Date of Award

2011

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Richard Niles

Second Advisor

W. Elaine Hardman

Third Advisor

Maiyon Park

Abstract

Thyroid cancer is the most prevailing malignancy of the endocrine system. Its incidence is rapidly rising at the second fastest rate of all malignancies in the United States, making it a significant health problem. Although the majority of thyroid cancer is slowly-growing and well-differentiated, available treatment options are very limited, and most of them require complete removal of the thyroid gland and surrounding tissues. Patients who have undergone thyroid removal have to take life-long hormone replacement therapy, which is very inconvenient and costly. Therefore, there is an urgent need to develop new treatments for this disease. As a prerequisite for designing a better therapy for thyroid cancer patients, we now must further our understanding on how thyroid cancer develops, especially its underlying molecular mechanisms. In this study, we have found that PITX2, a bicoid homeodomain transcription factor known to play a critical role in the left-right asymmetry formation as well as the development of multiple organs, is frequently expressed in human follicular cell-derived (papillary, follicular and anaplastic) thyroid cancer tissues but not in normal thyroids. This is the first finding that indicates over-activated PITX2 may contribute to the development of thyroid cancer. Following this exciting discovery, we performed cell-based and biochemical studies to uncover the molecular mechanism of PITX2 action in thyroid tumorigenesis. Knockdown of PITX2 gene expression in human thyroid cancer cells significantly reduced cell proliferation and soft-agar colony formation. Biochemical analysis of cell cycle regulators upon PITX2 knockdown revealed downregulation of Cyclin D1, Cyclin D2 and dephosphorylation of Rb. Chromatin immunoprecipitation and promoter reporter assay indicated that Cyclin D2 was a direct target gene of PITX2. Consistently, we observed that high expression levels of Cyclin D2 were frequently associated with PITX2 expression in follicular cell-derived thyroid cancer tissues. To confirm these findings in vivo, we took advantage of a mouse model of thyroid cancer (TRbetaPV/PV mouse). Consistently, the aberrant elevation of Pitx2 levels in the thyroid cancer of TRbetaPV/PV mice were accompanied by the upregulation of Cyclin D1, Cyclin D2 and increased phosphorylation of Rb. Taken together, these results provide the first evidence implicating an oncogenic role of PITX2 in human cancer. To better understand the role of PITX2 in the regulation of gene transcription, we aimed to decipher PITX2 regulating and interacting networks by genomic and proteomic approaches. As a result, we identified four novel PITX2-associated protein partners YB-1, hnRNP K, nucleolin and hnRNP U in mass spectrometry analysis. Overexpression of PITX2 resulted in upregulation of 868 genes (two-fold to twenty five-fold) and downregulation of 191 genes (two-fold to fifteen fold) in microarray analysis. Using semi-quantitative RT-PCR, we verified 16 potential PITX2 target genes. Interestingly, Cyclin A1, a male germ cell-specific gene essential for spermatogenesis, is among the most upregulated genes. We then investigated whether Cyclin A1 was a PITX2 target gene in the context of thyroid cancer cells. Remarkably, we found that Cyclin A1 indeed was expressed in papillary thyroid cancer but not in normal thyroids. Using promoter-driven reporter assays, an evolutionarily conserved DNA element responsible for PITX2-induced gene transcription was identified in the Cyclin A1 promoter. Intriguingly, further biochemical evidence demonstrated that PITX2 activated Cyclin A1 through a histone H3K4 methylation pathway. Collectively, our data reveal for the first time that PITX2 may play an oncogenic role in human thyroid tumorigenesis. Aberrant expression of PITX2 in thyroid cancer promotes cell proliferation by facilitating cell cycle progression. This oncogenic effect of PITX2 is at least in part mediated by its transcriptional target genes Cyclin D2 and Cyclin A1. This study furthers our understanding of the molecular mechanisms that govern thyroid carcinogenesis and provides a new perspective on the development of novel therapeutics for thyroid cancer patients.

Subject

Thyroid gland - Cancer

Subject

Genetic transformation

Subject

Protein-protein interactions

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