My research involves several aspects of hematopathology, with special emphasis on pathogenesis of malignant lymphomas, especially mantle cell lymphoma (MCL). MCL is an aggressive non-Hodgkin's lymphoma which brings together the worst characteristics of both high-grade and low-grade lymphomas: the course is usually aggressive and the disease is rarely curable. MCL is characterized by the chromosome translocation t(11;14)(q13;q32), which results in overexpression of cyclin D1. However, this translocation alone is not sufficient to result in lymphoma, and additional genetic alterations are necessary.
My first primary study of secondary genomic alterations are frequently detected in MCL, of which chromosome 13q31-q32 gain/amplification is one of the most frequent. Amplification at chromosome 13q31-q32 targets a microRNA cluster, miR-17~92, and overexpression of the cluster accelerates MYC-induced lymphomagenesis in mice. However, the functional role of miR-17~92 in MCL has not been examined. Based on gene expression profiling data, we found that high level expression of C13orf25, the primary transcript from which these microRNAs are processed, was associated with poor survival in MCL patients (p=0.0027). Enforced overexpression of miR-17~92 in retrovirally transduced MCL cell lines increased the phosphorylation of AKT and its downstream targets and reduced chemotherapy-induced apoptosis. PTEN was down-modulated in these cells, and our data suggest that PTEN is a direct target of miR-17~92 in MCL. Furthermore, we discovered that protein phosphatase PHLPP2, a negative regulator of the PI3K/AKT pathway, is also a direct target of miR-17~92. We demonstrated that PHLPP2 was deleted in Rec-1, a MCL cell line, and PHLPP2 mRNA expression was decreased in Rec-1 and several other MCL cell lines. Its downregulation may therefore act synergistically with PTEN downregulation to promote activation of the PI3K/AKT pathway in MCL. Also we demonstrated that BIM is a direct target of the miR-17~92. In summary, overexpression of miR-17~92 down-modulates multiple proteins involved in PI3K/AKT signaling and apoptosis, and these effects collaboratively enhance resistance to chemotherapy in MCL cells.
My secondary study is on miR-17~92, which is processed from the transcript of C13orf25, a gene amplified in some lymphomas and solid tumors. Recent studies have shown that C13orf25 is activated by MYC and E2F transcription factors; however, detailed examination of its transcriptional regulation has not been reported. Nested deletions of the promoter region were cloned into luciferase reporters. Examination in HEK293T cells and in NIH3T3 cells revealed the core promoter to extend approximately from -155 nt to +113 nt from the transcriptional start site. From 5' to 3', this region comprises several putative SP1 sites, sites for NF-Y, ETS, OCT, and MYC, and two tandem E2F sites. This region is highly conserved in vertebrates, and most of the putative transcription factor binding sites are conserved even in fish. Additional highly conserved sequences likely represent binding sites for unidentified transcription factors. Additional deletions of this core region reduced luciferase activity. Our studies indicate that the promoter is regulated by the collaborative activity of several transcription factors, each of which individually has only a moderate effect. Furthermore, our results suggest that C13orf25 is not only activated by E2F and MYC, but also repressed by complexes of E2F and RB1 family members and by repressors of the MYC family such as MNT and MXI1. In addition to transcriptional regulation of the promoter, the gene appears to be regulated by enhancers; at least two regions within the first intron were found to have enhancer activity. Because the miR-17~92 cluster acts as an important oncogene in several cancers and targets genes important in regulating cell proliferation and survival, further studies of its transcriptional control are warranted.
Timothy Greiner, M.D.
My research involves the study of the etiology and progression of lymphoma by examining the mutational spectra of oncogenes and tumor suppresser genes. My laboratory has been involved in characterizing mutations in ATM, p53 and bcl-6. We are correlating mRNA expression patterns in lymphoma with patient survival, mutation status, and morphological subtypes. We are currently examining the methylation status of DNA in diffuse large cell and mantle cell lymphomas. The emphasis on molecular epidemiology involves the identification of EBV subtypes, the gene expression, and the molecular abnormalities in post-transplant lymphoproliferative disorders.
Other Specialized Cancer Research
Julia Bridge, M.D.
My research involves several aspects of genetics with special emphasis on cancer genetics of bone and soft tissue tumors. Over the last two decades, we have identified a number of tumor-specific chromosomal abnormalities for both benign and malignant bone and soft tissue tumors. These abnormalities are important diagnostically and prognostically, as well as for defining treatment strategies. These data have also contributed significantly to our understanding of the histopathogenesis of many of these neoplasms. Following identification of the anomalies cytogenetically, the key chromosomal breakpoint locations and underlying involved genes are further characterized with FISH positional cloning analysis and molecular techniques. Moreover, we custom design probe sets from these defined breakpoints for molecular cytogenetic assays and primer sets for RT-PCR analysis as additional diagnostic adjuncts.
Samuel Cohen, M.D., Ph.D.
My research involves several aspects of carcinogenesis, with an emphasis on urinary bladder as a model system in rodents and extrapolation between rodent models and human diseases. We have postulated that agents increase cancer risk by either directly interacting with DNA or increasing cell proliferation in appropriate target cells, allowing for more opportunity for spontaneous mutations to occur during DNA replication. Genotoxic chemicals, such as aromatic amines, nitrofurans, nitrosamines, and acrolein, require metabolic activation, DNA adduct formation, and mutagenesis. Numerous nongenotoxic chemicals have been identified, enhancing carcinogenesis by increasing urothelial cell proliferation, including arsenic, sodium salts, amino acids, calculus-forming chemicals, and phenolic chemicals. Increased proliferation occurs either by direct mitogenesis, such as by high doses of Propoxur, or by cellular toxicity and consequent regenerative hyperplasia, such as occurs with formation of calculi by chemicals like uracil, or by processes involving more subtle cytotoxicity, such as following high doses of sodium saccharin or arsenic. We successfully demonstrated the mode of action of sodium saccharin and related sodium salts. Based on our investigations, it is unlikely that these pose a carcinogenic hazard to humans. Studies with these salts involve various aspects of toxicology, basic chemistry, cell kinetics, electron microscopy, pathology, in vivo bioassays, renal physiology and molecular biology. These findings led to the delisting of saccharin from the National Toxicology Program’s List of Carcinogens.
PPARγ and dual PPAR agonists, developed as anti-diabetic and antilipidemic drugs, frequently produce bladder cancer in rats and hemangiosarcomas in mice. We are investigating the mechanisms involved for both of these targets, already showing that for the bladder the mechanism involves indirect formation of calcium-containing urinary solids that produce urothelial cytotoxicity and regeneration. The mechanism does not occur in humans. A variety of other nongenotoxic bladder carcinogens are being investigated in animal and in vitro systems. More recently, we have investigated the bladder carcinogenicity of dimethylarsinic acid (DMAV), an organic arsenical, and inorganic arsenic in rodent models and in cell culture. DMA and its metabolites are non-DNA reactive, but orally consumed DMA produces urothelial necrosis with consequent regeneration. Urothelial cytotoxicity is produced in vitro by trivalent arsenicals at concentrations less then 1μM. Urinary levels of DMAIII following carcinogenic doses of DMAV suggest that it may be critical to the urothelial toxicity induced in vivo. The relationship to growth factors, receptors, and cell cycle control mechanisms is being evaluated. A similar process is being evaluated for inorganic arsenic - a known human bladder carcinogen.
Rakesh Singh, Ph.D.
The overall goal of our research is to define the mechanism(s) that regulate the process of metastasis. We hypothesize that metastasis is a highly selective process that is regulated by interrelated mechanisms whose outcome is dependent upon both the intrinsic properties of tumor cells and the host response. Using human tumors xenografted in nude mice and murine tumor models, these studies have demonstrated the role of host-derived factors in regulating angiogenesis, resulting in site-specific expression of angiogenic factors, including basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and metastasis. Further characterization of the cellular and molecular mechanisms underlying these processes are currently ongoing in our laboratory. In addition, we are investigating the mechanism(s) of organ-specific metastasis. Recent reports suggest specific organ tissues carry unique marker molecules accessible to circulating cells. We have identified the molecule(s) expressed in organ tissues, which might be important to organ-specific metastasis using phage display libraries. Further characterization of organ-specific signature molecules will be useful in designing novel, highly targeted therapeutic approaches against organ-specific metastasis. In addition, our current research activities have also been focused on designing the strategies for inhibiting tumor-induced angiogenesis and activating anti-tumor immunity with the potential for synergizing the outcome of conventional therapeutic approaches, as well as understanding the role of tumor-stromal interaction in tumor progression and metastasis.
James Talmadge, Ph.D.
The Laboratory of Transplantation Immunology is focused on the process of metastasis, and interventional strategies that augment the host response against primary and systemic disease. We have demonstrated that the process of metastasis is selective, controlled by intrinsic properties of the tumor, and the host response against the tumor. Our research emphasizes molecular and cellular immunology, vaccines, transplantation, gene therapy, hematology and oncology. The primary focus is on host-immune interactions and the regulation of tumor progression, metastasis and therapeutic intervention with the goal of developing preclinical, clinical and surrogate-based hypotheses. Clinical and translational research includes studies focus on the tumor microenvironment including myeloid derived suppressor cells (MDSCs), dendritic cells (DCs) and T-cells; and gene therapeutic vaccines with a focus on breast cancer and melanoma. This includes clinical studies with Dr. Ken Cowan and Dr. Elizabeth Reed at UNMC, Dr. Dmitry Gabrilovich at Moffitt Cancer Center and collaborations with Intrexon, Inc. Early-stage studies have also focused on immune recovery following stem cell transplantation, at present, primarily in collaboration with Dr. Greg Bociek, Internal Medicine, as regards non-myeloablative, allogeneic, stem cell transplantation.
Basic/translational research studies are focused on host-tumor interactions during tumor progression, metastasis and cytoreductive therapy. These studies include a program in molecular therapeutics that is focused on cyclooxygenase (COX)-2 inhibitors, tyrosine kinase inhibitors, all-trans-retinoic acid (ATRA) and vascular endothelial growth factor (VEGF) inhibition and their regulation of immune intervention. Our goal is to overcome tumor and iatrogenic suppression of DC function and T-cell responses to vaccines that is associated with MDSCs. As part of these studies, we have identified a critical regulatory role for a unique cellular phenotype strongly associated with tumor-associated immunosuppression that when removed slows tumor growth and prolongs survival. Studies into the mechanism of immunosuppression suggest a critical role for inducible nitric oxide synthase and arginase as a local/regional mediator of T-cell suppression. We are also very active (along with many others) in the development of a Biological Production Facility, which utilizes good manufacturing practices (GMPs) required for many of the above clinical studies. This includes the development of new vaccine manufacturing strategies and the validation of aspects of manufacture, such as release assays, freezing protocols, etc.
- Cancer Biology
Research Resources & Core Facilities
BSL3 Containment Laboratories
- Genome Assembly & Analysis
- Mammalian Cell Adhesion Molecule Database
- Tissue Procurement Shared Resource
- Tissue Sciences Facility