pathway mediates the effects of SPARC on ... - Wiley Online Library

ORIGINAL ARTICLE

Pigment Cell Melanoma Res. 24; 219–232

The p53⁄p21Cip1 ⁄ Waf1 pathway mediates the effects of SPARC on melanoma cell cycle progression Nina Fenouille1,2, Guillaume Robert1,2, Me´lanie Tichet1,2, Alexandre Puissant2,3, Maeva Dufies1,2, Ste´phane Rocchi1,2,4, Jean-Paul Ortonne1,4, Marcel Deckert2,5,6, Robert Ballotti1,2,4 and Sophie Tartare-Deckert1,2,4 1 INSERM, U895, Team 1, Nice, France 2 University of Nice – Sophia Antipolis, Nice, France 3 INSERM, U895, Team 2, Nice, France 4 Department of Dermatology, CHU Nice, Nice, France 5 INSERM, U576, Nice, France 6 Department of Clinical Hematology, CHU Nice, Nice, France

KEYWORDS melanoma ⁄ SPARC ⁄ p53 ⁄ cell cycle ⁄ cell migration PUBLICATION DATA Received 11 August 2010, revised and accepted for publication 13 October 2010, published online 18 October 2010

CORRESPONDENCE S. Tartare-Deckert, e-mail: [email protected]

doi: 10.1111/j.1755-148X.2010.00790.x

Summary Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, belongs to the family of matricellular proteins that modulate cell–matrix interactions and cellular functions. SPARC is highly expressed in melanoma, and we reported that SPARC promotes epithelial ⁄ mesenchymal-like changes and cell migration. Here, we used siRNA and conditional shRNA to investigate the contribution of tumor-derived SPARC to melanoma cell growth in vitro and in vivo. We found that depletion of SPARC induces G2 ⁄ M cell cycle arrest and tumor growth inhibition with activation of p53 and induction of p21Cip1 ⁄ Waf1 acting as a checkpoint, preventing efficient mitotic progression. In addition, we demonstrate that reduced mesenchymal features and the invasive potential of SPARC-silenced cells are independent of p21Cip1 ⁄ Waf1 induction and cell cycle arrest. Importantly, overexpression of SPARC reduces p53 protein levels and leads to an increase in cell number during exponential growth. Our findings indicate that in addition to its well-known function as a mediator of melanoma cell migration and tumor–host interactions, SPARC regulates, in a cell-autonomous manner, cell cycle progression and proliferation through the p53 ⁄ p21Cip1 ⁄ Waf1 pathway.

Introduction Members of the matricellular protein family are known to influence interactions between malignant cells and their microenvironment, and act as potent modulators of cellular functions (Bornstein and Sage, 2002; Clark and Sage, 2008). This growing family of secreted extra-

cellular matrix-associated proteins comprises thrombospondins, tenascins, osteopontin, CNN proteins and SPARC. Although they have no structural homology, they all share the functional property of modulating cell– matrix interactions. SPARC (also known as osteonectin or BM-40) is the prototype of this group of microenvironmental proteins. SPARC mediates diverse cellular

Significance Matricellular proteins, such as SPARC, are key regulators of tumor growth and metastasis. Some melanoma cells highly expressed SPARC and, clinically, its expression correlates with adverse outcome. SPARC has a well-established pro-invasive activity and is known to alter processes of transformation related to tumor–host interactions. Our study unveils a novel role for this matricellular protein in regulation of the p53 tumor suppressor pathway and progression into mitosis, and outlines the importance of SPARC in melanoma malignancy.

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processes such as de-adhesion, migration and inhibition of proliferation via its regulation of cell–matrix interactions and extracellular matrix production (Brekken and Sage, 2001). SPARC is involved in normal tissue remodeling processes such as bone resorption, wound healing and tissue injuries, as well as inflammation and cancer progression (Bradshaw and Sage, 2001; Clark and Sage, 2008; Sangaletti and Colombo, 2008). The role of SPARC in cancer biology is complex and dependent upon the origin of the tumor cells and the environment in which tumors evolve. SPARC exerts tumorsuppressive activities in neuroblastomas, pancreatic, colorectal and ovarian cancers, and certain myelodysplastic syndromes and acute myeloid leukemia, whereas it promotes aggressive tumor phenotype and metastatic behavior in prostate and breast cancers, glioma and melanoma (Clark and Sage, 2008; Podhajcer et al., 2008). The progression to malignant melanoma coincides with the altered expression of cell–matrix and cell–cell communication molecules such as SPARC (Haass et al., 2005; Miller and Mihm, 2006). SPARC has been shown to be an important marker for melanoma invasion and metastasis, and in patients with melanoma its expression correlates with the aggressiveness of the tumor and poor survival (Alonso et al., 2007; Massi et al., 1999; Sturm et al., 2002). SPARC production facilitates tumorigenicity by supporting the invasive behavior of melanoma cells and by inhibiting anti-tumor activity of certain immune effector cells (Alvarez et al., 2005; Ledda et al., 1997). In an earlier study, we demonstrated that autocrine SPARC signaling mediates melanoma invasion through repression of E-cadherin and induction of mesenchymal transition (Robert et al., 2006). In addition, reducing SPARC levels in human melanoma cells was shown to delay tumor growth in mouse xenografts and SPARC produced by melanoma cells regulates their in vitro proliferative capacity (Ledda et al., 1997; Prada et al., 2007). From these studies, it has been proposed that the growth capacity of human melanoma cells depends on SPARC levels. But the molecular mechanisms of this effect remain unclear. To address this issue and to determine the significance of SPARC in human melanoma, we analyzed its role in tumor cell growth in vitro and in vivo using RNA interference (RNAi) strategies. We found that SPARC expression confers a selective advantage for the growth of melanoma cells that contain wild-type p53. Depletion of SPARC induces G2 ⁄ M cell cycle arrest and promotes p53-dependent p21Cip1 ⁄ Waf1 induction. Interestingly, induction of p21Cip1 ⁄ Waf1 prevents cell cycle progression but not inhibition of migration and invasion of SPARCsilenced cells, indicating that their migratory defect is independent of the cell cycle arrest. Finally, we show that SPARC overexpressing cells displayed lower levels of p53 and an increased proliferative activity compared to control cells. Our findings indicate that tumor cellderived SPARC may overcome a p53 ⁄ p21Cip1 ⁄ Waf1220

dependent G2 ⁄ M checkpoint to facilitate loss of cell cycle control and melanoma tumorigenicity.

Results Silencing SPARC by RNAi had marked antiproliferative effects on melanoma cells and reduced tumorigenicity in vivo To gain insight into how SPARC might regulate melanoma cell growth, we downregulated expression of SPARC in melanoma cells using complementary RNAi strategies (Supporting Information Figure S1). First, we used short interfering RNA (siRNA) oligonucleotide transfection to deplete SPARC in A375P and 1205Lu melanoma cells, and we evaluated the time-response effect on cell viability using yellow tetrazolium salt (XTT) assay. We observed that transfection of siRNA targeting SPARC (siSPARC) resulted in almost complete reduction of SPARC protein levels. Reduction of SPARC in the A375P and 1205Lu culture media was concomitant with a time-dependent decrease in cell viability to the level of 40 and 60% of control siRNA-transfected A375P and 1205Lu cells, respectively (Figure 1A). As a second approach, we established subclones of A375P cells expressing doxycycline-inducible shRNA specific for SPARC (shSPARC; Figure S1). Immunoblots in Figure 1B show the time course for SPARC depletion following the addition of doxycycline. SPARC was significantly reduced after 3 days of treatment. Doxycyclineinduced depletion of SPARC markedly diminished the number of A375P cells in a time-dependent manner. As expected, doxycycline treatment of A375P cells transfected with an inducible shRNA lacZ (shlacZ) plasmid failed to induce a decrease of cell number. A second A375P clone stably depleted of SPARC was analyzed and a similar reduction of cell number was observed (Supporting Information Figure S3A). Because the growth of tumor cells in vitro may differ from their growth in vivo, we also looked at the ability of our conditional shSPARC cell model to grow subcutaneous tumors in mice (Figure 1C). Control A375P shlacZ- or shSPARC-expressing cells were injected subcutaneously into Nude mice. Mice were divided into two groups: one group was fed with doxycycline water to induce shRNA (+Dox) and the other group with regular water ()Dox) for 15 days. As expected, doxycycline administration had no effect on the tumor volume derived from control A375P shlacZ cells. However, the final volume of A375P shSPARC tumors in the doxycycline group was significantly reduced compared with the untreated group. Immunoblotting of the tumors excised at the end of the experiment confirmed effective SPARC knockdown upon doxycycline induction in the shSPARC tumors. These data are consistent with the in vitro studies and provide evidence that conditional knockdown of SPARC in tumors substantially inhibits their growth in vivo. ª 2010 John Wiley & Sons A/S

SPARC depletion promotes G2 ⁄ M cell cycle arrest

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Figure 1. RNAi depletion of SPARC decreases cell viability in melanoma cells and inhibits tumor growth in a xenograft model. (A) A375P and 1205Lu melanoma cells were transfected with control siRNA (siCTRL, open bars) or SPARC siRNA (siSPARC, filled bars), at a final concentration of 50 nM. Cell viability was measured by XTT assay at different times after transfection. Results are expressed in percent of control. Columns, mean of four independent determinations; error bars, SD. Immunoblots show SPARC levels in protein lysates and supernatants of these cells (bottom panels). HSP60 and Ponceau S-stained bands were used as loading controls in lysates and culture media, respectively. (B) A375P TRex cells stably expressing a control lacZ shRNA (shlacZ) or a doxycycline-inducible SPARC shRNA (shSPARC; clone #3F9) were grown for 7 days in doxycycline-free medium ()Dox, open bars) or doxycycline-containing medium (+Dox, filled bars). Viable cells were counted at the indicated times and data are expressed in percent of control. Columns, mean of two independent experiments performed in triplicate; error bars, SD. SPARC expression was analyzed by immunoblotting as described above. (C) Conditional RNAi knockdown of SPARC led to a statistically significant reduction of tumor development in mice. The amount of 5 · 106 A375P TRex cells expressing shlacZ (top panel) or shSPARC (clone #3F9; bottom panel) were subcutaneously inoculated in the left dorsal side of Nude mice (n = 5). For induction of shRNA synthesis in stable clones, 1 mg ⁄ ml doxycycline was added to the drinking water. Tumor growth was monitored for 15 days and the final tumor volume was calculated as described in Methods. Mean volumes are indicated by the horizontal line. *P < 0.005 (Student’s test). At the end of the study, A375P shlacZ and shSPARC tumors were collected, snap-frozen, resuspended in lysis buffer and subjected to immunoblot analysis using specific antibodies against human SPARC and HSP60 as loading control. The experiment was repeated twice with similar results.

Depletion of SPARC causes an accumulation of cells in G2 ⁄ M and modulates expression levels of G2 ⁄ M cell cycle-regulatory proteins Cell cycle profiles were examined with A375P cells, which were transiently transfected with control siRNA (siCTRL) or SPARC siRNA (Figure 2A and Supporting Information Figure S2A). Increasing amounts of siSPARC promoted a dose-dependent accumulation of cells in G2 ⁄ M phase, which was correlated with downregulation of SPARC protein levels. As a control, we exposed cells to bleomycin (BLM), a DNA-damaging agent that can block G2 ⁄ M transition (Baus et al., 2003). BLM induced a G2 ⁄ M accumulation that was comparable to the one observed in SPARC-depleted cells (Figure 2A). The percentage of BLM-treated A375P cells in G2 ⁄ M phase was 26%. Quantification and statistical analysis of the cell cycle data indicated that 23% of siSPARCtransfected cells were arrested in G2 ⁄ M, compared to 11% for the untransfected or siCTRL-transfected cells. A similar effect of the siSPARC on cell cycle was observed in 1205Lu melanoma cell line (Figure 2B, top ª 2010 John Wiley & Sons A/S

panel, and Figure S2B). Induction of SPARC knockdown following the addition of doxycycline in A375P shSPARC subclones also resulted in accumulation of cells in G2 ⁄ M phase (Figure 2B, bottom panel, and Figures S2B and S3B). As expected, doxycycline treatment had no effect on cell cycle distribution in A375P shlacZ cells. Taken together, these results suggest that cell growth suppression mediated by SPARC depletion results from G2 ⁄ M cell cycle arrest. To further characterize the G2 ⁄ M arrest induced by SPARC depletion, we examined the expression of genes important for cell cycle regulation using real-time Q-PCR-based assays. A375P cells transiently transfected with siCTRL or siSPARC were used for this mRNA expression profiling (Supporting Information Figure S4). This approach revealed several genes modulated by siSPARC, some of which have been verified at protein level. siRNA-mediated SPARC depletion selectively reduced expression of proteins regulating the progression from G2 to mitosis, such as cdk1, Cyclin B1 and Cdc25C, but had no significant effect on protein levels 221

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Figure 2. Depletion of SPARC causes a G2 ⁄ M cell cycle arrest in melanoma cells. (A) Dose-response of siSPARC on cell cycle. A375P cells were transfected, or not (NT), with 50 nM of siCTRL or increasing concentrations of siSPARC (from 10 to 50 nM). Four days after transfection, cells were stained with PI and analyzed for DNA content by flow cytometry. The percentage of cells with 4 N DNA content, representative of the G2 ⁄ M phase of the cell cycle, is indicated. A treatment for 5 h with bleomycin (BLM) at 10 lg ⁄ ml was used as positive control. Immunoblot analysis of SPARC expression in cell lysates is shown below the flow cytometry profiles. (B) Cell cycle distribution of A375P and 1205Lu cells depleted of SPARC after treatment with 50 nM siRNA, or A375P cells depleted of SPARC by stable expression of inducible shSPARC in the absence or presence of doxycycline for 7 days. Cell cycle profiles were analyzed by flow cytometry of PI-stained cells. Histograms represent the percentage of cells in different phases of the cell cycle. Columns, mean of five independent experiments; error bars, SD. (C) Expression levels of mitotic and interphasic Cyclin ⁄ cdk complexes in SPARC-depleted A375P cells. Four days after siRNA transfection, protein lysates were prepared and immunoblotting was performed with the indicated antibodies. A treatment for 5 h with bleomycin (BLM) at 10 lg ⁄ ml was used as positive control. NT, non-transfected condition.

of cdk4, 6 and 2 and Cyclin D1, E and A2, which act during interphasis (Figure 2C). All these changes are compatible with G2 ⁄ M cell cycle arrest in cells lacking SPARC. Also, decreased phosphorylation of Histone H3 at serine 10 further indicated the failure of mitotic progression in melanoma cells upon knockdown of SPARC and BLM treatment (Figure 2C). Depletion of SPARC results in elevated p53 and p21Cip1 ⁄ Waf1 levels in melanoma cells and xenografted tumors p53 and its target p21Cip1 ⁄ Waf1 have been implicated in G2 ⁄ M transition through inhibition of mitotic Cyclin B1 ⁄ cdk1 complex at both transcriptional and post-translational levels (Taylor and Stark, 2001). Treatment of the wild-type-p53-containing A375P cells with siSPARC promoted a concentration-dependent induction of p53 and p21Cip1 ⁄ Waf1 (Figure 3A). Similar changes in expression 222

levels of p53 and p21Cip1 ⁄ Waf1 as well as of cdk1 and Cyclin B1 could also be seen after siSPARC treatment of other melanoma cells characterized by wild-type p53 such as 1205Lu, 501mel and WM9, and upon induction of shSPARC with doxycycline in A375P shSPARC cells (Figure 3B). Immunoblotting of A375 shSPARC tumor samples obtained in our xenograft assays (see Figure 1C) revealed that steady-state levels of p53 and p21Cip1 ⁄ Waf1 were elevated following shSPARC induction in tumors from mice that received doxycycline treatment compared with untreated ones, suggesting that the p53 pathway was activated in tumors derived from SPARC-silenced cells (Figure 3C). These data indicate that targeting SPARC in vitro and in vivo leads to increased p53 and p21Cip1 ⁄ Waf1 expression levels. To check that upregulation of p21Cip1 ⁄ Waf1 was a consequence of specific inhibition of SPARC expression, the silencing of SPARC was rescued by exogenous SPARC ª 2010 John Wiley & Sons A/S


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Figure 3. Depletion of SPARC modulates key proteins in the G2 ⁄ M checkpoint pathway. (A) A375P cells were transfected with 50 nM of siCTRL or increasing concentrations of siSPARC (from 1 to 50 nM). After 4 days, total cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) A375P, 1205Lu, 501mel and WM9 melanoma cells were transfected for 4 days with siCTRL or siSPARC at 50 nM. A375P shlacZ and shSPARC (clone #3F9) cells were grown for 7 days in doxycycline-free medium ()Dox) or doxycycline-containing medium (+Dox). Total protein lysates from resulting cells were analyzed by immunoblotting with the indicated antibodies. (C) Expression levels of p21Cip1 ⁄ Waf1 and p53 in A375P shSPARC tumors depleted, or not, for SPARC after doxycyclin treatment. Protein lysates were prepared and immunoblotting was performed as decribed in Figure 1C. (D) Exogenously added SPARC normalizes p21Cip1 ⁄ Waf1 protein levels in SPARCdepleted cells. A375P cells were transfected with 50 nM of siCTRL or siSPARC. Twenty-four hours later, 20 lg ⁄ ml of full-length SPARC recombinant protein with MBP tag (MBP-SPARC) was added for 3 days. Immunoblots show SPARC and p21Cip1 ⁄ Waf1 expression levels in cell lysates. Culture supernatants were also collected and analyzed for endogenous SPARC and MBP-SPARC levels by immunoblotting with anti-SPARC antibodies. HSP60 and Ponceau S-stained bands were used as loading controls.

treatment. This was performed by addition of MBPSPARC fusion protein to siCTRL- and siSPARC-transfected cells. Immunoblotting confirmed the presence of recombinant protein in culture media and the expression of endogenous SPARC in cell lysates and culture media (Figure 3D). This experiment showed that siSPARC-mediated upregulation of p21Cip1 ⁄ Waf1 expression could be preventing by the addition of exogenous SPARC protein. p53 contributes to p21Cip1 ⁄ Waf1 induction mediated by SPARC depletion As p21Cip1 ⁄ Waf1 is mainly transcriptionally regulated by p53 (el-Deiry et al., 1993), we addressed the question of whether p53 was involved in p21Cip1 ⁄ Waf1 induction in SPARC-depleted cells. First, we evaluated steadystate levels of p21Cip1 ⁄ Waf1 (CDKN1A) mRNA levels in A375P cells transfected with siCTRL or siSPARC by real-time Q-PCR. Figure 4A shows that SPARC knockdown cells displayed elevated p21Cip1 ⁄ Waf1 mRNA levels. Reporter assays using a p21Cip1 ⁄ Waf1 promoterluciferase construct that contains p53-binding sites, revealed that treatment with SPARC siRNA led to a twofold induction of p21Cip1 ⁄ Waf1 promoter activity (Figure 4B). Interestingly, this induction was inhibited by addition of the specific p53 inhibitor pifithrin-a (PFTa). Also, cotransfection of the reporter gene with an ª 2010 John Wiley & Sons A/S

expression vector for p53 increased the activity of the p21Cip1 ⁄ Waf1 promoter in both control and siSPARC-treated cells, maintaining a twofold induction. Similar results were obtained using an artificial p53-dependent reporter gene (Figure 4C). In this case, the stimulation of the reporter gene by p53 was stronger than that by the p21Cip1 ⁄ Waf1 promoter. We next asked whether decreasing the level or activity of p53 could prevent accumulation of p21Cip1 ⁄ Waf1 protein in SPARC-depleted cells (Figure 4D). We observed that siRNA-mediated downregulation and pharmacological inhibition of p53 by PFTa almost completely reversed the stimulatory effect of SPARC depletion on p21Cip1 ⁄ Waf1 expression. Similarly, induction of p21Cip1 ⁄ Waf1 by BLM was suppressed in p53-depleted cells or PFTa-treated cells. These results support the conclusion that p21Cip1 ⁄ Waf1 upregulation mediated by SPARC depletion occurs mainly through activation of p53. Depletion of SPARC induces a p53 ⁄ p21Cip1 ⁄ Waf1dependent cell cycle arrest p21Cip1 ⁄ Waf1 is known to bind with and inhibit the activity of Cyclin ⁄ cdk complexes and thus regulates both G1 ⁄ S and G2 ⁄ M transitions (Abbas and Dutta, 2009; Xiong et al., 1993). To determine the role of elevated 223

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Figure 4. The tumor suppressor protein p53 is activated and regulates p21Cip1 ⁄ Waf1 expression in SPARC-depleted A375 cells. (A) Analysis of p21Cip1 ⁄ Waf1 (CDKN1A) mRNA levels. RNAs were prepared from A375P cells treated with 50 nM siCTRL or siSPARC for 4 days and then subjected to real-time Q-PCR analysis as described in Supporting Information. Data are expressed in arbitrary units as fold change between siSPARC-treated cells and control cells. Columns, mean of two independent amplifications performed in duplicate; error bars, SD. (B) p21Cip1 ⁄ Waf1 promoter activation. Cells were transfected with 50 nM siCTRL (open bars) or siSPARC (filled bars), and 24 h later with the human p21Cip1 ⁄ Waf1 promoter reporter (p21Cip1 ⁄ Waf1-luc) together with p53 expression construct or empty vector. After 3 days, luciferase activities were measured and normalized to b-galactosidase activities. A treatment for 2 days with the p53 inhibitor pifithrin-a (PFTa) at 20 lg ⁄ ml was used as control. Columns, mean of triplicates; errors bars, SD. *P < 0.05 (Student’s test). NS, not significant. (C) p53 transcriptional activity. Cells were transfected with 50 nM siCTRL (open bars) or siSPARC (filled bars), and 24 h later with a p53-responsive promoter reporter (PG13-luc) together with a p53 expression construct or empty vector. Measurement of luciferase activities was assessed as described above. (D) p21Cip1 ⁄ Waf1 induction is dependent on p53 activation. Cells were transfected for 4 days with control siRNA (siCTRL), p53 siRNA (sip53), SPARC siRNA (siSPARC) alone or in combination, at a final concentration of 50 nM (left panel). A375P cells transfected with siCTRL or siSPARC for 2 days were incubated with PFTa at 20 lg ⁄ ml or DMSO as a vehicle control for an additional 2 days (right panel). Cells were exposed for 5 h to bleomycin (BLM) at 10 lg ⁄ ml for control of p53 activation. Total protein lysates from resulting cells were analyzed by immunoblotting with the indicated antibodies.

p21Cip1 ⁄ Waf1 levels in the G2 ⁄ M arrest mediated by depletion of SPARC, we used siRNA to target p21Cip1 ⁄ Waf1 (sip21) in SPARC-depleted cells. sip21 normalized the cell cycle distribution and prevented cdk1 and Cyclin B1 downregulation protein levels in siSPARCtreated A375P cells (Figure 5A,B). Of note, sip21 did not affect upregulation of p53 induced by siSPARC. To determine whether increased levels of p21Cip1 ⁄ Waf1 were associated with the mitotic Cyclin B1 ⁄ cdk1 complex, we performed co-immunoprecipitation assays in response to siSPARC treatment. SPARC-depleted and control A375P lysates were subjected to immunoprecipitation with antibodies to p21Cip1 ⁄ Waf1 followed by cdk1 and Cyclin B1 immunoblotting (Figure 5C). The amount of cdk1 and Cyclin B1 present in p21Cip1 ⁄ Waf1 complex was increased within 2 days of siSPARC treatment. 224

Of note, the ability of p21Cip1 ⁄ Waf1 to associate with cdk1 in SPARC-depleted cells occurred before the decrease in cdk1 protein level. As p21Cip1 ⁄ Waf1 has been shown to inhibit the activating phosphorylation of cdk1 on threonine 161 (Smits et al., 2000), we analyzed the level of threonine 161 phosphorylation during the time-course of siSPARC. Consistent with an inhibition of cdk1 activity, we observed a decrease of the slower migrating phosphorylated form of cdk1 that co-immunoprecipitated with p21Cip1 ⁄ Waf1. Accordingly, a decrease in cdk1 phosphorylation on threonine 161 was seen in total lysates 3 days after siSPARC treatment (Figure 5C). Regarding a functional role for p53 in mediating p21Cip1 ⁄ Waf1 upregulation and subsequent G2 ⁄ M arrest in SPARC-silenced cells, melanoma cells harboring a ª 2010 John Wiley & Sons A/S


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Figure 5. The cdk-inhibitor p21Cip1 ⁄ Waf1 is required for the G2 ⁄ M arrest induced by depletion of SPARC. (A) p21Cip1 ⁄ Waf1 depletion bypasses cell cycle arrest after SPARC depletion. A375P cells were transfected with control siRNA (siCTRL), p21Cip1 ⁄ Waf1 siRNA (sip21), SPARC siRNA (siSPARC) alone or in combination, at a final concentration of 50 nM. Four days after transfection, cells were stained with PI and analyzed for DNA content by flow cytometry. Histograms represent the percentage of cells in different phases of the cell cycle. Columns, mean of two independent experiments; error bars, SD. (B) Expression levels of indicated proteins were determined by immunoblotting extracts of A375P cells transfected as described above. (C) A375P cells were transfected with 50 nM siCTRL or siSPARC. At various time points, p21Cip1 ⁄ Waf1 was immunoprecipitated (IP) from cell lysates, and the resulting immunocomplexes, as well as cell lysates, were analyzed by immunoblotting with the indicated antibodies. (D) p53-mutated melanoma cells (SKmel28 and MeWo), were transfected with 50 nM siCTRL or siSPARC. Four days after transfection, cells were stained with PI and analyzed for DNA content by flow cytometry. The percentage of cells with 4 N DNA content, representative of the G2 ⁄ M phase of the cell cycle is indicated (top panel). Total protein lysates from resulting cells were analyzed by immunoblotting with the indicated antibodies (bottom panel).

mutated TP53 gene (SKmel28 and MeWo cells) (Albino et al., 1994; O’Connor et al., 1997) exhibited resistance to SPARC siRNA-mediated abnormal accumulation in G2 ⁄ M (Figure 5D and Supporting Information Figure S5). In addition, p53 protein levels were unaltered upon SPARC depletion in these two melanoma cell lines. p21Cip1 ⁄ Waf1 protein was undetectable by immunoblotting in both control and SPARC-depleted SKmel28 and MeWo cells (data not shown). Our results show that inhibition of G2 ⁄ M progression induced by SPARC depletion occurs through a p53 ⁄ p21Cip1 ⁄ Waf1-dependent mechanism that involves an initial association of p21Cip1 ⁄ Waf1 with the Cyclin B1 ⁄ cdk1 complex and a secondary reduction in cdk1 and Cyclin B1 levels. Depletion of SPARC decreases in vitro cell migration and invasion independent of p53 and p21Cip1 ⁄ Waf1 Besides its relevance in cell cycle regulation, p21Cip1 ⁄ Waf1 may influence cell motility and breast cancer epithelial– mesenchymal transition (EMT) (Besson et al., 2008; Liu et al., 2009). Because SPARC knockdown cells exhibit ª 2010 John Wiley & Sons A/S

reduced mesenchymal behavior and motility (Robert et al., 2006), it is possible that elevated p21Cip1 ⁄ Waf1 levels may contribute to the impaired migratory phenotype. To test this hypothesis, A375P cells were co-transfected with siRNA targeting both SPARC and p21Cip1 ⁄ Waf1, and effects on in vitro cell migration and invasion were evaluated using modified Boyden Chamber assays (Figure 6A). SPARC-silenced cells exhibited a marked reduction in serum-stimulated cell motility and invasion through Matrigel (BD Biosciences, Le Pont de Claix, France) compared with control cells, consistent with previously published results (Ledda et al., 1997; Robert et al., 2006; Smit et al., 2007). However, when assayed in the presence of p21Cip1 ⁄ Waf1 siRNA, SPARC-depleted cells showed a similar reduction in their migration and invasion abilities. We also found that expression of the mesenchymal marker, fibronectin was markedly reduced in SPARC-depleted cells and inactivation of p21Cip1 ⁄ Waf1 upon SPARC knockdown did not restore its expression level (Figure 6B). In addition, when transwell assays were carried out in SKmel28 and MeWo cells containing mutant copies of p53, we still observed a reduction of their migration abilities upon SPARC knockdown 225

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Figure 6. Depletion of SPARC decreases cell migration and invasion independently of p53 status and induction of p21Cip1 ⁄ Waf1. (A) A375P cells were transfected with control siRNA (siCTRL), p21Cip1 ⁄ Waf1 siRNA (sip21), SPARC siRNA (siSPARC) alone or in combination, at a final concentration of 50 nM. Four days after transfection, serum-stimulated motility (left panel) and Matrigel cell invasion assays (right panel) were performed using Transwell inserts. Cells were left to migrate for 5 h or invade for 18 h, then fixed, stained and counted. Columns, means of triplicates from two independent experiments; error bars, SD. Representative images of the lower surface of membranes are shown. (B) The expression levels of indicated proteins were determined by immunoblotting extracts of A375P cells transfected as described above. (C) p53mutated melanoma cells (SKmel28 and MeWo), were transfected with 50 nM siCTRL or siSPARC for 4 days, before assessment of migration assays, as described above.

(Figure 6C). These results support the notion that migration defects of SPARC-depleted melanoma cells are independent of the functional status of p53 and induction of p21Cip1 ⁄ Waf1. Depletion of SPARC does not modulate MITF levels in melanoma cells Microphthalmia-associated transcription factor (MITF) is a key regulator of melanoma cell proliferation and migration and p21Cip1 ⁄ Waf1 expression (Carreira et al., 2005, 2006; Garraway et al., 2005). Therefore, we tested whether SPARC knockdown could alter MITF protein and mRNA levels in melanoma cells (Supporting Information Figure S6 and data not shown). No significant modulation of MITF levels was detected in response to siRNA-mediated SPARC depletion. Thus, it is unlikely that the functional effects of SPARC knockdown in melanoma cells were mediated by MITF. SPARC overexpression reduces p53 levels and moderately increases proliferation From the data presented so far, it appears that SPARC knockdown induces p53 accumulation and activation that leads to a transcriptional program involved in G2 ⁄ M cell cycle arrest. We next examined whether overexpression of SPARC could affect p53 protein levels 226

and cell proliferation. For these experiments, we used 501mel cells, which express relatively low levels of SPARC (Robert et al., 2006). We established 501mel cells expressing an Myc-tagged SPARC protein (501mel SPARC cells) and confirmed overexpression of SPARC by immunoblot analysis (Figure 7A). Interestingly, overexpression of SPARC in 501mel cells resulted in a marked decrease in p53 protein levels, suggesting a role for SPARC in p53 regulation. Moreover, as determined by counting the total number of cells, there was an increase of 20% in the number of growing cells in SPARC overexpressing cells compared with parental cells (Figure 7B).

Discussion SPARC is a matricellular protein produced by tumor and neighboring stromal cells that influences the development and metastatic behavior of various cancers. In some melanoma cells, SPARC is highly expressed and promotes an EMT-like behavior (Robert et al., 2006; Smit et al., 2007). Tumor cell-derived SPARC can also influence in vivo melanoma growth, immune evasion and inflammatory processes (Podhajcer et al., 2008). Suppression of SPARC in human melanoma cells was shown to potently limit tumor growth in mouse ª 2010 John Wiley & Sons A/S


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Figure 7. Overexpression of SPARC reduces p53 levels and moderately increases proliferation in melanoma cells. (A) Total protein lysates from 501mel cells expressing Myc-tagged human SPARC (501mel SPARC) or carrying an empty expression cassette of pcDNA3 vector (501mel CTRL) were analyzed by immunoblotting with indicated antibodies. (B) Proliferation curves of 501mel CTRL and 501mel SPARC cells. Cells were cultured for 7 days, and counted at the indicated days. Points, mean of six independent measurements; error bars, SD. Proliferation during exponential growth (from day 3 to day 5) was calculated from the tangent to the curve and expressed as the number of counted cells per day.

xenografts (Ledda et al., 1997). This reduction of melanoma growth was attributed to tumor rejection by activated host polymorphonuclear leukocytes, rather than an effect on cell proliferation and survival (Alvarez et al., 2005; Prada et al., 2007). Here, we provide evidence that SPARC produced by melanoma cells supports proliferation and cell cycle progression. We show that reducing SPARC levels by RNAi in different melanoma cell lines harboring wild-type p53 inhibits cellular proliferation through activation of a p53 ⁄ p21Cip1 ⁄ Waf1dependent G2 ⁄ M checkpoint. Additional experiments performed in p53-mutated melanoma cells confirmed the role of p53 in mediating G2 ⁄ M arrest upon SPARC silencing. Further, our results using conditional RNAi to knockdown SPARC in vivo extend earlier observations and support the notion that SPARC is a required autocrine factor for melanoma growth in tumors that contain wild-type p53. Importantly, our study indicates that suppression of SPARC in xenografted tumors engages the p53 tumor suppressor pathway. This agrees with the in vitro data and could account for the observed inhibition of growth of SPARC-depleted tumors compared to control tumors. Re-activation of p53 in liver carcinomas was shown to trigger innate immune response and tumor clearance in mice (Xue et al., 2007). Because of this, it would be interesting to study whether the increased infiltration and activation of host leukocytes observed in SPARC-silenced tumors (Alvarez et al., 2005; Prada et al., 2007) is a consequence of p53 activation in melanoma cells. Our work reveals an interesting link between SPARC and the p53 ⁄ p21Cip1 ⁄ Waf1pathway. We found that one consequence of ablating SPARC in melanoma cells is the triggering of a p53-dependent p21Cip1 ⁄ Waf1 signaling pathway. p53 functions as a transcriptional regulator of genes involved in cell cycle arrest and apoptosis pathways (Riley et al., 2008) and, unlike many other tumors, ª 2010 John Wiley & Sons A/S

the majority of melanomas express wild-type p53 (Albino et al., 1994; O’Connor et al., 1997; Weiss et al., 1993). p53 stabilization following SPARC depletion results in transcriptional activation of p21Cip1 ⁄ Waf1 and G2 ⁄ M cell cycle arrest. Mechanistic studies revealed that p21Cip1 ⁄ Waf1 is the principal mediator of the changes on cell cycle progression observed upon SPARC knockdown. However, despite a recent report showing that p21Cip1 ⁄ Waf1 can repress features of EMT and reduces migration of breast tumor cells, our findings indicate that it is unlikely that increased expression of p21Cip1 ⁄ Waf1 in SPARC-depleted A375 melanoma cells contributes to their impaired motility and mesenchymal behavior. This was determined by the fact that preventing p21Cip1 ⁄ Waf1 upregulation completely rescued proliferation defects of SPARC knockdown cells but not motility defects and changes of known EMT-related genes, such as fibronectin. In addition, our data in cells with p53 mutants indicate that anti-migratory effects of SPARC knockdown are not mediated through wild-type p53. Thus, decreased motility and invasion of SPARCdeficient melanoma cells is independent of cell cycle defect and p53 genetic status. This observation supports the hypothesis that SPARC depletion in human melanoma cells results in inhibition of cell cycle progression and reduction of invasive mesenchymal phenotype, each in part through the activation of distinct signaling pathways that are respectively dependent and independent of p53 and induction of p21Cip1 ⁄ Waf1. During the preparation of our manuscript, one study reported similar results on the induction of p21Cip1 ⁄ Waf1 and inhibition of cell proliferation upon SPARC silencing in melanoma cells (Horie et al., 2010). However, those authors indicated that the effect of SPARC depletion on cell cycle was independent of p21Cip1 ⁄ Waf1. In contrast, our findings show that p53-dependent induction of p21Cip1 ⁄ Waf1 is responsible for the proliferation defect 227

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and that p53 and p21Cip1 ⁄ Waf1 were induced in our tumor xenograft experiments. Thus, our study provides the first demonstration that activation of this tumor suppressor pathway is functionally related to the cell cycle defects of SPARC-depleted melanoma cells. Intriguingly, Horie and collaborators reported the induction of p53 and G1 cell cycle arrest in SPARC-depleted MeWo cells, whereas we found this p53-mutated cell line to be refractory to the effect of SPARC knockdown on p53 induction and cell cycle progression. At present, the reason behind this discrepancy remains unclear. A function of p53 and p21Cip1 ⁄ Waf1 in G2 checkpoint control has been well established (Bunz et al., 1998; Taylor and Stark, 2001). It is known that p21Cip1 ⁄ Waf1 can bind Cyclin B1 ⁄ cdk1 complexes and inhibits the activity of cdk1 that is required for G2 ⁄ M progression (Baus et al., 2003; Xiong et al., 1993). Also, accumulation of p21Cip1 ⁄ Waf1 was reported to inhibit phosphorylation of cdk1 on threonine 161 and consequently cdk1 activity (Smits et al., 2000). Thus, the increased binding of p21Cip1 ⁄ Waf1 to Cyclin B1 ⁄ cdk1 complexes and the decrease in threonine 161 phosphorylation of cdk1 could be a possible explanation for the observed early accumulation of SPARC-depleted cells in G2 ⁄ M phase. In

addition, p21Cip1 ⁄ Waf1 is essential for sustaining G2 arrest by the transcriptional repression of cdk1 and Cyclin B1 (Taylor and Stark, 2001). The mechanisms by which p21Cip1 ⁄ Waf1 decreases Cyclin B1 ⁄ cdk1 transcription were shown to be dependent on activation of pocket proteins of the retinoblastoma family (Flatt et al., 2000; Taylor et al., 2001). Consistently, we found that p21Cip1 ⁄ Waf1 is required for the decrease in the levels of these two mitotic regulators in SPARC knocked-down cells. We propose that the above-described mechanisms could act in concert to keep the cells arrested in G2 upon SPARC depletion (Figure 8). Our data strongly support the conclusion that p21Cip1 ⁄ Waf1 plays an important role in the control of the G2 ⁄ M checkpoint by p53 in SPARC-depleted melanoma cells. How depletion of SPARC can lead to activation of the p53 ⁄ p21Cip1 ⁄ Waf1 signaling pathway remains to be elucidated. Given that p53-dependent G2 ⁄ M arrest is a common checkpoint mechanism in response to DNA damage (Lakin and Jackson, 1999), we have analyzed the potential involvement of this pathway upon SPARC silencing. However, we did not observe phosphorylation of cdk1 on tyrosine 15, the main target of the G2 ⁄ M DNA damage checkpoint, or modulation of Chk1 and

Figure 8. Model for G2 ⁄ M checkpoint activation and cell cycle arrest in response to SPARC depletion in melanoma cells. Melanoma cells produce and secrete high levels of the matrix-associated SPARC protein in their microenvironment. Loss of SPARC production activates a G2 ⁄ M checkpoint, leading to stimulation of p53 signaling pathway and upregulation of the cdk inhibitor p21Cip1 ⁄ Waf1. p21Cip1 ⁄ Waf1 is essential for inactivation of the Cyclin B1 ⁄ cdk1 complex, a key initiator of mitosis. First, p21Cip1 ⁄ Waf1 inhibits cdk1 by blocking its phosphorylation on Thr161 and then it mediates the transcriptional repression of Cyclin B1 and cdk1 expression, which causes sustained G2 ⁄ M arrest and inhibition of proliferation of SPARC-depleted cells.

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Chk2 activities in SPARC-depleted cells (data not shown). These led us to suggest that SPARC depletion stimulates p53 independently of these DNA-damage effector checkpoint kinases. Another possibility is the involvement of the stress-activated kinases JNK1 ⁄ 2 in the effect of SPARC knockdown on p21Cip1 ⁄ Waf1 and G2 ⁄ M. These kinases were recently shown to support the growth of melanoma cells, and their inhibition in 1205Lu cells resulted in a G2 ⁄ M arrest through p53dependent induction of p21Cip1 ⁄ Waf1 (Alexaki et al., 2008). Thus, we can imagine that depletion of SPARC negatively regulates JNK signaling, leading to induction of p53 ⁄ p21Cip1 ⁄ Waf1 and G2 ⁄ M arrest. We are currently investigating whether SPARC knockdown modulates the JNK pathway in melanoma cells. The acquisition of abnormalities at the G2 ⁄ M checkpoint can lead to the genesis and progression of tumors (Elledge, 1996). Failures of this checkpoint allow cells with damaged DNA to enter M phase. We speculate that acquired expression of SPARC by melanoma cells during progression of the disease could contribute to inactivation of the p53 and p21Cip1 ⁄ Waf1-dependent G2 arrest, which could be responsible for malignant growth. In support of this, we found that overexpression of SPARC in 501mel cells leads to decreased p53 protein levels and that cell proliferation during exponential growth tends to be higher than that in the control group. This potential role for SPARC in G2 ⁄ M checkpoint is in agreement with a previous study showing that SPARC levels are modulated during the cell cycle and are increased in the G2 ⁄ M phase (Ford et al., 1993). As a whole, these findings fit with the idea that SPARC is not just a regulator of tumor–host interactions, but also contributes in a cell-autonomous manner to tumorigenesis. The molecular mechanism by which SPARC might alter the p53 transduction pathway is not yet known and awaits further investigation. However, our rescue experiments in which the RNAi effect was reversed through addition of SPARC recombinant protein in the culture media indicate that secreted SPARC, rather than the intracellular form of SPARC, likely mediates regulation of p21Cip1 ⁄ Waf11 in melanoma cells. Depending on the tumor cell type, SPARC differentially affects biological processes related to proliferation or apoptosis. The addition of SPARC induced apoptosis of colon and ovarian carcinoma cells and reduced proliferation of prostate cancer cell lines (Said et al., 2009; Tang and Tai, 2007; Yiu et al., 2001). Also, SPARC expression induced autophagy and subsequent apoptosis in neuroectodermal tumor cells (Bhoopathi et al., 2010). However, SPARC had the opposite effect on the survival of glioma cells (Shi et al., 2004, 2007) and our findings suggested that SPARC contributes to the proliferation of melanoma cells and inactivation of p53. This controversial evidence indicates that the role of SPARC in proliferation and cell cycle regulation is complex and dependent on the cellular context of its expression. It is ª 2010 John Wiley & Sons A/S

still unclear why SPARC has variable and opposing effects on tumor cell growth. However, an attractive possibility that arises from our study is that the function of SPARC to act as a tumor-promoting factor or tumor suppressor might be related to the p53 genotype of the tumor. It is tempting to speculate that the genetic status of p53 in a tumor might predict the response of the tumor to exogenous SPARC. In tumors, both malignant cells and surrounding stromal cells can produce SPARC, and the origin of SPARC in the tumor microenvironment contributes to the complexity of SPARC actions in tumorigenesis. It remains unclear whether normal stromal SPARC has different biological activities compared with tumor-derived SPARC (Clark and Sage, 2008; Podhajcer et al., 2008). Host-derived SPARC in tumor was associated with angiogenesis, anti-inflammatory properties and ECM deposition and limited the growth of neuroblastoma, ovarian, pancreatic and prostate tumors as well as lung adenocarcinoma (Brekken et al., 2003; Chlenski et al., 2002; Puolakkainen et al., 2004; Said et al., 2007, 2009), whereas it promoted growth and metastasis of breast carcinomas (Sangaletti et al., 2003, 2008). Also, stromal SPARC was correlated with a poor prognosis in pancreatic and non-small cell lung cancer (Infante et al., 2007; Koukourakis et al., 2003). From these studies, it was concluded that stromal host cells are likely to biologically the most relevant source of SPARC in the tumor environment. However, the situation seems different in melanoma, where fibroblast-derived SPARC and stroma reorganization induced by SPARC did not influence melanoma growth (Prada et al., 2007). More careful examination is needed of the significance and source of host-derived SPARC in the melanoma stroma, but our present study and the literature highlight the fact that tumor-derived SPARC, rather than host-derived SPARC, influence the growth of melanoma tumors (Alvarez et al., 2005; Ledda et al., 1997; Prada et al., 2007). In conclusion, our findings demonstrate that SPARC produced autonomously by tumor cells supports neoplastic cellular proliferation and tumorigenic phenotype and may function as a novel matrix regulator of p53 and p21Cip1 ⁄ Waf1-mediated cell cycle progression.

Methods Cell culture, antibodies, plasmids and reagents Human A375P, 1205Lu, 501mel, WM9, SKmel28 and MeWo cells with known p53 status were maintained as described previously (Bailet et al., 2009; Gaggioli et al., 2005). The 501mel cells expressing Myc-tagged human SPARC (501mel SPARC) or carrying an empty expression cassette of pcDNA3 vector (501mel CTRL) were constructed by stable transfection using Fugene lipofection reagent (Roche Applied Science, Indianapolis, IN, USA) and clonal selection with 2 lg ⁄ ml puromycin. Antibody to human SPARC was purchased from Haematologic Technologies (Essex Junction, VT, USA). Antibodies against HSP60, Myc, p53, Cyclin E, cdk2 and Cdc25C were from Santa

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Fenouille et al. Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p21Cip1 ⁄ Waf1, cdk1, phospho-cdk1 (Thr161), cdk4, cdk6, Cyclin D1, Histone H3 and phospho-Histone H3 (Ser10) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against fibronectin was from Sigma (St Louis, MO, USA). Antibodies against Cyclin B1 and Cyclin A2 were from Novocastra (Nanterre, France). Peroxidase-conjugated anti-mouse, anti-rabbit and anti-goat antibodies were from Dakopatts (Trappes, France). The p21Cip1 ⁄ Waf1 promoter construct fused to luciferase (p21Cip1 ⁄ Waf1-luc) was a gift of Dr. C. Goding (Carreira et al., 2005). The p53-responsive reporter (PG13-luc) was provided by Dr. B. Vogelstein (el-Deiry et al., 1993). The expression vector encoding p53 was from Stratagene (Santa Clara, CA, USA). SPARC recombinant protein with maltose binding protein (MBP) was purchased from GenWay Biotech (San Diego, CA, USA). Primers and culture reagents were obtained from Invitrogen (Carlsbad, CA, USA). All other chemicals were obtained from Sigma, unless indicated otherwise.

siRNA transfection The control and Stealth SPARC #686 siRNA duplexes were designed by Invitrogen and described in Figure S1. Human p53 and p21Cip1 ⁄ Waf1 siRNAs were purchased from Santa Cruz Biotechnology. Transfection of siRNA was carried out with Lipofectamine RNAiMAX (Invitrogen), at a final concentration of 50 nM.

Generation of doxycycline-inducible cell lines A375P cells stably expressing small hairpin RNA targeting SPARC (shSPARC) or lacZ (shlacZ) as a control were engineered with the BLOCK-iT inducible H1 RNAi expression system from Invitrogen. A375P cells were transfected with the pcDNA6 ⁄ TR vector encoding the tetracycline repressor, using Amaxa technology. Clones were selected by limited dilution using 10 lg ⁄ ml blasticidin, resulting in the isolation of A375P TRex cell lines. To significantly knockdown SPARC expression, we constructed three vectorbased shRNA, targeting different regions of SPARC mRNA (Figure S1A). The oligonucleotide sequences for the three SPARC shRNA were ligated into pENTR ⁄ H1 ⁄ TO behind a doxycyclineinducible H1 promoter. A shRNA sequence against lacZ gene was used as control. The vectors were tested for inducible knockdown of SPARC in HEK293 TRex cells and one was found to be very effective in suppressing SPARC expression (Figure S1B). The pENTR ⁄ H1 ⁄ TO-shRNA #338 construct was transfected into A375P TRex and clones were selected by limited dilution using 10 lg ⁄ ml blasticidin and 1 mg ⁄ ml zeocin. Antibiotic-resistant cells were isolated and 20 clonal lines were treated with 1 lg ⁄ ml doxycycline and analyzed for SPARC gene knockdown by immunoblotting. The A375P subclones named as #3F9 and #5H9 were chosen for further analysis.

In vivo tumor growth analysis A375P TRex cells (5 · 106) expressing shlacZ or shSPARC (clone #3F9) were subcutaneously inoculated in the left dorsal side of 6-week-old female athymic Nude nu ⁄ nu mice (Harlan, Gannat, France). For induction of the H1 promoter in stable clones, 1 mg ⁄ ml doxycycline was added to the drinking water of mice 2 days before injection, and the water with doxycycline was changed twice a week. Tumor size was assessed using calipers, and volume was calculated according to the formula: tumor volume (in mm3) = tumor width · tumor length2 · 0.5. After 2 weeks, mice were sacrificed and tumors excised, minced, put into liquid nitrogen and stored at )80C. Animal experiments were performed in accordance with relevant institutional regulations; research protocols were approved by relevant authorities.

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Cell viability assay Cell viability was determined by a colorimetric assay that is based on the cleavage of yellow tetrazolium salt (XTT) to form an orange formazan dye by mitochondrial dehydrogenases (Cell Proliferation Kit II; Roche Diagnostics, Indianapolis, IN, USA). The absorbance of the formazan dye was measured at 490 nm. All experimental values were determined from quadruplicate wells.

Cell cycle analysis Cell cycle profiles were determined by flow cytometric analysis of propidium iodide (PI)-stained cells. Briefly, cells were washed, fixed in citrate buffer and incubated at )20C for 1 h. Cells were then stained in buffer containing 40 lg ⁄ ml PI for 1 h at 4C. Cell cycle profiles were collected using a FACScan instrument and analyzed with the CELLQUEST software (Becton-Dickinson, Le Pont de Claix, France).

Protein analysis Cells were harvested and lysed in buffer containing 1% Triton X-100, supplemented with protease inhibitors and phosphatase inhibitors (Roche Diagnostics). Lysates were pelleted, and 50 lg protein was separated by SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and immunoblotted with primary antibodies, followed by peroxidase-conjugated IgG. Immunoreactivity was detected with the enhanced chemiluminescence system (Amersham, Orsay, France). For immunoprecipitation, 1 mg lysate was incubated overnight with 5 lg polyclonal rabbit anti-p21Cip1 ⁄ Waf1 antibody (Santa Cruz Biotechnology) and protein G-Sepharose at 4C. Beads were washed with lysis buffer and immunoprecipitated samples were resolved by SDS-PAGE.

Luciferase assay Cells were transfected with a combination of plasmids encoding firefly luciferase under the control of p53-responsive elements or p21Cip1 ⁄ Waf1 promoter (1.4 lg), and b-galactosidase (0.1 lg) under the CMV promoter, in the presence of the expression vector for p53 or empty vector control (0.4 lg). Luciferase activities were determined with the Luciferase assay system (Promega, Madison, WI, USA). The values were normalized for b-galactosidase readings in each sample. All experimental values were determined from triplicate wells.

Gene array analysis using real-time quantitative PCR (Q-PCR) The expression level of 90 genes related to p53-mediated signal transduction, growth arrest and apoptosis was evaluated from 2 lg cDNA by real-time Q-PCR using an ABI Biosystems 7900HT Sequence Detector System (Applied Biosystems, Carlsbad, CA, USA) and the SYBR Green dye detection protocol as described previously (Bailet et al., 2009). The relative expression level of target genes was normalized for RNA concentrations with four different housekeeping genes (GAPDH, actin, HPRT and ubiquitin) and calculated using the formula DCT(siSPARC))DCT(siCTRL) and expressed as fold over control (2DDCT). Values are the mean of duplicates and are representative of two independent experiments.

Migration and invasion assays Serum-stimulated chemotaxis assays were monitored using modified Boyden chambers containing polycarbonate membranes (8-lm pores, Transwell; Corning, Sigma). Cells were seeded on the upper side of the filters and chambers were placed on 24-well plates containing 10% FBS medium. Cells were allowed to migrate for 5 h at 37C in 5% CO2. Migratory cells on the lower


SPARC depletion promotes G2 ⁄ M cell cycle arrest membrane surface were fixed in 3% paraformaldehyde, stained with 0.1% crystal violet, and counted (five randomly per well). For invasion assays, the upper side of the filter was coated with 5 lm ⁄ ml Matrigel. Cells were allowed to migrate for 18 h and stained as described above.

Statistical analysis Unless stated otherwise, experiments shown were representative of at least three independent experiments. All data were presented as means ± standard deviation (SD). Statistical analysis was performed using the Student t-test with P < 0.05 deemed statistically significant.

Acknowledgements This work was supported by INSERM and ARC grant 1136. S. Tartare-Deckert is a recipient of a Contrat d’Interface Clinique, Service de Dermatologie, CHU de Nice. We thank Dr. M. Herlyn for melanoma cell lines, Dr. C. Goding and Dr. B. Vogelstein for plasmids, and Dr. G. Ponzio and Dr. S. Grosso for helpful discussions and technical advice.

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Supporting information Additional Supporting Information may be found in the online version of this article: Figure S1. Validation of shRNA sequences targeting human SPARC. Figure S2. Depletion of SPARC causes a G2 ⁄ M cell cycle arrest in melanoma cells. Figure S3. Effect of conditional RNAi-mediated depletion of SPARC on cell number and cell cycle progression. Figure S4. Modulation of cell cycle regulators by SPARC siRNA treatment. Figure S5. p53 is required for the G2 ⁄ M cell cycle arrest induced by SPARC depletion. Figure S6. Depletion of SPARC does not modulate MITF levels in melanoma cells. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
