**5. Aurora kinases and thyroid cancers**

which, depending on the post-mitotic checkpoint activation, may be unable to proceed in a new cell cycle or rather may proliferate and become polyploid. The exit from cell cycle is likely to generate viable quiescent cells, whereas endoreplicating cells have greater tendency to undergo apoptosis. Actually, the functional inhibition of Aurora kinases is considered a promising therapeutic option against those malignancies that do not respond to the available therapies [101–108]. Up to date, about 30 small-molecule inhibitors of Aurora kinases have been developed and some of them, reported in **Table 1**, are being evaluated in Phase I-II clinical trials [101–108]. Of some interest are the preclinical observations showing the ability of different Aurora kinase inhibitors to have additive or synergist effects when combined with other anticancer therapies [109, 110]. At example, among the pan-Aurora kinase inhibitors, the AMG-900 in combination with the HDAC (histone deacetylase) inhibitor vorinostat has been shown to synergistically reduce proliferation and survival of medulloblastoma and prostate cancer-derived cell lines [111, 112]. Similarly, the SNS-314 has been shown to possess additive inhibitory effects on the HCT 116 cell line when combined with either carboplatin, gemcitabine, 5-FU, daunomycin, docetaxel, or vincristine [113]. Also the MK-0457 has revealed additive effects when combined with docetaxel on ovarian cancer cell lines or with cisplatin on the HepG2 cell line [114, 115]. Finally, the pan-Aurora kinase inhibitor CCT 137690 has been shown to sensitize SW620 colorectal cancer cells to radiotherapy [116]. In clinical trials, disease stabilization and, less frequently, partial responses in patients with solid cancers have been witnessed with the majority of Aurora kinase inhibitors, while more encouraging observations have been made in patients with hematological malignancies [101–110]. On-target toxicity observed with these drugs included grade 3/4 neutropenia, leukopenia, and myelosuppression, while off-target effects included hypertension, somnolence, mucositis, stomatitis, proctalgia, and ventricular dysfunction [101–110]. For example, the MK-0457 has been employed in different clinical trials in which patients with advanced solid tumors have been enrolled. In a Phase I dose escalation study, the most common doselimiting toxicity observed was neutropenia and herpes zoster, and major adverse events include nausea, vomiting, diarrhea, and fatigue [117]. Although no objective tumor responses were observed in this trial, 12 of 27 patients experienced stable disease with a median duration of 75.5 days (range 38–328 days). Of the latter, one patient with ovarian cancer achieved prolonged stable disease for 11 months, and one patient with rectal cancer had stable

The MK-0457 was found to have off-target inhibitory effects on both wild-type and mutant Abl kinases and showed to be a potent inhibitor of the BCR-ABL T315I mutant, which mediates clinical resistance to imatinib, nilotinib, and dasatinib [118]. On these bases, a phase I/II dose escalation study of MK-0457 was performed in patients with leukemias [119, 120]. Patients with refractory hematologic malignancies received 1–21 cycles of MK-0457, and maximumtolerated doses were calculated for a 5-day short infusion as 40 mg. Mucositis and alopecia were the most common drug-related adverse events observed in these patients. Forty-four percent (8/18) of patients, positive for the BCR-ABL T315I mutation, affected by chronic myelogenous leukemia (CML) had hematologic responses, and 33% (1/3) of patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) obtained

disease over 7 months [117].

104 Anti-cancer Drugs - Nature, Synthesis and Cell

complete remission [119, 120].

Normal human thyrocytes express all three Aurora kinases in a cell cycle-dependent manner [98]. The expression of Aurora-A and Aurora-B in these cells is mainly regulated at the transcriptional level, while that of Aurora-C appears to be modulated at the posttranscriptional level [98]. An increased expression of all the Aurora kinases has been shown in various cell lines originating from different epithelial thyroid tumor histotypes, compared with normal thyrocytes, as well as in DTC and ATC tissues, compared with normal matched tissues [60, 98, 122]. In addition, a study aimed to evaluate the gene expression profile in ATC identified *AURKA* as one of the most frequently and most strongly overexpressed genes in these tumors [123]. In fact, gain of chromosome 20q, where *AURKA* is located (20q13.2), is frequently encountered in ATC [124]. Based on these findings, the potential therapeutic value of Aurora kinase inhibition on the proliferation and growth of PTC and ATC cells has been evaluated in preclinical studies [125–130]. In particular, different pan-Aurora kinase inhibitors, including the MK-0457 (VX-680), the SNS-314 mesylate, and the ZM447439 have been evaluated *in vitro* [126–129]. These molecules were found to inhibit proliferation of ATC cells in a time- and dosedependent manner and to impair cancer cell colony formation in soft agar. Cell cultures exposed to pan-Aurora inhibitors revealed an accumulation of tetra- and polyploid cells because of endoreplication events followed by the activation of caspase-3 and accumulation of a sub-G0/G1 cell population, both indicative of apoptosis [126–129]. Treated cells showed mitotic alterations consistent with the inhibition of Aurora kinases, including major impairment of centrosome functions, with abnormal spindle formation characterized by the presence of short microtubules, inhibition of histone H3 phosphorylation, and inability to complete the cytokinesis. The effects of a selective inhibition of either Aurora-A or Aurora-B have been also explored [125, 129, 131]. The selective inhibition of Aurora-B expression, by means of RNA interference, or function, by means of small-molecule compounds (e.g., AZD1152), has been reported to significantly reduce growth and tumorigenicity of ATC-derived cells, both *in vivo* and *in vitro* [125]. In the same way, functional inhibition of Aurora-A by MLN8054 in a panel of ATC-derived cell lines has been shown to block cell proliferation and to induce cell cycle arrest and apoptosis [129]. In xenograft experiments, the drug was capable of reducing tumor volume by 86% [129]. Interestingly, the combined treatment with MLN8054 and bortezomib, targeting the ubiquitin-proteasome system, showed additive effects on ATC-derived cell proliferation and apoptosis, compared with monotherapy [131]. More recently, pazopanib, a multi-target inhibitor of tyrosine kinases including the VEGFR (vascular endothelial growth factor receptor), shown to have impressive therapeutic activity in patients affected by radioactive iodine-refractory DTC, was tested in a phase II clinical trial on ATC patients [132, 133]. Despite several of them treated with pazopanib had a transient disease regression, no response evaluation criteria in solid tumors (RECIST) response was obtained [131]. Moreover, in a preclinical study on a panel of ATC-derived cell lines, pazopanib was found to potentiate the cytotoxic effects of paclitaxel *in vitro* and in xenograft experiments [134]. These pazopanib effects were attributed to an unexpected off-target inhibition of Aurora-A in ATC-derived cell lines. In fact, the same results were obtained when combining paclitaxel and MLN8237, a selective Aurora-A inhibitor. In the same study, the authors also showed that the combined administration of pazopanib and paclitaxel attained a marked and durable regression of lung metastasis in a single ATC patient [134].

In conclusion, the preclinical and clinical data so far available indicate that Aurora kinase inhibitors may have a therapeutic potential for the treatment of the more aggressive thyroid cancers either in monotherapy or, more likely, in combination therapy with antimicrotubule drugs.
