Discoveries of genetic alterations in lung and kidney cancers have provided the foundation for investigations carried out in our group. In lung cancers, our group discovered the SEMA3F gene and showed it was a potent tumor suppressor. Ongoing studies are examining its ability to block angiogenesis, and to develop approaches for use as a therapy. Loss of E-cadherin is another common event in lung cancers that is a critical step in metastasis. This loss is often mediated by transcriptional repressors like ZEB and SNAIL and our recent work has shown that ZEB inhibits SEMA3F. This apparent linkage between a tumor suppressor and a metastasis inducer is under active investigation. In kidney cancer, we discovered the TRC8 gene, which is a membrane-bound tumor suppressor with ubiquitin ligase activity disrupted in selected families with hereditary kidney cancer. We are actively searching for ubiquitylation targets, as this activity appears essential for growth suppression. TRC8 is regulated by sterols and it interacts with components of the lipid homeostatic machinery, and with a translation initiation factor. These data suggest that TRC8 provides a regulatory linkage between the lipid biosynthetic process, protein synthesis and growth control.

Dr. Greenberg’s research interests encompass three areas:

Basis Research:  Transglutaminases: Structure and Function
Clinical Research : Diagnosis and Treatment of Thrombosis
Translational Research:  Development of TGM-2 Inhibitors

Basic Science:  Transglutaminase Structure and Function.
Recombinant DNA and  protein biochemistry techniques are used to study the structure and function of Factor XIII and tissue transglutaminase (TGM-2). Collaborative studies with the Structural Biochemistry Lab at MUSC are studying mechanism of action of recently identified TGM-2 inhibitors.

Clinical Research: Diagnosis and Treatment of Thrombosis.
Earlier studies led to FDA approval of the D-dimer assay and PFA-100 instrument. Danapioid (Ong10172) a heparinoid was discovered to treat heparin-induced thrombocytopenia and thrombosis. The hep-PF4 assay identified patients at risk for poor clinical outcomes and new approaches to manage this disorder are being studied..  The  D-dimer antigen was found to be a surrogate marker for vascular damage in sickle cell disease and studies are in progress to monitor the effect of antithrombotics in sickle cell disease.  Clinical trials are in progress to test the safety and efficacy of newly approved antithrobotic drugs for prevent of deep vein thrombosis. New instrumentation that uses whole blood is being studied as an improvement in evaluating bleeding risk of liver disease patients in collaboration with MUSC Liver Center and Pathology Departments.

Translational Research: Development of TGM-2 Inhibitors.
Coagulation, inflammation and fibrosis are often associated with human disease processes.  The lab is developing inhibitors to TGM-2 and examining their effect on fibrosis and inflammation.  TGM-2 inhibitors were recently discovered to be effective therapy for the neurodegenerative process using Drosophila. The effect of these agents in other neurodegenerative diseases is also being studied in collaboration with the MUSC Neurosciences.

Selected Publications:
1: Adam SS, Key NS, Greenberg CS. D-dimer antigen: current concepts and future prospects. Blood. 2009 Mar 26;113(13):2878-87. Epub 2008 Nov 13. Review. PubMed PMID: 19008457.

2: Lai TS, Liu Y, Tucker T, Daniel KR, Sane DC, Toone E, Burke JR, Strittmatter WJ, Greenberg CS. Identification of chemical inhibitors to human tissue transglutaminase by screening existing drug libraries. Chem Biol. 2008 Sep 22;15(9):969-78. PubMed PMID: 18804034; PubMed Central PMCID: PMC2637080.

3: Parsons AC, Yosipovitch G, Sheehan DJ, Sangüeza OP, Greenberg CS, Sane DC. Transglutaminases: the missing link in nephrogenic systemic fibrosis. Am J Dermatopathol. 2007 Oct;29(5):433-6. PubMed PMID: 17890909.

4: Lai TS, Liu Y, Li W, Greenberg CS. Identification of two GTP-independent alternatively spliced forms of tissue transglutaminase in human leukocytes, vascular smooth muscle, and endothelial cells. FASEB J. 2007 Dec;21(14):4131-43. Epub 2007 Jul 3. PubMed PMID: 17609251; PubMed Central PMCID: PMC2157556.

5: Sane DC, Kontos JL, Greenberg CS. Roles of transglutaminases in cardiac and vascular diseases. Front Biosci. 2007 Jan 1;12:2530-45. Review. PubMed PMID: 17127261.

6: Bennett-Guerrero E, Slaughter TF, White WD, Welsby IJ, Greenberg CS, El-Moalem H, Ortel TL.

Preoperative anti-PF4/heparin antibody level predicts adverse outcome after cardiac surgery. J Thorac Cardiovasc Surg. 2005 Dec;130(6):1567-72. PubMed PMID: 16308000.

7: Lai TS, Tucker T, Burke JR, Strittmatter WJ, Greenberg CS. Effect of tissue transglutaminase on the solubility of proteins containing expanded polyglutamine repeats. J Neurochem. 2004 Mar;88(5):1253-60. PubMed PMID: 15009681.

8: Ariëns RA, Lai TS, Weisel JW, Greenberg CS, Grant PJ. Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood. 2002 Aug 1;100(3):743-54. Review. PubMed PMID: 12130481.

9: Slaughter TF, Sreeram G, Sharma AD, El-Moalem H, East CJ, Greenberg CS. Reversible shear-mediated platelet dysfunction during cardiac surgery as assessed by the PFA-100 platelet function analyzer. Blood Coagul Fibrinolysis. 2001 Mar;12(2):85-93. PubMed PMID: 11302482.

Our laboratory is focused on two project areas:

We continue to study the control of apoptosis in tumor cells using the Bcl2 antagonist ABT737.  This agent is currently under study in the clinic for treatment of lymphoid malignancies and small cell lung cancer.  We are studying the ability of this agent to induce senescence rather than apoptosis, and find that this is cell line specific.  Additional biochemical experiments are being carried out to understand how this agent works.

The second project under study in the laboratory is the role of the Pim protein kinase in malignancy.  To study this enzyme, we have developed specific small molecule inhibitors of Pim and investigated their activity against tumors.  In the study of hematopoietic malignancies we find that these inhibitors are most active against T-cell ALL while they have growth inhibitory activity against myeloid leukemias, as well.  Further evaluation of these agents as antileukemia treatments is continuing.

We have also investigated the role of Pim in regulating tumor growth through modulation of the mTOR pathway.  By using Pim knockout fibroblasts we find that the absence of Pim markedly decreases the mTOR pathway activity.  We find an increase in AMPK phosphorylation and a decrease in raptor protein to account for this change.  This inhibition of the pathway is paralleled by an increase in the AMP/ATP ratio that is responsible for stimulating AMPK.

Additional experiments are underway to look at the role of Pim during normal stem cell development and the ability of this protein kinase to regulate prostate tumor growth.  These studies are taking place using animal models and tissue culture experiments.