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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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uPA suppresses p53 promoter activity via binding to and attenuation of the Hoxa-5 function. (A) HEK293 cells were transfected with HOXA5-FLAG in pcDNA3.1 and uPA/pcDNA3.1 vector. Two days after transfection, cells were harvested, and nuclear extracts were prepared using the Novagen NucBuster Protein Extraction Kit. uPA and/or HOXA5 were immunoprecipitated using anti-uPA mouse monoclonal Abs (IMTEK, Moscow, Russia) and rabbit polyclonal anti-HOXA5 Abs (Santa Cruz Biotechnology). Immunoprecipitated proteins were subjected to Western blot analysis. Immunoprecipitated and coimmunoprecipitated uPA and/or HOXA5-FLAG were detected using anti-uPA rabbit polyclonal Abs (389; American Diagnostica, Stamford, CT) and mouse monoclonal HRP-conjugated anti-FLAG M2 Abs (Sigma-Aldrich). (B) MIA PaCa-2 and PANC-1 cancer cells were stably transfected with p53 promoter luciferase reporter plasmid. uPA expression was suppressed with puPA or uPA was overexpressed (uPAOE) in these cells. In parallel, cells were incubated with WT-uPA. p53 promoter activity was determined by measurement of luciferase activity as described in Materials and Methods. (C) MIA PaCa-2 and PANC-1 cells were stably transfected with p53-luc plasmid and sorted to obtain SP and ΔSP cells. uPA was suppressed or overexpressed or added to the cells, and luciferase activity was measured as described. (D) HEK293 cells were cotransfected with p53-luc, uPA/pcDNA3.1+ plasmid encoding human WT-uPA, or HOXA5-FLAG/pcDNA3.1 plasmid encoding C-terminus–tagged HOXA5-FLAG. Empty pcDNA3.1 was used as the negative control, and pRL TK plasmid encoding constitutively expressed Renilla luciferase was cotransfected to normalize the data. Luciferase activity was determined using a Promega Dual Luciferase Reporter Assay Kit. (E) Effect of uPA on DNA-binding capacity of HOXA5. HEK293 cells were transfected either with empty pcDNA3.1 (mock transfected) or with HOXA5-FLAG in pcDNA3.1 alone or in combination with uPA/pcDNA3.1 vector. Two days after transfection, cells were harvested. Nuclear extracts were prepared using the NucBuster Protein Extraction Kit. EMSA reactions were performed using biotinylated, double-stranded, p53 promoter-derived Hoxa-5–specific oligonucleotides. 1, No NE; 2, + mock-transfected NE; 3, + uPA-transfected NE; 4, + HOXA5-transfected NE; 5, + HOXA5-transfected NE + BSA; 6, + HOXA5-transfected NE + scuPA (500 ng); 7, + HOXA5-transfected NE + specific “cold” oligo duplex; 8, + HOXA5-transfected NE + scuPA + anti-uPA Abs; 9, + mock-transfected NE + scuPA (500 ng). S1, Probe shift, caused by HOXA5 overexpression; S2, DNA–protein complex, formed in presence of NE from the mock-transfected cells; SS1, probe supershift caused by HOXA5-bound anti-HOXA5 Ab.
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Figure 6: uPA suppresses p53 promoter activity via binding to and attenuation of the Hoxa-5 function. (A) HEK293 cells were transfected with HOXA5-FLAG in pcDNA3.1 and uPA/pcDNA3.1 vector. Two days after transfection, cells were harvested, and nuclear extracts were prepared using the Novagen NucBuster Protein Extraction Kit. uPA and/or HOXA5 were immunoprecipitated using anti-uPA mouse monoclonal Abs (IMTEK, Moscow, Russia) and rabbit polyclonal anti-HOXA5 Abs (Santa Cruz Biotechnology). Immunoprecipitated proteins were subjected to Western blot analysis. Immunoprecipitated and coimmunoprecipitated uPA and/or HOXA5-FLAG were detected using anti-uPA rabbit polyclonal Abs (389; American Diagnostica, Stamford, CT) and mouse monoclonal HRP-conjugated anti-FLAG M2 Abs (Sigma-Aldrich). (B) MIA PaCa-2 and PANC-1 cancer cells were stably transfected with p53 promoter luciferase reporter plasmid. uPA expression was suppressed with puPA or uPA was overexpressed (uPAOE) in these cells. In parallel, cells were incubated with WT-uPA. p53 promoter activity was determined by measurement of luciferase activity as described in Materials and Methods. (C) MIA PaCa-2 and PANC-1 cells were stably transfected with p53-luc plasmid and sorted to obtain SP and ΔSP cells. uPA was suppressed or overexpressed or added to the cells, and luciferase activity was measured as described. (D) HEK293 cells were cotransfected with p53-luc, uPA/pcDNA3.1+ plasmid encoding human WT-uPA, or HOXA5-FLAG/pcDNA3.1 plasmid encoding C-terminus–tagged HOXA5-FLAG. Empty pcDNA3.1 was used as the negative control, and pRL TK plasmid encoding constitutively expressed Renilla luciferase was cotransfected to normalize the data. Luciferase activity was determined using a Promega Dual Luciferase Reporter Assay Kit. (E) Effect of uPA on DNA-binding capacity of HOXA5. HEK293 cells were transfected either with empty pcDNA3.1 (mock transfected) or with HOXA5-FLAG in pcDNA3.1 alone or in combination with uPA/pcDNA3.1 vector. Two days after transfection, cells were harvested. Nuclear extracts were prepared using the NucBuster Protein Extraction Kit. EMSA reactions were performed using biotinylated, double-stranded, p53 promoter-derived Hoxa-5–specific oligonucleotides. 1, No NE; 2, + mock-transfected NE; 3, + uPA-transfected NE; 4, + HOXA5-transfected NE; 5, + HOXA5-transfected NE + BSA; 6, + HOXA5-transfected NE + scuPA (500 ng); 7, + HOXA5-transfected NE + specific “cold” oligo duplex; 8, + HOXA5-transfected NE + scuPA + anti-uPA Abs; 9, + mock-transfected NE + scuPA (500 ng). S1, Probe shift, caused by HOXA5 overexpression; S2, DNA–protein complex, formed in presence of NE from the mock-transfected cells; SS1, probe supershift caused by HOXA5-bound anti-HOXA5 Ab.

Mentions: To further elucidate the functions of uPA in the nucleus, we examined whether uPA binds directly to transcription factors (TFs), using a protein TF array. Supplemental Figure S5A shows that uPA binds to homeobox transcription factors Lhx2, HOXA5, and Hey. Binding of uPA to Lhx2 (Gorantla et al., 2011) and HOXA5 (Figure 6A) was confirmed by coimmunoprecipitation pull-down assays. HOXA5 up-regulates expression of the p53 tumor suppressor gene in breast cancer cells (Raman et al., 2000). It has been suggested that down-regulation of HOXA5 expression or loss of its function results in inhibition of p53 expression (Raman et al., 2000), which enhances tumorigenicity, EMT, and acquisition of stem-like phenotype by breast cancer cells (Mizuno et al., 2010). Loss of p53 function has also been linked to the induction of EMT and stemness in pancreatic cells (Keck and Brabletz, 2011; Pasi and Pelicci, 2011). Therefore we hypothesized that uPA further promotes stemness of pancreatic cancer cells by down-regulating p53 through interference with HOXA5. To assess whether uPA regulates activity of the p53 promoter, we stably transfected MIA PaCa-2 and PANC-1 cells with the human p53 promoter-driven luciferase reporter construct (Mia PaCa-2(p53-luc) and PANC-1(p53-luc) cells, respectively). Suppression of uPA expression in Mia PaCa-2(p53-luc) and PANC-1(p53-luc) cells caused activation of p53 promoter-driven luciferase expression, whereas uPA overexpression (uPAOE) inhibited p53 promoter-controlled luciferase activity (Figure 6B). Similar results were obtained using SP or ΔSP cells (Figure 6C). To determine whether uPA suppresses p53 promoter activity by interfering with the function of HOXA5, we used human embryonic kidney 293 cells (HEK293 cells), which express low levels of endogenous HOXA5 and uPA. HEK293 cells were cotransfected with the p53-luc construct and HOXA5- and/or uPA-encoding vectors. Figure 6D shows that coexpression of p53-luc and HOXA5 leads to activation of p53 promoter activity, whereas concomitant coexpression of uPA attenuates HOXA5-mediated activation of p53 promoter.


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)

uPA suppresses p53 promoter activity via binding to and attenuation of the Hoxa-5 function. (A) HEK293 cells were transfected with HOXA5-FLAG in pcDNA3.1 and uPA/pcDNA3.1 vector. Two days after transfection, cells were harvested, and nuclear extracts were prepared using the Novagen NucBuster Protein Extraction Kit. uPA and/or HOXA5 were immunoprecipitated using anti-uPA mouse monoclonal Abs (IMTEK, Moscow, Russia) and rabbit polyclonal anti-HOXA5 Abs (Santa Cruz Biotechnology). Immunoprecipitated proteins were subjected to Western blot analysis. Immunoprecipitated and coimmunoprecipitated uPA and/or HOXA5-FLAG were detected using anti-uPA rabbit polyclonal Abs (389; American Diagnostica, Stamford, CT) and mouse monoclonal HRP-conjugated anti-FLAG M2 Abs (Sigma-Aldrich). (B) MIA PaCa-2 and PANC-1 cancer cells were stably transfected with p53 promoter luciferase reporter plasmid. uPA expression was suppressed with puPA or uPA was overexpressed (uPAOE) in these cells. In parallel, cells were incubated with WT-uPA. p53 promoter activity was determined by measurement of luciferase activity as described in Materials and Methods. (C) MIA PaCa-2 and PANC-1 cells were stably transfected with p53-luc plasmid and sorted to obtain SP and ΔSP cells. uPA was suppressed or overexpressed or added to the cells, and luciferase activity was measured as described. (D) HEK293 cells were cotransfected with p53-luc, uPA/pcDNA3.1+ plasmid encoding human WT-uPA, or HOXA5-FLAG/pcDNA3.1 plasmid encoding C-terminus–tagged HOXA5-FLAG. Empty pcDNA3.1 was used as the negative control, and pRL TK plasmid encoding constitutively expressed Renilla luciferase was cotransfected to normalize the data. Luciferase activity was determined using a Promega Dual Luciferase Reporter Assay Kit. (E) Effect of uPA on DNA-binding capacity of HOXA5. HEK293 cells were transfected either with empty pcDNA3.1 (mock transfected) or with HOXA5-FLAG in pcDNA3.1 alone or in combination with uPA/pcDNA3.1 vector. Two days after transfection, cells were harvested. Nuclear extracts were prepared using the NucBuster Protein Extraction Kit. EMSA reactions were performed using biotinylated, double-stranded, p53 promoter-derived Hoxa-5–specific oligonucleotides. 1, No NE; 2, + mock-transfected NE; 3, + uPA-transfected NE; 4, + HOXA5-transfected NE; 5, + HOXA5-transfected NE + BSA; 6, + HOXA5-transfected NE + scuPA (500 ng); 7, + HOXA5-transfected NE + specific “cold” oligo duplex; 8, + HOXA5-transfected NE + scuPA + anti-uPA Abs; 9, + mock-transfected NE + scuPA (500 ng). S1, Probe shift, caused by HOXA5 overexpression; S2, DNA–protein complex, formed in presence of NE from the mock-transfected cells; SS1, probe supershift caused by HOXA5-bound anti-HOXA5 Ab.
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Figure 6: uPA suppresses p53 promoter activity via binding to and attenuation of the Hoxa-5 function. (A) HEK293 cells were transfected with HOXA5-FLAG in pcDNA3.1 and uPA/pcDNA3.1 vector. Two days after transfection, cells were harvested, and nuclear extracts were prepared using the Novagen NucBuster Protein Extraction Kit. uPA and/or HOXA5 were immunoprecipitated using anti-uPA mouse monoclonal Abs (IMTEK, Moscow, Russia) and rabbit polyclonal anti-HOXA5 Abs (Santa Cruz Biotechnology). Immunoprecipitated proteins were subjected to Western blot analysis. Immunoprecipitated and coimmunoprecipitated uPA and/or HOXA5-FLAG were detected using anti-uPA rabbit polyclonal Abs (389; American Diagnostica, Stamford, CT) and mouse monoclonal HRP-conjugated anti-FLAG M2 Abs (Sigma-Aldrich). (B) MIA PaCa-2 and PANC-1 cancer cells were stably transfected with p53 promoter luciferase reporter plasmid. uPA expression was suppressed with puPA or uPA was overexpressed (uPAOE) in these cells. In parallel, cells were incubated with WT-uPA. p53 promoter activity was determined by measurement of luciferase activity as described in Materials and Methods. (C) MIA PaCa-2 and PANC-1 cells were stably transfected with p53-luc plasmid and sorted to obtain SP and ΔSP cells. uPA was suppressed or overexpressed or added to the cells, and luciferase activity was measured as described. (D) HEK293 cells were cotransfected with p53-luc, uPA/pcDNA3.1+ plasmid encoding human WT-uPA, or HOXA5-FLAG/pcDNA3.1 plasmid encoding C-terminus–tagged HOXA5-FLAG. Empty pcDNA3.1 was used as the negative control, and pRL TK plasmid encoding constitutively expressed Renilla luciferase was cotransfected to normalize the data. Luciferase activity was determined using a Promega Dual Luciferase Reporter Assay Kit. (E) Effect of uPA on DNA-binding capacity of HOXA5. HEK293 cells were transfected either with empty pcDNA3.1 (mock transfected) or with HOXA5-FLAG in pcDNA3.1 alone or in combination with uPA/pcDNA3.1 vector. Two days after transfection, cells were harvested. Nuclear extracts were prepared using the NucBuster Protein Extraction Kit. EMSA reactions were performed using biotinylated, double-stranded, p53 promoter-derived Hoxa-5–specific oligonucleotides. 1, No NE; 2, + mock-transfected NE; 3, + uPA-transfected NE; 4, + HOXA5-transfected NE; 5, + HOXA5-transfected NE + BSA; 6, + HOXA5-transfected NE + scuPA (500 ng); 7, + HOXA5-transfected NE + specific “cold” oligo duplex; 8, + HOXA5-transfected NE + scuPA + anti-uPA Abs; 9, + mock-transfected NE + scuPA (500 ng). S1, Probe shift, caused by HOXA5 overexpression; S2, DNA–protein complex, formed in presence of NE from the mock-transfected cells; SS1, probe supershift caused by HOXA5-bound anti-HOXA5 Ab.
Mentions: To further elucidate the functions of uPA in the nucleus, we examined whether uPA binds directly to transcription factors (TFs), using a protein TF array. Supplemental Figure S5A shows that uPA binds to homeobox transcription factors Lhx2, HOXA5, and Hey. Binding of uPA to Lhx2 (Gorantla et al., 2011) and HOXA5 (Figure 6A) was confirmed by coimmunoprecipitation pull-down assays. HOXA5 up-regulates expression of the p53 tumor suppressor gene in breast cancer cells (Raman et al., 2000). It has been suggested that down-regulation of HOXA5 expression or loss of its function results in inhibition of p53 expression (Raman et al., 2000), which enhances tumorigenicity, EMT, and acquisition of stem-like phenotype by breast cancer cells (Mizuno et al., 2010). Loss of p53 function has also been linked to the induction of EMT and stemness in pancreatic cells (Keck and Brabletz, 2011; Pasi and Pelicci, 2011). Therefore we hypothesized that uPA further promotes stemness of pancreatic cancer cells by down-regulating p53 through interference with HOXA5. To assess whether uPA regulates activity of the p53 promoter, we stably transfected MIA PaCa-2 and PANC-1 cells with the human p53 promoter-driven luciferase reporter construct (Mia PaCa-2(p53-luc) and PANC-1(p53-luc) cells, respectively). Suppression of uPA expression in Mia PaCa-2(p53-luc) and PANC-1(p53-luc) cells caused activation of p53 promoter-driven luciferase expression, whereas uPA overexpression (uPAOE) inhibited p53 promoter-controlled luciferase activity (Figure 6B). Similar results were obtained using SP or ΔSP cells (Figure 6C). To determine whether uPA suppresses p53 promoter activity by interfering with the function of HOXA5, we used human embryonic kidney 293 cells (HEK293 cells), which express low levels of endogenous HOXA5 and uPA. HEK293 cells were cotransfected with the p53-luc construct and HOXA5- and/or uPA-encoding vectors. Figure 6D shows that coexpression of p53-luc and HOXA5 leads to activation of p53 promoter activity, whereas concomitant coexpression of uPA attenuates HOXA5-mediated activation of p53 promoter.

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