Histological evaluation of AMPK signalling in primary breast cancer

BackgroundAMP-activated protein kinase (AMPK) acts as a cellular fuel gauge that responds to energy stress by suppressing cell growth and biosynthetic processes, thus ensuring that energy-consuming processes proceed only if there are sufficient metabolic resources. Malfunction of the AMPK pathway may allow cancer cells to undergo uncontrolled proliferation irrespective of their molecular energy levels. The aim of this study was to examine the state of AMPK phosphorylation histologically in primary breast cancer in relation to clinical and pathological parameters.MethodsImmunohistochemistry was performed using antibodies to phospho-AMPK (pAMPK), phospho-Acetyl Co-A Carboxylase (pACC) an established target for AMPK, HER2, ERα, and Ki67 on Tissue Micro-Array (TMA) slides of two cohorts of 117 and 237 primary breast cancers. The quick score method was used for scoring and patterns of protein expression were compared with clinical and pathological data, including a minimum 5 years follow up.ResultsReduced signal, compared with the strong expression in normal breast epithelium, using a pAMPK antibody was demonstrated in 101/113 (89.4%) and 217/236 (91.9%) of two cohorts of patients. pACC was significantly associated with pAMPK expression (p = 0.007 & p = 0.014 respectively). For both cohorts, reduced pAMPK signal was significantly associated with higher histological grade (p = 0.010 & p = 0.021 respectively) and axillary node metastasis (p = 0.061 & p = 0.039 respectively). No significant association was found between pAMPK and any of HER2, ERα, or Ki67 expression, disease-free survival or overall survival.ConclusionThis study extends in vitro evidence through immunohistochemistry to confirm that AMPK is dysfunctional in primary breast cancer. Reduced signalling via the AMPK pathway, and the inverse relationship with histological grade and axillary node metastasis, suggests that AMPK re-activation could have therapeutic potential in breast cancer.

[1]  R. DePinho,et al.  The Kinase LKB1 Mediates Glucose Homeostasis in Liver and Therapeutic Effects of Metformin , 2005, Science.

[2]  L. Goldstein,et al.  Polychemotherapy for early breast cancer: results from the international adjuvant breast cancer chemotherapy randomized trial. , 2007, Journal of the National Cancer Institute.

[3]  J. McGarry.  Banting lecture 2001: dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. , 2002, Diabetes.

[4]  D. Hanahan,et al.  The Hallmarks of Cancer , 2000, Cell.

[5]  A. Thompson,et al.  Targeting AMPK: a new therapeutic opportunity in breast cancer. , 2008, Critical reviews in oncology/hematology.

[6]  W Godolphin,et al.  Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. , 1989, Science.

[7]  N. Sonenberg,et al.  Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. , 2007, Cancer research.

[8]  F. Cognetti,et al.  Re: Polychemotherapy for early breast cancer: results from the international adjuvant breast cancer chemotherapy randomized Trial. , 2007, Journal of the National Cancer Institute.

[9]  D. Vertommen,et al.  Activation of AMP-Activated Protein Kinase Leads to the Phosphorylation of Elongation Factor 2 and an Inhibition of Protein Synthesis , 2002, Current Biology.

[10]  David Carling,et al.  Supplemental Data LKB 1 Is the Upstream Kinase in the AMP-Activated Protein Kinase Cascade , 2003 .

[11]  Demetrios Vavvas,et al.  Malonyl-CoA, fuel sensing, and insulin resistance. , 1999, American journal of physiology. Endocrinology and metabolism.

[12]  S. Cannistra,et al.  Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas. , 2001, The American journal of pathology.

[13]  R. Ceddia,et al.  Direct metabolic regulation in skeletal muscle and fat tissue by leptin: implications for glucose and fatty acids homeostasis , 2005, International Journal of Obesity.

[14]  H. Morkoç,et al.  On the incorporation of Mg and the role of oxygen, silicon, and hydrogen in GaN prepared by reactive molecular beam epitaxy , 1997 .

[15]  D. Hardie,et al.  The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. , 2002, Diabetes.

[16]  D. Hardie,et al.  AMP-activated protein kinase is activated by low glucose in cell lines derived from pancreatic beta cells, and may regulate insulin release. , 1998, The Biochemical journal.

[17]  I. Ellis,et al.  Assessment of the new proliferation marker MIB1 in breast carcinoma using image analysis: associations with other prognostic factors and survival. , 1995, British Journal of Cancer.

[18]  D. Hardie,et al.  Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A. , 1997, Journal of applied physiology.

[19]  E. Araki,et al.  AMPK and cell proliferation – AMPK as a therapeutic target for atherosclerosis and cancer , 2006, The Journal of physiology.

[20]  D. Hardie,et al.  Management of cellular energy by the AMP‐activated protein kinase system , 2003, FEBS letters.

[21]  W. McGuire,et al.  Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. , 1987, Science.

[22]  Jérôme Boudeau,et al.  LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR‐1 , 2004, The EMBO journal.

[23]  B. Viollet,et al.  Mechanism of Action of A-769662, a Valuable Tool for Activation of AMP-activated Protein Kinase* , 2007, Journal of Biological Chemistry.

[24]  G. Lopaschuk,et al.  High Rates of Fatty Acid Oxidation during Reperfusion of Ischemic Hearts Are Associated with a Decrease in Malonyl-CoA Levels Due to an Increase in 5′-AMP-activated Protein Kinase Inhibition of Acetyl-CoA Carboxylase (*) , 1995, The Journal of Biological Chemistry.

[25]  L. Bertrand,et al.  Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes. , 2002, European journal of biochemistry.

[26]  K. Kim,et al.  Critical phosphorylation sites for acetyl-CoA carboxylase activity. , 1994, The Journal of biological chemistry.

[27]  J. McGarry.  Dysregulation of Fatty Acid Metabolism in the Etiology of Type 2 Diabetes , 2001 .

[28]  B. Kemp.  Faculty Opinions recommendation of Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. , 2003 .

[29]  N. Kimura,et al.  A possible linkage between AMP‐activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway , 2003, Genes to cells : devoted to molecular & cellular mechanisms.

[30]  S. Majumdar,et al.  Increased Cancer-Related Mortality for Patients With Type 2 Diabetes Who Use Sulfonylureas or Insulin , 2006, Diabetes Care.

[31]  A. Paradiso,et al.  Breast cancer: biological characteristics in postmenopausal type 2 diabetic women. Identification of therapeutic targets. , 2003, Current drug targets. Immune, endocrine and metabolic disorders.

[32]  Margaret S. Wu,et al.  Role of AMP-activated protein kinase in mechanism of metformin action. , 2001, The Journal of clinical investigation.

[33]  S. Chirala,et al.  The subcellular localization of acetyl-CoA carboxylase 2. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Hardie,et al.  Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets. , 1992, European journal of biochemistry.

[35]  K. Inoki,et al.  TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival , 2003, Cell.

[36]  Jérôme Boudeau,et al.  Complexes between the LKB1 tumor suppressor, STRADα/β and MO25α/β are upstream kinases in the AMP-activated protein kinase cascade , 2003, Journal of biology.

[37]  Martin M. Matzuk,et al.  Continuous Fatty Acid Oxidation and Reduced Fat Storage in Mice Lacking Acetyl-CoA Carboxylase 2 , 2001, Science.

[38]  D. Carling,et al.  Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia , 2000, Current Biology.

[39]  B. Turk,et al.  AMPK phosphorylation of raptor mediates a metabolic checkpoint. , 2008, Molecular cell.

[40]  Dario R Alessi,et al.  Metformin and reduced risk of cancer in diabetic patients , 2005, BMJ : British Medical Journal.

[41]  D. Hardie,et al.  AMPK: a key sensor of fuel and energy status in skeletal muscle. , 2006, Physiology.

[42]  Yosef Yarden,et al.  Signal transduction and oncogenesis by ErbB/HER receptors. , 2004, International journal of radiation oncology, biology, physics.

[43]  J. Menéndez,et al.  Antitumoral actions of the anti-obesity drug orlistat (XenicalTM) in breast cancer cells: blockade of cell cycle progression, promotion of apoptotic cell death and PEA3-mediated transcriptional repression of Her2/neu (erbB-2) oncogene. , 2005, Annals of oncology : official journal of the European Society for Medical Oncology.

[44]  D. Hardie,et al.  AMP‐activated protein kinase – development of the energy sensor concept , 2006, The Journal of physiology.

[45]  G L Rosner,et al.  Patterns and variability of tumor oxygenation in human soft tissue sarcomas, cervical carcinomas, and lymph node metastases. , 1995, International journal of radiation oncology, biology, physics.

[46]  D. Alessi,et al.  Identification of the sucrose non‐fermenting related kinase SNRK, as a novel LKB1 substrate , 2005, FEBS letters.

[47]  M. Kaminishi,et al.  Cell cycle regulation via p53 phosphorylation by a 5'-AMP activated protein kinase activator, 5-aminoimidazole- 4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line. , 2001, Biochemical and biophysical research communications.

[48]  D. Carling.  AMP-activated protein kinase: balancing the scales. , 2005, Biochimie.

[49]  M. Munday.  Regulation of mammalian acetyl-CoA carboxylase. , 2002, Biochemical Society transactions.

[50]  D. Hardie,et al.  C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest* , 2009, Journal of Biological Chemistry.

[51]  D. Hardie,et al.  AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy , 2007, Nature Reviews Molecular Cell Biology.

[52]  Christopher Poole,et al.  Polychemotherapy for Early Breast Cancer: Results From the International Adjuvant Breast Cancer Chemotherapy Randomized Trial , 2007 .

[53]  Nahum Sonenberg,et al.  Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells. , 2006, Cancer research.

[54]  H. Kuramoto,et al.  PTEN immunohistochemical expression is suppressed in G1 endometrioid adenocarcinoma of the uterine corpus , 2004, Journal of Cancer Research and Clinical Oncology.

[55]  C. Townsend,et al.  Synthesis and antitumor activity of an inhibitor of fatty acid synthase. , 2000, Proceedings of the National Academy of Sciences of the United States of America.