Transcription Factor Activity Imaging in Brain and Brain Tumors
John Laterra, M.D., Ph.D.
Kennedy Krieger Research Institute, Baltimore, MD
David Mahoney Neuroimaging Program
December 2004, for 3 years
Using Imaging to Identify Tumor Growth-Promoting Proteins as Therapeutic Targets
In this project, researchers will modify laboratory techniques so that they can be used to locate and quantify abnormal proteins, called “transcription factors,” that promote brain tumor growth. The techniques will be adapted for use first in animals, and ultimately in humans, as a means to evaluate therapies that halt tumor growth by destroying transcription factors.
Tumor growth is facilitated by abnormalities in genes that direct the production of transcription factor proteins. These proteins increase the growth and migration of brain tumors, and their invasion into surrounding brain tissues. Imaging tools, called FIAU- SPECT and FIAU-PET, currently are used to locate and quantify transcription factors in laboratory cells. The investigators will modify these tools for use in live animals and ultimately in patients. This would facilitate evaluation of brain tumor therapies directed at transcription factors, and also help to classify brain tumors at the molecular level.
Significance: If currently available laboratory tools that can locate and quantify tumor growth-promoting transcription factors can be successfully modified for human use, they could become an important aid in evaluating the effectiveness of experimental therapies designed to destroy or inhibit these proteins.
Transcription Factor Activity Imaging in Brain and Brain Tumors
The deregulation of specific cell signaling pathways and their downstream transcription factor targets plays an important pathogenic role in numerous brain disorders. Thus, having the capability to image second messenger and transcription factor activity in vivo will advance our understanding of disease mechanisms and our ability to target pathogenic pathways. In vivo transcription factor imaging has the exciting potential to allow for real time analysis of location and magnitude of specific transcriptional events within target tissues and will greatly facilitate the classification of brain diseases at the molecular level in vivo, the quantification of baseline activities of specific transcription factors/pathways, and the noninvasive monitoring of transcription factor responses to therapies that target transcription factors and their upstream regulators.
A primary role for dysfunctional transcription regulation in brain pathogenesis is well defined in primary brain tumors that are now the most common cause of cancer-related death in children and increasingly responsible for neurologic disability and death in middle-aged adults. The molecular mediators of dysfunctional brain tumor transcription are numerous, and many are considered promising therapeutic targets under active pre-clinical and clinical investigation. The clinical translation of these discoveries will be aided substantially by non-invasive transcription factor imaging. The hypothesis of this proposal is that reporter transgenes can be used to image and quantify the activity of endogenous brain tumor-associated transcription factors within brain at baseline and in response to therapeutics that alter transcription factor activity.
In Aim #1 we will engineer chimeric tri-reporter transgenes (coding for luciferase, red fluorescence protein, and a truncated herpes simplex sr39 thymidine kinase) for localizing and quantifying the activity of specific transcription factors (i.e. Gli and E2F) linked to brain tumor cell malignancy and validate their reporter function in vitro.
In Aim #2 we will establish that reporter transgenes can be used to quantify and localize transcription factor activity in intracranial tumor xenografts derived from tumor cell lines engineered to stably express the tri-reporters constructed and validated in aim #1. We will also image the effects of anti-tumor agents expected to down-regulate Gli- and E2F-dependent reporter expression in tumor xenografts using the HSV sr39 thymidine kinase (ttk) substrates 125I-FIAU (SPECT imaging) and 124I-FIAU (PET imaging).
In Aim #3 we will determine if reporter constructs delivered to wild-type brain tumor xenografts in vivo can be used to quantify tumor transcription factor activity and transcriptional responses to anti-tumor therapeutics. The successful completion of these experiments will establish the feasibility of monitoring endogenous disease-related transcription factors within brain and brain tumors in vivo. Establishing this capability will substantially impact upon the development of novel therapeutics that target cell signaling and transcriptional pathways.
Reporter transgenes can be used to image and quantify the activity of endogenous brain tumor-associated transcription factors within brain at baseline and in response to therapeutics that alter transcription factor activity.
1. To engineer chimeric tri-reporter transgenes for quantifying the activity of transcription factors linked to brain tumor cell malignancy in vivo and to validate their reporter function in vitro.
2. To establish that reporter transgenes can be used to image and quantify transcription factor activity within intracranial tumor xenografts in vivo.
3. To determine if reporter constructs delivered to wild-type intracranial tumors in vivo can be used to quantify transcription factor activity and responses to anti-tumor therapeutics.
We will engineer plasmid expression vectors consisting of transcription factor-specific 5'-enhancer sequences upstream of a chimeric tri-reporter cDNA (luciferase, red Fluorescent protein, and enhanced herpes simplex thymidine kinase) that will support multiple in vivo and in vitro applications. Focus will be placed on 5'-regulatory DNA consensus sequences for two candidate transcription factors (i.e. Gli, and E2F) that play critical roles in the formation and growth of primary brain tumors (e.g. medulloblastoma, glioma). Reporter functions will be validated in vitro in tumor cell models under conditions that regulate activity of the transcription factors of interest.
Human brain tumor cell lines will be stably transfected with the validated reporter plasmids (or controls) in vitro. Transfected cells will be implanted to the brains of mice and tumors will be imaged in vivo using single photon emission computer tomography (SPECT) and/or positron emission tomography (PET) to assess the location and magnitude of reporter expression. Brains will be recovered and analyzed using biochemical and histological techniques to independently verify the magnitude and location of reporter expression as determined non-invasively. The effects of anti-tumor agents expected to downregulate Gli and E2F on reporter expression will be quantified.
For transcription factor imaging to be successful in the clinical setting, reporter constructs must first be delivered to a patient's existing tumor. Validated reporter transgenes will be cloned into replication defective adenoviral vectors for high efficiency in vivo gene delivery to pre-established intracranial tumor xenografts. In vivo reporter expression will be quantified by PET and or SPECT imaging and then reporter expression will be verified in isolated brains. The effects of anti-tumor agents on reporter imaging as established in Aim #2 will also be determined in this clinically-translatable model system.
Bioluminescence imaging (BLI) is the most widely used method for imaging an array of biological, biochemical, and molecular events in cells and animals. Applications include monitoring tumor growth and therapeutic response, measuring protein-protein interactions, observing the trafficking and proliferation of immune cells, and measuring gene expression patterns. The method most commonly relies upon the administration of a molecule called D-luciferin to cultured cells or to animals harboring cells engineered to express the “reporter enzyme” luciferase. The interaction between luciferin and luciferase generates light that is used to quantify (or report upon) the process of interest. Experimental agents that unknowingly modulate luciferin levels in luciferase-expressing cells might alter BLI output. Therefore, understanding how luciferin accumulates in cells is critical to the reliable application of BLI. Our recent efforts to apply BLI to study several new inhibitors of a cancer promoting pathway led us to discover that BLI using D-luciferin as a substrate is influenced substantially by cell expression of a transporter protein called ABCG2/BCRP. ABCG2/BCRP is a very interesting cell surface pump that was already known to protect cancer cells from chemotherapy. We found that ABCG2/BCRP pumps D-luciferin out of cells. We then found that changes in the activity of ABCG2/BCRP substantially alter BLI results. Our findings show that ABCG2/BCRP expression and function must be considered in experimental studies that rely on BLI with D-luciferin. Our findings also establish the feasibility of developing rapid and efficient BLI-based methods to identify novel modulators of ABCG2/BCRP for future testing in cancer and other disorders.
Bioluminescence imaging (BLI) is becoming indispensable to study transgene expression in vivo and for high throughput drug screening in vitro. Because reaction of D-luciferin with firefly luciferase (fLuc) produces photons of sufficiently long wavelength to permit imaging in intact animals, use of this substrate and enzyme pair has become the method of choice for performing BLI in vivo. We now demonstrate that expression of the ATP-binding cassette (ABC) family transporter ABCG2/BCRP impacts upon BLI signal output from the substrate D-luciferin. Studies in vitro demonstrate that D-luciferin is a substrate for ABCG2/BCRP but not for the MDR1 P-glycoprotein (ABCB1/Pgp), multidrug resistance protein 1 (MRP1/ABCC1) or MRP2/ABCC2. D-Luciferin uptake within cells is shown to be modulated by ABCG2/BCRP inhibitors including the potent and selective ABCG2/BCRP inhibitor fumitremorgin C (FTC). Images of xenografts engineered to express transgenic ABCG2/BCRP as well as xenografts derived from the human prostate cancer cell line 22Rv1 that naturally expresses ABCG2/BCRP demonstrate that ABCG2/BCRP expression and function within regions of interest substantially influences D-luciferin dependent bioluminescent output in vivo. These findings highlight the need to consider ABCG2/BCRP effects during D-luciferin-based BLI and suggest novel high throughput methods for identifying new ABCG2/BCRP inhibitors.
Zhang Y., Bressler J.P., Neal J., Lal B., Bhang H-EC, Laterra J., Pomper M.G. ABCG2/BCRP expression modulates D-Luciferin-based bioluminescence imaging. Cancer Res, 67:9389-9397, 2007 . *contributed equally to this publication.