Scilit | Article - Clinical significance of decreased programmed cell death 4 expression in patients with gia [...] (2023)

Clinical significance of decreased programmed cell death 4 expression in patients with giant cell tumors of the bone

Abstract: Programmed cell death 4 (PDCD4) has been recognized as a novel tumor suppressor gene, which inhibits the activation and translation of activator protein (AP)‑1. Dysregulated expression of PDCD4 is also involved in various human tumors and is linked to tumor progression and development. However, the function and clinical implication of PDCD4 in giant cell tumors of the bone (GCTBs) has not been previously investigated. In the present study, PDCD4 expression was determined in 83 samples of GCTBs at mRNA and protein levels by quantitative reverse transcription‑polymerase chain reaction, western blotting and immunohistochemistry. The results demonstrated that PDCD4 mRNA expression was reduced in 63% of GCTB samples (17/27) and protein expression was decreased in 65% of samples (54/83), compared with adjacent non‑tumor tissues. Furthermore, decreased expression of PDCD4 was significantly associated with certain clinicopathological characteristics, including the Campanacci grade and recurrence. A strong negative correlation was determined between PDCD4 expression and the Ki‑67 positive rate in GCTBs (r=‑0.6392; P Introduction Giant cell tumor of the bone (GCTB) is generally a benign, but often locally aggressive osteolytic tumor which easily causes severe bone destruction at the meta-epiphyseal region of the long bones and more than half of GCTBs occur around the knee (1,2). With modern surgical techniques, more aggressive curettage may aid in avoiding higher recurrence rates (3). However, it has been reported that ~10–60% of GCTBs exhibit local postoperative recurrence (4–6), 10% of GCTBs undergo malignant transformation and up to 5% of GCTBs exhibit pulmonary metastases (7). GCTBs have diverse histological subtypes (8). Characterized by high proliferative abilities, GCT stromal cells are the major type in GCTBs (9). Although numerous studies have focused on cell proliferation and cell cycle regulation of GCTBs, which serve a key role in the decreased recurrence rates and improved clinical outcomes, there is little evidence of certain regulators or signaling pathways, which could be regarded as predictive markers for recurrence or metastasis. In this regard, it is necessary to investigate the biological and clinical function of certain molecules involved in the development and metastasis of GCTBs, which may be used as novel biomarkers for the diagnosis and prognosis of patients with giant cell tumors. Programmed cell death 4 (PDCD4) is a novel tumor suppressor and a promising candidate for a targeted molecular therapy for tumors based on regulating different cellular signal transduction pathways. PDCD4 could restrain the growth, malignant transformation and metastasis of tumor cells at mRNA, protein and cellular levels (10). It has been reported that nuclear PDCD4 inhibits the activity of transcription factor activator protein-1 (AP-1) and controls gene transcription in mouse epidermal JB6 cells (11,12). PDCD4 could also suppress cap-dependent translation of mRNAs with highly structured 5′-regions through interaction with the eukaryotic translation initiation factor 4A helicase (13). Furthermore, PDCD4 gene knockout mice developed spontaneous tumors of lymphoid origin (14). However, Jansen et al (15) observed a significant reduction in the carcinoma incidence and papilloma-to-carcinoma conversion frequency in PDCD4 transgenic mice compared with wild-type mice. Recently, numerous studies have identified a decreased expression of PDCD4 in multiple types of human cancer cell lines and primary tumors, including cervical cancer (16), gastric cancer (17), glioma (18), hepatocellular carcinoma (19), gastrointestinal stromal tumors (20) and nasal inverted papilloma (21). Certain studies have demonstrated that PDCD4 served a role in the progression of osteocarcinoma. Nevertheless, the precise regulation of PDCD4 in GCTBs remains largely unknown. In the present study, expression of PDCD4 was decreased in GCTBs compared with adjacent non-tumor tissues. In addition, it was demonstrated that abnormal PDCD4 expression level was associated with clinicopathological features, including the Campanacci grade and recurrence. Materials and methods Clinical specimens A total of 83 GCTB samples, including 27 frozen and 56 paraffin-embedded tissues, were collected from patients (median age, 40 years old), who underwent surgery at the Department of Orthopedics, Shandong Provincial Hospital Affiliated to Shandong University from September 2015 to March 2017. The specimens were immediately frozen in liquid N after surgery and stored at −80°C. Written informed consent was obtained from all participants. The present study was approved by the ethics guidelines of Chinese Medical Association. The protocol was completely approved by the Shandong Provincial Hospital Institutional Review Board (IRB). None of the patients had received immunotherapy, radiotherapy or chemotherapy prior to surgery. GCTBs were staged using the Campanacci grading system (22). RNA Extraction and quantitative reverse transcription-PCR (qRT-PCR) Total RNA was extracted from frozen tissues of primary GCTBs using a modified TRIzol one-step extraction method (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) (23,24). First-strand cDNA was synthesized from 3 µg total RNA using the Revert Aid First Strand c-DNA Synthesis kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. The PCR primer pairs specific for PDCD4 were as follows: Forward, 5′-CCAAAGAAAGGTGGTGCA-3′ and reverse, 5′-TGAGGTACTTCCAGTTCC-3′. The following thermocycling conditions were used for the PCR: Initial denaturation at 94°C for 2 min; 35 cycles of denaturation at 95°C for 90 sec, annealing at 66°C for 90 sec and extension at 72°C for 90 sec. Human β-actin was used as an internal control. The primers for β-actin were forward, 5′-CATGTACGTTGCTATCCAGGC-3′, and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′. Each sample was obtained from three independent experiments and used for analysis of relative normalized mRNA expression. Western blot analysis Protein lysates were separated by SDS-PAGE. The concentration of protein was determined using BCA Protein Assay (Beyotime Institute of Biotechnology, Haimen, China). Then, the protein were transferred onto polyvinylidene difluoride membranes and were blocked with 5% skim milk in TBST containing 0.1% Tween-20 for 1 h. The filters were incubated with primary antibodies against PDCD4 (1:5,000, cat. no. 9535; Cell Signaling Technology, Inc., Danvers, MA, USA) and β-actin (1:1,000, sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), followed by secondary antibody (1:2,000, Anti-rabbit IgG, HRP-linked Antibody cat. no. 7074; Cell Signaling Technology, Inc.) conjugated with peroxidase for 1 h at room temperature. The immune complexes were visualized by the enhanced chemiluminescence reagent (SuperSignal West Pico Chemiluminescent Substrate; Pierce; Thermo Fisher Scientific, Inc.). Western blot analysis was performed at least 3 times for each sample. Immunohistochemistry Tissue sections (4–6 µm) from frozen and paraffin blocks were dewaxed in xylene and rehydrated in alcohol. Antigen retrieval of sections was achieved by microwaving in citric saline and treatment with 3% hydrogen peroxide. Immunohistochemical staining using PDCD4 (1:100, PAB10308; Abnova, Taipei, Taiwan) and anti-Ki-67 antibodies (1:500, M724029, Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) was performed to delineate PDCD4 expression and cell proliferation in tumor samples. Sections were also stained with hematoxylin. The intensity and percentage area of PDCD4 staining was categorized into five grades: Score 0 (−), score 1 (+), score 2 (++), score 3 (+++), score 4 (++++) and score 5 (+++++). Scores of 4 or 5 indicated relatively high expression of PDCD4, while scores of 0–3 indicated relatively low expression. All staining experiments were performed in duplicate. Slides were evaluated by two independent pathologists. Statistical analysis All statistical analyses were performed using SPSS v.22.0 statistical software package (IBM Corp., Armonk, NY, USA). Two-way analysis of variance with Student-Newman-Keuls post hoc test or Student's t-test was performed to determine statistical significance. The significance of differences between groups was estimated by χ2 and Pearson's coefficient tests. The associations were analyzed by Spearman's correlation and multivariate regression analyses. All statistical analyses were two-sided and values are presented as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference. Statistical significance was evaluated with data from at least three independent experiments. Results Decreased expression of PDCD4 is observed in GCTBs at the mRNA level The present study first quantified the expression of PDCD4 mRNA in primary GCTBs by qRT-PCR. The mRNA level of PDCD4 was markedly decreased or absent in 63% (17/27) of the frozen GCTB samples compared with adjacent non-tumorous tissues (Fig. 1A). The results demonstrated that there was a significantly differential expression of PDCD4 between primary GCTBs and non-tumorous tissues at the mRNA level (Fig. 1B). a I 12 15 II 31 10 III 11 4 Recurrence 0.0426^a Yes 21 5 No 33 24 Ki-67 labeling index (%) 13.28±5.06 5.83±3.84 <0.0001^b a P<0.05 b P<0.001. PDCD4, Programmed cell death 4. PDCD4 inhibits malignant proliferation of GCTBs It was previously demonstrated that Ki-67 protein is closely associated with the malignant proliferation of GCTBs (25). Therefore, the present study further examined the Ki-67 expression at the protein level in GCTBs (Fig. 5) and confirmed the association between the Ki-67 LI (Ki-67 labeling index) and the expression of PDCD4. The Ki-67 LI of GCTBs with low expression of PDCD4 was markedly increased compared with GCTBs exhibiting high expression of PDCD4 (P<0.001; Fig. 6A). The results demonstrated that PDCD4 may have a negative association with the Ki-67 LI (r=−0.6392; P<0.001; Fig. 6B). All data suggested that PDCD4 may have an important effect on inhibition of the malignant proliferation of GCTBs. Figure 5. Immunohistochemistry analysis of Ki-67 in GCTB. GCTB samples exhibited different Ki-67 protein expression (magnification, ×400). GCTB, giant cell tumor of bone.

Keywords: programmed cell death 4 / expression / giant cell tumors of the bone / clinical significance