BCR041220P SURFACE TRAIL DECOY RECEPTOR-4 EXPRESSION CORRELATES
WITH TRAIL RESISTANCE IN MCF7 BREAST CANCER CELLS Ahter D Sanlioglu,
Ercument Dirice, Cigdem Aydin, Nuray Erin, Sadi Koksoy and Salih Sanlioglu
This rebuttal letter contains both the reviewers’ criticisms and responses to reviewers’
comments. Reviewer 1 is followed by a response to reviewer 1, reviewer 2 is followed by
response to reviewer 2 and so forth.
Reviewer 1
In this manuscript, authors investigate some mechanisms of resistance of breast cancer cells
to TRAIL. It is an important topic. Mechanisms of TRAIL resistance need to be elucidated if
TRAIL is to be used successfully in clinical setting.
Data regarding the role of NF-kappaB in resistance of MCF-7 cells to TRAIL is appropriate
and could be published with minor alterations. Data regarding the role of TRAIL-R4 is less
convincing. Authors investigated a single cell line that is resistant to TRAIL and demonstrated
that expression of TRAIL-R4 is high.. On the basis of this observation they claim that TRAILR4 expression correlates with TRAIL resistance. In my opinion, this claim is not justified. To
suggest correlation, the authors need to at least show low expression of TRAIL-R4 in TRAILsensitive cells. It would seem appropriate to just state that TRAIL-R4 is highly expressed in
MCF-7 cells and to confirm its role in TRAIL resistance (for example, by suppressing its
expression with siRNA) in the future work. Other minor concerns are summarized below:
1. MCF-7 is referred to as "advanced breast cancer cell line". In general, it is not
considered "advanced". As it does not matter much for this manuscript, I would omit
this description
2. The "NF-kappaB promoter based Luciferase system" needs a better description. I
assume it has NF-kappaB binding sited cloned upstream of the Luciferase coding
sequence, but it is not mentioned. It will also help to know the number of the binding
elements.
3. It would be useful to determine expression of the adenovirally-delivered proteins
under different experimental conditions (e.g. single infections, co-infections) by
Western blotting
4. The Annexin V data would be easier to interpret if the proportion of positive cells
was provided for each condition
Response to Reviewer 1:
We agree with the reviewer’s comments on TRAIL-R4. We have done further assays and
incorporated essential data on MDA-BM-231 breast cancer cell line onto the result section.
This cell line expresses low levels of TRAIL-R4 and was sensitive to TRAIL. One new figure
with three panels was added to the manuscript. These changes are also applied to the
discussion. We strongly believed that this result considerably strengthened our argument.
Other minor modifications asked by the reviewer are as follows:
1- The reviewer asked us to omit the “advanced” term from breast cancer. Even though
this was not the main point in the paper as accepted by the reviewer, but still we took
out all the “advanced” terms from our manuscript.
2- NF-kB promoter based Luciferase system is better described in the revised version of
the manuscript as asked by the reviewer. This change is incorporated into NF-kB
transcription activation assay under Materials and Methods section. We need to state
that AdNFkBLuc has already been described in detail as referenced in the manuscript
[1].
3- We always confirm the transgene expression from adenoviral vectors following the
making of a viral prep. The expression of IKKKA was confirmed by western blotting
and the expression of TRAIL was confirmed by flow cytometry. In order to avoid
concerns with viral doses we have always done dose titration assays testing multiple
doses to achieve an optimum effect. This in a way relinquishes the necessity of
performing expression analysis by western blotting. Even co-expression analysis
should not be required since TRAIL has a bystander effect. This means that as long
as cells are infected with AdIKKKA virus, the expression of TRAIL in that particular
cell is not required to observe an effect. Another words, TRAIL not only kills the cells
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infected with Ad5hTRAIL but also kills neighboring uninfected cells in which TRAIL is
not expressed. This is true as long as cell are sensitive to TRAIL.
4- Flow cytometry data are presented in different ways in manuscripts such as
histograms, bar charts, dot blots, density blots etc. Because we used a lot of different
constructs we found it easier to show our results in a histogram format rather than
providing just the numbers. In order to avoid confusion, each experimental parameter
is shown in a different color. Our figure clearly shows that significant proportions of
cells bind to Annexin V only when cells were coinfected with Ad5hTRAIL and
AdIKKKA revealing apoptosis. On the other hand, flow data on Figure 1 for example
are shown as bar graphs.
Reviewer 2
The paper submitted by Sanlioglu et al. shows strong data supporting a role for NFkB
activity in MCF-7 cells resistance to TRAIL, for this part of the paper the referee has only
minor comments (see remarks 1 to 3 bellow). In the second part of the paper a correlation
between TRAIL-R4 expression and TRAIL resistance in MCF-7 cells is shown, this last
part should be improved to provide more data about the importance of TRAIL-R4 in
TRAIL resistance (see remark 4. bellow).
1. The MCF-7 breast cancer cell line has been shown to be sensitive to recombinant
TRAIL treatment (Munoz-Pinedo, C. et al. J Biol Chem. 2003) (Ruiz-Ruiz, C. et aL
Biochem J. 2002) (MacFarlane, M. et al. J Biol Chem 2002). Since cells lines can
vary from lab to lab, the authors should comment whether this cell line is also
resistant to recombinat TRAIL or not.
2. Results from figure 3 and 4 are similar but just with two different MOI for Ad5hTRAIL,
these two figures should be combined in one.
3. In the text, page 13, the authors state that IKKßKA overexpression in MCF-7 cells
gradually reduced both the TRAIL-induce and basal NFkB activities and address the
reader to figure 5. In figure 5 A and B then, an additional column should be included
to display the results for the dowregulation of basal NFkB activity by IKKßKA
overexpression.
4. The authors should demonstrate the importance of the correlation of TRAIL-R4
expression on cell surface and TRAIL resistance:
a. Because TRAIL-R4 is a decoy receptor for TRAIL, is the resistance of these
cells to TRAIL due to TRAIL-R4 expression?. The authors must show
whether MCF-7 cells become more sensitive to TRAIL-induced apoptosis by
blocking TRAIL-R4 receptor at the cell surface.
b. Since there are contradictory results about whether TRAIL-R4 is capable to
activate NFkB upon biding to TRAIL (Harper, N. et al. J Biol Chem.
2001)(Degli-Esposti, MA. et al. Immunity 1997) it is important to show if
TRAIL-R4 activates NFkB upon biding to TRAIL in MCF-7 cells. This could be
done by blocking, in infected MCF-7 cells with AdNFkBLuc, the other TRAIL
receptors that have been shown to activate NFkB (TRAIL-R1 and TRAIL-R2)
and then checking NFkB activity after TRAIL treatment.
5. Reference 27 should be corrected.
Response to Reviewer 2:
This referee states that we have a strong data supporting a role for NFkB activity in MCF-7
cells resistance to TRAIL and he has only minor comments for this part. He wants the
connection between TRAIL-R4 expression and TRAIL resistance solidified with further
assays. In connection with this:
1- The referee asks if we could comment on whether MCF7 cell line is resistant to
soluble TRAIL or not. We did not speculate anything about the efficacy of soluble
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TRAIL on MCF7 cells in our manuscript. We believe that experiments conducted
using soluble form of TRAIL may not necessarily match experiments conducted using
viral vectors. There are even discrepancies just based on the source of TRAIL
purified. For example, the efficacy of yeast purified TRAIL was different than that of
bacterial purified form of TRAIL. These discrepancies have been reported before.
Contrary to the references cited by the referee, there are also reports which
suggested that MCF7 cells were found to be resistant to soluble form of TRAIL. One
such report was published by Keane et al [2]. This is in accordance with our results.
Our manuscript deals with the efficacy of adenovirus delivered TRAIL on breast
cancer cells.
This referee asks if we could combine Figure 3 and 4 as one figure. This is possible,
but considering panel size (duplicate fluorographs) and graphs in them, we believe it
is better to have them as separate figures. This is necessary for clarity purposes.
The reviewer asks that In figure 5 A and B, an additional column should be included
to display the results for the dowregulation of basal NFkB activity by IKKßKA
overexpression. We believe that there is misunderstanding here in this criticism.
There is no need for extra columns to show that AdIKKKA expression down
regulated basal levels of NF-kB activity, it has already been shown in the Figure itself.
As seen in Figure 5, endogenous NF-kB activity was measured around 600 RLU/g
protein. Even though rest of the columns has Ad5hTRAIL infected, only AdIKKKA
infection but not AdCMVLacZ takes down NF-kB activity to 200 RLU/g protein. This
implies that basal levels of NF-kB activity have also been down regulated by IKKKA
expression.
The referee asks that the authors should demonstrate the importance of the
correlation between TRAIL-R4 expression and TRAIL resistance. There are three
ways to demonstrate the importance of TRAIL-R4 in TRAIL resistance. One is to use
blocking antibodies against TRAIL-R4, the other is to use siRNA approach to lower or
inhibit TRAIL-R4 expression, and the last one is to find another breast cancer cell line
naturally displaying low TRAIL-R4 expression on surface. The last option was also
important for testing of our hypothesis in more than one breast cancer cell line
(MCF7). For this reason, we have done several assays which demonstrated that low
levels of TRAIL-R4 expression correlated with TRAIL sensitivity in MDA-MB-231
breast cancer cells. MDA-MB-231 and MCF7 cells both displayed significant levels of
death receptor expression (TRAIL-R1 and TRAIL-R2) on surface. None of these cell
lines expressed detectable levels of TRAIL-R3 on surface. Thus, the only difference
between these two cell lines was the level of TRAIL-R4 expression which was
documented using Real Time RT-PCR and flow cytometry assays. With these data
(Figure 8), we can clearly state that high levels of TRAIL-R4 expression is correlated
with TRAIL resistance in MCF7 breast cancer cell line. The other comment that the
referee asks the proof whether or not TRAIL-R4 is capable of activating NF-kB has
already been published. We did not claim that there were conflicting reports on NF-kB
activation by TRAIL-R4 in our manuscript. This is referee’s statement. What we stated
in our manuscript is that our observation is consistent with a previous report
suggesting that transient TRAIL-R4 overexpression protected target cells from TRAIL
induced cytotoxicity [3]. TRAIL R4 is known to protect cells from apoptosis by acting
both as a decoy receptor and an antiapoptotic signal provider. There are significant
numbers of publications in Pubmed which supports this. These results were also
supported by studies indicating that TRAIL sensitive target cells transfected with
either TRAIL-R3 or TRAIL-R4 decoy receptors displayed a reduction in apoptotic cell
death [4, 5] Intriguingly, TRAIL-R4 decoy receptor was more effective in protecting
cells from TRAIL mediated apoptosis than TRAIL-R3.[4] These last two publications
came out in Science. Nevertheless, studying TRAIL-R4 activation of NF-kB pathway
is out of the scope of this current manuscript. Lastly, regardless of NF-kB activation
TRAIL-R4 can still serve as a decoy receptor.
Reference 27 has been corrected. We would like to thank the referee for this
correction.
Reviewer 3
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In this study TRAIL receptor expression and TRAIL resistance in MCF7 breast cancer cells
was investigated. Exogenous expression of TRAIL was achieved by adenoviral infection of
MCF7 cells with Ad5hTRAIL. The effects of TRAIL activation and/or IKKß inhibition by
adenoviral expression of a dominant negativ mutant (IKKßKA) on NF-kB activation/
expression and apoptotic cell death determined by AnnexinV binding was analysed. Results:
MCF7/ Ad5hTRAIL transfectants do not display increased cell death. NF-kB expression was
slightly increased (about 30%) in MCF7/ Ad5hTRAIL transfectants and reduced NF-kB
expression was determined in MCF7/ Ad5hTRAIL/ AdIKKßKA transfectants as well as
increased apoptotic cell death shown by cell viability/cytotoxicity assays and AnnexinV
binding. Finally, TRAIL receptor expression (TR-1, TR-2 and decoy receptors TR-3, TR-4)
was determined in MCF7 cells: FACS analysis displays high TR-1/TR-2-, “substantial” TR-4
and no TR-3 expression whereas TR-4 expression was the highest and TR-1 expression was
low on the transcript level. The paper provides some interesting results but in my view
interpretations of results are not convincing and not sufficiently supported by the data.
Major critisisms:
Several papers describe the lack of caspase 3 activity in MCF7 cells which is not considered
or discussed by the authors. In view of the central roles of caspase 3 as the main caspase
(beside caspase 6, 7) in excecuting apoptosis signalling as well as the caspase-3 mediated
proteolytic inactivation of IKKßB, loss of NF-kB activation and concomitant loss of antiapoptotic gene expression has to be considered in the interpretation of the results.
A decrease of the viability of MCF7 cells was not observed in Ad5hTRAIL transfectants which
was interpreted as TRAIL resistance. This should be further supported by experiments
proving the “functionality” of TRAIL delivered to the cell after Ad5hTRAIL infection (TRAIL
expression and expression analysis of TR-1/TR-2 dependent target genes) and compared to
the exposure of MCF7 cells to increasing amounts of recombinant soluble TRAIL or TRAIL
agonistic TR antibodies.
Figures 3 and 4 summarize the results concerning cytotoxic effects of Ad5hTRAIL /
AdIKKßKA co-infection. Cytotoxicity was seen with 5000 MOI AdIKKßKA/ 1000 MOI
Ad5hTRAIL (Figure 3) and with 100/500 MOI AdIKKßKA/ 5000 MOI Ad5hTRAIL (Figure 4)
and it was concluded that inhibition of NF-kB activation sensitizes MCF7 cells to TRAILinduced apoptosis. Again, this results should be reproducible with recombinant soluble
TRAIL (or TRAIL agonistic TR antibodies). To exclude the possibility that this effect only or in
part depends on the increase of MOI s in combination with the block of NF-kB activation, cell
viability should be analysed in AdCMVLacZ/ AdIKKßKA transfectants as a negativ control.
In Figure 5 the inhibiting effect of the dominant negativ mutant IKKßKA on NF-kB activation is
clearly demonstrated in MCF7 cells confirming the results of cited papers (34, 36). However,
reduction of basal NF-kB activation was only seen with MOI 5000 AdIKKßKA whereas the
increased NF-kB activation in Ad5hTRAIL transfectants (MOI 5000) was already seen with
MOI 1000 AdIKKßKA. How can this be explained. In addition, the point of the triple
transfectant Ad5hTRAIL / AdCMVLacZ / AdNFkBLuc as a negativ control is not clear.
Figure 6 demonstrates that cell death of AdIKKßKA/ Ad5hTRAIL transfected MCF7 cells is
associated with AnnexinV binding and apoptosis. As mentioned above this should be shown
also for recombinant soluble TRAIL / TRAIL agonistic TR antibodies and AdCMVLacZ/
AdIKKßKA transfectants should be used as an additional negativ control.
Figure 7 represents the investigation of expression levels of the different TRAIL receptors.
The quantification of TRAIL receptors on the protein level by FACS analysis is not described
and the differences of RT-PCR and FACS data of TR1 as well as TR4 expression is not
discussed.
A “considerable” level of TR4 expression in a TRAIL resistant cell line do not sufficiently
support the conclusion that TRAIL resistance is “well correlated” with TR4 expression.
Therefore, the titel of this paper is only speculativ. To demonstrate a correlation of TR4
expression and TRAIL resistance a representative collection of (breast) cancer cell lines
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should be analysed or, much better, TRAIL resistance should be investigated in TR4 shRNA
knock down experiments.
Response to Reviewer 3:
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This reviewer suggested that we should discuss the importance of the lack of
caspase 3 expression and the outcome on NF-kB activation. However we have no
data on caspase 3 in our manuscript and we have not studied it, we did not even
mention it. Furthermore, restoration of caspase 3 gene expression did not influence
the cell viability of MCF7 breast cancer cells [6]. While we fully respect reviewer’s
comment, we would rather like to avoid making such predictions in our manuscript.
This reviewer asked us to confirm the functionality of TRAIL delivered by adenovirus
vectors. The expression of TRAIL from adenovirus construct has been confirmed by
flow cytometry as a routine viral prep preparation procedure. More importantly, the
addition of Figure 8 Panel C which shows that Ad5hTRAIL alone but not AdCMVLacZ
kills MDA-MB-231 breast cancer cells proves the functionality of Ad5hTRAIL vector.
While a significant degree of cell death was observed with Ad5hTRAIL in MDA-MB231 cells, no such degree of cell death was observed in MCF7 cells.
- This referee states that the TRAIL induced cell death under NF-kB inhibition
(Figures 3 and 4) should be reproducible using soluble TRAIL as well. Theoretically
speaking this might be what is expected, practically however, this may or may not
happen. But as described above, results obtained using soluble TRAIL may not
match to that of delivered by adenovirus vectors. There are even differences between
yeast purified TRAIL and bacterial purified form of TRAIL in terms of the efficacy.
Under these circumstances we believe that the results are not essential for the sake
of our argument. Not to forget, we are assessing the efficacy of adenovirus delivered
TRAIL not soluble purified form of TRAIL as a gene therapy approach. This reviewer
also asks in order to rule out high MOI values as causative agents in cell death, cell
viability should be analyzed in AdCMVLacZ/ AdIKKßKA transfectants as a negative
control. Actually this was done and the following statement was present on page 13 in
the manuscript. “On the other hand, AdCMVLacZ infection instead of AdIKKKA in
breast cancer cells revealed no TRAIL sensitization (data not shown).”
The reviewer states that “reduction of basal NF-kB activation was only seen with an
MOI 5000 AdIKKßKA whereas the increased NF-kB activation in Ad5hTRAIL
transfectants (MOI 5000) was already seen with MOI 1000 AdIKKßKA. “
Unfortunately, we do not understand what this question asks. We can only state that
MOI of 1000 DNA particles/cell of AdIKKKA virus was sufficient to decrease TRAIL
induced NF-kB activation only when MOI of Ad5hTRAIL was 5000 (Figure 5 Panel B).
With an MOI of 1000 of Ad5hTRAIL we did not observe significant levels of NF-kB
activation (Figure 5 Panel A). The reviewer also asks us to clarify the point of doing
triple infection (Ad5hTRAIL / AdCMVLacZ / AdNFkBLuc) as a negative control. The
point of doing this particular infection was to show that decrease in NF-kB activity
provided by AdIKKKA infection was not due to the adenovirus itself (adenoviral
proteins might influence NF-kB activity) but rather due to IKKKA expression. So this
served as negative control.
This reviewer asks us to perform Annexin V binding assays using soluble TRAIL as
well. As above mentioned reasons, we believe that this will not add anything more to
our paper. The reviewer also asks us to use AdCMVLacZ/ AdIKKßKA as a negative
control in Annexin V binding assay. Proper control for Ad5hTRAIL and AdIKKKA
coinfection is Ad5hTRAIL and AdCMVLacZ. The reason for this is because in order
to show that under the setting of NF-kB inhibition (IKK inhibition) TRAIL induces
apoptosis, we have to replace AdIKKKA with AdCMVLacZ. This means that when
NF-kB is not inhibited Ad5hTRAIL does not induce apoptosis. Furthermore any
reduction in cell viability observed in Ad5hTRAIL and AdCMVLacZ will be due to
adenovirus itself. These are our accepted proper controls.
The referee states that a description on flow cytometry procedure is not given and the
differences of RT-PCR and FACS data of TR1 as well as TR4 expression are not
discussed. We have incorporated flow cytometry procedure into Materials and
Methods section. We have included sufficient statements on RT-PCR and FACS data
in the discussion. Giving detailed descriptions were not necessary. We already stated
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that gene expressions on transcript levels may not correlate with protein expression
on surface in the discussion. Some of the differences on TRAIL-R1 and TRAIL-R4
can be deduced from the Figures as well. We only pointed out the major differences.
The referee mentioned that a correlation of TR4 expression and TRAIL resistance
should be demonstrated in a representative collection of (breast) cancer cell lines or
much better, TRAIL resistance should be investigated in TR4 shRNA knock down
experiments. We agree with the reviewer’s comment. Therefore we analyzed another
breast cancer cell line namely MDA-MB-231. This cell line exhibited lowest levels of
TRAIL-R4 RNA transcript on Real Time RT PCR assay (Figure 8 Panel A). Flow
cytometry analysis indicated that TRAIL-R4 expression was very low on the surface
and more interestingly this cell line displayed TRAIL sensitivity. This strengthens our
argument on TRAIL-R4 receptor expression and its connection to TRAIL resistance.
These results relinquish the necessity of doing siRNA assays since the purpose of
doing siRNA is to lower or inhibit TRAIL-R4 expression on surface. Instead we found
a breast cancer cell line which naturally displays TRAIL receptor expression profile
similar to that of MCF7 on surface with the exception of TRAIL-R4. We hope that our
additions will satisfy the reviewer’s comments.
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