Calreticulin as a potential diagnostic biomarker for lung cancer

Calreticulin (CRT) is an endoplasmic reticulum luminal Ca2+-binding chaperone protein. By immunizing mice with recombinant fragment (rCRT/39-272), six clones of monoclonal antibodies (mAbs) were generated and characterized. Based on these mAbs, a microplate chemiluminescent enzyme immunoassay (CLEIA) system with a measured limit of detection of 0.09 ng/ml was developed. Using this CLEIA system, it was found that soluble CRT (sCRT) level in serum samples from 58 lung cancer patients was significantly higher than that from 40 healthy individuals (only 9 were detectable, P < 0.0001). Among them, serum sCRT in the small cell lung cancer was lower than that in adenocarcinoma (P = 0.0085), while both were lower than that in the squamous cell carcinoma (P = 0.013, P = 0.0012, respectively). Moreover, it was found that sCRT in sera from the patients after chemotherapy was higher than that from the patients without chemotherapy (P = 0.042). Further study by immunohistochemistry showed that CRT was also highly expressed in the cytoplasm and on the membrane of the lung cancer cells, while there was a trace amount of CRT expression in normal lung cells. Correspondingly, the expression level of CRT on lung cancer cell membrane was associated with the tumor pathological grade. This study demonstrates that sCRT concentration in sera of lung cancer patients is higher than that in sera of healthy individuals, and CRT expression level on lung cancer cell membrane is associated with tumor pathological classification and grade. These findings suggest that CRT may be used as a biomarker in lung cancer prediction and diagnosis.

[1]  Yuanjie Sun,et al.  Highly sensitive microplate chemiluminescence enzyme immunoassay for the determination of staphylococcal enterotoxin B based on a pair of specific monoclonal antibodies and its application to various matrices. , 2010, Analytical chemistry.

[2]  N. Harashima,et al.  Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma , 2010, Cancer Immunology, Immunotherapy.

[3]  M. Michalak,et al.  Calreticulin: non‐endoplasmic reticulum functions in physiology and disease , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[4]  T. Isono,et al.  Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine. , 2004, Clinical chemistry.

[5]  S. Hanash,et al.  Cleavage of endoplasmic reticulum proteins in hepatocellular carcinoma: Detection of generated fragments in patient sera. , 2006, Gastroenterology.

[6]  L. Zitvogel,et al.  Molecular determinants of immunogenic cell death: surface exposure of calreticulin makes the difference , 2007, Journal of Molecular Medicine.

[7]  A. Bergner,et al.  Endoplasmic reticulum Ca2+-homeostasis is altered in small and non-small cell lung cancer cell lines , 2009, Journal of experimental & clinical cancer research : CR.

[8]  J. Gong,et al.  Establishment of an enzyme‐linked immunosorbent assay system for determining soluble CD96 and its application in the measurement of sCD96 in patients with viral hepatitis B and hepatic cirrhosis , 2009, Clinical and experimental immunology.

[9]  T. Isono,et al.  Urinary calreticulin in the diagnosis of bladder urothelial carcinoma , 2009, International journal of urology : official journal of the Japanese Urological Association.

[10]  M. Ho,et al.  Identification of Calreticulin as a Prognosis Marker and Angiogenic Regulator in Human Gastric Cancer , 2009, Annals of Surgical Oncology.

[11]  Wei Zhang,et al.  Involvement of damage-associated molecular patterns in tumor response to photodynamic therapy: surface expression of calreticulin and high-mobility group box-1 release , 2011, Cancer Immunology, Immunotherapy.

[12]  Lin Sun,et al.  Functional Analysis of Recombinant Calreticulin Fragment 39–272: Implications for Immunobiological Activities of Calreticulin in Health and Disease , 2010, The Journal of Immunology.

[13]  D. Lai,et al.  Calreticulin expression in neuroblastoma--a novel independent prognostic factor. , 2005, Annals of oncology : official journal of the European Society for Medical Oncology.

[14]  L. Zitvogel,et al.  Calreticulin exposure dictates the immunogenicity of cancer cell death , 2007, Nature Medicine.

[15]  A. Jemal,et al.  Global Patterns of Cancer Incidence and Mortality Rates and Trends , 2010, Cancer Epidemiology, Biomarkers & Prevention.

[16]  M. Michalak,et al.  Calreticulin enhances porcine wound repair by diverse biological effects. , 2008, The American journal of pathology.

[17]  L. Zitvogel,et al.  Calreticulin exposure is required for the immunogenicity of γ-irradiation and UVC light-induced apoptosis , 2007, Cell Death and Differentiation.

[18]  J. Casal,et al.  Proteomic expression analysis of colorectal cancer by two‐dimensional differential gel electrophoresis , 2005, Proteomics.

[19]  S. Grinstein,et al.  Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion , 1997, Nature.

[20]  L. Zitvogel,et al.  Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin , 2008, Cell Death and Differentiation.

[21]  P. van Endert,et al.  Mechanisms of pre‐apoptotic calreticulin exposure in immunogenic cell death , 2009, The EMBO journal.

[22]  N. Viñolas,et al.  Usefulness of Serum Tumor Markers, Including Progastrin-Releasing Peptide, in Patients with Lung Cancer: Correlation with Histology , 2009, Tumor Biology.

[23]  K. Öberg,et al.  Gene Transfer of Vasostatin, a Calreticulin Fragment, into Neuroendocrine Tumor Cells Results in Enhanced Malignant Behavior , 2006, Neuroendocrinology.

[24]  W. Janssen,et al.  Cell-Surface Calreticulin Initiates Clearance of Viable or Apoptotic Cells through trans-Activation of LRP on the Phagocyte , 2005, Cell.

[25]  Z. Milovanović,et al.  Effects of Humoral Immunity and Calreticulin Overexpression on Postoperative Course in Breast Cancer , 2009, Pathology & Oncology Research.

[26]  M. Michalak,et al.  The ins and outs of calreticulin: from the ER lumen to the extracellular space. , 2001, Trends in cell biology.

[27]  F. Khuri,et al.  Lung cancer: New biological insights and recent therapeutic advances , 2011, CA: a cancer journal for clinicians.

[28]  Ash A. Alizadeh,et al.  Calreticulin Is the Dominant Pro-Phagocytic Signal on Multiple Human Cancers and Is Counterbalanced by CD47 , 2010, Science Translational Medicine.

[29]  D. Hallahan,et al.  Radiation induces an antitumour immune response to mouse melanoma , 2009, International journal of radiation biology.

[30]  L. Zitvogel,et al.  Immunogenic cancer cell death: a key-lock paradigm. , 2008, Current opinion in immunology.

[31]  F. Chew,et al.  Clinicopathological significance of calreticulin in breast invasive ductal carcinoma , 2010, Modern Pathology.

[32]  W. Xiong,et al.  Thrombospondin Induces RhoA Inactivation through FAK-dependent Signaling to Stimulate Focal Adhesion Disassembly* , 2004, Journal of Biological Chemistry.

[33]  L. Zitvogel,et al.  Leveraging the immune system during chemotherapy: moving calreticulin to the cell surface converts apoptotic death from "silent" to immunogenic. , 2007, Cancer research.

[34]  A. Belldegrun,et al.  Dendritic Cell Surface Calreticulin Is a Receptor for NY-ESO-1: Direct Interactions between Tumor-Associated Antigen and the Innate Immune System1 , 2006, The Journal of Immunology.