Overexpression of carboxypeptidase X M14 family member 2 predicts an unfavorable prognosis and promotes proliferation and migration of osteosarcoma

Background Carboxypeptidase X, M14 family member 2 (CPXM2), has been associated with several human developmental disorders. However, whether CPXM2 is involved in oncogenesis or tumor progression remains unclear. Currently, the clinical relevance and function of CPXM2 in human osteosarcoma were investigated. Materials and methods The expression of CPXM2 in osteosarcoma cell lines and tissues were explored by immunohistochemistry and western blotting assays. A eukaryotic expression plasmid was transfected into fetal osteoblast cells to overexpress CPXM2 and the endogenous CPXM2 in osteosarcoma cells was silenced through an RNA interference (RNAi) method transfection. These transfections were validated via western blotting, and the expression levels of several key molecules involved in the epithelial mesenchymal transition was also determined via western blotting. The expression levels of CPXM2 in a fetal osteoblast cell line with CPXM2 overexpressing and an osteosarcoma CPXM2-knockout cell line was confirmed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunofluorescence. The malignant phenotype of osteosarcoma cells was indicated by the cholecystokinin octapeptide, colony formation assay, scratch wound healing assay, and Transwell® migration assay. Results We found that CPXM2 was overexpressed in osteosarcoma and that the overexpression was associated with an unfavorable prognosis and tumor node metastasis staging. The knockdown of CPXM2 in cultured osteosarcoma cells significantly impeded cell proliferation and migration. In addition, the upregulation of CPXM2 in fetal osteoblast cells significantly promoted cell proliferation and migration. Besides, western blotting results revealed that several key molecules involved in the epithelial mesenchymal transition (EMT) were regulated by CPXM2. Conclusion Taken together, these results imply an active role for CPXM2 in promoting tumor aggressiveness via epithelial to mesenchymal transition (EMT) modulation in osteosarcoma.


Background
Generally, proteins undergo post-translational modifications (PTMs) to impact the functions of the protein [1] . Proteolysis, a well-known PTM comprises more than 500 identified proteases and peptidases which can modulate or active the activity of proteins and peptides [2]. Release of C-terminal amino acids are a widespread process that plays a role in degradation, processing, and modulation of proteins and peptides [3]. Carboxypeptidases (CPs) were revealed to conduct numerous varied physiological functions via eliminating C-terminal amino acids from peptides and proteins [4]. For instance, carboxypeptidase D is the main contributor for antibody Cterminal lysine cleavage in Chinese hamster ovary cells [5]. Carboxypeptidase E (CPE) is a novel regulator of the canonical Wnt signaling pathway and have a potential role in cell proliferation, cell fate determination and tumorigenesis [6]. The genetic defects in CPs have been involved with numerous developmental diseases [7]. A recent study revealed that Carboxypeptidase E is essential for migration and dendrite arborization of cortical neuron.
Although not all are recognized to be active as peptidases, the M14 family proteins including 25 distinct family members of carboxypeptidase is the largest family of enzymes responsible for cleavage of C-terminal residues in most mammals [8,9]. The M14 family members performed diverse biological impacts owing to their variable tissue, cellular, and subcellular distributions and substrate specificities [5,10]. Carboxypeptidase X, M14 family member 2 (CPXM2) have been reported to be involved in cell-cell interactions and associated with developmental diseases [11,12], late-onset Alzheimer's disease, and cognitive decline in schizophrenia [13,14]. However, whether CPXM2 is involved in oncogenesis or tumor progression remains unclear.
The main reason for the poor prognosis of osteosarcoma is metastasis and recurrence, and the overall 5-year survival rate is less than 20% [15,16]. To date, restraining the recurrence and metastasis of osteosarcoma has been shown to be limited in the therapy of this disease, and once tumors progress into the metastatic stage, there has been no feasible efficient therapy until now [17]. Hence, it is of vital importance to elucidate the molecular mechanisms that lead to disease progression of osteosarcoma. In the current study, we found that CPXM2 was overexpressed in osteosarcoma compared to normal bone tissues. However, at present there are no reports documenting the impact of CPXM2 in osteosarcoma tumorigenesis. RNA extraction and the quantitative polymerase chain reaction (qPCR)

Antibodies
Total RNA was isolated from cell cultures or from snapfrozen tissues from osteosarcoma patients using RNAiso Plus (Takara Bio, Kusatsu, Japan) according to the manufacturer's instructions, reverse transcribed with HiScript Q Select RT SuperMix (R132-01, Vazyme, Jiangsu, China) according to the manufacturer's protocol, and subjected to real-time reverse transcription (RT)-PCR using the 2 -ΔΔCT method [18]. The thermocycling conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, 60°C for 32 s, 95°C for 15 s, 60°C for 60 s and 95°C for 15 s. Each sample was determined in duplicate. All PCR products were confirmed by 2.0% agarose gel electrophoresis. The sequences for RT-PCR primers were: CPXM2 forward primer, 5′-GTGCGCGGGAAGAAATGAC-3′; and reverse primer, 5′-CCTCCCTTGAGTGATGACACC-3′. The specificity of primers was validated by sequencing. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. Experiments were repeated three times in duplicate.

Western blotting
Total protein was extracted from cell cultures or homogenized tissues from osteosarcoma patients using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China) containing phenylmethylsulfonyl fluoride (Beyotime Biotechnology) and proteinase inhibitor cocktail solution (Roche, Basel, Switzerland), and quantitated using the bicinchoninic acid protein assay (Beyotime Biotechnology) as recommended by the manufacturers. Western blotting was performed according to standard methods as previously described [18]. Briefly, Proteins (30 μg) were separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. Subsequently, the membranes were blocked with 5% fat-free dry milk at room temperature for 1 h. Then, the blots were incubated with a rabbit anti-CPXM2 and β-actin antibody (1:1000 dilution; Abcam, ambridge, MA, USA), and rabbit monoclonal antibodies against E-Cadherin, N-Cadherin, Vimentin and ZEB1 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. The membranes were again washed with TBST and incubated with respective horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000; goat anti-rat cat. no. A0192, goat anti-mouse cat. no. A0216 and goat anti-rabbit cat. no. A0239, Beyotime, Jiangsu, China) at room temperature for 1 h. The proteins were finally examined by an enhanced chemiluminescence system (ECL) (P0018AS, Beyotime, Jiangsu, China). The grayscale values of protein bands were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Cell proliferation assay hFOB.1.19 or U2OS cells were seeded at a density of 3 × 10 5 cells/well into 36-well plates in triplicate and cultured at 37°C overnight in an incubator. A growth curve was drawn based on the growth every 12 h over 4 days as analyzed via a colorimetric water-soluble tetrazolium salt kit (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) in accordance with the manufacturer's protocol.

Cell colony formation assay
A plate colony formation assay was performed as previously described [19]. Briefly, stably transfected hFOB.1.19 or U2OS cells (5 × 10 2 cells/well) were seeded in six-well plates and cultivated in F12K or RPMI-1640 complete medium at 37°C for 14 days. The cell colonies were washed with phosphate-buffered saline (PBS) twice, fixed with methanol for 20 min, and stained with 0.1% crystal violet in PBS (Beyotime Biotechnology) for 15 min. The colonies containing > 50 cells were counted and the experiments were performed in triplicate.

Cell migration assay
A Transwell® migration assay was performed as previously reported [19]. In brief, hFOB.1.19 or U2OS cells (4 × 10 5 cells/ml) were seeded in serum-free RPMI1640 or F12K medium in the top chamber of a Transwell® insert. The medium containing 20% FBS in the lower chamber served as a chemoattractant. After incubation for 24 h at 37°C, the cells on the top side of the membrane were removed with a cotton swab, and those on the bottom side were fixed with methanol for 20 min and then stained with crystal violet (0.1% in PBS) for 15 min. Six randomly selected fields per well were photographed, and the numbers of migrated cells were counted.

Scratch wound healing assay
A monolayer scratch wound assay was employed as previously described [20]. Briefly, cells (4 × 10 5 cells/well) were seeded in 12 well plates and grown to nearly 100% confluence. A scratch wound was generated with a 200 μl pipette tip. Wound closure was photographed at 0 and 48 h.

Patients and tissue samples
Our research was performed with the approval of the Research Ethics Committee of First Hospital of Jilin University. The participants signed written informed consent prior this research. Tissues were gathered from 36 patients who underwent surgical resection at First Hospital of Jilin University between January 2006 and June 2013. The patients were chosen on account of the following principles: pathological diagnosis of OS; no prior or second tumor; no history of chemotherapy and radiotherapy. All excised tissues were immediately frozen in liquid nitrogen and stored at − 80°C for the following study. The histologic grade and histologic subtypes were classified according to the 2013 WHO classification of bone tumors. The clinicopathologic parameters of osteosarcoma patients, containing age, gender, distant metastasis, histologic grade, histologic subtypes, Enneking clinical and TNM stage, were summarized and shown as Table 1. Histologically non-cancerous bone tissue was gained from 36 knee arthritis patients who was treated at The Second Hospital of Jilin University between January 2007 and October 2013, including 60 men and 36 women with an average age of 64 years.

Immunohistochemistry
Sections were stained with an anti-CPXM2 polyclonal antibody using IHC in the Department of Pathology at our hospital. In brief, after deparaffinating, rehydrated in graded ethanol, antigen retrieval with Citrate Buffer pH 6.0 (1:300 dilution; ZLI-9065, ZSGB-Bio, Beijing, China) Survival analyses were conducted using the Kaplan-Meier method and differences in survival were examined using the log-rank test. Univariate and multivariate survival analyses were conducted using the Cox proportional hazards regression model. Statistical significance was defined as a value of P < 0.05. All statistical tests were two-sided.

CPXM2 is overexpressed in osteosarcoma cell lines
The expression of CPXM2 in osteosarcoma cell lines (Saos2, 143B, MG63, and U2OS) and a human fetal osteoblast cell line (hFOB.1.19) were examined via RT-qPCR and western blotting. It was determined that the CPXM2 were low expressed in hFOB.1.19 cells at both mRNA and protein levels, but high expressed in osteosarcoma cell lines Saos2, 143B, MG63, and U2OS (Fig. 1a-c).

Expression of CPXM2 in human osteosarcoma tissues was upregulated and predicts an unfavorable prognosis
Additionally, the CPXM2 expression was investigated in 36 osteosarcoma tissue samples and 36 noncancerous tissue samples. As it revealed in Fig. 2a, the CPXM2 is mainly expressed in the cytoplasm of the osteosarcoma cells. The cells that showed CPXM2 overexpression in noncancerous bone tissue were osteoblasts, and the tumor cells with CPXM2 expression derived from osteoblasts in tumor tissues. Also, CPXM2 was highly expressed in 58.33% (21/36) of osteosarcoma tissues and 27.78% (10/ 36) of non-neoplastic bone tissues ( Table 1); The association between CPXM2 and clinical features was also evaluated, and CPXM2 expression was not meaningfully associated with the age (P = 0.912), sex (P = 0.876), response to chemotherapy (P = 0.426), and history of trauma or bone fracture (P = 0.756) in osteosarcoma patients. However, CPXM2 expression was meaningfully associated with Enneking clinical stages (P = 0.003), TNM stage (P = 0.002), pulmonary metastasis at vascular level (P = 0.003) and, E-Cadherin expression (P = 0.004) in osteosarcoma patients. Furthermore, our data observed that the CPXM2 expression is mainly expressed (15/27, 71.4%) in the osteoblastic osteosarcoma (P = 0.002), and notably associated with histologic grade (P = 0.011) ( Table 1). Western blotting analysis was also performed with the patient tissues in order to analyze the variations between noncancerous tissues and osteosarcoma tissues in CPXM2 expression. As illustrated in Fig. 2b and d, CPXM2 was notably upregulated in the 36 osteosarcoma tissues vs. the 36 noncancerous bone tissues (P = 0.0005).
The association between CPXM2 and survival time were analyzed using the Kaplan-Meier survival curves and the log-rank test. As illustrated in Fig. 2c, patients with high level of CPXM2 expression (median survival, 34.27 months) had a notably shorter survival time than those whose with low expression level of CPXM2 protein (median survival, 46.24 months; P = 0.0053).
To determine the influence of CPXM2 on the proliferative capacity of fetal osteoblast cells, the CCK-8 was used to explore the proliferative capacity of hFOB.1.19 cells. As depicted in Fig. 3b, there was a noteworthy alteration between the proliferation rates of the hFOB.1.19-CPXM2 and empty vector groups (P = 0.004). We also determined the ability of CPXM2-overexpressing cells to form colonies in 2D monolayer cultures (Fig. 3c). The number of colonies formed by CPXM2-overexpressing cells was significantly higher than the number formed by the Vector-transfected cells (P = 0.0002). Moreover, a Matrigel invasion assay was conducted to assess the metastatic capacity of fetal osteoblast cells; the observations revealed a meaningfully increased number of invasive cells (P = 0.0001) in the hFOB.1.19 -CPXM2 group, versus the empty vector group (Fig. 3d), indicating that CPXM2 promotes the migration ability of fetal osteoblast cells in vitro. Furthermore, wound-healing assay was conducted to explore the influence of CPXM2 on cell migration ability; it was displayed that after 12 and 24 h, the migration capacity of hFOB.1.19-CPXM2 cells was meaningfully enhanced, versus the empty vector group (P = 0.0013 and P = 0.0022, respectively; Fig. 3e). Taken together, these evidences indicated that that CPXM2 plays an active role in promoting osteosarcoma tumor aggressiveness via EMT modulation.

Influence of CPXM2 knockdown on osteosarcoma cell metastasis
To explore the influence of CPXM2 on the metastatic ability of osteosarcoma cells, U2OS cells were transfected with p-EGFP-scramble or p-EGFP-CPXM2-RNAi plasmids. Western blotting was conducted to investigate the expression of CPXM2 and any alterations in the EMT process (Fig. 4a). The ratio of CPXM2 (P = 0.001), N-Cadherin (P = 0.0024), ZEB1 (P = 0.0026) and Vimentin (P = 0.0017) expression was significantly decreased while the expression of E-Cadherin (P = 0.0022) was significantly To determine the impact of CPXM2 on the malignant phenotype of osteosarcoma cell lines, the CCK8 method was used to draw the growth curve of the U2OS cell line. As depicted in Fig. 4b, the proliferation rate of CPXM2-RNAi transfected cells was significantly lower than that of the scramble-transfected cells (P = 0.0012). We also determined the ability of CPXM2-silenced cells to form colonies in 2D monolayer cultures (Fig. 4c). The number of colonies formed by CPXM2-silenced cells were significantly lower than the number formed by the scramble-transfected cells (P = 0.0030).
The results obtained from Matrigel invasion assay revealed that the amount of invasive U2OS cells in the CPXM2-silenced group was notably decreased (P = 0.0012; Fig. 4d), Moreover, Wound-healing assays (Fig. 4e) revealed that the migration distances of CPXM2-RNAi cells were significantly decreased after 12 and 24 h (P = 0.0017 and P = 0.0023, respectively) versus the scramble group.

Discussion
Our knowledge about CPXM2 is still quite limited. Most studies have related CPXM2 to developmental diseases, mental disorders, and neurodegenerative diseases [11][12][13][14]. By analyzing differentially expressed genes in dermal fibroblasts from patients with Apert syndrome and controls, Cetinkaya et al. showed CPXM2 to be a gene with gene ontology terms associated with extracellular matrix organization, which may regulate early differentiation of connective tissues [11]. Using both growth-restricted and normal-term placentas, Sabri et al. recognized CPXM2 as one of the most upregulated genes in fetal growth restriction. Using bioinformatics analysis, these authors also suggested a potential connection between fetal growth restriction and gastrointestinal diseases [12]. There are at least 25 carboxypeptidases, several of which have already been identified as potential tumor biomarkers [4,6,[22][23][24]. However, the underlying mechanism to explain the roles of these carboxypeptidases in modulating oncogenesis and tumor progression is still lacking. To date, a limited number of studies have surveyed the practical association between osteosarcoma carcinogenesis and CPXM2. Currently, we found that CPXM2 expression was overexpressed in osteosarcoma sections, and that the upregulation of CPXM2 promoted metastatic phenotype of fetal osteoblast cells. Furthermore, in the present study, we focused on the association between CPXM2 and 22 cases of pulmonary metastasis at the vascular level, our data revealed thatCPXM2 expression was significantly associated with distant metastasis in osteosarcoma patients. These observations suggested that abnormal expression of CPXM2 represented a potential target for genetic diagnosis, pathological staging, recurrence and prognosis in osteosarcoma. However, there are also 27.78% (10/36) of non-neoplastic bone tissues displayed   Loss of CPXM2 decreased the on the proliferation and migration ability of osteosarcoma U2OS cells. a Western blotting was used to examine the effects of silencing CPXM2 and EMT process in the osteosarcoma U2OS cells. b Growth curve of osteosarcoma cells detected by the CCK-8 assay. c The abilities of osteosarcoma cells to form colonies under 2D culture condition were determined through colony formation assay. d The Transwell chambers method was utilized to explore the impact of CPXM2 silence on the invasive ability of osteosarcoma U2OS cells in vitro. e The wound-healing assay was utilized to explore the migration ability of osteosarcoma cells in vitro. **P < 0.01, compared with the scramble group. CPXM2, Carboxypeptidase X, M14 family member 2; EMT, epithelial mesenchymal transition; OD, optical density CPXM2 expression. This result may be because CPXM2 is necessary for the proliferation of osteoblasts and is expressed in osteoblasts that are in the proliferative stage.
Currently, the specific impact of CPXM2 in osteosarcoma remains unclear. As EMT is a critical process that mediates tumor progression and metastasis, we postulate that CPXM2 may catalyze key molecules that regulate EMT in osteosarcoma. Our data represent that, CPXM2, an osteosarcoma proto-oncogene, significantly promoted the invasiveness of hFOB.1.19 cells. An initial investigation into the molecular mechanism of this effect was also performed, and it was determined that CPXM2 affects the EMT process via ZEB1, ultimately enhancing the metastatic capacity of fetal osteoblast cells. Moreover, an osteosarcoma cell line (U2OS) with a CPXM2knockout was also settled up and it is illustrated that CPXM2-silencing lead to a suppression on metastasis in U2OS cells via modifying EMT process. To the best of our knowledge, this study is the first to investigate the prognostic value and molecular function of CPXM2 in cancers.
There are several unanswered questions in this study. First, we do not know the substrates of CPXM2. Second, further studies are needed to confirm whether CPXM2 can be used as a serum prognostic biomarker for osteosarcoma patients. Third, it is noted that the small sample size is a flaw of the current study, and that the character of CPXM2 as a gene biomarker may be investigated further by assessing the data of both inpatients and outpatients, and by collecting a larger number of samples.

Conclusion
Currently, the specific impact of CPXM2 in osteosarcoma remains unclear. Our data represent that, CPXM2, as an osteosarcoma proto-oncogene, was found to accelerate tumor progression by promoting osteosarcoma cell migration via modulation of EMT process, indicating that a detailed study of this putative marker is warranted.