Much of the hospital's success in identifying and treating children with hearing loss is the result of the rapid transfer of research findings from laboratories to clinic and bedside.
Areas of Research - Hereditary Communication Disorders - Cell Signaling and Tumor Angiogenesis Laboratory: The Molecular Basis of Cell Adhesion to Extra Cellular Matrix Derived Molecules under Normal and Pathological Conditions
Akulapalli Sudhakar, M.Sc; M.Phil & Ph.D., Director
Overview: Akulapalli Sudhakar is interested in understanding the molecular basis of extra cellular matrix derived molecules in cell adhesion and the signaling mechanisms in human vascular diseases such as tumor angiogenesis and macular degeneration.
Background: Most cells in human body adhere to the neighboring cells and to the extra cellular matrix (ECM, a fibrillar meshwork like structure) in blood vessels. Cellular adhesion to ECM through integrins receptors, play important roles in the normal functions such as cellular organization, proliferation, migration, survival and metabolism including gene expression. During the embryonic development, cell adhesion is important for the correct movement of cells modeling the embryo. In the adult development, appropriate cell adhesion is necessary for numerous physiological processes that can be deranged in diseased conditions including tumor angiogenesis, macular degeneration, arthritis, cancer etc.
Our laboratory seeks to understand the ECM containing type IV collagen derived anti-angiogenic proteins that are present in normal circulation, which are involved in cell signaling and the way these proteins control adhesion and migration of cells in pathological processes. Type IV collagen derived anti-angiogenic proteins, by binding to cell surface integrins, transduce the signaling mechanisms in regulating anti-angiogenic activity. Thus, integrins serve as transmembrane linkers between the ECM and cytoskeleton for outside-in signaling. Both type IV collagen and several integrin receptors are essential for angiogenesis (a process in which new blood vessel formation). Presently, we are analyzing type IV collagen derived non-collagenous domains (NC1) binding to certain integrins and regulate anti-angiogenic and anti-tumorogenic actions specifically targeting tumor microvascular endothelial cells. Our recent work has led to the new interpretations on the efficacy of certain type IV collagen NC1 domains working as antiangiogenic and anti-tumorigenic drugs (Science 2002, 295, 140-143; PNAS 2003, 100, 4766-4771; JCI 2005, 115, 2801-2810; Blood 2007, 110; 1168-1177).
Cell Signaling and Tumor Angiogenesis Laboratory Facilities:
The laboratory consists of 1,200 sq. ft. of newly renovated space with a separate tissue culture room (150 sq. ft.) that contains modern cell culture incubators. It is located adjacent to all utilities; deionizer water runs from the tap. There is also a cold cabinet for chromatography and protein purification and space for storage for heavy equipment. Autoclaving and dishwashing are done in the building. Major equipment within the laboratory includes medium and low rate centrifuges, microfuges, refrigerated centrifuge, vertical and horizontal electrophoresis equipment, western blotting apparatus, equipment for handling and staining of slides, bacterial and two hepa filtered cell culture incubators, two refrigerators, three -200C and one -800C freezers, one liquid nitrogen storage tank (can store more than 2000 tubes), 1 gradient PCR thermal cyclers, one 37°C incubator, 4 magnetic stirrers, 2 Accumet pH meter (AB 15), 1 inverted microscope, 4 sets of electrophoresis equipment, separate tissue culture room with laminar hood and 2 CO2 incubators, one 270C (for Sf9 cells) and one 370C (for normal cell culture), 2 microbalances, 2 shakers, 3 water baths, 1 speed-vac, 1 micro plate reader (Bio-Rad) and other basic molecular biology equipment. All of the necessary minor equipment needed to carry out cell signaling work is also available. A Sorvall high-speed centrifuge, Beckman ultracentrifuge, Phosphoimager screens, high-pressure HPLC system, Spectronic 1001 spectrophotometer and Zeiss fluorescent microscope/camera are available in a common facility adjacent to the laboratory. Newly acquired equipment includes an ABI Prism 7000 real time PCR system, and a DAKO auto staining system for standardizing histochemical and immunohistochemical staining procedures and much of the equipments are shared with Dr. Cosgrove’s Laboratory.
The Cell Signaling Laboratory has 3 PCs and 3 Mac with all the required software. The computers provide access to the World Wide Web services. One exclusively dedicated color laser printer is located in the Cell Signaling Lab on the first floor. In addition, Dr. Sudhakar has exclusive use of portable MacPro Notebook for data analysis and manuscript preparation. This computer also has the Mac Vector 5.0 to perform primary and secondary structure analysis on DNA and protein sequences.
Staff
Chandra S. Boosani, Ph.D. (Research Associate I) Dr. Boosani is expert in molecular cloning and protein expression. His work in the laboratory includes cloning, expression, purification and in vitro and in vivo functional characterization of anti-angiogenic and anti-tumorigenic proteins (Combostatin, Malignostatin etc.) which were recently developed in the laboratory.
Duane C Delimont, M.S. (Technologist IV, Animal Care Facility) Duane is our departmental technician for maintaining α1(IV)NC1 transgenic mice. He is experienced and responsible for gene sequencing and genotyping.
Representative Publications from Cell Signaling and Tumor Angiogenesis Laboratory (2005 - onwards):
| 1. Sudhakar, A & Boosani, C.S. (2008). Inhibition of tumor angiogenesis by Tumstatin: insights into | |
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| 2. Boosani, C. S and Sudhakar, A (2008). “Molecular Cloning and functional characterization of | |
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| 3. Boosani, C.S., Mannam, A.P., Cosgrove. D., Silva, R., Hodivala-Dilke, K.M., Keshamouni, G.V. & | |
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| 4. Sudhakar, A (2007). Signaling mechanisms of collagen derived endogenous angiogenesis | |
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| 5. Sudhakar, A & Boosani, C.S. (2007). Signaling mechanisms of endogenous angiogenesis | |
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| 6. Keshamouni, V.G., Michailidis, G., Grasso, C.S., Anthwal, S., Strahler, J.R., Walker, A., | |
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| 7. Boosani, C.S. & Sudhakar, A. (2006). Cloning, purification and characterization of a | |
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| 8. Sudhakar, A*., Nyberg, P., Keshamouni, V.G., Mannam, A.P., Li, J., Sugimoto, H., Cosgrove, D.E. & | |
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| 9. Hamano, Y., Sugimoto, H., Soubasakos, M.A., Kiemar, M., Olsen, B.R., Lawler, J., | |
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| 10. I Sund, M., Hamano, Y., Sugimoto, H., Sudhakar, A., Soubasakos, M., Yerramalla, U., Benjamin, | |
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| 11. Hamano, Y., Zeisberg, M., Sugimoto, H., Lively, J., Maeshima, Y., Yang, C., Hynes, R.O., Werb, Z., | |
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| 12 . Sudhakar, A., Sugimoto, H., Yang, C.Q., Lively, J., Zeisberg, M. & Kalluri R. (2003). | |
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| 13. Maeshima, Y., Sudhakar, A., Lively, J.C., Ueki, K., Kharbanda, S., Kahn, C.R., Sonenberg, N., | |
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Summary of Research Program
For Physicians and Scientists
Our laboratory is working on anti-angiogenic type IV collagen non-collagenous (NC1) domains in intact animals through parallel studies in cell culture system with purified NC1 domains. These anti-tumor proteins are analyzed in detail through specific integrin binding interactions and signaling studies. This strategy offers great promise for the design of drugs to block specific signaling which promotes angiogenesis in a hope to combat vascular diseases such as cancer and macular degeneration. Our laboratory is expert in vitro and in vivo experiments with proven strategies to characterize molecules that are most important for inhibition of angiogenesis. The fundamental interest of our laboratory is translated in two major areas, 1) Tumor angiogenesis and 2) Macular degeneration.
Tumor angiogenesis: Cell Signaling and Tumor Angiogenesis Laboratory routinely performs molecular screening and identification of new anti-angiogenic or anti-cancer molecules from extra cellular matrix of type IV collagen. Our laboratory has cloned and expressed several of the anti-angiogenic molecules (NC1 domains from α1, α3 and α6 chains of type IV collagen) using baculovirus insect cells (Sf-9) system and tested in cell culture and in live mice in which solid tumors are induced. We identified several of these domains inhibiting hypoxia factors and these factors have been shown to be strongly associated with the anti-tumor activity in a variety of solid tumors. The mechanism of targeting tumor hypoxia and tumor angiogenesis with these endogenous angiogenesis inhibitors is under investigation. Our approach to understand the process of inhibition of tumor angiogenesis led new insights in constraining cancer progression. In addition we invented a new molecule and termed combostatin (four known anti-angiogenic protein functional domains were fused to engineer a fusion protein), this work is supported by Flight Attendant Medical Research Institute (FAMRI). The combostatin developed is being subjected to pre-clinical testing alongside the individual progenitor proteins for its ability to reduce growth of solid tumors in mice. These studies have a real potential for development of a novel treatment method for solid tumors associated with angiogenesis.
Macular Degeneration: The extracellular matrix (ECM), cell signaling and the emerging role of integrin signaling processes are important factors modulating macular degeneration. The new areas of research being developed in my laboratory include studies on the role of ECM derived angiogenesis inhibitors and their molecular signaling in macular degeneration. A multidisciplinary research team at the department of genetics (inter-collaboration with Drs. Cosgrove and Pang’s laboratory with Cell Signaling laboratory) focused on type IV collagen derived angiogenic inhibitors and combostatin role in vascular diseases such as age related macular degeneration in addition to tumor angiogenesis in cancer. My laboratory has developed α1(IV)NC1 transgenic mice, α1(IV)NC1 and α3(IV)NC1 adenovirus to test these in macular degeneration. We are testing antiangiogenic gene therapy for cancer and macular degeneration via systemic administration of adenovirus that secretes α1(IV)NC1 or α3(IV)NC1 molecules. In addition recently we have identified that these molecules also participating in the formation of a complex with pro form of matrix metallorpotenae-2 (MMP-2) and inhibiting MMP-2 activation. This opens the door for a large multidisciplinary effort aimed to illuminate the common mechanisms of regulation of MMP-2 activation in several diseases including cancer, macular degeneration and arthritis. This new area will include biochemical analysis of the MMP-2 and α1NC1 or α3NC1 complex formation to regulate MMP-2 activation inhibition and constitutes the foundation for a long-term research plan for the Cell Signaling and tumor angiogenesis laboratory.
For Families
Cancer is currently one of the most prevalent causes of death in the United States of America. Current therapeutic options aim only to slow the progression of cancer disease. Therefore, a renewed effort must be made to identify relevant endogenous cancer inhibitor molecules that could be exploited as therapeutic drugs. Cell Signaling and Tumor Angiogenesis laboratory is screening important anti-cancer molecules, which are released into circulating blood of cancer patients. Several of these endogenous circulating molecules were cloned in the laboratory and identified them as inhibitors of solid tumor growth (cancer). It should be noted that this method of treatment is not likely to have side effects, since these proteins are normally found in the body. Such a treatment option would be far superior to surgery, radiation, and chemotherapy that have quite severe side effects.
Age related macular degeneration (AMD) is also one of the major causes of blindness in the elderly people in the world. Researchers long ago noticed that progression of AMD related to abnormal growth of blood vessels and leakiness in the macula region or retina in the eye leads to this devastating eye disease. We have tested several of the endogenous circulating anti-angiogenesis molecules, and our combostatin to be inhibiting proliferation of endothelial cells and may be used to treat AMD in near future. We therefore expect that our laboratory studies on endogenous angiogenesis inhibitor molecules will advance cure for cancer and AMD.