Prognostic significance of AMPK activation in advanced stage colorectal cancer treated with chemotherapy plus bevacizumab

Colorectal cancer (CRC) is the third most common tumour worldwide and represents the second leading cause of cancer death in Europe and the USA. Approximately 20% of CRC patients will present distant metastasis (stage IV) at the time of diagnosis with 5-year survival <10% (Jessup et al, 1996). Management of metastatic CRC (mCRC) has substantially evolved over the past decade with the introduction of new chemotherapy combinations and biological agents. The US Food and Drug Administration approved the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab for first-line treatment of patients with mCRC in 2004 (Hurwitz et al, 2004). Since its approval, bevacizumab in combination with chemotherapy has become the standard first-line treatment option in mCRC, extending both progression-free survival (PFS) and overall survival (OS) (Hurwitz et al, 2004; Kabbinavar et al, 2005; Saltz et al, 2008). Despite clinical use of bevacizumab for nearly a decade, however, important questions still remain, including the prediction and management of potential adverse effects and selection of the ideal patients' population. In this regard, predictive biomarkers of response are still not available, although a number of potential systemic, circulating, tissue and imaging biomarkers have emerged from recently completed clinical trials (Jubb and Harris, 2010). Observations in patients (Faivre et al, 2007; Mehta et al, 2011) and preclinical findings (Nardo et al, 2011) both suggest that VEGF blockade may perturb the energy balance of cancer cells. Several groups have looked at metabolic perturbations associated with anti-angiogenic therapy by using either magnetic resonance spectroscopy (Keunen et al, 2011) or hyperpolarised 13C magnetic resonance spectroscopy (Bohndiek et al, 2012), reporting increased lactate levels and production of labelled malate from fumarate, an early indicator of cell necrosis. In line with these findings, we showed for the first time that anti-VEGF therapy causes metabolic perturbations in experimental tumours, including marked reduction in glucose and ATP levels (Nardo et al, 2011). Moreover, probably as consequence of marked glucose depletion and exhaustion of ATP levels, we also observed that anti-angiogenic therapy increased AMP-activated protein kinase (AMPK) activation in tumour xenografts and that tumour cells lacking AMPK activity undergo marked tumour necrosis following a short-term course of anti-VEGF therapy (Nardo et al, 2011). AMPK is a heterotrimeric serine/threonine protein kinase, and is activated following its phosphorylation by its upstream kinase liver kinase B1 (LKB1) (also known as STK11) in response to an increase in cellular AMP/ATP ratio (Shackelford and Shaw, 2009). AMPK has a central role in the regulation of energy metabolism in all eukaryotes, and governs glucose and lipid metabolism in response to alterations in nutrients and intracellular energy levels, contributing to maintain the steady-state levels of intracellular ATP (Hardie et al, 2012). Phosphorylation of acetyl-CoA carboxylases by AMPK contributes to regulation of fatty acid metabolism (Hardie et al, 2012) and is considered an indirect read-out of AMPK activation. We hypothesise that the integrity of signalling pathways involved in the control of cell metabolism, growth and quiescence, such as AMPK, could be important to sense changes in the tumour microenvironment caused by angiogenesis inhibition and to enable tumour cells adaptation. Accordingly, tumour cells lacking AMPK activity are expected to undergo necrosis following anti-angiogenic therapy, due to their incapacity to maintain ATP levels. Although in patients the association between tumour necrosis and outcome of anti-angiogenic therapy is not firmly established, in preclinical models necrosis often leads to increased recruitment into tumours of macrophages and other specialised subsets of myeloid cells which can promote tumour angiogenesis and leads to escape from VEGF blockade (Ferrara, 2010). Therefore, defective AMPK activity might be predicted to associate initially with improved tumour response, followed by increased resistance to VEGF neutralisation. Several studies investigated the prognostic role of AMPK in human malignancies, including colorectal, lung, ovarian, gastric cancer, renal cell carcinoma and hepatocarcinoma (Baba et al, 2010; Tsavachidou-Fenner et al, 2010; Buckendahl et al, 2011; Kang et al, 2012; William et al, 2012; Zheng et al, 2013). In most of these studies—except gastric cancer (Kang et al, 2012)—low levels of AMPK activation correlated with poor prognosis. With regard to targeted therapy, one study reported that low phosphorylated AMPK (pAMPK) levels are associated with poor response to bevacizumab and interferon-α therapy in renal cell carcinoma (Tsavachidou-Fenner et al, 2010). Based on this rationale and the paucity of studies investigating the predictive value of AMPK activation in patients treated with anti-angiogenic drugs, we designed a pilot retrospective study to analyse activation of the AMPK pathway in CRC patients treated with bevacizumab plus chemotherapy and to correlate it with patient clinical data and survival.