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

Kadera BE, Li L, Toste PA, Wu N, Adams C, Dawson DW, Donahue TR - PLoS ONE (2013)

Bottom Line: Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described.Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread.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. miR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion.

View Article: PubMed Central - PubMed

Affiliation: Division of General Surgery, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America.

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.

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microRNA-21 in the PDAC stroma is expressed in activated myofibroblasts.(A) miR-21 in situ hybridization and co-immunofluorescence for α-SMA and nestin on serial sections of two human PDAC tumors (Whipple resection - WR 22 & 28) reveals miR-21 expression in a subset of activated myofibroblasts, not exclusive to stellate cells (nestin positive). miR-21 positive staining is white in the merge. (B) High-power magnification (40x) again reveals miR-21 expression in a subset of α-SMA expressing myofibroblasts. Vimentin, a marker for quiescent fibroblasts, does not localize with miR-21.
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pone-0071978-g002: microRNA-21 in the PDAC stroma is expressed in activated myofibroblasts.(A) miR-21 in situ hybridization and co-immunofluorescence for α-SMA and nestin on serial sections of two human PDAC tumors (Whipple resection - WR 22 & 28) reveals miR-21 expression in a subset of activated myofibroblasts, not exclusive to stellate cells (nestin positive). miR-21 positive staining is white in the merge. (B) High-power magnification (40x) again reveals miR-21 expression in a subset of α-SMA expressing myofibroblasts. Vimentin, a marker for quiescent fibroblasts, does not localize with miR-21.

Mentions: The PDAC stroma is comprised of a diverse cell population [24], including fibroblasts, activated myofibroblasts, stellate cells [26], inflammatory cells, and endothelial cells. Activated myofibroblasts and stellate cells are associated with PDAC TC invasion and chemotherapy resistance [4], [27]. Based on our previous results that stromal miR-21 expression is associated with prognosis, we next sought to determine the specific cell type expressing miR-21. An ISH-immunofluorescence digital overlay for miR-21 (white), the activated myofibroblast marker α-smooth muscle actin (red, α-SMA), and the stellate cell marker nestin (green) (Figure 2A) reveals that miR-21 is expressed in a subset of α-SMA and nestin positive cells. A larger magnification view (Figure 2B) of miR-21 (white), α-SMA (red), and the fibroblast marker vimentin (green) confirms these findings. These results reveal that miR-21 is expressed in a subpopulation of activated myofibroblasts and stellate cells, as opposed to simply representing a surrogate marker of these cell types.


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

Kadera BE, Li L, Toste PA, Wu N, Adams C, Dawson DW, Donahue TR - PLoS ONE (2013)

microRNA-21 in the PDAC stroma is expressed in activated myofibroblasts.(A) miR-21 in situ hybridization and co-immunofluorescence for α-SMA and nestin on serial sections of two human PDAC tumors (Whipple resection - WR 22 & 28) reveals miR-21 expression in a subset of activated myofibroblasts, not exclusive to stellate cells (nestin positive). miR-21 positive staining is white in the merge. (B) High-power magnification (40x) again reveals miR-21 expression in a subset of α-SMA expressing myofibroblasts. Vimentin, a marker for quiescent fibroblasts, does not localize with miR-21.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750050&req=5

pone-0071978-g002: microRNA-21 in the PDAC stroma is expressed in activated myofibroblasts.(A) miR-21 in situ hybridization and co-immunofluorescence for α-SMA and nestin on serial sections of two human PDAC tumors (Whipple resection - WR 22 & 28) reveals miR-21 expression in a subset of activated myofibroblasts, not exclusive to stellate cells (nestin positive). miR-21 positive staining is white in the merge. (B) High-power magnification (40x) again reveals miR-21 expression in a subset of α-SMA expressing myofibroblasts. Vimentin, a marker for quiescent fibroblasts, does not localize with miR-21.
Mentions: The PDAC stroma is comprised of a diverse cell population [24], including fibroblasts, activated myofibroblasts, stellate cells [26], inflammatory cells, and endothelial cells. Activated myofibroblasts and stellate cells are associated with PDAC TC invasion and chemotherapy resistance [4], [27]. Based on our previous results that stromal miR-21 expression is associated with prognosis, we next sought to determine the specific cell type expressing miR-21. An ISH-immunofluorescence digital overlay for miR-21 (white), the activated myofibroblast marker α-smooth muscle actin (red, α-SMA), and the stellate cell marker nestin (green) (Figure 2A) reveals that miR-21 is expressed in a subset of α-SMA and nestin positive cells. A larger magnification view (Figure 2B) of miR-21 (white), α-SMA (red), and the fibroblast marker vimentin (green) confirms these findings. These results reveal that miR-21 is expressed in a subpopulation of activated myofibroblasts and stellate cells, as opposed to simply representing a surrogate marker of these cell types.

Bottom Line: Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described.Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread.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. miR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion.

View Article: PubMed Central - PubMed

Affiliation: Division of General Surgery, Department of Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus