title: Chemokine CXCL12 activates dual CXCR4 and CXCR7-mediated signaling pathways in pancreatic cancer cells.
abstract: BACKGROUND
  Previously assumed to be a select ligand for chemokine receptor CXCR4, chemokine CXCL12 is now known to activate both CXCR4 and CXCR7. However, very little is known about the co-expression of these receptors in cancer cells.
  
  
  METHODS
  We used immunohistochemistry to determine the extent of co-expression in pancreatic cancer tissue samples and immunoblotting to verify expression in pancreatic cancer cell lines. In cell culture studies, siRNA was used to knock down expression of CXCR4, CXCR7, K-Ras and β-arrestin -2 prior to stimulating the cells with CXCL12. Activation of the mitogen-activated protein kinase pathway (MAPK) was assessed using both a Raf-pull down assay and western blotting. The involvement of the receptors in CXCL12-mediated increases in cell proliferation was examined via an ATP-based proliferation assay.
  
  
  RESULTS
  First, we discovered frequent CXCR4/CXCR7 co-expression in human pancreatic cancer tissues and cell lines. Next, we observed consistent increases in ERK1/2 phosphorylation after exposure to CXCL12 or CXCL11, a CXCR7 agonist, in pancreatic cancer cell lines co-expressing CXCR4/CXCR7. To better characterize the receptor-mediated pathway(s), we knocked down CXCR4 or CXCR7, exposed the cells to CXCL12 and examined subsequent effects on ERK1/2. We observed that CXCR7 mediates the CXCL12-driven increase in ERK1/2 phosphorylation. Knockdown of CXCR4 expression however, decreased levels of K-Ras activity. Conversely, KRAS knockdown greatly reduced CXCL12-mediated increases in ERK1/2 phosphorylation. We then evaluated the role of β-arrestin-2, a protein directly recruited by chemokine receptors. We observed that β-arrestin-2 knockdown also inhibited increases in ERK1/2 phosphorylation mediated by both CXCR4 and CXCR7. Finally, we investigated the mechanism for CXCL12-enhanced cell proliferation and found that either receptor can modulate cell proliferation.
  
  
  CONCLUSIONS
  In summary, our data demonstrate that CXCR4 and CXCR7 are frequently co-expressed in human pancreatic cancer tissues and cell lines. We show that β-arrestin-2 and K-Ras dependent pathways coordinate the transduction of CXCL12 signals. Our results suggest that the development of therapies based on inhibiting CXCL12 signaling to halt the growth of pancreatic cancer should be focused at the ligand level in order to account for the contributions of both receptors to this signaling pathway.
authors.affiliation: Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA 91010, USA.
authors.forename: Eileen L
authors.initials: EL
authors.lastname: Heinrich
authors.forename: Wendy
authors.initials: W
authors.lastname: Lee
authors.forename: Jianming
authors.initials: J
authors.lastname: Lu
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Lowy
authors.forename: Joseph
authors.initials: J
authors.lastname: Kim
mesh_terms: D019390:Arrestins / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D054377:Chemokine CXCL12 / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005557:Formaldehyde; D055785:Gene Knockdown Techniques; D006801:Humans; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D016612:Paraffin Embedding; D010766:Phosphorylation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D054387:Receptors, CXCR / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D016707:Tissue Fixation; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D018631:ras Proteins / Q000378:metabolism
chemical_list: C501896:ACKR3 protein, human; C000605254:ARRB2 protein, human; D019390:Arrestins; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D054387:Receptors, CXCR; D019718:Receptors, CXCR4; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D005557:Formaldehyde; D048049:Extracellular Signal-Regulated MAP Kinases; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins