Mitsuhiko Ikura

Picture of Dr. Mitsuhiko Ikura

Professor

Canada Research Chair in Cancer Structural Biology
Ph.D. Hokkaido, Japan

MaRS Centre
Toronto Medical Discovery Tower
101 College Street, Room 4-804
Toronto, Ontario M5G 1L7

Phone: (416) 581-7550
Lab Phone: (416) 581-7551
Email Dr. Mitsuhiko Ikura

 

Structural Biology and Cancer Cell Signalling

Keywords : Cancer cells, Signal transduction, cell adhesion, cell polarity, gene transcription, cytoskeletal dynamics, calcium signalling, phospho-inositide signalling, mTOR signalling, NMR, X-ray crystallography, protein structure and function, live-cell fluorescence imaging.

Structural biology has become a mature field in modern biology, which has produced unparalleled details of insights into a variety of biological phenomena and systems. Structural biologists use nuclear magnetic resonance (NMR), X-ray, and electron microscopy (EM) to determine atomic-resolution structures of medically important proteins and other biomolecules, which helps our understanding of human diseases such as cancer. Ultimately this could lead to identifying a novel therapeutic approach against a specific type of cancers. Over the past two decades, the Ikura laboratory, located at the Toronto Medical Discovery Tower, has determined a number of 3D structures of proteins and has been investigating on molecular and structural mechanisms underlying a variety of cell signalling systems involved in human health and disease. Through our structural biology research, we hope to contribute to better understanding of cancer biology as well as developing anti-cancer therapeutics.

We know that cancer is caused by genetic mutations and/or epigenetic alternations, and that different tumours exhibit significant diversities in genotype and phenotype. From this viewpoint, it is clear that we need to treat cancer patients individually based on their specific genotypes and disease states. The concept of “personalized medicine” to treat cancer patients as individuals rather than phenotypic groups has become a reality with the development of highly selective and less toxic drugs such as Gleevec and Herceptin. While these compounds showed high potency in treating specific subtypes of leukemia and breast cancer, respectively, they benefit only a small fraction of current cancer patients, and are themselves only first generation drugs. Another challenge steams from cancer cell’s ability to quickly develop drug resistance. Identification and targeting of other signalling molecules and processes will lead to the next generation of selective cancer therapeutics. The future of molecularly-targeted therapy depends upon our greater understanding of the specific molecular genetic abnormalities that drive different subsets of cancer.

In our laboratory we are particularly interested in understanding how genetic alterations affect the 3D structures of molecules, and deregulate critical signalling pathways. Our experimental tools include NMR spectroscopy, X-ray crystallography, EM, fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence (TIRF) imaging microscopy. NMR, X-ray, and EM enable us to determine atomic-resolution 3D structures of key molecular players and analyze the dynamics and kinetics of their interactions, while in-cell FRET and TIRF microscopy let us visualize where and how the signalling molecules work in the cell in response to a stimulus. We combine structural and biochemical analyses with functional studies on mammalian cell lines and model organisms using advanced technologies such as gene knock-out and RNAi-based gene silencing. Presently our research focuses on signalling pathways involved in three key aspects of cancer: i) cancer cell signalling, ii) cancer metastasis, and iii) cancer homeostasis. We believe that our molecular studies of cancer cell signalling will contribute to a deeper understanding of this life-threatening human disease. Details of specific studies are summarized below:

1. Small GTPase-mediated signalling

The 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.

2. 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.

3. Calcium Signaling

Cancer-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 ( 2+ ) 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 Ca 2+ 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 Ca 2+ 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 (IP 3 ) receptor (IP 3 R), G-protein-coupled receptors and receptor tyrosine kinases. Recent studies have revealed that Ca 2+ high/STIM2low expression. These molecular alterations in cancer cells lead to remodeling of intracellular Ca 2+ 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 2+ -release-activated 2+ (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., 2+ channels and 2+ pumps).

Our current focus is the store-operated 2+ 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 2+ depletion of the endoplasmic reticulum (ER) by the IP 3 receptor, STIM1 senses the low concentration of 2+ in the ER and sends a signal to the Orai1 channel, which forms a pore in the plasma membrane, leading to the 2+ influx. We employ structural methodologies to elucidate the molecular basis of this 2+ signalling process in order to understand (i) how the ER 2+ depletion is trigged by IP 3 and its receptor IP 3 R, (ii) how STIM1 functions as an ER 2+ 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.

4. Transcription Regulation

Many cell signalling pathways ultimately control the transcription of specific genes in the nucleus. In humans, gene transcription is governed by RNA polymerases, general transcription factors (GTFs) and chromatin regulators, which are highly expressed in many cell types. Differentiation of cells into neurons or muscles, for example, depends upon numerous transcriptional activators and repressors, many of which are highly specific to certain tissue and cell types, and receive signals from cytoplasmic signalling events. For instance, a lineage of hematopoietic cells, including cancerous blood cells, is defined by cell-specific transcription factors. Some (~15%) patients with acute myeloid leukemia (AML) carry the t(8,21) chromosomal translocation in their white blood cells, which generates a promiscuous fusion oncoprotein AML1-ETO. We have determined the mechanism underlying how AML1-ETO suppresses the normal transcriptional activity of the transcriptional activator HEB (an E-protein critical in T-cell and B-cell development). Our study offered the structural basis for designing inhibitors of the abnormal function of AML1-ETO. We also investigate the transcription factor (TF) protein FOXO3a, which regulates many cellular processes including DNA repair. In the presence of DNA damage, it binds to specific DNA sequences to induce the expression of target genes that serve to delay replication and repair damaged DNA before it is copied. We have solved the structure of the DNA-binding forkhead (FH) domain of FOXO3a and characterized its interaction with the tumour suppressor protein p53. FOXO3a modulates gene expression through a conserved transactivation domain (CR3) that recruits a co-activator protein called CBP. Based on these and other results, we postulated a mechanistic mode of gene transcription regulation by this protein. We are currently pursuing structural studies on the regulatory mechanisms of transcription in order to provide detailed structural information that may provide the basis for novel anti-cancer drug development.

5. DNA Damage Response

In human cells, both normal metabolic activities and environmental factors (e.g., UV light and radiation) continually inflict DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day. These alterations can affect the expression of genes proximal to damaged sites, cause point mutations, or inflict gross structural damage to chromosomes. As described above, these alterations can lead to cancer development. Amazingly, our body’s DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, damaged DNA can trigger apoptosis. This has been exploited in anticancer therapeutics: cancer cells rely on a limited set of DNA repair pathways thus drugs that inhibit DNA repair can selectively target tumour cells with few side effects. There are a number of proteins involved in the DNA repair process, including poly-ADP ribose polymerase (PARP), a major player in the cellular response to DNA damage. PARP inhibitors have been shown to selectively kill cancer cells in BRCA1-/-or BRCA2-/- reast cancer patients, the first example demonstrating the significance of the “synthetic lethality” approach in anti-cancer therapy. Aprataxin PNK-Like Factor (APLF) participates in the DNA damage response by interacting with poly-ADP ribose as well as other nuclear proteins such as Ku70/80 proteins and histones. We will investigate the potential of these proteins as anti-cancer therapeutic targets. More specifically, we will elucidate the structures of APLF and APLF in complex with these factors in order to identify potential binding sites for inhibitors.

Acknowledgements:

Research in the Ikura laboratory has been supported by public and non-profit granting agencies including Canadian Institutes of Health Research (CIHR), Canadian Cancer Society Research Institute (CCSRI), Cancer Research Society (CRS), Heart and Stroke Foundation of Ontario (HSFO), and Canadian Foundation of Innovation (CFI).

For further information about our research, visit our laboratory website at: http://nmr.uhnres.utoronto.ca/ikura
For information on “Cellular Calcium Information Server”, visit: http://calcium.uhnres.utoronto.ca
For information about our NMR facility, visit: http://nmr.uhnres.utoronto.ca/ikura/nmrsuite/nmrsuite.html

 

List of Key Publications:

Link to Pubmed Publications
  • Yang Z, Xue B, Umitsu M, Ikura M, Muthuswamy SK, Neel BG. The signaling adaptor GAB1 regulates cell polarity by acting as a PAR protein scaffold. (2012) Mol Cell 47(3):469-83.

  • Wang F, Marshall CB, Yamamoto K, Li GY, Gasmi-Seabrook GM, Okada H, Mak TW, Ikura M. Structures of KIX domain of CBP in complex with two FOXO3a transactivation domains reveal promiscuity and plasticity in coactivator recruitment. (2012) Proc Natl Acad Sci U S A. 109(16):6078-83.

  • Meiri D, Marshall CB, Greeve MA, Kim B, Balan M, Suarez F, Bakal C, Wu C, Larose J, Fine N, Ikura M, Rottapel R. Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition. (2012) Mol Cell 45(5):642-55.

  • Seo MD, Velamakanni S, Ishiyama N, Stathopulos PB, Rossi AM, Khan SA, Dale P, Li C, Ames JB, Ikura M, Taylor CW. Structural and functional conservation of key domains in InsP3 and ryanodine receptors. (2012) Nature 483(7387):108-12.

  • Muik M, Fahrner M, Schindl R, Stathopulos P, Frischauf I, Derler I, Plenk P, Lackner B, Groschner K, Ikura M, Romanin C. STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation. (2011) EMBO J. 30(9):1678-89.

  • Zheng L, Stathopulos PB, Schindl R, Li GY, Romanin C, Ikura M. Auto-inhibitory role of the EF-SAM domain of STIM proteins in store-operated calcium entry. (2011) Proc Natl Acad Sci USA. 108(4): 1337-42.

  • Ishiyama N, Lee SH, Liu S, Li GY, Smith MJ, Reichardt LF, Ikura M. Dynamic and static interactions between p120 catenin and E-cadherin regulate the stability of cell-cell adhesion. (2010) Cell 141(1):117-28.

  • Smith MJ, Hardy WR, Li GY, Goudreault M, Hersch S, Metalnikov P, Starostine A, Pawson T, Ikura M. The PTB domain of ShcA couples receptor activation to the cytoskeletal regulator IQGAP1. (2010) EMBO J. 29(5):884-96.

  • Li GY, McCulloch RD, Fenton AL, Cheung M, Meng L, Ikura M, Koch CA. Structure and identification of ADP-ribose recognition motifs of APLF and role in the DNA damage response. (2010) Proc Natl Acad Sci U S A. 107(20):9129-34.

  • Amador FJ, Liu S, Ishiyama N, Plevin MJ, Wilson A, MacLennan DH, Ikura M. Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation "hot spot" loop. (2009) Proc Natl Acad Sci U S A. 106(27):11040-4.

  • Marshall CB, Ho J, Buerger C, Plevin MJ, Li GY, Li Z, Ikura M, Stambolic V. Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR. (2009) Science Signal. 2(55):ra3, 1-11.

  • Stathopulos PB, Zheng L, Li GY, Plevin MJ, Ikura M. Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry. (2008) Cell, 135(1): 110-22.

  • Hayashi I, Plevin MJ, Ikura M. CLIP170 autoinhibition mimics intermolecular interactions with p150Glued or EB1 (2007) Nat Struct Mol Biol .14(10):980-1.

  • Plevin, M.J., Zhang J., Guo C., Roeder R.G., Ikura M. The acute myeloid leukemia fusion protein AML1-ETO targets E proteins via a paired amphipathic helix-like TBP-associated factor homology domain (2006) Proc Natl Acad Sci USA . 103(27):10242-7.

  • Ikura, M. Ames, J.B. Genetic polymorphism and protein conformational plasticity in the calmodulin superfamily: two ways to promote multifunctionality (2006) Proc Natl Acad Sci USA . 103(5):1159-64.

  • Hayashi, I., Wilde A., Mal T. K., Ikura M. Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex (2005) Mol. Cell , Aug. 19: (19)4: 449-460.

  • Bosanac I, Yamazaki H, Matsu-Ura T, Michikawa T, Mikoshiba K, Ikura M. Crystal structure of the ligand binding suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor. (2005) Mol Cell , Jan. 21;17(2): 193-203.

  • Plevin MJ, Mills MM, Ikura M. The LxxLL motif: a multifunctional binding sequence in transcriptional regulation. (2005) Trends Biochem Sci . 2005 Feb;30(2) : 66-9.

  • Mizuno H, Mal TK, Tong KI, Ando R, Furuta T, Ikura M, Miyawaki A. Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. (2003) Mol Cell. Oct;12(4):1051-8.

  • Bosanac I, Alattia JR, Mal TK, Chan J, Talarico S, Tong FK, Tong KI, Yoshikawa F, Furuichi T, Iwai M, Michikawa T, Mikoshiba K, Ikura M. (2002) Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand. Nature 420 , 696-700.

  • Hoeflich KP, Ikura M. (2002) Calmodulin in action: diversity in target recognition and activation mechanisms. Cell 108: 739-742.

  • Truong K, Sawano A, Mizuno H, Hama H, Tong KI, Mal TK, Miyawaki A, Ikura M. (2001) FRET-based in vivo Ca 2+ imaging by a new calmodulin-GFP fusion molecule. Nat. Struct. Biol . 8, 1069-1073.

  • Tepass U, Truong K, Godt D, Ikura M, Peifer M. (2000) Cadherins in embryonic and neural morphogenesis. Nat. Rev. Mol. Cell. Biol . 1, 91-100.

  • Osawa M, Tokumitsu H, Swindells MB, Kurihara H, Orita M, Shibanuma T, Furuya T, Ikura M. (1999) A novel target recognition revealed by calmodulin in complex with Ca 2+ -calmodulin-dependent kinase kinase. Nat. Struct. Biol . 6, 819-824.

  • Liu, D., Ishima, R., Tong, K. I., Bagby, S., Kokubo, T., Muhandiram, D. R., Kay, L. E., Nakatani, Y., and Ikura, M. (1998) Solution Structure of a TBP-TAFII230 Complex: Protein Mimicry of the Minor Groove Surface of the TATA Box Unwound by TBP. Cell 94, 573-583.

  • Tanaka, T., Saha, S. K., Tomomori, C., Ishima, R., Liu, D., Tong, K. I., Park, H., Dutta, R., Qin, L., Swindells, M. B., Yamazaki, T., Ono, A. M., Kainosho, M., Inouye, M., and Ikura, M. (1998) NMR Structure of the Histidine Kinase Domain of the E. coli Osmosensor EnvZ. Nature 396, 88-92.

  • Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M. and Tsien, R. (1997) Cameleons: Fluorescent Indicators for Ca 2+ Based on Green Fluorescent Proteins and Calmodulin. Nature 388, 882-887.

  • Nagar B, Overduin M, Ikura M, Rini JM. (1996) Structural basis of calcium-induced E-cadherin rigidification and dimerization . Nature 380, 360-364.

  • Bagby, S., Kim, S., Maldonado, E., Tong, K. I., Reinberg, D., and Ikura, M. (1995) The Solution Structure of the Carboxy-terminal Core Domain of Human TFIIB: Similarity to Cyclin A and Interaction with the TATA Binding Protein, Cell 82, 857-867.

  • Tanaka, T., Ames, J.B., Harvey, T.S., Stryer, L. and Ikura, M. (1995) Sequestration of the Membrane-targeting Myristoyl Group of Recoverin in the Calcium-free State, Nature 376, 141-448.

  • Overduin M, Harvey TS, Bagby S, Tong KI, Yau P, Takeichi M, Ikura M. (1995) Solution structure of the epithelial cadherin domain responsible for selective cell adhesion. Science 267, 386-389.

 

Graduate Students:

  • Fernando Amador
  • Marc Balan
  • Mohammad Mazhab Jafari