Aurora kinase A in gastrointestinal cancers: time to target

Cancers can develop in any anatomical site of the GI system including esophagus, stomach, small intestine, colon, rectum, liver, gall bladder, and pancreas. A cancer in any of these organs represents a unique type where different molecular mechanisms of tumorigenesis can exist. Recent studies have shown that AURKA is frequently overexpressed and/or amplified in several GI cancers [25-28]. In the following sections, we will review and discuss some of the recently reported studies about AURKA functions in GI cancers.

Upper gastrointestinal (UGI) cancers include tumors of the stomach and esophagus. While esophageal squamous cell carcinoma (ESCC) is the predominant histological type of esophageal cancers in the world, there has been a significant increase in the incidence of gastroesophageal reflux-related esophageal adenocarcinomas (EAC) in the Western world [41]. The majority of gastric cancers are adenocarcinomas; other subtypes such as lymphomas and gastrointestinal stromal tumors are rare and will not be discussed in this review. Amplifications of chromosomal region 20q13, where AURKA is located, have been described in gastric and esophageal cancers [42,43]. Polymorphisms in the AURKA gene have also been reported in UGI cancers [44,45]. Several studies have shown frequent overexpression of AURKA at the mRNA and protein levels in ESCC, EAC, and gastric cancers [28,46-49]. It has also been suggested that the increased AURKA expression might be used as a prognostic marker of ESCC [50]. In addition, overexpression of AURKA in premalignant and malignant histological stages of reflux-induced Barrett’s tumorigenesis has been reported [51]. Similarly, overexpression of AURKA has been observed in early pre-neoplastic stages of gastric cancer in mouse models and human [49]. Taken together, these reports strongly suggest a critical role for AURKA in carcinogenesis.

Molecular studies have shown that AURKA promotes cell survival signaling while it suppresses the pro-apoptotic machinery in UGI cancer cells [52,53]. We and others have reported that AURKA promotes activation of the AKT pro-survival signaling pathway [47,54]. Furthermore, it has been shown that AURKA can regulate and suppress GSK3β kinase activity in gastric cancer cell lines [19]. The AURKA-GSK3β interaction activates β-catenin/TCF transcription complex, which leads to increased mRNA expression of several oncogenic proteins such as CCND1, c-MYC, c-MYC-binding protein, CLDN1, FGF18, and VEGF [19]. Pro-inflammatory signaling is a significant component in the etiology of tumor development and progression of UGI cancers. In this context, recent reports indicated that AURKA overexpression promotes activation of NF-κB [49] and STAT3 [55] pathways in these cancers. In these studies, AURKA inhibition abrogates the pro-survival and pro-inflammatory signaling mediated by NF-κB and STAT3 with suppression of key pro-survival target genes such as BCL-xL, BCL2, and MCL1 [49,55]. Some of the main features of cancer cells include resistance to pro-apoptotic stimuli and the ability to overcome genotoxic stress and chemotherapeutic-induced death. We and others have shown that AURKA can suppress p53 pro-apoptotic functions [48,56]. A recent study has shown that AURKA regulates the key ubiquitin ligase involved in degradation of p53, HDM2, whereby overexpression of AURKA enhanced HDM2 stabilization and promoted degradation of p53, thereby abrogating p53 pro-apoptotic function in response to chemotherapy [48]. Not only does AURKA circumvent p53 pathway, but it also provides a mechanism for cancer cells to evade apoptosis by suppressing p73, an important p53 family protein [52,56]. Collectively, these signaling mechanisms mediated by AURKA can promote cancer cell survival and provide a chemoresistance phenotype. These findings also suggest AURKA as part of a signaling hub controlling several key pathways that regulate the hallmarks of the cancer cell network.

Several AURKA inhibitors have been developed to overcome AURKA-mediated pro-survival and anti-apoptotic activities in cancer cells. Inhibition of AURKA using investigational MLN8054 or MLN8237 led to reversal of anti-apoptotic signaling cascades with activation of pro-apoptotic p73 in p53 deficient cells with re-expression of several pro-apoptotic proteins such as PUMA, NOXA, and p21 [52]. AURKA inhibition by small molecule MLN8237 as a single agent or in combination with Cisplatin or Docetaxel significantly enhanced cell death in esophageal adenocarcinoma xenograft mouse model [53,57]. These findings strongly indicate that AURKA could be an important therapeutic target in upper GI cancers.

Colorectal cancer (CRC) was one of the first cancers found to have AURKA amplification. A 1998 study showed that overexpression of AURKA resulted in centrosome amplification, chromosomal instability and transformation in mammalian cells, including colon cancer cells [34]. Genetic variations in the AURKA gene were also detected and associated with aneuploidy in human colon tumors and low penetrance CRC susceptibility factor [58,59]. AURKA overexpression was associated with the down-regulation of checkpoint with forkhead and ring finger domains (CHFR) in colorectal cancers with high microsatellite instability [60]. Importantly, recent reports indicated that AURKA is critical for tumorigenicity and chemoresistance in CRC stem cells [31], and suggested AURKA as a predictive marker for recurrence in stage III in colon tumors lacking microsatellite instability [61]. These studies, as well as other studies, focused mainly on the fact that overexpression of AURKA leads to chromosomal instability. In normal cells, expression of AURKA protein is predominantly confined to the nucleus and subject to spatio-temporal regulation during mitotic progression. In contrast, expression of AURKA protein is frequently observed in the cytoplasm, in addition to the nucleus, in cancer cells, suggesting an extended role of AURKA beyond regulation of mitosis [62]. This finding provides additional evidence supporting the wealth of signaling data for AURKA reported in upper GI cancers (summarized in the section above). For instance, Tseng and colleagues found that AURKA enhances formation and aggregation of mutant Ras (RasV12) through regulation of RAS/MEK/ERK pathways and AKT phosphorylation in colon cancer [63]. Aurora kinase pharmacological inhibition sensitized colon cancer cells to Tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) [64]. In addition, AURKA inhibition, using specific or pan Aurora kinase inhibitors, sensitized colon cancer cells to chemotherapy [65], TNF or TRAIL induced apoptosis [64] as well as mitotic failure and cell death when inhibited along with SRC [66]. Other reports showed that pharmacological inhibition of AURKA with MLN8054 in colon cancer HCT-116 xenografts induces senescence in vivo [36]. These findings suggest that more studies are needed to better characterize the role of AURKA in colon cancer signaling and therapeutics.

Overexpression of AURKA was reported in pancreatic cancer [26], in the early stages of abnormalities in pancreatic ducts and ductal dysplasia in transgenic mouse model for pancreatic cancer [67], and linked to chromosome instability and centrosome abnormality [68]. As observed in other GI cancers, suppression of AURKA using RNA interference [69] or small molecule inhibitor SNS-314 [70] suppresses tumor growth and enhances chemosensitivity. Similar results were obtained with specific AURKA inhibitor CYC3 in combination with Paclitaxel [71]. In addition, AURKA plays a role in protecting human pancreatic ductal adenocarcinoma cells from autophagic cell death induced by metabolic stress [72]. Interestingly, while it was shown in colorectal cancer that AURKA regulates MEK/ERK pathway [63], it was reported that AURKA is a downstream target of this pathway in pancreatic cancer [73], raising the possibility of an existing positive feedback loop between AURKA and MEK/ERK pathway. This signaling loop further underscores the potential therapeutic benefit of targeting AURKA in pancreatic cancer. Additionally, AURKA can phosphorylate RalA at Ser194 and regulate its subcellular localization, which is essential for Ras-mediated cellular transformation [74]. However, further studies by the same group failed to show correlation of RalA (S194) phosphorylation with responsiveness to MLN8237, indicating that the principal target of AURKA may not be RalA [75].

In human hepatocellular carcinoma (HCC), AURKA was found to be overexpressed at mRNA level in 61% of HCC samples [30], whereas gene amplification was recorded in only 3 out of 224 HCC samples [30]. Notably, high expression levels of AURKA were associated with high-grade, high-stage tumors and poor outcome. Microarray studies suggested that AURKA phosphorylates and stabilizes hepatoma up-regulated protein (HURP) [76], a cell cycle regulated gene that is overexpressed in human hepatocellular carcinoma (HCC) [77]. On the other hand, it was reported that hypoxia and hypoxia-inducible factor-1 (HIF-1) are important in the tumor microenvironment and could regulate AURKA at the transcriptional level [78,79]. Other studies showed that AURKA, can in turn, regulate HIF-1α through activation of AKT and p38-MAPK signaling pathways leading to malignant phenotypes of HCC [80], indicating a positive feedback loop sustaining continuous activation of AURKA. Several preclinical studies investigated the effects of AURKA inhibition in HCC. For example, a report showed that the pan Aurora inhibitor PHA-739358 inhibits HCC tumor growth in a tumor xenograft mouse model [81]. Further, AURKA inhibition with small molecule R1498 was found to target mitosis pathway and suppress angiogenesis [82]. These studies emphasize the multifaceted roles of AURKA in HCC and imply AURKA as a feasible druggable target in HCC.

The aforementioned preclinical studies shed light on the roles and contributions of AURKA in tumorigenesis, which serve as premise for the development of several pan Aurora kinase inhibitors (AKIs) and selective inhibitors of AURKA. In the following section, we will focus on the present investigational AURKA inhibitors in completed, ongoing, or planned clinical trials.

MLN8237 (alisertib) is a second generation derivative of the initial small molecule MLN8054, developed by Millennium Pharmaceuticals, Inc. Both generations work as highly specific ATP-competitive AURKA inhibitors with an IC50 of 2 nM in chronic myeloid leukemia (CML) for MLN8237 [83,84]. The MLN8237 IC50 values vary in other cancers, ranging from 6.7 nM in HeLa cells to 469 nM in DLD-1 colon cancer cell line [85]. MLN8237 can target AURKB at higher doses (396.5 nM) [35,85]. The first generation MLN8054 was terminated in phase 1 clinical trials due to off-target toxicities [86]. Both drugs are ATP competitive, where they bind and inhibit AURKA phosphorylation at T288. In murine xenograft models, MLN8237 was well tolerated with doses ranging from 15 to 40 mg/kg/d. MLN8237 was also found to be well absorbed and steady state plasma levels were reached within 7 days of twice daily dosing in phase 1 clinical trials [87]. Investigations of MLN8237 in preclinical studies have shown that it induces abnormal G2/M cell cycle arrest, abnormalities in mitotic spindles, and apoptosis in upper GI cancers [48,49,53]. In addition, MLN8237 down-regulates several oncogenic pathways such as NF-κB [49,88], AKT [19], and STAT3 [55] in cancer cells. On the other hand, several reports demonstrated that treatment with MLN8237 could activate p53 [47,48,89] and p73 [52,56] apoptotic pathways. The effects of MLN8237 were tested in several non-GI cancers. It has been shown that MLN8237 sensitized PC3 and DU145 prostate cancer cells to radiation, increased the DNA double-strand breaks, induced G2/M cell cycle arrest and polyploidy [90]. Treatment of OVCAR-5 and SKOV3ip2 ovarian cancer cells with MLN8237 resulted in inhibition of cytoskeletal regulatory SRC (assessed by decreased phosphorylation at Tyr-416) and decreased migration and adhesion [18]. Moreover, MLN8237 has been tested on many pediatric cancer cell lines like Ewing sarcoma, neuroblastoma, glioblastoma, rhabdomyosarcoma, ALL, and AML where it inhibited cell growth and delayed tumor growth [91]. In vitro and in vivo tumor xenografts mouse models showed that MLN8237 enhances anti-tumor activity of chemotherapeutics. For instance, dual treatment of MLN8237 with Docetaxel enhanced apoptosis and anti-tumor activity in lymphoma [92] and UGI adenocarcinomas cell models [53]. Similarly, a combination of Cisplatin with MLN8237 had robust anti-tumor activity in tumor xenograft models of esophageal adenocarcinomas [57]. In B-cell Non-Hodgkin Lymphoma, MLN8237 in combination with Vincristine and Rituximab showed robust cell death in vitro and had curative effects in vivo using mantle cell lymphoma (MCL) mouse xenograft model [93]. Collectively, the present data indicate that MLN8237 is an effective drug with high specificity and limited off target activity that works in various models. Notably, there are 49 clinical trials with MLN8237 in leukemia, breast, lung, colorectal, ovarian, and pancreatic cancers (clinicaltrials.gov; March 2015). Phase 1 clinical studies of the drug as a single agent demonstrate preliminary activity where 37% of patients achieved a best response of stable disease for more than 6 months in some solid tumors (colorectal cancer, chondrosarcoma, leiomyosarcoma, and liposarcoma) with modest toxicity such as nausea, stomatitis, and alopecia among other adverse effects [87]. The recommended phase 2 dose of MLN8237 is 50 mg orally given twice daily on days 1–7 of a 21-day cycle for various solid tumors and hematologic malignancies [94]. Phase 2 studies of MLN8237 as a single agent are ongoing in multiple tumor types including lung, breast, ovarian, UGI, and pancreatic cancers (Table 2). In some of the clinical trials, MLN8237 has been tested in combination with other drugs such as Cetuximab and Docetaxel among others (Table 2). A recent phase 2 clinical trial of MLN8237 as a single agent in advanced solid tumors revealed that 9% of patients (4 out of 47 patients) with gastro-esophageal adenocarcinoma partially responded to the treatment [95]. A newly registered phase 1 clinical trial (NCT02319018) is ongoing to investigate the side effects of MLN8237 in combination with other chemotherapeutic drugs (fluorouracil, leucovorin calcium, oxaliplatin). This clinical trial will be the first to evaluate MLN8237 exclusively in gastrointestinal tumors (clinicaltrials.gov, Table 2).

ENMD-2076 was originally developed by EntreMed, Inc. as a selective AURKA inhibitor with an IC50 of 14 nM in biochemical assays [96]. However, ENMD-2076 was found to inhibit several other oncogenic kinases at different IC50 including FLT3 (1.86 nM), SRC (20.2 nM), VEGFR3 (15.9 nM), VEGFR2 (58.2 nM), PDGFRα (56.4 nM), and FGFR1 (92.7 nM) [96]. This drug was shown to induce G2/M cell cycle arrest and apoptosis, consistent with AURKA inhibition responses. ENMD-2076 was found to be selectively toxic to multiple myeloma (MM) cells but minimally toxic to hematopoietic progenitor cells [97]. Interestingly, exposure as short as 6 hours induces apoptosis and reduces AURKA autophosphorylation at T288; however, this was achieved at doses that also inhibited AURKB and several other kinases [97]. In vitro studies in multiple myeloma showed that treatment with ENMD-2076 inhibited AKT phosphorylation, and thus, altered the activity of some of its targets, including GSK3β, survivin, and XIAP [97]. Additionally, ENMD-2076 was shown to down-regulate phosphorylation of STAT3 at Ser-727 [97]. In mouse xenograft models of multiple myeloma, ENMD-2076 showed good tolerability at doses up to 200 mg/kg, and immunohistochemistry analysis revealed reduced Ki67 and increased cleaved caspase-3 expression levels [97]. In a patient-derived xenograft model of CRC, ENMD-2076 inhibited tumor growth, which led to regression; this was associated with significant reduction in 18-FDG uptake at day 3 and 21 of treatment [98]. In breast cancer, ENMD-2076 was most effective against triple-negative tumors with increased mutant p53 expression [99]. In addition, cells lacking estrogen receptor expression and/or HER2 overexpression were the most sensitive cells to the drug [99]. A completed phase 1 clinical trial indicated that this drug is tolerable at 160 mg/m² daily administered orally in patients with advanced solid tumors [100]. ENMD-2076 is currently being evaluated in phase 1 and 2 clinical trials, specifically in breast and ovarian cancers (clinicaltrials.gov, Table 2).

MK-5018 (VX689) is a specific AURKA inhibitor (IC50: 0.046 nM) developed by Vertex Pharmaceuticals [101]. MK-5018 also inhibits AURKB and AURKC, although at higher doses (200 folds higher than AURKA) with various IC50 values among different cancer cell lines [101]. Tumor xenografts treated with MK-5018 showed significant tumor inhibition at doses of 15 or 30 mg/kg twice daily for 12 days, indicating the high tolerability of the drug [101]. In addition to a typical AURKA inhibition response (polyploidy and cell cycle arrest), inhibition of NF-κB activity and reduction of cytokine production were observed after treatment of ovarian cancer stem cells with MK-5108 [102]. Further, MK-5108 was investigated in combination with other therapeutic agents. For instance, MK-5108, in combination with histone deacetylase inhibitor, vorinostat, induced lymphoma cell death along with reduction of c-Myc, hTERT, and micro RNA levels in vitro [103]. In a colon cancer xenograft mouse model, MK-5108 was tested in combination with Docetaxel and showed additive anti-tumor activity as compared to animals that received Docetaxel alone [101]. A completed phase 1 clinical trial of MK-5108 as a single agent or in combination with Docetaxel to test its safety, side effects, and tolerability showed moderate toxicities with some clinical activity as a single agent in patients with advanced and/or refractory solid tumors [104] (clinicaltrials.gov, Table 2).