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. 2016 Aug 24;8(353):353ra113.
doi: 10.1126/scitranslmed.aad8949.

Thymidine Phosphorylase Exerts Complex Effects on Bone Resorption and Formation in Myeloma

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Free PMC article

Thymidine Phosphorylase Exerts Complex Effects on Bone Resorption and Formation in Myeloma

Huan Liu et al. Sci Transl Med. .
Free PMC article

Abstract

Myelomatous bone disease is characterized by the development of lytic bone lesions and a concomitant reduction in bone formation, leading to chronic bone pain and fractures. To understand the underlying mechanism, we investigated the contribution of myeloma-expressed thymidine phosphorylase (TP) to bone lesions. In osteoblast progenitors, TP up-regulated the methylation of RUNX2 and osterix, leading to decreased bone formation. In osteoclast progenitors, TP up-regulated the methylation of IRF8 and thereby enhanced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1 protein), leading to increased bone resorption. TP reversibly catalyzes thymidine into thymine and 2-deoxy-d-ribose (2DDR). Myeloma-secreted 2DDR bound to integrin αVβ3/α5β1 in the progenitors, activated PI3K (phosphoinositide 3-kinase)/Akt signaling, and increased DNMT3A (DNA methyltransferase 3A) expression, resulting in hypermethylation of RUNX2, osterix, and IRF8 This study elucidates an important mechanism for myeloma-induced bone lesions, suggesting that targeting TP may be a viable approach to healing resorbed bone in patients. Because TP overexpression is common in bone-metastatic tumors, our findings could have additional mechanistic implications.

Conflict of interest statement

The authors have no competing financial interests.

Figures

Fig. 1
Fig. 1. TP is highly expressed in myeloma
(A) Representative immunohistochemical images of bone marrow biopsies from tissue arrays from 14 healthy and 14 myeloma patients stained for CD138 and TP. (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). Scale bars, 50 μm. P values were determined by Student’s t test. (C) Western blot analysis of TP expression in normal plasma cells from 4 healthy donors, malignant plasma cells of 6 myeloma patients, and 6 established human myeloma cell lines. Primary plasma cells were isolated from the bone marrow aspirates of healthy donors or myeloma patients. β-Actin served as loading control. Data are representative of triplicate blots.
Fig. 2
Fig. 2. Association of TP expression and lytic bone lesion in myeloma
(A) Shown is the 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) Bone marrow 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) The correlation between TP staining in bone marrow biopsies and the numbers of bone lesions in myeloma patients. P values were determined by Pearson Coefficient. (C) Representative images of immunohistochemical staining show TP expression in myeloma cells and CD138+ infiltrated myeloma cells within bone marrow of the patient samples from (B) highlighted with red circles. Scale bar, 10 μm. (D to H) Based on 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’ bone marrow aspirates/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 (DG) and summarized data of the percentage of bone volumes vs 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), non-targeted shRNA (shCtrl), and TP shRNA (shTP)] and MM.1S [WT, control vector (Vec), and TP cDNA (TP)]. Data are averages ± SD (n = 5 mice/group, 3 replicate studies). P values were determined by Student’s t test.
Fig. 3
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’ bone marrow aspirates/group) or the femurs from SCID mice injected with myeloma cell lines ARP-1 (wild-type [WT], non-targeted 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) The percentage of bone surface eroded by osteoclasts (ES/BS) (A), the percentage of bone surface covered with osteoclasts (Oc.S/BS) (B), the percentage of osteoid surface (OS/BS) (C), and the 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 bar, 20 μm. All data are averages ± SD (n = 5 mice/group, 3 replicate studies). All P values were determined by Student’s t test.
Fig. 4
Fig. 4. TP inhibits the expression of RUNX2, osterix, and IRF8 through hypermethylation of their CpG islands
(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. TSS represents the transcription start site. The arrow indicates the translation-initiating ATG site. The CpG-rich test region is marked with a horizontal bar. (B to E) MSCs or preOCs were co-cultured with myeloma cells ARP-1 (wild-type [WT], non-targeted 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 methylation-specific PCR (MSP) or bisulfite sequencing PCR (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 co-cultured 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 are individual samples with averages ± SD (n = 5) of 3 experiments. P values were determined by Student’s t test. (F) Summary of BSP analysis shows 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 samples with averages ± SD (n = 5) of 3 experiments. P values were determined by Student’s t test.
Fig. 5
Fig. 5. 2DDR inhibits osteoblast differentiation and activates osteoclast differentiation by upregulating DNMT3A expression
Human MSCs or preOCs were cultured in medium without (0) or with 0.5, 1, or 2 mM of 2DDR for 48 hours. In some studies, MSCs or preOCs carried with non-targeted shRNAs (shCtrl) or DNMT3A shRNAs (shDNMT3A) were cultured with 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. (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
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) The percentage of bone volume to total volume (BV/TV) (B), bone surface eroded by osteoclasts (ES/BS) and 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/group, 3 replicate studies). (E) Dnmt3a mRNA expression in murine MSCs and preOCs isolated from bone marrow 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, 3 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/group, 3 replicate studies). All P values were determined by Student’s t test. (G) A depiction of signaling pathways involved in the myeloma TP-mediated suppression of osteoblastgenesis and activation of osteoclastgenesis.
Fig. 7
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 RPMI8226 or U266 were measured after 48 hours of culture. Data are relative to RPMI8226. Data are averages ± SD (n = 3) of 3 experiments. (B) PreOCs were cultured alone or co-cultured with RPMI8226 or U266 cells in medium without or with RANKL (10 ng/ml) for 1 week. preOCs alone without or with 10 ng/ml of RANKL 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 3 experiments. (C) MSCs were co-cultured with RPMI8226 or U266 in osteoblast medium for 2 weeks and then stained for Alizarin red S. The relative expression of osteoblast differentiation–associated genes BGLAP, ALP, and COL1A1 were determined in attached cells. mRNA expression was normalized to cells without myeloma (No MM, set to 1). Data are averages ± SD (n = 3) of 5 experiments. (D to F) RPMI8226 cells (5×105 cells/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), bone surface eroded by osteoclasts (ES/BS) and bone surface covered with osteoclasts (Oc. S/BS) (E), as well as percentage of osteoid surface (OS/BS) and bone surface lined with osteoblasts (Ob.S/BS) (F). Data are averages ± SD (n = 5 mice/group, 3 replicate studies). All P values were determined by Student’s t test.

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