Edward Chu MD
Deputy Director, Cancer Center; Chief, Section of Medical Oncology
Developmental therapeutics, cancer drug resistance, translational regulation, RNA-protein interactions, colorectal cancer, GI cancers, cancer drug development.
Current Projects1. Investigate the cis- and trans-acting elements that mediate the interactions between TS protein and its own TS mRNA
2. Investigate the cellular miRNAs that regulate TS mRNA translation
3. Develop siRNAs as novel therapeutic molecules to target TS expression
4. Develop Chinese herbal medicine as modulators of cancer chemotherapy in the treatment of GI cancers
My research efforts have focused on elucidation of the mechanisms
of cytotoxicity and resistance for the antimetabolite class of antineoplastic
agents. Specifically, my group has studied the fluoropyrimidines,
5-fluorouracil (5-FU) and capecitabine, the two most active single agents
currently available for the treatment of human colorectal cancer and used in
the treatment of a broad range of cancers, including breast and other GI
Our basic research studies have provided new insights as to how tumors become resistant to the effects of cancer chemotherapy, and my laboratory discovered a novel translational autoregulatory mechanism that controls the expression of the chemotherapeutic target thymidylate synthase. Based on these molecular studies, my research is also focused on the design and development of novel treatment approaches for human cancers, with a specific focus on colorectal cancer.
I have been actively involved in the clinical translational research of colorectal cancer, and his specific area of research interest has been on clinical drug development for colorectal cancer and other GI malignancies. As part of this effort, I have been focused on developing clinical trials that incorporate Chinese herbal medicines as part of standard cytotoxic chemotherapy in order to modulate clinical activity by reducing toxicity and enhancing efficacy.
Extensive Research Description
laboratory has focused on characterizing the translational autoregulation of
the folate-dependent enzymes, thymidylate synthase (TS) and dihydrofolate
reducatase (DHFR). TS plays a critical role in the process of DNA biosynthesis
as it catalyzes the reaction which provides the sole intracellular de novo
source of thymidylate, an essential nucleotide precursor for DNA biosynthesis.
This enzyme has served as a target for cancer chemotherapy for well over 40
years and remains an active area of investigation. DHFR plays an essential role
in the synthesis of tetrahydrofolate, which is the key one-carbon donor
required for de novo synthesis of purines, pyrimidines, and proteins. This
enzyme has also served as an important target in cancer chemotherapy.
Several investigators had made the observation that exposure of cultured human cancer cells, human tumors in in vivo model systems, and patient tumor specimens to TS inhibitor compounds, the best known of which is the fluoropyrimidine 5-fluorouracil, resulted in an acute induction of TS enzyme and protein. Moreover, this induction of TS protein appeared to correlate with the rapid development of cellular drug resistance to these TS inhibitor compounds. Our laboratory was the first to show that this induction of TS protein was mediated, at least in part, by a translational regulatory mechanism. We went on to show that TS expression is regulated by a translational autoregulatory mechanism in which TS protein binds to its own cognate TS mRNA. While this process of translational autoregulation has been well-described in bacteriophage and prokaryotes, this was the first demonstration of translational autoregulation in a eukaryotic organism. We have also demonstrated that dihdrofolate reductase (DHFR), is regulated in a similar fashion. Thus, our laboratory has demonstrated this relatively novel regulatory process in eukaryotic organisms for two different genes, thymidylate synthase and dihydrofolate reductase, and our laboratory is considered one of the leaders, if not the leader, in this important field of research (an important point to emphasize).
Our lab continues to focus on characterizing the molecular elements involved in mediating the interaction between TS protein and its own target TS mRNA. We have identified two novel cis-acting elements on the TS mRNA, one resides in the 5’-upstream region and includes the translational AUG start site, while the second element resides in the protein-coding region. Our work has involved a dissection of the critical nucleotides and secondary structure that play a key role in RNA recognition. In addition, we have recently shown using in vivo transfection experiments that each element can function independently of one another in controlling TS expression. However, it appears that both elements may be required for optimal translational regulation of TS expression in vivo. We developed an in vitro selection methodology to further characterize the critical nucleotide elements required for binding of TS protein to the TS mRNA. This is a powerful method to identify nucleic acid molecules with high-affinity binding for a wide range of targets including small molecules and proteins.
Using a completely random 25-nt oligoribonucleotide (ORN) library, we identified a single RNA ligand that can bind TS protein with significantly higher affinity (up to 20-fold) than wild-type TS RNA sequences. This interaction requires the presence of a 3-nt stem-loop structure. In vivo transfection experiments confirm that this selected RNA sequence requires a functionally intact TS for its biological activity. However, in contrast to the native TS RNA sequences previously identified which function as negative repressor elements, this in vitro-selected RNA sequence appears to function as an enhancer element in that its presence stimulates the synthesis of TS at the translational level. There are presently two examples of enhancer elements residing on the ferritin mRNA and hepatitis c mRNA. Thus, this is a relatively novel finding in that the majority of the well-characterized cis-acting translational regulatory sequences function as repressor elements. The importance of this work is that it represents a powerful approach to characterize the molecular basis underlying the TS mRNA-TS protein interaction. In addition, such an approach may provide the rational basis for molecular drug design to identify novel small molecules that can be used to inhibit and/or sequester TS enzyme activity and function. In its role as an RNA binding protein, TS is capable of directly interacting with other cellular RNA species in addition to its own TS mRNA.
Our lab developed a novel immunoprecipitation-RNA:random PCR method to isolate cellular RNA sequences that form ribonucleoprotein complexes with TS protein in human colon cancer cells. Using this approach, we identified nine different cellular mRNAs including those corresponding to the p53 tumor suppressor gene and the myc family of genes. These studies suggested that TS may be involved in the coordinate regulation of expression and/or function of these identified genes. Since coming to Yale, our lab has focused on characterizing the biological significance of the interaction of TS protein with the p53 mRNA, especially since p53 has been shown to play a critical role in cell cycle control, apoptosis, and as a key mediator of chemosensitivity. We have shown that TS binds to a sequence located in the protein-coding region of p53 mRNA and that this interaction results in translational repression, as determined by in vitro translation experiments. In vivo transfection experiments using either a tetracycline inducible system or a constitutive system where TS is overexpressed in human colon cancer cells that express a mutant and functionally inactive TS, show conclusively that overexpression of TS results in marked suppression of synthesis of p53 protein with no associated change in p53 mRNA levels. Thus, in the in vivo setting, TS directly binds to p53 mRNA resulting in translational repression.
As part of this work, our laboratory developed a novel immunoprecipitation-RT:PCR method to document the presence of TS protein-p53 mRNA complexes in intact biological systems. Further studies have shown that the TS protein-p53 mRNA interaction results in an inability of these TS-overexpressing cells to undergo cell cycle checkpoint following exposure to a host of DNA damaging agents. They also have an impaired ability to undergo apoptosis. Moreover, these TS-overexpressing cells with impaired expression of p53 develop resistance to UV- and gamma-irradiation as well as resistance to a host of unrelated anticancer agents including 5-FU, adriamycin, purine analogs such as fludarabine, and the platinum class of compounds. Thus, the regulation of p53 expression by TS may represent a novel mechanism by which the property for pleiotropic drug resistance may develop.
Our lab is now in the process of characterizing the molecular and biological downstream consequences of the interaction between TS protein and the p53 mRNA. Our lab is focused on characterizing the molecular elements underlying the interaction between TS protein and its own TS mRNA as well as the interaction between TS protein and the p53 mRNA. These studies are of fundamental importance from a basic biology point of view as they provide new insights into the elements that mediate RNA-protein interactions and the process of translational regulation. Moreover, they provide novel opportunities and strategies to develop new approaches for the treatment of human cancer.
As one example, we are working with a medicinal chemist to develop novel small molecules to optimally inhibit TS expression and function. As noted previously, one of the potential problems with the class of TS inhibitor compounds currently in clinical practice is that they result in the acute induction of new TS protein, a self-defeating process. Ideally, it would be nice to develop an inhibitor compound that would potently inhibit TS enzyme activity yet still allow the TS protein to bind its target TS mRNA, resulting in translational repression and inhibition of new TS protein synthesis. We have recently identified 3 pyrrolopyrimidine lead compounds that appear to fulfill these criteria. They do not seem to prevent the interaction of TS protein with target TS mRNA, and treatment of human colon cancer cells with these compounds results in sustained inhibition of TS activity with no acute induction of TS protein. We are now performing a structure-activity relationship study of these lead compounds in the hopes of identifying a candidate that can be eventually taken to the clinic.