TRANSLATIONAL STUDIES OF TARGETING THE THROMBOXANE A2 RECEPTOR IN CANCER.

Author

Viola Allaj, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2014

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Nie, Daotai

Abstract

Thromboxane A2 (TXA2) is an eicosanoid formed by the action of thromboxane A2 synthase (TXS) utilizing the cyclooxygenase product, prostaglandin H2, as the substrate. This prostanoid acts mainly by binding to its cognate thromboxane receptor (TP), a G protein coupled receptor (GPCR). GPCRs convey the majority of signal transduction across cell membranes. They are heptahelical serpentine receptors with many clinical implications. Aberrant expression or deregulated activity of GPCRs contributes to some of the most prevalent human diseases. TP activation can cause platelet activation, vasoconstriction, thrombosis and mitogenesis. A single copy of the TP gives rise to TPα and TPβ isoforms, which share the first 328 amino acids but differ at the carboxy-terminal domains. Previous studies in our lab have shown that the TXA2-TPβ signaling axis pathway regulates tumor cell cytoskeleton by inducing cell contraction during migration. The cytoskeleton reorganization observed was mediated through small RhoA GTPase activation. Metastasis is the major cause of death from cancer and acquisition of motility by cancer cells is a detrimental component in metastasis. Therefore, it is important to identify a natural TP antagonist that will serve as a therapeutic regimen for cancer by inhibiting the TPβ induced cell contraction. Our work has shown that SIU1315, a phytoestrogen, blocks cell contraction induced by the TP agonist U46619. The SIU1315 inhibitory concentration (IC50) of U46619 induced contraction was determined to be 10 uM and treatment with the compound alleviated RhoA activation. Moreover, SIU1315 reduces the migratory capacity and invasiveness of PC3 cells. Utilization of the compound to inhibit TP-mediated tumor cell motility and metastatic progression in an in vivo model of experimental metastasis had an unexpected outcome. Administration of SIU1315 in female SCID mice, that received tumor cells via tail vein injection, did not prevent the metastatic spread of malignant cells. On the contrary, mice that had been treated with SIU1315 were indicative of a higher number of metastatic lung lesions than the non-treated control group. Similar evidence was observed by luciferase readouts in other organs. It is suspected that SIU1315 stimulates the vasodilatory nitric oxide that further interferes with the metastatic process. SIU1315 could be used as an anti-metastatic agent in combination with compounds that would suppress activation of nitric oxide. Furthermore, the quest for TPβ specific antibodies, that would have the potential to distinguish between thromboxane receptor isoforms, led to investigation of a number of purified antibodies. Validation of TPβ specific antibodies demonstrated that none of the examined antibodies was specific for TPβ. Determination of the specific TPβ domain that is responsible for U46619 induced cell contraction proved rather difficult. Generation and utilization of TPβ mutants requires additional optimization studies. The function of TP receptors in lung cancer has not been extensively studied. Kaplan-Meier analysis indicated that patients with high levels of TP expression had significantly shorter disease free survival than patients with low TP expression. Generation of stable lung cancer cell lines expressing TP receptors revealed a higher proliferative advantage of cells expressing TPα when compared to the vector control. U46619 induced activation of TP receptors designated a time dependent stimulation of phosphorylated Erk in stable lung cancer cell lines. Evidence suggested the involvement of phosphorylated Akt in U46619 mediated TP activation. Data indicated differential activation of phosphorylated Erk isoforms. The significance of this observation requires further investigation. TP activation seemed to be positively correlated with β-catenin expression. In conclusion, translational studies on the thromboxane receptor reveal major functions in cancer pathogenesis. Therapeutic targeting of the TP receptor could prove beneficial for cancer patients.