Research interests: Functions of cGMP-dependent protein kinase (PKG, cGK) in inflammation and cancer

The Browning Laboratory                                                                                     

                                                                                                research opportunities

Education

BSc         1988        University of Toronto, Canada
MSc         1990        University of Toronto, Canada
PhD         1995        University of Toronto, Canada

Current Research

The Browning laboratory is focused on characterizing the functions of the cGMP-dependent protein kinases (PKG) in tumor biology, with the greater objective to exploit the potential of these enzymes as therapeutic targets for cancer prevention and treatment. Our broad approach incorporates contemporary biochemical and molecular tools to understand PKG signaling in engineered mouse models and in human cells grown in vitro and as xenografts in nude mice.

PKG expression and signaling in normal and tumor tissues.

The cGMP-dependent protein kinases exist as three different proteins encoded by two distinct genes. Our results indicate that PKG1β is found in normal epithelium from a variety of tissues but is downregulated in tumors. Understanding the mechanisms that mediate PKG silencing in colon cancer cells is an important step toward exploitation of the anti-tumor properties, and we are investigating epigenetic regulation of the type 1 PKG gene. Type 2 PKG has a more restricted tissue-expression pattern and is most widely known for its natriuretic functions in the intestinal epithelium. Although the major upstream activator of PKG2 in the intestine (uroguanylin) is silenced by tumors, we have found that PKG2 expression is maintained in colon cancer cells and may represent a novel target.

A central goal of our laboratory is to understand the basic signaling pathways controlled by PKG cancer cells in order to better understand the anti-tumor properties and to identify additional downstream therapeutic targets. This work has discovered novel cGMP-independent mechanisms of PKG activation and has demonstrated regulation of small-GTPase and MAPK pathways by PKG. We are currently characterizing the regulation of β-catenin and downstream effectors by PKG since these proteins have essential roles in the regulation of angiogenesis, proliferation and differentiation.

Key References:

Hou Y, GuptaN, Schoenlein P, Wong E, Martindale R, Ganapathy V. and Browning D. An Anti-Tumor Role For cGMP-Dependent Protein Kinase. Cancer Letters. 2006;240:60-68.

Hou Y, Ye Y and Browning D. Activation of the small GTPase Rac1 by cGMP-dependent Protein Kinase. Cell Signalling. 2004; 16(9):1061-9

Hou Y, Lascola J, Dulin NO, Ye RD, Browning DD. Activation of cGMP-dependent protein kinase by protein kinase C. J Biol Chem. 2003;278, 16706-12.

Browning DD, McShane MP, Marty C, Ye RD. Nitric oxide activation of p38 MAPK in 293T fibroblasts requires cGMP-dependent protein kinase. J Biol Chem. 2000;275, 2811-6.

The anti-tumor functions of PKG.
Several investigators have described anti-tumor properties of the cGMP signaling axis in colon cancer cells, but some controversy exists concerning the mechanism. Our group has pioneered investigations of PKG in tumor biology by engineering colon cancer cells that are inducible for type 1 PKG expression. Using these cells we have found that PKG is essential to the loss-of-adhesion-stimulated death pathway (anoikis) present in normal epithelial cells that is lost in malignant cells. Resistance to anoikis is essential for metastatic spreading, and we have shown that ectopic PKG not only re-established sensitivity to anoikis in colon cancer cells but completely blocked metastatic colonization of mouse lungs in an intravenous animal model. Future experiments aimed at determining the mechanism include in vitro models of metastasis and orthotopic transplantation in nude mice. We have also found that ectopic expression of type 1 PKG reduces the ability of colon cancer cells to grow as subcutaneous tumors in nude mice. More recently the anti-tumor effect of PKG was shown to be mediated by compromised angiogenesis, due in part to PKG-dependent attenuation of β-catenin and VEGF levels in these tumors. A key focus of our group is to confirm this tumor suppressive role of PKG in the intestine using genetically engineered mice. We are presently interested in examining tumorigenesis in mice with intestine-specific PKG-knockout or overexpression, particularly in context of the APCmin colon cancer model.

Key References:

Browning D. PKG as a therapeutic target for the treatment of metastatic colorectal cancer. Expert Opin Ther Targets. 2008;12, 367-76.

Kwon I-K, Schoenlein PV, Delk J, Liu K, Thangaraju M, Dulin N, Ganapathy V, Berger F and Browning D. Expression of PKG in Metastatic Colon Carcinoma Cells Blocks Tumor Angiogenesis. Cancer. 2008;112, 1462-70.

Hou Y, Wong E, Martin J, Schoenlein P, Dostmann W. and Browning D. A Role for Cyclic-GMP Dependent Protein Kinase in Anoikis. Cell Signalling. 2005;18(6):882-8.

The role of PKG in inflammation.
Chronic inflammation precedes the development of nearly all adult tumors and remains important for progression. Inappropriate immune-activation in the intestinal tract can lead to inflammatory bowel disease, which has significant correlation with intestinal tumorigenesis. Elevation of cGMP levels in the intestinal tract using GC-C agonists has been shown to have anti-inflammatory as well as tumor-suppressive effects, but the mechanism is not known. Our laboratory and others have found that type 1 PKG is important to the normal behavior of leukocytes, but it also has barrier-protective functions in cell monolayers. We hypothesize that activation of PKG in the intestinal epithelium mediates the anti-inflammatory effects of cGMP in these cells and is an important area of interest that will be characterized in our murine transgenic and knockout models.

Key References:

Ying L, Hofseth A, Browning D, Nagarkatti M, Nagarkatti P and Hofseth L. Nitric oxide inactivates the retinoblastoma pathway in chronic inflammation. Cancer Research. 2007;67(19):9286-93.

Browning DD, Windes ND, Ye RD. Activation of p38 mitogen-activated protein kinase by LPS in human neutrophils requires nitric oxide-dependent cGMP accumulation. J Biol Chem. 1999;274, 537-42.

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