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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 expression in the PDAC stroma is associated with poor prognosis.(A) Representative images of histoscores for miR-21 in situ hybridization in PDAC tumor cells and stroma. These two cellular compartments were scored as 0 negative (not depicted), 1 weakly positive, 2 moderately positive, 3 strongly positive. (B) Kaplan-Meier analysis reveals that high miR-21 stromal expression is associated with decreased overall survival (P = 0.04). miR-21 expression intensity was dichotomized into high (n = 73) vs. low (n = 72) based on the median score of all tumors.
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pone-0071978-g001: microRNA-21 expression in the PDAC stroma is associated with poor prognosis.(A) Representative images of histoscores for miR-21 in situ hybridization in PDAC tumor cells and stroma. These two cellular compartments were scored as 0 negative (not depicted), 1 weakly positive, 2 moderately positive, 3 strongly positive. (B) Kaplan-Meier analysis reveals that high miR-21 stromal expression is associated with decreased overall survival (P = 0.04). miR-21 expression intensity was dichotomized into high (n = 73) vs. low (n = 72) based on the median score of all tumors.

Mentions: PDAC is associated with a dense stroma that contributes to tumorigenesis [24]. We hypothesized that miR-21 expression in the PDAC stroma correlated with clinical progression of disease. The UCLA PDAC TMA contains samples from 153 resected early-stage PDACs and was stained for miR-21 utilizing ISH. Representative images for histoscoring of TC and stroma is shown in Figure 1A. 78.4% of patients had a median histoscore for peritumoral stroma of ≥1 (Figure S1). Patients were dichotomized into miR-21 high (n = 73) or low (n = 72) based on the median histoscore = 1.5. On Kaplan-Meier survival analysis, high miR-21 stromal expression correlated with shorter overall survival (P = 0.04, Figure 1B), while miR-21 TC expression did not (Figure S1). As a means to explain the underlying mechanism of worse survival, miR-21 stromal expression was correlated with various histopathologic factors previously shown to be associated with prognosis – on both this TMA and an independent PDAC patient cohort [25] (Table 1). Interestingly, miR-21 in PDAC stroma did not correlate with tumor grade. It was strongly correlated with lymph node (LN) positivity (P = 0.004); 67% of miR-21 high patients had positive LNs while only 42% with low miR-21 had LN involvement. On Cox proportional hazards multivariate analysis, even after controlling for clinicopathologic variables associated with survival, stromal miR-21 expression on the TMA remained significant (HR = 1.56, P = 0.023) (Table 2). Taken together, these results reveal that miR-21 expression in the PDAC stroma is prognostically significant because it is correlated with TC invasion and metastasis.


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 expression in the PDAC stroma is associated with poor prognosis.(A) Representative images of histoscores for miR-21 in situ hybridization in PDAC tumor cells and stroma. These two cellular compartments were scored as 0 negative (not depicted), 1 weakly positive, 2 moderately positive, 3 strongly positive. (B) Kaplan-Meier analysis reveals that high miR-21 stromal expression is associated with decreased overall survival (P = 0.04). miR-21 expression intensity was dichotomized into high (n = 73) vs. low (n = 72) based on the median score of all tumors.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750050&req=5

pone-0071978-g001: microRNA-21 expression in the PDAC stroma is associated with poor prognosis.(A) Representative images of histoscores for miR-21 in situ hybridization in PDAC tumor cells and stroma. These two cellular compartments were scored as 0 negative (not depicted), 1 weakly positive, 2 moderately positive, 3 strongly positive. (B) Kaplan-Meier analysis reveals that high miR-21 stromal expression is associated with decreased overall survival (P = 0.04). miR-21 expression intensity was dichotomized into high (n = 73) vs. low (n = 72) based on the median score of all tumors.
Mentions: PDAC is associated with a dense stroma that contributes to tumorigenesis [24]. We hypothesized that miR-21 expression in the PDAC stroma correlated with clinical progression of disease. The UCLA PDAC TMA contains samples from 153 resected early-stage PDACs and was stained for miR-21 utilizing ISH. Representative images for histoscoring of TC and stroma is shown in Figure 1A. 78.4% of patients had a median histoscore for peritumoral stroma of ≥1 (Figure S1). Patients were dichotomized into miR-21 high (n = 73) or low (n = 72) based on the median histoscore = 1.5. On Kaplan-Meier survival analysis, high miR-21 stromal expression correlated with shorter overall survival (P = 0.04, Figure 1B), while miR-21 TC expression did not (Figure S1). As a means to explain the underlying mechanism of worse survival, miR-21 stromal expression was correlated with various histopathologic factors previously shown to be associated with prognosis – on both this TMA and an independent PDAC patient cohort [25] (Table 1). Interestingly, miR-21 in PDAC stroma did not correlate with tumor grade. It was strongly correlated with lymph node (LN) positivity (P = 0.004); 67% of miR-21 high patients had positive LNs while only 42% with low miR-21 had LN involvement. On Cox proportional hazards multivariate analysis, even after controlling for clinicopathologic variables associated with survival, stromal miR-21 expression on the TMA remained significant (HR = 1.56, P = 0.023) (Table 2). Taken together, these results reveal that miR-21 expression in the PDAC stroma is prognostically significant because it is correlated with TC invasion and metastasis.

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