The histone deacetylase SIRT6 promotes glycolysis through the HIF-1α/HK2 signaling axis and induces erlotinib resistance in non-small cell lung cancer

Abstract

Erlotinib is a first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). Overcoming erlotinib resistance is crucial to improve the survival of advanced non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. It is also an important clinical problem that urgently needs a solution. In this study, we explored strategies to overcome erlotinib resistance from the perspective of energy metabolism. SIRT6 is a histone deacetylase. Here, we found that high expression of SIRT6 is associated with poor prognosis of lung adenocarcinoma, especially in EGFR-mutated NSCLC patients. The next cell experiment found that SIRT6 expression increased in erlotinib-resistant cells, and SIRT6 expression was negatively correlated with the sensitivity of NSCLC to erlotinib. Inhibition of SIRT6 promoted erlotinib-induced apoptosis in erlotinib-resistant cells, and glycolysis in drug-resistant cells was also inhibited. Functional studies have shown that SIRT6 increases glycolysis through the HIF-1α/HK2 signaling axis in drug-resistant cells and inhibits the sensitivity of NSCLC cells to erlotinib. In addition, the HIF-1α blocker PX478-2HCL attenuated the glycolysis and erlotinib resistance induced by SIRT6. More importantly, we confirmed the antitumor effect of SIRT6 inhibition combined with erlotinib in NSCLC-bearing mice. Our findings indicate that the cancer metabolic pathway regulated by SIRT6 may be a new target for attenuating NSCLC erlotinib resistance and has potential as a biomarker or therapeutic target to improve outcomes in NSCLC patients.

Keywords: EGFR-TKI; Erlotinib; Glycolysis; NSCLC; SIRT6.

Fig. 1

SIRT6 expression is upregulated in NSCLC tissues, and SIRT6 upregulation is associated with poor prognosis in NSCLC patients. SIRT6 is overexpressed in LUSC (A) and LUAD (B) tissues in comparison with normal lung tissues. mRNA expression data were obtained using UALCAN. Survival analysis of the association of SIRT6 with OS in lung cancer (C) and LUAD (D) patients from the Kaplan–Meier Plotter website. E NSCLC tissue sample information downloaded from the TCGA database. Mutation data, RNA-seq data and clinical information data of samples were integrated to obtain EGFR mutation data of NSCLC, and then the correlation between SIRT6 expression level and overall survival was analyzed. ***p < 0.001

Fig. 2

SIRT6 is highly expressed in erlotinib-resistant cells. A and B The sensitivity of PC9, PC9/ER, HCC827, and HCC827/ER NSCLC cells to erlotinib was detected using a CCK-8 kit after treatment or not with erlotinib for 48 h. C and D PC9, PC9/ER, HCC827, and HCC827/ER NSCLC cells were treated or not with 1 µM erlotinib for 48 h. Cell lysates were prepared and subjected to western blot analysis using the indicated antibodies. *p < 0.05, **p < 0.01

Fig. 3

Inhibition of SIRT6 attenuates the resistance of NSCLC cells to erlotinib and enhances the cell apoptosis induced by erlotinib. A The PC9/vector, HCC827/vector, PC9/SIRT6, and HCC827/SIRT6 cell lines were established using PC9 and HCC827 cells transfected with SIRT6-overexpressing vector and control vector. SIRT6 protein expression was analyzed using western blotting. B After transfecting si-NC, si-SIRT6-1 and si-SIRT6-2 in erlotinib-resistant cell lines (PC9/ER and HCC827/ER), the cell lines PC9/ER/si-SIRT6-1, PC9/ER/si-SIRT6-2 and HCC827/ER/si-SIRT6-1, HCC827/ER/si-SIRT6-2 and the corresponding control cell lines PC9/ER/NC and HCC827/ER/NC were established, and the protein levels of SIRT6 were analyzed using western blotting. The overexpression of SIRT6 in PC9 and HCC827 cells (C, D) and the influence of SIRT6 knockdown (E, F) on erlotinib sensitivity in PC9/ER and HCC827/ER cells were measured using the CCK-8 assay. G, H SIRT6-knockdown cells were treated with 1 µM erlotinib for 48 h, and erlotinib-mediated apoptosis was detected using flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Erlotinib-resistant NSCLC cells have a stronger ability to produce ATP and lactic acid. A Intracellular ATP contents of PC9, HCC827, PC9/ER, and HCC827/ER cells were measured. B After PC9, HCC827, PC9/ER, and HCC827/ER cells were treated with 1 µM erlotinib for 48 h, the intracellular ATP content was measured. C The lactate production of PC9, HCC827, PC9/ER, and HCC827/ER cells was measured. D After PC9, HCC827, PC9/ER, and HCC827/ER cells were treated with 1 µM erlotinib for 48 h, lactate production was tested. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

SIRT6 overexpression can enhance glycolysis in erlotinib-sensitive NSCLC cells, while SIRT6 knockdown attenuates glycolysis in erlotinib-resistant NSCLC cells. A The ATP content between SIRT6-overexpressing cells and the respective control cells was compared. B After treatment of SIRT6-overexpressing cells and the respective control cells with 1 µM erlotinib for 48 h, the ATP content was compared. C The lactic acid between SIRT6-overexpressing cells and the respective control cells was compared. D After treatment of SIRT6-overexpressing cells and the respective control cells with 1 µM erlotinib for 48 h, the lactic acid was compared. The same method was used to measure the intracellular ATP content (E, F) and lactic acid production (G, H) of the SIRT6-knockdown cell line and the corresponding control cell line. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

SIRT6 promotes glycolysis through the HIF-1α/HK2 signaling pathway to induce erlotinib resistance. A, B western blot showing the molecular changes in the SIRT6/HIF-1α/HK2 signaling pathway after overexpression or knockdown of SIRT6 in cells. C Changes in the gene levels of related molecules on the signal axis after treatment of PC9/SIRT6 and HCC827/SIRT6 cells with a HIF-1α inhibitor (PX-478 2HCl (Selleck Chem, Houston, USA)) at 20 µM for 48 h. D After treatment with 10 µM or 20 µM PX-478 2HCl for 48 h, the cell lysate was used for western blotting to analyze the changes in related molecules. After incubating PC9/SIRT6 and HCC827/SIRT6 cells with or without 20 µM PX-478 2HCl for 48 h, lactic acid production (E) and intracellular ATP content (F) were compared. After treatment with 20 µM PX-478 2HCl for 48 h in PC9/SIRT6 and HCC827/SIRT6 cells, the influence of the HIF-1α inhibitor on erlotinib sensitivity in SIRT6-overexpressing cells was evaluated using a CCK-8 assay (G). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

SIRT6 induces erlotinib resistance in vivo. A, B Two weeks after the establishment of the xenograft model, erlotinib was continuously administered for 14 days, the mice were euthanized, and the tumors were removed (n = 5 mice/group). C, D Tumor growth curves representing the mean ± SEM of tumor volumes of five mice in the indicated xenograft group. E A TUNEL assay (scale bars, 20 µm) was used to analyze cell apoptosis in tumor tissues. F The proportion of apoptotic cells in tumor tissues was measured by Image-Pro Plus software. G, H Immunohistochemical staining (scale bars, 50 µm) to confirm SIRT6, HIF-1α and HK2 protein expression in the designated tumor sample group and Ki-67 staining to measure tumor cell proliferation. I, J The percentage of SIRT6-, HIF-1α-, HK2-, and Ki-67-positive cells in different groups was analyzed with Image-Pro Plus. *p < 0.05, **p < 0.01, ***p < 0.001