Cytoplasmic APE1 promotes lung cancer aggressiveness and cisplatin resistance via the COX-2/Akt/β-catenin signaling pathway

We found that cytoplasmic APE1 expression was lower in cisplatin sensitive cells than in cisplatin-resistant cells, and the upregulation of cytoplasmic APE1 signicantly reduced cisplatin sensitivity in lung cancer cells. Gain-of-function studies demonstrated that cytoplasmic APE1 promoted lung cancer cell proliferation, migration and invasion in vitro and tumor growth in vivo, which were inhibited after cisplatin treatment. In patient samples, cytoplasmic APE1 in lung cancer tissues was an independent indicator of overall survive (OS) for lung cancer patients (P < 0.001). Mechanistic studies revealed that cytoplasmic APE1 promotes lung cancer malignancy by activating the COX-2/Akt/β-catenin pathway. Furthermore, the mutation of the APE1-C65 site caused upregulation of cytoplasmic APE1 and inhibited cell growth, migration and invasion of lung cancer cells.


Background
Lung cancer is the most common cancer in the world, and cisplatin-based chemotherapy is frequently used in clinical treatments. However, resistance to cisplatin is common during the treatment of lung cancer, leading to low overall survival (OS) and a low objective response rate (ORR) to cisplatin in lung cancer. Therefore, further investigation into the mechanism of cisplatin resistance is urgently warranted.
Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional enzyme that acts as a key AP endonuclease during base excision repair. APE1 redox activity maintains the activity of transcription factors [1,2]. APE1 contains a subcellular localization signal; the amino-acid sequence of human APE1 has an identi ed consensus nuclear localization signal (NLS) within its N-terminus, which has been con rmed to function in subcellular targeting [3]. The current data suggest a model in which unmodi ed, full-length APE1 is mainly targeted to the nucleus via its N-terminal elements [3]. More importantly, APE1 is widely overexpressed in most tumor tissues, and APE1 overexpression is associated with poor prognosis, chemoresistance or radioresistance, and increased angiogenesis [4][5][6]. In this study, cytoplasmic APE1 may mediate resistance to cisplatin in lung cancer, and upregulation of cytoplasmic APE1 induced by plasmid transfection increased resistance to cisplatin.
Here, we propose that cytoplasmic APE1 causes COX-2 mediated cisplatin resistance in lung cancer and that cytoplasmic APE1 expression is signi cantly higher in lung cancer patients who have poor OS after cisplatin-based chemotherapy. Cytoplasmic APE1 regulates cancer traits in lung cancer cells, including proliferation, metastasis and invasion. In addition, cytoplasmic APE1 overexpression activates the Akt/βcatenin signaling pathway by upregulating COX-2 in lung cancer cells.

Cell culture
Calu-1 and A549 cell lines were from the American Type Culture Collection (ATCC, USA). The cDDPresistant A549 (A549-cDDP) cell line was kindly provided by Xinqiao Hospital, Army Medical University.
A549-cDDP cells screening procedure: A549 cells growing in logarithmic phase was rstly treated with a concentration of 0.1 µM cisplatin for 48 hours. The cells were subsequently incubated with normal medium until reached to logarithmic phase again. This process was repeatedly conducted with increasing cisplatin concentration by 0.1 µM very cycle. Finally, these cells grew well in cisplatin at a concentration of 4 µM. Calu-1 and A549 cells were cultured in DMEM medium containing 10% FBS and 37℃ humidi ed incubator with 5% CO2. Transfection shRNA-APE1 (shAPE1: the 33 amino-acid N-terminal truncated form, NLS mutation of APE1) and shRNAcon (shCON, the full-length form) plasmid [7], shRNA-C65s (shC65S: C65 site mutation of APE1) and shRNA-con (shCON) plasmid [8], and siRNA-COX-2 (siCOX-2) and siRNA-con (siCON) plasmid were purchased from Genechem Corporation (Shanghai, China). Using the Silencer siRNA construction kit (Qiagen, Venlo, The Netherlands), the cells were performed with the RNAiFect reagents (The Netherlands) to ensure the stability of small RNAi transfection [9].

In vivo experiment
Female BALB/C nude mice were from the Daping Hospital (China), the mice were injected A549 cells that were stably transfected with either a plasmid containing APE1 having a NSL mutation (shAPE1) or the vehicle (shCON). Cisplatin (cDDP) was delivered via intraperitoneal injection for 3 weeks. Measuring tumor diameter and calculating tumor volume was as follows: ab2/2 mm 3 (a = length; b = width), where is the length and is the width of the tumor. Mice were randomized: shCON (PBS), shAPE1, and a combination of shAPE1 + cDDP [10].
Immuno uorescence (IF) and Immunohistochemistry (IHC) analysis For IF analysis, collected cell were permeabilized, blocked, and incubated with the rst antibody. Then, the cells were ooded and stained with DAPI. Zeiss microscope performed transmission electron microscopy analysis (Carl Zeiss GmbH, Oberkochen, Germany). Images were captured using a confocal microscope (40 × magni cation), data are observed from more than three microscopic elds of view per.
For IHC analysis, the slides were baked for 1 hour at 60℃, soaked with xylene for 30 minutes, soaked in different concentrations of alcohol for 5 seconds and were immersed in a pressure cooker containing citric acid buffer and heated to air jet for 2 minutes. The slides were incubated with PARP1 and p62 monoclonal antibody (1:100 and 1:50 dilution), and then with secondary antibody (1:50 dilution).
Cytoplasmic PARP1 and p62 staining score was performed. Weak staining and < 50% of stained cells were de ned as low expression [11].

Patients
We selected 124 patients with unresectable, locally advanced or metastatic lung cancer (table S1); the enrolled patients were treated between July 2006 and July 2015 at the Daping Hospital of the Army Medical University of China. Tissue samples of lung cancer patients before cisplatin basedchemotherapy were collected, and were evaluated the clinic effect of cytoplasmic APE1 and COX-2.

Statistical analysis
Kaplan-Meier method analyzed the OS and PFS, statistical analysis was analyzed using SPSS 20.0.0 (USA). Two-sided P-values < 0.05 were considered signi cant.

Overexpression of cytoplasmic APE1 induced cisplatin resistance in lung cancer
To explore the mechanism of cisplatin resistance, we constructed and identi ed cisplatin resistant A549 cells (A549-cDDP) and cisplatin sensitive A549 cells (A549-CON) (Fig. 1A). We found that both cytoplasmic and total APE1 expression levels were higher in the cisplatin-resistant cell line A549-cDDP than in A549-CON (Fig. 1B). These results were further demonstrated in more detail by immuno uorescence (Fig. 1C). It was clearly observed that the upregulation of APE1 expression was restricted to the cytoplasm rather than the nuclear area ( Figure S1A). The relationship between APE1 expression and cisplatin resistance has been widely con rmed [12]. We believe that cytoplasmic APE1 may play an important role in the mechanism of resistance to cisplatin. These results were further con rmed in another lung cancer cell line Calu-1 by overexpressing cytoplasmic APE1 through transfection of a plasmid expressing APE1 with an NLS inactive mutation. As shown in Fig. 1D and Figure S1B, cisplatin treatment increased cytoplasmic APE1 levels and total APE1 levels in Calu-1 cells in a similar manner to that observed in A549 cells. Overexpression of cytoplasmic APE1 signi cantly increased the cisplatin resistance and proliferation of Calu-1 cells ( Fig. 1E and F) after cisplatin treatment. These results indicated that the upregulation of cytoplasmic APE1 expression after cisplatin treatment was more pronounced in A549-CON cells than in A549-cDDP cells. Therefore, we proposed that cytoplasmic APE1 could be the major cause of cisplatin resistance.

Effects of cytoplasmic APE1 on cell proliferation, migration and invasion
Then, we investigated the role of cytoplasmic APE1 in cisplatin sensitivity. Overexpression of cytoplasmic APE1 in A549 and Calu-1 cells ( Fig. 2A) substantially increased cell growth, cell migration and invasive capacity following cisplatin treatment ( Figure. 2B-D). The cell growth, migration and invasion capacities were higher in the control group than in the cDDP alone group, and the level of cytoplasmic APE1 in the cDDP alone group was higher than that in the control group and lower than that in the group treated with shAPE1 and cisplatin, as shown in Figure. 1 and Figure. 2, so the cDDP alone group is not displayed. These results suggested that cytoplasmic APE1 promoted the growth, migration and invasion of lung cancer cells after cisplatin treatment.

Overexpression of cytoplasmic APE1 promoted tumor growth in vivo
We also researched the impact of cytoplasmic APE1 on cisplatin resistance in vivo by using xenograft tumors in nude mice consisting of A549 cells expressing either a control (shCON) or cytoplasmic APE1overexpression vector (shAPE1). We observed that overexpression of cytoplasmic APE1 signi cantly increased tumor growth and resistance to cisplatin in vivo ( Figure. 3A-B). Western blot results in xenograft tumors also revealed that NLS inactive mutated APE1 (shAPE1) increased cytoplasmic APE1 expression and that cisplatin induced the expression of cytoplasmic and total APE1 in vivo (Fig. 3C). These results were further veri ed by immunohistochemical analysis (Fig. 3D). Cisplatin induced the expression of cytoplasmic and total APE1, and cytoplasmic expression enhanced resistance to cisplatin in vivo.

The effect of cytoplasmic APE1 was affected by COX-2 levels
Our further studies showed that the overexpression of cytoplasmic APE1 signi cantly increased COX-2 expression in lung cancer cells (Fig. 4A). These results were veri ed by immunohistochemistry staining of COX-2 expression in xenograft tumors, and both cisplatin and cytoplasmic APE1 induced the expression of COX-2 ( Fig. 4B). To explore whether COX-2 is a functional target of cytoplasmic APE1, A549 cells were transfected with shRNA plasmids against COX-2 (siCOX-2). COX-2 expression was induced in siCON cells but remained depressed in siCOX-2 cells under cDDP treatment (Fig. 4C). As expected, cytoplasmic APE1 overexpression attenuated the inhibition of cell proliferation, migration, and invasion conferred by cisplatin treatment, and the downregulation of COX-2 attenuated the enhancement in cell proliferation, migration, and invasion conferred by cytoplasmic APE1 overexpression (Fig. 4D-F). The downregulation of COX-2 directly regulated cell proliferation, migration, and invasion associated with cisplatin treatment and cytoplasmic APE1 expression. These data revealed that COX-2 expression plays an important role in mediating the effects of cytoplasmic APE1.
Cytoplasmic APE1 promoted the Akt/β-catenin signaling pathway by targeting COX-2 in lung cancer As previously shown, COX-2 was located upstream of the Akt/β-catenin signaling pathway ( [13,14] and Fig. 4C). Accordingly, we sought to use cytoplasmic APE1 to probe the role of COX-2 in the Akt/β-catenin signaling in A549 cells. We demonstrated that overexpression of cytoplasmic APE1 increased β-catenin and phosphorylated Akt, but cisplatin treatment reduced β-catenin and phosphorylated Akt (Fig. 5A). As expected, both COX-2 inhibition and cisplatin treatment decreased the levels of β-catenin and phosphorylated Akt (Fig. 5B). Furthermore, western blot results further veri ed that cisplatin inhibited the activation of the Akt/β-catenin signaling pathway conferred by the overexpression of cytoplasmic APE1 in vivo (Fig. 5C). Overall, overexpression of cytoplasmic APE1 activated the COX-2/Akt/β-catenin signaling pathway, and cisplatin increased cytoplasmic APE1 but inhibited the COX-2/Akt/β-catenin signaling pathway. Therefore, we believe that cytoplasmic APE1 promotes cell proliferation and cisplatin resistance by activating the COX-2/Akt/β-catenin signaling pathway, reversing its inhibition by cisplatin.
Clinical prognostic values of cytoplasmic APE1 and COX-2 To evaluate the clinical value of cytoplasmic APE1 and COX-2 after cisplatin based chemotherapy in lung cancer patents, we analyzed cytoplasmic APE1 and COX-2 expression in 124 tissue blocks obtained from biopsies of lung cancer tissues by immunohistochemistry (table S1). Immunohistochemistry analyses showed that APE1 staining was localized in the cytoplasm and nucleus, while COX-2 was mostly localized to the cytoplasm. The intensity and percentage of cytoplasmic APE1 and COX-2 staining were determined for each of the clinical cases using a protocol previously described by our laboratory, as well as other investigators. Kaplan-Meier curves showed that high levels of cytoplasmic APE1 in the tumor sample signi cantly correlated with shorter OS (median time to progression of low-expression vs highexpression of cytoplasmic APE1: 19.0 months vs. 8.0, respectively, P < 0.005; Kaplan-Meier curves are shown in Fig. 5D) and that the COX-2 high-expression group had a shorter OS than the low-expression group (median time to progression of low-expression vs high-expression of COX-2: 14.0 months vs. 12.0, respectively, P < 0.005; Kaplan-Meier curves shown in Fig. 5E). These data revealed that both cytoplasmic APE1 and COX-2 are independent prognostic indicators in patients with lung cancer (Table  S2). Cytoplasmic APE1 was downregulated by mutation of APE1-C65 site Finally, we explored the molecular mechanisms responsible for cytoplasmic APE1 in lung cancer cells. The APE1-C65 site plays an important role in the redox function of APE1, and we next examined whether APE1-C65 mutations can impair cytoplasmic APE1 expression and therefore recover cisplatin sensitivity. We investigated the role of cytoplasmic APE1 in cisplatin sensitivity using a plasmid expressing APE1 with an APE1 C65 site inactive mutation (shC65S). shC65S induced cytoplasmic APE1 expression in A549 cells (Fig. 6A), which signi cantly reduced resistance to cisplatin, cell growth, cell migration and invasion ability. Moreover, the reduced cell growth, migration and invasion induced by shC65S were further suppressed after cisplatin treatment (Fig. 6B-D). Phosphorylated Akt and β-catenin downstream of the COX-2/Akt/β-catenin signaling pathway were also inhibited by shC65S and shC65S plus cisplatin treatment (Fig. 6E). Taken together, APE1-C65 site mutations could increase cytoplasmic APE1 expression and resistance to cisplatin in lung cancer cells.

Discussion
We reported that APE1 overexpression was associated with cisplatin resistance in lung cancer [12]. However, the detailed mechanism of APE1 in cisplatin resistance remains elusive. In this study, we have found that cytoplasmic APE1 can cause resistance to cisplatin by upregulating COX-2 expression in lung cancer. Additionally, cytoplasmic APE1 expression is important in the occurrence of poor prognosis in lung cancer. Thus, we conclude that cytoplasmic APE1 can predict certain prognostic outcomes for lung cancer patients. Speci cally, cytoplasmic APE1 expression can be used as a new method of identifying lung cancer patients with high tumor metastasis risk. These ndings underscore the role of cytoplasmic APE1 expression in the development and progression of lung cancer.
Several reports have documented the associations between cytoplasmic APE1 expression and lung tumor aggressiveness, patient survival and relapse in lung cancer; additionally, patient prognosis in human epithelial ovarian cancer and anti-in ammatory activity in cultured endothelial cells have also been linked to cytoplasmic APE1 levels [7,[15][16][17]. In this study, we explored the role of cytoplasmic APE1 in the malignant phenotype of lung cancer in vitro and in vivo. Our results demonstrated that cytoplasmic APE1 can promote lung cancer cell growth and aggressiveness by inactivating the Akt/β-catenin signaling pathway by increasing COX-2 levels.
Cytoplasmic APE1 activity could be the major factor in cisplatin resistance induced by the overexpression of APE1. In our results, cell lines with cisplatin resistance had high cytoplasmic APE1 levels, and upregulation of cytoplasmic APE1 in cisplatin-sensitive cells caused resistance to cisplatin. Lower expression of cytoplasmic APE1 in lung cancer tissues was linked to better OS in patient samples. These results are entirely consistent with our earlier research that showed that cisplatin treatment increases APE1 expression and that inhibiting APE1 expression signi cantly increases the sensitivity of lung cancer cells to cisplatin [11,18]. Clinical data analysis showed that survival prognosis was signi cantly better in lung cancer patients with low APE1 than in those with high APE1 expression levels [11]. The role of APE1 in cisplatin resistance can at least partially attributed to increased cytoplasmic APE1 expression.
The activated Akt/β-catenin signaling pathway plays an important role in enhancing the malignant phenotype of human cancer [19,20]. It has been reported that COX-2 indirectly regulates the Akt/β-catenin signaling pathway, while activated Akt/β-catenin signaling is strongly involved in cancer cisplatin resistance [21,22]. The associations between the inhibition of Cox-2 expression, COX-2 inhibitors and cisplatin therapy resistance have been universally con rmed. Akt/β-catenin is one of the downstream signaling molecules activated by COX-2 in lung cancer malignancy that is mediated by cytoplasmic APE1.
The main purpose of our study was to explore the role of cytoplasmic APE1 in cisplatin resistance. Two critical questions were raised. First, cytoplasmic APE1 may have two functions: overexpression of cytoplasmic APE1 activated the COX-2/Akt/β-catenin signaling pathway and promoted cell proliferation and invasion, but overexpression of cytoplasmic APE1 induced by cisplatin played another role. Therefore, we believe that cytoplasmic APE1 may not only promote cell proliferation and invasion, but also reduce the toxicity of cisplatin by other mechanisms. Second, the mechanism by which cisplatin increased the expression of cytoplasmic of APE1 may be through inducing APE1-C65 site mutations. C65 is located within adjacent strands in the middle of one of the beta sheets within APE1, and the APE1-C65s site mutation causes a signi cant loss of the redox activity of APE1, which may cause the expression of cytoplasmic APE1. Both the APE1-C65 site mutation and cisplatin increased the expression of cytoplasmic APE1 and inhibited the COX-2/Akt/β-catenin signaling pathway. Moreover, cisplatin enhanced APE1-C65 site mutation-mediated inhibition of the COX-2/Akt/β-catenin signaling pathway. Previous studies have suggested that mutation of the N-terminus of APE1 induces cytoplasmic APE1 expression, speci cally in the case of the C65 mutation, which is involved in cell proliferation [23]. However, our study con rmed that cytoplasmic APE1 can cause cisplatin resistance by activating the COX-2/Akt/β-catenin signaling pathway, and the detailed relationships among cisplatin, APE1-C65 site mutations and cytoplasmic APE1 required further study.
Our report describes cytoplasmic APE1 expression in human lung cancers. Increased expression of cytoplasmic APE1 may be important in tumor aggressiveness, poor prognostic phenotype and the acquisition of cisplatin resistance in lung cancer. In addition, overexpression of cytoplasmic APE1 promoted lung cancer cells aggressiveness by the COX-2 downstream signaling pathway. Thus, cytoplasmic APE1 could be a new prognostic marker and target for cisplatin resistance.

Declarations
Ethics approval and consent to participate: The data used to support the ndings of this study are available from the corresponding author upon request.

Competing interests:
None declared.