The Effect Of AMPK Activation On Tumor Biological Behavior And Anti-cancer Therapy

Background and Objective:Cancer is a significant health problem worldwide, which usually has high morbidity and mortality and has become the first of the five most-common deadly reasons in China. Recent data shows that cancer mortality rate reaches to 20%, which seriously threatens people’s health condition in China. With the development of society, obesity and metabolic diseases are getting more common, it also further increases the risk of tumors. Energy homeostasis is one of the protective mechanisms that can prevent the over-growth of tumor cells. However, tumor cells could specifically rely on the metabolic reorganization such as the Warburg effect to gain energy, which can overcome the energy crisis due to cell over-proliferation, which is an important characteristic of tumor cells. Therefore, the energy metabolic pathway has become a potential target of tumor therapy.Cellular energy metabolism could be regulated by many signaling pathways including AMPK pathway, which is involved in regulating the Warburg effect, the metabolism of fatty acids and glutamine. Meanwhile, AMPK may has cross-talk with PI3K-Akt and MAPK signaling pathways and many transcription factors which co-regulates cellular biological functions such as cell proliferation, growth and apoptosis. At the beginning of tumorigenesis, the deletion of AMPKα1 gene promotes the growth of malignant tumors. The loss of AMPK activity was thought to be one of the most important factors that cause cancer development. Therefore, many chemo- or biological- agents which can activate AMPK have been studied for the ability of inhibiting tumor growth. and the results are promising. Some of them might to become the promising candidates for cancer therapy, especially metformin.Despite AMPK can be activated by different ways, full activation of AMPK will not be fulfilled without AMPKK, which includes: LKB1, Ca MKKβ, TAK1 et al; The expression level of LKB1, the most important upstream activator of AMPK, is quite uneven based on the category of the cancer, so it would put impact on anticancer effect of AMPK activators such as metformin, and also the mechanism of stress-induced LKB1-AMPK activation still need to be elucidated. Moreover, AMPK was found to be activated under some stress conditions like cytokine stimulation or anticancer medicines without those classical AMPKK. These stress condition not only can induce cell apoptosis, but AMPK activation, but the mechanism remains unclear.As above, for up-regulating the AMPK activity in cancer cells, it is essential to study the LKB1-AMPK pathway and other pathway that involves in AMPK activation independent of LKB1.Thus, this study will focus on:(1) The expression level of p-AMPK and LKB1 in different cancer tissues.(2) The effect of LKB1 over-expression on tumor suppression and drug sensibility of etoposide(an common-used anti-cancer drug).(3) The effect of ATM and LKB1 on AMPK activation induced by etoposide, and the effect of AMPK phosporylation on anti-cancer drug sensitivity.(4) The effect of stress-induced MLK3 activation on AMPK activity and its possible mechaniam. Methods and materials1. The specimens enrolled in this study were obtained from the First Affiliated Hospital of Nanchang University(Ethical approval NO. 2014(025)), including 60 cases of gastric adenocarcinoma tissues and the corresponding adjacent-tumor tissues. The expression of p-AMPK–Thr172(phospho-adenosine protein kinase) and LKB1 were analyzed by using immunohistochemistry staining. In addition, 65 cases of lung adenocarcinoma tissues at different stages were also collected and the expression of p-AMPK–Thr172 was observed.2. We transfected SGC-7901 and A549(both are LKB1 absent) cells with functional wild-type LKB1 gene to establish stable cell lines which express LKB1. The growth and survival capacity were observed using MTT assay; cell migration was observed using cell wound scratch assay; Flow cytometry was used to detect the cell cycle, the proportion of CD44 positive cells, and the apoptotic ratio of the cells.3. C4-2 cells were incubated with etoposide in time- or dose- dependent way, then harvest the cells and the phosphorylation of AMPK and ATM was detected by western blot; The expression of ATM or LKB1 were interfered with si RNA transfection, then repeat the etoposide incubation in time- or dose-dependent way, the phosphorylation of AMPK and ATM were assessed.4. C4-2 DN stable Cell line were established by introducing dominant-negative mutant into C4-2 cells for inactivating AMPK, then C4-2DN cells were exposed to etoposide in time-dependent way, and then the apoptotic ratio of cells was detected by FACS, C4-2 were transfected with scramble RNA as control(C4-2 E). C4-2 DN and C4-2 E, A549 E and A549-LKB1(without and with LKB1 expression) were treated with etoposide in time-dependent way, and apoptosis related protein markers were detected by Western blot(Cleaved Caspase 3\Cleaved PARP).5. A549 E and A549LKB1(with or without LKB1 expression)were treated with different stimulators, then cells were collected and lysed for Western blot to check the expression of phosphorylation level of AMPK and JNK. Moreover, we screened the stimulators that can activate AMPK and JNK simultaneously by using western blot. MLK3 plasmid was incorporated into HEK-293 T cells, the phosphorylation level of AMPK was checked. The recombinant MLK3 and AMPK proteins are purified by GSH purification. In vitro enzymology experiments were used to show whether AMPK could be phosphorylated by MLK3 directly. In HEK-293 T cells, the interaction of recombinant MLK3 and AMPK a1 were observed by pull-down technique. GST-MLK3 was specifically bind to GSH beads, and western blot was used to detect endogenous AMPKα1 or AMPKα2 protein. Myc-AMPK?1 or ?2 was co-transfected with GST-MLK3 plasmid into HEK293 T cells, the cell lysates were incubated with GSH beads, and then centrifuged to harvest GSH-beads-MLK3 and captured protein, western blot was done with anti-GST and anti-Myc antibodies. Results 1. The expression of p-AMPK and LKB1 in adenocarcinoma tissues and corresponding adjacent-tumor tissues.(1) Expression level of p-AMPK-Thr172 and LKB1 in gastric adenocarcinoma was significantly lower than corresponding adjacent-tumors(p<0.001). The phosphorylation of AMPK was found to be:(-)20cases,(+) 33 cases,(++) 6 cases,(+++) 1 case in gastric adeno-carcinoma tissues;(-) 5 cases,(+) 11 cases,(++) 28 cases,(+++) 16 cases in adjacent-tumor tissues. The expression of LKB1 was found to be(-) 23 cases,(+) 25 cases,(++) 12 cases,(+++) 0 case in cancer tissues,(-) 9 cases,(+) 13 cases,(++) 27 cases,(+++)11 cases in adjacent-tumor tissues.(2) The expression of p-AMPK was decreased with the development of pulmonary adenocarcinoma, and the expression level showed stageⅠ>stageⅡ>stageⅢ(which has a correlation of-0.397 by Spearman correlation analysis, p<0.005). The phosphorylation of AMPK was found to be:(-)4 cases,(+) 5 cases,(++) 10 cases,(+++) 3 case in stage Ⅰ;(-)5 cases,(+) 10 cases,(++) 9 cases,(+++) 3 case in stage Ⅱ;(-)9 cases,(+) 6 cases,(++) 1 cases,(+++) 0 case in stage Ⅲ; 2. The effect of LKB1 expression on the biological behavior of SGC-7901 cells;Up-regulation of LKB1 expression in LKB1-absent cancer cell line SGC-7901 by inputting functional LKB1 gene restrained cell growth and migration ability, decreased the ratio of CD44+ cells, and increased sensibility to anti-cancer drugs(Eg. Oxaliplatin, 5-FU, irinotecan). 3. The role of ATM and LKB1 in etoposide-induced AMPK activation.⑴ Anticancer drug etoposide could activate ATM and AMPK in A549 cells in dose- and time-dependent way.⑵ We transfected ATM si RNA or scrambled si RNA into C4-2 cells, and 48 h later, treated them with different doses of etoposide. Western blot results revealed that knockdown of ATM abrogated AMPK activation, whereas ATM and AMPK were activated in control.⑶ We transfected LKB1 si RNA or scrambled si RNA into C4-2 cells, and 48 h later, treated them with different doses of etoposide. It revealed that si RNA knockdown of LKB1 abrogated AMPK activation, whereas did not affect ATM phosphorylation. In control group, ATM and AMPK were activated both.⑷ After the LKB1 expression was restored by introduction of wild type of LKB1 in A549, ATM and AMPK were activated simultaneously by etoposide. In control group without LKB1 expression, etoposide could only activate ATM, but not induce phosphorylation of AMPK.These results clearly indicate that ATM functions upstream of LKB1 that relays signal onto AMPK in response to etopoide. 4. The effect of AMPK activation on anti-tumor drug sensibility.⑴ After etoposide treatment, C4-2E cells(with AMPK activity) was more likely to be apoptotic than C4-2DN cells(without AMPK activity).⑵ After etoposide treatment, the expression of p-AMPK(Thr172), Cleaved Caspase 3 and Cleaved PARP in C4-2E was higher than C4-2DN cell; the similar results were obtained from A549-LKB1 cellss.⑶ This indicated that LKB1 was likely to contribute to AMPK activation and induce cell apoptosis. 5. The role of MLK3 on the activation of AMPK.⑴ TNF-?, anisomycin, H2O2 and sorbitol could activate AMPK and JNK. Interestingly, osmotic shock inducer sorbitol was able to upregulate p-AMPK(Thr172) and p-JNK significantly in LKB1-absent cell line A549-WT, indicating that sorbitol could activate AMPK and JNK independently of LKB1.⑵ After the cells were exposed to sorbitol again, phosphorylation of AMPK were up-regulated in A549-LKB1 and A549-WT both.⑶ After introduction of MLK3 c DNA into HEK293 cells, Over-expression of MLK3 could up-regulate p-AMPK(Thr172). MLK3 over-expression by incorporating MLK3 gene was able to increase AMPK phosphorylation significantly in HEK293 Tcells.⑷ In vitro enzymology experiments: AMP or LKB1 protein could not induce phosphorylation of AMPK respectively, while AMP and LKB1 protein together can induce phosphorylation AMPK. MLK3 not only could induce phosphorylation of AMPK with AMP, but phospholate AMPK alone.⑸ Whether MLK3 interacts directly with AMPK? It was shown that recombinant MLK3 could combine with endogenous AMPK?1 subunit by GST-pull down technique. Then, after co-transfection of both Myc-AMPK?1/?2 and p EBG-MLK3 into HEK293 T cell line, it was proved that exogenous MLK3 was also able to combine exogenous Myc-AMPK?1 using coimmuno-precipitation. Conclusion1. The expression of LKB1 and p-AMPK(Thr172) is significantly lower in tumor tissues than in adjacent tumor tissues and decreased gradually during tumor progression, indicating that both may be invoved in tumorigenesis and cancer progression. Therefore, recovery of LKB1 expression in LKB1-abscent cells may inhibit tumor cell viability and migration, and increase the anti-tumor drug sensibility of cancer cells.2. The process of AMPK activation induced by etoposide may need ATM and LKB1 both, which could enhance drug sensibility of cancer cells to anti-cancer agents.3. MLK3, the upstream activator of JNK, can combine with AMPK?1, and phosphorylate AMPK in LKB1-indipendent way.