AMPK Causes Cell Cycle Arrest in LKB1-Deficient Cells via Activation of CAMKK2

Abstract

The AMP-activated protein kinase (AMPK) is activated by phosphorylation at Thr172, either by the tumor suppressor kinase LKB1 or by an alternate pathway involving the Ca 2þ /calmodulindependent kinase, CAMKK2. Increases in AMP:ATP and ADP:ATP ratios, signifying energy deficit, promote allosteric activation and net Thr172 phosphorylation mediated by LKB1, so that the LKB1-AMPK pathway acts as an energy sensor. Many tumor cells carry loss-of-function mutations in the STK11 gene encoding LKB1, but LKB1 reexpression in these cells causes cell-cycle arrest. Therefore, it was investigated as to whether arrest by LKB1 is caused by activation of AMPK or of one of the AMPK-related kinases, which are also dependent on LKB1 but are not activated by CAMKK2. In three LKB1-null tumor cell lines, treatment with the Ca 2þ ionophore A23187 caused a G 1 arrest that correlated with AMPK activation and Thr172 phosphorylation. In G361 cells, expression of a truncated, Ca 2þ /calmodulin-independent CAMKK2 mutant also caused G 1 arrest similar to that caused by expression of LKB1, while expression of a dominant-negative AMPK mutant, or a double knockout of both AMPK-a subunits, also prevented the cell-cycle arrest caused by A23187. These mechanistic findings confirm that AMPK activation triggers cell-cycle arrest, and also suggest that the rapid proliferation of LKB1-null tumor cells is due to lack of the restraining influence of AMPK. However, cell-cycle arrest can be restored by reexpressing LKB1 or a constitutively active CAMKK2, or by pharmacologic agents that increase intracellular Ca 2þ and thus activate endogenous CAMKK2.Implications: Evidence here reveals that the rapid growth and proliferation of cancer cells lacking the tumor suppressor LKB1 is due to reduced activity of AMPK, and suggests a therapeutic approach by which this block might be circumvented.

Genotoxic Damage Activates the AMPK-α1 Isoform in the Nucleus via Ca2+/CaMKK2 Signaling to Enhance Tumor Cell Survival

“…7D). We have shown previously that A23187 treatment of G361 cells causes a G 1 arrest that is abolished in AMPK DKO cells (22). Supplementary Figure S4 confirms that, similar to HeLa cells, prior treatment with A23187 protected G361 cells against cell death induced by etoposide in clonogenic survival assays, with the effect being abolished in DKO cells.…”

“…AMPK activation using the pharmacologic activator 5-aminoimidazole-4-carboxamide ribonucleoside (46), or by glucose deprivation or overexpression of a mutant (T172D) AMPK kinase domain (47), causes cell-cycle arrest in G 1 phase. We have recently confirmed that these effects of AMPK activators, which are associated with increased expression of the cyclin-dependent kinase inhibitor p21 (CDKN1A), are AMPK dependent as they were abolished by a double knockout of AMPK (22). A cell-cycle arrest in G 1 phase would limit the entry of cells into S-phase, where they are particularly vulnerable to the generation of double-stranded DNA breaks induced by etoposide while DNA is being replicated.…”

“…Knockout of AMPK-a1 and a2 (PRKAA1 and PRKAA2) in G361 and HeLa cells was carried out using the CRISPR-Cas9 method, as described previously for G361 cells (22).…”

MARK2 inhibits the growth of HeLa cells through AMPK and reverses epithelial-mesenchymal transition

“…In addition, the HeLa cell line is severely impaired in LKB1 activity (30). The present study showed that inducible expression of MARK2 in HeLa cells was sufficient to mimic LKB1 signaling activation as previously reported (12)(13)(14), and further confirms that MARK2 is a critical downstream target of LKB1.…”

“…When cellular AMP:ATP ratios rise, LKB1 directly phosphorylates the threonine 172 (Thr172) in the activation loop of AMPK and results in an increase in ATP-producing activities and a decrease in ATP-consuming processes including essentially all biosynthetic pathways required for cell growth (10,11). Previous investigations have shown that activation of the LKB1-AMPK pathway is able to suppress cell growth by arresting the cell cycle in the G1 phase (12,13). In a similar manner, we confirmed and extended these observations by demonstrating that exogenous activation of LKB1-AMPK signaling induces downregulation of cyclins (cyclin D1 and D3) and upregulation of the cyclin-dependent kinase (CDK) inhibitors (p53, p21 and p16), and thus, inhibits G1/S cell cycle transition, even in cells with endogenous expression of LKB1 (14).…”