Protein Kinase C Signaling and Multidrug Resistance in Breast Cancer

            There are two major research foci for my research group. One relates to increasing our understanding of how the activity of the protein kinase C (PKC) family of kinases is regulated in cells. Disregulation of PKC isoenzymes is associated with many disease states, including cancer and diabetes. Our second research focus is "multidrug resistance", where tumours exhibit or acquire the ability to resist killing by a wide spectrum of structurally unrelated chemotherapy drugs. Our goals are to better understand the various mechanisms responsible for multidrug resistance and to improve the effectiveness of chemotherapy in patients. Current research projects include:

(1) Mapping of newly identified novel PKC inhibitory sites within the PKC Regulatory Domain: We have recently published evidence that the activity of the protein kinase C alpha catalytic domain can be potently inhibited by its regulatory (R) domain without requiring the "pseudosubstrate" site within the PKC R domain. Since there are no additional pseudosubstrate-like sites within this R domain, this observation challenges the central role of pseudo-substrates in the autoinhibition of protein kinases and suggests that additional site(s) are involved. Our most recent paper maps these novel autoinhibitory regions using tandem PCR-mediated mutagenesis approaches. We are currently using site-directed mutagenesis to identify the key residues involved autoinhibition.

(2) Modulation of PKC function by the Cytoskeletal Protein Calponin: The activity of "classical" PKCs (alpha, beta, and gamma) is regulated by a variety of hormonal stimuli that induce a translocation of the enzymes to specific phospholipid-abundant subcellular compartments, resulting in increased enzyme autophos-phorylation and catalytic activity. In collaboration with Kathy Morgan of Harvard University, we have found that the cytoskeletal protein calponin can directly activate PKC, without requiring the binding of traditional PKC activators in cells (phospholipids, lipids, or Ca++). Calponin also appears to increase the amount of PKC bound to membranes, suggesting that it also plays a role in recruitment of PKCs to membranes. We are currently using mutagenesis approaches to precisely map important regions within PKC involved in calponin-dependent activation.

(3) Multidrug Resistance in Breast Tumour Cells: We have established a panel of isogenic cell lines that have been selected for resistance to various chemotherapy drugs used in the treatment of human breast cancer. Since the cell lines all originate from the same source and were selected in an identical manner, they should be invaluable in examining the relationship between the expression of specific genes or proteins in breast tumour cells and drug resistance. Using cDNA micro-array and other approaches, we have identified a variety of genes, whose expression correlates with resistance of breast tumour cells to specific drugs in vitro. In an upcoming clinical trial by the National Cancer Institute of Canada, we will examine the utility of these genes as predictors of clinical response in patients undergoing chemotherapy for locally advanced breast cancer. We are also identifying agents that can effectively kill drug-resistant breast tumour cells. One such agent (calphos-tin C) is capable of killing cell lines that are > 4000-fold more resistant to drug than wildtype cell lines.

Selected Recent Publications:

Reed, K., Hembruff, S. L., Sprowl, J. A. and Parissenti, A. M. 
The temporal relationship between ABCB1 promoter hypomethylation, ABCB1 expression, and the acquisition of drug resistance. 
The Pharmacogenomics Journal, Feb 2, Epub ahead of print. (2010)

Parissenti A.M., Chapman, J.A., Kahn, H.J., Guo, B., Han, L., O'Brien, P., Clemons M.P., Jong, R., Dent, R., Fitzgerald, B., Pritchard, K.I., Shepherd, L.E., Trudeau, M.E.
Association of low tumor RNA integrity with response to chemotherapy in breast cancer patients. 
Breast Cancer Res. Treat., 119: 347-56. (2010) [PDF]