CXCL12/CXCR4 signaling axis induces SHH expression in pancreatic cancer cells via ERK- and Akt- mediated activation of NF-κB: implications for bidirectional tumor-stromal interactions Ajay P. Singh 1,2,* , Sumit Arora 1,# , Arun Bhardwaj 1,# , Sanjeev K. Srivastava 1 , Madhavi P. Kadakia 3 , Bin Wang 4 , William E. Grizzle 5 , Laurie B. Owen 1 , Seema Singh 1,* 1 Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA; 2 Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, Alabama, USA; 3 Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio, USA; 4 Department of Mathematics and Statistics, University of South Alabama, Mobile, Alabama, USA; 5 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA Running Title: CXCL12/CXCR4 signaling induces SHH expression * To whom correspondence should be addressed: Ajay P. Singh, Ph.D. or Seema Singh, Ph.D., Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, U.S.A. Phone: 251-445-9843; Fax: 251-460-6994, E-mail: asingh@usouthal.edu or seemasingh@usouthal.edu # These authors contributed equally to this manuscript. Key Words: CXCL12, CXCR4, SHH, NF-κB, pancreatic cancer, tumor-stromal interaction Background: CXCL12/CXCR4 and hedgehog pathways, predominantly acting in paracrine fashion, play important roles in pancreatic cancer pathobiology. Results: CXCL12/CXCR4 signaling regulates the expression of hedgehog ligand, the sonic hedgehog, in pancreatic cancer cells. Conclusion: Our findings indicate a novel molecular link between CXCL12/CXCR4 and hedgehog pathways. Significance: Our data provide molecular basis for an active bidirectional tumor-stromal interaction in pancreatic cancer. SUMMARY Recent evidence suggests a major role of tumor-stromal interactions in pancreatic cancer pathobiology. The chemokine CXCL12 [stromal cell-derived factor-1(SDF- 1)], abundantly produced by the stromal cells, promotes progression, metastasis and chemoresistance of pancreatic cancer cells. On the other hand, pancreatic tumor cell- derived sonic hedgehog (SHH) acts predominantly on the stromal cells to induce desmoplasia and thus has a paracrine effect on tumorigenesis and therapeutic outcome. In this study, we have examined the association between these two proteins of pathological significance in pancreatic cancer. Our data demonstrate that CXCL12 leads to a dose- and time-dependent upregulation of SHH in pancreatic cancer cells. CXCL12-induced SHH upregulation is specifically mediated through the receptor CXCR4 and is dependent on the activation of downstream Akt and extracellular regulated kinase (ERK) signaling pathways. Both Akt and ERK cooperatively promote nuclear accumulation of NF-κB by inducing the phosphorylation and destabilization of its inhibitory protein, IκB-α. Using dominant negative IκB-α, SHH promoter (deletion mutant)-reporter and chromatin immunoprecipitation assays, we demonstrate that CXCL12 exposure enhances direct binding of NF-κB to SHH promoter and suppression of NF-κB activation abrogates CXCL12-induced SHH expression. 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M112.409581 The latest version is at JBC Papers in Press. Published on September 20, 2012 as Manuscript M112.409581 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from by guest on February 8, 2016 http://www.jbc.org/ Downloaded from