Research ArticleCancer Metastasis

Thymidine phosphorylase exerts complex effects on bone resorption and formation in myeloma

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Science Translational Medicine  24 Aug 2016:
Vol. 8, Issue 353, pp. 353ra113
DOI: 10.1126/scitranslmed.aad8949
  • Fig. 1. TP is highly expressed in myeloma.

    (A) Representative immunohistochemical images of bone marrow (BM) biopsies from tissue arrays from 14 healthy and 14 myeloma patients stained for CD138 and TP. Scale bar, 50 μm. MM, multiple myeloma. (B) Densitometry analysis of CD138+ cells or TP+ cells in (A). Data are box plots showing the distribution and median value of quantitative staining (n = 14). P values were determined by Student’s t test. (C) Western blot analysis of TP expression in normal plasma cells from four healthy donors, malignant plasma cells of six myeloma patients (Pt), and six established human myeloma cell lines. Primary plasma cells were isolated from the BM aspirates of healthy donors or myeloma patients. β-Actin served as loading control. Data are representative of triplicate blots.

  • Fig. 2. Association of TP expression and lytic bone lesion in myeloma.

    (A) Correlation coefficient between the mRNA levels of TP and numbers of bone lesion in myeloma patients (n = 52). P values were determined by Pearson coefficient. (B and C) BM biopsy samples from n = 13 patients in (A) were labeled with an anti-TP antibody. TP staining was analyzed using the Image-Pro Plus. (B) Correlation between TP staining in BM biopsies and the numbers of bone lesions in myeloma patients. P values were determined by Pearson coefficient. (C) Representative images of immunohistochemical staining showing TP expression in myeloma cells and CD138+ infiltrated myeloma cells within BM of the patient samples from (B) highlighted with red circles. Scale bar, 10 μm. (D to H) On the basis of the levels of TP expression in myeloma cells, patients’ myeloma cells were separated into high and low TP expression groups (TPhigh and TPlow; n = 5 patients’ BM aspirates per group). In addition, myeloma cells were injected into the bone chips of SCID-hu mice or SCID mouse femurs. Shown are representative x-ray images (D to G) and summarized data of the percentage of bone volumes versus total volumes (BV/TV) (H) of lytic lesion in the implanted human bone chips of SCID-hu mice injected with TPhigh and TPlow cells or in the femurs of SCID mice injected with myeloma cell lines ARP-1 [wild-type (WT), nontargeted shRNA (shCtrl), and TP shRNA (shTP)] and MM.1S [WT, control vector (Vec), and TP cDNA (TP)]. Data are averages ± SD (n = 5 mice per group, three replicate studies). P values were determined by Student’s t test.

  • Fig. 3. Myeloma-expressed TP enhances osteoclast-mediated bone resorption and inhibits osteoblast-mediated bone formation in vivo.

    The implanted human bone chips from SCID-hu mice injected with TPhigh and TPlow cells (n = 5 patients’ BM aspirates per group) or the femurs from SCID mice injected with myeloma cell lines ARP-1 [WT, nontargeted shRNA (shCtrl), and TP shRNA (shTP)] and MM.1S [WT, control vector (Vec), and TP cDNA (TP)] were fixed, TRAP- or toluidine blue–stained, and analyzed by BIOQUANT OSTEO software. (A to D) Percentage of bone surface eroded by osteoclasts (ES/BS) (A), percentage of bone surface covered with osteoclasts (Oc. S/BS) (B), percentage of osteoid surface (OS/BS) (C), and percentage of total bone surface lined with osteoblasts (Ob. S/BS) (D) in myeloma-bearing human bone chips or mouse femurs. (E and F) Bone formation rate (BFR/BS) was measured by calcein injection, and the undecalcified bone sections were imaged and analyzed. Shown are representative images or summarized data of bone formation in the femurs from SCID mice injected with myeloma cell lines ARP-1 (shCtrl and shTP) and MM.1S (Vec and TP). Scale bars, 20 μm. All data are averages ± SD (n = 5 mice per group, three replicate studies). All P values were determined by Student’s t test. y, year.

  • Fig. 4. TP inhibits the expression of RUNX2, osterix, and IRF8 through hypermethylation of their CGIs.

    (A) Schematic diagrams of CpG-rich test regions on the promoter of RUNX2 or osterix in human MSCs and on the promoter of IRF8 in human preOCs. The arrow indicates the translation-initiating ATG site. The CpG-rich test region is marked with a horizontal bar. TSS, transcription start site. (B to E) MSCs or preOCs were cocultured with myeloma cells ARP-1 [WT, nontargeted shRNA (shCtrl), and TP shRNA (shTP)] and MM.1S [WT, control vector (Vec), and TP cDNA (TP)] in their respective medium for 7 days. After cultures, bisulfite-treated genomic DNA was subjected to MSP or BSP analysis. (B) DNA gel electrophoresis shows the unmethylated (U) and methylated (M) PCR products from MSP analysis. Sequencing results from BSP analysis shows percentage of methylation in the promoter of RUNX2 or osterix in MSCs and in the promoter of IRF8 in preOCs cocultured with myeloma cell lines ARP-1 and MM.1S (C), shCtrl or shTP ARP-1 cells (D), or Vec or TP MM.1S cells (E). Cultured MSCs or preOCs without myeloma (No MM) served as a control. Data in (C) to (E) are individual cultures with averages ± SD (n = 5) of three experiments. P values were determined by Student’s t test. (F) Summary of BSP analysis showing percentage of methylation in the promoter of RUNX2 or osterix in MSCs and in the promoter of IRF8 in preOCs, of healthy donors and TPhigh or TPlow patients. Data are individual patients’ samples with averages ± SD (n = 5) of three experiments. P values were determined by Student’s t test.

  • Fig. 5. 2DDR inhibits osteoblast differentiation and activates osteoclast differentiation by up-regulating DNMT3A expression.

    Human MSCs or preOCs were cultured in medium without (0) or with 0.5, 1, or 2 mM 2DDR for 48 hours. In some studies, MSCs or preOCs carried with nontargeted shRNAs (shCtrl) or DNMT3A shRNAs (shDNMT3A) were cultured with phosphate-buffered saline (PBS) or 1 mM 2DDR. (A to D) Expression of RUNX2 and osterix (A), DNMT3A mRNA expression and activity (B), methylation of CGIs in the promoter regions of RUNX2 and osterix (C), and ALP activity and Alizarin red S staining (D) in MSC-derived cells after 2DDR treatment. OD490, optical density at 490 nm. (E to H) Expression of IRF8 (E), DNMT3A mRNA expression and activity (F), methylation of CGIs in the promoter region of IRF8 (G), and the number of multiple nuclear (≥3) TRAP+ cells and secretion of TRAP5b (H) in preOC-derived cells after 2DDR treatment. mRNA expression was normalized to cells without 2DDR (set at 1). The levels of β-actin served as loading controls. Data are averages ± SD (n = 3). P values were determined by Student’s t test. Each experiment was repeated three times.

  • Fig. 6. Administration of TP inhibitor in myeloma-bearing mice reduces bone lesions and osteoclastogenesis and enhances osteoblastogenesis.

    ARP-1 cells were injected into the femurs of SCID mice. Mice without myeloma cells served as controls (No MM). After 3 weeks, mice were treated with PBS as vehicle controls or TP inhibitor 7DX (200 μg/kg) or TPI (300 μg/kg). After treatment, mice were scanned for radiography, and mouse femurs were subjected to toluidine blue staining or TRAP staining. (A) Representative x-ray images of mouse femurs. (B to D) Percentage of bone volume to total volume (BV/TV) (B), percentage of bone surface eroded by osteoclasts (ES/BS) and of bone surface covered with osteoclasts (Oc. S/BS) (C), and percentage of osteoid surface (OS/BS) and of bone surface lined with osteoblasts (Ob. S/BS) (D). Data are averages ± SD (n = 5 mice per group, three replicate studies). (E) Dnmt3a mRNA expression in murine MSCs and preOCs isolated from BM aspirates of ARP-1–bearing mice. Data are averages relative to no MM–bearing mice (No MM) treated with vehicle (set at 1) ± SD (n = 5 mice/group, three replicate studies). (F) 2DDR levels in the serum of ARP-1–bearing mice. Data are averages relative to that in no MM–bearing mice (No MM) treated with vehicle (set at 1) ± SD (n = 5 mice per group, three replicate studies). All P values were determined by Student’s t test. (G) Depiction of signaling pathways involved in the myeloma TP-mediated suppression of osteoblastogenesis and activation of osteoclastogenesis. OB, osteoblast.

  • Fig. 7. TP expressed by myeloma cells regulates osteoblast and osteoclast differentiation in vitro and in vivo.

    (A) Relative levels of 2DDR in human myeloma cell lines RPMI 8226 or U266 were measured after 48 hours of culture. Data are relative to RPMI 8226. Data are averages ± SD (n = 3) of three experiments. (B) PreOCs were cultured alone or cocultured with RPMI 8226 or U266 cells in medium without or with RANKL (10 ng/ml) for 1 week. PreOCs alone without or with RANKL (10 ng/ml) served as controls. Numbers of multinuclear (≥3) TRAP+ cells and the relative expression of osteoclast differentiation–associated genes TRAP, CALCR, and CTSK were measured. mRNA expression was normalized to cells without myeloma (No MM, set to 1). Data are averages ± SD (n = 3) of three experiments. (C) MSCs were cocultured with RPMI 8226 or U266 in osteoblast medium for 2 weeks and then stained with Alizarin red S. The relative expression of osteoblast differentiation–associated genes BGLAP, ALP, and COL1A1 was determined in attached cells. mRNA expression was normalized to cells without myeloma (No MM, set to 1). Data are averages ± SD (n = 3) of five experiments. (D to F) RPMI 8226 cells (5 × 105 cells per mouse) were injected into the femurs of SCID mice. Mice without myeloma cell injection served as controls (No MM). After 3 weeks, mice were intraperitoneally injected with PBS as vehicle control or the TP inhibitors 7DX (200 μg/kg) or TPI (300 μg/kg) three times per week for 2 weeks. After treatment, mice were scanned for radiography, and mouse femurs were subjected to toluidine blue staining or TRAP staining. We calculated the percentage of bone volume to total volume (BV/TV) (D), the percentage of bone surface eroded by osteoclasts (ES/BS) and of bone surface covered with osteoclasts (Oc. S/BS) (E), and the percentage of osteoid surface (OS/BS) and of bone surface lined with osteoblasts (Ob. S/BS) (F). Data are averages ± SD (n = 5 mice per group, three replicate studies). All P values were determined by Student’s t test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/353/353ra113/DC1

    Methods

    Fig. S1. Modulation of TP expression does not affect the growth and survival of myeloma cells.

    Fig. S2. Myeloma-expressed TP enhances RANKL-mediated osteoclast differentiation and activity in vitro.

    Fig. S3. Myeloma-expressed TP enhances NFATc1 expression and activity through inhibition of IRF8.

    Fig. S4. Myeloma-expressed TP inhibits osteoblast differentiation and activity in vitro.

    Fig. S5. TP inhibits the expression and activities of RUNX2 and osterix in vitro.

    Fig. S6. Myeloma-expressed TP enhances DNMT3A levels in MSCs and preOCs.

    Fig. S7. Myeloma cells with high TP expression secrete more 2DDR, which affects osteoclast and osteoblast differentiation in vitro.

    Fig. S8. 2DDR up-regulates DNMT3A through α5β1Vβ3-PI3K/Akt signaling pathways.

    Fig. S9. Knockdown of integrins or Akt1/2 abrogates the effects of 2DDR on DNMT3A expression.

    Table S1. Primers for reverse transcription PCR and quantitative PCR.

    Table S2. Primers for chromatin immunoprecipitation PCR.

    Table S3. Primers for MSP and BSP.

  • Supplementary Material for:

    Thymidine phosphorylase exerts complex effects on bone resorption and formation in myeloma

    Huan Liu, Zhiqiang Liu, Juan Du, Jin He, Pei Lin, Behrang Amini, Michael W. Starbuck, Nora Novane, Jatin J. Shah, Richard E. Davis, Jian Hou, Robert F. Gagel, Jing Yang*

    *Corresponding author. Email: jiyang{at}mdanderson.org

    Published 24 August 2016, Sci. Transl. Med. 8, 353ra113 (2016)
    DOI: 10.1126/scitranslmed.aad8949

    This PDF file includes:

    • Methods
    • Fig. S1. Modulation of TP expression does not affect the growth and survival of myeloma cells.
    • Fig. S2. Myeloma-expressed TP enhances RANKL-mediated osteoclast differentiation and activity in vitro.
    • Fig. S3. Myeloma-expressed TP enhances NFATc1 expression and activity through inhibition of IRF8.
    • Fig. S4. Myeloma-expressed TP inhibits osteoblast differentiation and activity in vitro.
    • Fig. S5. TP inhibits the expression and activities of RUNX2 and osterix in vitro.
    • Fig. S6. Myeloma-expressed TP enhances DNMT3A levels in MSCs and preOCs.
    • Fig. S7. Myeloma cells with high TP expression secrete more 2DDR, which affects osteoclast and osteoblast differentiation in vitro.
    • Fig. S8. 2DDR up-regulates DNMT3A through α5β1Vβ3-PI3K/Akt signaling pathways.
    • Fig. S9. Knockdown of integrins or Akt1/2 abrogates the effects of 2DDR on DNMT3A expression.
    • Table S1. Primers for reverse transcription PCR and quantitative PCR.
    • Table S2. Primers for chromatin immunoprecipitation PCR.
    • Table S3. Primers for MSP and BSP.

    [Download PDF]

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