Using transcriptomics to identify and validate novel biomarkers of human skeletal muscle cancer cachexia

BackgroundCancer cachexia is a multi-organ tissue wasting syndrome that contributes to morbidity and mortality in many cancer patients. Skeletal muscle loss represents an established key feature yet there is no molecular understanding of the disease process. In fact, the postulated molecular regulators of cancer cachexia originate largely from pre-clinical models and it is unclear how these translate to the clinical environment.MethodsRectus abdominis muscle biopsies were obtained from 65 upper gastrointestinal (UGI) cancer patients during open surgery and RNA profiling was performed on a subset of this cohort (n = 21) using the Affymetrix U133+2 platform. Quantitative analysis revealed a gene signature, which underwent technical validation and independent confirmation in a separate clinical cohort.ResultsQuantitative significance analysis of microarrays produced an 83-gene signature that was able to identify patients with greater than 5% weight loss, while this molecular profile was unrelated to markers of systemic inflammation. Selected genes correlating with weight loss were validated using quantitative real-time PCR and independently studied as general cachexia biomarkers in diaphragm and vastus lateralis from a second cohort (n = 13; UGI cancer patients). CaMKIIβ correlated positively with weight loss in all muscle groups and CaMKII protein levels were elevated in rectus abdominis. TIE1 was also positively associated with weight loss in both rectus abdominis and vastus lateralis muscle groups while other biomarkers demonstrated tissue-specific expression patterns. Candidates selected from the pre-clinical literature, including FOXO protein and ubiquitin E3 ligases, were not related to weight loss in this human clinical study. Furthermore, promoter analysis identified that the 83 weight loss-associated genes had fewer FOXO binding sites than expected by chance.ConclusionWe were able to discover and validate new molecular biomarkers of human cancer cachexia. The exercise activated genes CaMKIIβ and TIE1 related positively to weight-loss across muscle groups, indicating that this cachexia signature is not simply due to patient inactivity. Indeed, excessive CaMKIIβ activation is a potential mechanism for reduced muscle protein synthesis. Our genomics analysis also supports the view that the available preclinical models do not accurately reflect the molecular characteristics of human muscle from cancer cachexia patients.

[1]  C. Begg,et al.  Prognostic effect of weight loss prior to chemotherapy in cancer patients. Eastern Cooperative Oncology Group. , 1980, The American journal of medicine.

[2]  A. Russell,et al.  Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin‐1 , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[3]  Jan Nedergaard,et al.  Myogenic gene expression signature establishes that brown and white adipocytes originate from distinct cell lineages , 2007, Proceedings of the National Academy of Sciences.

[4]  Joseph R. Bertino,et al.  Prognostic effect of weight loss prior to chemotherapy in cancer patients. Eastern Cooperative Oncology Group. , 1980, The American journal of medicine.

[5]  M. Tarnopolsky,et al.  Gene expression profiling in human skeletal muscle during recovery from eccentric exercise. , 2008, American journal of physiology. Regulatory, integrative and comparative physiology.

[6]  E. Olson,et al.  Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation , 2000, Nature.

[7]  R. Tibshirani,et al.  Significance analysis of microarrays applied to the ionizing radiation response , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Claes Wahlestedt,et al.  Human muscle gene expression responses to endurance training provide a novel perspective on Duchenne muscular dystrophy , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  M. Rennie,et al.  Disassociation between the effects of amino acids and insulin on signaling, ubiquitin ligases, and protein turnover in human muscle , 2008, American journal of physiology. Endocrinology and metabolism.

[10]  A. Goldberg,et al.  Coordinate activation of autophagy and the proteasome pathway by FoxO transcription factor , 2008, Autophagy.

[11]  M. Raney,et al.  Evidence for the involvement of CaMKII and AMPK in Ca2+-dependent signaling pathways regulating FA uptake and oxidation in contracting rodent muscle. , 2008, Journal of applied physiology.

[12]  A. Goldberg,et al.  Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  Rafael A. Irizarry,et al.  Bioinformatics and Computational Biology Solutions using R and Bioconductor , 2005 .

[14]  J. McCubrey,et al.  Redox Regulation of the Calcium/Calmodulin-dependent Protein Kinases* , 2004, Journal of Biological Chemistry.

[15]  E. Richter,et al.  A Ca2+–calmodulin–eEF2K–eEF2 signalling cascade, but not AMPK, contributes to the suppression of skeletal muscle protein synthesis during contractions , 2009, The Journal of physiology.

[16]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[17]  A. Goldberg,et al.  Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[18]  C. Mammucari,et al.  Downstream of Akt: FoxO3 and mTOR in the regulation of autophagy in skeletal muscle , 2008, Autophagy.

[19]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[20]  T. Fahey,et al.  Nutritional support of the cancer patient , 1992, JPEN. Journal of parenteral and enteral nutrition.

[21]  R. Bellantone,et al.  Increased muscle ubiquitin mRNA levels in gastric cancer patients. , 2001, American journal of physiology. Regulatory, integrative and comparative physiology.

[22]  A. Steensberg,et al.  Interleukin-6 production by contracting human skeletal muscle: autocrine regulation by IL-6. , 2003, Biochemical and biophysical research communications.

[23]  Gordon K. Smyth,et al.  limma: Linear Models for Microarray Data , 2005 .

[24]  T. Ueno,et al.  LC3 conjugation system in mammalian autophagy , 2004, The International Journal of Biochemistry & Cell Biology.

[25]  Rafael A Irizarry,et al.  Exploration, normalization, and summaries of high density oligonucleotide array probe level data. , 2003, Biostatistics.

[26]  Thomas Lengauer,et al.  Improved scoring of functional groups from gene expression data by decorrelating GO graph structure , 2006, Bioinform..

[27]  E. Chin.  The role of calcium and calcium/calmodulin-dependent kinases in skeletal muscle plasticity and mitochondrial biogenesis , 2004, The Proceedings of the Nutrition Society.

[28]  M. Tisdale.  Mechanisms of cancer cachexia. , 2009, Physiological reviews.

[29]  J. Davis.  Bioinformatics and Computational Biology Solutions Using R and Bioconductor , 2007 .

[30]  H. Friess,et al.  Activity of the Akt-dependent anabolic and catabolic pathways in muscle and liver samples in cancer-related cachexia , 2007, Journal of Molecular Medicine.

[31]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[32]  A. Russell,et al.  Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease. , 2007, American journal of respiratory and critical care medicine.

[33]  G. Gibson,et al.  Skeletal muscle mRNA levels for cathepsin B, but not components of the ubiquitin-proteasome pathway, are increased in patients with lung cancer referred for thoracotomy. , 2002, Clinical science.

[34]  M. Marinelli,et al.  Ubiquitin ligases MuRF1 and MAFbx in human skeletal muscle atrophy. , 2008, Joint, bone, spine : revue du rhumatisme.

[35]  Carl Johan Sundberg,et al.  Modulation of extracellular matrix genes reflects the magnitude of physiological adaptation to aerobic exercise training in humans , 2005, BMC Biology.

[36]  D J Glass,et al.  Identification of Ubiquitin Ligases Required for Skeletal Muscle Atrophy , 2001, Science.

[37]  D. Leroith,et al.  Skeletal muscle. , 2005, Advances in experimental medicine and biology.

[38]  Thomas N. Sato,et al.  Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation , 1995, Nature.

[39]  J. York,et al.  The Endothelial Receptor Tyrosine Kinase Tie1 Activates Phosphatidylinositol 3-Kinase and Akt To Inhibit Apoptosis , 2002, Molecular and Cellular Biology.

[40]  C. Dejong,et al.  Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss. , 2005, The international journal of biochemistry & cell biology.

[41]  Simon W. Jones,et al.  Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[42]  A. Goldberg,et al.  FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. , 2007, Cell metabolism.

[43]  J. Timmons,et al.  Dysregulation of Mitochondrial Dynamics and the Muscle Transcriptome in ICU Patients Suffering from Sepsis Induced Multiple Organ Failure , 2008, PloS one.

[44]  O. Ljungqvist,et al.  Increased expression of inflammatory pathway genes in skeletal muscle during surgery. , 2009, Clinical nutrition.

[45]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[46]  A. Goldberg,et al.  FoxO3 controls autophagy in skeletal muscle in vivo. , 2007, Cell metabolism.

[47]  B. Schoser,et al.  Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long‐term bed rest with and without countermeasures , 2008, Journal of anatomy.

[48]  Martin Hägglund,et al.  Atrogin-1/MAFbx and MuRF1 are downregulated in aging-related loss of skeletal muscle. , 2006, The journals of gerontology. Series A, Biological sciences and medical sciences.

[49]  Wei Wei,et al.  Treatment of cultured myotubes with the calcium ionophore A23187 increases proteasome activity via a CaMK II-caspase-calpain-dependent mechanism. , 2004, Surgery.

[50]  I. Macdonald,et al.  Substrate availability limits human skeletal muscle oxidative ATP regeneration at the onset of ischemic exercise. , 1998, The Journal of clinical investigation.

[51]  A. Goldberg,et al.  What do we really know about the ubiquitin-proteasome pathway in muscle atrophy? , 2001, Current opinion in clinical nutrition and metabolic care.

[52]  G. Filippatos,et al.  Studies on apoptosis and fibrosis in skeletal musculature: a comparison of heart failure patients with and without cardiac cachexia. , 2003, International journal of cardiology.

[53]  Ole Winther,et al.  JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update , 2007, Nucleic Acids Res..

[54]  V. Sukhatme,et al.  Suppression of Tie‐1 in endothelial cells in vitro induces a change in the genome‐wide expression profile reflecting an inflammatory function , 2009, FEBS letters.

[55]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[56]  Robert Gentleman,et al.  Using GOstats to test gene lists for GO term association , 2007, Bioinform..

[57]  J. Wojtaszewski,et al.  Effect of endurance exercise training on Ca2+–calmodulin‐dependent protein kinase II expression and signalling in skeletal muscle of humans , 2007, The Journal of physiology.

[58]  M. Kutner,et al.  Total parenteral nutrition as an adjunct to chemotherapy of metastatic colorectal cancer. , 1981, Cancer treatment reports.

[59]  R. Souhami,et al.  Protein synthesis in muscle measured in vivo in cachectic patients with cancer. , 1984, British medical journal.

[60]  T. Scherstén,et al.  Lysosomal enzyme activity in muscle tissue from patients with malignant tumor , 1972, Cancer.

[61]  Rickard Sandberg,et al.  Improved precision and accuracy for microarrays using updated probe set definitions , 2007, BMC Bioinformatics.

[62]  M. Muscaritoli,et al.  Increased Muscle Proteasome Activity Correlates With Disease Severity in Gastric Cancer Patients , 2003, Annals of surgery.

[63]  Szymon M. Kielbasa,et al.  Regulation of Clock-Controlled Genes in Mammals , 2009, PloS one.