PMC:5337621 / 22030-30865 JSONTXT

{"project":"2_test","denotations":[{"id":"28218735-16158051-79088319","span":{"begin":105,"end":107},"obj":"16158051"},{"id":"28218735-17259342-79088320","span":{"begin":223,"end":224},"obj":"17259342"},{"id":"28218735-18160664-79088321","span":{"begin":226,"end":228},"obj":"18160664"},{"id":"28218735-24899685-79088322","span":{"begin":449,"end":451},"obj":"24899685"},{"id":"28218735-24587643-79088323","span":{"begin":976,"end":978},"obj":"24587643"},{"id":"28218735-15289843-79088324","span":{"begin":1200,"end":1201},"obj":"15289843"},{"id":"28218735-18311783-79088325","span":{"begin":1203,"end":1205},"obj":"18311783"},{"id":"28218735-21479365-79088326","span":{"begin":1207,"end":1209},"obj":"21479365"},{"id":"28218735-15289843-79088327","span":{"begin":1356,"end":1357},"obj":"15289843"},{"id":"28218735-18311783-79088328","span":{"begin":1492,"end":1494},"obj":"18311783"},{"id":"28218735-17974987-79088329","span":{"begin":2371,"end":2373},"obj":"17974987"},{"id":"28218735-16158051-79088330","span":{"begin":2802,"end":2804},"obj":"16158051"},{"id":"28218735-24899685-79088331","span":{"begin":2948,"end":2950},"obj":"24899685"},{"id":"28218735-24950801-79088332","span":{"begin":2999,"end":3001},"obj":"24950801"},{"id":"28218735-16185509-79088333","span":{"begin":3218,"end":3220},"obj":"16185509"},{"id":"28218735-23146905-79088334","span":{"begin":3222,"end":3224},"obj":"23146905"},{"id":"28218735-21852011-79088335","span":{"begin":3226,"end":3228},"obj":"21852011"},{"id":"28218735-26770398-79088336","span":{"begin":3230,"end":3232},"obj":"26770398"},{"id":"28218735-14720326-79088337","span":{"begin":3477,"end":3479},"obj":"14720326"},{"id":"28218735-19004025-79088338","span":{"begin":3578,"end":3580},"obj":"19004025"},{"id":"28218735-11069302-79088339","span":{"begin":3582,"end":3584},"obj":"11069302"},{"id":"28218735-22492986-79088340","span":{"begin":4202,"end":4204},"obj":"22492986"},{"id":"28218735-19111882-79088341","span":{"begin":4231,"end":4233},"obj":"19111882"},{"id":"28218735-17124180-79088342","span":{"begin":4615,"end":4617},"obj":"17124180"},{"id":"28218735-25778398-79088343","span":{"begin":4937,"end":4939},"obj":"25778398"},{"id":"28218735-12517802-79088344","span":{"begin":4941,"end":4943},"obj":"12517802"},{"id":"28218735-22306998-79088345","span":{"begin":5297,"end":5299},"obj":"22306998"},{"id":"28218735-25778398-79088346","span":{"begin":5348,"end":5350},"obj":"25778398"},{"id":"28218735-20418484-79088347","span":{"begin":5493,"end":5495},"obj":"20418484"},{"id":"28218735-25778398-79088348","span":{"begin":5670,"end":5672},"obj":"25778398"},{"id":"28218735-21924763-79088349","span":{"begin":6382,"end":6384},"obj":"21924763"},{"id":"28218735-17317845-79088350","span":{"begin":7575,"end":7577},"obj":"17317845"},{"id":"28218735-26770398-79088351","span":{"begin":7967,"end":7969},"obj":"26770398"},{"id":"28218735-24886669-79088352","span":{"begin":7971,"end":7973},"obj":"24886669"}],"text":"Discussion\nMitotic kinases, including AURKA, are key signaling components of genotoxic response pathways.32 Previous studies have documented aberrant expression of AURKA in various carcinomas and hematological malignancies.7, 33 In addition, our earlier data has also suggested that AURKA was highly expressed in epirubicin resistance breast tumor initiating cells and contributed to the maintenance of stemness and drug resistance in breast cancer.34 Given the essential roles of AURKA overexpression in cancer progression, targeting AURKA offers an attractive approach for cancer therapy. In the present study, we found that AURKA could override DNA-damaging agent-induced cell death in gastric cancer cell lines, resulting in the development of drug resistance which was at least partially mediated through Survivin stabilization.\nGastrointestinal adenocarcinoma responds poorly to conventional chemotherapy and has an unfavorable outcome if diagnosed in an advanced stage.35 Therefore, it is vital to identify early prognostic biomarkers and effective therapeutic targets for gastric adenocarcinoma management. A number of epidemiological studies have assessed AURKA expression in gastric cancer.9, 36, 37, 38 For example, in an analysis of 88 human primary gastric tumor specimens, Kamada et al. reported positive staining for AURKA in 41% of samples.9 Similarly, in an analysis of 130 gastric cancer subjects, \u003e50% AURKA positivity was reported in upper gastrointestinal adenocarcinoma.36 In the present study, we analyzed a large cohort of 240 gastric cancer patient specimens and found that AURKA was highly overexpressed in gastric cancer tissues as assessed by immunohistochemistry and western blotting (Figure 1a). Moreover, immunohistochemical analysis showed that AURKA overexpression was correlated with tumor stage, lymph node metastasis and distant metastasis, but not with gender, age, tumor size, tumor site and tumor grading, suggestive of a role of AURKA in gastric cancer progression. In agreement, high AURKA expression was inversely associated with overall survival of gastric cancer patients (Figure 1b) and had been shown to be an independent prognostic factor for gastric cancer (Figure 3c). This was consistent with our previous study in laryngeal squamous cell carcinoma patients that elevated AURKA expression predicted poor overall survival.39 Resistance to DNA-damaging agent-induced apoptosis is a major mechanism of poor chemotherapeutic response. Our study indicated that the limited chemotherapy efficacy might be due to high expression levels of AURKA in gastric cancer. Consistent with this notion, overexpression of AURKA in gastric cells could overcome DNA damage-induced apoptotic cell death (Figure 1d), which again was in agreement with another previous study.32 Recent reports showed that AURKA overexpression was essential for the tumorigenic capacity and drug resistance of breast tumor initiating cells34 as well as chemoresistance in lung cancer cells,40 supporting our study that deregulated overexpression of AURKA in gastric cancer led to clinical chemoresistance.\nAs a member of IAPs, Survivin can confer drug resistance and is correlated with drug refractory tumors.18, 19, 41, 42 Survivin expression is significantly upregulated in gastric cancers compared with the tissues of normal mucosa, atrophic gastritis and intestinal metaplasia, and is negatively associated with OS of patients who received CDDP-based chemotherapy.43 Mechanistically, Survivin is upregulated by upstream factors, such as p34/cdc2-cyclin B1 and Plk1.44, 45 In the present work, we demonstrated that inhibition of AURKA kinase activity by VX-680 or depletion of total AURKA, suppressed Survivin expression, whereas overexpression of AURKA upregulated Survivin in gastric cancer cells (Figures 2a and b). These findings are further corroborated by the finding that AURKA and Survivin co-expressed in gastric cancer patients' specimens (Figure 2d). In addition, we also found that AURKA regulated Survivin expression through suppression of its ubiquitylation and proteasomal degradation (Figure 4). In previous reports, AURKA has been shown to stabilize LIMPK2 in breast cancer46 and N-myc in neuroblastoma26 by inhibiting their ubiquitylation and degradation.\nIn dissecting molecular mechanism that leads to Survivin upregulation in response to AURKA overexpression, we found that Survivin upregulation by AURKA was not regulated at the transcriptional level (Supplementary Figure 2). Previous studies have shown that p53 represses Survivin transcription through promoter hypermethylation.47 In our studies, VX-680 significantly suppressed Survivin protein levels in both the wild-type and p53 knockout MEFs, indicating the regulation of Survivin expression by AURKA was unlikely to be dependent on p53.\nSurvivin stabilization and upregulation by post-translational modifications have been described previously.48, 49 In a mass spectrometry analysis of Survivin co-immunoprecipitates, we found FBXL7 E3 ubiquitin ligase acted as a Survivin-interacting protein. FBXL7 belongs to the leucine-rich repeats containing F-box family of proteins and is a part of SCFs (SKP1-Cul1-F-box) E3 ligase complex. FBXL7 has been shown to induce the ubiquitylation of AURKA during mitosis50 and Survivin in a cell cycle-independent manner.48 FBXL7 is a potential tumor suppressor gene as a previous study found an association between SNPs in FBXL7 and an increased breast cancer risk.51 We showed that in unsynchronized cells, FBXL7 promoted Survivin degradation by proteasomal pathway in our experimental setting, which was in agreement with a previous report,48 while AURKA expression remained unaffected in response to FBXL7 overexpression. Indeed, we found that both the FBXL7 mRNA and protein levels increased in response to AURKA depletion in both AGS and BGC823 cells. Basal levels of FBXL7 mRNA and protein were low in gastric cancer cells and only became detectable after AURKA depletion. This led us to speculate that FBXL7 was a potential tumor suppressor gene and its expression was repressed by AURKA in gastric cancer. We also found the FOXP1 transcription factor to be an AURKA-interacting partner in the regulation of FBXL7 transcription. This finding was in agreement with a previous ChIP-sequencing analysis that FOXP1 bound to the FBXL7 promoter in vivo.52 In concordance, we found that FOXP1 could activate FBXL7 promoter activity, while AURKA repressed the FOXP1-mediated induction in FBXL7 promoter activity. Our In silico analysis showed that FOXP1 was a potential AURKA substrate that led us to speculate that AURKA could post-translationally modify FOXP1 to alter its transcriptional activity. Since AURKA depletion did not affect FOXP1 cytoplasmic/nuclear localization and shuttling, we concluded that AURKA modulated FOXP1 without affecting its (Supplementary Figure 3b and 3c) translocation. Collectively, our data suggested that AURKA negatively regulated FBXL7 expression through modulating the activity of the FOXP1 transcription factor and thereby, restricting Survivin degradation by the FBXL7-ubiquitinylation complex (Figure 8).\nNotably, inhibition of AURKA using the inhibitor VX-680 limited cell proliferation and that VX-680 synergized with the DNA-damaging agent doxorubicin in suppressing cell proliferation in BGC823 cells (Figure 3a). These results are concordant with previous findings that AURKA suppression increased chemosensitivity to docetaxel in both in vitro and in vivo models of esophageal squamous cell carcinoma;53 however, the mechanism underlying the AURKA-mediated chemotherapeutic resistance remains enigmatic. Previous studies have shown that Survivin is upregulated because of its cytoprotective function in response to several anti-cancer agents, including doxorubicin, suggesting that tumor cells enhance Survivin expression to counteract the apoptotic signals induced by chemotherapeutic agents.42, 54 In our work, we found that doxorubicin alone induced Survivin expression, while the combination of VX-680 and doxorubicin induced apoptotic cell death synergistically in the human gastric BGC823 cells, accompanied by a decrease in AURKA activation and Survivin expression (Figure 3b). Collectively, our data suggest that the synergistic cytotoxic effect of AURKA inhibition and doxorubicin is possibly attributable to a convergence of signals that ultimately lead to the downregulation of the expression of the anti-apoptotic protein Survivin. This data proposes the inclusion of AURKA inhibitors as therapeutic agents for gastric cancer management. In conclusion, our data suggest that AURKA limits Survivin ubiquitylation and degradation in gastric cancer and provide a novel therapeutic target to promote the efficacy of DNA-damaging agent in gastric cancer."}