Mitsuhiko Ikura, PhD
- Small GTPase SignallingThe small GTPase K-Ras is one of the most frequently mutated oncogene products, with activating mutations most prevalent in human pancreatic (95%), thyroid (55%), colorectal (35%), and lung (35%) carcinomas. We intend to identify and characterize druggable target molecules that play a critical role in the Ras signalling pathway in cancer cells that are “addicted” to K-Ras activating mutations (for instance, G12V). The Ras signalling pathway involves many proteins, some of which may offer targets for synthetic lethality with K-Ras mutations in cancer cells. Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) are obvious candidates for therapeutic targets, as they directly alter the activity of small GTPase proteins and hence effector proteins downstream in the signalling pathway. By disrupting or inhibiting one of those signalling proteins, it may be possible to specifically kill the oncogene-addicted cells by virtue of “synthetic lethality”. We believe that molecular and structural studies of these key signalling molecules are extremely important to better understand how the signalling pathways in certain cancers are “wired” for cell survival, and how we could exploit cancer-specific molecular pathways to selectively kill tumour cells.We have recently developed a new real-time NMR methodology to assay small GTPase nucleotide exchange and hydrolysis, as well as the catalysis of these reactions by GEFs and GAPs, respectively. This assay uses the natural ligands GTP and GDP and has been successfully applied to a number of small GTPases including Ras, RhoA, and Rheb. Rheb, a member of the Ras sub-family of small GTPases, functions as an activator of mammalian Target of Rapamycin (mTOR), which is implicated in cell growth, proliferation, and tumourgenesis. We have characterized a number of mutations in the tuberous sclerosis complex 2 (TSC2) protein, a Rheb-specific GAP implicated in the congenital genetic disorder TSC in humans. Using NMR spectroscopy and X-ray crystallography, we are currently investigating regulatory mechanisms of several small GTPases such as Rheb, Ras and RhoA, and addressing questions of how somatic mutations found in GTPases or their GAPs/GEFs deregulate the GTPase cycle, thereby affecting normal cell function and leading to tumourgenesis. Examples include the RhoGEFs, PDZRhoGEF, and Lfc/GEF-H1, as well as RasGAPs and RasGEFs. Our NMR-based GTPase assays could offer a highly specific and reliable tool to perform functional assays for biomarkers and drug development.
- Cell Adhesion and Polarity
The main cause of mortality in cancer patients is secondary tumours, which are mediated by metastatic epithelial cancer cells. As a matter of fact, epithelial cancers constitute ~85% of all cancers including primary tumours. For these reasons, a detailed understanding of epithelial cells is crucial to combat cancer. Like many other cell types, epithelial cells have the ability to adhere to one another and form a specific multicellular structure (epithelium), which is fundamental to normal cell development and morphogenesis of human tissues. Cancer disrupts normal cell adhesion and polarity functions, permitting the spread of cancer cells from the primary cancer site to other locations in the body (metastasis). Cell adhesion and polarity are both coupled with intracellular signalling and cytoskeletal assembly. Together these cellular processes control the shape and motility of cells in a dynamic manner. The cell-cell adhesion molecule cadherin and its associated factors called catenins play critical roles in maintaining the stable cell-cell interaction. Loss of E-cadherin function has been implicated with tumourgenesis and metastatic potential of cancers.
Not surprisingly, recent studies have revealed that the cadherin adhesion system is coupled with cell polarity proteins that co-localize at the apical side of the epithelium. Using X-ray crystallography and NMR spectroscopy, we plan to determine the structural basis of the cadherin-mediated cell-cell interaction, and are working to determine the mechanisms by which the cadherin function is regulated through interaction with intracellular proteins and cytoskeletal components. Both E-cadherin and p120-catenin are well-known targets for Src protein kinases. We are investigating how Src-dependent protein phosphorylation alters the structure and function of the cadherin-catenin complex. We are also studying the Par complex, which consists of a number of cell polarity proteins. Structural characterization of these protein-protein interactions would be crucial in understanding how cell polarity and cell adhesion phenomena are dynamically regulated. We hope to reveal novel insights to support the development of therapeutics to reduce or abolish the metastatic potential of cancer cells.
- Calcium SignalingCancer-causing mutations disrupt the homeostasis of normal cells, and remodel it to promote survival of the cancerous cells. Among many factors that control cellular homeostasis, the calcium ion (Ca2+) is a vital second messenger in the cell, whose concentration is tightly regulated, such that it generates cellular signals in response to external stimuli. Utilizing Ca2+signal pathways, metastatic cancer cells develop an enhanced propensity to migrate, and effectively detach from the tumour, enter the circulation system, and re-establish secondary growth in distant organs.The Ca2+ signalling pathway responsible for cancer metastasis appears to involve Orai1, and stromal interaction molecule 1 (STIM1), which are downstream of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), G-protein-coupled receptors and receptor tyrosine kinases. Recent studies have revealed that Ca2+ high/STIM2 low expression. These molecular alterations in cancer cells lead to remodeling of intracellular Ca2+ homeostasis, resulting in altered gene expression in the cell. Interestingly, a point mutation of Orai1 (R91W) was identified in patients with the severe combined immune deficiency syndrome (SCID) and this mutation was found to impair Ca2+-release-activated Ca2+ (CRAC) activity. In order to understand how the concentration of 2+ in the cell is regulated in both a spatial and a temporal manner, and how this affects gene expression in cancer cells, we need to better understand the roles and precise regulatory mechanisms of various 2+ transporters (i.e., Ca2+ channels and Ca2+ pumps).Our current focus is the store-operated calcium entry (SOCE) mechanism, which was initially demonstrated to play a pivotal role in lymphocyte (T cell) activation, but is now recognized as a ubiquitous process fundamental to many cell types. Recent studies identified STIM1 and Orai1 as key components of the CRAC channels that mediate SOCE in T-cells. Upon the Ca2+depletion of the endoplasmic reticulum (ER) by the IP3 receptor, STIM1 senses the low concentration of Ca2+ in the ER and sends a signal to the Orai1 channel, which forms a pore in the plasma membrane, leading to the Ca2+ influx. We employ structural methodologies to elucidate the molecular basis of this Ca2+ signalling process in order to understand (i) how the ER Ca2+depletion is trigged by IP3 and its receptor IP3R, (ii) how STIM1 functions as an ER Ca2+ sensor, (iii) how STIM1 communicates with the plasma-membrane Orai1 channel to activate the CRAC function, and (iv) how the SCID mutation may lead to disruption of the normal T cell function. We hope to understand the specific roles of Orai1 and STIM1 in cancer cells and how we may be able to manipulate their deregulated functions in cancer cells.
Research in the Ikura laboratory has been supported by public and non-profit granting agencies including the Canadian Institutes of Health Research, the Canadian Cancer Society Research Institute, the Heart & Stroke Foundation of Ontario, the Canada Foundation for Innovation and The Princess Margaret Cancer Foundation.
Director (Interim), Research Institute, Princess Margaret Cancer Centre