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

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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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Acceleration of gap closure by asTF over-expressorsA) Comparison of gap closure ability of Pt45.P1/asTF+ cells versus Pt45P1 cells under early and advanced stage conditions. B,C) Quantification of cell motility. The graph shows the area unoccupied by Pt45.P1/asTF+ or Pt45.P1 cells under both early and advanced stages at various time intervals (0 h, 18 h, 24 h, 48 h). D,E) Effect of thymidine on cell motility. This assay was performed to compare the gap closure ability of asTF over-expressors and Pt45.P1 cells in the presence of thymidine (2 μM), a G1/S blocker. The graph represents the area unoccupied by Pt45.P1/asTF+ and Pt45.P1 cells under both normoxia and hypoxia at the designated time intervals. The error bars are SEM. *indicates significance at p < 0.05.
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Figure 5: Acceleration of gap closure by asTF over-expressorsA) Comparison of gap closure ability of Pt45.P1/asTF+ cells versus Pt45P1 cells under early and advanced stage conditions. B,C) Quantification of cell motility. The graph shows the area unoccupied by Pt45.P1/asTF+ or Pt45.P1 cells under both early and advanced stages at various time intervals (0 h, 18 h, 24 h, 48 h). D,E) Effect of thymidine on cell motility. This assay was performed to compare the gap closure ability of asTF over-expressors and Pt45.P1 cells in the presence of thymidine (2 μM), a G1/S blocker. The graph represents the area unoccupied by Pt45.P1/asTF+ and Pt45.P1 cells under both normoxia and hypoxia at the designated time intervals. The error bars are SEM. *indicates significance at p < 0.05.

Mentions: To measure the capacity for directed migration, a gap closure (“wound healing”) assay was performed, whereby we assessed the ability of asTF-over-expressing versus control Pt45.P1 cells to close a gap created by disrupting a monolayer through scratching the center of a well. asTF over-expression facilitated cell motility as evidenced by a complete restoration of the Pt45.P1/asTF+ monolayer within 24 hours, whereas Pt45.P1 cells still showed a visible gap after 48 hours (Figure 5A–C). To ensure that the migration result was not compromised by the different growth rates between Pt45.P1/asTF+ cells and the parental cell line Pt45.P1, we also performed the experiment in the presence of 2 μM thymidine, which blocks cell cycle progression at G1/S phase 26. The results were comparable, corroborating the enhancing effect of asTF on cell motility (Figure 5D–E).


Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions
Acceleration of gap closure by asTF over-expressorsA) Comparison of gap closure ability of Pt45.P1/asTF+ cells versus Pt45P1 cells under early and advanced stage conditions. B,C) Quantification of cell motility. The graph shows the area unoccupied by Pt45.P1/asTF+ or Pt45.P1 cells under both early and advanced stages at various time intervals (0 h, 18 h, 24 h, 48 h). D,E) Effect of thymidine on cell motility. This assay was performed to compare the gap closure ability of asTF over-expressors and Pt45.P1 cells in the presence of thymidine (2 μM), a G1/S blocker. The graph represents the area unoccupied by Pt45.P1/asTF+ and Pt45.P1 cells under both normoxia and hypoxia at the designated time intervals. The error bars are SEM. *indicates significance at p < 0.05.
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Related In: Results  -  Collection

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Figure 5: Acceleration of gap closure by asTF over-expressorsA) Comparison of gap closure ability of Pt45.P1/asTF+ cells versus Pt45P1 cells under early and advanced stage conditions. B,C) Quantification of cell motility. The graph shows the area unoccupied by Pt45.P1/asTF+ or Pt45.P1 cells under both early and advanced stages at various time intervals (0 h, 18 h, 24 h, 48 h). D,E) Effect of thymidine on cell motility. This assay was performed to compare the gap closure ability of asTF over-expressors and Pt45.P1 cells in the presence of thymidine (2 μM), a G1/S blocker. The graph represents the area unoccupied by Pt45.P1/asTF+ and Pt45.P1 cells under both normoxia and hypoxia at the designated time intervals. The error bars are SEM. *indicates significance at p < 0.05.
Mentions: To measure the capacity for directed migration, a gap closure (“wound healing”) assay was performed, whereby we assessed the ability of asTF-over-expressing versus control Pt45.P1 cells to close a gap created by disrupting a monolayer through scratching the center of a well. asTF over-expression facilitated cell motility as evidenced by a complete restoration of the Pt45.P1/asTF+ monolayer within 24 hours, whereas Pt45.P1 cells still showed a visible gap after 48 hours (Figure 5A–C). To ensure that the migration result was not compromised by the different growth rates between Pt45.P1/asTF+ cells and the parental cell line Pt45.P1, we also performed the experiment in the presence of 2 μM thymidine, which blocks cell cycle progression at G1/S phase 26. The results were comparable, corroborating the enhancing effect of asTF on cell motility (Figure 5D–E).

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