiated by the binding
of T-cell receptors (TCR) to intrathymic MHC molecules are now largely accepted
as critical for lineage choice, the intra-cellular effectors of this process
have remained mysterious. To gain insight into this question, we hypothesized
that genes encoding such effectors would be upregulated during lineage choice,
and we conducted a microarray screen to compare gene expression in T-cell precursors
during and immediately before lineage differentiation. One top candidate that
emerged from this search encodes a zinc finger DNA binding protein known as
cKrox, a member of a large family of transcription factors involved in many
differentiation processes. We found that cKrox was specifically upregulated
during the differentiation of CD4 but not of CD8 T cells in the thymus, and
that its expression remained CD4 specific in mature T cells. 