Thyroid Cancer Overview - Signaling Pathway. Diagnostics Marker. Targeted Therapy and Clinical Trials.

An Introduction to Thyroid Cancer

Thyroid cancer (TC) remains the most common endocrine malignancy worldwide and its incidence and mortality has increased steadily over the last four decades. Improvements in diagnostic techniques that allow a more efficient detection of small tumors may account in part for this increase, although a true rise in the occurrence of thyroid cancer is suggested by the findings that the number of tumors of larger size and that are detectable by palpation has also risen during this period. Thyroid carcinomas develop from two different cell types in the thyroid gland: follicular cells and parafollicular (C) cells. The American Cancer Society’s most recent estimates for thyroid cancer in the United States for 2018 are: about 53,990 new cases of thyroid cancer (40,900 in women, and 13,090 in men) and about 2,060 deaths from thyroid cancer (1,100 women and 960 men). Thyroid cancer is commonly diagnosed at a younger age than most other adult cancers. Therefore, in order to improve the benefit of screening and the survival rate of patients, it is very important to establish a system of early diagnosis and targeted therapy for thyroid cancer. The development, progression, invasion, and metastasis are closely associated with multiple signaling pathways and the functions of related molecules, such as Src, Janus kinase (JAK)-signal transducers and activators of transcription (STAT), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt, NF-κB, thyroid stimulating hormone receptor (TSHR), Wnt-β-catenin and Notch signaling pathways. Each of the signaling pathways could exert its function singly or through network with other pathways. These pathways could cooperate, promote, antagonize, or interact with each other to form a complex network for the regulation. Dysfunction of this network could increase the development, progression, invasion, and metastasis of thyroid cancer.

1 Main Signaling Pathways in Thyroid Cancer Therapy

Fig.1 Thyroid cancer signaling pathway. Targeted agents (listed in orange boxes) include those in clinical use (colored in red) and those in preclinical or early phase development (colored in green) for the treatment of advanced stage thyroid cancer.

Thyroid cancer has become the most common cancer among all the malignancies. Several treatment modalities including surgical resection, radioactive iodide therapy, and hormone-suppressive therapy could result in good prognosis in most patients with differentiated thyroid cancer (DTC); however, such conventional treatment methods are not effective in treating patients with medullary thyroid cancer (MTC) or anaplastic thyroid cancer (ATC). Although conventional treatment methods are not effective for such patients, researches in molecular biology could bring new chances. The development, progression, invasion, and metastasis of thyroid cancer are closely associated with multiple signaling pathways and functions of related molecules. Any changes in these signaling pathways could be used as biomarkers to help diagnosis and predict the prognosis of thyroid cancer; in addition, potential treatment targets could also be found in these pathways.

1.1 Src Signaling Pathway

Src family kinase (SFK) is a family of non-receptor tyrosine kinases. SFK could be activated by a variety of signals including tyrosine kinase, G protein-coupled receptors, steroid receptor, and signal transducers and activators of transcription (STAT). SFK could then be involved in cell proliferation, growth, motility, migration, angiogenesis, and intracellular transport. Both the direct and indirect activations of SFKs are associated with the progression and metastasis of malignancies. Activated Src pathway could consequently activate several other pathways including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), FAK, and STAT pathways; these activations finally affect the growth, invasion, and metastasis of thyroid cancer.

1.2 RTK Signaling Pathway

RTK signaling pathway includes two major signaling pathway branches. MAPK pathway could be activated by receptor tyrosine kinase, G protein-coupled receptors, and cytokines. It can then transduce extracellular signals into the cells and nuclei to induce biological responses including cell proliferation, differentiation, and apoptosis. Overactivation of MAPK pathway could upregulate the expression of numerous oncoproteins including chemokines, vascular endothelial growth factor A (VEGFA), matrix metalloproteinases (MMPs), prohibitin, vimentin, MET, NF-κB, HIF1α, EG-VEGF, transforming growth factor-β (TGFβ), and thrombospondin 1 (TSP1). PI3K/Akt pathway is a very important intracellular signaling pathway for mammals, which is closely associated with cell proliferation, transformation, metabolism, motility, and development and progression of tumors. PI3K-activated Akt could in turn phosphorylate a series of downstream target proteins including Bad, caspase 9, forkhead, Par-4, p2l, and mammalian target of rapamycin (mTOR) to activate or inhibit their functions, which finally promote cell survival.

1.3 NF-κB Signaling Pathway

The association between NF-κB and development of tumor mainly relies on the fact that NF-κB could inhibit cell apoptosis. NF-κB could influence cell apoptosis by regulating cytokines including TNF-α, IL-1β, IL-6, and IL-8; it could also inhibit apoptosis by inducing or upregulating the expression of antiapoptotic gene such as bcl-2 or by inducing the expression of TNF-α receptor family (TRAF1 and TRAF2), cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2), and zinc finger protein A20. Specifically, for thyroid cancer, NF-κB activation was found in PTCs, FTCs, and ATCs, and researchers suggested that activation of NF-κB could promote dedifferentiation of PTCs and FTCs and thus play important roles in each stage of thyroid cancer.

1.4 TSH Receptor (TSHR) Signaling Pathway

TSHR could combine with TSH and thus stimulate the growth of thyrocytes directly or indirectly by stimulating growth factors including autocrine growth factors and VEGF. Two intracellular signaling pathways, namely Gsα-mediated adenylyl cyclase-cyclic AMP (cAMP) signaling pathway and Gq- or G11-mediatedphospholipase C β-inositol 1,4,5-trisphosphate-intracellular Ca²⁺ signaling pathway, could be activated after the combination of TSHR with TSH. Each signaling molecule in these pathways could interact with the signaling molecules of other pathways including Wnt, PI3K, and MAPK pathways to form a network. Although TSHR’s genetic and epigenetic alterations do not directly lead to carcinogenesis, it has a crucial role in tumor growth, which is initiated by several oncogenes.

2 Thyroid Cancer Diagnosis

Thyroid cancer is the most common endocrine malignancy with steadily increasing incidence over the past few decades. Although standard strategies for the management of thyroid cancer offer optimal outcomes in thyroid cancer patients with favorable histological types at early stage, challenges arising from diagnosis and therapy still exist during clinical practice. A number of genetic alterations have been described in thyroid cancer, which provides an unprecedented opportunity for the identification of novel diagnostic and prognostic molecular markers as well as novel therapeutic targets. Molecular-targeted therapies, which have been investigated recently with increasing success, may prove to be a breakthrough in patients with advanced, radioiodine-refractory thyroid cancers. Early diagnosis and appropriate treatment can improve prognosis and reduce mortality.

2.1 Molecular Markers for Thyroid Cancer

Thyroid cancer progresses through a series of genetic and epigenetic alterations, including activating and inactivating somatic mutations, changes in gene expression patterns, microRNA dysregulation, and aberrant gene methylation. Most mutations are somatic mutations, either point mutations or chromosome alterations. Genetic alterations involving the mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway occur in thyroid tumors have been reported. These non-overlapping genetic alterations, BRAF, RAS, and RET/PTC, are found in 70% of PTCs, while RAS and PAX8/PPARc are found in 70% of FTC. Additional mutations known to occur in poorly differentiated and anaplastic carcinomas involve the TP53 and CTNNB1 genes. MTCs, both familial and sporadic, frequently carry point mutations located in the RET gene or in RAS genes. These mutational markers, as well as altered gene expression and miRNA profiles, have been explored for diagnostic use in thyroid nodules, and some are already in clinical use. BRAF is the most widely studied mutation and the most common genetic alteration in TC. Various methodologies are available to the clinical laboratory that allow detection of molecular mutations on several types of specimens, including, but not limited to, blood, buccal swabs, fresh snap-frozen tissue, FNA biopsy material, and formalin-fixed paraffin-embedded tissue. Commonly utilized analysis techniques found in the clinical laboratory include DNA sequencing, immunocytochemistry (ICC), fluorescence in situ hybridization, PCR, and its numerous variations including reverse transcription PCR, real-time PCR, and single-strand conformation polymorphism analysis PCR. Several studies have demonstrated that normal thyroid cells have a unique profile of miRNA expression and many miRNAs are dysregulated in thyroid cancer cells. A subset of these miRNAs, including miR-221, miR-222, miR-146b, miR-155, and miR-187, has been consistently found to be upregulated in thyroid papillary carcinoma. miRNA dysregulation is a common finding in malignancy, and there is mounting evidence supporting a role of miRNAs in carcinogenesis.

2.2 Protein Markers for Thyroid Cancer

The actual diagnosis of thyroid cancer is made with a biopsy. However, this might not be the first test done for thyroid diseases, there is a need for additional methods for early diagnosis. In order to overcome this challenge, it is essential to discover novel, highly sensitive, and specific biomarker. Therefore, accurate identification of the histological type of cancer and respective protein biomarkers is crucial for adequate therapy. Proteins are the most suitable biomarkers for thyroid cancer diagnosis because of their involvement in cellular processes. ICC analysis of candidate protein markers is an appealing diagnostic adjunct as the technique is readily available at low cost, although variability in techniques and result interpretation can occur. The most frequently studied marker is Galectin3 (GAL3), which is implicated in cell adhesion, cell cycle regulation, apoptosis, and tumor progression. In metaanalysis of the most frequently utilized single markers for ICC analysis, GAL3 appeared to have the highest sensitivity (85%) and specificity (90%) for TC. In addition, thyroid-stimulating hormone (TSH), T3 and T4 (thyroid hormones), thyroglobulin and carcinoembryonic antigen (CEA) are commonly used protein markers in early diagnosis and prognosis of thyroid cancer.

3 Targeted Therapy for Thyroid Cancer

For the past several decades thyroid cancer has been the most common endocrine tumor, with a~5% increase in incidence each year in the USA. The standard therapeutic approach to all thyroid cancers includes surgery, with radioactive iodine (RAI) being offered to some patients with follicular cell-derived thyroid cancers. Despite the favorable prognosis of this disease, 15–20% of differentiated thyroid cancer (DTC) cases and most anaplastic types, remain resistant to standard treatment options, including radioactive iodine (RAI). In addition, around 30% of medullary thyroid cancer (MTC) cases show resistance after surgery. A small fraction (< 10%) of DTC as well as many MTCs and almost all ATCs are not cured by standard therapy, instead spreading to distant metastatic sites. Recently, a number of scientific advances have illuminated some of the molecular pathways responsible for thyroid cancer. This growing knowledge raises the hope that it will soon be possible to develop specific therapeutics tailored to these molecular changes. In Table 1-8, selected clinical trials of novel therapeutic targets for the treatment of thyroid cancer are presented.

3.1 Thyroid Cancer Therapy for Src Signaling Pathway

Src family kinase (SFK) is a family of non-receptor tyrosine kinases. Src inhibitors, such as PP2, SU6656, and dasatinib, could effectively inhibit cell proliferation and expression of P-Src and P-FAK in papillary thyroid carcinoma (PTC) cell lines. Dasatinib could inhibit the growth of cancer cells, induce apoptosis and cell cycle arrest, and prevent tumor growth and metastasis. Bosutinib could effectively reduce the tumor growth, invasion, and pulmonary metastasis of thyroid cancer in Thrb(PV/PV)Pten(+/-) mice. Similarly, ADZ0530 could effectively inhibit the cell growth and invasion of PTC and ATC by inhibiting Src-FAK pathway.

Table 1 Clinical trials of Src inhibitor Dasatinib

Nct id	Status	Lead sponsor	Study first posted
NCT02465060	Recruiting	National Cancer Institute (NCI)	June 8, 2015

3.2 Thyroid Cancer Therapy for RTK Signaling Pathway

RTKs comprise the largest family of dominant oncogenes. It regulates cell proliferation, differentiation, invasion, angiogenesis and apoptosis through various signaling pathways and serves as a poor prognostic factor in cancer. Tyrosine kinase inhibitors (TKIs) including sorafenib, pazopanib, axitinib, sunitinib, and motesanib could not only inhibit the MAPK pathway, but also inhibit several targets. Currently, many drugs including temsirolimus, everolimus, rapamycin, INK-128, OSI027, AZD8055, GSK690693, MK-2206, CAL-101, BYL719, PX866, GDC0941, BKM120, and ZSTK474 that target PI3K/Akt have been investigated in phases I to III clinical trials, among which, temsirolimus and everolimus have been found with remarkable efficacies in treating thyroid cancers.

Table 2 Clinical trials of BRAF inhibitor Sorafenib

Nct id

Status

Lead sponsor

Study first posted

Table 3 Clinical trials of VEGFR inhibitor Pazopanib

Nct id	Status	Lead sponsor	Study first posted
NCT01813136	Recruiting	Centre Leon Berard	March 18, 2013
NCT00625846	Active, not recruiting	National Cancer Institute (NCI)	February 28, 2008
NCT01236547	Active, not recruiting	National Cancer Institute (NCI)	November 8, 2010

Table 4 Clinical trials of VEGFR inhibitor Sunitinib

Nct id	Status	Lead sponsor	Study first posted
NCT00381641	Active, not recruiting	National Cancer Institute (NCI)	September 28, 2006
NCT02465060	Recruiting	National Cancer Institute (NCI)	June 8, 2015

Table 5 Clinical trials of mTOR inhibitor Temsirolimus

Nct id

Status

Lead sponsor

Study first posted

Table 6 Clinical trials of mTOR inhibitor Everolimus

Nct id

Status

Lead sponsor

Study first posted

Table 7 Clinical trials of mTOR inhibitor Rapamycin

Nct id

Status

Lead sponsor

Study first posted

Table 8 Clinical trials of mTOR inhibitor INK-128

Nct id	Status	Lead sponsor	Study first posted
NCT02244463	Recruiting	Dana-Farber Cancer Institute	September 19, 2014
NCT03430882	Recruiting	M.D. Anderson Cancer Center	February 13, 2018
NCT02465060	Recruiting	National Cancer Institute (NCI)	June 8, 2015

3.3 Thyroid Cancer Therapy for NF-κB Signaling Pathway

Researchers suggested that activation of NF-κB could promote dedifferentiation of PTCs and FTCs and thus play important roles in each stage of thyroid cancer. Inhibition of NF-κB activation could affect the growth, apoptosis, and invasion of thyroid cancer cells. Small-molecule triptolide, an NF-κB inhibitor, was used to treat two human ATC cell lines, namely TA-K cells and 8505C cells. The expression of cyclinD1, VEGF, and uPA was effectively inhibited, which in turn effectively reduced the angiogenesis and invasion of ATC. Bortezomib, an inhibitor of proteasome, could increase the expression of p21(CIP1/WAF1) and thus inhibit cell growth, increase cell apoptosis, and arrest cells at G2-M phase in two ATC cell lines, namely C643 and SW1736 cells.

3.4 Thyroid Cancer Therapy for TSHR Signaling Pathway

Targeting TSHR is an optimal method to induce the redifferentiation of poorly differentiated carcinoma of the thyroid and increase the iodide uptake of iodine-refractory thyroid carcinomas by increasing the NIS expression. There are two research directions for targeted therapy for TSHR signaling pathways, including small molecule ligands and antibodies, such as small molecule ligand agonists to the TSHR - Org 41841, NCG00161870, MS437 and MS438; small molecule TSH antagonists - NCGC242595, NCGC00242364, NCGC00229600 and Org274179-0; as well as TSHR antibodies - M22, K1-18 and K1-70. However, these targeted therapeutic small molecules and antibodies are still in the preclinical stage.