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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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ASPH gene was subcloned into pcDNA3 with 5′ c-Myc tagged vector. Inclusion of a c-Myc-tag allowed discrimination of exogenous expressed protein from endogenous cellular levels(a) “Wild type” (WT) ASPH construct; (b) ASPH mutant (H675Q) construct where enzymatic activity was reduced to 20% of “wild type” protein level; (c) Western blot analysis of WT and mutant proteins with antibodies to ASPH and c-Myc. A direct physical interaction of ASPH with (d) Notch1 or (e) JAG2 by co-immunoprecipitation (IP) was observed in MIA PaCa2 cells. ASPH, Notch1, and JAG2 immunoprecipitates were fractionated by SDS-PAGE and analyzed by Western blot using the FB-50 monoclonal antibody. The negative control (Neg) corresponds to an IP result using non-relevant antibody to hepatitis B virus. (f) Microscopy showing increased nuclear translocation of activated Notch1 ICN in the nuclei of MIA PaCa2 cells stably expressing WT-ASPH. Note that the mutant H675Q ASPH substantially reduces nuclear localization of Notch1 ICN comparable to that in vector transfected MIA PaCa2 cells, which suggests that the enzymatic activity of the protein is critical for Notch activation.
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Figure 4: ASPH gene was subcloned into pcDNA3 with 5′ c-Myc tagged vector. Inclusion of a c-Myc-tag allowed discrimination of exogenous expressed protein from endogenous cellular levels(a) “Wild type” (WT) ASPH construct; (b) ASPH mutant (H675Q) construct where enzymatic activity was reduced to 20% of “wild type” protein level; (c) Western blot analysis of WT and mutant proteins with antibodies to ASPH and c-Myc. A direct physical interaction of ASPH with (d) Notch1 or (e) JAG2 by co-immunoprecipitation (IP) was observed in MIA PaCa2 cells. ASPH, Notch1, and JAG2 immunoprecipitates were fractionated by SDS-PAGE and analyzed by Western blot using the FB-50 monoclonal antibody. The negative control (Neg) corresponds to an IP result using non-relevant antibody to hepatitis B virus. (f) Microscopy showing increased nuclear translocation of activated Notch1 ICN in the nuclei of MIA PaCa2 cells stably expressing WT-ASPH. Note that the mutant H675Q ASPH substantially reduces nuclear localization of Notch1 ICN comparable to that in vector transfected MIA PaCa2 cells, which suggests that the enzymatic activity of the protein is critical for Notch activation.

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)

ASPH gene was subcloned into pcDNA3 with 5′ c-Myc tagged vector. Inclusion of a c-Myc-tag allowed discrimination of exogenous expressed protein from endogenous cellular levels(a) “Wild type” (WT) ASPH construct; (b) ASPH mutant (H675Q) construct where enzymatic activity was reduced to 20% of “wild type” protein level; (c) Western blot analysis of WT and mutant proteins with antibodies to ASPH and c-Myc. A direct physical interaction of ASPH with (d) Notch1 or (e) JAG2 by co-immunoprecipitation (IP) was observed in MIA PaCa2 cells. ASPH, Notch1, and JAG2 immunoprecipitates were fractionated by SDS-PAGE and analyzed by Western blot using the FB-50 monoclonal antibody. The negative control (Neg) corresponds to an IP result using non-relevant antibody to hepatitis B virus. (f) Microscopy showing increased nuclear translocation of activated Notch1 ICN in the nuclei of MIA PaCa2 cells stably expressing WT-ASPH. Note that the mutant H675Q ASPH substantially reduces nuclear localization of Notch1 ICN comparable to that in vector transfected MIA PaCa2 cells, which suggests that the enzymatic activity of the protein is critical for Notch activation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: ASPH gene was subcloned into pcDNA3 with 5′ c-Myc tagged vector. Inclusion of a c-Myc-tag allowed discrimination of exogenous expressed protein from endogenous cellular levels(a) “Wild type” (WT) ASPH construct; (b) ASPH mutant (H675Q) construct where enzymatic activity was reduced to 20% of “wild type” protein level; (c) Western blot analysis of WT and mutant proteins with antibodies to ASPH and c-Myc. A direct physical interaction of ASPH with (d) Notch1 or (e) JAG2 by co-immunoprecipitation (IP) was observed in MIA PaCa2 cells. ASPH, Notch1, and JAG2 immunoprecipitates were fractionated by SDS-PAGE and analyzed by Western blot using the FB-50 monoclonal antibody. The negative control (Neg) corresponds to an IP result using non-relevant antibody to hepatitis B virus. (f) Microscopy showing increased nuclear translocation of activated Notch1 ICN in the nuclei of MIA PaCa2 cells stably expressing WT-ASPH. Note that the mutant H675Q ASPH substantially reduces nuclear localization of Notch1 ICN comparable to that in vector transfected MIA PaCa2 cells, which suggests that the enzymatic activity of the protein is critical for Notch activation.
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