Lingyu Luo

AMPK Inhibits the Stimulatory Effects of TGF-β on Smad2/3 Activity, Cell Migration, and Epithelial-to-Mesenchymal Transition

AMP-activated protein kinase (AMPK), an important downstream effector of the tumor suppressor liver kinase 1 (LKB1) and pharmacologic target of metformin, is well known to exert a preventive and inhibitory effect on tumorigenesis; however, its role in cancer progression and metastasis has not been well characterized. The present study investigates the potential roles of AMPK in inhibiting cancer-cell migration and epithelial-to-mesenchymal transition (EMT) by regulating the canonical transforming growth factor β (TGF-β) signaling pathway, an important promoting factor for cancer progression. Our results showed that activation of AMPK by metformin inhibited TGF-β–induced Smad2/3 phosphorylation in cancer cells in a dose-dependent manner. The effect of metformin is dependent on the presence of LKB1. A similar effect was obtained by expressing a constitutive active mutant of AMPKα1 subunit, whereas the expression of a dominant negative mutant of AMPKα1 or ablation of AMPKα subunits greatly enhanced TGF-β stimulation of Smad2/3 phosphorylation. As a consequence, expression of genes downstream of Smad2/3, including plasminogen activator inhibitor-1, fibronectin, and connective tissue growth factor, was suppressed by metformin in a LKB1-dependent fashion. In addition, metformin blocked TGF-β–induced inteleukin-6 expression through both LKB1-dependent and -independent mechanisms. Our results also indicate that activation of LKB1/AMPK inhibits TGF-β–stimulated cancer cell migration. Finally, TGF-β induction of EMT was inhibited by phenformin and enhanced by knockdown of LKB1 expression with shRNA. Together, our data suggest that AMPK could be a drug target for controlling cancer progression and metastasis.

ATM and LKB1 dependent activation of AMPK sensitizes cancer cells to etoposide-induced apoptosis.

The present study aims to determine the effect of AMPK on etoposide-induced apoptosis of cancer cells. Our results revealed that etoposide induced AMPK activation in prostate C4-2 cancer cells, an event that was attenuated by ATM siRNA. In A549 cells that lack LKB1, AMPK was unable to be activated by etoposide, which was restored by introduction of LKB1. Likewise, silencing LKB1 in C4-2 cells impaired AMPK activation. Finally, etoposide displayed a potent pro-apoptotic effect in cancer cells with functional LKB1 and AMPK. Thus, our results establish a linear relationship of ATM, LKB1 and AMPK in response to the DNA damage drug.

Role of the LKB1/AMPK pathway in tumor invasion and metastasis of cancer cells (Review)

Liver kinase B1 (LKB1), also known as serine/threo-nine kinase 11 (STK11), is a tumor suppressor that is inactivated in Peutz-Jeghers familial cancer syndrome. LKB1 phosphorylates and activates AMP-activated protein kinase (AMPK), which negatively regulates cancer cell proliferation and metabolism. However, recent evidence demonstrates that the LKB1/AMPK pathway is involved in the process of tumor invasion and migration, which is an important hallmark of carcinoma progression to higher pathological grades of malignancy. This review focuses on the function of the LKB1/AMPK pathway in the invasion and migration of cancer cells and provides an overview of therapeutic strategies aimed at this pathway in malignant tumors.

MLK3 Phophorylates AMPK Independently of LKB1

Emerging evidence has shown that cellular energy metabolism is regulated by the AMPK and MLK3-JNK signaling pathways, but the functional link between them remains to be determined. The present study aimed to explore the crosstalk between MLK3 and AMPK. We found that both JNK and AMPK were phosphorylated at their activation sites by TNF-α, Anisomycin, H2O2 and sorbitol. Interestingly, sorbitol stimulated phosphorylation of AMPK at T172 in LKB1-deficient cells. Following the screening of more than 100 kinases, we identified that MLK3 induced phosphorylation of AMPK at T172. Our in vitro analysis further revealed that MLK3-mediated phosphorylation of AMPK at T172 was independent of AMP, but addition of AMP caused a mobility shift of AMPK, an indication of autophosphorylation, suggesting that AMP binding and phosphorylation of T172 leads to maximal activation of AMPK. GST-pull down assays showed a direct interaction between AMPKα1 subunit and MLK3. Altogether, our results indicate that MLK3 serves as a common upstream kinase of AMPK and JNK and functions as a direct upstream kinase for AMPK independent of LKB1.

Negative regulation of Bmi-1 by AMPK and implication in cancer progression

Bmi-1 is a transcriptional regulator that promotes tumor cell self-renewal and epithelial to mesenchymal transition and its upregulation is associated with tumor progression, AMPK is an intracellular fuel-sensing enzyme and plays important roles in tumor cell growth and progression. Thus, the present study aims to examine the regulation of Bmi-1 by AMPK. First, our data revealed that, as compared to adjacent normal tissue, Bmi-1 was highly expressed in gastric cancer, whereas phosphorylation of AMPK (p-AMPK) was reduced. Similar findings were observed in lung adenocarcinomas and appeared that the expression of Bmi-1 was correlated with pathological grades of the cancer, where opposite changes were found in p-AMPK. Second, Metformin, a pharmacological AMPK activator and anti-diabetic drug, or ectopic expression of LKB1, diminished expression of Bmi-1 in cancer cells, an event that was reversed by silencing LKB1. Third, knockdown of LITAF, previously identified as a downstream target of AMPK, upregulated Bmi-1, associated with increased cell viability, colony formation, and migration of cancer cells in vitro. Fourth, metformin increased the abundance of miR-15a, miR-128, miR-192, and miR-194, which was prevented by knockdown of LITAF. Accordingly, transfection of these individual miRNAs downregulated Bmi-1. Altogether, our data for the first time suggest a regulatory axis in cancer cells: AMPK upregulates LITAF, which in turn increases miRNAs, leading to attenuation of Bmi-1 expression.

Metformin and berberine, two versatile drugs in treatment of common metabolic diseases

Metformin has been used as a glucose lowering drug for several centuries and is now a first-line drug for type 2 diabetes mellitus (T2DM). Since the discovery that it activates AMP-activated protein kinase (AMPK) and reduces risk of cancer, metformin has drawn great attentions. Another drug, berberine, extracted from berberis vulgaris L. (root), was an ancient herbal medicine in treating diarrhea. Ongoing experimental and clinical studies have illuminated great potential of berberine in regulation of glucose and lipid homeostasis, cancer growth and inflammation. Furthermore, the lipid lowering effect of berberine is comparable to those conventional lipid drugs but with low toxicity. Therefore, it is right time to transform beneficial effects of berberine into therapeutic practice. Metformin and berberine share many features in actions despite different structure and both could be excellent drugs in treating T2DM, obesity, cardiac diseases, tumour, as well as inflammation. Since these disorders are often connected and comprise common pathogenic factors that could be targeted by the two drugs, understanding their actions can give us rationale for expansion of their clinical uses.

Down-regulation of adenosine monophosphate–activated protein kinase activity: A driver of cancer

Adenosine monophosphate–activated protein kinase (AMPK), a serine/threonine protein kinase, is known as “intracellular energy sensor and regulator.” AMPK regulates multiple cellular processes including protein and lipid synthesis, cell proliferation, invasion, migration, and apoptosis. Moreover, AMPK plays a key role in the regulation of “Warburg effect” in cancer cells. AMPK activity is down-regulated in most tumor tissues compared with the corresponding adjacent paracancerous or normal tissues, indicating that the decline in AMPK activity is closely associated with the development and progression of cancer. Therefore, understanding the mechanism of AMPK deactivation during cancer progression is of pivotal importance as it may identify AMPK as a valid therapeutic target for cancer treatment. Here, we review the mechanisms by which AMPK is down-regulated in cancer.

Mechanisms of AMPK in the maintenance of ATP balance during energy metabolism: AMPK and ATP balance

AMP‐activated protein kinase (AMPK) is a conserved sensor of cellular energy change and is activated by increased AMP/ATP and/or ADP/ATP ratios. AMPK maintains the energy balance by decreasing the ATP‐consuming processes such as transcription of synthetic fat genes and rRNA, the translation of ribosomal proteins, synthesis of cholesterol and fatty acid, while the metabolic pathways such as glucose and fatty transport, fatty acid oxidation, autophagy, mitochondrial synthesis and oxidative metabolism are increased to preserve ATP during energy deficiency. Recent advance has demonstrated that AMPK activity has a close association with the initiation and progression in various cancers. Here we review the mechanisms that AMPK controls energy metabolism through regulating ATP synthesis and consumption, and further discuss the deregulation of AMPK in cancers.

AMPK downregulates ALK2 via increasing the interaction between Smurf1 and Smad6, leading to inhibition of osteogenic differentiation

Activin A receptor type I or activin receptor-like kinase 2 (ACVRI/ALK2) belongs to type I TGF-β family and plays an important role in bone development. Activating mutations of ALK2 containing the R206 to H mutation, are present in 95% in the rare autosomal genetic disease fibrodysplasia ossificans progressiva (FOP), which leads to the development of ectopic bone formation in muscle. The effect of AMP-activated protein kinase (AMPK) activation on ALK2R206H-mediated signaling in fibroblasts obtained from a FOP patient was assessed in the present study. The activity of the mutated ALK2 was suppressed by pharmacological AMPK activators such as metformin and aspirin, while their actions were blocked by the dominant negative mutant of AMPK and mimicked by the constitutively active mutant of AMPK. Furthermore, activation of AMPK upregulated Smad6 and Smurf1 and thereby enhanced their interactions, resulting in its proteosome-dependent degradation of ALK2. In contrast, knockdown of Smad6 or Smurf1 prevented metformin-induced reduction of ALK2. To evaluate the biological relevance of AMPK action on ALK2 activity, we induced FOP fibroblasts into iPS cells and found that their osteogenic differentiation in vitro was inhibited by metformin. Our studies provide novel insight into potential approaches to treatment of FOP, since several AMPK activators (e.g. metformin, berberine, and aspirin) are already in clinical use for the treatment of diabetes and metabolic syndromes.

Clinical significance and role of LKB1 in gastric cancer.

Liver kinase B1 (LKB1) functions as a tumor suppressor gene, and loss in the expression of LKB1 contributes to human carcinogenesis and tumor progression. The present study investigated the association between LKB1 and gastric cancer. SGC‑7901 gastric cancer cell lines and 63 patients with gastric cancer were examined in the present study, and lentivirus transfection, reverse transription‑quantitative polymerase chain reaction and flow cytometric analyses were performed. By examining the expression of LKB1 using immunohistochemical analyses, the present study found that the expression of LKB1 was reduced in the gastric cancer tissues, and restoration of the expression of LKB1 reduced tumor cell viability, migration rate and the expression of CD44, induced cell cycle arrest at the G2 phase of the cell cycle, and increased the sensitivity of the gastric cancer cells to anticancer drugs. LKB1 protein is a tumor‑suppressor in gastric cancer and may be potentially be developed as a novel gene therapy target in the treatment of gastric cancer.