Thyroid hormone has been shown to induce proliferation of a variety of human cancer cell lines via a cell surface binding site on integrin αvβ3. Clinical studies indicate that thyroid hormone may support brain tumors, breast cancer, head-and-neck cancers, renal cell carcinoma and several other cancers (1). The effect of microenvironmental thyroid hormone on growth of colorectal cancer has not been investigated fully, but the hormone enhances experimental induction of colon cancer, e.g., with azoxymethane (2) and supports P-glycoprotein gene (MDR1) upregulation—and thus, chemoresistance — in colon cancer cell lines (3). Abnormalities of intracellular thyroid hormone signaling have also been documented in many human colon carcinomas and may relate to K-ras mutations (4). High-mobility group AT-hook 2 (HMGA2) is associated with increased risk of metastasis and with reduced survival rates in patients with colorectal cancer. Preliminary results presented in this proposal indicate that thyroid hormone stimulates β-catenin, HMGA2 expression and cell proliferation in colorectal cancer lines. The mechanisms and, specifically, the signaling pathways, activated by thyroid hormone in its induction of HMGA2 are not understood. The preparatory crosstalk between integrin αvβ3 and Wnt/β-catenin signal and downstream signaling required for expression of HMGA2 need to be clarified. A thyroid hormone derivative, tetraiodothyroacetic acid (tetrac), inhibits proliferation in many types of human cancer cells in vitro via the thyroid hormone receptor on integrin αvβ3. In this proposal, we will investigate the relationships among integrin αvβ3, Wnt, β-catenin and HMGA2 that are regulated by thyroid hormone. In addition, pharmacodynamic (PD) studies of tetrac on xenografts of colorectal cancer are explore that may offer the possibility of a new regimen of treatment for colorectal cancer. The SPECIFIC AIMS are: 1. To define the molecular mechanism involved in thyroid hormone-induced, β-catenin-HMGA2-dependent proliferation of human colorectal cancer cell lines in vitro; 2. To establish the molecular basis by which thyroid hormone analogue, tetrac, acts to block HMGA2-dependent proliferation of human colorectal cancer cells in an in vitro bellows perfusion cell culture system we have developed; and 3. To verify that the observed effects of tetrac-induced anti-proliferation expressed via HMGA2 in human colorectal cancers are also demonstrable in murine xenografts. The Methods used will include: (i) suppression with shRNA to identify the critical role of integrin αvβ3 in the contributions of β-catenin, APC, and HMGA2 to thyroid hormone-induced colon cell proliferation and the phosphorylation status of the several signaling pathway proteins will be examined (Aim 1); (ii) RT-PCR and immunoblotting of integrin αvβ3 and signal proteins in thyroid hormone/tetrac-treated cells as well as the MTT and TUNEL assay (Aims 1 and 2); (iii) reporter assays to identify expression levels/interactions with HMGA2 related gene expression, such as HIF-1α (Aims 2); (iv) Affymetrix arrays to identify gene signatures of thyroid hormone-treated primary colorectal cancer cells with overexpressed HMGA2 and control cancer cells (Aims 1 and 3); (v) definition of PD of tetrac in a perfusion cell culture system (Aim 2) in preparation for (vi) xenograft studies to confirm the effects of tetrac on anti-proliferation (Aim 3). By understanding the mechanisms behind the pathogenic effects of thyroid hormone and anti-proliferative actions of tetrac, we will propose and develop new treatment approaches to colorectal cancers.
|Effective start/end date||8/1/15 → 7/31/16|