ORIGINAL ARTICLE Activation of the SDF1/CXCR4 pathway retards muscle atrophy during cancer cachexia GB Martinelli 1 , D Olivari 1 , AD Re Cecconi 1 , L Talamini 1 , L Ottoboni 2 , SH Lecker 3 , C Stretch 4 , VE Baracos 4 , OF Bathe 5 , A Resovi 6 , R Giavazzi 1 , L Cervo 7 and R Piccirillo 1 Cancer cachexia is a life-threatening syndrome that affects most patients with advanced cancers and causes severe body weight loss, with rapid depletion of skeletal muscle. No treatment is available. We analyzed microarray data sets to identify a subset of genes whose expression is specifically altered in cachectic muscles of Yoshida hepatoma-bearing rodents but not in those with diabetes, disuse, uremia or fasting. Ingenuity Pathways Analysis indicated that three genes belonging to the C-X-C motif chemokine receptor 4 (CXCR4) pathway were downregulated only in muscles atrophying because of cancer: stromal cell-derived factor 1 (SDF1), adenylate cyclase 7 (ADCY7), and p21 protein-activated kinase 1 (PAK1). Notably, we found that, in the Rectus Abdominis muscle of cancer patients, the expression of SDF1 and CXCR4 was inversely correlated with that of two ubiquitin ligases induced in muscle wasting, atrogin-1 and MuRF1, suggesting a possible clinical relevance of this pathway. The expression of all main SDF1 isoforms (α, β, γ) also declined in Tibialis Anterior muscle from cachectic mice bearing murine colon adenocarcinoma or human renal cancer and drugs with anticachexia properties restored their expression. Overexpressing genes of this pathway (that is, SDF1 or CXCR4) in cachectic muscles increased the fiber area by 20%, protecting them from wasting. Similarly, atrophying myotubes treated with either SDF1α or SDF1β had greater total protein content, resulting from reduced degradation of overall long-lived proteins. However, inhibiting CXCR4 signaling with the antagonist AMD3100 did not affect protein homeostasis in atrophying myotubes, whereas normal myotubes treated with AMD3100 showed time- and dose-dependent reductions in diameter, until a plateau, and lower total protein content. This further confirms the involvement of a saturable pathway (that is, CXCR4). Overall, these findings support the idea that activating the CXCR4 pathway in muscle suppresses the deleterious wasting associated with cancer. Oncogene (2016) 35, 6212–6222; doi:10.1038/onc.2016.153; published online 23 May 2016 INTRODUCTION Cancer cachexia is a highly debilitating syndrome, involving severe body weight loss (BWL) due to depletion of skeletal muscle, with or without fat tissue loss. 1 It cannot be corrected by nutritional supplementation 2,3 and affects up to 80% of patients with advanced cancers. The rapid loss of muscle mass is the main cause of functional impairment, fatigue and respiratory complica- tions, ultimately leading to death in 20–48% of cases. To date, no treatment is available. Various circulating inflammatory factors have been implicated in cancer cachexia, for example, tumor necrosis factor-α (TNFα) and interleukin 6 (IL6) that is increased in the serum of patients with advanced non-small-cell lung cancer, 4 colon cancer, 5 prostate cancer 6 and esophageal squamous cell cancer, 7 but anticytokine therapies are ineffective. 8,9 Moreover, these factors are common to other wasting syndromes, such as that caused by chronic kidney diseases, 10 and not restricted to cancer cachexia. To identify a genetic signature of muscle wasting typical of cancer and not of other systemic (for example, uremia) or local (for example, muscle disuse) dysfunctions, we re-analyzed previous gene expression profiles generated by microarray. 11 In that study, the gene expression comparison of muscles atrophying because of cancer, diabetes, fasting, disuse or uremia led to the important discovery of the atrogenes, in other words, genes that are differentially expressed in all kinds of muscle atrophy. Among them, Forkhead box-containing, subfamily O3 (FoxO3) is the main transcription factor driving the expression of most of the atrogenes, such as those implied in the lysosomal and proteaso- mal pathways, which promote overall proteolysis. 12,13 Two muscle-restricted ubiquitin ligases, atrogin-1 and muscle RING finger protein 1 (MuRF1), are dramatically upregulated by FoxO3 in all settings of muscle wasting, including cancer cachexia. 14,15 Molecules that block this activation of proteolysis or increase muscle protein synthesis might serve as pharmacological agents to combat wasting. Re-analysis of the same microarray datasets enabled us to identify the genes specifically altered in rodent muscles because of cancer and not because of diabetes, fasting, disuse or uremia. Interestingly, three were drastically downregulated only in the atrophying gastrocnemius of Yoshida hepatoma-bearing rats: stromal cell-derived factor 1 (SDF1) also named CXCL12, adenylate cyclase 7 (ADCY7), and p21 protein-activated kinase 1 (PAK1). Ingenuity Pathway Analysis associated all of them to the C-X-C chemokine receptor type 4 (CXCR4) pathway. 1 Department of Oncology, IRCCS - Mario Negri Institute for Pharmacological Research, Milan, Italy; 2 San Raffaele Scientific Institute, INSpe, Milan, Italy; 3 Beth Israel Deaconess Center, Boston, MA, USA; 4 Department of Oncology, University of Alberta, Edmonton, Alberta, Canada; 5 Department of Surgery and Oncology, University of Calgary, Calgary, Alberta, Canada; 6 Department of Oncology, Tumor Angiogenesis Unit, IRCCS - Mario Negri Institute for Pharmacological Research, Bergamo, Italy and 7 Department of Neuroscience, IRCCS - Mario Negri Institute for Pharmacological Research, Milan, Italy. Correspondence: Dr R Piccirillo, Department of Oncology, IRCCS - Mario Negri Institute for Pharmacological Research, Via G La Masa 19, 20156 Milan, Italy. E-mail: rosanna.piccirillo@marionegri.it Received 3 September 2015; revised 5 February 2016; accepted 11 March 2016; published online 23 May 2016 Oncogene (2016) 35, 6212 – 6222 © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0950-9232/16 www.nature.com/onc