EFFECTS OF METFORMIN AND GLUCOSE RESTRICTION ON THE INVASIVE CAPACITY OF HCC CELLS: MODULATING AMPK ACTIVATION

ALEJANDRO PEDRO PARIAN; FLORENCIA HIDALGO; MARÍA CECILIA LAROCCA; FACUNDO MAURO TONUCCI; CRISTIÁN FAVRE; EVANGELINA ALMADA; ANABELA CECILIA FERRETTI

Congreso; Reunión de Sociedades de Biociencias; 2017

AMPK is an antioncogenic kinase whose dysfunction has been relatedto hepatocellular carcinoma (HCC) etiology. We have demonstratedthat AMPK induces cell-cycle arrest and death in HCC cellsundergoing glucose restriction, and that AMPK is inhibited by PKAphosphorylation (S173), which prevents apoptotic death. AMPK decreasesmigration/invasion in different cell lines, but its effects inHCC cells remain unclear. We have previously shown that metforminand other AMPK activators decrease HCC cells migration. Metforminpossesses promising antitumor effects via AMPK and inhibitsPKA, as well.We aim to study AMPK role on migration/invasion of HCC cellstreated with metformin and its modulation by PKA.Invasion assays showed that 1 (M1) and 5 (M5) mM metformindecreased invasion of C3A (M1:52.3±11.8*, M5:16.5±9.3* %) andHuH-7 (M1:67.5±4.9*, M5:24.8±12.1* % vs C) cells. AMPK knockdownprevented this effect. Besides, metformin induced apoptoticdeath.When C3A cells were subjected to glucose starvation (GS), decreaseof invasion (M1GS:24.8±14.1* % vs M1) and apoptosis percentage(M5GS:71.5±3.4* vs M5:52.4±6.8 %) were enhanced.P-AMPKα(T172) and PKA phosphorylated substrates in celllysates were analyzed. Metformin increased AMPK activation ina dose dependent manner, and decreased PKA activity at 5 mM.AMPK activation was significantly potentiated when cells were incubatedwith M5 combined with GS, whilst PKA activity was almostundetectable.We performed invasion assays in C3A cells stable expressing WTand S173C forms of AMPK. S173C cells had lower invasion levels inresponse to AMPK activation (S173C: GS:36.5±3.9*, M1:16.5±1.3*,AICAR:4.0±0.4* % vs WT: GS:62.6±3.7, M1:51.9±7.4, AICAR:23.2±2.8%).We demonstrated that effects of metformin are enhanced byglucose restriction, and this is related, at least in part, to a greateractivation of AMPK associated to PKA inhibition. We conclude thatAMPK(S173) phosphorylation by PKA prevents AMPK anti-invasiveeffects. *p