The AMPK signalling pathway in cancer and DNA damage

Abstract

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and metabolism, both at the cellular and at the whole body level. It acts as a cellular fuel gauge, sensing changes in AMP: ATP or ADP: ATP ratios. Once activated by energy deficit, AMPK switches on catabolic processes, generating ATP, and inhibits biosynthetic processes, consuming ATP, thus conserving cellular energy. AMPK also regulates metabolism at the whole-body level, for example, by controlling appetite and entraining feeding with the circadian clock. AMPK regulates the cell cycle and modulates neuronal excitability. In recent years, it has been found that AMPK acts downstream of the tumour suppressor LKB1, that the AMPK signalling pathway is down-regulated in some tumours, and that diabetics treated with the AMPK-activating drug metformin exhibit a lower incidence of cancer. These results suggest that AMPK may itself be a tumour suppressor, although direct evidence has so far been lacking.

This thesis provides genetic evidence that AMPK functions as a tumour suppressor in vivo. A T cell-specific mouse tumour model was developed to delineate the role of AMPK in cancer. T cell-specific deletion of PTEN in mice causes development of T cell lymphomas at a median age of 85 days (43-214 days), while T cell-specific deletion of AMPK as well as PTEN caused development of lymphomas much sooner, at a median age of 65 days (46-87days, p<0.0001, hazard ratio = 6.3). The distribution of tumours was also significantly different between the two groups; the PTEN-null mice developed lymphomas in the thymus as well as in peripheral lymphoid organs such as lymph nodes and spleen, while the PTEN and AMPK double null lymphomas were largely confined to the thymus (p<0.001). Before the onset of tumours, the PTEN AMPK double null T cells were larger and had greatly increased phospho-S6 levels compared to wild type and PTEN null T cells. These results suggest that AMPK delays tumour onset in cells with an overactive PI3K pathway by downregulating the mTOR pathway.

Recent studies have also suggested a role for AMPK in the response to DNA damage, produced by treatments such as ionising radiation and the anti-cancer drug etoposide. However, the molecular mechanism underlying this activation was unclear. This thesis shows that etoposide activates AMPK in a CaMKKβ-dependent manner in HeLa cells and G361 cells, both of which lack LKB1. Activation is restricted to the AMPK-α1 isoform within the nucleus, and this is associated with increased nuclear calcium flux. Taken together the results suggest that release of intra-nuclear calcium activates CaMKK-β, which in turn activates AMPK-α1. In addition, activation of AMPK by the calcium ionophore A23187 increased the survival of mouse embryonic fibroblasts treated with etoposide in an AMPK-dependent manner.

This thesis also investigates the role of C-terminal phosphorylation of LKB1 on Ser-325 and Ser-431. The results presented in this thesis show that mutation of these sites to alanine, did not affect activation of AMPK by LKB1. It also shows that ERK2 does not phosphorylate LKB1 on Ser-325 efficiently, and that phosphorylation of Ser431 by p90RSK does not affect activation of AMPK. These results do not support the view that phosphorylation of these two residues in the C-terminal tail of LKB1 inhibits AMPK activation.

Overall, the studies performed in this thesis demonstrate that AMPK can be both a “friend” and a “foe” in cancer; On the one hand, it functions as a tumour suppressor delaying the onset of cancer, while on the other hand, it increases the survival of cancer cells treated with the anti-cancer drug etoposide. These distinct effects of AMPK might be exploited therapeutically, both in the prevention and in the treatment of cancer.

Date of Award	2013
Original language	English
Sponsors	Wellcome Trust
Supervisor	Grahame Hardie (Supervisor)