Wilms’ Tumor Gene 1 (WT1) Silencing Inhibits Proliferation of Malignant Peripheral Nerve Sheath Tumor sNF96.2 Cell Line

Wilms’ tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. WT1 expression has been variably reported in both benign and malignant peripheral nerve sheath tumors (MPNSTs) by means of immunohistochemistry. The aim of the present study was to characterize its potential pathogenetic role in these relatively uncommon malignant tumors. Firstly, immunohistochemical analyses in MPNST sNF96.2 cell line showed strong WT1 staining in nuclear and perinuclear areas of neoplastic cells. Thus, we investigated the effects of silencing WT1 by RNA interference. Through Western Blot analysis and proliferation assay we found that WT1 knockdown leads to the reduction of cell growth in a time- and dose-dependent manner. siWT1 inhibited proliferation of sNF96.2 cell lines likely by influencing cell cycle progression through a decrease in the protein levels of cyclin D1 and inhibition of Akt phosphorylation compared to the control cells. These results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. Thus, WT1 is suggested to serve as a potential therapeutic target for MPNSTs.


Introduction
Malignant peripheral nerve sheath tumor (MPNST) is an aggressive and rare type of sarcoma, usually arising from peripheral nerves. They can occur sporadically or more frequently (up to 50% of cases) from pre-existing neurofibromas in the context of Neurofibromatosis type 1 (NF1) [1], representing the major cause of mortality in this syndrome [2][3][4][5]. Nevertheless its pathogenesis is poorly understood. Although genes involved in regulating the cell cycle and growth signal transduction have been reported to be deregulated mainly in MPNST [6][7][8], there is still an urgent need to identify other molecular actors in order to plan new therapeutic approaches.
WT1 expression has been also reported in various neuroepithelial tumors including peripheral nerve sheath tumors (neurofibromas and schwannomas) [45][46][47]. By real-time RT-PCR, WT1 overexpression has been described also in MPNST [28] even if correlation with grade of malignancy has not been defined [45].
Recently, functional in vitro studies showed that WT1 silencing through an antisense oligomer results in growth inhibition in different cancer cell lines, including breast [48][49], lung [50], melanoma [51][52], glioblastoma [53][54], as well as various types of solid tumors [55] cell lines. Moreover, WT1 silencing reduced in vivo the number and growth of visible metastatic tumor foci in the lungs through aerosol delivery of PEI-WT1 RNAi complexes [56].
As the role of WT1 in MPNST is not established, reducing its level by specific RNA interference (RNAi) in established MPNST cell line is helpful for a better understanding of its role in the pathogenesis of these tumors.
In this study, we used a human MPNST cell line (sNF96.2) to investigate whether WT1 silencing by RNAi is capable to suppress the growth of this cell line. Moreover, to understand the molecular mechanisms by which WT1 plays its role, some pathways involved in the regulation of cell cycle were examined. The results show that RNAi directed against the human WT1 gene inhibited effectively the WT1 expression and suppressed the growth of the MPNST cells in both dose-and time-dependent manner. This effect occurs through the down-regulation of PI3K/ Akt/cyclin D1 signaling pathway, regulating cell cycle progression, cell proliferation and cell transformation.
Cells were then washed with PBS for 20 min, followed by a 1-hour incubation with the appropriate fluorescent dye-conjugated secondary antibody. Chromosomal DNA was stained with 49, 6-diamidino-2-phenylindole (DAPI) and imaging was performed with a Leica fluorescence microscope connected to a digital camera (Spot, Diagnostic Instruments, Sterling Heights, USA) and adjusted for contrast in Corel Draw version 9.
4˚C. The proteins were extracted with a lysis buffer (10 mM Tris-HCl plus 10 mM KCl, 2 mM MgCl 2 , 0.6 mM PMSF and 1% SDS, pH 7.4) enriched with protease and phosphatase inhibitor cocktail tablets (Roche Applied Science). For the subcellular fractionation, aliquots of 10 6 cells were suspended in 150 ml of buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonylfluoride), incubated on ice for 15 min and homogenized by 15 passages through a 25 gauge needle, followed by centrifugation at 12000 rpm for 40 s at 4˚C. The supernatants were collected and stored as a cytoplasmic fraction, whereas the pelleted nuclei were washed in 70 ml of buffer A and re-suspended in buffer B (20 mM Hepes, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride) supplemented with 1X of protease inhibitor cocktail (Roche Applied Science). After 30 min incubation on ice, the nuclear extracts were collected by centrifugation at 12000 rpm for 5 min. The extracts were rapidly frozen and stored at 280˚C until processed for Western blot. Before freezing, the protein concentration was estimated using the bicinchoninic acid assay (Pierce).

Cell proliferation assay
After transfection, siRNA-transfected cells were harvested at specific time points. The total viable cell number was assessed by trypan blue exclusion assay and counted by a hemacytometer under an inverted microscope (Leica).

Statistical Analysis
In this study, the results are expressed as the means ¡ standard deviation (SD). All experiments were repeated at least three times. Statistical significance was determined by the two-tailed Student's t-test, and P-values,0.05 were considered to indicate statistically significant differences.

Cell morphology analysis and immunocytochemistry
To investigate the WT1 localization at cellular level, MPNST sNF96.2 cell line was used (ATCC) [57][58]. Cells had spindle-shaped morphology and were immunopositive for S-100 indicating Schwann cell lineage.
For immunocytochemistry two different antibodies directed against the Cterminal (C- 19, sc-192,) or the N-terminal (clone 6F-H2) portion of the WT1 molecule, respectively, were used. Both antibodies showed similar immunoreactivity even if the specificity of antibody against the N-terminal portion (clone 6F-H2) was higher than WT1 C-19 antibody (Fig. 1). The results revealed that WT1 protein is strongly expressed in the nucleus and in the cytoplasmic area around the nucleus (Fig. 1). To confirm the intracellular distribution of WT1, cellular proteins were separated into nuclear and cytoplasmic fractions using the total cellular lysate as a control. Western blot analysis revealed that WT1 was predominantly located in the nuclear fraction compared to cytoplasmic one, which showed a very weak expression of WT1 (Fig. 2).

Effects of siRNA WT1 on apoptosis
To test the effect of WT1 silencing on apoptosis, we examined the activity of caspases 3, which are effector of the apoptotic pathway. Therefore, we used Western blot analysis to measure the expression of active caspase 3 in sNF96.2 cells treated with siWT1 compared to negative control (Fig. 5). We found that the cleaved caspase-3 expression did not change at 50 nM siWT1 after both 48 and 72 hours of treatment (Fig. 5).

Effects of siRNA WT1 on cell cycle
In order to assess the influence of siRNA WT1 on cell cycle, we analyzed the PI3K/ Akt/cyclin D1 signaling pathway, which regulates cell cycle progression and is implicated in the cell proliferation and transformation [59][60]. Therefore, we used Western blot analysis to measure the expression of PI3K, AKT/pAKT and Cyclin D1 in sNF96.2 cells treated with siWT1 compared to negative control (  Fig. 6). At 50 nM siWT1 the expression of PI3K, pAKT and Cyclin D1 proteins slightly decreased after 48 hours to reach a maximum decrement after 72 hours of treatment (Fig. 6). Discussion WT1 involvement in human cancer is very complex, acting as a tumor suppressor in some contexts and as an oncogene in others [61]. Its variable involvement in a  siRNA WT1 in MPNST Cell Line large series of tumors is likely due to the complex nuclear/cytoplasmic roles played by WT1 [13,62]. In fact, besides to the well-known role in transcriptional regulation, WT1 is likely involved in RNA metabolism, translational regulation and association with translating polysomes [62]. The different facets of WT1 are in line with the different nuclear/cytoplasmic expression patterns detected in various tumors by using antibodies directed against the C-terminal portion (WT1 C- 19) or the N-terminal portion (WT1 clone 6F-H2) of the molecule [17,29].
In this study, the expression profile of WT1 in MPNST cell line and the effect of siWT1 on cell growth suggest an articulate work planning performed by WT1 in this specific malignancy.
We showed firstly that WT1 is expressed predominantly in nuclear and perinuclear areas and weaker in the cytoplasm of MPNST cells. These results are in line with WT1 involvement in diverse cellular activities and variable behavior due to the fact that it regulates many genes and it can be modulated by a number of cofactors [13,63]. Of particular interest is the relationship with actin, described in both nucleus and cytoplasm [64]. Notably, perturbation of the actin cytoskeleton is essential for malignant transformation and WT1 was named as one of the proteins implicated in actin cytoskeletal changes in cancer cells [64][65]. WT1 might be a specific adaptor protein that links a specific subset of mRNAs to actin for transporting to the target location and, in turn, actin may act as a cytoplasmic anchor for WT1 [64]. Again, Rong [66] described an intriguing interaction of WT1 with Signal transducers and activators of transcription 3 (STAT3), which is overexpressed or constitutively activated in a variety of human malignancies. Synergistically overexpression of WT1 and STAT3 in tumor development, including Wilms' tumor, increases the expression level of STAT3 target genes, including cyclin D1 and Bcl-xL, resulting in an advantage of cell proliferation. It is noteworthy that the locations of STAT3 and WT1 protein in primary Wilms' tumor cells were found mostly located in the nucleus compared to prevalent cytoplasm expression in control normal cells near the tumor [66]. The strong WT1 expression in nuclear compartment of MPNST cell line suggests a similar model in this neoplasm. In support of this data, recently it has been showed that EGFR-STAT3 pathway is necessary for MPNST transformation as demonstrated by the results that STAT3 knockdown by shRNA prevented MPNST formation in vivo, and pSTAT3 fall in vivo by reduced EGFR activity [67]. Finally, the concentration of WT1 in the perinuclear zone of MPNST cell lines is consistent with its function close to the nucleus to act in nuclear-cytoplasmic shuttling under appropriate conditions [62]. It is evident that the field of action of WT1 is very broad. Further studies are required to identify the interaction partners of WT1 and to explore the functional relevance of such cooperation for planning new experimental approaches.
In this study we further investigated the significance of WT1 expression in MPNST cell line by WT1 silencing experiments. WT1 knockdown performed in different cancer lines showed to impede cell proliferation and viability by a multitude of mechanisms. Firstly, WT1 downregulation induced mitochondrial damage and resultant apoptosis in different solid tumors [50][51][52]55]. Numerous studies have demonstrated that WT1 regulates apoptosis by targeting directly or indirectly bcl-2 family members, including the pro-apoptotic family members Bak and Bax, and the anti-apoptotic family member Bfl-1/A1 critically depending on cell lineage analyzed and specific WT1 isoform acting [68]. Differently, WT1 silencing in glioblastoma causes decreased viability by IFG-1R overexpression, which causes a non-apoptotic, non-autophagic programmed cell death termed ''paraptosis'' [54]. Silencing of WT1 causes decreased proliferation and viability in most cancer cell lines including K562 and MM6 leukemia [69], MCF-7 breast cancer [48][49], A549 lung cancer [50], B16F10 melanoma lung metastasis [51], and U251MG human multiform glioblastoma [53]. Accordingly, in MPNST cell line we showed that silencing of WT1 effectively inhibited WT1 protein expression. While activation of apoptosis was not shown, we found a reduced proliferative capacity likely due to WT1 effect on cell cycle progression through a decrease in the protein levels of the key components of PI3K/Akt/Cyclin D1 pathway. The result is in line with previous data showing WT1 effects on the cell cycle both directly and indirectly by regulating genes involved in cell cycle regulation [70][71][72][73]. PI3K/Akt/Cyclin D1 pathway is now recognized as one of the most important pathways in regulating cell survival and proliferation [74]. Activation of PI3K, an intracellular signal transducer enzyme, can phosphorylate phosphatidylinositol 4,5-biphosphate (PIP2) into phosphatidylinositol 3,4,5triphosphate (PIP3). As a second messenger, PIP3 recruits Akt to the cell membrane where Akt is fully activated by phosphorylation at position Ser473 [75]. After activation, Akt translocates to the cytoplasm and nucleus to phosphorylate its substrates and promote cell proliferative and survival signals through the upregulation of cyclinD1 [76][77][78][79]. In conclusion, our results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. In models of neuronal differentiation, it has been proposed that WT1 could maintain cells in an undifferentiated state [22,53,80]. In this way, silencing of WT1 has been suggested to promote a more differentiated phenotype of astrocytoma cells with a lower proliferative capacity [53]. Likewise, maintaining Schwann cells in more a undifferentiated state might be caused by WT1 in vivo overexpression in human MPNSTs as suggested by the in vitro growth inhibition and reduced cyclin D1 protein levels, caused by WT1 silencing and the expression profile during peripheral nervous system development where the tumor is found.
In conclusion, the present study showed that in MPNST, at least in vitro, WT1 acts as an oncogene rather than a tumor suppressor. It is noteworthy that, AKT and PI3K pathways were recently found to be highly activated in MPNST cell lines so that AKT activation blockade, either by inhibition of the PI3K upstream or directly through AKT inhibitors, may potentially be pursued as a systemic anti-MPNST approach [81][82]. Our results suggest that, WT1, intimately influencing pAKT/Cyclin D1 pathway, also could be tested as potential agent for a genetargeted therapy approach for the treatment of MPNSTs, which represent a human inauspicious disease without still effective targeted therapies.

Author Contributions
Conceived and designed the experiments: RP GM. Performed the experiments: