**4. Aurora kinases and cancer**

Chromosomal instability is thought to represent the mean by which premalignant cells acquire novel functional capabilities responsible for cancer cell growth and tumor progression [73]. In fact, aberrations in chromosome number and structure, likely resulting from a combination of ineffective checkpoints and anomalous cellular divisions, occur in the majority of human cancers [74]. Given the crucial tasks of Aurora kinases in all mitotic stages, their dysfunction and/or dysregulation is believed to greatly contribute to aneuploidy. Whether Aurora kinases may have a role in cancer initiation is still a matter of debate. It has been reported that the overexpression of either Aurora-A, Aurora-B, or Aurora-C induces cell malignant transformation [75–77]. In different studies, however, the transforming ability of either Aurora-A or Aurora-B overexpression could not be confirmed [78, 79].

Aurora-A kinase has been often implicated in cancer progression, and its hyperactivation has been demonstrated to induce resistance to microtubule-targeted chemotherapy [80–82]. The *AURKA* gene is amplified in many malignancies, and its overexpression has been reported to be significantly associated with a higher tumor grade and a poor prognosis in a number of cancers, including chondrosarcoma, nasopharyngeal carcinoma, breast cancer, glioblastoma, colorectal cancer, gastric cancer, and ovarian carcinoma [83–89]. In addition, somatic mutations located within the catalytic domain of Aurora-A, altering kinase activity and subcellular localization, have been described in human cancer cells [90]. The oncogenic potential of Aurora-A derives from a sum of several spatially and temporally distinct actions. Unlike normal cells, in many cancer cells the expression of Aurora-A becomes constitutive throughout the cytoplasm, regardless of the cell cycle phase; this can trigger a plethora of improper interactions, phosphorylations, and mislocalizations. Aurora-A may also represent the downstream target of mitogenic pathways, such as MAPK/ERK (mitogen-activated protein kinases), and be overexpressed because of their constitutive activation in tumors [81]. The Aurora-A excess interferes with different cell cycle checkpoints, that is, the late G2 checkpoint, which restrains genetically aberrant cells to enter mitosis, the spindle assembly checkpoint, which blocks the metaphase–anaphase transition in cells with defective spindles, and the postmitotic G1 checkpoint, which arrests cell cycle in aneuploid cells [81, 83]. Centrosome amplification and unrestrained multinucleation, leading to abnormal mitotic spindle, are also observed in Aurora-A overexpressing cells [91]. Moreover, Aurora-A may significantly contribute to tumor progression by interacting with and inhibiting several tumor suppressor proteins such as p53, BRCA1 (breast cancer 1), and Chfr (checkpoint with forkhead and ring finger domains). Interestingly, activation of the MAPK signaling pathways has been found to induce accumulation of Aurora-A kinase in ERα+ breast cancer cells, and epithelial-tomesenchymal transition (EMT) [92, 93]. In these cells, Aurora-A has been shown to promote SMAD5 phosphorylation and nuclear translocation, upregulation of stemness gene SOX-2, and acquisition of a stem cell-like phenotype [92, 93].

Aurora-B plays a less clear role in tumorigenesis. An increased level of Aurora-B in normal cells induces premature chromosome separation and segregation errors, promotes generation of tetraploid/aneuploid cells, and potentiates Ras oncogenic activity [77, 80, 94–96]. Neither amplification nor specific mutations of *AURKB* gene have been shown to occur in human malignancies; nevertheless, Aurora-B overexpression has been demonstrated in several cancer types, including hepatocellular and oral squamous cell carcinomas, where it correlates with poor prognosis [80, 94–96].

At present, very little is known about the role of Aurora-C in cancer progression. Although Aurora-C is almost not detectable in normal somatic cells, it is highly expressed in various tumor cell lines [97–100]. One study has described the transforming potential of overexpressed Aurora-C in NIH-3T3 cells, and a correlation between the level of active kinase and tumor aggressiveness of the cells injected in nude mice [77].

#### **4.1. Aurora kinase inhibitors**

to recruit condensin proteins (**Figure 3**) [67, 68]. From early G2 phase to prophase, Aurora-B phosphorylates histone H3, but its physiological meaning remains unclear. From late prophase to metaphase CPC localizes to the inner centromere, playing a role in formation and stability of the bipolar mitotic spindle, kinetochore assembly, correction of non-bipolar chromosomespindle attachments, and control of the spindle checkpoint (**Figure 3**). In anaphase, the CPC relocates to the midzone of the mitotic spindle and to the cell cortex, remaining evident in the midbody of telophasic cells where it modulates the activity of several proteins involved in spindle dynamics, cleavage furrow formation and completion of cytokinesis (**Figure 3**) [49, 50,

Aurora-B activation requires the auto-phosphorylation and binding to INCENP, while all CPC components are necessary for its proper localization during mitosis. Several kinases, such as BubR1 and Bub1 (checkpoint kinases), monopolar spindle 1 (Mps1), checkpoint kinase 1 (Chkl), Tousled-like kinase-1, Plk1, and TD-60/RCC2 (regulator of chromosome condensation 2), have been shown to be involved in Aurora-B activation. The phosphorylation status and

The expression of Aurora-C is maximal during the G2/M phase. This kinase seems to have a prominent role in the meiotic division, as it is expressed at relative high levels in germ cells during spermatogenesis and oogenesis, and at very low levels in somatic cells. Aurora-C is highly similar to Aurora-B in sequence (61% identity), which may explain why the two kinases display similar localization patterns and share interacting proteins and substrates such as INCENP, Survivin, Borealin, and others [49, 70]. Interestingly, when ectopically expressed in cells depleted of Aurora-B, Aurora-C is capable of rescuing the Aurora-B-dependent mitotic functions [40]. It is also worth to note that Aurora-C has been shown to interact with and

Chromosomal instability is thought to represent the mean by which premalignant cells acquire novel functional capabilities responsible for cancer cell growth and tumor progression [73]. In fact, aberrations in chromosome number and structure, likely resulting from a combination of ineffective checkpoints and anomalous cellular divisions, occur in the majority of human cancers [74]. Given the crucial tasks of Aurora kinases in all mitotic stages, their dysfunction and/or dysregulation is believed to greatly contribute to aneuploidy. Whether Aurora kinases may have a role in cancer initiation is still a matter of debate. It has been reported that the overexpression of either Aurora-A, Aurora-B, or Aurora-C induces cell malignant transformation [75–77]. In different studies, however, the transforming ability of either Aurora-A or

Aurora-A kinase has been often implicated in cancer progression, and its hyperactivation has been demonstrated to induce resistance to microtubule-targeted chemotherapy [80–82]. The

activity of Aurora-B are controlled by PP1 and PP2A phosphatases [69].

phosphorylate TACC1 in thyroid cells in the cytokinetic bridge [71, 72].

Aurora-B overexpression could not be confirmed [78, 79].

67–69].

102 Anti-cancer Drugs - Nature, Synthesis and Cell

**3.3. Aurora-C**

**4. Aurora kinases and cancer**

The overexpression of Aurora kinases in human cancers and their relevance in controlling the mitotic process have led to the development of small-molecule inhibitors as anticancer drugs. Aurora inhibition results in cytokinesis failure and generation of tetraploid cells, 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 disease over 7 months [117].

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 complete remission [119, 120].


**Table 1.** Aurora kinase inhibitors in clinical trials (adapted with permission from Ref. [14]).

Another multicenter phase II study evaluated the safety and efficacy of MK-0457 on 52 patients affected by CML or Ph+ ALL with BCR-ABL T315I mutation [121] (Seymour et al. 2014). Patients were treated with a 5-day continuous infusion of MK-0457 administered every 14 days at 40, 32, or 24 mg. The most common adverse events were neutropenia and febrile neutropenia. Eight percent (4/52) of patients achieved major cytogenetic response and 6% (3/52) had a complete or a partial response. Thirteen percent (2/15) of patients with chronic phase CML achieved complete hematologic response. None of the patients with advanced CML or Ph+ ALL achieved major hematologic response [121].

A comprehensive description of clinical trials performed with the different Aurora kinase inhibitors has been recently reported [109, 110].
