Lysophosphatidic Acid Acyltransferase β (LPAATβ) Promotes the Tumor Growth of Human Osteosarcoma

Background Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated. Methodology/Principal Findings Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma. Conclusions/Significance Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors.


Introduction
Osteosarcoma (OS) is the most common primary malignancy of bone and accounts for ,5% of childhood tumors in the United States with incidence peaking during the second decade of life [1][2][3][4][5]. The molecular pathogenesis underlying OS development remains to be thoroughly elucidated [4,6]. At presentation, approximately 80% of patients are afflicted by some degrees of metastasis mandating management through chemotherapy and surgical resection [7][8][9][10]. Pulmonary metastasis remains the main cause of death in patients with OS [4,11]. Many variants of OS are relatively resistant to current chemotherapy regimens [5,12,13]. We and others have reported that numerous genetic alterations may be found in OS tumors [4][5][6][14][15][16]. However, it remains challenging to identify common genetic alterations that lead to OS development given the diversity and complexity in its pathogenesis [4,[17][18][19][20][21].
Lysophosphatidic acid acyltransferase (LPAAT, aka, 1-acylglycerol-3-phosphate O-acyltransferase 2, Agpat2) comprises a family of trans-membrane proteins consisting of at least six isoforms. The biological role of LPAAT is to convert lysophosphotidic acid (LPA) into phosphatidic acid (PA) [22], while only the a and b isoforms have significant acyltransferase activity [23]. LPAATb expression is specific to heart, liver, adipose and pancreas [24][25][26]. Inherited mutation of LPAATb is associated with lipodystrophy type 1, an autosomal recessive condition characterized by impaired triglyceride synthesis, low body fat percentage and insulin resistance [27].
PA is an important metabolite involved in phospholipid biosynthesis and membrane remodeling [28]. PA is considered an important secondary messenger capable of modulating pathways responsible for cell survival and proliferation, such as the mTOR and Raf-1 signaling cascade [29][30][31][32]. Increasing evidence indicates that enhanced lipid biosynthesis is a characteristic feature of cancer and deregulated lipogenesis plays an important role in tumor cell survival [33][34][35][36]. In fact, the oncogenic nature of lipogenesis closely depends on the activity and/or expression of key cancer-related oncogenes, such as HER2 [34]. Targeted gene deletion of LPAATb in mice results in a complete absence of both white and brown adipose tissue [37], providing a biochemical link between the triglyceride synthesis pathway and adipogenesis in the liver and adipose tissue. Thus, it is conceivable that LPAATb may play an important role in regulating cancer-related lipid metabolism.
We sought to investigate the role of LPAATb in regulating OS cell proliferation and tumor growth. LPAATb expression is readily detected in most OS cell lines. Exogenous expression of LPAATb promotes OS cell proliferation and migration, while silencing LPAATb expression inhibits cell proliferation and migration.
Using an orthotopic xenograft model of human OS, we have demonstrated that exogenous expression of LPAATb effectively promotes OS tumor growth, while knockdown of LPAATb reduces OS tumor volume in the xenograft model. Taken together, our results strongly suggest that LPAATb expression may be associated with aggressive phenotypes of human OS and that LPAATb may play an important role in regulating OS cell proliferation and tumor growth. Therefore, targeting LPAATb may be exploited as a novel therapeutic strategy for the clinical management of OS.

Tumor lines, cell culture and chemicals
No human subjects were used in the reported studies. However, tumor cell lines derived from patients were used, which has been approved by the Institutional Review Board (protocol #17043B). HEK293 and human OS lines 143B, SaOS2, MG63 and TE85 were purchased from ATCC (Manassas, VA). MG63.2 cell line was established by serially passaging the parental MG63 cells in mice followed by extraction and culture of pulmonary metastasis as previously described [38,39]. Primary OS cells were isolated from resected OS specimens as previously described [15,40,41]. Cells were maintained in complete Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FCS (fetal calf serum, HyClone, Logan, UT) at 37uC in 5% CO2. Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich (St Louis, MO) or Fisher Scientific (Pittsburgh, PA).

RNA isolation and semi-quantitative RT-PCR analysis
Total RNA was isolated using TRIZOL Reagents (Invitrogen) and cDNA was generated via reverse transcription reaction with hexamer and Superscript II RT (Invitrogen). The first strand cDNA products were further diluted 5-to 10-fold and used as PCR templates. Semi-quantitative RT-PCR was carried out using PCR primers designed by using the Primer3 program to amplify PCR reactions were carried out as previously described [15,41,48,49,[52][53][54]. PCR products were separated via 1.2% agarose gels. The resulting bands were analyzed in Kodak ImageStation 440CF using Kodak 1D 3.6 software.

Crystal violet staining
Subconfluent cells were seeded in 12-well plates and infected with an optimal titer of AdR-LPAATb, AdR-siLPAATb or AdRFP. Cells were subjected to crystal violet staining at 5 days after infection as previously described [41,55]. Images were taken from the plates relative staining intensities were analyzed by using ImageJ software.

Cell proliferation (MTT) assay
MTT assay was used to quantitatively analyze cell proliferation as previously described [15,41,56]. Briefly, cells were seeded in 96well plates and infected with AdR-LPAATb, AdR-siLPAATb or AdRFP. Cell proliferation was assessed by MTT assay daily for five days. Optical absorbance was measured at 570 nm using a 96well microplate reader. All experiments were performed in triplicate.

Cell migration and wound healing assay
The experiments were carried out essentially as described previously [15,16,39,41,53,57]. Subconfluent cells were plated into 6-well plates and infected with AdR-LPAATb, AdR-siLPAATb or AdRFP in 1% FCS containing medium. At 16 h after infection, the monolayer of cells was wounded using micropipet tips. Marks were created on the plates using 18-gauge needles as reference points for serial imaging. Bright field images of the same field were taken at 4, 12, 24, and 36 hours. The results were repeated in at least two batches of experiments.

Orthotopic tumor model of human osteosarcoma
The reported animal work was carried out according to the guidelines approved by the Institutional animal Care and Use Committee (Protocol #71328). Athymic nude (nu/nu) mice (4-6 week-old, male) were used (5 mice per group, Harlan Laboratories, Indianapolis, IN). Human OS cells (e.g., 143B or MG63) were stably transduced with a retroviral vector expressing firefly luciferase (namely 143B-Luc or MG63-Luc). Cells were seeded in 100 mm cell culture dishes and infected with AdR-LPAATb, AdR-siLPAATb or AdRFP. At 36-48 h after infection cells were collected and resuspended in PBS to a final concentration of 2610 7 cells/ml. 50 ul of the resuspened cells (i.e., 10 6 cells/ injection) were periosteally injected posterior to the proximal tibia using a 25 gauge needle.

Xenogen bioluminescence imaging
Xenogen imaging was carried out as described [15,41]. Animals were anesthetized with isoflurane. Approximately 10 min prior to using Xenogen IVIS 200 imaging system, animals were injected (ip) with D-Luciferin sodium salt (Gold BioTechnology) at 100 mg/kg in 0.1 ml sterile PBS. The pseudoimages were obtained by superimposing the emitted light over the gray-scale photographs of the animal. Quantitative analysis was done with Xenogen's Living Image V2.50.1 software.

Tumor volume measurement
The dimensions of the primary tumor sites were measured every 3-4 days. The volume was calculated as previously reported [38,39], by using the following equation: volume = (L+W)(L)(W)(0.2618). The width (W) was an average of the distance at the proximal tibia at the level of the knee joint in the anterior-posterior and medial-lateral planes. The length (L) was the distance from the most distal extent of the calf musculature or distal tumor margin to the knee joint or proximal tumor margin [39].

Histological analysis
Animals were sacrificed and the primary tumors were harvested at necropsy and fixed in 10% formalin or decalcification solution (Cal-Ex, Fisher Scientific). The fixed samples were embedded in paraffin. Sections were stained with hematoxylin and eosin, and analyzed under a microscope.

Immunohistochemical staining
Immunohistochemical analysis was carried out as previously described previously [14][15][16]39,41]. Briefly, paraffin sections were deparaffinized, rehydrated and probed with anti-LPAATb, anti-PCNA, or isotype IgG, followed by incubation with secondary antibodies conjugated with HRP. The expression of expected proteins was visualized by DAB staining and examined under a microscope. Stains without the primary antibody, or with control IgG, were used as negative controls.

Statistical analysis
Microsoft Excel was used to calculate standard deviations (SD) and statistically significant differences between samples using the two-tailed Student's t-test. For all quantitative assays, each assay condition was performed in triplicate and the results were repeated in at least three independent experiments. All collected data were subjected to statistical analysis. A p-value ,0.05 was defined as statistically significance.

LPAATb is expressed in most human OS cells
In order to understand the possible role of LPAATb in human OS tumorigenesis and progression, we analyzed the endogenous expression of LPAATb in a panel of human OS cell lines. We first assessed the expression of LPAATb in the four commercially available OS lines (i.e., MG63, 143B, TE85, and SaOS2) and the line MG63.2 derived from MG63 [38]. As shown in Fig. 1A, expression of LPAATb was readily detected in three of the five lines. Interestingly, expression of LPAATb is more apparent in lines with higher xenogenic tumor growth potential, such as 143B, MG 63, and MG63.2 as compared to lines with less tumorigenic capacity, such as TE85 and SaOS2 [39]. We next examined the expression of LPAATb in five OS lines derived from OS patients [15,40,41]. Three of the five patient-derived OS lines exhibited apparent expression of LPAATb while other two lines showed weak but detectable expression of LPAATb (Fig. 1B). Our previous studies indicated that UCHOS4 and UCHOS15 lines were more tumorigenic than UCHOS11 cells [15,40,41]. The expression of LPAATb at protein level was further confirmed in xenograft tumors formed by 143B and MG63.2 cells (Fig. 1C). Taken together, these results indicate that LPAATb is commonly expressed in human OS cells and that LPAATb expression is seemingly correlated with aggressive phenotype of OS cell lines'.

LPAATb promotes cell viability and proliferative activity of OS cells
In order to further investigate the functional role of LPAATb in OS proliferation and tumor growth, we sought to generate recombinant adenoviruses that can effectively over-express LPAATb (AdR-LPAATb) and siRNAs targeting human LPAATb (AdR-siLPAATb) using the AdEasy technology [44,45]. We constructed an adenoviral vector that over-expresses mouse LPAATb driven by a CMV promoter ( Fig. 2A). The siRNA expressing adenoviral vectors were constructed using the pSOS system [46,47,49], for which four target sites were chosen (Fig. 2B). The generated adenoviruses, which also express the RFP maker, were shown to effectively transduce 143B cells (Fig. 2C) and other OS lines (data not shown). We further demonstrated that AdR-LPAATb effectively overexpressed mouse LPAATb mRNA while human LPAATb mRNA was significantly knocked down by AdR-siLPAATb (Fig. 2D), indicating that the adenoviral vectors are functional.
Using these adenoviral vectors, we investigated the effect of overexpression or knockdown of LPAATb on OS cell viability and proliferation. Using the crystal violet viability assay, we found that over-expression of LPAATb promoted 143B proliferation and increased viable cells, whereas knockdown of LPAATb led to a decrease in cell proliferation and viability of 143B cells (Fig. 3A). Quantitative analysis of the crystal violet staining results indicated that the increase in cell viability and proliferation resulted from LPAATb overexpression or the decrease in cell viability and proliferation caused by LPAATb knockdown was statistically significant when compared with the control cells (Fig. 3B). The effect of LPAATb on OS cell proliferation was further assessed by MTT assay. Consistent with the results obtained from crystal violet staining assay, overexpression of LPAATb promoted OS cell proliferation while silencing LPAATb expression inhibited cell proliferation (Fig. 3C). These results were reproducible in other OS cell lines (data not shown). Taken the above findings together, LPAATb expression can enhance OS cell viability and proliferation in vitro.

LPAATb enhances OS cell migration in vitro
The ability of tumor cells to migrate is considered an important indicator of tumor cell's aggressiveness. To assess the effect of LPAATb on OS cell migration in vitro, we conducted the commonly-used cell wounding experiment [15,16,39,41,53,57]. We found that over-expression of LPAATb effectively promoted a faster wound healing than that of the control cells (Fig. 4A vs. 4B). Conversely, knockdown of LPAATb expression inhibited cell migration and wound healing, at least in the duration of the testing period (Fig. 4C vs. 4B). Similar results were obtained from MG63.2 and other OS cell lines (data now shown). Thus, the above results strongly suggest that LPAATb expression may be related to OS cell migration-related aggressiveness phenotype.

Overexpression of LPAATb promotes tumor growth in an orthotopic model of human OS tumors
We further analyzed the effect of LPAATb expression on OS tumor growth in vivo. Using our previously established orthotopic tumor model of human OS [15,38,39,41], we found that 143B cells transduced with LPAATb formed much larger tumors than that of the control group's, whereas knockdown LPAATb led to the inhibition of tumor growth (Fig. 5A). Quantitative analysis of the Xenogen bioluminescence imaging data revealed that overexpression of LPAATb significantly promoted OS tumor growth, especially at the late stage (p,0.05) while knockdown of LPAATb did not significantly affect Xenogen imaging signal (Fig. 5B). Although Xenogen bioluminescence imaging is sensitive and quantitative, the imaging results may not be reliable when tumors have necrosis and/or are less vascularized. Thus, we also measured the tumor sizes during the course of the study, and found that the overall tumor growth trends were in general consistent with that of Xenogen imaging analysis (Fig. 5C).
However, the tumor volume was reduced in the LPAATb knockdown group (p,0.05). The calculated doubling time was 9.30 days for RFP control group, 11.99 days for the siLPAATb group, and 6.64 days for the LPAATb group, indicating that tumors with LPAATb over-expression had a significantly shorter doubling time than that of the control group (p,0.02). Characterization of AdR-LPAATb-mediated overxpression and AdR-siLPAATb-mediated knockdown. Subconfluent 143B cells were infected with AdR-LPAATb, AdR-siLPAATb, or AdRFP control for 48 h. Total RNA were collected and subjected to RT-PCR. The resultant cDNA was subjected to semiquantitative PCR using primers specific for mouse LPAATb mRNA (for AdR-LPAATb infection) or human LPAATb mRNA (for AdR-LPAATb infection). GAPDH was used as an internal control to normalize all samples. doi:10.1371/journal.pone.0014182.g002 Figure 3. Effect of LPAATb on osteosarcoma cell proliferation. A. Crystal violet staining assay for cell viability. 143B cells were seeded in 12well plates and infected with an optimal titer of AdR-LPAATb, AdR-LPAATb, or AdRFP control. Viable cells were subjected to crystal violet staining at 5 days after infection. Representative duplicate staining is shown. B. Quantitative analysis of the crystal violent staining assay. Relative staining intensities were measured by using ImageJ software. ''*'' p-value ,0.05. C. MTT cell proliferation. 143B cells were seeded in 96-well plates and infected with an optimal titer of AdR-LPAATb, AdR-LPAATb, or AdRFP control (in triplicate). At the indicated time points after infection, cells were subjected to MTT assay to determine relative proliferative activity. ''*'' p-value ,0.01. doi:10.1371/journal.pone.0014182.g003 We further conducted histologic examination on the retrieved tumor samples. H & E staining revealed that the LPAATb overexpression group exhibited increased cell numbers with relatively higher nuclear to cytoplasmic ratio (1.7), while the LPAATb knockdown group has demonstrates relatively lower nuclear to cytoplasmic ratio (0.73) When compared to RFP group (1.2) (Fig. 6 top panel). Proliferative activity of the tumor cells was assessed by anti-PCNA immunohistochemical staining. Consistent with H & E staining, the LPAATb overexpression group exhibited increased cell proliferation whereas the LPAATb knockdown group showed relatively low proliferative activity (Fig. 6 bottom  panel). Taken together, these in vivo results strongly suggest that LPAATb may play an important role in promoting OS tumor growth.

Discussion
OS is the most common primary malignancy of bone [1][2][3][4]58]. Although clinical management of OS has significantly improved, survival rate in the last few decades has plateaued. This bottle-neck has been a consequence of our poor understanding of the molecular mechanism underlying OS development and progression. Here, we investigate the role of LPAATb in OS cell proliferation and tumor growth. LPAATb expression is readily detected in most OS cell lines. Exogenous expression of LPAATb promotes OS cell proliferation and migration, while silencing LPAATb expression inhibits cell proliferation and migration.
Using an orthotopic xenograft model of human OS, we demonstrate that exogenous expression of LPAATb effectively promotes OS tumor growth, while knockdown of LPAATb reduces OS tumor volume in the xenograft model. These results suggest that LPAATb may play an important role in regulating OS cell proliferation and tumor growth.
Our in vitro results suggest that the expression level of LPAATb may correlate with OS malignant characteristics. We have found an increased level of LPAATb in tumors with highly malignant OS lines, such as MG63.2, 143B and UCHOS4, whereas LPAATb expression level is lower in OS lines that are more differentiated and with lower malignant characteristics, such as TE85 and SaOS [15,39]. However, further studies on LPAATb expression level needs to be carried out on a large set of clinical OS samples in order to determine if LPAATb expression may serve as an indicator of OS aggressive phenotypes.
LPAATs are a family of enzyme that catalyses the biosynthesis of PA [22]. Overexpression of LPAATb has been shown to transform cells in vitro [59]. LPAATb is highly expressed in advanced ovarian tumors and is associated with aggressive histology and decreased overall survival [60][61][62][63]. Chemical inhibitors of LPAATb exhibit anti-tumor activity and promote apoptosis in lymphomas, acute leukemia, and multiple myeloma [59,[64][65][66][67]. Inhibition by either genetic means or by isoformspecific small molecules results in a block to cell signaling pathways and apoptosis [59].
PA is a versatile lipid second-messenger that functions as a cofactor in several critical signaling pathways in cancer cells. For example, the full activation of c-Raf-1 and B-raf is manifested only when phosphatidic acid physically interacts with a polybasic amino acid segment in these kinases [28]. This interaction is required for the translocation of Raf kinases to the plasma membrane where they phosphorylate and activate their targets. Moreover, binding of phosphatidic acid to a polybasic domain in mTOR is essential for its activation [30]. Thus, the role of phosphatidic acid is central to the regulation of proteins in both proliferative and survival pathways in tumor cells. We found that silencing LPAATb expression in osteosarcoma cells resulted in decreased expression of c-Myc, cyclin D1, c-Fos, and MDM2, but an increased Rb expression (data not shown). Nonetheless, the exact role of LPAATb in OS development remains to be investigated.
It has been reported that inhibition of LPAATb with small interfering RNA or selective inhibitors CT32521 and CT32228 induces apoptosis in human ovarian and endometrial cancer cell  lines in vitro and enhances the survival of mice bearing ovarian tumor xenografts [61]. Previous studies have also demonstrated that inhibition of LPAATb expression with siRNA in mammalian cells suppresses basal Erk phosphorylation [68]. Inhibition of LPAATb with small-molecule antagonists prevents the translocation of Raf to the plasma membrane and subsequent Erk phosphorylation [68]. These inhibitors also suppress the activation of proteins in the phosphoinositide-3-kinase/Akt pathway, including Akt, mTOR, and S6 kinase [68]. Therefore, it is conceivable that LPAATb may be exploited as a novel cancer drug target for OS clinical management.
Taking together, we have conducted in vitro and in vivo studies to investigate the role of LPAATb in OS cell proliferation and tumor growth. Our results indicate that exogenous expression of LPAATb promotes OS cell proliferation and migration, while silencing LPAATb expression inhibits cell proliferation and migration. Furthermore, exogenous expression of LPAATb effectively promotes OS tumor growth, while knockdown of LPAATb reduces OS tumor volume in an orthotopic xenograft model of human OS. These results strongly suggest that LPAATb expression may be associated with aggressive phenotypes of human OS and that LPAATb may play an important role in regulating OS cell proliferation and tumor growth. Therefore, targeting LPAATb may be exploited as a novel therapeutic strategy for OS clinical management. This is especially attractive given the availability of selective pharmacological inhibitors.