Abstract
Background: Secreted protein acidic and rich in cysteine (SPARC) has been shown to play an integral role in the progression of numerous malignancies. The aim of this study was to investigate the expression of SPARC in uveal melanoma (UM). Materials and Methods: SPARC expression was assessed in UM cell lines using RT-PCR and immunocytochemistry. Small interfering RNA directed against SPARC was used to transfect each of the cell lines, which were subsequently run in proliferation assays. SPARC expression was further investigated in 19 cases of human UM and 11 primary and 8 metastatic tumors from a rabbit xenograft model. Results: The cell lines transfected with SPARC siRNA showed a significant decrease in proliferation compared to controls. All cases of human uveal melanoma demonstrated positive staining for SPARC as did all primary and metastatic tumors from the xenograft model. Conclusion: SPARC may represent a novel target to inhibit growth of UM.
- Uveal melanoma
- SPARC
- immunohistochemistry
- tumor biology
Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. It occurs in the uveal tract, with the majority of tumors arising from the choroid. The traditional procedure for treating patients was to enucleate the affected eye in an attempt to prevent the formation of metastases. This, unfortunately, did not seem to have any effect on the incidence of metastatic disease, suggesting that malignant cells had already seeded distant sites at the time of primary tumor diagnosis and enucleation. Although some patients are still enucleated, others are candidates for plaque radiotherapy, a more conservative and less traumatizing treatment option (1). Regardless of the primary tumor management strategy pursued, many patients inevitably develop metastatic disease. Thus, there is an imminent need to discover novel therapeutic targets to arrest local and systemic disease progression.
Recent efforts have investigated the potential of various agents to reduce the proliferative and invasive capacity of UM cells in vitro (2, 3). Our lab has previously demonstrated the efficacy of a COX-2 inhibitor in preventing primary tumor formation in a xenograft rabbit model of UM (4). Additionally, animals treated with the COX-2 inhibitor developed metastases later than the untreated control group, suggesting that this approach may have some potential clinical impact in UM progression. Other groups have looked at the potential of immunotherapy to treat disease (5) and, similar to the COX-2 inhibition results, these seem promising. There is need, however, to investigate other targets that could serve as candidates for primary or adjuvant therapies in managing primary tumor and metastasis development. One such target that has emerged in some other malignancies is secreted protein acidic and rich in cysteine (SPARC), although there seems to be some discrepancy about its role, being correlated with good prognosis in some types of cancer (6, 7) and poor prognosis in others (8, 9), including cutaneous melanoma (10-13).
SPARC (also known as osteonectin or BM-40) is a non-structural extracellular matrix component that has a normal physiological role in modulating cell-matrix interactions during tissue development, repair, and remodeling. It acts primarily as a de-adhesive molecule while also acting as a cell-cycle inhibitor and modulator of cytokine activities (14, 15).
The purpose of this study was to investigate the role of SPARC in proliferation of UM cell lines in vitro and to further examine its expression in vivo. Elucidation of this role may provide insight into potential therapeutic significance of targeting SPARC in UM patients.
Materials and Methods
Cell culture. Three human UM cell lines (SP6.5, MKTBR, OCM-1) and one transformed human melanocyte cell line (UW-1) were incubated at 37°C in a humidified 5% CO2-enriched atmosphere. Cells were cultured with RPMI-1640 medium (Invitrogen, Burlington, Ontario, Canada), supplemented with 5% heat-inactivated fetal bovine serum (FBS; Invitrogen), 1% fungizone (Invitrogen), and 1% penicillin-streptomycin (Invitrogen). Cells were cultured as a monolayer in 25 cm2 flasks (Fisher, Whitby, Ontario, Canada) and observed twice weekly, at every media change, for normal growth by phase contrast microscopy. The cultures were grown to confluence and passaged by treatment with 0.05% trypsin in EDTA (Fisher) at 37°C and washed in 7 ml RPMI-1640 media before being centrifuged at 120 xg for 10 minutes to form a pellet. Cells were then suspended in 1 ml of media and counted using the Trypan Blue dye exclusion test. The UM cell lines SP6.5 and MKT-BR were established by Dr. Pelletier (Laval University, Quebec, Canada) and Dr. Belkhou (CJF INSERM, France), respectively. Dr. Albert (University of Wisconsin-Madison, USA) established the OCM-1 and UW-1 cell lines.
Transfection of SPARC siRNA. SPARC HP genome-wide designed siRNA was purchased and used for these experiments (cat # SI02630306; Qiagen, Ontario, Canada). Fluorescently labeled non-silencing control siRNA (cat # 1022079; Qiagen) was used for detection of transfection efficiency as well as controls. Transfection of the cell lines was performed as per the manufacturer's protocol. Briefly, cells were seeded at a density of 5×104 cells per well in a 6-well plate and allowed to adhere overnight in complete growth media. Afterwards, the media was replaced with serum-free 1640-RPMI with HiPerfect reagent (Qiagen) and either target or control siRNA (5 nM). The complexes were allowed to incubate for four hours before complete growth media was added. Cells were then allowed to incubate for 24 hours before they were harvested for use in experiments.
Quantitative real-time PCR. Primers for SPARC (QuantiTect® Primer Assays) were used with QuantiTect SYBR® green kits (Qiagen) for quantitative real-time PCR. Total mRNA was extracted from the four cell lines transfected with SPARC siRNA and from the corresponding control cell lines using an RNeasy RNA extraction kit (Qiagen). A Chromo4 thermocycler (MJ Research) was used and all results were analyzed using the GeneEx software. Beta-actin was used as a housekeeper gene for purposes of normalization.
In vitro proliferation assay. The sulforhodamine-B-based assay kit (TOX-6, Sigma-Aldrich) was performed as per the National Cancer Institute protocol. Briefly, the four cell lines were seeded into wells at a density of 5×103 cells per well, in eight wells per cell line, either control or target siRNA transfected (5 nM). A row of 8 wells exposed to only RPMI-1640 media was used as a control. Cells were allowed to incubate for 48 hours following cell seeding. Following this 48-hour period, cells were fixed to the bottom of the wells using a solution of 50% trichloroacetic acid (TCA) for 1 hour at 4°C. Plates were then rinsed with distilled water, to remove TCA and media, and air dried. The sulforhodamine-B dye was added to each well and allowed to stain for 25 minutes. The sulforhodamine-B dye was subsequently removed by washing with a 10% acetic acid solution and once more allowed to air dry. The dye that was incorporated into the fixed cells at the bottom of the wells was solubilized in a 10 mM solution of Tris. The absorbance of the solute was measured using a microplate reader at a wavelength of 510 nm. This gave a comparison between control cell proliferation rates over 48 hours with the proliferation rate of cells transfected with SPARC siRNA over the same time period.
Immunocytochemistry. Immunocytochemistry was performed on three human UM cell lines (SP6.5, MKTBR, OCM-1) and one transformed human melanocyte cell line (UW-1) using the Ventana BenchMark fully automated machine (Ventana Medical System Inc., AZ, USA). The fully automated processing of bar code labeled slides included baking of the slides, solvent-free deparaffinization, and CC1 (Tris/EDTA buffer pH 8.0) antigen retrieval. Slides were incubated with a monoclonal mouse anti-human SPARC antibody (Abcam, Cambridge, MA; 1:50 dilution) for 30 min at 37°C, followed by application of biotinylated secondary antibody (8 min at 37°C) and an avidin/streptavidin enzyme conjugate complex (8 min at 37°C). Finally, the antibody was detected by Fast Red chromogenic substrate and counterstained with hematoxylin. Cytospins were independently graded as either positive or negative for SPARC expression by two different pathologists and no discrepancies were found.
Immunohistochemistry
Primary human UM and control eyes. Formalin-fixed, paraffin-embedded samples from 19 enucleated eyes of UM patients were collected from the archives of the Henry C. Witelson Ocular Pathology Laboratory and Registry, McGill University, Montreal, Canada.
SPARC expression was investigated using a mouse anti-human monoclonal antibody (Abcam). Immunostaining was performed using the Ventana Benchmark system. Sections of placenta were used as positive controls for SPARC expression while primary antibody was omitted in serial sections to serve as negative controls. Tumors were graded by extent (focal if <30% staining positive; diffuse if >30% positive) and intensity (weak, moderate or strong) of staining. Additionally, expression of SPARC was assessed in juxtatumoral retinal pigment epithelial (RPE) cells, choroidal melanocytes and choroidal endothelial cells. Additionally, eyes from four donors (aged 17, 53, 87 and 90 years) were obtained from the Eye Bank of Canada and stained for SPARC expression.
Animal model. Archived tissue from an experimental rabbit model of human UM was obtained for immunohistochemical analysis. Sections from 11 rabbit eyes with primary tumors and 8 rabbit lungs with metastatic UM were immunostained with an anti-SPARC antibody. Prmary tumors were graded by extent and intensity while metastases were graded by intensity only. Serial sections of each metastatic specimen were previously stained for the melanoma marker HMB-45; staining in these sections was compared to SPARC staining to confirm that any presence or absence of staining observed was in metastatic cells and not rabbit lung tissue.
Results
Quantitative real-time PCR. The efficacy of siRNA knockdown for SPARC was assessed using quantitative real-time PCR analysis of isolated mRNA. All cell lines showed similar expression of SPARC mRNA. The cells transfected with SPARC siRNA showed a significant decrease (p<0.05) in SPARC mRNA; this decrease varied between the cell lines. The smallest abrogation of expression was seen in the UW-1 cell line while the three UM cell lines demonstrated stronger abrogation of expression (Figure 1).
Proliferation assays. Proliferation of controls varied between the four cell lines. A statistically significant decrease (p<0.05) in proliferation was seen in all four cell lines transfected with SPARC siRNA compared to their respective controls (Figure 2). The largest decrease in proliferation occurred in the SP6.5 cell line.
Immunocytochemistry. All of the cell lines showed strong staining for SPARC (Figure 3). The percentage and intensity of staining appeared to be consistent across all cell lines and we were consequently unable to rank the cell lines according to expression. Staining was predominantly cytoplasmic, although a number of cells stained positively in their nuclei.
Immunohistochemistry of primary tumors and control eyes. All of the 19 tumors stained positively for SPARC and had a diffuse staining pattern (Figure 4). Four out of 19 (21%) tumors had strong staining, 13 of 19 (68%) moderate staining, and 2 of 19 (11%) weak staining. Juxtatumoral RPE cells stained either moderately or strongly in all cases, while staining for RPE of control eyes ranged from weak to strong (Figure 5). Choroidal melanocytes were negatively stained in one UM case and moderately or strongly in the remaining 18 cases. Similarly, choroidal melanocytes in control eyes were moderately or strongly positive in all cases (Figure 6). Four out of 19 (21%) tumors had moderate positive staining in choroidal endothelial cells, while 15 out of 19 (79%) tumors were negative. In contrast, moderately positive staining was seen in choroidal endothelial cells in all control eyes.
Immunohistochemistry of rabbit model tissue. Six out of 11 (55%) primary tumors stained strongly positive for SPARC, 3 (27%) moderately positive, and 2 (18%) weakly positive. The two tumors that were weakly positive showed a focal pattern of staining, while the remaining tumors all had diffuse staining. Juxtatumoral RPE was moderately positive in all cases. Six out of 8 (75%) lung metastases were strongly positive for SPARC, while 2 out of 8 (25%) were moderately positive (Figure 7).
Discussion
SPARC expression has previously been reported to play a role in facilitating or abrogating tumor cell proliferation in various types of cancer (6-11). Due to the apparent cancer-specific role of SPARC, it is necessary to investigate its significance in UM rather than inferring this from published data in other cancer types. An initial investigation of SPARC expression in isolated human primary UM tumors was reported by Ordonez et al., with the authors demonstrating that SPARC immunoreactivity was detected in each of the 27 specimens assayed (16). The current study corroborates the results of Ordonez and colleagues by demonstrating immunoreactivity for SPARC in 19 primary UM cases and in four cell lines. Additionally, the results of the proliferation assays - in which down-regulation of SPARC reduced the proliferation rates of all four cell lines - provide direct evidence that SPARC may be important in tumor proliferation. Notably, SP6.5, the most proliferative cell line assayed (17), had the greatest percentage decrease in proliferation after SPARC siRNA transfection.
The primary and metastatic tumors from the UM xenograft model all stained positively for SPARC. The primary tumors had similar staining profiles as the primary human UM, ranging from weak to strong expression. This similarity suggests that the xenograft model used would be ideal for investigating future anti-SPARC therapies. Moreover, the expression of SPARC was either moderate or strong in all of the lung metastases in the animal model, suggesting that both primary and metastatic tumors could potentially be treated with a single anti-SPARC approach.
Previous transcriptional profiling of a UM cell line was performed by our group and recently reported (18). This study investigated changes in expression patterns of various genes from the primary cell line to intraocular tumor to metastasis in a xenograft model. Results from these microarray analyses demonstrated an increase in SPARC expression from cell line to intraocular tumor. The increased expression of SPARC in the intraocular tumor compared to the cell line may reflect its role in tumor proliferation. This would be in agreement with findings by Sangaletti et al. who reported that SPARC-null mice presented with reduced tumor growth in a model of mammary carcinoma (19). Other groups, however, have seen the opposite: enhanced growth of tumors in SPARC-null mice, attributable in part to decreased tumor encapsulation, reduced tumor cell apoptosis, and decreased tumor cell macrophage infiltration (6, 20). In the current study, however, all tumors from human patients and from the xenograft model expressed SPARC, suggesting that expression of this protein, rather than absence thereof, may contribute to UM tumorigenesis.
Figure 4D highlights nuclear staining in some cells within the tumor. This pattern of staining was observed in a number of cases and its significance is the subject of current investigations. Koukourakis and colleagues similarly noted a nuclear pattern of SPARC staining in fibroblasts of tumor-associated stroma in cases of non-small cell lung cancer (9). An earlier report looking at SPARC localization in the chicken embryo concluded that the function of SPARC in the nuclear matrix may be analogous to its known functions in the extracellular matrix (21). Further investigation will shed some light on the significance of nuclear localization of SPARC in UM.
The general findings for SPARC that we report here reflect the trend of findings in cutaneous melanoma (10-13). Expression of SPARC appears to be associated in both diseases with neoplastic progression. Consequently, therapeutic targeting of SPARC may represent one approach to managing primary and systemic UM. Furthermore, the observation that all tumors in this study expressed SPARC suggests that this protein may represent a broad therapeutic target in UM irrespective of tumor characteristics.
- Received February 11, 2009.
- Accepted May 21, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved