Although most thyroid carcinomas are relatively indolent, there have been no significant improvements in survival over the last two decades. In fact, the trend in US cancer mortality with associated annual percentage change (APC) for cancer of the thyroid is disturbing; while an average 3% decrease in mortality from thyroid cancer was seen in males and females from 1975–1985, such a decrease has been absent in either gender since 1985 (NCI 2008
). More intriguing, Surveillance Epidemiology and End Results (SEER) data indicate a 6.2% increased incidence of thyroid cancer during the same two decades (NCI 2008
). Clearly, insight into the molecular pathogenesis of HCC, MTC, ATC, and other aggressive forms of thyroid cancer may lead to the development of more targeted therapies in the future.
Fortunately, the molecular basis of well-differentiated thyroid carcinoma, MTC, and anaplastic thyroid cancer is currently under investigation. With the application of current molecular techniques, nearly two decades of research have begun to elucidate critical genetic pathways involved in the development of specific thyroid tumor types. In particular, chromosomal inversions or translocations causing mutations in the RET
proto-oncogene may play an important role in approximately 40% of patients with PTC (Vidal et al 2005
; Carlomagno et al 2002
). Likewise, mutations that activate RET
activity can lead to several hereditary cancer syndromes, including MEN types 2A and 2B and FMTC. Some studies suggest that differentiated thyroid carcinoma cell invasion is regulated through epidermal growth factor receptor-dependent (EGFR) activation of matrix metalloproteinases (Yeh et al 2006
). Anaplastic thyroid carcinoma is postulated to develop secondary to dedifferentiation from a more differentiated tumor and has been associated with the loss of the p53 tumor suppressor protein (Sherman 2003
; Moretti et al 1997
). Many of these findings have paved the way for the development of phase II/III clinical trials, several of which may be found in . Fortunately, the importance of these genetic pathways, as well as a myriad of others, continue to be described today.
National Cancer Institute/US National Institutes of Health list of clinical trials now accepting participants
The RET receptor tyrosine kinase (RTK)
The RET gene encodes a 120-kDa transmembrane tyrosine kinase that functions as a receptor for the glial-derived neurotrophic factor (GDNF) family of growth factors (Manie et al 2001
). Mutations in the RET proto-oncogene have been implicated in patients with hereditary MTC associated with MEN types 2A and 2B and familial MTC (FMTC). Likewise, nearly 40% of patients with PTC have rearrangements of the RET gene that join the promoter and NH2
-terminal domains of unrelated genes to the COOH-terminal fragment of RET (Vidal et al 2005
). The cumulative result of the point mutations associated with MEN 2A and 2B and FMTC and the chromosomal rearrangements associated with PTC is activation of the RET receptor tyrosine kinase (RTK).
While MTC tends to be a slow-growing tumor primarily treated with surgical resection, it also frequently metastasizes to the liver and regional lymph nodes, precluding patients from a curative resection. Clearly, there is a great need for novel therapeutic and palliative strategies to treat these patients with metastatic MTC. RTK inhibitors are likely to be beneficial for patients with hereditary MTC, where currently there are no effective chemotherapy or radiotherapy options.
ZD6474 (vandetanib) is a low-molecular-weight tyro-sine kinase inhibitor that has demonstrated potent and selective inhibition of RTK in vitro (Carlomagno et al 2002
; Wedge et al 2002
). In additional pre-clinical studies, Carlomagno et al (2005)
have shown that ZD6474 blocks in vivo phosphorylation and signaling of the RET/PTC3 and RET/MEN2B oncoproteins, inhibits the proliferative autonomy of RET/PTC3-transformed cells, and prevents the growth of two human PTC cell lines that carry spontaneous RET/PTC1 rearrangements. Based on phase I studies, ZD6474 was generally well tolerated at doses ≤300 mg/day and adverse events were generally mild and limited by dose adjustment. The most common monotherapy-related adverse outcomes included diarrhea, asymptomatic QTc prolongation, and a skin rash (Holden et al 2005
; Tamura et al 2006
). Currently, promising data in patients with MTC have led to ZD6474 being assigned orphan drug designation by the US Food and Drug Administration. These data are based on studies in which ZD6474 has demonstrated clinical activity in a single-arm Phase II study in 30 patients with metastatic hereditary MTC (Wells et al 2007
). Concomitantly, an international, randomized, placebo-controlled phase II study of ZD6474 monotherapy in metastatic MTC is currently recruiting patients.
In addition to ZD6474, several other novel RTK inhibitors, sorafenib and sunitinib, for example, are under investigation for treatment of RET-dependent thyroid carcinomas. Utilizing an orthotopic anaplastic thyroid carcinoma xenograft model in nude mice, Kim et al have shown that sorafenib, a multikinase inhibitor of RTK, VEGFR, and BRAF kinase, inhibits proliferation of ATC cell lines and inhibits tumor angiogenesis via induction of endothelial apoptosis (Kim et al 2007
). Similarly, the orally administered multitarget tyrosine kinase inhibitor, SU11248 (sunitinib), has been shown to be a novel potent inhibitor of thyroid oncogenic RET/papillary thyroid cancer kinases (Kim et al 2006
EGFR as a therapeutic target in human thyroid carcinoma
Epidermal growth factor (EGF) is a 6-kDa polypeptide which has been shown to stimulate the proliferation of normal and malignant thyroid cells and inhibit cellular differentiation Hoelting et al 1994
). Overexpression of the EGF receptor (EGFR), a transmembrane 170-kDa glycoprotein tyrosine kinase, is frequently detected in thyroid carcinomas, and though controversial (Mitsiades et al 2006
), this pathway has been proposed to be important for thyroid carcinoma proliferation and metastasis. Recent studies with gefitinib and erlotinib, well-known EGFR kinase inhibitors, have resulted in objective responses in patients with non-small cell lung cancers (NSCLC), tumors in which EGFR is also frequently overexpressed. This has led some to believe that novel therapeutic compounds targeting the EGFR pathway may have future palliative and therapeutic potential in patients with refractory or metastatic thyroid carcinomas. In fact, the aforementioned compound ZD6474 is, in addition to its role in RTK inhibition, a potent and selective inhibitor of vascular endothelial growth factor receptor (VEGFR) and EGFR. As above, this compound is currently in phase II clinical trials to measure its efficacy and clinical impact in patients with hereditary MTC.
Interestingly, many of the novel RTK inhibitors, ZD6474, sorafenib, and sunitinib, for example, appear to non-specifically inhibit multiple signaling pathways critical in thyroid carcinogenesis, suggesting a potential synergistic effect to these novel drugs; however, the question of whether this lack of specificity will be advantageous or disadvantageous in the long run for patients remains unanswered.
More recently, studies have demonstrated that differentiated thyroid cancer cell invasion in vivo is regulated through EGFR-dependent activation of matrix metalloproteinase (MMP)-2/gelatinase A (Yeh et al 2006
). Undoubtedly, the role of MMP inhibitors as effective anti-tumor agents for the treatment of aggressive thyroid carcinomas is intriguing.
BRAF mutations in PTC
Kimura et al studied 124 snap frozen tumor samples, including 78 papillary carcinomas, using DNA isolation and single-strand conformational polymorphism (SSCP) analyses (Kimura et al 2003
). Overall, 28 of 124 cancers demonstrated mutations in exon 15 of the B-type Raf kinase (BRAF
) gene; all had the same thymine-to-adenine transversion at nucleotide 1796, resulting in a valine-to-glutamate substitution at residue 600 (currently designated as V600E). More importantly, all of the BRAFV600E
mutations were in papillary carcinomas (Kimura et al 2003
). By contrast, all 46 of the follicular neoplasms were wild-type. Since this initial report, Xing has shown that the BRAF activating mutation is the most common genetic alteration in thyroid cancer (Xing 2005
), occurring in 18% to 87% of thyroid cancers (Trovisco et al 2006
); importantly, the BRAFV600E
mutation occurs in nearly 45% of sporadic cases of PTC (Xing 2005
) and 24% of cases of anaplastic carcinoma (Salvatore et al 2006
). Recently, Kebebew and colleagues sought to determine the association of BRAF
mutations with indicators of poor prognosis for PTC and patient outcome in an attempt to determine whether BRAFV600E
is associated with an aggressive tumor phenotype (Kebebew et al 2007
). In patients with conventional PTC, patients with the BRAFV600E
mutation were associated with a more aggressive tumor phenotype and were characterized by older age at the time of initial presentation, the presence of lymph node and/or distant metastases, higher TNM stage, and recurrent or persistent disease with a median follow-up of 6 years (Kebebew et al 2007
). Genetic analysis for BRAF mutations in patients with PTC may enable better prediction of those tumors associated with a poor prognosis and may be useful in selecting initial therapeutic modalities in these patients.
The raf-1/mitogen-regulated extracellular kinase (MEK)/extracellular regulated kinase (ERK) pathway in MTC
The raf-1/MEK/ERK pathway has long been recognized for its role in cancer biology. Although activation of this signaling pathway is commonly considered to be growth promoting in several cancers, in certain cell-specific subtypes, raf-1 activation results in growth suppression (Kunnimalaiyaan et al 2007b
). Specifically, activation of the raf-1 signaling pathway in MTC has been shown to lead to significant morphologic differentiation and growth reduction both in vitro and in a mouse xenograft TT model in vivo (Ravi et al 1998
; Park et al 2003
; Vaccaro et al 2006
). Similarly, we have shown that raf-1 activation results in a reduction of the neuroendocrine tumor markers achaete-scute complex-like 1 (ASCL1), chromogranin A (CgA), and calcitonin in vitro (Park et al 2003
; Kunnimalaiyaan and Chen 2006
; Chen et al 2005
; Sippel et al 2003
Until recently, the mechanism by which raf-1 pathway activation inhibits MTC cell growth was unclear; in fact, conflicting observations had been reported regarding the regulation of ERK1/2 in MTC cancer cells. Recent studies into the role of GSK-3β and its downstream relationship to Raf-1 have begun to elucidate the mechanisms responsible for growth inhibition in MTC cells.
Inactivation of glycogen synthase kinase- 3β (GSK-3β) in MTC
GSK-3β is a multifunctional serine/threonine protein kinase that plays an important role in cellular metabolism, cell fate determination, proliferation, and survival (Hardt and Sadoshima 2002
; Harwood et al 1995
). The activity of this kinase is inhibited by phosphorylation of a single serine residue (Ser9). Thus, unlike other kinases, GSK-3β is highly active when non-phosphorylated (in unstimulated cells), and becomes inactivated (phosphorylated) in response to signaling cascades, including the raf-1/MEK/ERK pathway (Cohen and Frame 2001
). At our institution, Kunnimalaiyaan et al (2007b)
found that raf-1 activation in human MTC cells (TT cells) results in phosphorylation of GSK-3β (Kunnimalaiyaan et al 2007b
). Furthermore, inactivation of GSK-3β by phosphorylation results in MTC growth inhibition both in vitro and in vivo. Inactivation of GSK-3β in TT cells with well known GSK-3β inhibitors such as lithium chloride (LiCl) and SB216763 is associated with a significant decrease in neuroendocrine markers such as human-achaete-scute complex-like 1 (ASCL1) and chromogranin A (CgA). LiCl has been utilized clinically for more than fifty years as an adjunctive psychiatric medication for the treatment of bipolar disorder and has shown only minimal adverse side effects. As such, the efficacy of LiCl therapy in patients with MTC is currently being investigated at our institution in phase II clinical trials.
Overexpression of the Notch1 intracellular domain in MTC
Notch1 is a multifunctional transmembrane receptor that regulates cellular differentiation, proliferation, and survival (Kadesh 2004
; Maillard and Pear 2003
; Yoon and Gaiano 2005
). Binding of any one of the Notch ligands (Delta1 (DLL-1) or Jagged1 (JAG-1), for example) promotes a sequence of proteolytic cleavages resulting in the activated Notch intracellular domain (NICD). The active NICD then translocates to the nucleus and binds with the DNA-binding protein complex CSL (CBF1, Su (H), and LAG-1), resulting in the transcriptional activation of target genes such as hairy enhancer of split-1 (HES-1) (Kunnimalaiyaan et al 2007a
). In human cancer cells, Notch1 has a dual role as either a tumor suppressor or an oncogene. It has been shown that Notch1 is upregulated in many types of cancer, including pancreatic cancer, colon cancer, non-small cell cancer, cervical cancer, renal cell carcinoma, and several lymphomas. It has been suggested that expression of Notch1 signaling prevents cellular differentiation and inhibits apoptosis in certain cancers. Conversely, Notch1 signaling is very minimal or absent in prostate cancer and NETs such as small-cell lung cancer (SCLC), pancreatic carcinoid, and MTC (Kunnimalaiyaan and Chen 2007
). These apparent but paradoxical functions clearly indicate that the role of Notch signaling is dependent on its cellular context.
Using a doxycycline-inducible NICD in MTC cells (TT cells), we have recently shown that overexpression of Notch1 in MTC cells – Notch1 signaling is normally absent in MTC cells – results in a dose-dependent reduction in NE tumor markers and growth (Kunnimalaiyaan et al 2006
; Kunnimalaiyaan and Chen 2007
). These findings indicate that identification of compounds capable of pharmacologically activating the Notch1 signaling may have a therapeutic role in treating NE diseases caused by aberrant expression of ASCL1 and other hormones. In fact, our laboratory has recently shown the histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) to be strong Notch1 activators in NE tumors, including MTC. Moreover, we have shown these compounds to limit growth and reduce neuroendocrine markers both in vitro and in vivo. The efficacy of these HDAC inhibitors as part of a comprehensive therapy in patients with MTC is currently being investigated at our institution in ongoing phase II clinical trials.
Mutation of the p53 tumor suppressor gene
The tumor suppressor gene, p53, plays a critical role in cell cycle control, DNA repair, and synthesis, apoptosis, and many other cellular processes. Though it is believed that nearly 50% of all human malignancies are due to inactivating mutations of the p53 tumor suppressor gene, the significance of the p53 gene in thyroid carcinogenesis is less clear (Malaguarnera et al 2007
). In a review of the International Agency for Research on Cancer (IARC) TP53 tumor suppressor database, the largest comprehensive database of its kind in existence, Olivier et al showed inactivating mutations of the p53 gene have been found in only 10% of thyroid carcinomas and mainly in poorly differentiated and aggressive histotypes (Olivier et al 2002
). Similarly, in a cohort of nearly 100 patients, Saltman et al clearly demonstrated the presence of aberrant p53 expression in more aggressive phenotypes of thyroid carcinoma; the study demonstrated a gradual increase in p53 immunopositivity rate along the spectrum of thyroid carcinoma progression with a statistically significant difference between well-differentiated PTC and anaplastic phenotypes (0% vs 31.8%, respectively; p < 0.001) (Saltman et al 2006
). Clearly, studies elucidating the role of inactivating mechanisms in the early stages of thyroid cancer are needed to help facilitate a better understanding of the complex network of p53 family member isoforms and their role in thyroid carcinogenesis.