CXCR4 Pathway Retards Muscle Atrophy During Cancer Cachexia
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Oncogene (2016) 35, 6212–6222 © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE Activation of the SDF1/CXCR4 pathway retards muscle atrophy during cancer cachexia GB Martinelli1, D Olivari1, AD Re Cecconi1, L Talamini1, L Ottoboni2, SH Lecker3, C Stretch4, VE Baracos4, OF Bathe5, A Resovi6, R Giavazzi1, L Cervo7 and R Piccirillo1 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 of cancer, diabetes, fasting, disuse or uremia led to the important Cancer cachexia is a highly debilitating syndrome, involving discovery of the atrogenes, in other words, genes that are severe body weight loss (BWL) due to depletion of skeletal muscle, differentially expressed in all kinds of muscle atrophy. Among with or without fat tissue loss.1 It cannot be corrected by them, Forkhead box-containing, subfamily O3 (FoxO3) is the nutritional supplementation2,3 and affects up to 80% of patients main transcription factor driving the expression of most of the with advanced cancers. The rapid loss of muscle mass is the main atrogenes, such as those implied in the lysosomal and proteaso- cause of functional impairment, fatigue and respiratory complica- mal pathways, which promote overall proteolysis.12,13 Two tions, ultimately leading to death in 20–48% of cases. To date, no muscle-restricted ubiquitin ligases, atrogin-1 and muscle RING treatment is available. finger protein 1 (MuRF1), are dramatically upregulated by FoxO3 in Various circulating inflammatory factors have been implicated all settings of muscle wasting, including cancer cachexia.14,15 in cancer cachexia, for example, tumor necrosis factor-α (TNFα) Molecules that block this activation of proteolysis or increase and interleukin 6 (IL6) that is increased in the serum of patients muscle protein synthesis might serve as pharmacological agents with advanced non-small-cell lung cancer,4 colon cancer,5 prostate to combat wasting. cancer6 and esophageal squamous cell cancer,7 but anticytokine Re-analysis of the same microarray datasets enabled us to therapies are ineffective.8,9 Moreover, these factors are common identify the genes specifically altered in rodent muscles because to other wasting syndromes, such as that caused by chronic of cancer and not because of diabetes, fasting, disuse or uremia. kidney diseases,10 and not restricted to cancer cachexia. Interestingly, three were drastically downregulated only in the To identify a genetic signature of muscle wasting typical of atrophying gastrocnemius of Yoshida hepatoma-bearing rats: cancer and not of other systemic (for example, uremia) or local (for stromal cell-derived factor 1 (SDF1) also named CXCL12, adenylate example, muscle disuse) dysfunctions, we re-analyzed previous cyclase 7 (ADCY7), and p21 protein-activated kinase 1 (PAK1). gene expression profiles generated by microarray.11 In that study, Ingenuity Pathway Analysis associated all of them to the C-X-C the gene expression comparison of muscles atrophying because chemokine receptor type 4 (CXCR4) pathway. 1Department of Oncology, IRCCS - Mario Negri Institute for Pharmacological Research, Milan, Italy; 2San Raffaele Scientific Institute, INSpe, Milan, Italy; 3Beth Israel Deaconess Center, Boston, MA, USA; 4Department of Oncology, University of Alberta, Edmonton, Alberta, Canada; 5Department of Surgery and Oncology, University of Calgary, Calgary, Alberta, Canada; 6Department of Oncology, Tumor Angiogenesis Unit, IRCCS - Mario Negri Institute for Pharmacological Research, Bergamo, Italy and 7Department 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: [email protected] Received 3 September 2015; revised 5 February 2016; accepted 11 March 2016; published online 23 May 2016 CXCR4 pathway has a role in cancer cachexia GB Martinelli et al 6213 CXCR4 is a Gαi-coupled member of G protein-coupled receptors conditions of muscle atrophy tested (uremia, disuse, fasting, (GPCRs) whose ligand SDF1 has a role in angiogenesis by diabetes) (P40.05; Figure 1a). recruiting endothelial progenitor cells from the bone marrow We identified 52 cancer cachexia-specific genes or expressed through CXCR4.16 SDF1 also has strong chemotactic activity for sequence tags that were further examined by Ingenuity Pathway lymphocytes, and CXCR4 was initially identified as the receptor Analysis, showing that CXCR4 pathway had the most significant required for HIV cell entry17 and found mutated in a rare enrichment (P = 0.012, ratio 3/158). Three genes of this pathway immunodeficiency disorder in humans.18 Antagonists of the (SDF1, ADCY7 and PAK1) were consistently downregulated in CXCR4 pathway are on the market and are used to mobilize cachectic muscles only during cancer (Figures 1b and c). Each one hematopoietic progenitor cells from bone marrow or under encodes for a protein of a different subcellular compartment. 19 investigation to restrain HIV infection and tumor dissemination. SDF1 is the extracellular ligand of CXCR4, ADCY7 is a calcium- Various splicing variants of SDF1 exist (α, β, γ), but their different inhibitable membrane-bound enzyme, while PAK1 is a cytosolic 20 functional roles have not yet been fully elucidated. The SDF1/ kinase downstream of the CXCR4 pathway.27 They were down- 21 CXCR4 pathway has a role in muscle formation in zebrafish, in regulated to 34, 1 and 20% of controls, respectively (Figure 1c). Xenopus22 and also in muscle regeneration in rodents23 as well as in muscle differentiation in vitro.24 However, nothing is known SDF1 expression is attenuated in the cachectic Tibialis Anterior about its role in muscle atrophy induced by cancer. muscle of C26-bearing mice Combining genetic approaches in adult murine muscles with fl pharmacological strategies in cultured myotubes, we investigated The suppression of this in ammatory pathway in cachectic fl whether and how the SDF1/CXCR4 pathway could have a muscles was surprising, as cancer cachexia involves in amma- 28 fi fi functional role in muscle wasting during cancer cachexia. Finally, tion. Thus, we rst validated this nding in the cachectic tibialis as we25 and others26 have reported that some angiogenic anterior (TA) of another species (Mus musculus) with another inhibitors reversed or even prevented cachexia in vivo without tumor (C26 colon adenocarcinoma) injected by a different route affecting tumor growth, we also assessed whether upregulation of (subcutaneous). Eight-week-old female and male C26-bearing muscular and/or circulating SDF1 was among the mechanisms by mice undergo BWL about 13 days after injection of C26 cells in the which these drugs might counteract cachexia. dorsal subcutis (Figure 2a and data not shown, respectively). In accordance with institutional guidelines, animals were killed when at least four out of five signs of distress (loss of mobility, kyphosis, RESULTS ruffled fur, low body temperature, tremor) were present or when Genes belonging to the CXCR4 pathway are downregulated 420% of BW was lost in 72 h. All C26-bearing animals had to be specifically in the atrophying gastrocnemius muscle of Yoshida killed by 18 days from tumor injection, when the tumor reached hepatoma-bearing rats about 600 mm3 (Figures 2b and c). We analyzed a previously generated mouse microarray data set By contrast,