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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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Stem cell–like properties of the SP cells derived from pancreatic cancer cells. (A) Mixed populations of MIA PaCa-2 and PANC-1 cells (2 × 106) were sorted by density-based flow cytometry (10,000 cells sorted per treatment condition, with three replications) to separate SP and ΔSP cells. Acquisition was performed on a FACSCalibur flow cytometer, and viable cells were analyzed with CellQuest software. (B) Cell lysates prepare from the sorted SP and ΔSP cells were immunoblotted for CD24 and CD44 to elucidate expression of cancer stem cell markers. (C) SP, ΔSP, and MP cells were implanted subcutaneously in nude mice (10,000 cells/mouse), and the tumor volumes in treated groups were quantified and represented graphically (mean ± SD; n = 5 and p < 0.001). (D) Subcutaneous tumors grown as in C were implanted orthotopically in the pancreas of nude mice as described in Materials and Methods and allowed to grow for 40 d. At the end of this period, pancreatic tissues were harvested and processed for paraffin sectioning. Expression levels of uPA were determined by immunohistochemistry using anti-uPA and control immunoglobulin G. Brown color denotes uPA-antibody–positive reaction. Normal pancreatic tissue was also sectioned and immunoprobed for uPA. (E) Proliferation and formation of the neurospheres by untreated SP cells derived from MIA-PA Ca-2 and PANC-1 cells (left). Right, disintegration of the neurospheres after exposure to shRNA specific for uPA (puPA).
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Figure 1: Stem cell–like properties of the SP cells derived from pancreatic cancer cells. (A) Mixed populations of MIA PaCa-2 and PANC-1 cells (2 × 106) were sorted by density-based flow cytometry (10,000 cells sorted per treatment condition, with three replications) to separate SP and ΔSP cells. Acquisition was performed on a FACSCalibur flow cytometer, and viable cells were analyzed with CellQuest software. (B) Cell lysates prepare from the sorted SP and ΔSP cells were immunoblotted for CD24 and CD44 to elucidate expression of cancer stem cell markers. (C) SP, ΔSP, and MP cells were implanted subcutaneously in nude mice (10,000 cells/mouse), and the tumor volumes in treated groups were quantified and represented graphically (mean ± SD; n = 5 and p < 0.001). (D) Subcutaneous tumors grown as in C were implanted orthotopically in the pancreas of nude mice as described in Materials and Methods and allowed to grow for 40 d. At the end of this period, pancreatic tissues were harvested and processed for paraffin sectioning. Expression levels of uPA were determined by immunohistochemistry using anti-uPA and control immunoglobulin G. Brown color denotes uPA-antibody–positive reaction. Normal pancreatic tissue was also sectioned and immunoprobed for uPA. (E) Proliferation and formation of the neurospheres by untreated SP cells derived from MIA-PA Ca-2 and PANC-1 cells (left). Right, disintegration of the neurospheres after exposure to shRNA specific for uPA (puPA).

Mentions: Side-population (SP) cells play a crucial role in tumorigenesis and cancer recurrence (Zhang et al., 2013). We first determined whether the side populations of pancreatic cancer cells we studied contain subpopulations of stem-like cells. Because culturing cancer cells under serum-free condition promotes the growth of cancer stem cells (Gou et al., 2007), we cultured MIA PaCa-2 and PANC-1 cells in complete or serum-free media with appropriate growth factors. We then detached the cells with trypsin and sorted them for density and size by standard flow cytometry. Cells cultured under serum-free conditions showed a side population of cells (25–36%) with lower density and size (Figure 1A) that characterize the CSC phenotype (Gou et al., 2007). To confirm this inference, protein extracts from the sorted populations of MIA PaCa-2 and PANC-1 cells grown under serum-free conditions were immunoprobed for the known cancer stem cell markers CD44 and CD24 (Lonardo et al., 2010; Moriyama et al., 2010; Rausch et al., 2010). The SP cells were positive for both CD44 and CD24, whereas the “residual” cells were positive only for CD44 (Figure 1B). These data indicate that the SP cells possess the cancer stem cell surface phenotype (Supplemental Figure S1). To further validate the stem cell character of MIA PaCa-2 SP cells and SP–depleted cells (ΔSP), we implanted these cells subcutaneously in nude mice (10,000 cells per mouse). The inoculates were allowed to grow for 40 d and then scored for the presence or absence of measurable (>1 mm in size) tumors. We observed that in 9 of 10 mice implanted with SP cells, tumors became visually evident within 40 d, whereas none of the mice implanted with CD24-negative cells (10,000 ΔSP cells) formed tumors over that time. When implanted with mixed population (MP) of MIA PaCa-2 cells (10,000 cells/mouse), 4 of 10 mice developed visually evident tumors (Figure 1C). Thus these in vivo studies indicate that the SP cells or cancer stem–like cells have a greater tumorigenicity potential than ΔSP or unseparated cancer cells. To obtain the orthotopic tumors derived from these subcutaneous tumors, we implanted naive nude mice orthotopically in the pancreas with fragments of these subcutaneous tumors as described previously (Fu et al., 1992) and allowed the tumors to develop for an additional 40 d. Forty days after implantation, pancreatic tissues were harvested and processed for paraffin sectioning and immunohistochemical analysis. Because increased expression of uPA is associated with higher “aggressiveness” for multiple tumor types, including pancreatic adenocarcinoma (Ceccarelli et al., 2010; Markl et al., 2010; Bekes et al., 2011; Provost et al., 2012), we studied expression levels of uPA in these orthotopic tumors using immunohistochemistry. We observed that orthotopic tumors grown from the implanted SP cell–derived subcutaneous tumors expressed uPA at much higher levels than those grown from implanted MP cells (Figure 1D). In contrast, normal pancreatic tissue expressed moderate-to-low levels of uPA (Figure 1D). To further assess the role of uPA in establishing the cancer stem cell phenotype, we overexpressed uPA in both SP and ΔSP MIA PaCa-2 cells (uPAOE-SP and uPAOE-ΔSP, respectively) and compared their proliferation and growth patterns using the sphere formation assay. We observed that SP cells possessed greater sphere-forming ability (p < 0.001) than ΔSP cells. Overexpression of uPA induced sphere formation in ΔSP cells (Supplemental Figure S2). The sphere-forming ability of SP cells was attenuated when uPA expression was suppressed with uPA-specific short hairpin RNA (shRNA; Mia PaCa-2(uPA-) and PANC-1(uPA-) cells), which led to significant disintegration of the pancreatospheres (Figure 1E). Fluorescence-activated cell sorting analysis of the mixed populations of MIA PaCa-2 and PANC-1 cells revealed that uPA overexpression (uPAOE) increased the proportion of SP cells (Supplemental Figure S3). Together these data indicate that uPA promotes 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)

Stem cell–like properties of the SP cells derived from pancreatic cancer cells. (A) Mixed populations of MIA PaCa-2 and PANC-1 cells (2 × 106) were sorted by density-based flow cytometry (10,000 cells sorted per treatment condition, with three replications) to separate SP and ΔSP cells. Acquisition was performed on a FACSCalibur flow cytometer, and viable cells were analyzed with CellQuest software. (B) Cell lysates prepare from the sorted SP and ΔSP cells were immunoblotted for CD24 and CD44 to elucidate expression of cancer stem cell markers. (C) SP, ΔSP, and MP cells were implanted subcutaneously in nude mice (10,000 cells/mouse), and the tumor volumes in treated groups were quantified and represented graphically (mean ± SD; n = 5 and p < 0.001). (D) Subcutaneous tumors grown as in C were implanted orthotopically in the pancreas of nude mice as described in Materials and Methods and allowed to grow for 40 d. At the end of this period, pancreatic tissues were harvested and processed for paraffin sectioning. Expression levels of uPA were determined by immunohistochemistry using anti-uPA and control immunoglobulin G. Brown color denotes uPA-antibody–positive reaction. Normal pancreatic tissue was also sectioned and immunoprobed for uPA. (E) Proliferation and formation of the neurospheres by untreated SP cells derived from MIA-PA Ca-2 and PANC-1 cells (left). Right, disintegration of the neurospheres after exposure to shRNA specific for uPA (puPA).
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Related In: Results  -  Collection

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Figure 1: Stem cell–like properties of the SP cells derived from pancreatic cancer cells. (A) Mixed populations of MIA PaCa-2 and PANC-1 cells (2 × 106) were sorted by density-based flow cytometry (10,000 cells sorted per treatment condition, with three replications) to separate SP and ΔSP cells. Acquisition was performed on a FACSCalibur flow cytometer, and viable cells were analyzed with CellQuest software. (B) Cell lysates prepare from the sorted SP and ΔSP cells were immunoblotted for CD24 and CD44 to elucidate expression of cancer stem cell markers. (C) SP, ΔSP, and MP cells were implanted subcutaneously in nude mice (10,000 cells/mouse), and the tumor volumes in treated groups were quantified and represented graphically (mean ± SD; n = 5 and p < 0.001). (D) Subcutaneous tumors grown as in C were implanted orthotopically in the pancreas of nude mice as described in Materials and Methods and allowed to grow for 40 d. At the end of this period, pancreatic tissues were harvested and processed for paraffin sectioning. Expression levels of uPA were determined by immunohistochemistry using anti-uPA and control immunoglobulin G. Brown color denotes uPA-antibody–positive reaction. Normal pancreatic tissue was also sectioned and immunoprobed for uPA. (E) Proliferation and formation of the neurospheres by untreated SP cells derived from MIA-PA Ca-2 and PANC-1 cells (left). Right, disintegration of the neurospheres after exposure to shRNA specific for uPA (puPA).
Mentions: Side-population (SP) cells play a crucial role in tumorigenesis and cancer recurrence (Zhang et al., 2013). We first determined whether the side populations of pancreatic cancer cells we studied contain subpopulations of stem-like cells. Because culturing cancer cells under serum-free condition promotes the growth of cancer stem cells (Gou et al., 2007), we cultured MIA PaCa-2 and PANC-1 cells in complete or serum-free media with appropriate growth factors. We then detached the cells with trypsin and sorted them for density and size by standard flow cytometry. Cells cultured under serum-free conditions showed a side population of cells (25–36%) with lower density and size (Figure 1A) that characterize the CSC phenotype (Gou et al., 2007). To confirm this inference, protein extracts from the sorted populations of MIA PaCa-2 and PANC-1 cells grown under serum-free conditions were immunoprobed for the known cancer stem cell markers CD44 and CD24 (Lonardo et al., 2010; Moriyama et al., 2010; Rausch et al., 2010). The SP cells were positive for both CD44 and CD24, whereas the “residual” cells were positive only for CD44 (Figure 1B). These data indicate that the SP cells possess the cancer stem cell surface phenotype (Supplemental Figure S1). To further validate the stem cell character of MIA PaCa-2 SP cells and SP–depleted cells (ΔSP), we implanted these cells subcutaneously in nude mice (10,000 cells per mouse). The inoculates were allowed to grow for 40 d and then scored for the presence or absence of measurable (>1 mm in size) tumors. We observed that in 9 of 10 mice implanted with SP cells, tumors became visually evident within 40 d, whereas none of the mice implanted with CD24-negative cells (10,000 ΔSP cells) formed tumors over that time. When implanted with mixed population (MP) of MIA PaCa-2 cells (10,000 cells/mouse), 4 of 10 mice developed visually evident tumors (Figure 1C). Thus these in vivo studies indicate that the SP cells or cancer stem–like cells have a greater tumorigenicity potential than ΔSP or unseparated cancer cells. To obtain the orthotopic tumors derived from these subcutaneous tumors, we implanted naive nude mice orthotopically in the pancreas with fragments of these subcutaneous tumors as described previously (Fu et al., 1992) and allowed the tumors to develop for an additional 40 d. Forty days after implantation, pancreatic tissues were harvested and processed for paraffin sectioning and immunohistochemical analysis. Because increased expression of uPA is associated with higher “aggressiveness” for multiple tumor types, including pancreatic adenocarcinoma (Ceccarelli et al., 2010; Markl et al., 2010; Bekes et al., 2011; Provost et al., 2012), we studied expression levels of uPA in these orthotopic tumors using immunohistochemistry. We observed that orthotopic tumors grown from the implanted SP cell–derived subcutaneous tumors expressed uPA at much higher levels than those grown from implanted MP cells (Figure 1D). In contrast, normal pancreatic tissue expressed moderate-to-low levels of uPA (Figure 1D). To further assess the role of uPA in establishing the cancer stem cell phenotype, we overexpressed uPA in both SP and ΔSP MIA PaCa-2 cells (uPAOE-SP and uPAOE-ΔSP, respectively) and compared their proliferation and growth patterns using the sphere formation assay. We observed that SP cells possessed greater sphere-forming ability (p < 0.001) than ΔSP cells. Overexpression of uPA induced sphere formation in ΔSP cells (Supplemental Figure S2). The sphere-forming ability of SP cells was attenuated when uPA expression was suppressed with uPA-specific short hairpin RNA (shRNA; Mia PaCa-2(uPA-) and PANC-1(uPA-) cells), which led to significant disintegration of the pancreatospheres (Figure 1E). Fluorescence-activated cell sorting analysis of the mixed populations of MIA PaCa-2 and PANC-1 cells revealed that uPA overexpression (uPAOE) increased the proportion of SP cells (Supplemental Figure S3). Together these data indicate that uPA promotes 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