1. NNK could strengthen stemness and chemoresistance in pancreatic cancer cells.
To address the potential role of NNK in pancreatic cancer cells, we first treated BxPC-3 and Panc-1 cells with NNK and found that NNK significantly increased the colony formation ability of pancreatic cancer cells (Fig. 1A), which meane that NNK may have a pro-stemness effect on pancreatic cancer cells. To further confirm this hypothesis, we then examined the effect of NNK on pancreatic cancer spherogenesis, and the results showed that the number of pancreatic cancer stem cell spheres (PCSCs) formed in the NNK-treated group was significantly higher than that in the control group (Fig. 1B). Since SRY-Box Transcription Factor 2 (SOX-2), Octamer-Binding Protein 4 (OCT-4) and Nanog Homeobox (Nanog) are three common indicators of cell stemness in pancreatic cancer cells, we examined their mRNA and protein expression levels in control group and NNK-treated PCSCs group. The results showed that SOX-2, OCT-4 and Nanog were all significantly upregulated by NNK stimulation (Fig. 1C). In summary, the above findings suggest that NNK could strengthen the stemness of pancreatic cancer cells.
Increasing evidence suggested that cell stemness is often accompanied by an increase in chemoresistance in pancreatic cancer. Hence, we next aimed to verify whether NNK is associated with gemcitabine resistance in pancreatic cancer cells. We performed a CCK-8 assay and found that pancreatic cancer proliferation was significantly decreased by gemcitabine, while this phenomenon was partly eliminated by NNK (Fig. 1D). Additionally, using a colony formation assay, we confirmed the above findings (Fig. 1E). In summary, the above findings suggest that NNK could promote the chemoresistance of pancreatic cancer cells.
2. NNK could induce autophagy in pancreatic cancer cells.
Notably, the activation of autophagy and related pathways may play an essential role in cancer cell stemness and chemotherapy resistance. Hence, we aimed to determine whether NNK influences autophagy in pancreatic cancer. Firstly, we used immunofluorescence to stain LC3 autophagy spots and found that NNK significantly increased the number of LC3 autophagy spots in Panc-1 cells (Fig. 2A). In addition, western blotting also showed that NNK can influence the protein expression levels of LC3 and p62 which were belong to representative cell autophagy markers in a time- and dose-dependent manner (Fig. 2B). It seemed that NNK may be preliminarily related to autophagy. Secondly, we applied the mRFP-GFP-LC3 autophagy double-labelled adenovirus system to observe the effect of NNK on autophagy more intuitively and accurately. After 24 h, fluorescence microscopy revealed that the red and green autophagy spots in the transfected cells were significantly increased after NNK intervention compared with that in the control group, and the yellow spots formed by the overlap of red and green spots were also significantly increased. In addition, the proportion of single red spots in cells in the NNK intervention group was significantly higher than that in the control group (Fig. 2C), which suggested that NNK promotes autophagy again. Thirdly, we examined the effect of NNK on autophagy-related pathways and found that NNK obviously modulated the mRNA expression of autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and Beclin1 in a time- and dose-dependent manner (Fig. 2D). Moreover, these phenomena were also confirmed by western blotting (Fig. 2E). Finally, to further analyse the role of the above autophagy-related pathways in NNK-induced autophagy, siRNA was used to knock down the expression ATG5, ATG7, and Beclin (Fig. 2F). Western blotting results showed that the increase in LC3 levels caused by NNK was inhibited by si-ATG5, ATG7 and Beclin1. Additionally, the number of LC3 autophagy spots in cells was also reduced (Fig. 2G). In conclusion, the above findings indicated that NNK could promote autophagy in pancreatic cancer cells in an ATG5-, ATG7- and Beclin1-dependent manner.
3. NNK could enhance the stemness and chemoresistance of pancreatic cancer cells by activating autophagy.
Notably, researchers have demonstrated that the stemness and chemoresistance of cancer cells were related to cell autophagy. Hence, to determine whether the increase in stemness/chemoresistance of pancreatic cancer cells induced by NNK was due to its effect on cell autophagy, we conducted a series of experiments.
We firstly conducted colony formation assays and transwell assays to evaluate the influence of autophagy on pancreatic cancer single-cell malignancy/stem cell-like behaviour with NNK treatment. The results showed that CQ, a classic autophagy inhibitor, decreased the number of Panc-1 colonies, indicating that autophagy may benefit the colony formation ability of single pancreatic cancer cells (Fig. 3A). In addition, NNK-induced promotion of Panc-1 colonies can also be significantly inhibited by CQ. Moreover, Transwell assay also confirmed similar phenomena in the invasion/migration of single pancreatic cancer cells (Fig. 3B); hence, the above findings suggest that NNK-mediated strengthening of stem cell-like behaviour in pancreatic cancer cells is autophagy dependent. Next, to illustrate the effect of NNK and downstream autophagy in pancreatic cancer cells stemness more directly, we measured the spherogenesis of Panc-1 cells, and the results showed that CQ inhibited sphere formation. NNK-induced promotion of Panc-1 spherogenesis was also significantly inhibited by CQ (Fig. 3C). We also found the similar variation as above in the expression of SOX-2, OCT-4 and Nanog stemness markers (Fig. 3D). In summary, the previous results revealed that NNK may enhance the stemness of pancreatic cancer cells by activating autophagy.
We next examined whether NNK-induced pancreatic cancer chemoresistance was mediated by cell autophagy. By using the CCK-8 assay, we found that autophagy inhibition sensitized the killing effect of gemcitabine on pancreatic cancer cells, and this phenomenon was evident even in the presence of NNK (Fig. 3E). Moreover, the colony formation assay also suggested the same phenomenon (Fig. 3F). These findings revealed that NNK enhanced the chemoresistance of pancreatic cancer cells by activating autophagy
4. NNK could promote autophagy through the modulation of β2AR and Akt.
We previously suggested that NNK is an analogue of catecholamines that can activate βAR efficiently, so we hypothesized that NNK promoted autophagy by activating the βAR signaling pathway in pancreatic cancer cells. To test this hypothesis, a series of experiments was designed. Firstly, β2AR (the main isoform of βAR expressed in pancreatic cancer) was efficiently knocked down by using a specific siRNA (Fig. 4A). Immunofluorescence analysis revealed that β2AR knockdown significantly reduced LC3 autophagy spots (induced by NNK) in pancreatic cancer cells (Fig. 4B), and a similar effect was also confirmed by examining the expression of ATG5, ATG7 and Beclin1 autophagy markers (Fig. 4C), which suggests that the autophagy-promoting effect of NNK partly depends on β2AR.
As a G protein-coupled receptor embedded the cell membrane, βAR senses the stimulation of extracellular ligands such as NNK and transduction signals through various intracellular pathways. Next, we aimed to determine the possible downstream autophagy-related pathways of βAR after stimulation with NNK. Through continuous screening and testing, we found that Akt, an important pro-pancreatic cancer kinase, may mediate this process. To verify this hypothesis, we used immunofluorescence and western blotting and found that knockdown of Akt significantly reduced LC3 autophagy spots and LC3 protein levels (induced by NNK) in PC cells (Fig. 4D, E). Moreover, a similar effect was also confirmed by examining the expression of the ATG5, ATG7 and Beclin1 autophagy markers (Fig. 4F). These inhibitory functions were similar to those of LY294002 Akt inhibitor (a PI3K inhibitor that can block Akt activation), which suggested that the autophagy-promoting effect of NNK was partly dependent on Akt.
5. NNK could form a potential β2AR-Akt feedback loop in pancreatic cancer cells autophagy.
The above findings suggested that the autophagy-promoting effect of NNK was β2AR- and Akt-dependent. Next, clearly elucidating the up- and downstream relationship between β2AR-Akt and NNK-induced pancreatic cancer cells autophagy was critical to our research. In general, membrane receptors are the initiators of signalling pathways when the cell is externally stimulated. Hence, we hypothesised that Akt may be downstream of β2AR. To test this, we first aimed to exclude the influence of β1AR on NNK-induced pancreatic cancer βAR activation, and the results showed that β1AR knockdown (Fig. 5A) had no effect on the activation of Akt (Fig. 5B); however, β2AR knockdown and inhibition significantly suppressed Akt activation (phosphorylation) and LC3 maturation (Fig. 5B, C), which suggested that the β2AR-Akt axis was the main pancreatic cancer cells autophagy inducer after NNK stimulation. However, we accidentally discovered that β2AR expression was modulated by NNK in pancreatic cancer cells (Fig. 5C), which reminded us that there may be a potential β2AR-Akt-autophagy feedback loop underlying the effects of NNK. To explore this hypothesis, we treated Panc-1 and BxPC-3 cells with NNK for different time periods and found that the mRNA expression of β2AR (rather than β1AR) was increased in a time-dependent manner (Fig. 5D), and this trend can be blocked by inhibitors (Fig. 5E). Moreover, at the protein level, NNK also promoted β2AR expression and Akt activation (Fig. 5F). In conclusion, these findings suggested that NNK activates autophagy through the β2AR-Akt pathway and increases β2AR expression levels to further strengthen this autophagy activation process, thus forming a potential β2AR-Akt feedback loop in pancreatic cancer cells autophagy.