Notch Signaling Activation Suppresses v-Src-Induced Transformation of Neural Cells by Restoring TGF-β-Mediated Differentiation

Background We have been investigating how interruption of differentiation contributes to the oncogenic process and the possibility to reverse the transformed phenotype by restoring differentiation. In a previous report, we correlated the capacity of intracellular Notch (ICN) to suppress v-Src-mediated transformation of quail neuroretina (QNR/v-srcts) cells with the acquisition by these undifferentiated cells of glial differentiation markers. Methodology/Principal Findings In this work, we have identified autocrine TGF-β3 signaling activation as a major effector of Notch-induced phenotypic changes, sufficient to induce transition in differentiation markers expression, suppress morphological transformation and significantly inhibit anchorage-independent growth. We also show that this signaling is constitutive of and contributes to ex-vivo autonomous QNR cell differentiation and that its down-regulation is essential to achieve v-Src-induced transformation. Conclusions/Significance These results support the possibility that Notch signaling induces differentiation and suppresses transformation by a novel mechanism, involving secreted proteins. They also underline the importance of extracellular signals in controlling the balance between normal and transformed phenotypes.


Introduction
Differentiation is a multi-step process resulting from a cascade of alternate activation and extinction of tissue-specific signaling pathways. Progression through this cascade is generally mediated by a sequence of extracellular signals, initiated by the differentiating cells themselves or by their environment. As a consequence of oncogenic events, this process is often interrupted and differentiating cells can no longer exit the cell cycle. Hence, blocking differentiation constitutes an important step in neoplastic transformation. However, understanding its contribution can be achieved only through the use of adequate experimental models. The rationale for a differentiation therapy is based on the assumption that cancer cells have retained the potential to respond to appropriate differentiation signals, which in turn would be sufficient to restore a normal phenotype. This could be achieved either because the transformed cells undergo growth arrest or because they no longer respond to oncogenic stimuli. Developing models for the latter would be useful to identify pathways essential for transformation and possibly result in new therapeutic approaches. However, the task of reverting a cancer cell to its normal state, in response to differentiation signals, has been only reached in a very few clinical or experimental instances (for review, [1]).
We previously reported that stable expression of Notch intra cellular domain (ICN) suppresses transformation of embryonic quail neuroretina (QNR) cells induced by a temperature sensitive v-Src (QNR/v-src ts ), without altering oncoprotein expression nor its downstream signaling activity. This remarkable phenotypic change is correlated with a differentiation switch, as these undifferentiated transformed cells acquire markers of glial cells [2]. Several reasons support the choice of this ex-vivo model to study how activation of differentiation signals could result in transformation suppression. QNR cells dissected from 7-day old embryos progressively cease to divide and autonomously execute glial and neuronal differentiation programs [2][3][4]. As a consequence of v-Src activity, they acquire sustained proliferative capacity [5,6], display all characteristics of oncogenic transformation [7,8] and repress their autonomous differentiation potential [2,9,10].
We have selected Notch as an instructive signal, because of its important contribution to neuroretina development. At early stages, it maintains progenitor cells in an undifferentiated state by inhibiting their neuronal differentiation [11], whereas at later stages it promotes glial differentiation [12,13]. We were also interested in this signaling pathway because of its dual contribution to either oncogenesis or tumor suppression, depending on the cell model (for review, [14]). We also showed that both suppression of transformation and switch in differentiation markers expression were mediated by its transcription factor partner, CBF [2]. Therefore, these results demonstrated that activating differentiation signals was sufficient to abolish cell response to oncogenic stimuli, thus lending further experimental basis to the differentiation therapy concept.
Our previous work also indicated that interference of constitutive Notch signaling with transformation possibly involved a secreted factor(s). Culture medium from revertant cells, stably expressing the Notch intracellular domain (QNR/v-src ts /ICN) or an activated human CBF (RBPJ-k), contains a paracrine activity which inhibits transformation of QNR/v-src ts cells [2]. This suggested that secreted factors could play a key role at the cross-roads between transformation and differentiation, in this cell system. Therefore, we undertook to identify this activity and investigate its possible autocrine effect on QNR cell transformation and differentiation.
In this report, we identified autocrine activation of TGF-b3 signaling as a major effector of the phenotypic changes induced by ICN signaling, sufficient to suppress transformation of QNR/vsrc ts cells and promote their acquisition of glial differentiation markers, in presence of an active oncoprotein. We also show that this signaling is activated during QNR cell ex-vivo differentiation and that its down-regulation by v-Src is essential to block differentiation and achieve transformation. Taken together, our results provide a potentially novel mechanism by which Notch signaling suppresses oncogenic transformation. They also underline the importance of extracellular signals in maintaining the balance between the normal and transformed phenotypes.

TGF-b3 mRNA is upregulated in QNR/v-src ts cells stably expressing ICN
To understand the mechanisms by which Notch signaling activation suppressed cell transformation, we compared the transcription profile of QNR cells transformed by a v-src mutant encoding a temperature sensitive (ts) oncoprotein (QNR/v-src ts ), with that of cells stably expressing ICN (QNR/v-src ts /ICN). mRNA were prepared from cells maintained at permissive (37uC) or restrictive (41uC) temperature. For each temperature, cDNA from QNR/v-src ts cells were probed with that of QNR/v-src ts /ICN cells, on microarrays spotted with 13,000 cDNA from chicken EST collections [15]. This analysis revealed 209 genes displaying, at least, a 1.5 fold increase in QNR/v-src ts /ICN, as compared to QNR/vsrc ts cells. As we previously showed that suppression of QNR/v-src ts cell transformation possibly involved a secreted factor(s) [2], we were primarily interested in genes encoding potentially secreted proteins. Among 18 such genes, we identified, as a most highly induced one, the gene encoding the TGF-b3 ligand, the expression of which was increased 8 to 16 times in QNR/v-src ts /ICN cells. We focused our study on this protein, because of its important role during oncogenesis (for review, [16]).
Gene array results were validated by QPCR experiments. These experiments showed a 13-time induction of normalized TGF-b3 mRNA levels in QNR/v-src ts /ICN cells, compared to QNR/vsrc ts cells at 37uC. At 41uC, TGF-b3 mRNA amounts were 10 times higher in QNR/v-src ts /ICN cells than in QNR/v-src ts cells, at the same temperature (Fig. 1). However, in QNR/v-src ts /ICN cells, transcript levels were more elevated at 41uC than at 37uC. A similar difference was observed in QNR/v-src ts cells, suggesting that TGF-b3 mRNA levels were regulated by v-Src activity. Increased levels of TGF-b3 mRNA were also found in QNR/vsrc ts cells stably expressing a constitutively activated human RBP-Jk, indicating that this increase was mediated by the ICN/CBF complex (data not shown). Therefore, it was likely that the presence of higher TGF-b3 mRNA levels would lead to an activation of TGF-b signaling in QNR/v-src ts /ICN cells.

QNR/v-src ts /ICN cells secrete and respond to mature TGF-b3 activity
We first investigated whether mature TGF-b was secreted by QNR/v-src ts /ICN cells. Therefore, we collected cell-free medium from these cells (ICN medium) maintained at 41uC (v-Src inactive) and from cells transferred to 37uC (v-Src active) for 72 hours, and examined its capacity to activate phosphorylation of the TGF-b signaling effector Smad2 [17], in QNR/v-src ts cells. Control medium was obtained from transformed QNR/v-src ts cells (v-Src medium), maintained at either temperature. Phosphorylated Smad2 was barely detectable in QNR/v-src ts cells cultured at 37uC and was not increased when these cells were treated with their own medium collected at either temperature ( Fig. 2A), ruling out both TGF-b signaling activation in transformed cells and TGF-b release by these cells at detectable levels. In contrast, incubating QNR/v-src ts cells in ICN medium, collected at either temperature, markedly increased the levels of phosphorylated Smad2. Furthermore, in agreement with our comparative Q-PCR results, medium collected at 41uC was significantly more efficient in activating Smad2 phosphorylation ( Fig. 2A).
To confirm that this increase in Smad2 phosphorylation was due to TGF-b activity, we treated QNR/v-src ts cells with ICN medium in presence of the TGF-bRI kinase inhibitor, SB431542 [18], at concentrations ranging between 0.25 and 10 mM. We observed a dose dependent reduction of Smad2 phosphorylation in presence of the inhibitor (Fig. 2B). QNR/v-src ts and QNR/v-src ts /ICN cells were incubated 72 hrs at 37uC or 41uC before extraction of total RNA. After reverse transcription of 1 mg of RNA, TGF-b3 cDNA was amplified by QPCR using specific primers ( In agreement with transcriptome analysis, we detected, by Western blotting, the presence of a specific mature TGF-b3 band, migrating at about 25 kDa, in ICN medium, collected at either temperature (Fig. 2C). This band was present in higher amounts in ICN medium collected at 41uC and was not found in v-Src medium collected under either temperature condition. To confirm the contribution of TGF-b3 to Smad2 phosphorylation, we incubated QNR/v-src ts cells with ICN medium, in the presence of increasing amounts of TGF-b3 blocking antibody. In agreement with SB431542 treatment, the levels of phosphorylated Smad2 were decreased as a function of antibody concentration (Fig. 2D), indicating that Smad2 phosphorylation was essentially due to secreted TGF-b3.
Finally, we examined the activation of TGF-b signaling in QNR/ v-src ts /ICN cells by measuring endogenous Smad2 phosphorylation in presence or absence of an active v-Src. Phosphorylated Smad2 was detected in QNR/v-src ts /ICN cells at 37uC and at higher levels at 41uC (Fig. 2E). In both cases, the presence of phosphorylated Smad2 was sensitive to that of the SB431542 inhibitor ( Fig. 2E), indicating that activation of TGF-b signaling resulted from an autocrine response of these cells to mature TGF-b3 activity.
TGF-b signaling was previously shown to directly regulate TGF-b3 transcription [19]. Therefore, we examined whether activation of this pathway in QNR/v-Src ts /ICN cells could be associated with an auto-regulation of TGF-b3 expression. We found that treatment of QNR/v-Src ts cells with recombinant TGF-b3 induced an increase of TGF-b3 mRNA levels (Fig. 3A). Conversely, inhibition of TGF-b signaling in QNR/v-Src ts /ICN cells resulted in a decrease of TGF-b3 mRNA amounts (Fig. 3B). Therefore, it appears that TGF-b3 mRNA induction results in both mature TGF-b3 secretion and autocrine signaling activation, which in turn initiates a positive feed-back control maintaining the levels of TGF-b3 mRNA in QNR/v-src ts /ICN cells.

TGF-b signaling suppresses v-Src-induced morphological transformation by restoring cytoskeleton organization
To determine whether suppression of morphological transformation was due to TGF-b3 activity, we examined the effects of recombinant TGF-b3 on cell morphology. QNR/v-src ts cultures, maintained at 37uC, are typically composed of refractile round or fusiform transformed cells. Their morphology was markedly changed within 24 hours, following treatment with 1-10 ng/ml of recombinant TGF-b3 (Fig. 4 a-c). Cells became flat, transparent with a polygonal, epithelial like morphology. The extent of these changes reflected a major cytoskeleton reorganization. Transformation of QNR/v-src ts cells is defined by the loss of actin stress fibers (Fig. 4 d). In contrast, the cytoskeleton of TGF-b3 treated cells became well organized, with abundant stress fibers ( Fig. 4

e, f).
QNR/v-src ts /ICN cells maintained at 41uC exhibit a normal morphology. When transferred to 37uC, they retain an organized cytoskeleton underlined by the presence of numerous stress fibers [2]. We investigated whether autocrine TGF-b signaling was responsible for maintaining their normal morphology, in presence of an active oncoprotein. Treatment of these cells with increasing concentrations of SB431542 induced morphological changes, including a marked increase in the number of refractile cells extending processes (Fig. 5 a-e). Actin fibers became thinner and Figure 2. QNR/v-src ts /ICN cells secrete and respond to mature TGF-b3. (A) Media were collected after a 30-hour incubation of QNR/vsrc ts (v-src ts medium) and QNR/v-src ts /ICN (ICN medium) cells at 37uC or 41uC. QNR/v-src ts cells were incubated at 37uC in normal medium (BME) or conditioned media for 1 hour, before lysis. Phosphorylated Smad2 was detected by western-blot. b-actin was used for protein level normalization. (B) QNR/v-src ts cells were treated at 37uC with increasing concentrations of SB431542 inhibitor for 2 hrs and then incubated in ICN medium during 1 hour in presence of inhibitor. Effects of this treatment on Smad2 phosphorylation were analyzed by western-blot. (C) Comparison of mature TGF-b3 levels in v-Src and ICN media. Cells were plated at 2.10 6 cells per 100 mm dish at 37uC or 41uC. One day after plating, growth media were replaced with serum-free BME. After a 30-hour incubation, media were collected and concentrated 50 times using Vivaspin6 columns. 20 mg of proteins were migrated under nondenaturing conditions. Mature TGF-b3 migrates as 25 kDa band. (D) QNR/v-src ts cells were treated, one hour before lysis, with ICN medium preincubated at 37uC for one hour with increasing amounts of neutralizing antibodies directed against TGF-b3. Levels of Smad2 phosphorylation were analyzed by western-blot. Erk was used to normalize protein loading. (E) Levels of phosphorylated Smad2 in QNR/ v-src ts /ICN cells treated at 37uC or 41uC with DMSO or 10 mM of SB431542 inhibitor for 24 hrs. doi:10.1371/journal.pone.0013572.g002 less organized in treated cells (Fig. 5 h-j). These changes were not detected in control cells treated with DMSO ( Fig. 5 f, g). Similar treatment of QNR/v-src ts cells had no detectable effect on their morphology (data not shown). These results indicated a dominant interference of TGF-b signaling with cytoskeleton organization, to restore a normal cell morphology.
TGF-b signaling suppresses morphological transformation by increasing a2-actin expression and Myosin Light Chain phosphorylation TGF-b activity was previously shown to interact with signaling pathways targeting cytoskeleton components (for review [20]). Accordingly, our comparative RNA analysis showed a 25-fold increase of a2-actin mRNA in QNR/v-src ts cells stably expressing ICN, as compared to v-Src-transformed cells. This was validated by QPCR (Fig. 6A), suggesting that TGF-b signaling contributes to cytoskeleton restoration, in part by increasing the abundance of a-actin fibers. Moreover, treatment of QNR/v-src ts cells with recombinant TGF-b3 significantly increased the levels of a2-actin mRNA, as determined by QPCR (Fig. 6B). Conversely, treatment of QNR/v-src ts /ICN cells with SB431542 resulted in a decreased a2-actin expression (Fig. 6C), indicating a significant contribution of TGF-b signaling in regulating the levels of this mRNA in ICN containing cells.
We previously correlated suppression of morphological transformation, in QNR/v-src ts /ICN cells, with an increase of Myosin Light Chain (MLC) phosphorylation, as compared to v-Src transformed cells [2]. To assess the contribution of TGF-b signaling to this phosphorylation, we treated QNR/v-src ts cells with increasing doses of recombinant TGF-b3. This treatment increased the levels of MLC phosphorylation in QNR/v-src ts cells, as compared to untreated cells (Fig. 6D).
Taken together, these results indicate that TGF-b signaling contributes to cytoskeleton reorganization both by increasing expression of a2-actin and favoring its polymerization into stress fibers by inducing MLC phosphorylation [21].
Autocrine TGF-b signaling contributes to anchorageindependent growth inhibition of QNR/v-src ts /ICN cells In addition to morphological transformation, v-Src transformed QNR cells display anchorage-independent growth capacity [8], a characteristic marker of ex-vivo oncogenic transformation. QNR/vsrc ts cells efficiently give rise to colonies in soft agar containing medium, whereas QNR/v-src ts /ICN cells fail to divide under the same conditions. Moreover, treatment of QNR/v-src ts cells with ICN medium also inhibits their anchorage-independent growth capacity [2]. We investigated whether autocrine TGF-b activity was also responsible for the loss of anchorage-independent growth by QNR/v-src ts /ICN cells. Therefore, we tested their capacity to form colonies in soft agar containing medium, in presence or absence of the SB431542 inhibitor. We found that while untreated cells could not give rise to colonies, their anchorage-independent growth capacity was restored when TGF-b signaling was inhibited. However, this restoration was only partial, as the number and size of colonies were smaller than those observed in QNR/v-src ts cells (Fig. 7).
Therefore, activation of autocrine TGF-b signaling, following ICN stable expression, is predominantly responsible for suppressing v-Src-induced transformation of QNR cells, as defined by the loss of both morphological transformation and anchorageindependent growth capacity.
TGF-b3 signaling is required to induce and maintain expression of glial markers in QNR/v-src ts /ICN cells We previously showed that primary dissociated QNR cultures are composed of a mixture of progenitor and differentiated cells. Accordingly, they contain neuronal and glial markers, indicating that they can autonomously proceed into differentiation (32). As a result of transformation, this process is interrupted, as the vast majority of QNR/v-src ts cells express only the marker combination of progenitor cells. In QNR/v-src ts /ICN cells, the loss of precursor cell markers was combined with the acquisition of neuroretina glial cell markers, indicating a switch in differentiation program (32).
To determine the contribution of TGF-b signaling to these changes, we treated QNR/v-src ts cells with recombinant TGF-b3 and examined the relative expression of two markers: Pax6, predominantly present in progenitor and transformed cells [2] and glutamine synthetase (GS), a marker of mature glial cells [22]. We observed a marked decrease of Pax6 protein levels together with an induction of GS expression, in treated cells (Fig. 8A). Thus, activation of TGF-b signaling is sufficient to inverse the balance of differentiation markers in QNR/v-src ts cells and recapitulates the opposite evolution of Pax6 and GS markers, as observed both during QNR cell differentiation and in QNR/v-src ts /ICN cells [22,23].
We next examined whether autocrine TGF-b signaling was responsible for inducing and maintaining glial like differentiation of QNR/v-src ts /ICN cells. Therefore, we treated these cells with SB431542, once ICN stable expression was achieved, and examined whether they could be induced to reexpress Pax6. We found that while the number of nuclear Pax6 positive cells was initially low in these cultures, the proportion of cells containing this marker markedly increased, following TGF-b signaling inhibition (Fig. 8B). Conversely, GS expression was suppressed in treated cells (data not shown). These results indicate that autocrine activation of TGF-b signaling, as a consequence of ICN stable expression, is effective in upsetting the balance of markers expression in QNR/v-Src ts cells.

TGF-b3 mRNA levels increase during ex-vivo QNR cell differentiation
We then examined whether TGF-b3 activation in QNR/v-src ts / ICN cells was merely due to ectopic ICN expression or whether it belonged to restoration of the QNR ex-vivo differentiation program, as a consequence of Notch signaling. Therefore, we investigated the presence of TGF-b3 mRNA in QNR cells dissected from 7 day-old embryos (E7) and cultured for various time lengths. As mentioned above, these cells undergo an intrinsic differentiation program defined by the progressive loss of precursor cell markers and the acquisition of glial and neuronal cell markers [2][3][4]. We found that,  while TGF-b3 mRNA was barely detectable in undifferentiated precursor cells one day after dissection (E7+1), its level progressively increased between 8 (E7+8) and 18 (E7+18) days, thereafter (Fig. 9A). These (E7+18) long term cultures essentially contain cells expressing only markers of glial differentiation (Fig. 9C). This indicated that steady state levels of TGF-b3 mRNA autonomously increase in cultured QNR cells and that this increasing parallels the course of their ex-vivo differentiation.

v-Src-mediated transformation correlates with inhibition of ex-vivo differentiation and TGF-b3 down-regulation
Results described above indicated that TGF-b3 expression was down-regulated in both QNR progenitors and undifferentiated transformed QNR/v-Src ts cells. The latter may simply reflect the possibility that precursor cells constitute a preferential target for v-Src-induced transformation and are subsequently maintained in their undifferentiated state, as a consequence of their sustained division. To determine whether v-Src activity would affect differentiation markers and TGF-b3 expression once established, we infected (E7+18) QNR cells, with RSVts68 and selected for transformed cells by passaging. While Pax6, a marker of precursor QNR cells, was no longer detected in (E7+18) uninfected cells (Fig. 9B a), the great majority of transformed cells reexpressed Pax6 (Fig. 9B b). In turn, the number of Pax6 positive cells progressively declined between 7 and 15 days, following v-Src thermal inactivation (Fig. 9B c,d), as observed during intrinsic ex-vivo differentiation (data not shown). Conversely, the GS glial marker, present in (E7+18) QNR cells, was no longer found in transformed cells, but became again detectable following v-Src inactivation (Fig. 9C). We also found that the level of TGF-b3 mRNA was markedly reduced in transformed cells, as compared to that of uninfected cells (Fig. 9D), and progressively resumed following v-Src inactivation at 41uC (Fig. 9E). To determine the contribution of TGF-b signaling in regulating expression of these two markers, we treated (E7+18) QNR transformed cells with SB431542, prior to their transfer to the nonpermissive temperature and maintained TGF-b signaling inhibition during 15 days. This resulted in delaying the autonomous downregulation of Pax6 (Fig. 9B, e-g) and reexpression of GS (Fig. 9C).
Taken together, these results indicate that v-Src-mediated transformation of QNR cells, already displaying characteristics of glial cells, leads to repression of the GS marker and, interestingly, to the reemergence of undifferentiated cells expressing the Pax6 precursor marker. Conversely, oncoprotein inactivation allows these cells to resume their autonomous differentiation. Moreover, concomitant down-regulation of TGF-b3 expression appears to be strictly correlated with transformation of QNR cells and partially mediate the differentiation block. Therefore, concomitant arrest of differentiation and down-regulation of TGF-b3 appear essential to achieve transformation of QNR/vsrc ts cells. Finally, they suggest that TGF-b3 expression, which is restored in QNR/v-src ts /ICN cells, is constitutive of and effects their switch toward glial like cells.

Discussion
We have been investigating how blocking differentiation contributes to the oncogenic transformation of neural cells and the possibility to activate instructive signals, as a mean to reverse the oncogenic process. In this work, we reported that v-Src-  mediated transformation of QNR cells is strictly correlated with interruption of differentiation and maintenance of transformed cells in an undifferentiated state. In addition, transformation can be suppressed by restoring endogenous signaling pathways involved in determining cell identity. We identified autocrine activation of TGF-b3 signaling as a key effector of ICN-mediated switch in expression of differentiation markers and suppression of transformation.
Ex-vivo differentiating QNR cells undergo phenotypic changes that reflect the execution of an intrinsic program, characterized by the progressive loss of precursor cell markers (e.g. Pax6) and the acquisition of neuronal and glial differentiation markers [2][3][4]. Long term cultures become essentially composed of flat epithelial like cells, expressing only markers of glial differentiation (e.g. GS and vimentin). We showed that a marked increase in TGF-b3 mRNA steady state levels is part of this autonomous process and partially contributes to its execution. However, these quiescent QNR cultures display remarkable plasticity, as they can be transformed by v-Src, reenter the cell cycle and reexpress Pax6. Upon v-Src ts thermal inactivation, their capacity to differentiate is again restored. Switching up and down the ratio of Pax6 to GS markers, depending on v-Src activity, correlates with down or upregulation of TGF-b3 expression. Thus, in this cell system, concomitant down-regulation of differentiation markers and TGF-b3 expression proved essential for v-Src-induced transformation. Similar down-regulation of TGF-b3 transcription was reported in v-Src transformed chicken fibroblasts, without further assessment of its contribution to transformation [24]. The mechanisms leading to TGF-b3 down-regulation, as a consequence of v-Src activity, are presently unknown. This contrasts with other cell systems, where v-Src was shown instead to activate TGF-b1 transcription essentially through AP1 sites on its promoter [25] and with reports showing increased TGF-b activity in oncogene transformed cells [26,27].
In contrast with v-Src-transformed cells, TGF-b3 mRNA is upregulated in QNR/v-src ts /ICN cells, once ICN stable expression is established, and is maintained through multiple cell passaging despite the presence of an active oncoprotein. Our results indicate that TGF-b3 expression is regulated at multiple levels. First, TGF-b3 mRNA level in QNR/v-src ts /ICN cells remains partially under v-Src control, as it is significantly higher when the oncoprotein is inactive. This suggests the existence of a dominant negative interference of Notch signaling with v-Src-dependent TGF-b3 down-regulation. Second, increased TGF-b3 expression also appears to be a consequence of Notch signaling per se, as its mRNA is maintained at a significantly higher level in QNR/vsrc ts /ICN as compared to QNR/v-src ts cells, when v-Src ts is thermally inactivated and Notch signaling is acting alone. However, we found that TGF-b3 mRNA levels were not significantly diminished when ICN expression was down-regulated by specific siRNA, in QNR/v-Src ts /ICN cells. Similarly, forced ICN expression in QNR/v-Src ts cells did not increase these levels in transient transfection assays (data not shown). Therefore, TGF-b3 mRNA up-regulation in QNR/v-Src ts /ICN cells is not a direct consequence of ICN expression but is likely to belong to the restoration of their autonomous differentiation program, as observed in normal QNR cells, and is subsequently maintained through a positive feed-back control. Although the mechanism involved in TGF-b3 mRNA up-regulation remains undefined, our work provides the first example of Notch and TGF-b signaling cooperation in suppressing transformation.
Mature TGF-b3 is secreted by QNR/v-src ts /ICN cells and initiates an autocrine loop to activate endogenous TGF-b signaling. Analyzing how this signaling contributes to the phenotypic changes induced by ICN, we have established a strong correlation between its capacity to suppress transformation and induce the switch in differentiation markers. Thus, treatment of precursor like QNR/v-src ts cells with recombinant TGF-b3 is sufficient to convert them into glial like cells, by down-regulating expression of Pax6 and inducing that of GS. This is concomitant with the loss of their transformed phenotype. Our data also point to a major role of TGF-b signaling in the control of cell morphology and cytoskeleton organization, as also reported in other cell systems (for review [20]). Maintaining normal cell morphology may also be essential for differentiation. This is supported by previous data showing that cytoskeleton depolymerization inhibits expression of the GS marker of Müller cell differentiation [28]. Thus, TGF-b signaling would control differentiation by acting both on cell markers expression and cytoskeleton organization. Therefore, it appears that the majority of the phenotypic changes, induced by ICN stable expression, are achieved by restoring endogenous TGF-b signaling, repressed as a consequence of v-Src activity.
Both Notch and TGF-b signaling play a dual role in oncogenesis, depending on the cell context (for review [14,29]. Notch activation contributes to oncogenesis by promoting survival or proliferation of undifferentiated precursor cells. The role of TGF-b signaling is more complex and impacts on various steps of tumor progression (for review [16,30]. When acting as tumor suppressors, both pathways promote growth arrest by upregulating expression of the cyclin/CDK inhibitor p21 cip1/waf1 , which in turn favors differentiation [31,32]. Our results indicate that suppression of QNR/v-src ts cell transformation by the conjunction of both signaling pathways involves distinct mechanisms, in various aspects. First, in this cell system, QNR/v-src ts cells reversion to a normal phenotype, in presence of an active v-Src, is compatible with their sustained division. Moreover, ICN stable expression confers extended growth capacity upon QNR/v-src ts /ICN cells when v-Src ts is thermally inactivated, whereas QNR/v-src ts cells stop dividing under these non permissive conditions (32). This strongly suggests that, while QNR/v-src ts /ICN cells have acquired glial specific markers, they are not mature glial cells, as their terminal differentiation would normally require extinction of Notch signaling [33,34]. Second, we also reported a strong cytoplasmic accumulation of p21 cip1/waf1 in dividing QNR/vsrc ts /ICN cells, suggesting for this protein a role distinct from that of a cell cycle inhibitor [2]. Third, all these phenotypic changes take place in presence of an active oncoprotein, in contrast to other models where oncogene inactivation is required for differentiation [35][36][37].
Tumor cells generally do not respond to extracellular signals that normally control their growth or differentiation [38]. A likely reason would be that they down-regulate expression of genes encoding such secreted proteins or their receptors. Alternatively, proteins secreted by tumor cells could be endowed with pleiotropic properties and instead favor tumor progression, as described with TGF-b signaling (for review [16,39]. However, it was also shown that certain tumor cells display remarkable plasticity when placed in appropriate environments (for review, [40,41]. Therefore, analyzing the response of tumor cells to extracellular signals, potentially capable of modulating the transformed phenotype is required to understand crucial events in transformation and develop specific therapeutic approaches.

Reagents
The TGF-b type I receptor kinase inhibitor, SB431542 was purchased from Sigma (Sigma-Aldrich, Lyon, France) and