Louise C. Showe

Immunopathogenesis and therapy of cutaneous T cell lymphoma

Cutaneous T cell lymphomas (CTCLs) are a heterogenous group of lymphoproliferative disorders caused by clonally derived, skin-invasive T cells. Mycosis fungoides (MF) and Sezary syndrome (SS) are the most common types of CTCLs and are characterized by malignant CD4(+)/CLA(+)/CCR4(+) T cells that also lack the usual T cell surface markers CD7 and/or CD26. As MF/SS advances, the clonal dominance of the malignant cells results in the expression of predominantly Th2 cytokines, progressive immune dysregulation in patients, and further tumor cell growth. This review summarizes recent insights into the pathogenesis and immunobiology of MF/SS and how these have shaped current therapeutic approaches, in particular the growing emphasis on enhancement of host antitumor immune responses as the key to successful therapy.

Classification and prediction of survival in patients with the leukemic phase of cutaneous T cell lymphoma.

We have used cDNA arrays to investigate gene expression patterns in peripheral blood mononuclear cells from patients with leukemic forms of cutaneous T cell lymphoma, primarily Sezary syndrome (SS). When expression data for patients with high blood tumor burden (Sezary cells >60% of the lymphocytes) and healthy controls are compared by Students t test, at P < 0.01, we find 385 genes to be differentially expressed. Highly overexpressed genes include Th2 cells–specific transcription factors Gata-3 and Jun B, as well as integrin β1, proteoglycan 2, the RhoB oncogene, and dual specificity phosphatase 1. Highly underexpressed genes include CD26, Stat-4, and the IL-1 receptors. Message for plastin-T, not normally expressed in lymphoid tissue, is detected only in patient samples and may provide a new marker for diagnosis. Using penalized discriminant analysis, we have identified a panel of eight genes that can distinguish SS in patients with as few as 5% circulating tumor cells. This suggests that, even in early disease, Sezary cells produce chemokines and cytokines that induce an expression profile in the peripheral blood distinctive to SS. Finally, we show that using 10 genes, we can identify a class of patients who will succumb within six months of sampling regardless of their tumor burden.

Editing of Epstein-Barr virus-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency.

Certain primary transcripts of miRNA (pri-microRNAs) undergo RNA editing that converts adenosine to inosine. The Epstein-Barr virus (EBV) genome encodes multiple microRNA genes of its own. Here we report that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited in latently EBV-infected cells. Editing of wild-type pri-miR-BART6 RNAs dramatically reduced loading of miR-BART6-5p RNAs onto the microRNA-induced silencing complex. Editing of a mutation-containing pri-miR-BART6 found in Daudi Burkitt lymphoma and nasopharyngeal carcinoma C666-1 cell lines suppressed processing of miR-BART6 RNAs. Most importantly, miR-BART6-5p RNAs silence Dicer through multiple target sites located in the 3′-UTR of Dicer mRNA. The significance of miR-BART6 was further investigated in cells in various stages of latency. We found that miR-BART6-5p RNAs suppress the EBNA2 viral oncogene required for transition from immunologically less responsive type I and type II latency to the more immunoreactive type III latency as well as Zta and Rta viral proteins essential for lytic replication, revealing the regulatory function of miR-BART6 in EBV infection and latency. Mutation and A-to-I editing appear to be adaptive mechanisms that antagonize miR-BART6 activities.

KLF17 is a negative regulator of epithelial-mesenchymal transition and metastasis in breast cancer

Metastasis is a complex multistep process, which requires the concerted action of many genes and is the primary cause of cancer death. Both pathways that regulate metastasis enhancement and those that regulate its suppression contribute to the tumour dissemination process. To identify new metastasis suppressors, we set up a forward genetic screen in a mouse model. We transduced a genome-wide RNA interference (RNAi) library into the non-metastatic 168FARN breast cancer cell line and orthotopically transplanted the cells into mouse mammary fat pads. We then selected cells that could metastasize to the lung and identified an RNAi for the KLF17 gene. Conversely, we demonstrate that ectopic expression of KLF17 in a highly metastatic 4T1 breast cancer cell line inhibits the ability of cells to metastasize from the mammary fat pad to the lung. We also show that suppression of KLF17 expression promotes breast cancer cell invasion and epithelial–mesenchymal transition (EMT), and that KLF17 protein functions by directly binding to the promoter region of Id1 (which encodes a key metastasis regulator in breast cancer) to inhibit its transcription. Finally, we demonstrate that KLF17 expression is significantly downregulated in primary human breast cancer samples and that the combined expression pattern of KLF17 and Id1 can serve as a potential biomarker for lymph node metastasis in breast cancer.

Gene Expression Profiles in Peripheral Blood Mononuclear Cells Can Distinguish Patients with Non–Small Cell Lung Cancer from Patients with Nonmalignant Lung Disease

Early diagnosis of lung cancer followed by surgery presently is the most effective treatment for non-small cell lung cancer (NSCLC). An accurate, minimally invasive test that could detect early disease would permit timely intervention and potentially reduce mortality. Recent studies have shown that the peripheral blood can carry information related to the presence of disease, including prognostic information and information on therapeutic response. We have analyzed gene expression in peripheral blood mononuclear cell samples including 137 patients with NSCLC tumors and 91 patient controls with nonmalignant lung conditions, including histologically diagnosed benign nodules. Subjects were primarily smokers and former smokers. We have identified a 29-gene signature that separates these two patient classes with 86% accuracy (91% sensitivity, 80% specificity). Accuracy in an independent validation set, including samples from a new location, was 78% (sensitivity of 76% and specificity of 82%). An analysis of this NSCLC gene signature in 18 NSCLCs taken presurgery, with matched samples from 2 to 5 months postsurgery, showed that in 78% of cases, the signature was reduced postsurgery and disappeared entirely in 33%. Our results show the feasibility of using peripheral blood gene expression signatures to identify early-stage NSCLC in at-risk populations.

Alternative transcription exceeds alternative splicing in generating the transcriptome diversity of cerebellar development

Despite our growing knowledge that many mammalian genes generate multiple transcript variants that may encode functionally distinct protein isoforms, the transcriptomes of various tissues and their developmental stages are poorly defined. Identifying the transcriptome and its regulation in a cell/tissue is the key to deciphering the cell/tissue-specific functions of a gene. We built a genome-wide inventory of noncoding and protein-coding transcripts (transcriptomes), their promoters (promoteromes) and histone modification states (epigenomes) for developing, and adult cerebella using integrative massive-parallel sequencing and bioinformatics approach. The data consists of 61,525 (12,796 novel) distinct mRNAs transcribed by 29,589 (4792 novel) promoters corresponding to 15,669 protein-coding and 7624 noncoding genes. Importantly, our results show that the transcript variants from a gene are predominantly generated using alternative transcriptional rather than splicing mechanisms, highlighting alternative promoters and transcriptional terminations as major sources of transcriptome diversity. Moreover, H3K4me3, and not H3K27me3, defined the use of alternative promoters, and we identified a combinatorial role of H3K4me3 and H3K27me3 in regulating the expression of transcripts, including transcript variants of a gene during development. We observed a strong bias of both H3K4me3 and H3K27me3 for CpG-rich promoters and an exponential relationship between their enrichment and corresponding transcript expression. Furthermore, the majority of genes associated with neurological diseases expressed multiple transcripts through alternative promoters, and we demonstrated aberrant use of alternative promoters in medulloblastoma, cancer arising in the cerebellum. The transcriptomes of developing and adult cerebella presented in this study emphasize the importance of analyzing gene regulation and function at the isoform level.

Microarray data on gene modulation by HIV-1 in immune cells: 2000-2006.

Here, we review 34 HIV microarray studies in human immune cells over the period of 2000–March 2006 with emphasis on analytical approaches used and conceptual advances on HIV modulation of target cells (CD4 T cell, macrophage) and nontargets such as NK cell, B cell, and dendritic cell subsets. Results to date address advances on gene modulation associated with immune dysregulation, susceptibility to apoptosis, virus replication, and viral persistence following in vitro or in vivo infection/exposure to HIV‐1 virus or HIV‐1 accessory proteins. In addition to gene modulation associated with known functional correlates of HIV infection and replication (e.g., T cell apoptosis), microarray data have yielded novel, potential mechanisms of HIV‐mediated pathogenesis such as modulation of cholesterol biosynthetic genes in CD4 T cells (relevant to virus replication and infectivity) and modulation of proteasomes and histone deacetylases in chronically infected cell lines (relevant to virus latency). Intrinsic challenges in summarizing gene modulation studies remain in development of sound approaches for comparing data obtained using different platforms and analytical tools, deriving unifying concepts to distil the large volumes of data collected, and the necessity to impose a focus for validation on a small fraction of genes. Notwithstanding these challenges, the field overall continues to demonstrate progress in expanding the pool of target genes validated to date in in vitro and in vivo datasets and understanding the functional correlates of gene modulation to HIV‐1 pathogenesis in vivo.

The p130 Isoform of Angiomotin Is Required for Yap-Mediated Hepatic Epithelial Cell Proliferation and Tumorigenesis

The p130 isoform of Amot exerts two oncogenic activities that promote liver cancer. Oncogenic Times 2 Proteins in the angiomotin (Amot) family can either promote or inhibit tumorigenesis through their actions on the Hippo-Yap pathway. Yi et al. found that the p130 isoform of Amot enhanced the activity of the transcription factor Yap to promote liver tumorigenesis. Mice with a liver-specific deficiency in Amot exhibited normal liver development but had reduced incidence of liver tumors in two models of hepatic cancer. By binding Yap and inhibiting its phosphorylation, Amot-p130 increased the nuclear translocation of Yap. In the nucleus, Amot-p130 enhanced the transcriptional activity of Yap for a subset of target genes, including those associated with tumorigenesis. Thus, the p130 isoform of Amot performs dual oncogenic functions by promoting Yap nuclear translocation and augmenting the activity of Yap at cancer-associated genes. The Hippo-Yap signaling pathway regulates a number of developmental and adult cellular processes, including cell fate determination, tissue growth, and tumorigenesis. Members of the scaffold protein angiomotin (Amot) family interact with several Hippo pathway components, including Yap (Yes-associated protein), and either stimulate or inhibit Yap activity. We used a combination of genetic, biochemical, and transcriptional approaches to assess the functional consequences of the Amot-Yap interaction in mice and in human cells. Mice with a liver-specific Amot knockout exhibited reduced hepatic “oval cell” proliferation and tumorigenesis in response to toxin-induced injury or when crossed with mice lacking the tumor suppressor Nf2. Biochemical examination of the Amot-Yap interaction revealed that the p130 splicing isoform of Amot (Amot-p130) and Yap interacted in both the cytoplasm and nucleus, which involved binding of PPxY and LPxY motifs in Amot-p130 to WW domains of Yap. In the cytoplasm, Amot-p130 prevented the phosphorylation of Yap by blocking access of the WW domains to the kinase Lats1. Within the nucleus, Amot-p130 was associated with the transcriptional complex containing Yap and Teads (TEA domain family members) and contributed to the regulation of a subset of Yap target genes, many of which are associated with tumorigenesis. These findings indicated that Amot acts as a Yap cofactor, preventing Yap phosphorylation and augmenting its activity toward a specific set of genes that facilitate tumorigenesis.

Stem Cells with Neural Crest Characteristics Derived from the Bulge Region of Cultured Human Hair Follicles

In this study, we demonstrate that we can isolate stem cells (SCs) with neural crest characteristics from the bulge area of cultured human hair follicles (HFs). These SCs can proliferate in situ and form spheroid structures attached to the bulge area of HFs, and they express immature neural crest cell markers but not differentiation markers. An expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. These human SCs are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. In addition, these SCs show differentiation potential toward mesenchymal lineages, and they can be differentiated into adipocyte, chondrocyte, and osteocyte lineages. Neuronal differentiation of these cells induces global gene expression changes with a significantly increased expression of neuron-associated genes. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. The presence of SCs with neural crest characteristics in HFs may offer new opportunities for the use of these cells in regenerative medicine.

Circulating Monocytes in HIV-1-Infected Viremic Subjects Exhibit an Antiapoptosis Gene Signature and Virus- and Host-Mediated Apoptosis Resistance

Mechanisms that may allow circulating monocytes to persist as CD4 T cells diminish in HIV-1 infection have not been investigated. We have characterized steady-state gene expression signatures in circulating monocytes from HIV-infected subjects and have identified a stable antiapoptosis gene signature comprised of 38 genes associated with p53, CD40L, TNF, and MAPK signaling networks. The significance of this gene signature is indicated by our demonstration of cadmium chloride- or Fas ligand-induced apoptosis resistance in circulating monocytes in contrast to increasing apoptosis in CD4 T cells from the same infected subjects. As potential mechanisms in vivo, we show that monocyte CCR5 binding by HIV-1 virus or agonist chemokines serves as independent viral and host modulators resulting in increased monocyte apoptosis resistance in vitro. We also show evidence for concordance between circulating monocyte apoptosis-related gene expression in HIV-1 infection in vivo and available datasets following viral infection or envelope exposure in monocyte-derived macrophages in vitro. The identification of in vivo gene expression associated with monocyte resistance to apoptosis is of relevance to AIDS pathogenesis since it would contribute to: 1) maintaining viability of infection targets and long-term reservoirs of HIV-1 infection in the monocyte/macrophage populations, and 2) protecting a cell subset critical to host survival despite sustained high viral replication.