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Aspartate β-Hydroxylase expression promotes a malignant pancreatic cellular phenotype.

Dong X, Lin Q, Aihara A, Li Y, Huang CK, Chung W, Tang Q, Chen X, Carlson R, Nadolny C, Gabriel G, Olsen M, Wands JR - Oncotarget (2015)

Bottom Line: The transforming properties of ASPH depend on enzymatic activity.ASPH links PC growth factor signaling cascades to Notch activation.A small molecule inhibitor of β-hydroxylase activity was developed and found to reduce PC growth by downregulating the Notch signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island, Kingston, RI, USA.

ABSTRACT
Pancreatic cancer (PC) is one of the leading causes of cancer related deaths due to aggressive progression and metastatic spread. Aspartate β-hydroxylase (ASPH), a cell surface protein that catalyzes the hydroxylation of epidermal growth factor (EGF)-like repeats in Notch receptors and ligands, is highly overexpressed in PC. ASPH upregulation confers a malignant phenotype characterized by enhanced cell proliferation, migration, invasion and colony formation in vitro as well as PC tumor growth in vivo. The transforming properties of ASPH depend on enzymatic activity. ASPH links PC growth factor signaling cascades to Notch activation. A small molecule inhibitor of β-hydroxylase activity was developed and found to reduce PC growth by downregulating the Notch signaling pathway. These findings demonstrate the critical involvement of ASPH in PC growth and progression, provide new insight into the molecular mechanisms leading to tumor development and growth and have important therapeutic implications.

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Related in: MedlinePlus

The β-hydroxylase activity of ASPH is required for its transforming activityMIA PaCa2 cells were stably transfected with empty vector (EV), “wild type” (WT)-ASPH, or H675Q-ASPH mutant and the levels of expression were confirmed by Western blot. There is low-level endogenous ASPH in MIA PaCa2 cells as shown here and in Fig. 1. Effects of EV, WT-ASPH and H675Q-ASPH on (a) cell proliferation, (b) migration, (c) invasion, and (d) colony formation are significantly different. (e and f) represents a Western blot demonstrating WT-ASPH induced activation of Notch signaling as determined by increased levels of Notch1 ICN, JAG2, as well as increased expression of downstream responsive genes HES1, HEY1, EpCAM, CD44, c-Myc, MMP2/9, cyclin D3 and PCNA. In contrast, the mutant H675Q ASPH construct shows significantly reduced activated Notch1 ICN, JAG2, as well as HES1, HEY1, EpCAM, CD44, c-Myc, MMP2/9, cyclin D3 and PCNA. The results suggest that the β-hydroxylase activity of ASPH is essential for Notch signaling activation. *p<0.05; **p<0.01; ***p<0.001.
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Figure 5: The β-hydroxylase activity of ASPH is required for its transforming activityMIA PaCa2 cells were stably transfected with empty vector (EV), “wild type” (WT)-ASPH, or H675Q-ASPH mutant and the levels of expression were confirmed by Western blot. There is low-level endogenous ASPH in MIA PaCa2 cells as shown here and in Fig. 1. Effects of EV, WT-ASPH and H675Q-ASPH on (a) cell proliferation, (b) migration, (c) invasion, and (d) colony formation are significantly different. (e and f) represents a Western blot demonstrating WT-ASPH induced activation of Notch signaling as determined by increased levels of Notch1 ICN, JAG2, as well as increased expression of downstream responsive genes HES1, HEY1, EpCAM, CD44, c-Myc, MMP2/9, cyclin D3 and PCNA. In contrast, the mutant H675Q ASPH construct shows significantly reduced activated Notch1 ICN, JAG2, as well as HES1, HEY1, EpCAM, CD44, c-Myc, MMP2/9, cyclin D3 and PCNA. The results suggest that the β-hydroxylase activity of ASPH is essential for Notch signaling activation. *p<0.05; **p<0.01; ***p<0.001.

Mentions: We have established WT-ASPH and H675Q mutant expression constructs (Fig. 4a, b, c). The human H675Q mutant ASPH has an 80% reduction in enzymatic activity and was subsequently examined to determine if there would be a loss in ability to promote cell proliferation, migration, invasion, and colony formation compared to the “wild type” protein. Figure 5 illustrates the stimulatory effects of WT-ASPH overexpression in PC cells on proliferation, migration, invasion, and colony formation, as well as the activation of Notch responsive genes. However, the mutant ASPH H675Q inhibited cell proliferation, migration, invasion, and colony formation as well as reduced HES1, HEY1, CD44, EpCAM, c-Myc, MMP2/9, cyclin D3, and PCNA gene expression in PC cells.


Aspartate β-Hydroxylase expression promotes a malignant pancreatic cellular phenotype.

Dong X, Lin Q, Aihara A, Li Y, Huang CK, Chung W, Tang Q, Chen X, Carlson R, Nadolny C, Gabriel G, Olsen M, Wands JR - Oncotarget (2015)

The β-hydroxylase activity of ASPH is required for its transforming activityMIA PaCa2 cells were stably transfected with empty vector (EV), “wild type” (WT)-ASPH, or H675Q-ASPH mutant and the levels of expression were confirmed by Western blot. There is low-level endogenous ASPH in MIA PaCa2 cells as shown here and in Fig. 1. Effects of EV, WT-ASPH and H675Q-ASPH on (a) cell proliferation, (b) migration, (c) invasion, and (d) colony formation are significantly different. (e and f) represents a Western blot demonstrating WT-ASPH induced activation of Notch signaling as determined by increased levels of Notch1 ICN, JAG2, as well as increased expression of downstream responsive genes HES1, HEY1, EpCAM, CD44, c-Myc, MMP2/9, cyclin D3 and PCNA. In contrast, the mutant H675Q ASPH construct shows significantly reduced activated Notch1 ICN, JAG2, as well as HES1, HEY1, EpCAM, CD44, c-Myc, MMP2/9, cyclin D3 and PCNA. The results suggest that the β-hydroxylase activity of ASPH is essential for Notch signaling activation. *p<0.05; **p<0.01; ***p<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359229&req=5

Figure 5: The β-hydroxylase activity of ASPH is required for its transforming activityMIA PaCa2 cells were stably transfected with empty vector (EV), “wild type” (WT)-ASPH, or H675Q-ASPH mutant and the levels of expression were confirmed by Western blot. There is low-level endogenous ASPH in MIA PaCa2 cells as shown here and in Fig. 1. Effects of EV, WT-ASPH and H675Q-ASPH on (a) cell proliferation, (b) migration, (c) invasion, and (d) colony formation are significantly different. (e and f) represents a Western blot demonstrating WT-ASPH induced activation of Notch signaling as determined by increased levels of Notch1 ICN, JAG2, as well as increased expression of downstream responsive genes HES1, HEY1, EpCAM, CD44, c-Myc, MMP2/9, cyclin D3 and PCNA. In contrast, the mutant H675Q ASPH construct shows significantly reduced activated Notch1 ICN, JAG2, as well as HES1, HEY1, EpCAM, CD44, c-Myc, MMP2/9, cyclin D3 and PCNA. The results suggest that the β-hydroxylase activity of ASPH is essential for Notch signaling activation. *p<0.05; **p<0.01; ***p<0.001.
Mentions: We have established WT-ASPH and H675Q mutant expression constructs (Fig. 4a, b, c). The human H675Q mutant ASPH has an 80% reduction in enzymatic activity and was subsequently examined to determine if there would be a loss in ability to promote cell proliferation, migration, invasion, and colony formation compared to the “wild type” protein. Figure 5 illustrates the stimulatory effects of WT-ASPH overexpression in PC cells on proliferation, migration, invasion, and colony formation, as well as the activation of Notch responsive genes. However, the mutant ASPH H675Q inhibited cell proliferation, migration, invasion, and colony formation as well as reduced HES1, HEY1, CD44, EpCAM, c-Myc, MMP2/9, cyclin D3, and PCNA gene expression in PC cells.

Bottom Line: The transforming properties of ASPH depend on enzymatic activity.ASPH links PC growth factor signaling cascades to Notch activation.A small molecule inhibitor of β-hydroxylase activity was developed and found to reduce PC growth by downregulating the Notch signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island, Kingston, RI, USA.

ABSTRACT
Pancreatic cancer (PC) is one of the leading causes of cancer related deaths due to aggressive progression and metastatic spread. Aspartate β-hydroxylase (ASPH), a cell surface protein that catalyzes the hydroxylation of epidermal growth factor (EGF)-like repeats in Notch receptors and ligands, is highly overexpressed in PC. ASPH upregulation confers a malignant phenotype characterized by enhanced cell proliferation, migration, invasion and colony formation in vitro as well as PC tumor growth in vivo. The transforming properties of ASPH depend on enzymatic activity. ASPH links PC growth factor signaling cascades to Notch activation. A small molecule inhibitor of β-hydroxylase activity was developed and found to reduce PC growth by downregulating the Notch signaling pathway. These findings demonstrate the critical involvement of ASPH in PC growth and progression, provide new insight into the molecular mechanisms leading to tumor development and growth and have important therapeutic implications.

Show MeSH
Related in: MedlinePlus