Our previous study had demonstrated that SIRT6 was upregulated in PTC and promoted tumor invasion and migration via inducing epithelial mesenchymal transformation(21). We took a note that SIRT6 can induce both Warburg effect and autophagy in other disease from other researches. However, in tumors, especially in thyroid cancer, the role of SIRT6 on these two effects have been less studied. Therefore, we determined whether SIRT6 induce Warburg effect and autophagy in PTC firstly.
We successfully generated SIRT6-upregulated PTC cells TPC1-SIRT6 and negative controls TPC1-NC. In order to study effect of SIRT6 on autophagy, Western blotting, RT-qPCR was performed to detect key autophagy gene expression. ratio of LC3Ⅱ/Ⅰ, expression of Beclin1 and ATG5 showed significantly upregulation, whereas P62 showed significantly downregulation in TPC1-SIRT6 compared with TPC1-NC. IF and TEM was further performed to observe autophagy flux and autophagosomes, and stronger autophagy flux, more autophagosomes were observed in TPC1-SIRT6. All these results indicated SIRT6 induced autophagy in TPC-1. In the further study on the mechanism of SIRT6 induced autophagy, we speculated that signaling pathways play an important role in this interaction at first. Two main common pathways: Pi3k/Akt/mTOR, MAPK/Erk/mTOR associated with Warburg effect and autophagy were detected. Although p-Akt was inhibited in TPC1-SIRT6, there was no significant change in its downstream p-mTOR. ERK/mTOR signaling pathway was not activated at all. We then turned our attention to oxidative stress, and ROS production of TPC1-SIRT6 showed significant increase compared with TPC1-NC. ER stress is closely related with ROS, as well as autophagy(22). ER stress s is characterized by incorrect folding and aggregation of unfolded proteins in endoplasmic reticulum lumen and disturbance of calcium balance. It can activate signal pathways such as unfolded protein response, endoplasmic reticulum overload response. PERK, ATF6, Irel are three key regulation pathways of ER- stress(23). ROS-related ER stress is most correlated with PERK signaling pathway, loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress(24). In addition, previous studies had shown that PERK, eIF2α, ATF4, CHOP all work as regulation factors of autophagy(25–27). Therefore, we further detected downstream of PERK pathway: eIF2α/ATF4/CHOP, and obvious activation was found in TPC1-SIRT6 compared with TPC1-NC. To determine causality between ROS and ER stress, TPC1-SIRT6 was treated with ROS inhibitor NAC and ER-stress inhibitor 4-PBA respectively. ROS inhibitor successfully repressed activation of eIF2α/ATF4/CHOP, whereas, ER-stress inhibitor had no effect in ROS. This indicated upregulation of ROS is the upstream of ER stress. Furthermore, both ROS and ER stress inhibitor restrained upregulation of autophagy in TPC1-SIRT6. All these results confirmed SIRT6 induced ER stress via PERK pathway, then upregulates ROS production and finally induces autophagy. Thus far, we have confirmed SIRT6 induced ER-stress via upregulating ROS, and subsequently promoted autophagy. However, we were still wondering how SIRT6 increase ROS. As SIRT6 is a member of the class III histone deacetylases that have been demonstrated inhibited H3K56ac and H3K9ac(28). We suggested SIRT6 targeted ROS regulator at transcriptional level via H3K56ac or H3K9ac. We consulted the CHIP-sequence datasets GSE102813 which studied SIRT6 depletion in BRAFV600E melanoma cells. SIRT6 binds to the promoter of NRROS accompanied with H3K56ac peak. Interestingly, BRAFV600E is also frequent mutation in PTC similar as melanoma(17). In addition, NRROS is a recent reported ROS negative regulator, which inhibits ROS via interacting with NOX2. Coincidentally, NRROS and NOX2 located in endoplasmic reticulum, which may meet ER-stress described above. Through Western blotting, RT-qPCR and CHIP analysis, we succeeded in verifying our suspect. SIRT6 binds to the promoter of NRROS and inhibited H3K56ac, thereby inhibited its expression at transcriptional level. Oppression of endoplasmic reticulum located NRROS protein promoted ROS and ER-stress sequentially.
In order to study effect of SIRT6 on Warburg effect, Western blotting, RT-qPCR was performed to detect key Warburg effect gene expression. Glut1, PKM, LDHA was significantly decreased in TPC1-SIRT6 compared with TPC1-NC; whereas GAPDH, HK2, PGK showed no change. Then we further detect glucose uptake, lactate production and ATP content of PTC cells. All these measurements decreased significantly in TPC1-SIRT6 compared with TPC1-NC. Therefore, we infirmed SIRT6 inhibited Warburg effect in TPC-1. There are many similarities between Warburg effect and autophagy effect on the biological function of tumors. Previous studies have also reported that there is a close relationship between Warburg effect and autophagy effect. In hepatocellular carcinoma, phosphorylation of autophagy associated gene ATG4B at Ser34 promotes Warburg effect in by inhibiting mitochondrial function and participating in metabolic reorganization(29). In prostate cancer, co-targeting hexokinase 2-mediated Warburg effect and ULK1-dependent autophagy cause tumor regression in xenograft, leads to a near-complete tumor suppression and remarkably extends survival in Pten −/p53-deficiency-driven mouse model(30). In A549 alveolar adenocarcinoma cells, the downregulation of Warburg effect gene PKM2 induced apoptosis and autophagy and this autophagy protected the cells from apoptotic cell death(31). In summary, Warburg effect and autophagy are closely related to the occurrence and development of tumors, and their regulatory networks overlap to some extent. However, there are few studies on the relationship between Warburg effect and autophagy in thyroid cancer. As we had infirmed to SIRT6 inhibited Warburg effect and induced autophagy in PTC, we further focused our attention in interaction mechanism between SIRT6 induced autophagy and Warburg effect.
To further study interaction of Warburg effect and autophagy, TPC1-SIRT6 were treated with NAC(5 mM) and autophagy inhibitor CQ respectively to detect Warburg effect indicators. Both NAC and CQ could rescued inhibited Warburg effect in TPC1-SIRT6. Taken together, SIRT6 induced autophagy via upregulating ROS, and upregulated autophagy further inhibited Warburg effect. As autophagy-lysosome pathway a main mechanism for protein degradation, we suggest SIRT6 inhibited Warburg effect protein via autophagic degradation. As expect, lysosome inhibitor CQ but not proteasome inhibitor MG-132 recue the expression of Glut1. Furthermore, SIRT6 depleted membrane expression of Glut1 and promoted its co-location with autophagosome marker LC3. Therefore, we infer SIRT6 inhibited Warburg effect via autophagic degradation of Glut1.
Here we find some contradictions with previous studies. ROS is a class of oxygen atoms or clusters formed by oxygen and is always reported as activator of Warburg effect via AMPK pathway(32, 33). Whereas in our research, upregulated ROS was accompanied with inhibited Warburg effect. We further increased concentration of NAC for treatment of TPC1-SIRT6. Interestingly, 15 mM NAC further inhibited autophagy, as well as restrained Warburg effect. We found that NAC at 5 mM had no effect on AMPK pathway, but at 10–15 mM, NAC significantly inhibited AMPK pathway. After treated TPC1-SIRT6 + NAC (15 mM) with AMPK activator, Warburg effect rescued again. Furthermore, we also found AMPK activation could increase autophagy directly.
Warburg effect is an important basis for rapid growth of tumors and PET/CT examination. The uptake rate and concentration of contrast media (such as 18F-FDG) in tumors with active Warburg effect are higher(34). Therefore, we learnt effect of SIRT6-ERstress-Autophagy-Warburg effect axis in growth of xenografts via animal model combined 18F-FDG PET/CT. Overexpression of SIRT6 significantly inhibited Warburg effect as well as tumor growth; however, CQ rescued the inhibition. Previous studies have suggested that chloroquine is a drug that treats cancer by inhibiting autophagy or other mechanisms(35, 36). Whereas our research found CQ promotes tumor growth via enhancing Warburg effect in SIRT6 upregulated PTC cell. Therefore, CQ should be more cautious applicated in the treatment of PTC patients and SIRT6 expression needs to be assessed. Similarly, NAC plays a dual role in the Warburg effect and growth of tumors. 50 mg/kg NAC significantly enhanced Warburg effect and tumor growth, whereas 150 mg/kg NAC restrained Warburg effect and tumor growth instead. In previous studies, NAC has been considered as an anti-cancer drug, which inhibits the growth of tumors through anti-oxidative stress and other mechanisms(37, 38). However, above mishap happened in TPC1-SIRT6. Therefore, the application of NAC treatment of PTC patients with overexpression of SIRT6, we may need to increase the concentration to prevent counteraction.