Professor Marc Lippman
Editor in Chief, Breast Cancer Research and Treatment
Dear Prof. Lippman,
Enclosed please find the revised manuscript BREA-D-14-01022: "First trimester
human placental factors induce breast cancer cell autophagy".
We have carefully read the reviewers’ comments and revised the manuscript
accordingly. Our responses to all reviewers' comments are detailed below. In an
attached file, you will find the revised manuscript. All changes are marked in yellow.
We hope you will find the revised form suitable for publication in "Breast Cancer
Research and Treatment".
Sincerely yours,
Dr. Shelly Tartakover-Matalon
Oncogenetic Laboratory
Meir Medical Center
Kfar Saba, Israel
Tel: 972-9-7472841; Fax: 972-9-747114 ;
E-mail: Matalon.shelly@clalit.org.il
Reviewer #1:
1. "M & M page 5. 10 mM of the autophagy formation inhibitor 3MA is used.
This is 10^5 M, and seems like a lot. What is the evidence for specificity at
this high concentration?"
The 3MA is widely used as an autophagy inhibitor. We used 10-5M (10µM) 3MA in
our biological systems. This concentration was previously used by other groups [1, 2]
and in our laboratory [3] in order to inhibit autophagy, and the results were published
in leading journals. Somehow, in the previous version the "µ" was misplaced with the
"m" in the Methods section. This issue was corrected.
2. "page 6. It is stated that LC3BII is a known marker of autophagy.
No
reference is given. Ideally, the authors should include a biochemical assay to
demonstrate that autophagy is actually occurring, rather than simply showing
an increase in a marker. This would greatly strengthen the findings."
During autophagy, a cytosolic form of LC3 (LC3-I, microtubule-associated protein 1
light chain 3) is bound to phosphatidylethanolamine to form conjugate (LC3BII),
which is recruited to the autophagosomal membranes. This process is essential for the
formation of the autophagosomes [4].Then the autophagosomes fuse with lysosomes
to form autolysosomes and at the same time, the LC3BII in the autolysosomal lumen
is degraded. Thus, lysosomal turnover of the autophagosomal marker LC3BII reflects
autophagic activity and detecting it by immunoblotting or immunofluorescence has
become a reliable method for monitoring autophagy [5, 6]. The reviewer was right for
stating that the reference was missing. Therefore, references were added to the first
paragraph of the results section in page 7.
In addition to the detection of the LC3BII marker, we added the 3MA, which is a well
accepted autophagy inhibitor [1, 2]. Autophagy and tumor cell invasion are often
interlinked [7, 8]. They were also found to be linked in EVT cells [9]. Therefore, our
observation that 3MA inhibited BCCL migration, further supports that the cancer cells
underwent autophagy. This information was also added to the manuscript in the
discussion section in page 10 and references regarding 3MA role as an autophagy
inhibitor were added to the manuscript in page 7.
3. "Figure 1. Despite the graph, the increase in LC3BII seen in the blot is
minimal and barely noted with "eyeball densitometry", which is often more
credible than a graphic representation of the data."
We agree with the reviewer that "eyeball densitometry" is sometimes the best
indicator. Our previous photograph contained both LC3BI and LC3BII bands. Only,
LC3BII which is the lower band is the indicator for autophagy. In this photograph the
LC3BII band of cells that were exposed to the placenta was clearly stronger than the
control. Nevertheless, almost no difference was observed in the upper band of LC3BI
between experiment and control, which may have misled the reviewer. We understand
that showing both bands may mislead other readers as well and therefore we decided
to show only the lower band in all our figures including figure 1 and 2. This way, the
photograph reflects the values in the graphs.
4. Figure 2. Again, the western blot fails to show substantial differences between
control and placenta, despite the graphs and the statistical notation.
Please read our previous answer.
5. In Figure 2E, by what biochemical mechanism do the authors explain their
findings
Autophagy and tumor cell invasion are often linked [7, 8]. We added 3MA, an
autophagy inhibitor, to the placenta –BCCL coculture and to the placental soluble
factors and demonstrated that it inhibited the BCCL migration. By this, we
demonstrated that the autophagy was interlinked to the cancer cell migration in our
biological system. Our results showed that placental factors induced HIF1α and
BNIP3 expression in the BCCLs. HIF1α is a transcription factor that can promote
autophagy through its target BNIP3 [10]. Indeed we demonstrated that the HIF1α
inhibitor (Vitexin) prevented the elevation of LC3BII and the migration of BCCL that
were exposed to the placental factors and by that proved that this pathway was
involved in promoting the autophagy in our system. We also showed that the PR
inhibitor (RU486) reduced the placenta induced elevation of HIF1α and LC3BII,
demonstrating that progesterone was at least one of the triggers that promoted the
autophagy process. These results fit to Zielniok et al. results that demonstrated
connection between E2 and progesterone to the expression of the autophagy markers
ATG3, ATG5, BECN1 [11]. Recently, Maes H et al. demonstrated that BNIP3
regulates the plasticity of the actin cytoskeletone, which is integral to cell migration
[12]. His group showed that BNIP3 has a pro-tumorigenic role, which drives
melanoma cancer cell migration [12]. Therefore, during the last month we also
analyzed the effect of placental soluble factors on BNIP3 expression and found that it
was indeed elevated. These results and the Maes et al. study may provide explanation
for the link between autophagy to cell migration in our biological system and suggest
that activation of the PR-HIF1α-BNIP3 axis was responsible for the placental induced
BCCL autophagy and elevated cancer cell migration. The new data regarding the
HIF1α inhibition on LC3BII expression and migration and the effect of placental
supernatant on BNIP3 expression was added to the result section in pages 8-9. The
explanation concerning the biochemical mechanism that mediates the connection
between BCCL autophagy and cell migration was added to the discussion section on
pages 10-11. Furthermore, we added a small scheme, which explains the mechanism
responsible for the placenta induced BCCL autophagy (Figure 5).
6. Figure 3. The images are not convincing. Further, the authors are claiming
an increase in HIF1a and signaling. Although they show that 9 genes are
linked to the HIF1a transcription network, no biochemical/mechanistic data
are presented to substantiate the array data. At this point, their results are
correlative.
Some direct demonstration of HIF1a signaling should be
presented, and perhaps some experiments with a HIF1a inhibitor.
We replaced the images to more convincing ones. In order to establish a mechanism
that connects between HIF1α to the autophagy, the HIF1α inhibitor (Vitexin) was
added to the placental supernatants and its effects on LC3BII expression and
migration were tested. Indeed, we found that the inhibitor prevented the induction of
LC3BII expression and the MCF-7 migration, and by that proved that HIF1α
regulates the autophagy/migration in our biological system. These results were added
to the result section on page 9, and were also discussed on pages 10-11.
7. Figure 4 shows effects of a PG inhibitor. Differences in the western blot are
barely perceptible. Given the understandable variability in placental samples
that the authors mention at the outset, have they considered measuring the
levels of PG in their supernatants? This would strengthen their data. In
addition, perhaps they could modulate the experimental system by the addition
of exogenous PG.
Progesterone levels were not analyzed in this study. Therefore, we cannot perform a
correlation test between the Progesterone levels and the LC3BII levels in the BCCLs.
Nevertheless, The E2 and Progesterone concentrations in the placental culture media
were previously tested by us and were found to be high and similar to those measured
during pregnancy [13]. Furthermore, we demonstrated that 1) cancer cells consumed
the progesterone [14] 2) the combination of Progesterone and E2 (in placental
supernatant concentrations) was sufficient to induce cancer cell motility [14] 3) the
PR inhibitor prevented BCCL migration [14] and the autophagy process. Therefore,
we believe that our results clearly prove the association between progesterone to
BCCL motility and autophagy. Moreover, although we demonstrated that
progesterone was involved in mediating the autophagy process, it does not exclude
the possibility that other mediators were also involved in this process. Therefore,
progesterone level analysis will not necessarily provide a perfect explanation.
8. The Discussion of triple negative breast cancers is a bit confusing since these
tumors are PR negative. They cite a recently accepted article on EMT, but
how this relates to the present study with PG from placenta is not clear. They
also mention STAT3, but this pathway is not developed in the current
manuscript. These concepts need to be tied together somehow, or deleted.
We agree that these topics were confusing, and thus they were deleted. Instead, we
added a new section connecting our results to published data regarding the association
between HIF1α and autophagy to breast cancer disease progression and metastasis
creation.
Reviewer #2:
"The manuscript is rich in experimental techniques and interesting results, however,
1- The manuscript needs to be rewritten in a more clearer language".
We gave our revised manuscript to a professional English editor.
2. "The introduction is poorly written and lacks a clear aim of the work".
As claimed above, we gave our revised manuscript (including the introduction) to a
professional English editor. Moreover, we added a clear and defined aim and a
sentence that summarizes the conclusion of the manuscript to the end of the
introduction (page 4).
3. The message that needs to be conveyed to the reader should be clear from the
introduction.
A clear aim and a sentence that summarizes the main conclusion which is the message
of the manuscript were added to the end of the introduction (page 4).
4. Did you do duplicate, triplicates of your experiments?
5. How many times did you repeat the experiments?
The experiments were repeated at least 3 times. Most of the experiments, such as the
cell elimination from the placenta, scratch tests and the qPCR were also done in
triplicates in each experiment. This information was added to the methods section
(pages 5-6).
6. "I would suggest you to study the immunohistochemical expression of markers of
autophagy in human breast cancer tissues taken as biopsies during pregnancy".
Although this suggestion is very interesting and should be done in the future, it is
technically very difficult to perform within the next few months. Breast cancer during
pregnancy is a rare event. A physician in our hospital meets such patients once-twice
a year. Therefore, collecting several examples of such tumors from pregnant women
in order to perform the immunohistochemistry staining may require several years.
Moreover, such experiments need an additional ethics committee approval which is
currently unavailable. We feel that the contribution of this manuscript to the cancer
and pregnancy area is important even without having this additional information.
Therefore, although we appreciate the benefit of this idea we wish to publish this
basic science manuscript without the suggested staining. In the future, we intend to
cooperate with other hospitals in order to have an adequate number of tumors to
establish our findings in tumors from pregnant women.
7. The discussion is poorly write with no highlight or comparison to similar work.
We added a new part to the discussion which contains the biochemical mechanism
that stands behind our findings. We also compared this mechanism to previous
publications. The new information is discussed and summarized on page 11.
References
1. Singh R, Xiang Y, Wang Y, Baikati K, Cuervo AM, Luu YK et al. Autophagy
regulates adipose mass and differentiation in mice. J Clin Invest. 2009;119(11):332939. doi:39228 [pii]
10.1172/JCI39228 [doi].
2. Zhou R, Yazdi AS, Menu P, Tschopp J. A role for mitochondria in NLRP3
inflammasome activation. Nature. 2011;469(7329):221-5. doi:nature09663 [pii]
10.1038/nature09663 [doi].
3. Zismanov V, Lishner M, Tartakover-Matalon S, Radnay J, Shapiro H, Drucker L.
Tetraspanin-induced death of myeloma cell lines is autophagic and involves increased
UPR signalling. Br J Cancer. 2009;101(8):1402-9. doi:6605291 [pii]
10.1038/sj.bjc.6605291 [doi].
4. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy.
Annu Rev Genet. 2009;43:67-93. doi:10.1146/annurev-genet-102808-114910.
5. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS et al.
Guidelines for the use and interpretation of assays for monitoring autophagy in higher
eukaryotes. Autophagy. 2008;4(2):151-75. doi:5338 [pii].
6. Tanida I, Ueno T, Kominami E. LC3 and Autophagy. Methods Mol Biol.
2008;445:77-88. doi:10.1007/978-1-59745-157-4_4 [doi].
7. Amaravadi RK. Autophagy and tumor cell invasion. Cell Cycle. 2012;11(20):37189. doi:22147 [pii]
10.4161/cc.22147.
8. Macintosh RL, Timpson P, Thorburn J, Anderson KI, Thorburn A, Ryan KM.
Inhibition of autophagy impairs tumor cell invasion in an organotypic model. Cell
Cycle. 2012;11(10):2022-9. doi:20424 [pii]
10.4161/cc.20424.
9. Saito S, Nakashima A. Review: The role of autophagy in extravillous trophoblast
function under hypoxia. Placenta. 2013;34 Suppl:S79-84. doi:S0143-4004(12)004614 [pii]
10.1016/j.placenta.2012.11.026.
10. Bellot G, Garcia-Medina R, Gounon P, Chiche J, Roux D, Pouyssegur J et al.
Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction
of BNIP3 and BNIP3L via their BH3 domains. Mol Cell Biol. 2009;29(10):2570-81.
doi:MCB.00166-09 [pii]
10.1128/MCB.00166-09 [doi].
11. Zielniok K, Motyl T, Gajewska M. Functional interactions between 17 beta estradiol and progesterone regulate autophagy during acini formation by bovine
mammary epithelial cells in 3D cultures. Biomed Res Int. 2014;2014:382653.
doi:10.1155/2014/382653.
12. Maes H, Van Eygen S, Krysko DV, Vandenabeele P, Nys K, Rillaerts K et al.
BNIP3 supports melanoma cell migration and vasculogenic mimicry by orchestrating
the actin cytoskeleton. Cell Death Dis. 2014;5:e1127. doi:cddis201494 [pii]
10.1038/cddis.2014.94 [doi].
13. Schindler AE. Endocrinology of pregnancy: consequences for the diagnosis and
treatment of pregnancy disorders. J Steroid Biochem Mol Biol. 2005;97(5):386-8.
14. Epstein Shochet G, Tartakover Matalon S, Drucker L, Pomeranz M, Fishman A,
Rashid G et al. Hormone-dependent placental manipulation of breast cancer cell
migration. Hum Reprod. 2012;27(1):73-88. doi:der365 [pii]
10.1093/humrep/der365 [doi].