Downregulation of ASPP2 in pancreatic cancer cells contributes to increased resistance to gemcitabine through autophagy activation

ASPP2 plays a key role in apoptosis regulation in the intrinsic and extrinsic apoptotic pathways [23, 24] and widely expressed in many human tissues.[25] The expression of ASPP2 can be enhanced in response to DNA damage,[13] and was down-regulated in both metastatic and invasive cells as compared to normal breast epithelium.[26] To exam the levels of expression of ASSP2 in the pancreatic cancer tissues and para-cancerous tissues, we determined ASSP2 expression level with RT-qPCR assay or immunoblotting assay, the ASSP2 expression level was less abundant in pancreatic cancer tissues compared with para-cancerous tissues (Fig. 1a and b). We also analyzed the patients’ samples with IHC assay (Table 1). As presented in Fig. 1c (left panel, 1# and 11# samples as the representative samples), the results suggest that ASSP2 protein expression was much lower in cancerous tissues (Fig. 1c, right panel). Furthermore, the ASPP2 low expression patients were found to exhibit high Histopathological grade (Table 1). More importantly, significant differences in both overall survival and disease-free survival were found among ASPP2 low/high groups (Fig. 1d). Taken together, all the data suggest that ASSP2 expression is lower in cancerous tissues than papa-cancerous tissues and it could be an independent prognostic predictor for pancreatic cancer.

To study the role of ASSP2 in cancer, we firstly analyzed its expression in series of pancreatic cancer cell lines using NCBI GEO database [27] and the data suggest that ASSP2 expression significantly differs in different PC cell lines. Low ASPP2 expression cell line BxPC-3 and high ASPP2 expression cell line SW1990 were used for further research (Fig. 2a). We organized ASPP2 stably over-expression cell lines in BxPC-3 and ASPP2 stably knockdown cell lines in SW1990, the mRNA and protein expression of ASPP2 were shown (Fig. 2b, c and Additional file 1: Figure S1A). The #2 of ASPP2 knockdown cell lines was used for next experiments. Forced ASPP2 over-expression resulted in a significant decrease of cancer cell proliferation, Knockdown of ASPP2 enhanced proliferative activity (Fig. 2d). Consistently, over-expression of ASPP2 attenuated cancer cell clone formation and vice versa (Fig. 2e). Together, these results reveal that down-regulation of ASPP2 could enhance cell proliferation of pancreatic cancer.

Recently, two groups found that ASPP2 could regulate survival autophagy and autophagic apoptosis through different ways in hepatoma cells,[21, 22] which indicated that ASPP2 could be the Indispensable regulator in autophagy. In pancreatic cancer, autophagy play essential function in cell growth under basal condition.[28] To examine its regulation on basal autophagy, firstly, we analyzed the autophagic activities of pancreatic cancer cell lines. Unexpectedly, BxPC-3 cells with low ASPP2 expression expressed higher LC3-II, the marker of autophagosomes,[29] sw1990 cells with high ASPP2 expression formatted less LC3-II (Fig. 3a). For directly observing the autopahgy process, we also transfected GFP-LC3 into these four PC cell lines and analyze the formation of autophagosomes,[29] we found that more autophagosomes were formed in the BxPC-3 cells than SW1990, PANC1and MiAPaca-2 cells (Fig. 3b). All the data presented suggest that autophagy negatively correlated with the expression of ASPP2.

Based on these data, we hypothesized that ASSP2 can negatively regulate basal autophagy in pancreatic cancer cells. To test this hypothesis, we checked the Autophagy-Related Gene Family in BxPC-3 cells with ASPP2 overexpression and in SW1990 cells with ASPP2 knockdown. Overexpression of ASPP2 could significantly decrease the amount of LC3-II and ATG family expression, and knockdown of ASPP2 enhanced it (Fig. 3c). With GFP-LC3 system, we confirmed the regulation of ASPP2 in autophagy (Fig. 3d). In addition, as presented in Fig. 3e, the accumulation of autophagic vacuoles was dramatically decreased in ASPP2 overexpression BxPC3 cells and silencing of ASPP2 reversed it. Furthermore, for detecting the autophagic flux, we examined mRFP-GFP-LC3 puncta formation with fluorescence microscope. Consistently, ASPP2 decreased the autophagosomal-lysosomal fusion process, which also supports ASPP2 as the negative regulator for autophagy under basal condition (Fig. 3f). As a P53 interacting-protein,ASPP2 often regulate cell function in P53-dependent way. P53 was also reported as the core-regulator in autophagy.[30] to exclude the function of P53 in the regulation of autophagy, we also check the basal autophagy with P53 inhibitor, pifithrin-α [31]. However, inhibition of P53 could not reverse the function of ASPP2 in autophagy, which suggested that ASPP2 regulated basal autophagy in P53-indepdent way (Additional file 1: Figure S1B).

AMPK is a key cellular energy sensor and functions to maintain energy homeostasis upon nutrient starvation,[32] it could regulate autophagy through mTOR pathway.[33] Firstly, we checked the phosphorylation of AMPK and TSC2. Down-regulation of ASPP2 could increase the phosphorylation of AMPK and TSC2 and vice versa (Fig. 4a). For confirming the ASPP2 regulate autophagy through AMPK, we knockdown the AMPK expression with shRNA (Fig. 4b),and found that AMPK knockdown could significantly inhibit the autophagic flux upregulation induced by ASPP2 knockdown (Fig. 4c,d,e). These results indicate that ASPP2 regulate autophagy through AMPK-mTOR pathways.

Gemcitabine is currently the standard chemotherapy treatment at all stages of pancreatic cancer.[34] Survival benefit and clinical impact however remain moderate due to a high degree of drug resistance. To dissect the effect of ASPP2 on gemcitabine mediated cell apoptosis, we treated these cells with gemcitabine. As shown in Fig. 5a, overexpression of ASPP2 can promote the gemcitabine induced cell death and knockdown of ASPP2 could inhibit it. To further define the role of ASPP2 in determining the sensitivity of PC to gemcitabine, we checked the cell apoptosis induced by gemcitabine, which is consistently with the cell death results (Fig. 5b). Apoptosis protein expression also proved it (Fig. 5c). All the results suggest that ASPP2 expression level can determine the susceptibility of the cells to gemcitabine treatment.

As ASPP2 could interact with P53 to Specifically Stimulate the Apoptotic Function of p53, we firstly checked that whether ASPP2 regulated gemcitabine-resistance in pancreatic cancer. With pifithrin-α treatment, we found that inhibition of P53 could not reverse the ASPP2-inudced cell apoptosis, which suggested that ASPP2 regulated gemcitabine-induced cell death in P53-indepdent way (Fig. 5d,e). As shown previously, ASPP2 can negatively regulate autophagic activity, so we next exam whether ASPP2 determining the susceptibility of cells to gemcitabine is dependent on its regulation of autophagy. To answer this question, we treated ASPP2 shRNA transduced SW1990 cells with gemcitabine in the presence or absence of CQ and 3-MA, which are the widely used autophagy inhibitors, and cell death and cell apoptosis were analyzed. The protection of knockdown of ASPP2 in SW1990 cells from gemcitabine induced apoptosis was dramatically impaired in the presence of autophagy inhibitors, suggesting that ASPP2 regulate gemcitabine induced cell apoptosis via autophagy (Fig. 6a, b, c). To dissect the ASPP2 role in regulation of gemcitabine induced cell apoptosis in vivo, we inoculated SW1990 cells with ASPP2 knockdown into nude mice and determine the tumor size at indicated time point post-inoculation. As shown in Fig. 6d and e, ASPP2 knockdown cells were more resistant to gemcitabine treatment; however, this is significantly impaired following treatment of CQ, further establishing the essential role of autophagy in determining the resistance to gemcitabine in vivo. Collectively, all the data presented here demonstrate that decrease expression of ASPP2 can confer cell the insensitivity to gemcitabine, which is dependent on its activation of autophagy.