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Multifunctional roles of urokinase plasminogen activator (uPA) in cancer stemness and chemoresistance of pancreatic cancer.

Asuthkar S, Stepanova V, Lebedeva T, Holterman AL, Estes N, Cines DB, Rao JS, Gondi CS - Mol. Biol. Cell (2013)

Bottom Line: Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA).In the present study we examine the role of uPA in the generation of PDAC CSC.Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is almost always lethal. One of the underlying reasons for this lethality is believed to be the presence of cancer stem cells (CSC), which impart chemoresistance and promote recurrence, but the mechanisms responsible are unclear. Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA). In the present study we examine the role of uPA in the generation of PDAC CSC. We observe a subset of cells identifiable as a side population (SP) when sorted by flow cytometry of MIA PaCa-2 and PANC-1 pancreatic cancer cells that possess the properties of CSC. A large fraction of these SP cells are CD44 and CD24 positive, are gemcitabine resistant, possess sphere-forming ability, and exhibit increased tumorigenicity, known characteristics of cancer stemness. Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA. We observe that uPA interacts directly with transcription factors LIM homeobox-2 (Lhx2), homeobox transcription factor A5 (HOXA5), and Hey to possibly promote cancer stemness. uPA regulates Lhx2 expression by suppressing expression of miR-124 and p53 expression by repressing its promoter by inactivating HOXA5. These results demonstrate that regulation of gene transcription by uPA contributes to cancer stemness and clinical lethality.

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Lhx2 is the predicted target for miR-124, which negatively regulates Lhx2. (A) Sequence alignment of miR-124 and predicted sequence pairing with a region of Lhx2 mRNA 3′-UTR. The nucleotides within the Lhx2 3′-UTR region that may interact with miR-124 are framed. (B) Alignment of nucleotide sequences of Lhx2 3′-UTR corresponding to the targets for miR-124 from several mammalian species. A high level of conservation suggests a functional role for these sequences. (C) Luciferase reporter assay. Interaction of miR-124 with Lhx2 3′-UTR luciferase reporter vector was transfected in the control and puPA-treated MIA PaCa-2 and PANC-1 cells alone and/or in combination with hsa-miR-124 and miR-124 inhibitor (anti–miR-124). Luciferase activity, which reflects extent of inhibition of Lhx2 3′-UTR reporter by miR-124, was quantified and normalized as described in Materials and Methods. The y-axis denotes relative luciferase units (RLU; mean ± SD; n = 3; *p < 0.05; **p < 0.01). (D) Western blot analysis of cell lysates (40 μg of total protein) obtained from control and hsa-miR-124– and/or anti–miR-124–treated MIA PaCa-2 and PANC-1 cells. Separated proteins were probed with anti-Lhx2 antibodies, followed by HRP-conjugated anti-rabbit secondary Abs and the bands were visualized with the chemiluminescent substrate. Anti-GAPDH Abs were used as a loading control. Fibrin zymography (bottom) was performed to determine uPA activity in the conditioned media of the cultured cells as described in Materials and Methods.
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Figure 4: Lhx2 is the predicted target for miR-124, which negatively regulates Lhx2. (A) Sequence alignment of miR-124 and predicted sequence pairing with a region of Lhx2 mRNA 3′-UTR. The nucleotides within the Lhx2 3′-UTR region that may interact with miR-124 are framed. (B) Alignment of nucleotide sequences of Lhx2 3′-UTR corresponding to the targets for miR-124 from several mammalian species. A high level of conservation suggests a functional role for these sequences. (C) Luciferase reporter assay. Interaction of miR-124 with Lhx2 3′-UTR luciferase reporter vector was transfected in the control and puPA-treated MIA PaCa-2 and PANC-1 cells alone and/or in combination with hsa-miR-124 and miR-124 inhibitor (anti–miR-124). Luciferase activity, which reflects extent of inhibition of Lhx2 3′-UTR reporter by miR-124, was quantified and normalized as described in Materials and Methods. The y-axis denotes relative luciferase units (RLU; mean ± SD; n = 3; *p < 0.05; **p < 0.01). (D) Western blot analysis of cell lysates (40 μg of total protein) obtained from control and hsa-miR-124– and/or anti–miR-124–treated MIA PaCa-2 and PANC-1 cells. Separated proteins were probed with anti-Lhx2 antibodies, followed by HRP-conjugated anti-rabbit secondary Abs and the bands were visualized with the chemiluminescent substrate. Anti-GAPDH Abs were used as a loading control. Fibrin zymography (bottom) was performed to determine uPA activity in the conditioned media of the cultured cells as described in Materials and Methods.

Mentions: We next investigated the mechanism by which uPA up-regulates the expression of Lhx2. We first performed miRNA target prediction analysis using the MiRanda (Enright et al., 2003) and PITA (Kertesz et al., 2007) algorithms. We found that Lhx2 mRNA is a potential target of hsa-mir-124 (miR-124) (Figure 4A). Specifically, we identified an 8-mer (CGUGCCUU) motif in the 3′ untranslated region (UTR) of Lhx2 that is highly conserved in multiple mammalian species as a potential binding site for miR-124 (Figure 4B). To validate this in silico prediction, we developed a reporter construct in which luciferase expression is controlled by the human Lhx2 3′-UTR DNA fragment containing the putative miR-124 interaction sequence. The Lhx2 3′-UTR reporter construct was transiently transfected into MIA PaCa-2 and PANC-1 cells alone or together with hsa-miR-124 again alone or in combination with the miR-124 inhibitor (anti–miR-124). Overexpression of miR-124 repressed the Lhx2 3′-UTR reporter (Figure 4C), whereas anti–miR-124 prevented the miR-124–mediated repression of Lhx2 3′-UTR luciferase activity in both MIA PaCa-2 and PANC-1 cells, confirming the specificity of miR-124 toward the 3′-UTR region of Lhx2. We then examined expression levels of miR-124 in human pancreatic cancer tissues versus normal (unaffected) pancreas using human pancreatic cancer tissue arrays. Figure 5A shows hematoxylin and eosin (H&E) staining of normal and pancreatic ductal adenocarcinoma (PDAC). We also performed in situ hybridization (Figure 5B) to examine the expression of miR-124 in human pancreatic cancer tissue arrays. We observed that tumor tissues did not express miR-124 (Figure 5C), in contrast to its expression in normal pancreatic tissues (Figure 5D). This suggests that overexpression of Lhx2 in pancreatic cancer tissues (Figure 3) might be due to suppression of miR-124. Because 1) enhanced miR-124 expression has inhibitory effects on cancer stem–like traits and invasiveness (Xia et al., 2012), 2) miR-124 targets Lhx2 transcript, and 3) uPA up-regulates Lhx2 in pancreatic cancer cells, we examined the relationship of uPA and miR-124. To determine whether uPA affects the expression of miR-124 and thereby regulates Lhx2, we used miRNA-specific stem loop PCR to examine expression of miR-124 in control MIA PaCa-2 and PANC-1, MIA PaCa-2(uPA-), and PANC-1(uPA-) cells cultured in absence or presence of exogenously added WT-uPA. We observed that down-regulation of uPA expression (MIA PaCa-2(uPA-) and PANC-1(uPA-) cells) induced expression of miR-124 compared with control MIA PaCa-2 and PANC-1 cells, whereas addition of WT-uPA inhibited miR-124 expression (Figure 5F). Furthermore, cells treated with shRNA specific for uPA (MIA PaCa-2(uPA-) and PANC-1(uPA-) cells) and transfected with the Lhx2 3′-UTR luciferase reporter construct showed significant decrease in luciferase activity, suggesting that uPA up-regulates Lhx2 by suppressing expression of miR-124. To determine whether down-regulation of uPA induces expression of miR-124 in vivo, we orthotopically implanted MIA PaCa-2 SP cells that exhibit cancer stem cell–like characteristics (Figure 1A and Supplemental Figures S2 and S4) into the pancreas of nude mice, which were then injected intraperitoneally with shRNA targeting uPA (puPA; plasmid expressing shRNA targeting uPA). In vivo suppression of uPA resulted in significant (p = 0.02) increase in expression of miR-124 in tumor tissue after 40 d but not in normal tissue (Figure 5E). Of interest, hsa-miR-124 also suppressed expression of both Lhx2 and uPA in MIA PaCa-2 and PANC-1 cells, whereas transfection of these cells with anti–miR-124 enhanced expression of Lhx2 and uPA (Figure 4D). Together these data suggest the existence of a negative feedback loop between uPA and miR-124, which may regulate expression of Lhx2 and pancreatic cancer cell stemness.


Multifunctional roles of urokinase plasminogen activator (uPA) in cancer stemness and chemoresistance of pancreatic cancer.

Asuthkar S, Stepanova V, Lebedeva T, Holterman AL, Estes N, Cines DB, Rao JS, Gondi CS - Mol. Biol. Cell (2013)

Lhx2 is the predicted target for miR-124, which negatively regulates Lhx2. (A) Sequence alignment of miR-124 and predicted sequence pairing with a region of Lhx2 mRNA 3′-UTR. The nucleotides within the Lhx2 3′-UTR region that may interact with miR-124 are framed. (B) Alignment of nucleotide sequences of Lhx2 3′-UTR corresponding to the targets for miR-124 from several mammalian species. A high level of conservation suggests a functional role for these sequences. (C) Luciferase reporter assay. Interaction of miR-124 with Lhx2 3′-UTR luciferase reporter vector was transfected in the control and puPA-treated MIA PaCa-2 and PANC-1 cells alone and/or in combination with hsa-miR-124 and miR-124 inhibitor (anti–miR-124). Luciferase activity, which reflects extent of inhibition of Lhx2 3′-UTR reporter by miR-124, was quantified and normalized as described in Materials and Methods. The y-axis denotes relative luciferase units (RLU; mean ± SD; n = 3; *p < 0.05; **p < 0.01). (D) Western blot analysis of cell lysates (40 μg of total protein) obtained from control and hsa-miR-124– and/or anti–miR-124–treated MIA PaCa-2 and PANC-1 cells. Separated proteins were probed with anti-Lhx2 antibodies, followed by HRP-conjugated anti-rabbit secondary Abs and the bands were visualized with the chemiluminescent substrate. Anti-GAPDH Abs were used as a loading control. Fibrin zymography (bottom) was performed to determine uPA activity in the conditioned media of the cultured cells as described in Materials and Methods.
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Related In: Results  -  Collection

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Figure 4: Lhx2 is the predicted target for miR-124, which negatively regulates Lhx2. (A) Sequence alignment of miR-124 and predicted sequence pairing with a region of Lhx2 mRNA 3′-UTR. The nucleotides within the Lhx2 3′-UTR region that may interact with miR-124 are framed. (B) Alignment of nucleotide sequences of Lhx2 3′-UTR corresponding to the targets for miR-124 from several mammalian species. A high level of conservation suggests a functional role for these sequences. (C) Luciferase reporter assay. Interaction of miR-124 with Lhx2 3′-UTR luciferase reporter vector was transfected in the control and puPA-treated MIA PaCa-2 and PANC-1 cells alone and/or in combination with hsa-miR-124 and miR-124 inhibitor (anti–miR-124). Luciferase activity, which reflects extent of inhibition of Lhx2 3′-UTR reporter by miR-124, was quantified and normalized as described in Materials and Methods. The y-axis denotes relative luciferase units (RLU; mean ± SD; n = 3; *p < 0.05; **p < 0.01). (D) Western blot analysis of cell lysates (40 μg of total protein) obtained from control and hsa-miR-124– and/or anti–miR-124–treated MIA PaCa-2 and PANC-1 cells. Separated proteins were probed with anti-Lhx2 antibodies, followed by HRP-conjugated anti-rabbit secondary Abs and the bands were visualized with the chemiluminescent substrate. Anti-GAPDH Abs were used as a loading control. Fibrin zymography (bottom) was performed to determine uPA activity in the conditioned media of the cultured cells as described in Materials and Methods.
Mentions: We next investigated the mechanism by which uPA up-regulates the expression of Lhx2. We first performed miRNA target prediction analysis using the MiRanda (Enright et al., 2003) and PITA (Kertesz et al., 2007) algorithms. We found that Lhx2 mRNA is a potential target of hsa-mir-124 (miR-124) (Figure 4A). Specifically, we identified an 8-mer (CGUGCCUU) motif in the 3′ untranslated region (UTR) of Lhx2 that is highly conserved in multiple mammalian species as a potential binding site for miR-124 (Figure 4B). To validate this in silico prediction, we developed a reporter construct in which luciferase expression is controlled by the human Lhx2 3′-UTR DNA fragment containing the putative miR-124 interaction sequence. The Lhx2 3′-UTR reporter construct was transiently transfected into MIA PaCa-2 and PANC-1 cells alone or together with hsa-miR-124 again alone or in combination with the miR-124 inhibitor (anti–miR-124). Overexpression of miR-124 repressed the Lhx2 3′-UTR reporter (Figure 4C), whereas anti–miR-124 prevented the miR-124–mediated repression of Lhx2 3′-UTR luciferase activity in both MIA PaCa-2 and PANC-1 cells, confirming the specificity of miR-124 toward the 3′-UTR region of Lhx2. We then examined expression levels of miR-124 in human pancreatic cancer tissues versus normal (unaffected) pancreas using human pancreatic cancer tissue arrays. Figure 5A shows hematoxylin and eosin (H&E) staining of normal and pancreatic ductal adenocarcinoma (PDAC). We also performed in situ hybridization (Figure 5B) to examine the expression of miR-124 in human pancreatic cancer tissue arrays. We observed that tumor tissues did not express miR-124 (Figure 5C), in contrast to its expression in normal pancreatic tissues (Figure 5D). This suggests that overexpression of Lhx2 in pancreatic cancer tissues (Figure 3) might be due to suppression of miR-124. Because 1) enhanced miR-124 expression has inhibitory effects on cancer stem–like traits and invasiveness (Xia et al., 2012), 2) miR-124 targets Lhx2 transcript, and 3) uPA up-regulates Lhx2 in pancreatic cancer cells, we examined the relationship of uPA and miR-124. To determine whether uPA affects the expression of miR-124 and thereby regulates Lhx2, we used miRNA-specific stem loop PCR to examine expression of miR-124 in control MIA PaCa-2 and PANC-1, MIA PaCa-2(uPA-), and PANC-1(uPA-) cells cultured in absence or presence of exogenously added WT-uPA. We observed that down-regulation of uPA expression (MIA PaCa-2(uPA-) and PANC-1(uPA-) cells) induced expression of miR-124 compared with control MIA PaCa-2 and PANC-1 cells, whereas addition of WT-uPA inhibited miR-124 expression (Figure 5F). Furthermore, cells treated with shRNA specific for uPA (MIA PaCa-2(uPA-) and PANC-1(uPA-) cells) and transfected with the Lhx2 3′-UTR luciferase reporter construct showed significant decrease in luciferase activity, suggesting that uPA up-regulates Lhx2 by suppressing expression of miR-124. To determine whether down-regulation of uPA induces expression of miR-124 in vivo, we orthotopically implanted MIA PaCa-2 SP cells that exhibit cancer stem cell–like characteristics (Figure 1A and Supplemental Figures S2 and S4) into the pancreas of nude mice, which were then injected intraperitoneally with shRNA targeting uPA (puPA; plasmid expressing shRNA targeting uPA). In vivo suppression of uPA resulted in significant (p = 0.02) increase in expression of miR-124 in tumor tissue after 40 d but not in normal tissue (Figure 5E). Of interest, hsa-miR-124 also suppressed expression of both Lhx2 and uPA in MIA PaCa-2 and PANC-1 cells, whereas transfection of these cells with anti–miR-124 enhanced expression of Lhx2 and uPA (Figure 4D). Together these data suggest the existence of a negative feedback loop between uPA and miR-124, which may regulate expression of Lhx2 and pancreatic cancer cell stemness.

Bottom Line: Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA).In the present study we examine the role of uPA in the generation of PDAC CSC.Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is almost always lethal. One of the underlying reasons for this lethality is believed to be the presence of cancer stem cells (CSC), which impart chemoresistance and promote recurrence, but the mechanisms responsible are unclear. Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA). In the present study we examine the role of uPA in the generation of PDAC CSC. We observe a subset of cells identifiable as a side population (SP) when sorted by flow cytometry of MIA PaCa-2 and PANC-1 pancreatic cancer cells that possess the properties of CSC. A large fraction of these SP cells are CD44 and CD24 positive, are gemcitabine resistant, possess sphere-forming ability, and exhibit increased tumorigenicity, known characteristics of cancer stemness. Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA. We observe that uPA interacts directly with transcription factors LIM homeobox-2 (Lhx2), homeobox transcription factor A5 (HOXA5), and Hey to possibly promote cancer stemness. uPA regulates Lhx2 expression by suppressing expression of miR-124 and p53 expression by repressing its promoter by inactivating HOXA5. These results demonstrate that regulation of gene transcription by uPA contributes to cancer stemness and clinical lethality.

Show MeSH
Related in: MedlinePlus