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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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Nuclear uPA regulates expression of Lhx2 in pancreatic cancer cells. (A) MIA-PaCa-2 and Capan-2 cells were left untreated or incubated with 20 nM of recombinant WT-uPA for 1 h, fixed in MeOH, and stained with anti-uPA rabbit polyclonal Abs and Alexa 488–conjugated anti-rabbit secondary Abs. Nuclei were counterstained with propidium iodide (red). Green staining denotes cytoplasmic and nuclear localization of uPA. (B) MIA PaCa-2 and PANC-1 cells plated and grown on chamber slides were transfected with pSV (scrambled vector) or puPA to lower uPA or uPA-encoding plasmid for uPA overexpression (pUPAOE). Nontransfected cells were also incubated with exogenously added WT-uPA protein. Cells were immunoprobed for uPA (green) and Lhx2 (red) and mounted with DAPI-containing mounting medium, and fluorescent photomicrographs were obtained as described (Stepanova et al., 2008). (C) Human pancreatic cancer tissue array (± cancer) was stained with H&E or immunoprobed for uPA or Lhx2 (A1, C1, E1, and F2 are malignant pancreatic adenocarcinoma tissues, and F7 is normal pancreatic tissue).
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Figure 3: Nuclear uPA regulates expression of Lhx2 in pancreatic cancer cells. (A) MIA-PaCa-2 and Capan-2 cells were left untreated or incubated with 20 nM of recombinant WT-uPA for 1 h, fixed in MeOH, and stained with anti-uPA rabbit polyclonal Abs and Alexa 488–conjugated anti-rabbit secondary Abs. Nuclei were counterstained with propidium iodide (red). Green staining denotes cytoplasmic and nuclear localization of uPA. (B) MIA PaCa-2 and PANC-1 cells plated and grown on chamber slides were transfected with pSV (scrambled vector) or puPA to lower uPA or uPA-encoding plasmid for uPA overexpression (pUPAOE). Nontransfected cells were also incubated with exogenously added WT-uPA protein. Cells were immunoprobed for uPA (green) and Lhx2 (red) and mounted with DAPI-containing mounting medium, and fluorescent photomicrographs were obtained as described (Stepanova et al., 2008). (C) Human pancreatic cancer tissue array (± cancer) was stained with H&E or immunoprobed for uPA or Lhx2 (A1, C1, E1, and F2 are malignant pancreatic adenocarcinoma tissues, and F7 is normal pancreatic tissue).

Mentions: We demonstrated previously that uPA is found within the nuclei of various types of proliferating cells (Stepanova et al., 2008). We therefore asked whether uPA localizes to the nuclei in pancreatic cancer cells. Immunocytochemical analysis of MIA Pa Ca-2, Capan-2, and PANC-1 cells revealed partial nuclear localization of uPA, which is significantly (p = 0.40) increased when recombinant uPA protein is added exogenously (Figure 3A and Supplemental Figure S4A). More recently, we reported that uPA binds to the transcription factor Lhx2 within the nuclei of pancreatic cancer cells and knockdown of uPA suppresses Lhx2 expression (Gorantla et al., 2011). Because Lhx2 is known to be involved in maintenance of stem/progenitor cell phenotype (Dahl et al., 2008; Tornqvist et al., 2010; Mardaryev et al., 2011; Nadal et al., 2012), we next investigated whether uPA–Lhx cross-talk regulates the maintenance of stem/progenitor cell phenotype in pancreatic cancer cells. To further decipher the role of nuclear uPA in the regulation of Lhx2 expression, we knocked down endogenous uPA expression in MIA PaCa-2 and PANC-1 cells using the puPA plasmid or uPA siRNA (MIA PaCa-2(uPA-) and PANC-1(uPA-) cells, respectively) and added wild-type (WT) uPA exogenously. We observed that addition of exogenous WT-uPA and overexpression of uPA (uPAOE) in MIA PaCa-2(uPA-) and PANC-1(uPA-) cells induced the expression of Lhx2 (Figure 3B). To determine whether uPA positively regulates Lhx2 expression in human tissues, we immunoprobed a human pancreatic tissue array for uPA and Lhx2. In malignant tissues, high levels of expression of both uPA with Lhx2 were observed. In contrast, normal pancreatic tissues showed no detectable expression of uPA and low expression levels of Lhx2 throughout (Figure 3C). Our previous studies indicated that uPAR is not essential for translocation of uPA to the nucleus. A uPA mutant lacking the uPAR-binding domain (ΔGFD-uPA) also translocates to cell nuclei, whereas a kringle-deficient uPA mutant (ΔK-uPA) did not, despite its ability to bind to uPAR (Stepanova et al., 2008). We found identical requirements for translocation of uPA in pancreatic cancer cells (Supplemental Figure S4, A and B). To address the role of uPAR in the regulation of Lhx2 expression by uPA specifically, we then studied the effect of these uPA deletion mutants on expression levels of Lhx2. PANC-1(uPA-) cells overexpressed Lhx2 in response to exogenously added WT-uPA and ΔGFD-uPA, whereas levels were unchanged after addition of identical concentrations of ΔK-uPA (Supplemental Figure S4C).


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)

Nuclear uPA regulates expression of Lhx2 in pancreatic cancer cells. (A) MIA-PaCa-2 and Capan-2 cells were left untreated or incubated with 20 nM of recombinant WT-uPA for 1 h, fixed in MeOH, and stained with anti-uPA rabbit polyclonal Abs and Alexa 488–conjugated anti-rabbit secondary Abs. Nuclei were counterstained with propidium iodide (red). Green staining denotes cytoplasmic and nuclear localization of uPA. (B) MIA PaCa-2 and PANC-1 cells plated and grown on chamber slides were transfected with pSV (scrambled vector) or puPA to lower uPA or uPA-encoding plasmid for uPA overexpression (pUPAOE). Nontransfected cells were also incubated with exogenously added WT-uPA protein. Cells were immunoprobed for uPA (green) and Lhx2 (red) and mounted with DAPI-containing mounting medium, and fluorescent photomicrographs were obtained as described (Stepanova et al., 2008). (C) Human pancreatic cancer tissue array (± cancer) was stained with H&E or immunoprobed for uPA or Lhx2 (A1, C1, E1, and F2 are malignant pancreatic adenocarcinoma tissues, and F7 is normal pancreatic tissue).
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Figure 3: Nuclear uPA regulates expression of Lhx2 in pancreatic cancer cells. (A) MIA-PaCa-2 and Capan-2 cells were left untreated or incubated with 20 nM of recombinant WT-uPA for 1 h, fixed in MeOH, and stained with anti-uPA rabbit polyclonal Abs and Alexa 488–conjugated anti-rabbit secondary Abs. Nuclei were counterstained with propidium iodide (red). Green staining denotes cytoplasmic and nuclear localization of uPA. (B) MIA PaCa-2 and PANC-1 cells plated and grown on chamber slides were transfected with pSV (scrambled vector) or puPA to lower uPA or uPA-encoding plasmid for uPA overexpression (pUPAOE). Nontransfected cells were also incubated with exogenously added WT-uPA protein. Cells were immunoprobed for uPA (green) and Lhx2 (red) and mounted with DAPI-containing mounting medium, and fluorescent photomicrographs were obtained as described (Stepanova et al., 2008). (C) Human pancreatic cancer tissue array (± cancer) was stained with H&E or immunoprobed for uPA or Lhx2 (A1, C1, E1, and F2 are malignant pancreatic adenocarcinoma tissues, and F7 is normal pancreatic tissue).
Mentions: We demonstrated previously that uPA is found within the nuclei of various types of proliferating cells (Stepanova et al., 2008). We therefore asked whether uPA localizes to the nuclei in pancreatic cancer cells. Immunocytochemical analysis of MIA Pa Ca-2, Capan-2, and PANC-1 cells revealed partial nuclear localization of uPA, which is significantly (p = 0.40) increased when recombinant uPA protein is added exogenously (Figure 3A and Supplemental Figure S4A). More recently, we reported that uPA binds to the transcription factor Lhx2 within the nuclei of pancreatic cancer cells and knockdown of uPA suppresses Lhx2 expression (Gorantla et al., 2011). Because Lhx2 is known to be involved in maintenance of stem/progenitor cell phenotype (Dahl et al., 2008; Tornqvist et al., 2010; Mardaryev et al., 2011; Nadal et al., 2012), we next investigated whether uPA–Lhx cross-talk regulates the maintenance of stem/progenitor cell phenotype in pancreatic cancer cells. To further decipher the role of nuclear uPA in the regulation of Lhx2 expression, we knocked down endogenous uPA expression in MIA PaCa-2 and PANC-1 cells using the puPA plasmid or uPA siRNA (MIA PaCa-2(uPA-) and PANC-1(uPA-) cells, respectively) and added wild-type (WT) uPA exogenously. We observed that addition of exogenous WT-uPA and overexpression of uPA (uPAOE) in MIA PaCa-2(uPA-) and PANC-1(uPA-) cells induced the expression of Lhx2 (Figure 3B). To determine whether uPA positively regulates Lhx2 expression in human tissues, we immunoprobed a human pancreatic tissue array for uPA and Lhx2. In malignant tissues, high levels of expression of both uPA with Lhx2 were observed. In contrast, normal pancreatic tissues showed no detectable expression of uPA and low expression levels of Lhx2 throughout (Figure 3C). Our previous studies indicated that uPAR is not essential for translocation of uPA to the nucleus. A uPA mutant lacking the uPAR-binding domain (ΔGFD-uPA) also translocates to cell nuclei, whereas a kringle-deficient uPA mutant (ΔK-uPA) did not, despite its ability to bind to uPAR (Stepanova et al., 2008). We found identical requirements for translocation of uPA in pancreatic cancer cells (Supplemental Figure S4, A and B). To address the role of uPAR in the regulation of Lhx2 expression by uPA specifically, we then studied the effect of these uPA deletion mutants on expression levels of Lhx2. PANC-1(uPA-) cells overexpressed Lhx2 in response to exogenously added WT-uPA and ΔGFD-uPA, whereas levels were unchanged after addition of identical concentrations of ΔK-uPA (Supplemental Figure S4C).

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