The Inhibitory Role Of AMPK On TGF-β Signalingand Function In Cancer Cells

Background and Objective: Cancer has become the most important threaten to human health mainly due to it’s invasive and metastatic properties. Cancer stem cells(CSCs) are the seed cells of invasion and metastasis, which are able to plant, survive and reproduce themselves. Epithelial--mesenchymal transition(EMT) is the initial step of invasion and metastasis, in which cancer cells obtain stem-cell properties, such as self renewal, proliferation and differentiation. Therefore, CSC/EMT has been thought as the basis of tumor metastasis, resistance and recurrence. TGF-β, an inducer of EMT, is closely associated with cancer progression, angiogenesis, pathologic staging and prognosis. In multiple types of cancers, TGF-β protein and its downstram genes have been found to be over expressed; Bmi-1, a target gene of TGF-β signaling, is involved the regulation of EMT and self-renewal of cancer stem cells and associated with tumor malignancy, metastasis, and prognosis. Thus, many scientists propose that inhibition of EMT/CSC can repress the development of cancer by regulating TGF-β/Smad signaling and Bmi-1 expression. The reprogramming of metabolism, which is one of the hallmarkers of cancer cells, is crucial for cancer cell survival. Epidemiological data show that, the risk of incidence of certain cancers is higher in fat and diabetic individuals compared to those without diabetes. However, application of metformin reduce cancer incidence. These studies have demonstrated that, AMPK activators(AICAR, Phenformin, A769962) play inhibitory effect on proliferation and migration of cancer cells; LKB1, an AMPK upstream kinase, also has inhibitory effect on metastasis, AMPK have been acknowledged as a tumor suppressor. AMPK inactivation because of genetic(LKB1 mutation) and microenviroment alteration may cause tumorigenesis and metastasis. Activated AMPK enforces the inhibitory effect of the checkpoint of energy and cell cycle. However, the role of AMPK on metastasis and EMT has not been elucidated. In addition, the role of AMPK on TGF-β and Bmi-1 expression in cancer cells has not been well characterized.Therefore, we investiagated:(1) whether dynamic TGF-β signaling was associated with inactivation of AMPK;(2) whether AMPK activation could inhibit TGF-β signaling, EMT and Bmi-1 expression.Methods and materials 1. Cell lines:AGS, SGC-7901, A549, Bean, MEF, MDA-MB-231, NMu MG, MCF10 A. 2. To construct stable cell lines with abscent/high LKB1 expression, LKB1 sh RNA or functional LKB1 gene were transfected into the cells to silence or recover the LKB1 expression respectively, these cells then would be incubated with AMPK activators(metformin et al.)and TGF-β, Western blot was performed to test phosphorylation of AMPK, phosphorylation of Smads,and EMT markers. 3. After incorporation of AMPK dominate negative mutant or AMPK constitutive active mutant into the cells, Western blot was performed to check the phosphorylation of AMPK, phosphorylation of Smad,and EMT markers. 4. Q-PCR was performed to deterimine the effect of LKB1 expression or AMPK activators on the m RNA expression of TGF-β target genes. 5. Wound healing assay and Transwell assay were performed to compare cell migration ability. 6. The serum was collected from:(1)the patients with 2-DM which were devided into two groups based on metformin administration,(2) the mice before and after metformine intake, then ELISA assay was performed to determine TGF-β level in human and mouse serum. 7. Luciferase Assay was performed to determine the effect of AMPK activation on TGF-β transcription and Smad function.Results 1. AMPK can inhibit the TGF-β1 expression Luciferase analysis showed that the trancriptional activity of TGF-β decreases 50% after the treatment of metformin and phenformin. ELISA analysis of TGF-β1 level in human serum demonstrated that the TGF-β1 level in patients with metformin treatment is lower than those without metformin treatment. There is no statistic difference between them might be due to limited samples. In vivo, the blood TGF-β1 level was obviously decreased after the treatment of metformin in mice. 2. The inhibition of LKB1/AMPK on TGF-β signal transduction In A549-LKB1 cells and MCF10 A cells, metformin can diminish phosphorylation of Smad2/3 by activating of AMPK. Moreover, The phosphorylation of Smad2/3 was decreased after introduction of the constitutive active AMPK mutant into the A549, while the phosphorylation of AMPK is increased. After introduction of functional LKB1 gene into A549 and MDA-MB-231 which both are lack of LKB1 expression, the phosphorylation of Smad2/3 induced by TGF-β was decreased, revealing that LKB1 can repress TGF-β signal transduction. AMPK inactivation could increase TGF-β signal transduction; TGF-β induced Smad2/3 phosphorylation was shown to be enhaced after introduction of dominant negative AMPKα1 mutant into C4-2 cells, AMPKα1andα2 knockout in MEF cells, or LKB1/AMPKα1 knowout in MCF-10 A. 3. Inhibitory effect of LKB1/AMPK on the cell migration induced by TGF-β. Wound healing and transwell assay showed that, TGF-β expression promoted the migration ability of A549, NMu MG and AGS cells, which could be diminished by AMPK activator, such as Metformin and A769962. However, after the AMPK activity was suppressed by introduction of AMPKa1 dominant negative mutant into C4-2, the migration of C4-2 became faster, which could be reversed again by the induction of doxycycline treatment that can recover AMPK expression. In addition, knockout of LKB1 by sh RNA transfection could enhance the migration rate of BEAS-2B. 4. Inhibitory effect of AMPK on EMT induced by TGF-β After the stimulation of TGF-β with A549 cells, the epithelial marker E-cadherin was decreased, and mesenchymal marker N-cadherin and Slug were increased; To give phenformin and TGF-β1 simultaneously could reverse TGF-β1induced EMT; these findings were also proved in gastric cancer cell line AGS and mammary epithelial cells NMu MG. 5. LKB1/AMPK inhibitsthe expression of TGF-β targeted genes Q-PCR demonstrated that TGF-β could promote the expression of PAI-1, FN, CTGF and IL-6 genes but not metformin. However, metformin can inhibit theexpression of TGF-β targeted genes, especially in the cells with functional LKB1 gene. 6. The inhibition of AMPK on Bmi-1 expression p-AMPK expression was increased in A549 cells treated by metformin, while Bmi-1 epxression is diminished. In addition, ectopic expression of LKB1 diminished expression of Bmi-1, which was reversed by silencing LKB1. In addition, LITAF, AMPK expression, was increased in SGC-7901 and AGS after being treated with metformin. Metformin could upregulat the expression of four type of micro RNAs, including mi R-15 a, mi R-128, mi R-192 and mi R-194, which could repress Bmi-1 expression.Conclusion TGF-β is an important factor to promote cancer metastasis; LKB1/AMPK could inhibit TGF-β signaling transduction and its function. Activation of AMPK can repress TGF-β signaling, TGF-β mediated EMT, and inhibit the expression of metastasis related genes such as Bmi-1 and β-catinin, then delay tumor metastasis progression.