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Inhibition of ERBB2-overexpressing Tumors by Recombinant Human Prolidase and Its Enzymatically Inactive Mutant.

Yang L, Li Y, Bhattacharya A, Zhang Y - EBioMedicine (2015)

Bottom Line: ERBB2 is an oncogenic receptor tyrosine kinase overexpressed in a subset of human breast cancer and other cancers.We recently found that human prolidase (PEPD), a dipeptidase, is a high affinity ERBB2 ligand and cross-links two ERBB2 monomers.Several anti-ERBB2 agents are on the market but are hampered by drug resistance and high drug cost. rhPEPD(G278D) may synergize with these agents and may also be highly cost-effective, since it targets ERBB2 with a different mechanism and can be produced in bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemoprevention, Roswell Park Cancer Institute, Buffalo, NY 14263, United States.

ABSTRACT

ERBB2 is an oncogenic receptor tyrosine kinase overexpressed in a subset of human breast cancer and other cancers. We recently found that human prolidase (PEPD), a dipeptidase, is a high affinity ERBB2 ligand and cross-links two ERBB2 monomers. Here, we show that recombinant human PEPD (rhPEPD) strongly inhibits ERBB2-overexpressing tumors in mice, whereas it does not impact tumors without ERBB2 overexpression. rhPEPD causes ERBB2 depletion, disrupts oncogenic signaling orchestrated by ERBB2 homodimers and heterodimers, and induces apoptosis. The impact of enzymatically-inactive mutant rhPEPD(G278D) on ERBB2 is indistinguishable from that of rhPEPD, but rhPEPD(G278D) is superior to rhPEPD for tumor inhibition. The enzymatic function of rhPEPD stimulates HIF-1α and other pro-survival factors in tumors, which likely attenuates its antitumor activity. rhPEPD(G278D) is also attractive in that it may not interfere with the physiologic function of endogenous PEPD in normal cells. Collectively, we have identified a human protein as an inhibitory ERBB2 ligand that inhibits ERBB2-overexpressing tumors in vivo. Several anti-ERBB2 agents are on the market but are hampered by drug resistance and high drug cost. rhPEPD(G278D) may synergize with these agents and may also be highly cost-effective, since it targets ERBB2 with a different mechanism and can be produced in bacteria.

No MeSH data available.


Related in: MedlinePlus

The effect of rhPEPD and rhPEPDG278D on ERBB2-ERBB3 heterodimer interaction. [A] CHO-K1/ERBB2 cells were transfected with human ERBB3 for 24 h and then treated with rhPEPD (5 nM), followed by immunoblotting. [B & C] CHO-K1/ERBB2 cells were transfected with human ERBB3 for 24 h and then treated with solvent, rhPEPD or rhPEPDG278D (each at 5 nM) for 1 h, with or without pretreatment with NRG-1 (20 nM, 15 min). Cell lysates were immunoprecipitated using an ERBB2 antibody or an ERBB3 antibody, followed by immunoblotting. For the immunoblots shown in B, sample loading was adjusted to contain an equal amount of ERBB2.
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f0025: The effect of rhPEPD and rhPEPDG278D on ERBB2-ERBB3 heterodimer interaction. [A] CHO-K1/ERBB2 cells were transfected with human ERBB3 for 24 h and then treated with rhPEPD (5 nM), followed by immunoblotting. [B & C] CHO-K1/ERBB2 cells were transfected with human ERBB3 for 24 h and then treated with solvent, rhPEPD or rhPEPDG278D (each at 5 nM) for 1 h, with or without pretreatment with NRG-1 (20 nM, 15 min). Cell lysates were immunoprecipitated using an ERBB2 antibody or an ERBB3 antibody, followed by immunoblotting. For the immunoblots shown in B, sample loading was adjusted to contain an equal amount of ERBB2.

Mentions: In BT-474 tumors (Fig. 3B), neither rhPEPD nor rhPEPDG278D downregulated ERBB3, consistent with our recent finding that the agents do not bind to ERBB3, but both agents caused ERBB3 to dephosphorylate along with loss of PI3K activity. In cells overexpressing both ERBB2 and ERBB3 (CHO-K1/ERBB2 + ERBB3), with no expression of other ERBBs, rhPEPD (5 nM) caused ERBB3 dephosphorylation without protein downregulation, along with transient increase in ERBB2 phosphorylation, rapid ERBB2 protein downregulation and rapid SRC dephosphorylation, whereas PI3K expression remained unchanged (Fig. 5A). As mentioned before, SRC is activated upon binding to activated ERBB2, whereas PI3K is activated by binding to activated ERBB3. In CHO-K1/ERBB2 + ERBB3 cells, both rhPEPD and rhPEPDG278D disassembled the ErbB2–ErbB3 signaling unit, causing dissociation of ERBB3 from ERBB2 along with dissociation of SRC and PI3K from the ERBBs (Fig. 5B and C), whether the signaling complex formed spontaneously or was stimulated by NRG-1 (an ERBB3 ligand). Moreover, both agents caused NRG-1-bound ERBB3 to dissociate from ERBB2 (Fig. 5C). It is possible that the agents may bind to ERBB2 in the heterodimer to force ERBB2 homodimerization at the expense of heterodimerization and/or disrupt the heterodimer by causing ERBB2 depletion.


Inhibition of ERBB2-overexpressing Tumors by Recombinant Human Prolidase and Its Enzymatically Inactive Mutant.

Yang L, Li Y, Bhattacharya A, Zhang Y - EBioMedicine (2015)

The effect of rhPEPD and rhPEPDG278D on ERBB2-ERBB3 heterodimer interaction. [A] CHO-K1/ERBB2 cells were transfected with human ERBB3 for 24 h and then treated with rhPEPD (5 nM), followed by immunoblotting. [B & C] CHO-K1/ERBB2 cells were transfected with human ERBB3 for 24 h and then treated with solvent, rhPEPD or rhPEPDG278D (each at 5 nM) for 1 h, with or without pretreatment with NRG-1 (20 nM, 15 min). Cell lysates were immunoprecipitated using an ERBB2 antibody or an ERBB3 antibody, followed by immunoblotting. For the immunoblots shown in B, sample loading was adjusted to contain an equal amount of ERBB2.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4465122&req=5

f0025: The effect of rhPEPD and rhPEPDG278D on ERBB2-ERBB3 heterodimer interaction. [A] CHO-K1/ERBB2 cells were transfected with human ERBB3 for 24 h and then treated with rhPEPD (5 nM), followed by immunoblotting. [B & C] CHO-K1/ERBB2 cells were transfected with human ERBB3 for 24 h and then treated with solvent, rhPEPD or rhPEPDG278D (each at 5 nM) for 1 h, with or without pretreatment with NRG-1 (20 nM, 15 min). Cell lysates were immunoprecipitated using an ERBB2 antibody or an ERBB3 antibody, followed by immunoblotting. For the immunoblots shown in B, sample loading was adjusted to contain an equal amount of ERBB2.
Mentions: In BT-474 tumors (Fig. 3B), neither rhPEPD nor rhPEPDG278D downregulated ERBB3, consistent with our recent finding that the agents do not bind to ERBB3, but both agents caused ERBB3 to dephosphorylate along with loss of PI3K activity. In cells overexpressing both ERBB2 and ERBB3 (CHO-K1/ERBB2 + ERBB3), with no expression of other ERBBs, rhPEPD (5 nM) caused ERBB3 dephosphorylation without protein downregulation, along with transient increase in ERBB2 phosphorylation, rapid ERBB2 protein downregulation and rapid SRC dephosphorylation, whereas PI3K expression remained unchanged (Fig. 5A). As mentioned before, SRC is activated upon binding to activated ERBB2, whereas PI3K is activated by binding to activated ERBB3. In CHO-K1/ERBB2 + ERBB3 cells, both rhPEPD and rhPEPDG278D disassembled the ErbB2–ErbB3 signaling unit, causing dissociation of ERBB3 from ERBB2 along with dissociation of SRC and PI3K from the ERBBs (Fig. 5B and C), whether the signaling complex formed spontaneously or was stimulated by NRG-1 (an ERBB3 ligand). Moreover, both agents caused NRG-1-bound ERBB3 to dissociate from ERBB2 (Fig. 5C). It is possible that the agents may bind to ERBB2 in the heterodimer to force ERBB2 homodimerization at the expense of heterodimerization and/or disrupt the heterodimer by causing ERBB2 depletion.

Bottom Line: ERBB2 is an oncogenic receptor tyrosine kinase overexpressed in a subset of human breast cancer and other cancers.We recently found that human prolidase (PEPD), a dipeptidase, is a high affinity ERBB2 ligand and cross-links two ERBB2 monomers.Several anti-ERBB2 agents are on the market but are hampered by drug resistance and high drug cost. rhPEPD(G278D) may synergize with these agents and may also be highly cost-effective, since it targets ERBB2 with a different mechanism and can be produced in bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemoprevention, Roswell Park Cancer Institute, Buffalo, NY 14263, United States.

ABSTRACT

ERBB2 is an oncogenic receptor tyrosine kinase overexpressed in a subset of human breast cancer and other cancers. We recently found that human prolidase (PEPD), a dipeptidase, is a high affinity ERBB2 ligand and cross-links two ERBB2 monomers. Here, we show that recombinant human PEPD (rhPEPD) strongly inhibits ERBB2-overexpressing tumors in mice, whereas it does not impact tumors without ERBB2 overexpression. rhPEPD causes ERBB2 depletion, disrupts oncogenic signaling orchestrated by ERBB2 homodimers and heterodimers, and induces apoptosis. The impact of enzymatically-inactive mutant rhPEPD(G278D) on ERBB2 is indistinguishable from that of rhPEPD, but rhPEPD(G278D) is superior to rhPEPD for tumor inhibition. The enzymatic function of rhPEPD stimulates HIF-1α and other pro-survival factors in tumors, which likely attenuates its antitumor activity. rhPEPD(G278D) is also attractive in that it may not interfere with the physiologic function of endogenous PEPD in normal cells. Collectively, we have identified a human protein as an inhibitory ERBB2 ligand that inhibits ERBB2-overexpressing tumors in vivo. Several anti-ERBB2 agents are on the market but are hampered by drug resistance and high drug cost. rhPEPD(G278D) may synergize with these agents and may also be highly cost-effective, since it targets ERBB2 with a different mechanism and can be produced in bacteria.

No MeSH data available.


Related in: MedlinePlus