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Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions

View Article: PubMed Central - PubMed

ABSTRACT

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of Tissue Factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced Tissue Factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-over-expressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early stage tumors, and in the hypoxic environment of late-stage cancers. asTF over-expression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF over-expression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of Carbonic Anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor micro-environment.

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Effect of CAIX inhibition on gap closureA) Structure of the CAIX inhibitor U-104, 1-(4-fluorophenyl)-3-(4-sulfamoylphenyl)urea. B) Gap closure ability of Pt45.P1/asTF+ cells over 48 hours in the presence of the CAIX inhibitor U-104 (75 μM) or of vehicle control (0.15% DMSO) under advanced stage or early stage conditions. env. represents the environment, under which the cells were kept, with hyp indicating hypoxic/low glucose and norm indicating normoxic/high glucose. C–G) Quantification of the gap closure abilities by Pt45.P1 and Pt45.P1/asTF+ cells in the presence or absence of U-104. The unoccupied area at 0 hours is set to 100%. C) Pt45.P1/asTF+ cells in early or advanced environments without U-104 treatment. D) Pt45.P1/asTF+ cells in the presence of U-104 under early or late stage environments. E) Pt45.P1/asTF+ cells in the early stage environment in the presence of U-104 or vehicle control. F) Pt45.P1/asTF+ cells in the presence of U-104 or vehicle control under late stage environment. G) Pt45.P1 cells in the presence of U-104 under early or advanced environments. *indicates significance at p < 0.05.
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Figure 6: Effect of CAIX inhibition on gap closureA) Structure of the CAIX inhibitor U-104, 1-(4-fluorophenyl)-3-(4-sulfamoylphenyl)urea. B) Gap closure ability of Pt45.P1/asTF+ cells over 48 hours in the presence of the CAIX inhibitor U-104 (75 μM) or of vehicle control (0.15% DMSO) under advanced stage or early stage conditions. env. represents the environment, under which the cells were kept, with hyp indicating hypoxic/low glucose and norm indicating normoxic/high glucose. C–G) Quantification of the gap closure abilities by Pt45.P1 and Pt45.P1/asTF+ cells in the presence or absence of U-104. The unoccupied area at 0 hours is set to 100%. C) Pt45.P1/asTF+ cells in early or advanced environments without U-104 treatment. D) Pt45.P1/asTF+ cells in the presence of U-104 under early or late stage environments. E) Pt45.P1/asTF+ cells in the early stage environment in the presence of U-104 or vehicle control. F) Pt45.P1/asTF+ cells in the presence of U-104 or vehicle control under late stage environment. G) Pt45.P1 cells in the presence of U-104 under early or advanced environments. *indicates significance at p < 0.05.

Mentions: To evaluate the functional significance of CAIX, which acts downstream of asTF in pancreatic cancer, the cell migration assays were performed in the absence or presence of 75 μM U-104. Under late-stage conditions and in the presence of U-104, the ability of Pt45.P1/asTF+ cells to close the gap was significantly reduced as compared to untreated and/or DMSO treated cells (Figure 6). Of note, U-104 neither affected the motility of Pt45.P1 cells (in early- or late-stage environments), nor of Pt45.P1/asTF+ cells under early stage conditions (Figure 6G). Thus, the hypoxia-specific upregulation of CAIX by asTF-over-expressing cells imparts them with a more aggressive phenotype under conditions that represent late-stage tumors.


Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions
Effect of CAIX inhibition on gap closureA) Structure of the CAIX inhibitor U-104, 1-(4-fluorophenyl)-3-(4-sulfamoylphenyl)urea. B) Gap closure ability of Pt45.P1/asTF+ cells over 48 hours in the presence of the CAIX inhibitor U-104 (75 μM) or of vehicle control (0.15% DMSO) under advanced stage or early stage conditions. env. represents the environment, under which the cells were kept, with hyp indicating hypoxic/low glucose and norm indicating normoxic/high glucose. C–G) Quantification of the gap closure abilities by Pt45.P1 and Pt45.P1/asTF+ cells in the presence or absence of U-104. The unoccupied area at 0 hours is set to 100%. C) Pt45.P1/asTF+ cells in early or advanced environments without U-104 treatment. D) Pt45.P1/asTF+ cells in the presence of U-104 under early or late stage environments. E) Pt45.P1/asTF+ cells in the early stage environment in the presence of U-104 or vehicle control. F) Pt45.P1/asTF+ cells in the presence of U-104 or vehicle control under late stage environment. G) Pt45.P1 cells in the presence of U-104 under early or advanced environments. *indicates significance at p < 0.05.
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Figure 6: Effect of CAIX inhibition on gap closureA) Structure of the CAIX inhibitor U-104, 1-(4-fluorophenyl)-3-(4-sulfamoylphenyl)urea. B) Gap closure ability of Pt45.P1/asTF+ cells over 48 hours in the presence of the CAIX inhibitor U-104 (75 μM) or of vehicle control (0.15% DMSO) under advanced stage or early stage conditions. env. represents the environment, under which the cells were kept, with hyp indicating hypoxic/low glucose and norm indicating normoxic/high glucose. C–G) Quantification of the gap closure abilities by Pt45.P1 and Pt45.P1/asTF+ cells in the presence or absence of U-104. The unoccupied area at 0 hours is set to 100%. C) Pt45.P1/asTF+ cells in early or advanced environments without U-104 treatment. D) Pt45.P1/asTF+ cells in the presence of U-104 under early or late stage environments. E) Pt45.P1/asTF+ cells in the early stage environment in the presence of U-104 or vehicle control. F) Pt45.P1/asTF+ cells in the presence of U-104 or vehicle control under late stage environment. G) Pt45.P1 cells in the presence of U-104 under early or advanced environments. *indicates significance at p < 0.05.
Mentions: To evaluate the functional significance of CAIX, which acts downstream of asTF in pancreatic cancer, the cell migration assays were performed in the absence or presence of 75 μM U-104. Under late-stage conditions and in the presence of U-104, the ability of Pt45.P1/asTF+ cells to close the gap was significantly reduced as compared to untreated and/or DMSO treated cells (Figure 6). Of note, U-104 neither affected the motility of Pt45.P1 cells (in early- or late-stage environments), nor of Pt45.P1/asTF+ cells under early stage conditions (Figure 6G). Thus, the hypoxia-specific upregulation of CAIX by asTF-over-expressing cells imparts them with a more aggressive phenotype under conditions that represent late-stage tumors.

View Article: PubMed Central - PubMed

ABSTRACT

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of Tissue Factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced Tissue Factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-over-expressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early stage tumors, and in the hypoxic environment of late-stage cancers. asTF over-expression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF over-expression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of Carbonic Anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor micro-environment.

No MeSH data available.


Related in: MedlinePlus