Thyroid cancer is the most common endocrine malignancy. With a rapidly rising incidence in recent years, novel efficient management strategies are increasingly needed for this cancer. Remarkable advances have occurred in understanding several major biologic areas of thyroid cancer, including the molecular alterations for the loss of radioiodine avidity of thyroid cancer, the pathogenic role of the MAP kinase and PI3K/Akt pathways and their related genetic alterations, and the aberrant methylation of functionally important genes in thyroid tumorigenesis and pathogenesis. These exciting advances provide unprecedented opportunities for the development of molecular-based novel diagnostic, prognostic, and therapeutic strategies for thyroid cancer.
Thyroid cancer is the most common endocrine malignancy, with a rapidly rising incidence in recent years. Effective management of thyroid cancer is based on molecular understanding of the tumorigenesis and progression of this cancer. Remarkable progress has occurred in exploring the molecular biology of thyroid cancer, particularly in its genetic alterations and related signaling abnormalities. One striking example is the BRAF mutation, which is the most common genetic alteration in thyroid cancer. Through aberrant activation of the MAP kinase pathway, the BRAF mutation has a fundamental role in thyroid tumorigenesis. Likewise, driven by genetic alterations discovered in recent years, the PI3K/Akt pathway has an instrumental role in thyroid tumorigenesis. More recent data suggest that genetically based coactivation of the MAP kinase and PI3K/Akt pathways is an important molecular mechanism that drives progression from differentiated to undifferentiated thyroid cancer. With these recent molecular discoveries, many of which are novel cancer markers and therapeutic targets, it is expected that more effective diagnostic, prognostic, and therapeutic approaches for thyroid cancer will be developed.
Thyroid cancer and its clinical challenges
The incidence of thyroid cancer is rising rapidly in the world . In the United States, the rise in the incidence of thyroid cancer is the fastest among cancers in many patient populations, particularly in women and elderly patients of both genders, with an estimated 2008 incidence of 37,340 cases and a prevalence of more than 350,000 cases . The major histologic types of follicular cell-derived thyroid cancer are papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), and anaplastic thyroid cancer (ATC) . Benign thyroid adenoma (BTA) is a common endocrine tumor. Medullary thyroid cancer derived from parafollicular cells is a relatively rare malignancy and is not discussed herein. PTC and FTC are generally differentiated, indolent, and highly curable with current treatments. ATC is an undifferentiated and rapidly lethal thyroid cancer . In fact, ATC is among the most aggressive and deadly human cancers. There are also poorly differentiated thyroid cancers that have a high incurability and mortality, albeit with a better prognosis than ATC. Poorly differentiated thyroid cancer can progress into ATC, and both can derive from PTC and FTC or occur de novo. Thyroid cancers, particularly poorly differentiated thyroid cancer and ATC, can loose the ability to take up iodide and consequently do not respond to radioiodine treatment; they become incurable if they are also surgically inoperable. The loss of radioiodine avidity is currently a major therapeutic challenge for thyroid cancer and a the main cause of thyroid cancer-related morbidity and mortality.
Several diagnostic challenges are often encountered in the clinical management of thyroid cancer. One is the diagnostic dilemma associated with “indeterminate cytology” on the widely used fine-needle aspiration biopsy (FNAB) in the evaluation of thyroid nodules. About 300,000 cases of thyroid nodules, which are mostly BTA, are diagnosed annually in the United States . About 20% to 30% of FNABs show “indeterminate” cytologic findings, a pattern that has remained essentially unchanged over the last 2 decades . Currently, virtually all of these patients pursue thyroid surgery to definitely reveal the nature of the nodules, although the vast majority of them are surgically proved to have benign nodules. Another diagnostic dilemma in the management of thyroid cancer is the difficulty in monitoring thyroid cancer recurrence using the standard serum thyroglobulin (Tg) testing when Tg antibodies are positive, which occurs in about 20% to 30% of cases, rendering the testing results unreliable in these patients . Careful risk stratification is a key step in the decision-making process for appropriate surgical and medical management of patients with thyroid cancer. This risk evaluation is conventionally based on clinicopathologic factors, which are often unreliable and are mostly unavailable before thyroid surgery. Recent advances in understanding the molecular biology of thyroid cancer show great promise for the development of novel molecular-based strategies to effectively tackle these diagnostic, prognostic, and therapeutic obstacles of thyroid cancer.
Impaired iodide metabolism in thyroid cancer
The unique and fundamental function of the follicular epithelial thyroid cells is to use iodide as a substrate to synthesize thyroid hormones to meet the normal metabolism of the body, a process that involves several thyroid-specific, iodide-handling protein molecules . In this process, iodide is transported from the blood stream into the thyroid cell through the sodium/iodide symporter (NIS) in the basal membrane, followed by its transportation into the follicular lumen through transporters such as pendrin (also called SLC26A4) in the apical membrane of the cell. Through thyroid peroxidase (TPO), iodide is oxidized and organified into Tg through iodination of tyrosine residues in Tg for the formation of thyroid hormones. The thyroid-specific transcription factors TTF-1, TTF-2, and Pax-8 have an important role in the regulation of these thyroid genes. The entire process is up-regulated by thyroid-stimulating hormone (TSH) that acts by binding to the TSH receptor (TSHR) on the cell membrane.
Expression of thyroid molecules, such as TSHR, NIS, TPO, Tg, and pendrin, is often lost in thyroid cancers , resulting in impaired or loss of the ability of thyroid cancer cells to concentrate radioiodine. Consequently, such cases of thyroid cancer fail radioiodine ablation therapy, the mainstay of medical treatment for this cancer. This loss represents a major challenge in the current treatment of thyroid cancer, particularly in poorly differentiated thyroid cancer and ATC . The specific molecular mechanism underlying the silencing of thyroid-specific genes in thyroid cancer is largely unclear. Our group demonstrated that BRAF mutation was closely associated with loss of radioiodine avidity in PTC . Recent studies by the author’s and other groups have demonstrated that thyroid-specific genes can be silenced by induced expression of BRAF mutant and the consequent overactivation of the Ras→ Raf→ MEK→ MAP kinase/ERK pathway (MAP kinase pathway) in thyroid cell lines . The genes can be re-expressed by removing the activated MAP kinase pathway signaling . We also showed that methylation of the TSHR gene was a mechanism in its silencing promoted by the BRAF mutant . Very recently, we have shown that the PI3K/Akt pathway may also have an important role in the regulation of thyroid genes (Hou and Xing, unpublished data, 2008). With this knowledge of the molecular events associated with thyroid-specific genes in thyroid cancer, it can be expected that unique treatment strategies targeting these molecular abnormalities will be developed to restore the radioiodine avidity of thyroid cancer.
Impaired iodide metabolism in thyroid cancer
The unique and fundamental function of the follicular epithelial thyroid cells is to use iodide as a substrate to synthesize thyroid hormones to meet the normal metabolism of the body, a process that involves several thyroid-specific, iodide-handling protein molecules . In this process, iodide is transported from the blood stream into the thyroid cell through the sodium/iodide symporter (NIS) in the basal membrane, followed by its transportation into the follicular lumen through transporters such as pendrin (also called SLC26A4) in the apical membrane of the cell. Through thyroid peroxidase (TPO), iodide is oxidized and organified into Tg through iodination of tyrosine residues in Tg for the formation of thyroid hormones. The thyroid-specific transcription factors TTF-1, TTF-2, and Pax-8 have an important role in the regulation of these thyroid genes. The entire process is up-regulated by thyroid-stimulating hormone (TSH) that acts by binding to the TSH receptor (TSHR) on the cell membrane.
Expression of thyroid molecules, such as TSHR, NIS, TPO, Tg, and pendrin, is often lost in thyroid cancers , resulting in impaired or loss of the ability of thyroid cancer cells to concentrate radioiodine. Consequently, such cases of thyroid cancer fail radioiodine ablation therapy, the mainstay of medical treatment for this cancer. This loss represents a major challenge in the current treatment of thyroid cancer, particularly in poorly differentiated thyroid cancer and ATC . The specific molecular mechanism underlying the silencing of thyroid-specific genes in thyroid cancer is largely unclear. Our group demonstrated that BRAF mutation was closely associated with loss of radioiodine avidity in PTC . Recent studies by the author’s and other groups have demonstrated that thyroid-specific genes can be silenced by induced expression of BRAF mutant and the consequent overactivation of the Ras→ Raf→ MEK→ MAP kinase/ERK pathway (MAP kinase pathway) in thyroid cell lines . The genes can be re-expressed by removing the activated MAP kinase pathway signaling . We also showed that methylation of the TSHR gene was a mechanism in its silencing promoted by the BRAF mutant . Very recently, we have shown that the PI3K/Akt pathway may also have an important role in the regulation of thyroid genes (Hou and Xing, unpublished data, 2008). With this knowledge of the molecular events associated with thyroid-specific genes in thyroid cancer, it can be expected that unique treatment strategies targeting these molecular abnormalities will be developed to restore the radioiodine avidity of thyroid cancer.
MAP kinase signaling pathway and BRAF mutation in thyroid cancer
The MAP kinase pathway is a classical conserved intracellular signaling pathway that has a fundamental role in cell proliferation, differentiation, apoptosis, and survival and, when aberrantly activated, tumorigenesis . In thyroid cancer, RET/PTC rearrangement is a common activator of the MAP kinase pathway . Activating Ras mutations which can activate the MAP kinase pathway are also common in thyroid cancer . BRAF mutation is a major cause of aberrant activation of the MAP kinase pathway in human cancers . Among the three known Raf kinases, A-Raf, B-Raf (BRAF), and C-Raf, BRAF is the most potent activator of the MAP kinase pathway . The T1799A point BRAF mutation accounts for more than 90% of the more than 40 mutations identified in the BRAF gene . This mutation causes a V600E amino acid change in the BRAF protein, resulting in constitutive and oncogenic activation of the BRAF kinase .
The discovery and characterization of the T1799A BRAF mutation in thyroid cancer represents one of the most exciting advances in the molecular biology of thyroid cancer in recent years . In fact, this mutation is the most common known genetic alteration in thyroid cancer. A few other activated BRAF mutants are rarely found in thyroid cancer. These mutants include the BRAF K601E , AKAP9-BRAF , BRAF V600E+K601del , BRAF V599ins , and V600D+FGLAT601-605ins, which results from an insertion of 18 nucleotides at nucleotide T1799 . The T1799A mutation is the most common and virtually the only BRAF mutation identified in thyroid cancer (hereafter referred to as “ BRAF mutation”). Our previous studies showed that the BRAF mutation was not a germline mutation in familial non-medullary thyroid cancers , and, as a somatic genetic alteration, it occurs exclusively in PTC and PTC-derived ATC, with an average prevalence of about 45% in the former and 25% in the latter. It does not occur in FTC or other types of thyroid tumors . The transgenic mouse model and our recent cell line and xenograft tumor studies demonstrated the tumorigenic ability of the BRAF mutation and its requirement to maintain cancer cell growth and proliferation. Numerous studies have demonstrated an association of BRAF mutation with aggressive clinicopathologic outcomes, including tumor invasion, metastasis, and recurrence of PTC . We also demonstrated an interesting association of BRAF mutation with loss of radioiodine avidity in recurrent PTC and its failure to be cured . This association is consistent with our finding of BRAF mutant–promoted silencing of thyroid iodide-handling genes and the reversal of this process by silencing the expression of BRAF mutant in thyroid cells . Numerous studies have demonstrated a close association of BRAF mutation with de-differentiation of PTC as reflected by decreased expression of thyroid-specific genes in PTC, including NIS , TPO , pendrin , and Tg ; therefore, the BRAF mutation is a novel powerful molecular prognostic marker for a poorer prognosis of thyroid cancer . We recently demonstrated that testing for this marker on preoperative FNAB specimens can preoperatively predict lymph node metastasis, extrathyroidal invasion, and tumor recurrence of PTC; therefore, such testing is uniquely helpful in guiding the initial surgical and medical treatments (Xing and colleagues, unpublished data, 2008).
PI3K/Akt signaling pathway and genetic alterations in thyroid cancer
The PI3K/Akt Pathway in Human Cancers
Like the MAP kinase pathway, the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway (PI3K pathway) has a fundamental role in the regulation of cell growth, proliferation, and survival and in human tumorigenesis . Among the several classes of PI3Ks, class I is the best characterized and is composed of heterodimers of a regulatory subunit, particularly p85, and one of the several p110 catalytic subunits. The α-type (PIK3CA) and β-type (PIK3CB) p110 subunits are widely expressed in different tissues, whereas other types of p110 subunits are only expressed in limited tissues. PIK3CA and PIK3CB belong to class IA that is activated by receptor tyrosine kinases. The p110 subunits contain a binding site for the regulatory subunit, through which various signals can be integrated from membrane receptors and activate the catalytic subunits of PI3K. The p110 catalytic subunits also contain a Ras-binding site through which Ras is involved in the PI3K/Akt signaling. Upon activation by signaling from various membrane growth factor receptors, such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular epithelial growth factor (VEGF) receptor, c-MET, and c-KIT, the p110 catalytic subunits (PIK3CA and PIK3CB) phosphorylate phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ), which localizes the Ser/Thr kinase Akt to cell membrane where it becomes phosphorylated and activated by the phosphoinositide-dependent kinases (PDK), particularly PDK-1. There are three types of Akts: Akt-1, Akt-2, and Akt-3. Activated Akt phosphorylates down-stream protein effectors and amplifies the signaling cascade, promoting cell proliferation and inhibiting apoptosis. Signaling of the PI3K/Akt pathway is antagonized by the tumor suppressor gene PTEN product, PTEN, which is a phosphatase that dephosphorylates PIP 3 , terminating the signaling of the PI3K/Akt pathway .
The PIK3CA gene frequently harbors mutations and amplifications in human cancers . Genetic alterations, including mutation, deletion, and aberrant methylation are common mechanisms in the inactivation of the PTEN gene in human cancers. Mutations are also found in the EGFR , PDGFR , c-MET , KIT , PDK-1 , and Akt-2 genes in the PI3K/Akt pathway in certain human cancers, usually in the kinase domains of these proteins . In addition to the PIK3CA amplification, genomic amplifications are found in the Akt-2 , EGFR , and PDGFRα genes in human cancers. Genetic alterations are important driving forces for the aberrant signaling of the PI3K/Akt pathway.
The PI3K/Akt Pathway in Thyroid Cancer
Previous studies showed common activation of PI3K signaling in thyroid cancers . Among the three isoforms of Akt, Akt-1 and Akt-2 were the most abundant and important in thyroid cancer . We first reported genomic copy gain and amplification of the PIK3CA gene in thyroid tumors, particularly FTC and ATC . We and others have also shown that PIK3CA mutation is particularly common in ATC and is relatively uncommon but can occur in differentiated thyroid cancer . Ras mutation is commonly found in thyroid tumors, particularly FTC and BTA . We have previously analyzed a number of genetic alterations in the PI3K pathway, including PIKCA mutation and amplification, Ras mutation, and PTEN mutation in various thyroid tumors, and found a relatively high prevalence of them, particularly in FTC and ATC . Coexistence of some of these genetic alterations and their coexistence with BRAF mutation were more frequently seen in aggressive thyroid cancers, particularly ATC . We have recently expanded these studies to a large panel of genetic alterations, including mutations and genetic amplifications in about 20 genes in this pathway, and have found at least one genetic alteration in 46 of 48 (96%) ATCs and coexistence of two or more genetic alterations in 37 of 48 (77%) ATCs . Interestingly, genetic alterations that could activate both the MAP kinase and PI3K pathways were found in most (81%) ATCs, which was in good correlation with elevated phosphorylation of ERK and Akt. These data provide the strongest genetic evidence for an extensive role of dual involvement of the MAP kinase and PI3K pathways in the pathogenesis of ATC, supporting a recent hypothesis that targeting multiple signaling pathways, particularly the MAP kinase and PI3K/Akt pathways, may be an effective and necessary therapeutic strategy for thyroid cancer .
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