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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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PDAC tumor cells induce tumor-associated fibroblasts (TAFs) to express microRNA-21.(A) Representative images of miR-21 in situ hybridization (ISH) on a patient-matched primary tumor and lymph node metastasis reveals high peritumoral expression of miR-21 in the stroma, decreasing in a radial gradient away from the tumor cells. (B) From miR-21 ISH on 23 patient-matched samples, miR-21 expression in the primary TAFs correlates with that of TAFs found in lymph node metastases (p = 0.06). miR-21 expression was dichotomized into high (n = 12) vs. low (n = 11) based on the median intensity level. (C) miR-21 expression in early-passage (p2) primary TAFs derived from resected human PDACs is elevated >8 times that of human pancreas fibroblasts (HPF) from noncancerous tissue. (D) Co-culture of HPFs with MiaPaCa tumor cells reveals a >5 fold increase in miR-21 in HPFs versus co-culture with normal human ductal epithelial (HPDE) cells. miR-21 expression was normalized to RNU6B. Error bars ± SD. Data are representative of 3 independent experiments.
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pone-0071978-g003: PDAC tumor cells induce tumor-associated fibroblasts (TAFs) to express microRNA-21.(A) Representative images of miR-21 in situ hybridization (ISH) on a patient-matched primary tumor and lymph node metastasis reveals high peritumoral expression of miR-21 in the stroma, decreasing in a radial gradient away from the tumor cells. (B) From miR-21 ISH on 23 patient-matched samples, miR-21 expression in the primary TAFs correlates with that of TAFs found in lymph node metastases (p = 0.06). miR-21 expression was dichotomized into high (n = 12) vs. low (n = 11) based on the median intensity level. (C) miR-21 expression in early-passage (p2) primary TAFs derived from resected human PDACs is elevated >8 times that of human pancreas fibroblasts (HPF) from noncancerous tissue. (D) Co-culture of HPFs with MiaPaCa tumor cells reveals a >5 fold increase in miR-21 in HPFs versus co-culture with normal human ductal epithelial (HPDE) cells. miR-21 expression was normalized to RNU6B. Error bars ± SD. Data are representative of 3 independent experiments.

Mentions: PDAC TCs recruit supportive cells to their environment during tumor initiation and progression [27]. While the source of TAFs is unclear, it has been previously shown that TAFs do not possess genomic mutations but are activated through interactions with TCs [28]. We next sought to determine how miR-21 is expressed in TAFs and hypothesized that TCs induce them to upregulate the oncomir. To begin to answer this question, we identified and assembled 23 patient-matched cores of LN metastasis from TMA primary tumors. Figure 3A reveals that miR-21 expression in primary and LN TAFs is strongest in the region immediately surrounding the malignant ducts. The expression decreases along a radial gradient away from TCs. Strikingly, miR-21 expression in primary tumor TAFs showed a near significant correlation with miR-21 expression in TAFs from patient-matched LN metastases (P = 0.06, Figure 3B). Moreover, primary human PDAC TAFs isolated via the outgrowth method [19] express >8 fold higher miR-21 than noncancerous human fibroblasts isolated from a region of the pancreas remote from the cancer (Figure 3C). As more direct mechanistic evidence, miR-21 expression in normal HPFs was >5 fold higher when co-cultured with MiaPaCa TCs than when co-cultured with normal HPDE cells (Figure 3D). Validation that the primary TAFs are not derived from TCs included sequencing of KRAS and in situ immunofluorescence staining for specific epithelial and mesenchymal markers. These primary cell types are indeed KRAS wild-type, pan-cytokeratin negative, and express α-SMA, Vimentin, and GFAP (Figure S2–S3). To ensure that this primary culture did not include a contaminating population of tumor cells that had undergone epithelial-to-mesenchymal transition (EMT), we performed a tumorigenesis assay in an immunocompromised xenograft. At a count of 3.5×105 cells, primary TAFs did not form tumors when injected in NOD/SCID IL2Rγ mice (Figure S4). These results suggest that TCs could induce TAFs in both the primary tumor and LNs to express miR-21.


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)

PDAC tumor cells induce tumor-associated fibroblasts (TAFs) to express microRNA-21.(A) Representative images of miR-21 in situ hybridization (ISH) on a patient-matched primary tumor and lymph node metastasis reveals high peritumoral expression of miR-21 in the stroma, decreasing in a radial gradient away from the tumor cells. (B) From miR-21 ISH on 23 patient-matched samples, miR-21 expression in the primary TAFs correlates with that of TAFs found in lymph node metastases (p = 0.06). miR-21 expression was dichotomized into high (n = 12) vs. low (n = 11) based on the median intensity level. (C) miR-21 expression in early-passage (p2) primary TAFs derived from resected human PDACs is elevated >8 times that of human pancreas fibroblasts (HPF) from noncancerous tissue. (D) Co-culture of HPFs with MiaPaCa tumor cells reveals a >5 fold increase in miR-21 in HPFs versus co-culture with normal human ductal epithelial (HPDE) cells. miR-21 expression was normalized to RNU6B. Error bars ± SD. Data are representative of 3 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750050&req=5

pone-0071978-g003: PDAC tumor cells induce tumor-associated fibroblasts (TAFs) to express microRNA-21.(A) Representative images of miR-21 in situ hybridization (ISH) on a patient-matched primary tumor and lymph node metastasis reveals high peritumoral expression of miR-21 in the stroma, decreasing in a radial gradient away from the tumor cells. (B) From miR-21 ISH on 23 patient-matched samples, miR-21 expression in the primary TAFs correlates with that of TAFs found in lymph node metastases (p = 0.06). miR-21 expression was dichotomized into high (n = 12) vs. low (n = 11) based on the median intensity level. (C) miR-21 expression in early-passage (p2) primary TAFs derived from resected human PDACs is elevated >8 times that of human pancreas fibroblasts (HPF) from noncancerous tissue. (D) Co-culture of HPFs with MiaPaCa tumor cells reveals a >5 fold increase in miR-21 in HPFs versus co-culture with normal human ductal epithelial (HPDE) cells. miR-21 expression was normalized to RNU6B. Error bars ± SD. Data are representative of 3 independent experiments.
Mentions: PDAC TCs recruit supportive cells to their environment during tumor initiation and progression [27]. While the source of TAFs is unclear, it has been previously shown that TAFs do not possess genomic mutations but are activated through interactions with TCs [28]. We next sought to determine how miR-21 is expressed in TAFs and hypothesized that TCs induce them to upregulate the oncomir. To begin to answer this question, we identified and assembled 23 patient-matched cores of LN metastasis from TMA primary tumors. Figure 3A reveals that miR-21 expression in primary and LN TAFs is strongest in the region immediately surrounding the malignant ducts. The expression decreases along a radial gradient away from TCs. Strikingly, miR-21 expression in primary tumor TAFs showed a near significant correlation with miR-21 expression in TAFs from patient-matched LN metastases (P = 0.06, Figure 3B). Moreover, primary human PDAC TAFs isolated via the outgrowth method [19] express >8 fold higher miR-21 than noncancerous human fibroblasts isolated from a region of the pancreas remote from the cancer (Figure 3C). As more direct mechanistic evidence, miR-21 expression in normal HPFs was >5 fold higher when co-cultured with MiaPaCa TCs than when co-cultured with normal HPDE cells (Figure 3D). Validation that the primary TAFs are not derived from TCs included sequencing of KRAS and in situ immunofluorescence staining for specific epithelial and mesenchymal markers. These primary cell types are indeed KRAS wild-type, pan-cytokeratin negative, and express α-SMA, Vimentin, and GFAP (Figure S2–S3). To ensure that this primary culture did not include a contaminating population of tumor cells that had undergone epithelial-to-mesenchymal transition (EMT), we performed a tumorigenesis assay in an immunocompromised xenograft. At a count of 3.5×105 cells, primary TAFs did not form tumors when injected in NOD/SCID IL2Rγ mice (Figure S4). These results suggest that TCs could induce TAFs in both the primary tumor and LNs to express miR-21.

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