The Division of Cancer Prevention focuses on the development of novel methods to control cancer through its prevention; recently, we started developing therapeutic methods as well.

Our Division consists of two main laboratories, one directed by Dr. Rigas, who is also the Division Chief and the other by Dr. Cao. Below, we provide a brief overview of the two labs.

RIGAS LAB

Dr. RIGAS’ LAB focuses on developing novel methods for the prevention of cancer. This goal is being pursued through a multidisciplinary approach encompassing work on basic mechanistic aspects of chemoprevention, animal studies and human clinical trials. Our recent work on nitric oxide-donating aspirin is an example of this approach.

Current work in our Lab is the natural extension of over 15 years of investigative work on cancer prevention by Dr. Rigas and his co-workers. Starting at Cornell University in the late 80’s, it was continued at Rockefeller University and at the Institute for Cancer Prevention (formerly known as The American Health Foundation). Since 2004 we have based our investigative efforts at Stony Brook University, which, given its scientific and clinical breadth and depth, is a perfect place to conduct this type of research.

Highlights of our research in cancer chemoprevention include the first demonstration that   

• Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation and induce apoptosis in cancer cells [1-3]


• NSAIDs alter the cell cycle of colonocytes; their effect on the cell cycle machinery has been mapped extensively, e.g. [4-6]


• The effect of NSAIDs can be independent of an effect on cyclooxygenase. This “unconventional” finding has now been amply confirmed by many investigators and has helped greatly expand our understanding of the mechanisms of chemoprevention [7] 


• Nitric Oxide-donating NSAIDs prevent cancer. For example, we have demonstrated the profound chemopreventive effect of NO-donating aspirin on colon and pancreatic carcinogenesis. (reviewed in [8]), they culminated to an NIH-funded clinical trial of NO-donating aspirin for the prevention of colon cancer.

Current work involves understanding the general mechanisms of action of chemopreventive agents and the study and development of novel agents. Particular emphasis is placed on redox regulation. For example, we have

• Suggested a mechanism of action of chemopreventive agents against cancer based on the induction of oxidative stress [9]
• Initiated studies of the underlying mechanism focused on the thioredoxin system [10]

In collaboration with our colleagues in the Department of Materials Science & Engineering, we

• have developed novel nanoparticles with a strong effect against breast cancer
• have developed an innovative approach to the rapid detection of protein biomarkers both in vivo and in vitro (novel sensors based on surface molecular imprinting), and
• are working on a novel drug delivery system

Finally, we are studying in depth a series of novel anticancer compounds, e.g. [11], including their detailed preclinical evaluation (structure-activity studies, cell signaling, molecular targets, efficacy in animal models, pharmacokinetic and pharmacodynamic evaluation).

References

1. Shiff, S.J., Koutsos, M.I., Qiao, L. and Rigas, B. (1996) Nonsteroidal antiinflammatory drugs inhibit the proliferation of colon adenocarcinoma cells: effects on cell cycle and apoptosis. Exp Cell Res, 222, 179-88.
2. Shiff, S.J., Qiao, L., Tsai, L.L. and Rigas, B. (1995) Sulindac sulfide, an aspirin-like compound, inhibits proliferation, causes cell cycle quiescence, and induces apoptosis in HT-29 colon adenocarcinoma cells. J Clin Invest, 96, 491-503.
3. Shiff, S.J. and Rigas, B. (1999) Aspirin for cancer [news; comment]. Nat Med, 5, 1348-9.
4. Goldberg, Y., Nassif, I.I., Pittas, A., Tsai, L.-L., Dynlacht, B.D., Rigas, B. and Shiff, S.J. (1996) The anti-proliferative effect of sulindac and sulindac sulfide on HT-29 colon cancer cells: Alterations in tumor suppressor and cell cycle-regulatory proteins. Oncogene, 12, 893-901.
5. Qiao, L., Hanif, R., Sphicas, E., Shiff, S.J. and Rigas, B. (1998) Effect of aspirin on induction of apoptosis in HT-29 human colon adenocarcinoma cells. Biochemical Pharmacology, 55, 53-64.
6. Qiao, L., Shiff, S.J. and Rigas, B. (1998) Sulindac sulfide alters the expression of cyclin proteins in HT-29 colon adenocarcinoma cells. Int J Cancer, 76, 99-104.
7. Hanif, R., Pittas, A., Feng, Y., Koutsos, M.I., Qiao, L., Staiano-Coico, L., Shiff, S.I. and Rigas, B. (1996) Effects of nonsteroidal anti-inflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin-independent pathway. Biochem Pharmacol, 52, 237-45.
8. Rigas, B. (2007) The use of nitric oxide-donating nonsteroidal anti-inflammatory drugs in the chemoprevention of colorectal neoplasia. Curr Opin Gastroenterol, 23, 55-9.
9. Rigas, B. and Sun, Y. (2008) Induction of oxidative stress as a mechanism of action of chemopreventive agents against cancer. Br J Cancer, 98, 1157-60.
10. Sun, Y. and Rigas, B. (2008) The thioredoxin system mediates redox-induced cell death in human colon cancer cells: Implications for the mechanism of action of anticancer agents. Cancer Res, in press.
11. Rigas, B. and Kozoni, V. (2008) The novel phenylester anticancer compounds: Study of a derivative of aspirin (phoshoaspirin). Int J Oncol, 32, 97-100.

CAO LAB

Dr. CAO'S LAB is interested in the biology and prevention of cancer metastasis. Recognizing that invasiveness is an early critical step for cancer metastasis, Dr. Cao’s lab has been focused on understanding the mechanism of cancer invasion and on developing a new screening approach for anti-cancer invasion drug discovery.

Current projects in the lab fall into three separate but related categories:

• The first category of projects seeks to better understand the role of proteases, especially matrix metalloproteinases (MMPs) in cancer metastasis. Cao’s lab is interested in the roles and mechanisms of matrix metalloproteinases, e.g. MMP-1, -2, -9, -14, and -28 in extracellular matrix degradation, cell migration and epithelial-to-mesenchymal transition (EMT). We are examining the role of the MMPs in initiating tumor invasion, by studying the host of cellular events that accompany MMP expression, such as oxidative stress and upregulation of genes associated with metastasis. In addition, we study the roles of the various domains of the secreted MMPs, such as MMP-1, MMP-2, and MMP-9, in tumor cell migration and invasion.
• The second research category explores a novel endoplasmic reticulum protein in cancer cell migration/invasion. We study its expression in human tumor tissues, as well as its function in cells in order to ascertain this novel gene’s role in cancer metastasis.
• The third research category involves developing a novel three dimensional invasion assay with high throughput potential for anti-cancer drug discovery.

Research Accomplishments:

• Identification of the first membrane type matrix metalloproteinase (MT1-MMP) gene in activation of proMMP-2: There has been increasing experimental and clinical evidence demonstrating that MMP-2 plays a critical role in human cancer invasion and metastasis. MMP-2 is secreted as a zymogen (latent form) and needs to be activated in order to perform its pathophysiological function. During the 1980s, a proMMP-2 activator was proposed to be localized at the plasma membrane of a cancer cell; however, the physiological activator of proMMP-2 was still a mystery until 1994. While working in the laboratory of Prof. M. Seiki at the Kanazawa University Cancer Institute in Japan, my colleagues and I cloned a novel MMP gene using an RT-PCR approach with primers that recognize the conserved sequences within the catalytic domain of MMPs. The most striking feature of this new MMP family member is the C-terminal transmembrane domain [1] that provided the name membrane type-MMP (MT1-MMP) for this new MMP. We further demonstrated that MT1-MMP activates proMMP-2 [2], promotes cancer cell migration, invasion and metastasis [3].
• Characterization of the role of MT1-MMP in early cancer dissemination: Most human tumors are epithelial in origin. Transition to a malignant phenotype often involves the loss of the epithelial phenotype and the acquisition of a fibroblastic or mesenchymal phenotype (EMT). This transition has emerged as a critical step in the conversion of early stage tumors into invasive cancers that disseminate widely into the systemic circulation. To explore the role of MT1-MMP in early stage cancer progression, we have employed a three-dimensional (3D) cell culture model. Expression of MT1-MMP cDNA in less invasive human LNCaP prostate cancer and MCF-7 breast cancer cells leads to a change in cell morphology from a cuboidal-like epithelial shape to a fibroblast-like shape; these cells displayed a scattered growth pattern in 3D type I collagen gels. Changes in cell morphology were accompanied by a decrease in epithelial markers and an increase in mesenchymal markers, both in vitro and in vivo, consistent with the EMT phenotypic change. We further demonstrated that MT1-MMP-induced phenotypic changes were dependent upon upregulation of Wnt5a, which has been implicated in EMT. We concluded that MT1-MMP plays an important role in early cancer dissemination by converting epithelial cells to migratory mesenchymal-like cells [4].
• Discovery of an alternative approach to drug targeting of specific MMPs: MMPs were originally identified as matrix digesting enzymes. In addition to their proteolytic activity, MMPs have been implicated as important regulatory molecules in cancer dissemination. We recently demonstrated that overexpression of the latent form of MMPs in non-malignant COS-1 cells resulted in enhanced cell migration, suggesting a non-proteolytic role of MMPs in cell migration [5]. By examining structure-function relationships, we found that the hemopexin (PEX) domains of MMP-9 and MT1-MMP play a critical role in enhancing cancer cell migration/invasion [5,6]. Furthermore, targeting the PEX domain of MT1-MMP with a polyclonal antibody against the PEX domain prevents cancer cell scattering and phenotypic changes in a 3D culture model [4]. Because of the failure of MMP inhibitors in clinical trials (targeting the conserved catalytic sites of MMPs), our discovery of the important biologic role of the PEX domain provides a novel molecular target and an alternative drug targeting approach to abrogate MMPs in cancer.
• Identification of a novel surrogate marker in cancer cell migration/invasion: Using a PCR-select subtraction hybridization approach to identify factors involved in cancer invasion, we have cloned and begun to characterize a gene (named C43) that we identified in concanavalin A-stimulated human HT1080 fibrosarcoma cells. The C43 gene is normally expressed in the central nervous system, including the brain and spinal cord. By mining DNA microarray databases from the Gene Expression Omnibus in NCBI and Oncomine in the Cancer Profiling Database, we identified the upregulation of C43 in human breast cancer tissues. We noted a correlation of C43 upregulation with cancer grade, stage, recurrence, estrogen receptor (ER) status, and HER-2 status. In preliminary studies, we have demonstrated that the C43 gene is highly expressed in various disseminated carcinoma cell lines compared to their less aggressive counterparts. Downregulation of C43 in human MDA-MB-231 and MDA-MB-435 breast cancer cells using a short hairpin RNA (shRNA) approach resulted in inhibition of cancer cell migration and invasion. Overexpression of this genein non malignant COS-1 cells or weakly aggressive human MCF-7 breast cancer cells enhanced random cell migration. The distribution of C43 gene was limited to the endoplasmic reticulum. The C43 gene was also found to be correlated with the cell dissemination. Our data indicate that the C43 gene may be a novel surrogate marker in cancer cell migration/invasion (patent application is pending; manuscript in preparation).
• Development of a novel three-dimensional (3D) high throughput phenotypic screening for an anti-cancer drug program: Based on DNA microarray analysis, it has been suggested that 3D cell culture environments can closely mimic in vivo growth conditions. The most commonly used 3D cell culture system, the multicellular spheroids, has been proposed as a drug screening tool. However, high throughput screening (HTS) of the multicellular spheroid assay is hampered by a lack of standardized and rapid procedures. In an effort to expedite the overall drug screening process, we have developed a novel 3D invasion assay which allows quantitative determination of cancer cell migration/invasion in a 3D matrix. Our preliminary data demonstrated that this novel invasion assay is simple, precise, easy to replicate, and has multiple applications. To increase the throughput and to standardize and automate the readout using imaging software, we have designed and tooled a 96-well prototype plate. This tooled plate permits automated microscopic imaging of cell invasion (manuscript in preparation). Our pilot studies indicate that this novel invasion assay may be useful for different applications including: 1) screening a small molecule compound library for identification of novel candidates for interfering with cancer cell migration/invasion; 2) screening an antibody library for identification of inhibitory antibodies; 3) validating selected compounds identified in chemical-based screening; 4) determining the effect of selected compounds on different types of cancers; 5) studying genes involved in cancer invasion; and screening a compound library against cell migration in other disease-related studies, e.g. macrophage migration in atherosclerosis. Our ongoing anti-cancer drug development using this novel technology for screening the NCI diversity compound library proves that our 3D invasion screening assay can be incorporated into a drug discovery program (Patent application is pending).

These research areas, resembling the three sides of a triangle, constantly challenge the Lab's understanding of cancer pathobiology. The long-term goal of Dr. Cao's lab is to better understand early cancer metastasis and to develop inhibitors to prevent cancer dissemination.

References

1. Cao, J., Sato, H., Takino, T., and Seiki, M. (1995). The C-terminal region of membrane type matrix metalloproteinase is a functional transmembrane domain required for pro-gelatinase A activation. J Biol Chem 270, 801-5.
2. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994). A matrix metalloproteinase expressed on the surface of invasive tumour cells [see comments]. Nature 370, 61-5.
3. Cao, J., Chiarelli, C., Kozarekar, P., and Adler, H. L. (2005). Membrane type 1-matrix metalloproteinase promotes human prostate cancer invasion and metastasis. Thromb.Haemost. 93, 770-778.
4. Cao, J., Chiarelli, C., Richman, O., Zarrabi, K., Kozarekar, P., and Zucker, S. (2008). Membrane type 1 matrix metalloproteinase induces epithelial-to-mesenchymal transition in prostate cancer. J.Biol.Chem. 283, 6232-6240.
5. Dufour, A., Sampson, N. S., Zucker, S., and Cao, J. (2008). Role of the hemopexin domain of matrix metalloproteinases in cell migration. J.Cell Physiol.
6. Cao, J., Kozarekar, P., Pavlaki, M., Chiarelli, C., Bahou, W. F., and Zucker, S. (2004). Distinct roles for the catalytic and hemopexin domains of membrane type 1-matrix metalloproteinase in substrate degradation and cell migration. J.Biol.Chem. 279, 14129-14139.