**2. The Aurora kinases**

**1. Introduction**

96 Anti-cancer Drugs - Nature, Synthesis and Cell

The incidence of thyroid cancer (TC) has increased from about five new cases per 100,000 persons observed in the early 90s to 15 new cases per 100,000 persons recorded in 2012 [1]. This increase is mainly due to the improved ability to detect malignancy in small thyroid nodules [2, 3]. TC represents about 96% of all endocrine malignancies and one of the most frequent cancers in women [1]. Based on histological and clinical criteria, TC are classified as well-differentiated TC (DTC), which includes the papillary (PTC) and follicular (FTC) histotypes, poorly differentiated TC (PDTC), and anaplastic TC (ATC). The PTC accounts for about 86% of all epithelial TC. It appears as a mass of branching papillae covered by cells with eosinophilic cytoplasm and enlarged nuclei and typically metastasizes via lymphatic vessels to local lymph nodes [4]. The FTC accounts for approximately 9% of all TC. It resembles the normal microscopic pattern of the thyroid and is characterized by hematogenous spread producing lung and bone metastases [4]. The less differentiated and more aggressive PDTC and ATC, each of which accounts for 1–2% of all TC, are thought to develop from the dedifferentiation of DTC, according to the multistep model of thyroid carcinogenesis [4–8]. The PDTC was included as a separate entity in the WHO classification of TC in 2004. PDTC retains sufficient differentiation to produce scattered small follicular structures and some thyroglobulin but generally lacks the usual morphologic characteristics of DTC, showing an intermediate clinical behavior between DTC and ATC. In addition, it is characterized by highgrade features such as widely infiltrative growth, necrosis, vascular invasion, and numerous mitotic figures [6, 9]. The ATC is composed of disseminated fleshy masses with areas of necrosis and hemorrhage. The cells have an undifferentiated phenotype with marked cytological atypia and high mitotic activity, and they are negative for thyroglobulin [4].

Established risk factors for TC include radiation exposure, family history of TC, lymphocytic thyroiditis, reduced iodine intake, and female gender [10]. All of them are thought to induce chromosome instability (CIN) in thyrocytes through still poorly defined direct and indirect mechanisms [10–13]. Actually, number and frequency of chromosomal abnormalities increase from DTC to PDTC and ATC [13]. CIN is also sustained by alterations in cell cycle regulators, frequently encountered in TC [10]. In particular, a deregulated control of the G1/S transition, following either an increased expression of promoting factors (cyclin D1 and E2F) or the downregulation or presence of loss-of-function mutations of factors inhibiting the G1/S transition (retinoblastoma, p16INK4A, p21CIP1, p27KIP1, and p53), has been documented in TC [10]. In addition, the aberrant expression of mitotic kinases, such as the polo-like kinase and the three members of the Aurora kinase family, is held co-responsible for abnormal cell

About 80% of PTC are characterized by mutually exclusive activating somatic mutations of genes encoding for proteins involved in the mitogen-activated protein kinase (MAPK) signaling pathway [4, 16]. These include rearrangements of the *RET* (rearranged during transformation) (RET/PTC) and neurotrophic tyrosine kinase receptor 1 (*NTRK1*) genes, and activating point mutations of the three *RAS* oncogenes (*HRAS*, *KRAS*, and *NRAS*) and *BRAF* [16]. In addition, mutations of genes encoding key players of the phosphoinositide 3-kinase

divisions and the establishment of aneuploid TC cells [14, 15].

The Aurora kinases belong to a family of serine/threonine kinases having in the Ipl1p (Increase in ploidy 1) gene, subsequently named Aurora gene, the founding member discovered in the budding yeast *Saccharomyces cerevisiae* during a genetic screening for mutations causing defective chromosomal segregation [35–38]. In mammals, the Aurora kinase family includes three proteins: Aurora-A, Aurora-B, and Aurora-C [39]. Structurally, they are characterized by three domains: a N-terminal domain with little similarity among the three Aurora kinases, instrumental in determining their different intracellular localizations, substrate specificity and functions; a catalytic domain, containing the activation loop and highly related in sequence among the three proteins; and a short C-terminal domain of 15–20 residues (**Figure 1**). Aurora kinase expression is tightly regulated during cell cycle, being low in the G1/S phase and maximal in the G2/M phase.

**Figure 1.** Schematic representation of Aurora kinase proteins. D-Box, destruction box; DAD, D box activating domain; KEN motif, amino acidic K-E-N, which serves as targeting signal for the Cdh1–anaphase promoting complex (adapted with permission from Ref. [14]).

#### **2.1. Aurora-A**

The Aurora-A is encoded by the *AURKA* gene located on the chromosome 20q13.2 and containing 11 exons (Gene ID: 6790). The *AURKA* promoter contains a putative TATA box at −37 to −14 and two CCAAT boxes at −101 to −88 and at −69 to −56 [Eukaryotic Promoter Database, Swiss Institute of Bioinformatics]. Two distinct cis-regulatory elements have been identified [40]. Of these, one positively regulates *AURKA* gene transcription, while the other is a cell cycle-dependent transcriptional repressor [40]. The former, essential for the gene expression, is a 7-bp sequence located at −85 to −79 that binds the transcription factor E4TF1. The second is formed by two repressor elements: a cell cycle-dependent element (CDE) located at −44 to −40, and a cell cycle gene homology region (CHR) located at −39 to −35 [40]. Over the last few years, a number of transcription factors capable of repressing or inducing *AURKA* gene expression have been identified. These include the p53, the HIF-1, and the INI1/hSSNF5, all reported to regulate negatively the activity of the *AURKA* promoter [41–43]. Conversely, other transcription factors have been shown to induce *AURKA* expression, among which the ΔEGFR/STAT5, the oncogene MYCN, and the MAPK via Ets2 transcription factors [44–47]. The Aurora-A protein consists of 403 amino acids with a predicted molecular mass of 45.8 kDa (**Figure 1**). In the activation loop, an Aurora kinase signature (xRxTxCGTx) is present in which the autophosphorylation of the Thr288 is required for kinase activation [48]. In addition, the Thr288 is positioned within a protein kinase A (PKA) consensus sequence, and *in vitro* experiments indicated a potential role of PKA in Aurora-A phosphorylation [49, 50]. The phosphatase PP1 has been shown to dephosphorylate and inactivate Aurora-A [16]. The Cterminal located destruction box (D box), containing the motif RxxLxxG, and the N-terminal A-box/D-box-activating domain (DAD), containing the motif RxLxPS, play an essential role in Aurora-A degradation by the anaphase promoting complex/cyclosome (APC/C)-ubiquitinproteasome pathway. Aurora-A degradation occurs in late mitosis/early G1 phase, when the D box is targeted by Fizzy-related proteins that transiently interact with the APC, and it is dependent from the APC/C activator protein Cdh1 [49–52]. In the N-terminal region the amino acidic sequence K-E-N, known as KEN motif, is also present, which serves as targeting signal for Cdh1-APC-mediated degradation of several mitotic proteins such as Nek2 and B99 [53]. However, this does not seem to be crucial for Aurora-A degradation [53]. Phosphorylation of the serine residue (Ser51) in the DAD domain has been shown to prevent Aurora-A degradation [54, 55].
