Behind the scenes of thyroid tumors : underlying genetic mechanisms

Abstract

Thyroid cancer is the most frequently observed malignancy involving endocrine tissues. This group of diseases is also the major cause of death related to neoplasia of the endocrine system. Tumors in the thyroid gland are common in the general population and the majority represents benign follicular adenoma (FTA). Thyroid follicular cell derived tumors encompass FTA, follicular thyroid carcinoma (FTC), papillary thyroid carcinoma (PTC), and poorly-differentiated thyroid carcinoma (PDTC). The parafollicular calcitonin-producing C-cells give rise to medullary thyroid carcinoma (MTC). The anaplastic thyroid carcinoma (ATC), is an undifferentiated, highly aggressive cancer largely of unknown etiology. The overall goal of this thesis was to further characterize the molecular genetic mechanisms leading to thyroid tumorigenesis and to explore the possibility of thyroid tumor progression.

MTC can occur in sporadic as well as in heritable forms. Human familial MTC (FMTC) is dominantly inherited, either alone or as part of a Multiple Endocrine Neoplasia type 2 (MEN 2) syndrome. Activating germ-line RET mutations is the cause of the disease in up to 95% of FMTC and MEN 2. Paper I demonstrates the first dog pedigree with familial clustering of thyroid cancer. The affected individuals demonstrated calcitonin positive thyroid cancers, hypothyroidism and chronic dermatitis. Based on the overlapping features with human FMTC, the dog Ret was analyzed as a candidate gene after in silico prediction of its genomic sequence. The lack of demonstrable Ret mutation in this pedigree suggests that the MTC predisposition does not share the genetic etiology commonly underlying FMTC phenotypes in human.

The molecular pathogenesis of ATC is mainly unknown partly as a result of tissue degeneration following pre-operative treatment. Therefore ATC cell lines serve as good models for delineation of genetic mechanisms in the tumor development. In Paper II, two novel ATC cell lines (HTh 104 and HTh 112) were presented and together with six other frequently used ATC lines (HTh 7, HTh 74, HTh 83, C 643, KAT-4 and SW 1736) were further characterized cytogenetically. Novel non-random breakpoints at 1p36 and 17q24-25 as well as 3p21-22 and 15q26 that are also implicated in well-differentiated thyroid cancers were revealed. Frequent gain of 20q, including the UBCH10 gene in 20q13.12 was observed, indicating that this region is important for the pathogenesis of or progression to ATC.

In Paper III, bisulphite Pyrosequencing of Long Interspersed Nucleotide Elements-1 and LUminometric Methylation Assay were used to quantify global methylation in a series of 21 FTC and their corresponding normal thyroid tissues. No significant difference of global methylation was detected between FTC and normal tissues. However RASSF1A promoter hypermethylation, reduced mRNA expression and allelic loss (AI) were observed in the same material. These findings suggest RASSF1A is frequently inactivated in FTC by promoter hypermethylation and AI, but is not coupled to global methylation changes.

In further deciphering the molecular genetic pathogenesis of ATC, array-CGH was performed in a series of 27 primary ATC in Paper IV. Amplicons 11q13, 20q11.2 and 20q13.12 were identified and subsequent fluorescence in situ hybridization revealed recurrent locus gain of CCND1 in 11q13 and UBCH10 in 20q13.12. Homozygous loss encompassing the CDKN2A locus in 9p21.3 was detected in one case. Cyclin D1 was found to be expressed in 67% of ATC while p16 protein expression was undetectable in 89% of the tumors. Subsequently, the effect of CCND1/Cyclin D1 on thyroid cell proliferation was assessed in vitro in ATC cells by means of siRNA and in thyroid cells after CCND1 transfection. Furthermore three ATCs harboured the common BRAF mutation V600E.