Yonsei Med J.  2017 May;58(3):505-513. 10.3349/ymj.2017.58.3.505.

Cyclized Oligopeptide Targeting LRP5/6-DKK1 Interaction Reduces the Growth of Tumor Burden in a Multiple Myeloma Mouse Model

• Park BM
• Kim EJ
• Nam HJ
• Zhang D
• Bae CH
• Kang M
• Kim H
• Lee W
• Bogen B
• Lim SK

• KMID: 2419106
• DOI: http://doi.org/10.3349/ymj.2017.58.3.505

Abstract

PURPOSE
Dickkopf 1 (DKK1) has been extensively investigated in mouse models of multiple myeloma, which results in osteolytic bone lesions. Elevated DKK1 levels in bone marrow plasma and serum inhibit the differentiation of osteoblast precursors. Present pharmaceutical approaches to target bone lesions are limited to antiresorptive agents. In this study, we developed a cyclized oligopeptide against DKK1-low density lipoprotein receptor-related protein (LRP) 5/6 interaction and tested the effects of the oligopeptide on tumor burden.
MATERIALS AND METHODS
A cyclized oligopeptide based on DKK1-LRP5/6 interactions was synthesized chemically, and its nuclear magnetic resonance structure was assessed. Luciferase reporter assay and mRNA expressions of osteoblast markers were evaluated after oligopeptide treatment. MOPC315.BM.Luc cells were injected into the tail vein of mice, after which cyclized oligopeptide was delivered subcutaneously 6 days a week for 4 weeks.
RESULTS
The cyclized oligopeptide containing NXI motif bound to the E1 domain of LRP5/6 effectively on surface plasmon resonance analysis. It abrogated the Wnt-β-catenin signaling inhibited by DKK1, but not by sclerostin, dose dependently. RT-PCR and alkaline phosphatase staining showed increased expressions of osteoblast markers according to the treatment concentrations. Bioluminescence images showed that the treatment of cyclized oligopeptide reduced tumor burden more in oligopeptide treated group than in the vehicle group.
CONCLUSION
The cyclized oligopeptide reported here may be another option for the treatment of tumor burden in multiple myeloma.

Keyword

Multiple myeloma; DKK1; oligopeptide; tumor; burden; Wnt signaling

MeSH Terms

Animals
Bone Marrow/*metabolism
Cell Differentiation/drug effects
Cell Line, Tumor
Disease Models, Animal
Intercellular Signaling Peptides and Proteins/*metabolism
Mice
Multiple Myeloma/*complications/pathology/*physiopathology
Oligopeptides/*pharmacology
Osteoblasts/*drug effects/pathology
Signal Transduction
Tumor Burden/*drug effects
Wnt Proteins/metabolism
beta Catenin
Intercellular Signaling Peptides and Proteins
Oligopeptides
Wnt Proteins
beta Catenin

Figure

• 

  Fig. 1 NMR structure of cyclized oligopeptide. (A) 2D TOCSY spectrum of DKK1 (1 mM) in 100% DMSO at pH 7.0, 283 K, recorded with a spin-lock mixing time of 70 ms. The spectrum shows the identification of amino acid spin systems based on scalar correlation of the backbone amide protons of (ω2), with the respective side-chain protons (ω1) of each spin system. (B) A backbone superposition of the energy-minimized average structure (〈SA〉kr) over the family of 20 final SA structures (〈SA〉kr). (C) A ribbon diagram of DKK1 with side-chain orientations is displayed as a stick model. NMR, nuclear magnetic resonance; 2D, two-dimensional; TOCSY, total correlation spectroscopy; DKK1, dickkopf 1; SA, simulated annealing.

• 

  Fig. 2 Luciferase reporter activity of cells treated with cyclized oligopeptide and ProteOn XPR36 analysis. MC3T3-E1-S-Top cells were treated with Wnt3a, DKK1, sclerostin, and oligopeptide. (A) This graph shows the oligopeptide bound directly to the LRP5 E1 domain and the activity response increased dose dependently. (B) Graph on the left shows the dose increasing with luciferase reporter activity as the concentration of the oligopeptide increased. However, in the graph on the right, when treated with sclerostin, the same trend is not seen. (C) The same cells were treated with Wnt7a, DKK1, sclerostin and oligopeptide. DKK1, dickkopf 1; LRP, lipoprotein receptor-related protein; SOST, sclerositn.

• 

  Fig. 3 Reverse transcription-PCR and Alkaline phosphatase staining assay. (A) ST2 cells were treated with Wnt7a, DKK1, and oligopeptide then the mRNA expression of both ALP and OPG assessed. (B) When Wnt7a alone was used, the staining was stronger than when Wnt7a and DKK1 were used together. The staining intensity also increased dose dependently with increasing concentration of oligopeptide treatment. Original magnification ×40. (C) The expressions of DKK1 and β-catenin were checked in the MOPC.315.BM.Luc cells prior to the experiment. DKK1, dickkopf 1; ALP, alkaline phosphatase; OPG, osteoprotegerin.

• 

  Fig. 4 Tumor growth was reduced in the MOPC315.BM.Luc cell induced multiple myeloma mouse model. (A) A brief timeline of the animal study is shown. (B) One representative mouse from each group was chosen based on the closest radiance activity to the median value. Images taken on day 0 and day 28 are shown and compared. On day 0, BLI was taken before the oligopeptide treatment. No radiance was detected in the untreated mouse. (C) Bioluminescence radiance fold changes of each group from day 0 to day 28 are shown. Each group was comprised of 10 mice. (D) Mice numbers according to the fold changes of bioluminescence radiance are shown. Fold changes were calculated simply by dividing the final value by the initial value. *p<0.05 and ***p<0.0001 by Mann-Whitney U test. MM, multiple myeloma.

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