MicroRNA-21 in pancreatic ductal adenocarcinoma tumor-associated fibroblasts promotes metastasis

PLoS One. 2013 Aug 22;8(8):e71978. doi: 10.1371/journal.pone.0071978. eCollection 2013.

Abstract

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is projected to rise to the second leading cause of U.S. cancer-related deaths by 2020. Novel therapeutic targets are desperately needed. MicroRNAs (miRs) are small noncoding RNAs that function by suppressing gene expression and are dysregulated in cancer. miR-21 is overexpressed in PDAC tumor cells (TC) and is associated with decreased survival, chemoresistance and invasion. Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described. In this study, we show that miR-21 expression in TAFs promotes TC invasion.

Methods: In-situ hybridization for miR-21 was performed on the 153 PDAC patient UCLA tissue microarray and 23 patient-matched lymph node metastases. Stromal and TC histoscores were correlated with clinicopathologic parameters by univariate and multivariate Cox regression. miR-21 positive cells were further characterized by immunofluorescence for mesenchymal/epithelial markers. For in vitro studies, TAFs were isolated from freshly resected human PDAC tumors by the outgrowth method. miR-21 was overexpressed/inhibited in fibroblasts and then co-cultured with GFP-MiaPaCa TCs to assess TC invasion in modified Boyden chambers.

Results: miR-21 was upregulated in TAFs of 78% of tumors, and high miR-21 significantly correlated with decreased overall survival (P = 0.04). Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread. Co-immunofluorescence revealed that miR-21 colocalized with peritumoral fibroblasts expressing α-smooth muscle actin. Moreover, expression of miR-21 in primary TAFs correlated with miR-21 in TAFs from patient-matched LN metastases; evidence that PDAC tumor cells induce TAFs to express miR-21. miR-21 expression in TAFs and TCs promotes invasion of TCs and is inhibited with anti-miR-21.

Conclusions: miR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion. Anti-miR-21 may represent a novel therapeutic strategy for dual targeting of both tumor and stroma in PDAC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / genetics*
  • Adenocarcinoma / metabolism
  • Adenocarcinoma / pathology
  • Aged
  • Animals
  • Base Sequence
  • Carcinoma, Pancreatic Ductal / genetics*
  • Carcinoma, Pancreatic Ductal / metabolism
  • Carcinoma, Pancreatic Ductal / pathology
  • Cell Line
  • Cell Line, Tumor
  • Cell Movement / genetics
  • Female
  • Fibroblasts / metabolism*
  • Fibroblasts / pathology
  • Gene Expression Regulation, Neoplastic
  • Humans
  • In Situ Hybridization
  • Kaplan-Meier Estimate
  • Lymphatic Metastasis
  • Male
  • Mice
  • Mice, Inbred NOD
  • Mice, Knockout
  • Mice, SCID
  • MicroRNAs / genetics*
  • Middle Aged
  • Pancreatic Neoplasms / genetics*
  • Pancreatic Neoplasms / metabolism
  • Pancreatic Neoplasms / pathology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Tumor Cells, Cultured

Substances

  • MIRN21 microRNA, human
  • MicroRNAs

Grant support

This work was supported by the Hirshberg Foundation (http://www.pancreatic.org), Concern Foundation (http://www.concernfoundation.org), Oppenheimer Foundation (http://oppenheimer.healthsciences.ucla.edu), California Institute for Regenerative Medicine TG2-01169 (http://www.cirm.ca.gov), CURE (http://www.cure.med.ucla.edu), Stop Cancer Foundation (www.stopcancer.org), Susan E. Riley Foundation and the Gerald S. Levey Surgical Research Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.