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Alisertib induces cell cycle arrest and autophagy and suppresses epithelial-to-mesenchymal transition involving PI3K/Akt/mTOR and sirtuin 1-mediated signaling pathways in human pancreatic cancer cells

Pancreatic cancer is the most aggressive cancer worldwide with poor response to current therapeutics. Alisertib (ALS), a potent and selective Aurora kinase A inhibitor, exhibits potent anticancer effects in preclinical and clinical studies; however, the effect and underlying mechanism of ALS in the pancreatic cancer treatment remain elusive. This study aimed to examine the effects of ALS on cell growth, autophagy, and epithelial-to-mesenchymal transition (EMT) and to delineate the possible molecular mechanisms in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that ALS exerted potent cell growth inhibitory, pro-autophagic, and EMT-suppressing effects in PANC-1 and BxPC-3 cells. ALS remarkably arrested PANC-1 and BxPC-3 cells in G2/M phase via regulating the expression of cyclin-dependent kinases 1 and 2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. ALS concentration-dependently induced autophagy in PANC-1 and BxPC-3 cells, which may be attributed to the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal-regulated kinases 1 and 2 (Erk1/2) but activation of 5′-AMP-dependent kinase signaling pathways. ALS significantly inhibited EMT in PANC-1 and BxPC-3 cells with an increase in the expression of E-cadherin and a decrease in N-cadherin. In addition, ALS suppressed the expression of sirtuin 1 (Sirt1) and pre-B cell colony-enhancing factor/visfatin in both cell lines with a rise in the level of acetylated p53. These findings show that ALS induces cell cycle arrest and promotes autophagic cell death but inhibits EMT in pancreatic cancer cells with the involvement of PI3K/Akt/mTOR, p38 MAPK, Erk1/2, and Sirt1-mediated signaling pathways. Taken together, ALS may represent a promising anticancer drug for pancreatic cancer treatment. More studies are warranted to investigate other molecular targets and mechanisms and verify the efficacy and safety of ALS in the treatment of pancreatic cancer.


Introduction
Pancreatic cancer is the most aggressive cancer worldwide with a 5-year survival rate of less than 6%. 1,2 Pancreatic cancer is the 12th most common cancer, and there were 338,000 cases diagnosed with pancreatic cancer in 2012. 1,2 In US, pancreatic cancer accounts for approximately 3% of all cancers and approximately 7% of cancer-related deaths. 1,2 In 2013, there were 45,220 cases diagnosed with pancreatic cancer, and there will be 46,420 new cases of pancreatic cancer in 2014. 2 In People's Republic of China, pancreatic cancer is the sixth leading cause of cancer-related death with a A/B/C (AURKA/B/C). Aurora kinases have a pivotal role in the regulation of mitosis, and the expression and activity of Aurora kinases are tightly regulated. 15 Abnormalities in the expression and/or activity of Aurora kinases can cause genetic instability, aneuploidy, turmorigenesis, and cell death. 15 AURKA is a key cell cycle regulator critical for mitotic events. 16 Aberrant expression and activity of AURKA often occur in various malignancies, including the bladder, breast, colon, liver, ovaries, pancreas, stomach, and esophagus cancer. 17 Thus, targeting AURKA has been proposed to be a very promising strategy for cancer treatment and a number of AURKA inhibitors have been developed. 18 Alisertib (ALS, MLN8237, Figure 1A), an investigational small-molecule inhibitor, selectively inhibits AURKA. 19 Currently, ALS is under various Phase I and Phase II clinical trials for advanced solid tumors and hematologic malignancies, although the underlying mechanism is not fully understood. 20 To date, there is only one report on the anticancer effect of ALS in the treatment of pancreatic ductal adenocarcinoma. 21 The effect of ALS on anchorageindependent growth in pancreatic ductal adenocarcinoma cell lines and growth of patient-derived xenografts was variable. RalA Ser194 phosphorylation levels in pancreatic ductal adenocarcinoma cell lines or patient-derived xenografted tumors did not correlate with the response to ALS. Notably, Ki67 (the cell proliferation-related antigen) was identified as a possible early biomarker for predicting the response to ALS in patient-derived xenografts. 21 These findings indicate that ALS is an effective anticancer drug for a subgroup of patients with pancreatic ductal adenocarcinoma via signaling pathways independent of RalA Ser194 phosphorylation. However, this study did not investigate how ALS kills pancreatic cancer cells via inhibition of AURKA and why RalA Ser194 phosphorylation is not required for the ALS's killing effect. There are only few data on the anticancer effect and underlying mechanisms of ALS in the treatment of pancreatic cancer. In this regard, we hypothesized that ALS inhibited the growth of pancreatic cancer cells via phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt)/mTOR and Sirt1-mediated signaling pathways. To test this hypothesis, we investigated the anticancer effects and possible mechanisms of ALS in human pancreatic cancer PANC-1 and BxPC-3 cells with a focus on the effects of ALS on cell cycle, autophagy, EMT, and redox homeostasis.

cell cycle distribution analysis
The effect of ALS on the cell cycle distribution of PANC-1 and BxPC-3 cells was determined using flow cytometry as described previously. 22 Briefly, PANC-1 and BxPC-3 cells were seeded into 60 mm Petri dishes. After cells were seeded for 24 hours, the cells reached ~75% confluence and were then treated with ALS at concentrations of 0.

Quantification of cellular autophagy
To examine the effect of ALS on autophagy in PANC-1 and BxPC-3 cells, cellular autophagy was first detected using flow cytometry as described previously. 23 Briefly, PANC-1 and BxPC-3 cells were seeded into 60 mm Petri dishes. After cells were seeded for 24 hours, the cells reached ~75% confluence and were then treated with fresh medium alone and ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours. Following the ALS treatment, cells were detached and resuspended in 250 µL of phenol red-free culture medium containing 5% FBS. Following that, 250 µL of the diluted Cyto-ID ® Green stain solution was added to each sample. Cells were incubated for 30 minutes at 37°C in the dark and then collected by centrifugation at 250× g. The cell pellet was washed with 1× assay buffer given in Cyto-ID ® Autophagy detection kit and resuspended in 500 µL fresh 1× assay buffer. Cells were analyzed using the green (FL1) channel of a flow cytometer.

Confocal fluorescence microscopy examination
The cellular autophagy level was further detected by examining using confocal fluorescence microscopy. Briefly, PANC-1 and BxPC-3 cells were seeded into eight-well chamber slide. The cells were treated with ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours. After the ALS treatment, the cells were washed with 1× assay buffer given in Cyto-ID ® Autophagy detection kit, followed by incubation with 100 µL of microscopy dual detection reagent for 30 minutes at 37°C in the dark. After the incubation, the cells were washed with 1× assay buffer to remove detection reagent and then the cells were examined using a Leica TCS SP2 laser scanning confocal microscopy (Leica Microsystems, Wetzlar, Germany) using a standard FITC filter set for imaging the autophagic signal at wavelengths of 405/488 nm.

Western blotting analysis
To examine the effect of ALS on the expression of various cellular proteins, the Western blotting assays were performed as described previously. 23 The PANC-1 and BxPC-3 cells were incubated with ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours. After ALS treatment, cells were washed with precold PBS and lysed with the RIPA buffer containing the protease inhibitor and phosphatase inhibitor cocktails. Protein concentrations were measured by Pierce BCA protein assay kit. Equal amount of protein sample (20 µg) was electrophoresed on 7% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis mini-gel after thermal denaturation for 5 minutes at 95°C. Following that, proteins were transferred onto methonal-activated PVDF membrane at 100 V for 2 hours at 4°C. Subsequently, membranes were blocked with 5% skim milk and probed with indicated primary antibody overnight at 4°C and then blotted with respective secondary antibody. Visualization was performed using Bio-Rad system. The blots were analyzed using ImageLab 3.0 (Bio-Rad) and protein level was normalized to the matching densitometric value of β-actin.

statistical analysis
Data are expressed as the mean ± standard deviation. Multiple comparisons were evaluated by one-way analysis of variance followed by Tukey's multiple comparison. A value of P0.05 was considered statistically significant. Assays were performed at least three times independently.

als regulates cell cycle distribution of Panc-1 and BxPc-3 cells
We next examined the effect of ALS on cell cycle distribution in PANC-1 and BxPC-3 cells using flow cytometry. ALS significantly induced G 2 /M phase arrest in both cell lines ( Figure 3). Treatment of cells with ALS concentrationdependently increased the percentage of PANC-1 and BxPC-3 cells in G 2 /M phase ( Figure 3A and B). The percentage of PANC-1 in G 2 /M phase was increased from 30.3% at basal level to 46.0%, 77.5%, and 80.9% when treated with ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours, respectively, and the percentage of BxPC-3 cells in G 2 /M phase was raised from 29.9% at basal level to 41.2%, 84.9%, and 85.0% when treated with ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours, respectively ( Figure 3A and B  Figure 3C and D). The results show that ALS treatment remarkably induces PANC-1 and BxPC-3 cells arrest in G 2 /M phase, which may be attributable, at least in part, to the inhibition of the AURKA activation.

als modulates key cell cycle regulators in Panc-1 and BxPc-3 cells
Since we have observed the inducing effect of ALS on cell cycle arrest in PANC-1 and BxPC-3 cells, we further examined the effect of ALS on the expression of cell cycle regulators, including CDK1/CDC2, CDK2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53 in PANC-1 and BxPC-3 cells using Western blotting assay. Incubation of PANC-1 cells with ALS at 0.1 µM, 1 µM, and 5 µM resulted in varying alterations in the expression levels of cell cycle regulators (Figure 4). CDC2 and cyclin B1 are two key regulators for G 2 -to-M phase transition. 24 The expression of CDC2, cyclin B1, and CDK2 was significantly suppressed in both cell lines with the treatment of ALS at concentrations of 0.1 µM, 1 µM, and 5 µM for 24 hours. There was a 18.3%, 26.6%, and 51.1% reduction in the expression level of CDC2, 7.50%, 12.9%, and 48.4% decline in the expression level of cyclin B1, and 9.60%, 17.6%, and 23.3% decrease in the expression level of CDK2 in PANC-1 cells when treated with ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours, respectively ( Figure 4A and B). Similarly, there was a 18.3%, 26.6%, and 51.1% reduction in the expression level of CDC2, 12.4%, 29.6%, and 60.9% decrease in the expression level of cyclin B1, and 12.0%, 21.3%, and 31.7% decline in the expression level of CDK2 in BxPC-3 cells when treated with ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours, respectively ( Figure 4A and B). For cyclin D1, there was a 16.3%, 22.7%, and 30.8% reduction in PANC-1 cells, but there was a 1.25fold, 1.32-fold, and 1.41-fold increase in BxPC-3 cells with the treatment of 0.1 µM, 1 µM, and 5 µM ALS, respectively ( Figure 4A and B). Furthermore, p21 Waf1/Cip1, p27 Kip1, and p53 play an important role in the regulation of cell cycle. The tumor suppressor protein p21 Waf1/Cip1 acts as an inhibitor of cell cycle progression, and it serves to inhibit kinase activity and block progression through G 1 /S in association with CDK2 complexes. 25 Phosphorylated p53 upregulates p21 Waf1/Cip1 transcription via a p53-responsive element, and activation of p53 leads to either cell cycle arrest and DNA repair or apoptosis. The expression of p21 Waf1/Cip1, p27 Kip1, and p53 was significantly increased in both cell lines with the treatment of ALS at concentration of 0.

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Wang et al 5 µM led to a 1.6-fold, 1.8-fold, and 2.4-fold increase in the expression of p21 Waf1/Cip1, 1.1-fold, 1.6-fold, and 1.8-fold rise in the expression of p27 Kip1, and 1.2-fold, 1.3-fold, and 1.7-fold elevation in the expression of p53, respectively ( Figure 4A and B). There was a similar regulatory effect of ALS on the expression levels of p21 Waf1/Cip1, p27 Kip1, and p53 in BxPC-3 cells. Treating cells with ALS at 0.1 µM, 1 µM, and 5 µM increased the expression level of p21 Waf1/Cip1 by 1.3-fold, 1.5-fold, and 1.9-fold, elevated the expression level of p27 Kip1 by 1.2-fold, 1.3-fold, and 1.4-fold, and increased the expression level of p53 by 5.6fold, 6.6-fold, and 7.1-fold compared to the control cells, respectively ( Figure 4A and B). These results indicate that ALS significantly downregulates the expression of CDC2, cyclin B1, and CDK2 but upregulates p21 Waf1/Cip1, p27 Kip1, and p53 in both PANC-1 and BxPC-3 cells, which contributes, at least in part, to the observed cell cycle arrest phenomenon in both cell lines.

als induces autophagy in Panc-1 and BxPc-3 cells
Autophagy is one of the predominant programmed cell death routes, and its role in cancer cell survival and death is controversial. 10,26 Recently, it has been reported that induction of autophagy is an emerging strategy for the treatment of pancreatic cancer. 26,27 As such, we examined the effect of ALS on autophagy in PANC-1 and BxPC-3 cells using flow cytometry and confocal microscopy. As shown in Figure 5, the percentage of autophagic cells at basal level is 3.7% and 1.5% for PANC-1 and BxPC-3 cells, respectively. Treating cells with ALS significantly increased autophagy in a concentration-dependent manner. In PANC-1 cells, there was a 2.0-fold, 3.1-fold, and 4.3-fold increase in autophagy when treated with 0.1 µM, 1 µM, and 5 µM ALS for 24 hours compared to the control cells ( Figure 5A and B). In BxPC-3 cells, incubation of cells with 1 µM and 5 µM ALS for 24 hours gave a 5.2-fold and 6.1-fold rise in the percentage of autophagic cells, respectively ( Figure 5A and B). Treating BxPC-3 cells with 0.1 µM ALS for 24 hours did not significantly induce autophagy. We further examined the autophagyinducing effect of ALS in PANC-1 and BxPC-3 cells using confocal microscopy. As shown in Figure 5C and D, ALS treatment results in a significant increase in autophagy in PANC-1 and BxPC-3 cells, compared to the control. There was a 1.1-fold and 1.1-fold increase in the autophagic death of PANC-1 cells when treated with ALS at 1 µM and 5 µM for 24 hours, respectively. In BxPC-3 cells, there was a 1.2fold, 1.2-fold, and 1.4-fold increase in autophagy when treated with 0.1 µM, 1 µM, and 5 µM ALS for 24 hours ( Figure 5C and D). Taken together, these results demonstrate that ALS induces autophagy in both PANC-1 and BxPC-3 cells.

als suppresses Pi3K/akt/mTOr axis in Panc-1 and BxPc-3 cells
Since we have observed the autophagy-inducing effect of ALS in PANC-1 and BxPC-3 cells, we further investigated the possible mechanisms for the autophagy-inducing effect of ALS. First, we examined the phosphorylation of PI3K, an upstream signaling molecule of Akt/mTOR pathway with an important role in the regulation of cell proliferation and autophagy, 5,10,28 which catalyzes the formation of phosphatidylinositol-3,4,5-triphosphate, initiating the activation of a number of signaling pathways related to cell proliferation, cell migration, cell survival, and cell death. 29 As shown in Figures 6-8, S1A-P, and S2A-P, ALS treatment causes significant alterations in the expression and phosphorylation levels of key functional proteins regulating autophagy signaling pathway. There was a concentration-dependent decrease in the phosphorylation levels of PI3K, Akt, and mTOR in PANC-1 and BxPC-3 cells with ALS treatment at 0.1 µM, 1 µM, and 5 µM. There was a 18.9%, 37.3%, and 57.8% decline in the ratio of p-PI3K over PI3K, 52.6%, 73.4%, and 71.1% decrease in the ratio of p-Akt over Akt, and 18.1%, 29.8%, and 47.7% reduction in the ratio of p-mTOR over mTOR in PANC-1 cells when treated with ALS at 0.1 µM, 1 µM, and 5 µM, respectively (Figures 6 and 7). Similarly, there was a 44.1%, 50.3%, and 58.8% decrease in the ratio of p-PI3K over PI3K, 39.1%, 43.0%, and 53.7% decline in the ratio of p-Akt over Akt, and 29.6%, 44.2%, and 53.2% reduction in the ratio of p-mTOR over mTOR in BxPC-3 cells treated with ALS at 0.1 µM, 1 µM, and 5 µM, respectively (Figures 6 and 8). Furthermore, there was a concentration-dependent increase in the phosphorylation level of Erk1 and Erk2, after the cells were incubated with ALS. In comparison to the control cells, incubation of PANC-1 cells with ALS at 0.1 µM, 1 µM, and 5 µM led to a 1.5-fold, 1.9-fold, and 2.2fold increase in the ratio of p-Erk1 over Erk1 and 1.5-fold, 1.7-fold, and 2.0-fold increase in the ratio of p-Erk2 over Erk2, respectively (Figures 6 and 7). There was a similar regulatory effect of ALS on the phosphorylation of Erk1 and Erk2 in BxPC-3 cells. Treatment of cells with ALS at 0.1 µM, 1 µM, and 5 µM increased the ratio of p-Erk1 over Erk1 1.4-fold, 2.0-fold, and 2.5-fold and elevated the ratio of p-Erk2 over Erk2 1.9-fold, 3.4-fold, and 4.6-fold compared to the control cells, respectively (Figures 6 and 8).

als inhibits eMT phenotype in Panc-1 and BxPc-3 cells
EMT is a critical process playing an important role in cancer invasion and metastasis, with a reduction in the expression of cell adhesion molecules. 11,12,32 Therefore, the effect of ALS on the expression of key EMT markers and regulators was examined in PANC-1 and BxPC-3 cells. ALS treatment results in differential effects on the expression level of EMT-related proteins (Figures 9-11). Incubation of PANC-1 cells with ALS at 0.1 µM, 1 µM, and 5 µM increased by 1.2-fold, 1.4-fold, and 1.5-fold the expression level of E-cadherin but decreased by 11.0%, 18.3%, and 24.2% the expression level of N-cadherin, respectively. Besides, treating PANC-1 cells with 5 µM ALS significantly decreased the expression level of slug, snail, and TCF-8 by 34.5%, 46.8%, and 31.1%, respectively, whereas, there was a 1.6-fold, 1.5-fold, and 1.5-fold increase in the expression level of Zona occludin-1 (ZO-1), β-catenin, and claudin 1 when PANC-1 cells were treated with 5 µM ALS, respectively (Figures 9-11). There was no significant change in the expression level of vimentin when PANC-1 cells were treated with ALS at 0.1 µM, 1 µM, and 5 µM.
As shown in Figures 9-11, ALS exhibits a similar modulating effect on the expression of the key EMT markers and regulators in BxPC-3 cells. The expression level of E-cadherin was increased 1.1-fold, 2.4-fold, and 2.5-fold, but the expression level of N-cadherin was reduced 5.3%, 32.7%, and 58.4%, when cells were treated with ALS at 0.1 µM, 1 µM, and 5 µM, respectively. There was a 24.4%, 32.5%, and 37.8% reduction in the expression level of slug, 24.1%, 26.3%, and 35.9% decrease in the expression level of snail, and 28.2%, 58.8%, and 73.7% decline in the expression level of TCF-8 in BxPC-3 cells when treated with ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours, respectively (Figures 9-11). In addition, incubation of BxPC-3 cells with ALS at 0.1 µM, 1 µM, and 5 µM increased 1.2-fold, 1.3-fold, 1.4-fold and 1.2-fold, 2.1-fold, and 3.0-fold the expression level of β-catenin and vimentin compared to the control, respectively. Treating BxPC-3 cells with 5 µM ALS increased 1.8-fold the expression level of claudin 1. Taken together, the results show that ALS inhibits the expression of key functional proteins regulating EMT, contributing to the anticancer effect of ALS in PANC-1 and BxPC-3 cells.

als suppresses the expression of PBeF and sirt1 in Panc-1 and BxPc-3 cells
Recently, accumulating evidence shows that Sirt1-mediated signaling pathway plays an important role in the regulation of cellular autophagy and cancer development. 33,34 PBEF, a rate limiting enzyme responsible for the NAD + biosynthesis pathway, plays an essential role in the regulation of Sirt1 activation. 35 Recently, studies have shown that Sirt1 deacetylates both histone and nonhistone proteins, such as p53, and deacetylation of p53 and inhibition of p53-regulated cell death indicate that Sirt1 is a negative regulator of p53. 36 We speculate that ALS may regulate expression of PBEF and Sirt1 and acetylation of p53 (Ac-p53) in PANC-1 and BxPC-3 cells. Thus, we examined the effect of ALS on the expression level of PBEF, Sirt1, and Ac-p53 in both cell lines. There was a significant decrease in the expression level of PBEF and Sirt1 in PANC-1 and BxPC-3 cells when treated with ALS ( Figure 12A and B). There was a 13.1%, 23.1%, and 31.1% reduction in the expression level of PBEF, and 8.4%, 16.3%, and 27.8% decline in the expression of Sirt1 in PANC-1 cells when treated with ALS at 0.1 µM, 1 µM, and 5 µM, respectively ( Figure 12A and B). Similarly, there was a 15.2%, 24.9%, and 25.7% decrease in the expression of PBEF, and 12.8%, 24.6%, and 36.0% decline in the expression of Sirt1 in BxPC-3 cells when treated with ALS at 0.1 µM, 1 µM, and 5 µM, respectively ( Figure 12A and B). In contrast, there was a significant increase in the level of Ac-p53 in PANC-1 and BxPC-3 cells when treated with ALS ( Figure 12A and B). Treatment of PANC-1 cells with ALS at 0.1 µM, 1 µM, and 5 µM resulted in 1.8-fold, 2.0-fold, and 1.7-fold increase in the level of Ac-p53, and 1.4-fold, 1.4-fold, and 1.6-fold rise in the ratio of Ac-p53 over p53 compared to the control cells, respectively. In BxPC-3 cells, incubation of cells with ALS at 0.1 µM, 1 µM, and 5 µM led to a 1.2-fold, 1.2-fold, and 1.3-fold increase in the level of Ac-p53, and 1.2-fold, 1.3-fold, and 1.4-fold elevation in the ratio of Ac-p53 over p53 compared to the control, respectively ( Figure 12A

and BxPc-3 cells
Nrf2 is a critical nuclear transcript factor controlling a wide range of antioxidant response element-dependent genes to regulate the physiological and pathophysiological outcomes of oxidant exposure, including cell death and EMT. 37-39 Thus, we further determined the effects of ALS on its expression. As shown in Figure 13A and B, there was a concentrationdependent reduction in the expression of Nrf2. Incubation of PANC-1 cells with ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours reduced the expression level of Nrf2 by 25.1%, 32.8%, and 41.2%, respectively. Similarly, treatment with ALS at 0.1 µM, 1 µM, and 5 µM for 24 hours significantly suppressed the expression level of Nrf2 by 9.9%, 25.9%, and 38.2% in BxPC-3 cells, respectively ( Figure 13A and B). These results suggested that the anticancer effect of ALS in PANC-1 and BxPC-3 cells may be ascribed, at least in part, to its regulatory effect on Nrf2-mediated signaling pathway.

Discussion
The pancreatic cancer is the most aggressive cancer with a very poor prognosis in patients with this disease, and the treatment of pancreatic cancer remains a major challenge. Therefore, there is an urgent need for new therapeutic strategies for pancreatic cancer treatment. Recently, AURKA has become a promising therapeutic target for cancer treatment, 20 which plays an important role in centrosome maturation and separation, bipolar spindle assembly, and mitotic entry. Abnormalities in the expression and activities of AURKA have been implicated in the initiation, development, and progression of various cancers, including the breast, colon, pancreatic, ovarian, and gastric cancers. 40 With accumulating evidence showing the role of the Aurora kinases in the pathogenesis of cancer, AURKA becomes a more important therapeutic target in the treatment of cancer. It has been reported that there is a correlation of the expression and gene amplification of AURKA with tumor proliferation rates and prognostic markers. 41 Furthermore, increasing studies show that AURKA can induce chemotherapeutic resistance and regulate several key signaling pathways related to cell cycle, autophagy, and EMT in cancer cells, 16,41,42 suggesting its pivotal role in cancer cell signaling. 43 Currently, there are a number of AURKA inhibitors that have been developed and are being tested in different Phase I and Phase II trials for cancer treatment. 18 In the present study, we have investigated the effect of ALS, a potent and selective inhibitor of AURKA, on pancreatic cancer cells. Our findings show that ALS exhibits potent inhibitory effects on pancreatic cancer cell growth and cell cycle progression. ALS also exerts a potent pancreatic cancer cell-killing effect via autophagic cell death with the involvement of PI3K/Akt/ mTOR, p38 MAPK, AMPK, and Sirt1-mediated signaling pathways. Moreover, ALS shows a substantial suppressing effect on EMT in pancreatic cancer cells.  AURKA exerts an important role in the regulation of chromosome assembly and segregation during mitosis. 44 It regulates the G 2 -to-M transition and inactivation of AURKA results in G 2 /M arrest. 45 In the present study, our findings clearly showed that ALS treatment remarkably decreased the ratio of p-AURKA over AURKA in PANC-1 and BxPC-3 cells, suggesting that ALS may induce cell cycle arrest in G 2 /M phase. Indeed, we have observed the inducing effect on cell cycle arrest in G 2 /M phase in PANC-1 and BxPC-3 cells treated with ALS. Notably, it has been reported that the regulatory effect of Aurora kinases on cell cycle distribution is associated with p53. 46,47 Activation of AURKA instigates Mdm2-mediated destabilization and inhibition of p53, 46 and AURKB phosphorylates and induces degradation of p53. 47 In our study, the phosphorylation level of p53 was significantly decreased, but the total expression level of p53 was remarkably increased in PANC-1 and BxPC-3 cells when treated with ALS, which in turn results in a reduction in the ratio of p-p53 over p53. Furthermore, our findings show that ALS treatment can significantly increase the expression levels of p21 Waf1/Cip1 that is a tumor suppressor protein and serves as an inhibitor of cell cycle progression via the inhibition of CDK activity. 25 Therefore, increased expression of p21 Waf1/Cip1 contributes to cell cycle arrest in PANC-1 and BxPC-3 cells. Moreover, treating PANC-1 and BxPC-3 cells with ALS also results in a significant increase in the expression level of p27 Kip1 that is a member of the Cip/ Kip family of CDK inhibitors, inducing cell cycle arrest via its inhibitory binding to CDK2/cyclin E and other CDK/ cyclin complexes. 48 Taken together, inducing effect of ALS on cell cycle arrest can prevent cell proliferation by modulation of a number of cell cycle regulators in PANC-1 and BxPC-3 cells, which can eventually lead to pancreatic tumor regression. [49][50][51] Recently, the role of autophagy in pancreatic cancer cell death is controversial. 26 Increasing evidence shows that targeting autophagy is a promising strategy for pancreatic cancer treatment via regulation of autophagy-related key functional proteins and signaling pathways. 9,26,52 Autophagy is a complicated process executed through multiple steps from intracellular membrane/vesicle reorganization to form double-membraned autophagosomes that fuse with lysosome to form autophagolysosomes, which eventually degrade the contents via acidic lysosomal hydrolases. 5,10,53 Increasing evidence demonstrates that the PI3K/Akt/mTOR signaling pathway is a central signaling pathway regulating cellular autophagy and that targeting this signaling pathway has been proposed to be a promising strategy for cancer treatment. [54][55][56] In the present study, our findings show that ALS exhibits a significant inhibitory effect on PI3K/Akt/mTOR signaling pathway in both PANC-1 and BxPC-3 cells, contributing to promoting effect of ALS on autophagy in both PANC-1 and BxPC-3 cells. It suggests that ALS may be a promising agent, which can be used to induce autophagic cell death for pancreatic cancer treatment. Notably, AMPK and p38 MAPK are two upstream signaling molecules, which can regulate cellular autophagy via the activation of p53, inhibition of mTOR, and other related signaling pathways. 5

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Wang et al our findings show that ALS significantly inactivates p38 MAPK signaling, which is responsive to a variety of stress stimuli and regulates autophagy. 5,54 Our previous study also showed that activation of AMPK and inactivation of p38 MAPK signaling contribute to the autophagic cell death in PANC-1 and BxPC-3 cells. 57 Collectively, AMPK and p38 MAPK contribute to the mTOR-mediated autophagy in PANC-1 and BxPC-3 cells.
Increasing number of studies have shown that modulation of EMT may represent an emerging therapeutic strategy for the treatment of pancreatic cancer, 12,32 due to the role in initiation, development, progression, invasion, migration, and metastasis and drug resistance in pancreatic cancer. 12,14,58,59 Thus, the key functional molecules that regulate EMT may serve as a therapeutic target. Cadherins are a superfamily of transmembrane glycoproteins regulating calcium-dependent cell-cell adhesion, including N-, P-, R-, B-, and E-cadherins. 11,12 E-cadherin acts as a suppressor of invasion and migration of many epithelial cancers, 11,12,32 and E-cadherin-mediated calcium-dependent cell-cell adhesion is negatively regulated by snail/slug. Inhibition of snail/slug expression can suppress the EMT progression in pancreatic cancer. 60 Our findings show that ALS treatment significantly increases the expression of E-cadherin but suppressed the expression of snail and slug in PANC-1 and BxPC-3 cells. It suggests that ALS can inhibit EMT, reducing drug resistance and suppressing pancreatic cancer progression. Moreover, it has been reported that activation of Erk1/2 signaling pathway is correlated with EMT progress in pancreatic cancer. 60 We have observed that ALS inactivates Erk1/2 signaling pathways with a marked inhibition on the phosphorylation of Erk1/2 in PANC-1 and BxPC-3 cells, which also may contribute to the inhibitory effect of ALS on EMT. Therefore, ALS may act as a putative EMT-targeting agent that will be clinically helpful to reduce the morbidity and mortality of pancreatic cancer.
There is emerging evidence showing that Sirt1 promotes pancreatic cancer cell viability and instigates drug resistance in pancreatic cancer, 61,62 indicating that inhibition on the activity of Sirt1 and Sirt1-mediated signaling pathway may be a novel strategy for pancreatic cancer treatment via reverse of the chemoresistance and promotion of cancer cell death in pancreatic cancer. Sirt1, an NAD + -dependent DNA repair enzyme originally discovered in yeast (Sir2), plays an important role in the regulation of autophagy and EMT via deacetylation of a number of functional proteins, such as p53, claudin 1, TCF-8, and FoxOs. 36,63-65 PBEF/ visfatin, a rate-limiting enzyme in the NAD + biosynthesis salvage pathway, is responsible for the catalysis of nicotinamide with 5-phosphoribosyl-1-pyrophosphate, producing nicotinamide mononucleotide. 66 PBEF/visfatin is important in NAD + biosynthesis which is essential for Sirt1-mediated signaling pathway. 66 In the present study, ALS decreased the expression levels of PBEF/visfatin and Sirt1 in PANC-1 and BxPC-3 cells. Notably, there was a significant rise in the level of Ac-p53 and the ratio of Ac-p53 over p-p53 in both cells treated with ALS ( Figure 12). Taken together, the results show that ALS-induced autophagy and EMT inhibition in pancreatic cancer cells may be attributed, partially, to the Sirt1-mediated pathway. In addition, it has been reported that enhanced Nrf2 activity facilitates the tumorigenesis in pancreatic cancer 67 and that overexpression of Nrf2 promotes pancreatic cancer cell viability and drug resistance. 68 Therefore, manipulation of the expression level and/or activity of Nrf2 may represent a potential strategy to reduce pancreatic tumor growth and to increase sensitivity to therapeutics. In our study, we also found that ALS significantly decreased the expression of Nrf2 in PANC-1and BxPC-3cells, suggesting that inhibition of Nrf2 expression contributes, at least in part, to the anticancer effect of ALS in pancreatic cancer treatment.
In summary, we have observed the anticancer effect of ALS and investigated the potential molecular mechanisms of its cancer cell-killing effect in human pancreatic cancer PANC-1 and BxPC-3 cells. The mechanisms of action of ALS were mainly ascribed to cell cycle arrest, autophagy induction, and EMT inhibition, and their related signaling pathways. ALS induced the inhibition of PI3K/Akt/mTOR, p38 MAPK, and Erk1/2 signaling pathways, but activation of AMPK signaling pathway contributes to the autophagyinducing and EMT-suppressing effects of ALS in PANC-1 and BxPC-3 cells. Collectively, ALS may represent a promising anticancer drug that can be used for pancreatic cancer treatment. More studies are warranted to reveal other potential targets and potential mechanisms of ALS in the treatment of pancreatic cancer. Abbreviations: als, alisertib; Pi3K, phosphatidylinositol 3-kinase; aMPK, 5′-aMP-dependent kinase; MaPK, mitogen-activated protein kinase; akt, protein kinase B; mTOr, mammalian target of rapamycin; erk, extracellular signal-regulated kinase; lc3, light chain 3; sD, standard deviation; anOVa, analysis of variance.

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