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Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reduction in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.
Medulloblastoma is the most familiar brain tumor in children’s, with a prevalence of 0.6 per 1×105 patient-years according to the Central Brain Tumor Registry of the USA . Even though multimodal treatment including surgery, radiation, and chemotherapy, tumor reappearance is frequent, and the most of these patients eventually die from progressive tumor . These treatments are also toxic and can lead to long-term disabilities . Consequently, finding novel ways to suppress the tumor growth and low-toxicity therapies for children with medulloblastoma is a major goal of several cancer laboratories.
Secreted protein, acidic and rich in cysteine (SPARC) has been shown to be involved in multiple biological functions, including cell proliferation . Even though the behaviors ascribed to SPARC are many, but the cellular and molecular mechanisms of SPARC that mediate these processes are largely unknown.
Constitutive activation of STAT proteins are members of a ubiquitously expressed family of transcription factors activated in response to growth factors and cytokines . STAT3 has been shown to be an oncogene , and many types of human cancer express constitutively active STAT3 [7–9]. STAT3 correlates with cell proliferation in breast carcinoma  and non-small cell lung cancer , and also inhibits apoptosis . Conversely, inhibition of JAK/STAT signaling suppresses cancer cell growth and induces apoptosis in various cancers [9;12]. Previously we have shown that SPARC induced neuronal like phenotype in medulloblastoma cells through its inhibitory effect on IL-6-regulated suppression of Notch-mediated STAT3 signaling pathway . It was also demonstrated by others that siRNA mediated inhibition of STAT3-induced p16, p21 and p27 mediated cell cycle arrest and apoptosis in colorectal cancer cells . Further, p21 known to induces G2/M phase arrest by inhibiting Cyclin-B/Cdc2 . Cdc25 is required for removal of inhibitory phosphotyrosines on Cyclin-B/Cdc2 and Cyclin-A/Cdk2 kinase complexes that mediate entry into mitosis .
In the present study, study, we investigated the effects of SPARC expression on cellular growth, and cell cycle distribution. We demonstrate that SPARC expression suppresses proliferation of medulloblastoma cells via the stimulation of G2/M phase arrest. We also provide experimental and mechanistic evidences that abnormalities of STAT3 signaling contribute to the SPARC induced cell cycle arrest.
Primary antibodies against SPARC, Cdc2, phospho-Cdc2 (T14/Y15), Cyclin-B1, Chk1, Chk2, STAT3 and phospho-STAT3 (Y705), GAPDH, HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), 3,3′-diaminobenzidine peroxidase (DAB), propidium iodide (Sigma, St.Louis, MO), MTT reagent (R&D Systems, Minneapolis, MN), and ECL system Thermo scientific, Rockford, IL) were used in this study.
Adenovirus vector carrying full length human SPARC cDNA (Ad-DsRed-SP) and an empty vector (Ad-DsRed) were constructed using Adeno-X ViraTrak Expression System-2 (Clontech Laboratories, Mountain View, CA) and amplified as described previously [13;16]. Constitutively active STAT3 Plasmid (pSTAT3C) obtained from Addgene (Cambridge, MA).
D425 and UW228 cells were kindly provided by Dr. Darell D. Bigner and Dr. Ali-Osman (Duke University Medical Center) respectively, and cells were cultured as described previously . Cells were cultured either in culture flasks (D425) or in petridishes (UW228) for 24hrs and infected with mock, Ad-DsRed or Ad-DsRed-SP as described previously . For constitutively active STAT3C (pSTAT3C) and vector (pEV; empty vector) transfections, FuGENE HD transfection reagent (Roche, Indianapolis, IN) was used as per manufacturer’s instructions.
RT-PCR was performed as described previously . The PCR primers used for this study are: SPARC sense 5’-GGAAGAAACTGTGGCAGAGG-3’ and antisense 5’-ATTGCTGCACACCTTCTCAA-3’ and GAPDH sense 5’-AGCCACATCGCTCAGACACC-3’and antisense 5’-GTACTCAGCGGCCAGCATCG-3’. The expected PCR products were resolved on 2% agarose gels and visualized using ethidium bromide staining.
Cells were infected with mock, 100MOI of Ad-DsRed or 50 and 100 MOI of Ad-DsRed-SP for 24hrs, and then lysed with lysis buffer containing protease inhibitors. Equal amounts of protein aliquots were resolved over SDS-PAGE gels and transferred on to PVDF membrane. Blots were blocked with nonfat dry milk for 1hr at room temperature and incubated with primary antibodies for overnight at 4°C followed by appropriate secondary antibody conjugated with HRP. The membranes were developed by using ECL reagent protocol. The blots were further probed for GAPDH, which was used as a loading control.
Cell proliferation was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as previously described . Cells (2×103/well) were plated and infected with mock, Ad-DsRed, or Ad-DsRed-SP as described above. After 12,24,36,48,60,72,84 and 96hrs MTT reagent was added to each well and absorbance of the formazan from each sample was measured at a test wavelength 550nm and reference wavelength 655nm. The % of viability was calculated as 100 × mean absorbance of sample divided by mean absorbance of mock.
The allocation of cells in the cell cycle phases was determined using FACS analysis of DNA content. The cells were transfected with pSTAT3C plasmid and then incubated for 24hrs. Then cells were treated with Ad-DsRed-SP for further 24hrs. Cells were collected and fixed in 70% ethanol and stained with propidium iodide for 30 min and analyzed for DNA content by using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
The animal experiments were carried out as described previously . D425 (1×105 cells/10μl) cells were stereotactically implanted. After 14 days of tumor cell implantation, the animals were randomized into 3 groups (10 mice/group). Each mouse received 3 intracranial injections (10μl) on days 15, 17, and 19 with PBS, Ad-DsRed or Ad-DsRed-SP (5×107 PFU). Animals were monitored for up to 90 days, which is when we arbitrarily terminated the experiment. Mice brains were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections (5μ) were obtained from the paraffin blocks and stained with hematoxylin and eosin (H&E) by using standard histologic techniques. Tumor volume was assessed by methods described previously .
We used a previously described protocol with minor changes . Briefly, the tissue sections (5μ) were deparaffinized, rehydrated, permeabilized, and incubated overnight with primary antibodies, HRP-conjugated secondary antibodies, followed by DAB substrate solution, and then counterstained with hematoxylin and mounted. The images were processed as described previously .
ImageJ software (NIH) was used to quantify the mRNA and protein band intensities. Statistical comparisons were performed using analysis of variance for analysis of significance between different values using GraphPad Prism software (San Diego, CA). Values are expressed as mean of triplicate experiments ± S.D. from at least three separate experiments, and differences were considered significant at a p value of less than 0.05.
To study the effects of SPARC overexpression, we infected UW228 and D425 cell lines with 50 and 100MOI of adenoviral vector harboring SPARC cDNA sequence (Ad-DsRed-SP) driven by a CMV promoter. PCR analysis was carried out using the primers for SPARC and GAPDH. Clear amplification of SPARC transcript up to 2.5-4 folds was observed in Ad-DsRed-SP-infected cells when compared to mock or Ad-DsRed-infected cells. The levels of GAPDH remained unchanged in all treatments, indicating uniform levels of mRNA taken in all cases (Fig. 1A). Immunoblot analysis confirmed SPARC overexpression increased up to 2–3 fold in cells infected with indicated 100MOI of Ad-DsRed-SP compared to mock and Ad-DsRed-infected cells (Fig. 1B).
To determine whether SPARC overexpression affected the growth of D425 and UW228 cells, the growth rates of SPARC-overexpressed cells were compared with those of mock and vector control. A very minimal decrease in proliferation was observed at 24hrs (5-8%). At 48hrs, there was a 15–20% decrease in proliferation in Ad-DsRed-SP-infected cells (100MOI), compared with mock and Ad-DsRed-infected controls. Finally, at 96hrs, there was a 65–75% inhibition of cell proliferation in Ad-DsRed-SP-infected cells (100MOI) compared with mock and Ad-DsRed-infected cells (Fig. 1C).
To determine whether SPARC expression induces cell cycle arrest in medulloblastoma cells, we analyzed DNA content by FACS analysis. FACS analysis indicated that cells treated with Ad-DsRed-SP accumulated in G2/M phase cells. Around 60% (50MOI) and >65% (100MOI) of Ad-DsRed-SP-infected cells were arrested in G2/M, compared to mock or Ad-DsRed-infected controls (Fig. 2A). These results indicate that Ad-DsRed-SP-induced cell cycle arrest at G2/M phase in medulloblastoma cell.
We next explored potential mechanisms of the altered cell cycle arrest profile. We determined p21 protein level in the total cell lysates of SPARC overexpressed cells by immunoblotting. SPARC expression induced p21 protein levels by 2–3 folds compared to controls (Fig. 2B). G2/M arrest was shown to involve an initial inhibition of Cyclin-B1/Cdc2 activity by p21 and a subsequent reduction of Cyclin-B1 and Cdc2 . To study the disparity in G2/M cell cycle molecule expression with SPARC expression, we carried out immunoblot analysis for various cell cycle controlling proteins. We found that protein levels of Chk1, Chk2, Cdc25A, Cdc25C, Cyclin-B1 and Cdc2 were down-regulated in the Ad-DsRed-SP-infected cells compared to mock and Ad-DsRed-infected cells (Fig. 2C).
We found that treatment of cells with Ad-DsRed-SP blocked STAT3 phosphorylation (Tyr-705) in a dose-dependent fashion . Activation of STAT3 was shown to play an important role in cell cycle transition with the concomitant downregulation of p21 . Constitutive activation of STAT3 directly contributes to oncogenesis by promoting proliferation and/or by suppressing apoptosis [6;20]. Further, inactivation of JAK3/STAT pathway in cancer cells was shown to increase p21 expression . We next investigated whether STAT3 signaling might have a role in mediating p21 associated cell growth arrest in SPARC overexpressed cells. Constitutively active STAT3 in Ad-DsRed-SP-infected cells led to suppression p21 (Fig. 3A). FACS analysis indicated that constitutively active STAT3 suppressed SPARC induced G2/M arrest. Percent of cells in G2/M cells was >65% in the cells treated with Ad-DsRed-SP alone in cells, whereas ~40% in G2/M phase in cells transfected with pSTAT3C prior to Ad-DsRed-SP-infection (Fig. 3B). These results indicate that STAT3 plays a role Ad-DsRed-SP-induced G2/M arrest in medulloblastoma.
We have previously shown that SPARC expression inhibits medulloblastoma tumor growth in vivo in an intracranial model [13;16]. To evaluate the effect of SPARC on tumor formation and STAT3 expression in vivo, we stereotactically implanted D425 cells intracranially in nude mice. The tumors that arose were challenged with intratumoral injections of Ad-DsRed-SP. There was significant decrease in the tumor volume in mice treated with Ad-DsRed-SP compared to mice treated with mock and Ad-DsRed. Histological analysis of H&E stained tumor sections showed about 75% reduction of tumor volume in the brains of mice treated with Ad-DsRed-SP as compared to mock and Ad-DsRed controls (Fig. 4A).
To determine whether SPARC altered STAT3 and p21 expression in vivo, tumor sections were immunoassayed for SPARC, phosphor-STAT3 (Tyr-705) and p21. Consistent with our in vitro observations, tumor sections from Ad-DsRed-SP-treated mice showed very minimal expression of phosho-STAT3 and increased staining for SPARC and p21 (Fig. 4B).
Even though there is a large body of information available for the SPARC in cancer research, relatively few data has been published concerning the role of SPARC in cell cycle arrest. The possible roles of SPARC in cell cycle arrest appear to have received more attention than any other tumor suppressor processes . SPARC are known for their multitalented functions in cell cycle progression, cell development and differentiation [22;13]. Here, we demonstrate that SPARC expression suppress STAT3 signaling leading to cell cycle arrest in medulloblastoma cells.
In this study, we demonstrated that SPARC overexpression is able to inhibit cell proliferation in medulloblastoma cell lines. The evidence for the role of SPARC in the regulation of cell proliferation has been demonstrated before . For example, Both SPARC and TGF-β were an inhibitor of cell cycle progression and proliferation in several types of cells [4;23]. SPARC negatively regulates breast cancer cell proliferation without stimulating metastasis , and alters the proliferation of stromal but not tumor cells in melanoma . They also observed that SPARC inhibited endothelial cell proliferation, spreading, and migration in five different sources of cells. In fact, low expression of integrins following exposure to SPARC considerably reduced cell proliferation and adhesion, in part by down-regulating the activation of Akt, FAK, MAPK 44/42 and Src . Over-expression of SPARC protein and mRNA were frequently seen in non-proliferating, but actively secreting, Leydig, Sertoli cells and migrating cells , it showed that this SPARC might direct a metabolic pathway additional to the cell cycle. Even though no study has claimed SPARC to be stimulatory to cellular development, this statement showed that SPARC might suppress proliferation. SPARC is stimulated after cells have primarily proliferated and might function to withdraw cells temporarily from the cell cycle in preparation for migration . In the present study, we demonstrate that expression of genes controlling cell-cycle p21 and Cycin-B1 are affected by SPARC expression. We show that SPARC expression suppressed Cyclin-B1 levels in medulloblastoma cells. Cyclin-B1, the regulatory subunit of Cdk1, is essential for the transition from G2 phase to mitosis. . Cyclin-B1 is highly expressed in various primary tumors and associated with poor prognosis [30;31]. Overexpression of Cyclin-B1 could contribute to the chromosomal instability in cancer cells through an alteration of spindle checkpoint . Previous studies demonstrated that downregulation of Cyclin-B1 is an effective and specific approach to inhibit proliferation in variety of tumor cells .
The STAT3 gene is known for its proliferation activities in several types of cancers and is involved in different cellular processes, including regulation of cell-cycle [34;19]. Blocking the pathway by either antisense oligonucleotides or dominant-negative proteins induced growth retardation and apoptosis in DU145 cells . Since STAT3 plays an important role in cell cycle transition with the concomitant downregulation of p21 , we analyzed the expression and biologic significance of STAT3 in SPARC mediated suppression of tumor cell proliferation. We demonstrated that treatment of cells with Ad-DsRed-SP resulted in downregulation of STAT3 and its phosporylation form. By utilizing constitutively active mutant of STAT3, we demonstrate that STAT3 plays a critical in SPARC induced G2/M arrest. Inactivation of JAK3/STAT pathway in cancer cells was shown to increase p21 expression . Thus, it is likely that observed induction of p21 by AdDsRed-SP in medulloblastoma cells is a consequence of SPARC-induced STAT3 inactivation. Our findings are in good agreement with the recent publication reporting that the functional inactivation of STAT3 resulted in significant inhibition of anchorage-dependent and -independent proliferation in vitro and reduced tumor growth in vivo due to inhibition of cyclin-dependent kinase 2 activity based on increased expression of p21WAF1 in vitro and in vivo .
In summary, signal transduction pathways are likely essential therapeutic targets that are aberrantly operated in various cancers. Intervention of activated STAT3 is a promising therapy to treat malignant cancers [37;38]. Our findings clearly indicate that SPARC targets STAT3 signaling network induce growth arrest and that manipulating its tumor-suppressive activity may be a useful for rational cancer therapy.
This research was supported by National Cancer Institute Grant CA132853 (to S.S.L.).
We also thankful to Dr. Darell D. Bigner and Dr. Ali-Osman, (Duke University Medical Center for providing D425 and UW228 cells, respectively. The authors also thankful to Prof. J.E. Darnell, Jr., for providing constitutively active STAT3 (Addgene plasmid: #8722).
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