COMBINING PROTEOMICS and SYSTEMS BIOLOGY to IDENTIFY THEAPEUTIC TARGETS & PATHWAYS OF CANCER DRUGS

Open Access
Author:
Kuzu, Omer Faruk
Graduate Program:
Genetics
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
None
Committee Members:
  • Gavin Peter Robertson, Thesis Advisor
  • Jiyue Zhu, Thesis Advisor
  • Jin Ming Yang, Thesis Advisor
  • Philip Lazarus, Thesis Advisor
Keywords:
  • Leelamine
  • Melanoma
  • Alox5
Abstract:
Melanoma is the malignancy of pigment producing melanocyte cells. It is the most lethal form of the skin cancer that takes the life of one person per hour in United States (U.S)[1]. It is estimated that more than 70 thousand new melanoma cases will be diagnosed in the U.S. in 2011 and at least one melanoma diagnosed patient in ten projected to die due to this deathful disease[2]. Moreover, about 40-60% of patients with advanced melanoma will have brain metastasis and after its detection in the brain, median survival time is only 3 months[3]. Unfortunately, melanoma is poorly responsive to chemotherapy. Although there are many drugs in clinical trials, ipilimumab is the second most promising drug which is recently approved by FDA, 35 years after the approval of the dacarbazine (DTIC) [4, 5]. However, the response rate for both DTIC and ipilimumab is lower than ~18% [6, 7]. Thus there is an urgent need for development of therapeutic drugs against malignant melanoma. We have previously identified leelamine (dehydroabietylamine), a diterpene molecule extracted from pine trees, as a potential melanoma tumor growth inhibitor from natural chemical library screening (unpublished data). We have shown that leelamine inhibits melanoma tumor growth and induces apoptosis both in vitro and in vivo. However, the mechanism of action of leelamine was not revealed. In here, we aimed to illuminate the molecular pathways and targets of leelamine with the help of proteomics and systems biology. To achieve our objective, we first investigated the activities of the two known targets of leelamine (PDK1 and CB1/CB2 receptors) on melanoma cells. We found that any of these targets directly contribute to the anti-tumor activity of leelamine. Thus to identify the actual target(s), we did antibody array analyses to investigate the expression of more than 800 proteins. We analyzed the data with the Ingenuity pathway analyses tool, which revealed that the PI3K-Akt pathway is the major target of leelamine. After validation of the deregulations in the members of the Akt signaling by western blot analyses we focused on upstream targets that regulate the Akt signaling. Combinational treatment of phosphotase inhibitor with leelamine eliminated the possibility of the regulation through activation of phosphatases. After that, we ran a second antibody array that was specifically designed to evaluate the activities of the 42 different receptor tyrosine kinases which are one of the major subfamily of proteins that regulate Akt signaling [8]. This array analyses revealed that activity of PDGFR is significantly decreased by leelamine treatment. We validated this inhibition by western blot analyses; however, several groups have reported that inhibition of PDGFR was not enough to decrease melanoma cell growth [9, 10]. Our siRNA mediated knockdown of the PDGFR also supported these reports by not decreasing the growth of UACC903 cells. Thus, PDGFR inhibition seemed to be an indirect effect of leelamine treatment. We continued our search by using GeneGo’s MetaDrug software, which is designed for evaluation of biological effects of small molecule compounds based on their structures. Basically, this tool compared the structure of leelamine with structures in its own database, which contains more than 600.000 compounds and interactions [11]. This analysis gave rise to two possible targets: NMDA receptors and ALOX5. Combinational treatment of leelamine with agonists or antagonists of these targets revealed that leelamine does not act through NMDA receptors. However, baicalein, an ALOX5/12/15 antagonist protected UACC903 cells from leelamine induced cell death in a concentration dependent manner. Even though, this information draws up us very close to our objective, we finished our thesis by proposing several hypotheses, which we will investigate in future. In the first chapter of this thesis, we review some of the current proteomic approaches and systems biology tools. In the second chapter we summarize the molecular background of the melanoma development. We briefly give information about the pathways and mutations that regulate melanoma development. The 3rd chapter is the major part of this thesis and include the rationale and results of our studies. It also includes a discussion of the possible future approaches and hypothesis to complete the identification of drugs target. The last chapter is dedicated to future directions in proteomics, systems biology and melanoma therapeutics.