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The Interconnectedness of All Things

Approximately ten years ago, Sheue-Yann Cheng, Ph.D., Head of the Gene Regulation Section in CCR’s Laboratory of Molecular Biology, teamed with an unusual lab partner—a mutant mouse. She made this mouse to study a rare inherited disease, resistance to thyroid hormone (RTH), caused by a mutation in one of two thyroid hormone receptor genes. RTH had been recognized for many years as a paradoxical deficit in thyroid hormone signaling that is seen despite elevated levels of thyroid hormone itself. But the disorder was only relatively recently traced to a receptor mutation that prevents hormone binding and the resulting transcriptional regulation. The mice she used to study the effects of this mutation turned out to be an important window into multiple physiological systems, including cancer.

Normally, laboratory mice live quite happily for 18 to 24 months, and yet Cheng’s mice were starting to die at six months. “Our mice were dying!” exclaimed Cheng, “so we did autopsies.” The Cheng team discovered that these mice had massively enlarged thyroid carcinomas.

Cheng immediately jumped on the opportunity to study what proved to be a model of follicular thyroid cancer. Thyroid cancers are one of the few cancers with a rising incidence around the world, particularly in women. Follicular thyroid cancer accounts for about 15 percent of the total thyroid cancer disease burden but has a poorer prognosis as compared to the dominant papillary variety. “Cancer is such a devastating disease. I felt that since I had been in the thyroid hormone field for so long, perhaps I could make a unique contribution.”

Spontaneous Tumor Generation

Thyroid hormones operate in a tightly controlled feedback loop involving the hypothalamus, pituitary, and thyroid glands. Activation of normal thyroid hormone receptors encoded by one of two genes—THRA or THRB—results in the downregulation of thyroid-stimulating hormone (TSH). TSH, as its name implies, encourages the growth and activation of cells in the thyroid gland.

Thyroid cancers are one of the few cancers with a rising incidence around the world.

The PV mutation that Cheng studies was derived from an RTH patient and encodes a mutation that shifts the translation of DNA by a single base pair near one end of THRB. This frameshift mutation results in a complete loss of binding of thyroid hormone receptor β to the thyroid hormone T3. In addition, the PV mutation acts in a dominant negative fashion, suppressing the function of the remaining normal TRβ receptor. Mice bearing this mutation, like people with RTH, have growth retardation and other hallmarks of reduced thyroid hormone signaling. RTH patients typically only have one mutated copy of the THRB gene; there has only been one report of a patient who had mutations in both copies. But homozygous mice bearing two copies of the PV mutation develop thyroid cancer.

“When we developed this mouse model, there wasn’t any other spontaneous mouse model of metastatic thyroid cancer,” said Cheng. “As they got older, they just developed cancers. Eventually 100 percent of these mice develop thyroid cancer.”

The hypothalamic-pituitary-thyroid (HPT) axis consists of a complex signaling network that regulates multiple organ systems. TSH = thyroid-stimulating hormone; TRH = thyrotropin-releasing hormone; T3, T4 = thyroid hormones; TR = thyroid hormone receptor. Image adapted from O’Shea et al., Nuclear Receptor Signaling (2006) 4, e011.
Image shows that the hypothalamic-pituitary-thyroid (HPT) axis consists of a complex signaling network that regulates multiple organ systems. TSH = thyroid-stimulating hormone; TRH = thyrotropin-releasing hormone; T3, T4 = thyroid hormones; TR = thyroid hormone receptor. Image adapted from O’Shea et al., Nuclear Receptor Signaling (2006) 4, e011.

Several studies later, Cheng and her colleagues have characterized the progression and molecular changes associated with their mouse model of thyroid cancer. The cellular progression of the disease resembles the human situation. “We have studied so many of these mice,” said Celine Guigon, Ph.D., a Postdoctoral Fellow in Cheng’s laboratory. “And they all develop goiter around two months of age and then go on to develop cancer.” Remarkably, even metastatic progression is observed reliably in these animals. “This mouse has a similar frequency of metastases to that seen in humans—around 25 to 30 percent for follicular carcinoma,” said Cheng.

They have also found very strong correlations between the additional mutations observed in human follicular thyroid carcinoma and those seen in their PV mice. For example, they have established that the tumor suppressor PPARγ has reduced expression and activity in their mice and, intriguingly, that administration of a PPARγ agonist, rosiglitazone, blocked the development of metastasis.

Having validated their model as recapitulating many of the hallmarks of follicular thyroid carcinoma, Cheng and her colleagues hope to use the power of mouse genetics and molecular biology to gain novel insights into the disease. For example, their work has already shed light on the controversial role of TSH in these cancers. “Some patients who have elevated TSH have a high incidence of thyroid cancer; however, some patients with aggressive cancer express lower levels of TSH receptor,” explained Cheng.

Cheng’s team has found that in mice that are homozygous for the PV mutation, TSH levels are elevated about 200 times above levels in mice with only a single copy of the mutation. “TSH stimulates the proliferation of thyrocytes,” said Cheng. “Together with the thyroid hormone receptor β mutation, these two altered signals stimulate cancer.” Cheng and her colleagues have shown that elevated levels of TSH alone do not cause cancer, nor does the dominant negative action of the PV receptor mutation alone. Both are required. “As you know, cancer is a multigenetic disease,” said Cheng. Her goal is to dissect the multiple molecular interactions critical to thyroid cancer formation and progression and bring the findings back to the clinic.

Scanning electron micrographs (SEM) of trabecular bone architecture in normal mice (left panel) and mice with mutations in the thyroid hormone receptor TRα. Image adapted from Bassett et al., Scanning, (Author manuscript; available in PMC 2009 July 6).
Image shows scanning electron micrographs (SEM) of trabecular bone architecture in normal mice (left panel) and mice with mutations in the thyroid hormone receptor TRα. Image adapted from Bassett et al., Scanning, (Author manuscript; available in PMC 2009 July 6).

Fat and Bones

“Why do we need two thyroid hormone receptor genes?” asked Cheng. THRA and THRB encode TRα and TRβ receptors, respectively. TRα and TRβ are known to have different distributions in the body, and mice lacking the genes for each of these receptor subtypes have distinct functional deficits. But no case of RTH had ever been reported to be the result of a THRA mutation. “So we decided to target the PV mutation to the THRA gene and see what happens in the mouse,” explained Cheng.

The lab is now studying lipid metabolism in these mice conferred by either THRA or THRB mutations; they are finding differences in regulation of both white adipocytes and lipid content of the liver. “We are focusing on lipid metabolism not only because our mice have distinct phenotypes, but because it is very important to know how thyroid hormone regulates lipid metabolism,” said Cheng. Drug companies are interested in developing thyroid hormone analogs to accomplish therapeutic goals like lowering cholesterol. However, because of its pleiotropic actions on different receptor subtypes, a simple thyroid hormone analog would have too many side effects. “So there is a drive to devise analogs that are TR-subtype specific.” Cheng hopes that her work will shed light on the potential effects of such specific analogs.

Meanwhile, Cheng has several collaborators who are using her mice to study diverse topics. “I cannot study everything in these mice,” said Cheng. “They have a phenotype of interest to lots of investigators.”

Graham Williams, Ph.D., Professor of Endocrinology at Imperial College in London, has worked with Cheng for several years.

“We had identified TRα as the major thyroid hormone receptor expressed in bone, and I developed an interest in in vivo models to investigate the molecular and physiological mechanisms of thyroid hormone action in bone,” said Williams. “After seeing the early phenotype descriptions of the PV mutants, I was sure they would be very informative to our understanding of the skeleton. So, at one of the American Thyroid Association meetings, I introduced myself to Dr. Cheng and suggested that we work together on analyzing the skeletal phenotypes.”

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