Comparative proteomics analysis of human osteosarcomas and benign tumor of bone

Abstract

We conducted comparative proteomic analysis of osteosarcoma, with hopes of identifying the specific protein markers of osteosarcoma and improve the understanding of tumorigenesis and progression of osteosarcoma. Proteins extracted from osteosarcoma tissue and benign bone tumors, including osteoblastoma, chondroblastoma, and giant cell tumor of bone, were examined using two-dimensional gel electrophoresis followed by mass spectrometry analysis and database searches. We also validated the expression levels of interesting proteins by Western blotting assay and immunohistochemical staining. Intensity alterations of 30 spots were detected in osteosarcoma, and 18 of these spots were finally identified, including 12 up-regulated proteins and 6 down-regulated ones. The up-regulated proteins include VIM, TUBA1C, ZNF133, EZR, ACTG1, TF, and so on. The six down-regulated proteins include ADCY1, ATP5B, TUBB, RCN3, ACTB, and YWHAZ. Subsequent immunohistochemical staining and Western blotting assay for TUBA1C and ZNF133 in osteosarcoma samples confirmed the observation obtained by proteomic analysis. Our results suggest that these identified proteins may be potential biomarkers for osteosarcoma tumorigenesis and therapeutics. Aberrant expression of cytoskeletal- and microtubule-associated proteins in osteosarcoma may provide an advantage for tumor invasion and metastasis by affecting the stability of microtubule, which consequently influences the prognosis of patients.

Introduction

Osteosarcoma is a malignant bone tumor that usually occurs in young patients during the first two decades of life [1]. The tumor cells are likely to invade the surrounding tissues or metastasize to distant sites in the period of tumor progression, which is one of the major causes for poor prognosis of this tumor. The present standard treatment for high-grade osteosarcoma includes neoadjuvant chemotherapy followed by surgical resection and postoperative chemotherapy. Although this standard treatment for osteosarcoma has significantly improved the survival rate in recent years, its prognosis continues to remain less optimistic [2]. In clinical practice, it has been observed that about one third of patients die of respiratory failure due to frequent progressive pulmonary metastases and poor response to multiagent chemotherapy [3].

Osteosarcoma is characterized by complex chromosomal abnormalities that vary widely from cell to cell. This tumor exhibits high degree of aneuploidy, gene amplification, and multiple unbalanced chromosomal rearrangements. Accumulated data have demonstrated that genetic changes of osteosarcoma frequently occur in chromosomes 1, 6p, 8q, 9, 11, 13, 17p, 19, 20, and 21. Previous studies highlight the highly unstable nature of the osteosarcoma genome [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. A recent study has revealed that varied chromosomal instability patterns may reflect different phenotypes of osteosarcoma, such as response to chemotherapy [14]. That indicates those aberrant genomic alterations may be involved in the mechanism of osteosarcoma development and result in the identification of prognostic markers and therapeutic targets. Without a uniform criterion for specimen collection and data analysis, however, different studies usually come to different conclutions. So far, there is not accurate chromosomal abnormality described in osteosarcoma. In addition, previous investigators have identified various factors associated with a poor prognosis in patients with osteosarcoma, including proliferation rate of tumor, P53 gene alteration, and, especially, angiogenesis-related factors [15], [16], [17]. Among those potential prognostic factors for osteosarcoma, the most consistent factor identified is a poor response (<90% necrosis) after neoadjuvant chemotherapy [18], [19]. Factually speaking, there is no specific marker to predict the prognosis of patients before chemotherapy or to apply clincally as a therapeutic target. It still remains largely unknown whether or not the osteosarcoma-specific molecules exist and how do they influence the development, progression, and prognosis of osteosarcoma.

At this time, proteomic expression profiling of human tumors has provided a better understanding of the molecular basis of neoplastic diseases and has identified novel biomarkers and new therapeutic targets [20]. Comparative proteomic analysis combined with two-dimensional gel electrophoresis (2-D) and mass spectrometry has been applied in studies of various cancers, including ovarian, lung, and prostate cancer [21], [22]. Although genetic alterations associated with the pathogenesis of osteosarcoma have been investigated in several studies, proteomic expression profiling of osteosarcoma has just been started. Several proteomic studies targeting osteosarcoma cell and osteoblastic cell recently revealed that the expression of AHA1, SLP-2, UQCRC1, and UCH-L1 was significantly elevated in osteosarcoma cells, suggesting that these identified proteins may serve as potential molecular targets for diagnosis and therapy of osteosarcoma [23], [24]. A preliminary investigation on the difference of expressed plasma proteins between osteosarcoma and osteochondroma demonstrated that an increased level of serum amyloid protein A in plasma might be used to differentiate malignant bone tumors from benign bone tumors for early detection of osteosarcoma [25]. In more recent studies, significantly increased expressions of CRYAB and ezrin have been found in osteosarcoma, especially in advanced stages of the disease, indicating that these proteins might be involved in the process of metastasis and chemoresistance in pediatric osteosarcoma [26]. To our knowledge, however, there are little proteomic studies concerning primary osteosarcoma tissues in comparison to benign osteogenenic tumor. Thus, it is necessary to identify the specific markers for development and progression of osteosarcoma, as well as its specific biologic behavior.

In this study, we focus on the proteomic analysis of classic osteosarcoma cases. We have collected primary osteosarcoma and benign bone tumor samples. The biologic behaviors of benign bone tumors are different compared with osteosarcoma, including the absence of the ability for tissue invasion and distant metastasis. The aim of this study is to identify the more specific markers of osteosarcoma. We question whether these identified proteins can elucidate the mechanism involved in the development and progression of osteosarcoma, as well as explain the specific biologic behaviors exhibited by osteosarcoma. We assume that these proteins can help clinicians improve the prognostic prediction of this malignant tumor. Furthermore, developing new therapeutic approaches to treat this malignant tumor by intervening in the production of these specific proteins appears to be promising.