Reviewer's report
Title:Epithelial to Mesenchimal transition in the liver field: the double face of
Everolimus in vitro
Version:2Date:1 December 2014
Reviewer: Raymond Yeung
Reviewer's report:
The manuscript by Masola et al. highlights the complexity of the biologic effects
when cells are exposed to varying concentrations of the rapamycin analog,
everolimus. EMT was noted only when cells were exposed to high concentration
of everolimus, which correlated with elevation in phospho-AKT. However, there
are a number of inconsistencies that need to be considered and explained.
Major Compulsory Revisions
1. While the data nicely showed that EVE(100nM) induces markers of EMT along
with increased AKT phosphorylation, there is a substantial volume of published
data showing the effects of high dose rapamycin in suppressing AKT activity
through its inhibitory effects on mTORC2 (e.g., disassembly). On the other hand,
low-dose rapamycin is associated with AKT activation through its interference
with mTORC1-mediated feedback inhibition. Therefore, the authors should
explain or demonstrate the reasons for the apparent discrepancy; this is crucial
to the conclusion of the manuscript.
2. The sequence of events needs to be clarified. AKT phosphroylation increases
after 24 hrs while TGFbeta expression rises after 6 hrs. This would suggest that
TGFbeta is not the results of AKT activation.
3. The findings of increased expression of FN, a-SMA, vimentin in EVE-treated
HSC do not represent EMT since HSC are not epithelial cells to begin with, as
stated on page 11, last line. EMT, however, would apply to HepG2 cells.
Minor Essential Revisions
1. More emphasis in the Discussion should highlight that the concentration (e.g,
100 nM) necessary to induce EMT is NOT clinically relevant, thus diminishes the
significance of the findings.
2. The authors imply that EMT is the result of AKT activation, but there is no data
to support this in the manuscript. The findings are purely an association. It would
be nice to provide stronger experimental evidence to link the two by manipulating
AKT genetically and pharmacologically in the cells.
3. Introduction, 1st paragraph: EVE belongs to the rapamycin class of drugs that
are allosteric inhibitors of mTORC1 (i.e., rapalogs). This is distinct from mTORi
that typically refers to mTOR kinase inhibitors, which suppress both mTORC1
and mTORC2.
4. Methods, Western Blot: Identification and source of antibodies need to be
stated (e.g., which p-AKT antibody was used?)
5. Discussion, 1st paragraph: "EVE remains a potent blocker of liver fibrosis at
therapeutic concentration....". This reviewer did not come across any evidence to
support this statement.
6. Methods: What is used as "untreated" control?
7. Reference 36 should be updated with recent publication of EVOLVE-1 trial.
Discretionary Revisions:
1. Figure 5: Why is there significant expression of p-AKT in 'untreated' cells after
24 hr starvation in serum free medium? What is activating AKT?
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:Epithelial to Mesenchimal transition in the liver field: the double face of
Everolimus in vitro
Version:2Date:1 December 2014
Reviewer: Raymond Yeung
Reviewer's report:
The manuscript by Masola et al. highlights the complexity of the biologic effects
when cells are exposed to varying concentrations of the rapamycin analog,
everolimus. EMT was noted only when cells were exposed to high concentration
of everolimus, which correlated with elevation in phospho-AKT. However, there
are a number of inconsistencies that need to be considered and explained.
Major Compulsory Revisions
1. While the data nicely showed that EVE(100nM) induces markers of EMT along
with increased AKT phosphorylation, there is a substantial volume of published
data showing the effects of high dose rapamycin in suppressing AKT activity
through its inhibitory effects on mTORC2 (e.g., disassembly). On the other hand,
low-dose rapamycin is associated with AKT activation through its interference
with mTORC1-mediated feedback inhibition. Therefore, the authors should
explain or demonstrate the reasons for the apparent discrepancy; this is crucial
to the conclusion of the manuscript.
2. The sequence of events needs to be clarified. AKT phosphroylation increases
after 24 hrs while TGFbeta expression rises after 6 hrs. This would suggest that
TGFbeta is not the results of AKT activation.
3. The findings of increased expression of FN, a-SMA, vimentin in EVE-treated
HSC do not represent EMT since HSC are not epithelial cells to begin with, as
stated on page 11, last line. EMT, however, would apply to HepG2 cells.
Minor Essential Revisions
1. More emphasis in the Discussion should highlight that the concentration (e.g,
100 nM) necessary to induce EMT is NOT clinically relevant, thus diminishes the
significance of the findings.
2. The authors imply that EMT is the result of AKT activation, but there is no data
to support this in the manuscript. The findings are purely an association. It would
be nice to provide stronger experimental evidence to link the two by manipulating
AKT genetically and pharmacologically in the cells.
3. Introduction, 1st paragraph: EVE belongs to the rapamycin class of drugs that
are allosteric inhibitors of mTORC1 (i.e., rapalogs). This is distinct from mTORi
that typically refers to mTOR kinase inhibitors, which suppress both mTORC1
and mTORC2.
4. Methods, Western Blot: Identification and source of antibodies need to be
stated (e.g., which p-AKT antibody was used?)
5. Discussion, 1st paragraph: "EVE remains a potent blocker of liver fibrosis at
therapeutic concentration....". This reviewer did not come across any evidence to
support this statement.
6. Methods: What is used as "untreated" control?
7. Reference 36 should be updated with recent publication of EVOLVE-1 trial.
Discretionary Revisions:
1. Figure 5: Why is there significant expression of p-AKT in 'untreated' cells after
24 hr starvation in serum free medium? What is activating AKT?
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Epithelial to Mesenchimal transition in the liver field: the double face of
Everolimus in vitro
Version:2Date:1 December 2014
Reviewer: Raymond Yeung
Reviewer's report:
The manuscript by Masola et al. highlights the complexity of the biologic effects
when cells are exposed to varying concentrations of the rapamycin analog,
everolimus. EMT was noted only when cells were exposed to high concentration
of everolimus, which correlated with elevation in phospho-AKT. However, there
are a number of inconsistencies that need to be considered and explained.
Major Compulsory Revisions
1. While the data nicely showed that EVE(100nM) induces markers of EMT along
with increased AKT phosphorylation, there is a substantial volume of published
data showing the effects of high dose rapamycin in suppressing AKT activity
through its inhibitory effects on mTORC2 (e.g., disassembly). On the other hand,
low-dose rapamycin is associated with AKT activation through its interference
with mTORC1-mediated feedback inhibition. Therefore, the authors should
explain or demonstrate the reasons for the apparent discrepancy; this is crucial
to the conclusion of the manuscript.
2. The sequence of events needs to be clarified. AKT phosphroylation increases
after 24 hrs while TGFbeta expression rises after 6 hrs. This would suggest that
TGFbeta is not the results of AKT activation.
3. The findings of increased expression of FN, a-SMA, vimentin in EVE-treated
HSC do not represent EMT since HSC are not epithelial cells to begin with, as
stated on page 11, last line. EMT, however, would apply to HepG2 cells.
Minor Essential Revisions
1. More emphasis in the Discussion should highlight that the concentration (e.g,
100 nM) necessary to induce EMT is NOT clinically relevant, thus diminishes the
significance of the findings.
2. The authors imply that EMT is the result of AKT activation, but there is no data
to support this in the manuscript. The findings are purely an association. It would
be nice to provide stronger experimental evidence to link the two by manipulating
AKT genetically and pharmacologically in the cells.
3. Introduction, 1st paragraph: EVE belongs to the rapamycin class of drugs that
are allosteric inhibitors of mTORC1 (i.e., rapalogs). This is distinct from mTORi
that typically refers to mTOR kinase inhibitors, which suppress both mTORC1
and mTORC2.
4. Methods, Western Blot: Identification and source of antibodies need to be
stated (e.g., which p-AKT antibody was used?)
5. Discussion, 1st paragraph: "EVE remains a potent blocker of liver fibrosis at
therapeutic concentration....". This reviewer did not come across any evidence to
support this statement.
6. Methods: What is used as "untreated" control?
7. Reference 36 should be updated with recent publication of EVOLVE-1 trial.
Discretionary Revisions:
1. Figure 5: Why is there significant expression of p-AKT in 'untreated' cells after
24 hr starvation in serum free medium? What is activating AKT?
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I declare that I have no competing interests.
assistant