SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

Abstract Skp1‐Cul1‐F‐box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma‐associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F‐box and leucine‐rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17‐mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17–Sufu axis in the pathogenesis of medulloblastoma.

Thank you for submitting your manuscript on Sufu regulation by Fbxl17 to The EMBO Journal. We have now received feedback from three expert referees, whose reports are copied below for your information. I am pleased to inform you that all referees consider your findings interesting and potentially important, and therefore in principle suitable for publication in our journal. Nevertheless, they do raise a number of major issues that would need to be satisfactorily addressed before eventual acceptance. As you will see, the majority of these issues, especially in the reports of referees 1 and 3, are of technical nature, referring mainly to the conclusiveness of the biochemical data and knockdown analyses, and so would appear overall straightforward to address.
However, there are also some more significant conceptual concerns that would also need to be taken into account, especially relating to the functional significance of Sufu degradation via Fbxl17. In this respect, it would be important to extend/validate at least some of the cellular assays in physiologically more relevant (cell culture) settings; as well as to better place Fbxl17 into the context of Smo-mediated Hh signaling (see referee 2, as well as ref 3 point 15), ideally by including at least some follow-up investigation on how Fbxl17 may be regulated by Smo activity and/or able to swiftly overcome the inhibitory effect of Sufu phosphorylation.
In this light, I would like to invite you to prepare a revised version of the manuscript, whichpending adequate answering of the discussed issues -we would be happy to consider further for publication in The EMBO Journal.
Overall the work is of novel and of good technical quality, it would have been significantly strengthened had the authors pursued further validation in more relevant systems. There are a number of points that need to be experimentally addressed. Much of the work is conducted in non-biologically relevant systems, although I acknowledge the inclusion of the one medulloblastoma line.
Overall immunoblotting quantification is hard to evaluate as control bands (e.g. GAPDH) are significantly overexposed. I often insist on the need to develop dilution curves to better quantify changes.
In fig. 1G first point evaluated is 6 hours, so half-life could be significantly shorter in this system The essential role of Fbxl17 for Sufu ubiquitylation could be limited to Standard representation for proliferation curves should be cell growth over time (fig 5C A, B). Fbxl17 non-targetable cDNA rescue should have been supplied.
Referee #2: In this paper, the authors describe the identification of the SuFu protein as an interacting partner with the E3 ubiquitin ligase Fbxl17 through LC-MS/MS analysis of proteins immunoprecipitated from HEK293T cells. Following up on this finding, they perform a number of studies to test the hypothesis that Fbxl17 controls SuFu protein levels in response to Hh signaling. They demonstrate that Fbxl17 binds directly to SuFu to promote its ubiquitylation and degradation and that this binding is inhibited by phosphorylation of SuFu and potentiated by Gli1. The data in support of these conclusions look quite good; however, quantitative analysis of the Western blots would be appropriate, along with an indication of the number of replicates performed for each assay. The authors also investigate the functional consequences of SuFu regulation by Fbxl17; they present evidence that knock down of Fbxl17 abrogates Gli1 transcription factor activity in Ptch1 MEFs, in which the Hh pathway is constitutively activated, as well as in PC3 cells. In addition, they show that the response of medulloblastoma cells to Hh pathway activation can be abrogated by Fbxl17. The authors suggest that Fbxl17 may play a similar role in regulating SuFu levels to that played by the Fused kinase in Drosophila (though they later suggest on page 15 that Fbxl17 may play an analogous role in Drosophila). However, while it is well established that Fused activity is regulated in response to Smo activation, the authors provide no indication as to whether or how Smo activity might regulate Fbxl17, save for ruling out an effect at the transcriptional level. It is quite possible that Fbxl17 acts passively, binding to SuFu in response to its dephosphorylation -the authors hint at this in the Discussion when they state that "upon Hh ligand binding to Ptch1, PKA and GSK3beta are inhibited" -though they provide no justification of this statement. A better discussion of this important issue is warranted. It is also not clear to me why simply overexpressing Fbxl17 is sufficient to inhibit SuFu -this implies that simply increasing the concentration of Fbxl17 is sufficient to overcome the inhibitory effect of SuFu phosphorylation, but this is could be explicitly investigated.

Referee #3:
Raducu et al., propose that the SCF(Fbxl17) E3 ligase complex targets Sufu for poly-ubiquitination and subsequent degradation in a Hh activation-dependent manner. They describe that phosphorylation events of Sufu regulate the interaction between Fbxl17 and Sufu, and the Fbxl17mediated Sufu degradation leads to enhancement of Gli transcriptional activity. The authors further demonstrated that depletion of Fbxl17 results in Sufu accumulation, leading to attenuation of medulloblastoma tumor growth. Indeed, Fbxl17 and Gli mRNA expression levels are significantly elevated in clinical samples of the Shh subtype medulloblastoma. This study may provide a molecular link between Sufu alterations and cancer development/progression in various tumors, especially in medulloblastoma. The animal and clinical studies are convincing, however the biochemical data presented are somewhat premature and therefore further analyses should be carried out.
Specific comments: 1. Fig. 1B and 1C: WCL lane needs to be provided in the same panel with IP samples to show the MW of obtained Fbxl17 bands in WCL are equal to the IP bands. In addition, a WB panel of IgG bands needs to be provided to indicate the same amount of IgG were used in control and Fbxl17 IP reactions.
2. Fig 1E and 1G: The effects of Fbxl17 knockdown are not convincing. Adding Hh stimulation may be beneficial to demonstrate a significant Sufu stabilization following Fbxl17 depletion.
3. Fig 1E, 1G, 2A, 4A, 4E, 5A-5C and 6E: Fbxl17 blots should be provided to show relevant Fbxl17 knockdown at protein level. This should be relatively straight forward as the Fbxl17 antibody for WB analysis has been validated in Fig. 1B and 1C. 4. Fig. 1G: It would be helpful to present the data in a graph by quantifying the band intensities of three independent experiments. 5. Fig. 2A: WB panels of input (HA, Fbxl17 and GAPDH blots) should be included. 6. Fig. 2: In vitro ubiquitination assay needs to be performed to prove that SCF(Fbxl17) can directly transfer polyubiquitin chain to Sufu on K257. 9. Fig 3D: It is difficult to interpret the data as the panels are confusing and not clearly or appropriately labeled.
10. Fig 3E: The strong polyubiquitination band, which is observed in the last lane, is not supposed to appear, as Fbxl17 is absent in this lane. Is it a contaminating band derived from polyubiquitinated Gli1 protein? The authors need to exclude this possibility. 11. Fig. 5B: Sufu and Fbxl17 blots need to be provided. 12. Fig. 6: Did the authors examine the frequency of Sufu mutations at the S352, S342 and S346 phosphorylation sites in the cohorts of the 285 medulloblastoma clinical samples?
13. EV3A-3B: Sufu Δ350-425 mutants should be included for the mapping. As authors mention that Fbxl proteins recognize a larger surface rather than a linear degron motif on substrates, it is critical to use a minimal deletion mutant. Extensive deletion might cause mis-folding, leading to a nonspecific interaction.
14. EV3C: S342/346 mutant need to be included as a positive control to set the criteria of what extent of enhancement is significant. 15. The authors propose the failsafe model through which PKA and GSK3beta phosphorylate Sufu to prevent unscheduled Sufu degradation by Fbxl17. However, upon Hh activation, the cells require a swift response to transmit the Hh signals into nucleus. Regarding the dephosphorylation of Sufu, which molecular mechanisms are involved in efficiently releasing the failsafe system? The authors conduct a number of studies to confirm their findings and to also show that a mutation in Sufu identified in patients affected by medulloblastoma in Gorlin syndrome, increases Sufu turnover through Fbxl17-mediated ubiquitylation, leading to enhanced HH pathway activation. The authors also provide a convincing study correlating Fbxl17 expression with the Shh subtype of medulloblastoma.
Overall the work is of novel and of good technical quality, it would have been significantly strengthened had the authors pursued further validation in more relevant systems. There are a number of points that need to be experimentally addressed.

1.
Much of the work is conducted in non-biologically relevant systems, although I acknowledge the inclusion of the one medulloblastoma line.

Answer 1:
One of the main limitations in the Hedgehog signaling field is the poor availability of cell lines, which maintain Hedgehog pathway active or in which the pathway can be modulated by treatments with agonists/antagonists. There are controversial opinions about the cell systems suitable for studying molecular regulation of Hedgehog signaling. For this reason, we conducted an extensive validation of pathway activation in DAOY in Fig. EV7. In addition to this, we have confirmed the functional role of Fbxl17-Sufu axis in PC3 cell lines ( Fig EV5 and 6), which do not rely on a ligand dependent mechanism of Hedgehog pathway activation but show regulation of Sufu by proteolysis and transcriptional regulation of Gli, in accordance to previous findings (Zhang et al, 2007). Mouse embryonic fibroblasts (MEFs) deficient in either Ptch1 (a major negative regulator of Hedgehog signaling) or Sufu have been extensively used in Hedgehog signaling to assess pathway activation. These have been used in our study in Figure 4 and 7F.
Most importantly, to establish a role for Fbxl17 in SHH medulloblastoma development, we show in Fig EV 10 A, B, C and D that Fbxl17 has an important role in the proliferation of Granule Cell Progenitors, the cells of origin of SHH medulloblastoma (Marino et al, 2000;Oliver et al, 2005;Schuller et al, 2008;Wechsler-Reya & Scott, 1999).

2.
Overall immunoblotting quantification is hard to evaluate as control bands (e.g. GAPDH) are significantly overexposed. I often insist on the need to develop dilution curves to better quantify changes.

Answer 2:
We do agree that some of the immunoblots contained oversaturated loading control bands and we apologize for that. To overcome this issue, some of these immunoblots were repeated and new panels containing less exposed loading control bands were included in Fig 1B, 1C, 1E, 2B and 5A. Relative quantifications have been modified accordingly without substantial changes in the significance of findings.
3. In fig. 1G first point evaluated is 6 hours, so half-life could be significantly shorter in this system

Answer 4:
We have performed ubiquitylation assay of Sufu in a more relevant cell system (DAOY cells) and this is shown now in Figure 2A. Absent polyubiquitylated species of Sufu upon Fbxl17 depletion reinforce the essential role for Fbxl17 in Sufu ubiquitylation in DAOY medulloblastoma cancer cell lines.

Answer 5:
Sufu was found to be phosphorylated on S352 in the following previous publication using LC/MS (Hsu et al, 2011). We have performed LC/MS analysis of Sufu secondary modifications in FigEV4A, where it is shown that the corresponding peptide could contain numerous modifications. Furthermore, we raised an antibody against a peptide 347-360 of Sufu containing S352 and T353 phosphorylated. We detected Sufu phosphorylation on Sufu WT but not on a mutant of Sufu where S352 and T353 were substituted to alanine (Fig.3C). This shows that Sufu is phosphorylated in vivo on S352/T353.
6. Standard (rescue) controls for siRNAs and shRNAs are not being provided Fbxl17 protein quantification upon silencing is not provided

Answer 6:
Rescue of cell proliferation and Sufu protein levels is observed after siRNA of Fbxl17 and rescue upon expressing Fbxl17 full length in DAOY cells (Fig 5A, 5C, Fig EV8A and B) and PC3 cells (Fig EV5C-E). Protein levels of Fbxl17 are presented in Fig 5C and Fig EV5D. Due to antibody limitation we measured the extent of Fbxl17 siRNA by QPCR in Fig 5B and E and Fig EV 5F.

8.
Standard representation for proliferation curves should be cell growth over time (fig 5C A, B).

Answer 8:
Given the nature of growth of DAOY cells we perform few determinations of cell numbers, which are not well represented using cell growth over time. This representation has been used in previous publications (McKee et al, 2012). The effect of Fbxl17 on cell proliferation has been followed with different methods, which confirm the validity of findings: 1. Measurements of relative cell proliferation 2. The use of an orthotopic rat model of medulloblastoma to monitor cell growth at different time points by MRI scan, which gives accurate determination of tumour volume and size superior to bioluminescence. 3. Using marker of cell proliferation such as Ki67. 4. Using BrdU incorporation in GCPs.

Answer 9:
Rescue using cDNA for Fbxl17 is provided in: Fig 5A and C, Fig EV8A and B and in Fig EV5C-E. Referee #2: 1. In this paper, the authors describe the identification of the SuFu protein as an interacting partner with the E3 ubiquitin ligase Fbxl17 through LC-MS/MS analysis of proteins immunoprecipitated from HEK293T cells. Following up on this finding, they perform a number of studies to test the hypothesis that Fbxl17 controls SuFu protein levels in response to Hh signaling. They demonstrate that Fbxl17 binds directly to SuFu to promote its ubiquitylation and degradation and that this binding is inhibited by phosphorylation of SuFu and potentiated by Gli1. The data in support of these conclusions look quite good; however, quantitative analysis of the Western blots would be appropriate, along with an indication of the number of replicates performed for each assay.

2.
The authors also investigate the functional consequences of SuFu regulation by Fbxl17; they present evidence that knock down of Fbxl17 abrogates Gli1 transcription factor activity in Ptch1 MEFs, in which the Hh pathway is constitutively activated, as well as in PC3 cells. In addition, they show that the response of medulloblastoma cells to Hh pathway activation can be abrogated by Fbxl17. The authors suggest that Fbxl17 may play a similar role in regulating SuFu levels to that played by the Fused kinase in Drosophila (though they later suggest on page 15 that Fbxl17 may play an analogous role in Drosophila). However, while it is well established that Fused activity is regulated in response to Smo activation, the authors provide no indication as to whether or how Smo activity might regulate Fbxl17, save for ruling out an effect at the transcriptional level. It is quite possible that Fbxl17 acts passively, binding to SuFu in response to its dephosphorylation -the authors hint at this in the Discussion when they state that "upon Hh ligand binding to Ptch1, PKA and GSK3beta are inhibited" -though they provide no justification of this statement. A better discussion of this important issue is warranted.

Answer 2:
We have now extended the discussion to clarify this point. We do agree that Fbxl17 acts passively after Sufu dephosphorylation. It has been reported that Sufu phosphorylation promotes its retention within the cilium (Chen et al, 2011) thus establishing a spatio-temporal determinant of Sufu dephosphorylation and ubiquitylation after pathway activation. However, the regulation of Fbxl17-Sufu axis is likely to be complex and different in the diverse tissue and models analyzed. For instance it is tantalizing to speculate that cells with a functional cilium could also regulate Fbxl17. These studies could not be undertaken in the current work, which focuses on Sufu regulation by Fbxl17. We don't think that Fbxl17 is playing an analogue role in Drosophila in Sufu degradation since the role of Sufu is substantially different in this model system and Fbxl17 has a little to poor sequence conservation to CG31633 (the postulated homologue). However, the essential role of CG31633 in Drosophila development emphasizes the importance of Fbxl17 during embryogenesis. We hope that our comments will stimulate studies on CG31633, which will clarify its role in this fascinating model system. We have changed the discussion to clarify our statement.

3.
It is also not clear to me why simply overexpressing Fbxl17 is sufficient to inhibit SuFu -this implies that simply increasing the concentration of Fbxl17 is sufficient to overcome the inhibitory effect of SuFu phosphorylation, but this is could be explicitly investigated.

Answer 3:
From previous literature it is clear that Sufu dephosphorylation facilitate its relocalization outside the cilium (Chen et al, 2011). Our data are in accordance to a passive model whereby the dephosphorylated fraction of Sufu is polyubiquitylated by Fbxl17. This mechanism is similar but opposite to the regulation of Gli2 and Gli3 operated by SCF bTrcp (Bhatia et al, 2006;Wang & Li, 2006). In both cases proper activation of Hh signaling needs reversal of phosphorylation mediated by PKA and GSK3b. Of note, while dephosphorylation is a central mechanism to proper Hh signaling (Eisner et al, 2015), the players mediating Sufu dephosphorylation are unknown.
Sufu can inhibit Hedgehog signaling also in the absence of cilia (Jia et al, 2009). In cancer cell lines, lacking cilia, a pool of Sufu, which is not phosphorylated could be present, due to constitutive pathway activation. In PC3 and DAOY activating mutation in Hedgehog signaling components could generate a pool of Sufu readily degradable by Fbxl17. This could explain the effect of Fbxl17 overexpression on Sufu levels. Importantly, in MEFs with two copies of Ptch1, in which the pathway is inactive, siRNA of Fbxl17 does not induce alteration in Sufu levels.

Referee #3:
Raducu et al., propose that the SCF(Fbxl17) E3 ligase complex targets Sufu for poly-ubiquitination and subsequent degradation in a Hh activation-dependent manner. They describe that phosphorylation events of Sufu regulate the interaction between Fbxl17 and Sufu, and the Fbxl17mediated Sufu degradation leads to enhancement of Gli transcriptional activity. The authors further demonstrated that depletion of Fbxl17 results in Sufu accumulation, leading to attenuation of medulloblastoma tumor growth. Indeed, Fbxl17 and Gli mRNA expression levels are significantly elevated in clinical samples of the Shh subtype medulloblastoma. This study may provide a molecular link between Sufu alterations and cancer development/progression in various tumors, especially in medulloblastoma. The animal and clinical studies are convincing, however the biochemical data presented are somewhat premature and therefore further analyses should be carried out.
Specific comments: 1. Fig. 1B and 1C: WCL lane needs to be provided in the same panel with IP samples to show the MW of obtained Fbxl17 bands in WCL are equal to the IP bands. In addition, a WB panel of IgG bands needs to be provided to indicate the same amount of IgG were used in control and Fbxl17 IP reactions.

Answer 1:
This has been provided as requested in the new Fig 2B and C. It is important to note that the commercially available antibody that we use recognize Fbxl17 only after enriching its levels by immunoprecipitation of Sufu. As a reference we have immunoprecipitated exogenous Fbxl17 which migrates at the same molecular weight as the endogenous. A panel containing IgG bands was also introduced to indicate the amount of IgG used for each immunoprecipitation. Fig 1E and 1G: The effects of Fbxl17 knockdown are not convincing. Adding Hh stimulation may be beneficial to demonstrate a significant Sufu stabilization following Fbxl17 depletion.

Answer 2:
After Hedgehog stimulation using SAG ( Fig 1E, and quantified in Fig EV1A) the effects were more significant and panels have been changed accordingly. We do agree that a proper activation of Hedgehog signaling is a limiting factor for Sufu stabilization upon Fbxl17 depletion. A better effect can be observed in Fig 4A, where Ptch1-depleted cells, with a constitutive Hh signaling activation, were used. Fig 1E, 1G, 2A, 4A, 4E, 5A-5C and 6E: Fbxl17 blots should be provided to show relevant Fbxl17 knockdown at protein level. This should be relatively straight forward as the Fbxl17 antibody for WB analysis has been validated in Fig. 1B and 1C.

Answer 3:
Please note (Fig 1B and C) that the antibody against Fbxl17 did not work on endogenous protein but only after enriching by immunoprecipitation of Sufu. We have performed qPCR of Fbxl17 for the experiments in Fig 1E (see Fig 1D), 1G (see Fig EV1F), 2A and B (see Fig 2C), 4A (see Fig 4B), 4E (see EV 4B), 5A (see Fig 5B), Fig 5D (see Fig 5E), Fig 6B (see Fig 6A), Fig EV5A (see Fig EV5F) and Fig EV6A (see Fig EV 6D). All the qPCR analysis show a drastic reduction of Fbxl17 mRNA in all cases. Fig. 1G: It would be helpful to present the data in a graph by quantifying the band intensities of three independent experiments.

Answer 4:
This has been presented in figure EV1E. Fig. 2A: WB panels of input (HA, Fbxl17 and GAPDH blots) should be included.

Answer 5:
In the new figure 2B, Western blot of Sufu and GAPDH have been included, and also QPCR of Fbxl17 has been reported in Fig 2C. 6. Fig. 2: In vitro ubiquitination assay needs to be performed to prove that SCF(Fbxl17) can directly transfer polyubiquitin chain to Sufu on K257.

Answer 6:
In vitro ubiquitylation assay of Sufu WT and Sufu K257R mutant was introduced in Fig EV2C. 7. Fig. 3A: The phosphorylation mimetic S to D mutant should be added in this analysis as performed in Fig. 2B.

Answer 7:
This has been added in Figure EV3D 8. Fig. 3B: It would be better to include Flag-Gli1 in the assay as performed in Fig. 2A.

Answer 8:
A new western blot has been introduced in Fig EV3E. 9. Fig 3D: It is difficult to interpret the data as the panels are confusing and not clearly or appropriately labeled.

Answer 9:
We apologize for this and have modified the figure for improved clarity. Fig 3E: The strong polyubiquitination band, which is observed in the last lane, is not supposed to appear, as Fbxl17 is absent in this lane. Is it a contaminating band derived from polyubiquitinated Gli1 protein? The authors need to exclude this possibility.

Answer 10:
This band derives from polyubiquitylation of Sufu mediated by endogenous Fbxl17. Sufu polyubiqutylation by endogenous Fbxl17 can also be observed in Fig 2A and B. Fig. 5B: Sufu and Fbxl17 blots need to be provided.

Answer 11:
As we mentioned earlier, due to antibody limitations, levels of endogenous Fbxl17 could not be assessed by Western blot. For Fig 5B (now Fig 5D), qPCR showing downregulation in Fbxl17 mRNA levels has been added in Fig 5E. Also, a representative image of three independent experiments along with the corresponding relative quantification has been added for Sufu protein in Fig EV8A and Fig EV8B. 12. Fig. 6: Did the authors examine the frequency of Sufu mutations at the S352, S342 and S346 phosphorylation sites in the cohorts of the 285 medulloblastoma clinical samples?

Answer 12:
This cohort does not contain information on Sufu mutations.
13. EV3A-3B: Sufu Δ350-425 mutants should be included for the mapping. As authors mention that Fbxl proteins recognize a larger surface rather than a linear degron motif on substrates, it is critical to use a minimal deletion mutant. Extensive deletion might cause mis-folding, leading to a nonspecific interaction. Thank you for submitting your revised manuscript for our consideration by the EMBO Journal. It has now been seen again by two of the original referee (see comments below). I am please to inform you that both of them are satisfied with the responses and revisions and have no further objections towards publication. We are therefore happy to accept your manuscript in principle at this stage.
- Mann--Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section;  are tests one--sided or two--sided?  are there adjustments for multiple comparisons?  exact statistical test results, e.g., P values = x but not P values < x;  definition of 'center values' as median or average;  definition of error bars as s.d. or s.e.m.
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4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe. 4.b. For animal studies, include a statement about blinding even if no blinding was done 5. For every figure, are statistical tests justified as appropriate?
Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it. Is there an estimate of variation within each group of data?
Is the variance similar between the groups that are being statistically compared?
6. To show that antibodies were profiled for use in the system under study (assay and species), provide a citation, catalog number and/or clone number, supplementary information or reference to an antibody validation profile. e.g., Antibodypedia (see link list at top right), 1DegreeBio (see link list at top right). 7. Identify the source of cell lines and report if they were recently authenticated (e.g., by STR profiling) and tested for mycoplasma contamination.
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