Correlations of DKK1 with pathogenesis and prognosis of human multiple myeloma

Abstract

OBJECTIVE:

Human multiple myeloma (MM) is a kind of common tumor in middle-aged and elderly people, in which the osteolytic lesion is formed mainly through inhibiting osteoblast (OB) differentiation and promoting osteoclast (OC) differentiation. Dickkopf-1 (DKK1) is a soluble Wnt inhibitor, which has an important correlation with the pathogenesis of human MM. Therefore, the correlations of DKK1 with pathogenesis and prognosis of human MM were investigated in this study.

METHODS:

The DKK1 expression in tissues and serum of myeloma patients was detected via immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Correlation between DKK1 expression and survival time of patients was analyzed via Kaplan-Meier analysis. To further study the mechanism of DKK1 expression in pathogenesis and prognosis of human MM, MM cells were treated with DKK1 neutralizing antibody (BHQ880) or transfected with DKK1-small-interfering ribonucleic acid (siRNA) to study its effects on OB differentiation, osteocalcin level, $\beta$ -catenin and interleukin-6 (IL-6) secretion. Moreover, the effect of DKK1-siRNA transfection on the activity of U266 cells was detected via methyl thiazolyl tetrazolium (MTT) assay.

RESULTS:

The DKK1 expression in tissues and serum of myeloma patients was significantly higher than that in control group ( $p<$ 0.01). In terms of survival time, the median survival time (45 months) in patients with low DKK1 expression was significantly longer than that in patients with high DKK1 expression (only 22 months). The DKK1 neutralizing antibody (BHQ880) and DKK1-siRNA significantly reduced the DKK1 level in MM cells, promoted the OB differentiation, increased the osteocalcin deposition, promoted the $\beta$ -catenin expression and decreased the IL-6 expression and $\beta$ -catenin phosphorylation. DKK1-siRNA could also reduce the proliferative activity of MM cells.

CONCLUSION:

DKK1 is closely related to the pathogenesis and prognosis of human MM, which might be a potential biomarker for the diagnosis of MM.

1. Introduction

Human multiple myeloma (MM) mainly leads to the blocked osteoblast (OB) differentiation and development of osteolytic lesion due to decline in osteocalcin deposition, whose mechanism mainly depends on the osteolysis caused by the imbalance between OB development and osteoclast (OC) differentiation [1, 2]. Generally, the osteolytic lesion cannot be repaired through the chemically-synthesized drugs [3]. A large number of studies have demonstrated that the pathogenesis of human MM is related to the inhibition of OB differentiation [4, 5].

In recent years, the secretory protein Dickkopf-1 (DKK1) regulated by Wnt has been widely studied in osteolytic lesion of MM [6, 7, 8]. The Wnt pathway plays an important role in cell proliferation and differentiation. The declined DKK1 level in classic Wnt pathway is related to the OB differentiation and osteocalcin deposition in several cancers such as prostate cancer and colon cancer [9, 10]. The Wnt signaling pathway exerts functions through regulating $\beta$ -catenin. According to recent studies, DKK1 neutralizing antibody can inhibit the occurrence of osteolytic lesion [11, 12]. In addition, experiments have demonstrated that inhibiting DKK1 can help reduce bone destruction in arthritis [13]. The latest studies have shown that silencing DKK1 in MM cell via small-interfering ribonucleic acid (siRNA) has important influences on the proliferation and differentiation of MM cells, which can reduce the OC proliferation and differentiation and increase the OB proliferation and osteocalcin deposition [14, 15].

In addition, poor prognosis has been reported in elderly patients with MM [16], and DKK1 is an important factor affecting the pathogenesis of human MM. In this study, therefore, the serum DKK1 in patients with myeloma was detected, and the correlation between the DKK1 expression level and prognosis was analyzed. Moreover, the effects of DKK1 on the biological behaviors of MM U266 cells in vitro were also evaluated.

2. Materials and methods

2.1 Materials

2.1.1 Main reagents and consuming materials

DKK1 neutralizing antibody (BHQ880) was purchased from Beijing Novartis Pharmaceutical Co., Ltd. DKK1-siRNA transfection kit was purchased from Wuhan Booute Biotechnology Co., Ltd. Human DKK1 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Sangon, Shanghai. Interleukin-6 (IL-6) quantitative detection kit was bought from Beijing Hotgen Biotechnology Co., Ltd. Radioimmunoprecipitation assay (RIPA) lysis solution was bought from Beijing Biolab Technology Co., Ltd. Anti-DKK1 antibody, anti- $\beta$ -catenin antibody, anti-p- $\beta$ -catenin antibody and $\beta$ -actin antibody were purchased from Wuhan AmyJet Scientific Co., Ltd. Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG antibody was bought from Beijing Biolab Technology Co., Ltd.

2.1.2 Data source and cell lines

The data of 58 MM patients were obtained from the Gansu Provincial Hospital, and patients were aged 60–70 years old with an average of 66 years old. Meanwhile, 45 age and gender-matched healthy individuals were recruited as a control group. Fifty-eight patients were followed up for 5 years (60 months), the prognosis was observed according to the follow-up conditions, and the survival time was analyzed based on the follow-up results. The MM U266 cell lines were purchased from the China Center for Type Culture Collection (Wuhan University), and cultured in the RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) under 5% CO ${}_2$ at 37 ${}^{\circ }$ C. This study was approved by the ethnic committee of Gansu Provincial Hospital and informed consents were obtained from all individuals.

2.2 Methods

2.2.1 Detection of DKK1 via ELISA

ELISA was performed according to instructions of the kit. Briefly, the kit was firstly warmed up at room temperature for 2 h, and samples were added into the DKK1 monoclonal antibody-coated plate, followed by reaction at room temperature for 45 min and washing with the washing solution for 3 times. Then the biotinylated anti-DKK1 antibody and horseradish peroxidase (HRP)-labeled avidin were added in order, followed by reaction at room temperature for 1 h and washing with the washing solution for 3 times. After the plate was patted dry, the color developing solution was added for color development for 10 min under dark followed by measuring the optical density (OD) at 450 nm. The standard curve was plotted based on the standard sample, and the DKK1 concentration in sample was calculated.

2.2.2 Hematoxylin-eosin (HE) staining

The bone tissues were fixed in the pre-cooled absolute ethanol at $-$ 20 ${}^{\circ }$ C overnight, washed with 70% ethanol and demineralized with 15% ethylene diamine tetraacetic acid (EDTA)-phosphate buffered saline (PBS). After treatment, samples were embedded into paraffin and sliced into 5 $\mu$ m-thick sections using a microtome. After HE staining, sections were observed and photographed under a transmission microscope.

2.2.3 Determination of osteocalcin deposition

The preosteoblasts were cultured for 3 weeks using the OB differentiation medium added with $\beta$ -glycerophosphate and ascorbic acid. The experimental group and control group were set before culture. Then the deposition amount of osteocalcin was detected via ELISA after alizarin red staining. The osteocalcin content was calculated according to the standard curve plotted based on the standard sample.

2.2.4 Determination of cell activity

The proliferative activity of MM U266 cells was detected via methyl thiazolyl tetrazolium (MTT) assay. U266 cells transfected with siRNA and control cells without transfection with siRNA were inoculated into a 24-well plate (1 $\times$ 10 ${}^5$ cells/well), followed by incubation at 37 ${}^{\circ }$ C. Twenty $\mu$ L MTT was added and then removed at 24, 48 and 72 h after culture, DMSO was added (200 $\mu$ L/well), and the OD was detected at 490 nm.

2.2.5 Detection of IL-6

The IL-6 protein content in the cell supernatant was detected via double-antibody sandwich ELISA according to instructions of the kit. Briefly, the samples to be tested were added into the IL-6 monoclonal antibody-coated ELISA plate, and the antigen in samples bound to the antibody to form the antibody-antigen complex. Then HRP-labeled IL-6 monoclonal antibody was added to form the antibody-antigen-HRP complex, followed by blue reaction under the action of tetramethylbenzidine (TMB) substrate and yellow reaction under the action of stop buffer. The OD was detected at 450 nm, and the IL-6 concentration was calculated according to the standard curve plotted based on the standard sample.

2.2.6 Western blotting

After treatment under different conditions, cells were fully lysed using the RIPA lysis solution, added with the protein loading buffer, boiled in water for 10 min and pre-cooled at 4 ${}^{\circ }$ C. The 12% polyacrylamide gel was prepared according to the formula, and samples were added into the corresponding wells, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under the constant pressure of 120 V. When the blue gel reached the bottom of electrophoresis pool, the electrophoresis was terminated, the gel was removed, and the excess part was cut off. According to the gel size, the filter paper and nitrocellulose membrane slightly larger than the gel were cut and placed in order, followed by membrane transfer. After that, the protein was sealed with 5% skim milk-PBS for 2 h and incubated with primary antibodies, including anti-DKK1 antibody (1:500), anti- $\beta$ -catenin and p- $\beta$ -catenin (1:400) and anti- $\beta$ -actin antibody (1:500), overnight. After washing, the HRP-labeled secondary antibody was added for reaction at room temperature for 3 h, followed by color development using the ECL solution and photography.

2.2.7 Immunofluorescence detection

U266 cells were silenced using DKK1-siRNA till the logarithmic growth phase, and cultured for another 3 d, followed by immunofluorescence detection of DKK1 protein. Briefly, cells were washed gently with PBS for 3 times and fixed with pre-cooled absolute ethanol at $-$ 20 ${}^{\circ }$ C for 15 min. Then the solution was discarded, and cells were washed with PBS for 3 times, sealed with 5% skim milk diluted using PBS for 2 h and washed again with PBS for 3 times. Then the anti-DKK1 murine antibody (1:400) was added, followed by reaction at room temperature for 1 h and washing with PBS for 5 times. The FITC-labeled goat anti-mouse IgG antibody (1:500) was added, followed by reaction at 37 ${}^{\circ }$ C for 45 min and washing with PBS for 5 times (5 min/time). Finally, cells were sealed with 10% glycerol-PBS, and observed and photographed under a laser scanning confocal microscope.

2.2.8 Statistical analysis

GraphPad Prism5 software was used for data processing in this study, and results were expressed as mean $\pm$ standard deviation (SD). The survival curve was plotted using the Kaplan-Meier analysis method, and the correlation between the survival time of myeloma patients and the DKK1 expression level was analyzed. Student $t$ test was adopted for other significance analyses. $p<$ 0.05 suggested that the difference was significant.

Figure 1.

DKK1 expression in myeloma in clinical cases.

3. Results

3.1 DKK1 expression in patients with multiple myeloma

The DKK1 expression was detected via ELISA in 58 myeloma patients and 45 healthy individuals. Results revealed that the serum DKK1 expression in myeloma patients was significantly higher than that in control group ( $p<$ 0.01) (Fig. 1).

3.2 Effect of DKK1 expression on survival time of patients

The correlation between survival time and DKK1 expression level in 58 myeloma patients was analyzed via Kaplan-Meier analysis. Fifty-eight myeloma patients were divided into DKK1 low-expression group ( $<$ 10 ng/mL, $n=$ 24) and DKK1 high-expression group ( $>$ 10 ng/mL, $n=$ 34) with 10 ng/mL as the cutoff value. As seen in Fig. 2, the median survival time (45 months) in DKK1 low-expression group was significantly longer than that (22 months) in DKK1 high-expression group (Fig. 2).

Figure 2.

Correlation between DKK1 expression and survival time of patients.

Figure 3.

Effect of DKK1 neutralizing antibody on OB differentiation.

Figure 4.

Detection of DKK1 expression after U266 cells are transfected with DKK1-siRNA via Western blotting and immunofluorescence assay.

3.3 Effect of DKK1 neutralizing antibody (BHQ880) on osteocalcin deposition

MM usually inhibits OB differentiation and increases OC differentiation. Osteocalcin is an important sign of OB differentiation, therefore the osteocalcin deposition was determined to reflect OB differentiation in this study. OB group and OB $+$ MM group were set up in this study. After treatment with BHQ880 and alizarin red staining, osteocalcin deposition was determined via ELISA. As shown in Fig. 3, after BHQ880 treatment, osteocalcin deposition in OB group (no MM cells) was significantly increased. For cells without BHQ880 treatment, the osteocalcin content in OB $+$ MM group was significantly lower than that in OB group, and DKK1 secreted by MM cells significantly inhibited the formation of osteocalcin in OB. In addition, the osteocalcin content was higher in OB $+$ MM group after BHQ880 treatment than that without BHQ880 treatment, further indicating that BHQ880 neutralizes DKK1 secreted by MM cells. To sum up, DKK1 secreted by MM cells can significantly reduce the osteocalcin deposition in OB, forming the osteolytic disease.

3.4 Silencing of DKK1 expression via DKK1-siRNA transfection into U266 cells

To further verify the effect of DKK1 on MM, MM U266 cells were transfected with DKK1-siRNA. The DKK1 expression after silencing was detected to verify the effect of siRNA interference on DKK1 expression. Results of Western blotting, ELISA and immunofluorescence assay revealed that the DKK1 expression was significantly declined in U266 cells transfected with DKK1-siRNA ( $p<$ 0.01), suggesting that siRNA interference successfully silenced the DKK1 expression in U266 cells (Fig. 4).

Figure 5.

Detection of changes in IL-6 content and $\beta$ -catenin expression after siRNA interference via ELISA and Western blotting.

3.5 Effect of siRNA interference on IL-6 and $\beta$ -catenin expressions

The IL-6 content after siRNA interference was detected by IL-6 ELISA kit, and the $\beta$ -catenin expression was detected via Western blotting. In the experiment, OB was cultured together with MM U266 cells. siRNA interference could significantly reduce the IL-6 expression compared with control group ( $p<$ 0.01) (Fig. 5). However, the IL-6 level in OB cultured with U266 cells was significantly higher than that in group without U266 cells, suggesting that MM cells can promote the IL-6 expression in OB ( $p<$ 0.05).

Moreover, the regulation on $\beta$ -catenin expression was studied after siRNA interference of DKK1. Results of Western blotting manifested that inhibition of DKK1 expression by siRNA interference remarkably promoted the $\beta$ -catenin protein expression compared with that in control group, thus reducing the phosphorylation level of $\beta$ -catenin ( $p<$ 0.01).

3.6 Effect of DKK1 inhibition on the activity of U266 cells

To identify the effect of DKK1 on the proliferative activity of U266 cells, we measured the proliferative activity of U266 cells at different time points (24 h, 48 h and 72 h) after siRNA interference of DKK1 by MTT assay. The activity of U266 cells was significantly reduced in DKK1-siRNA group compared with that in control group, which was decreased to about 50% after 72 h (Fig. 6), indicating that DKK1 inhibition significantly reduces the activity of U266 cells ( $p<$ 0.05).

Figure 6.

Detection of activity of U266 cells after siRNA interference in DKK1 via MTT assay.

Figure 7.

Effect of siRNA interference in DKK1 on OB differentiation.

3.7 Effect of DKK1 inhibition on OB differentiation

To investigate whether DKKI inhibition affects OB differentiation, we performed in-vivo mouse test through injection of U266 cells with siRNA interference and control cells. The serum osteocalcin was detected via ELISA, and OB differentiation was detected using the immunohistochemical method. OB differentiation in siRNA transfection group was significantly higher than that in control group, and results of ELISA showed that the osteocalcin content was significantly increased in siRNA transfection group compared with that in control group ( $p<$ 0.01) (Fig. 7), indicating that DKK1 inhibition can significantly increase osteocalcin deposition and promote OB differentiation.

4. Discussion

Human MM mainly causes the osteolytic disease, and its treatment method is the intervention targeting OC currently, such as bisphosphonates [17]. However, the treatment targeting OB differentiation remains vacant. Therefore, the effects of DKK1 on pathogenesis and prognosis of human MM were investigated in this study. In recent years, many studies have demonstrated that there is a certain correlation between DKK1 and pathogenesis of myeloma. Kaiser et al. studied the correlation between the serum DKK1 level and the pathogenesis of myeloma in patients in 2008 [18]. In 2009, Heider studied and showed that the serum DKK1 level in patients was significantly higher before treatment, and significantly declined after treatment [19].

In this study, we showed that serum DKK1 in myeloma patients was significantly higher than that in healthy individuals and the DKK1 expression level was correlated with the survival time of patients with longer survival time in patients with low DKK1 level.

To further study the effect of DKK1 on OB in the progression of MM, DKK1 neutralizing antibody and siRNA interference technique were used. We found BHQ880 promoted the osteocalcin deposition, and MM cells had a certain inhibitory effect on the osteocalcin deposition. Similar results were obtained via siRNA interference. After siRNA interference, OB differentiation and osteocalcin deposition were promoted, levels of IL-6 and $\beta$ -catenin phosphorylation were reduced, and $\beta$ -catenin expression was increased, suggesting that DKK1 may promote the proliferation of MM cells and reduce the OB differentiation and osteocalcin deposition through increasing the IL-6 level and $\beta$ -catenin phosphorylation, ultimately leading to the osteolytic disease. As DKK1 is a negative regulator of Wnt signaling pathway, in the present study we found DKK1 inhibition decreases cell proliferation which might be due to the diverse biological functions of DKK-1 in cancer biology as showed by loss of DKK1 expression as a tumor-suppressive activity in human cancers such as malignant melanoma and colon cancer [20, 21], and overexpression of DKK1 in myriad tumor types including hepatocellular carcinoma and osteosarcoma [22, 23]. Therefore, the biological functions of DKK1 in cancer biology might depend on the cancer cell types and the tumor niche rather than through affecting Wnt signaling alone.

5. Conclusion

Higher DKK1 expression is found in patients with myeloma and is associated with the median survival time. Inhibition of DKK1 promotes OB differentiation and increases osteocalcin deposition, suggesting therapeutic targeting DKK1 might be a novel approach for the treatment of myeloma.