1
August 24, 2012
POINT BY POINT LETTER
Article Title: PCA3 noncoding RNA is involved in the control of prostate-cancer cell
survival and modulates androgen receptor signaling
MS: 5252293497320221
Response to reviewers:
We thank the reviewers for their latest comments on our revised manuscript.
Considering the additional suggestions from one of the reviewers, in the current point-bypoint letter we have answered and discussed some points and appropriately modified the
main manuscript, which is being sent as a newly revised version. We also made an
additional English review. Modifications are highlighted throughout the revised
manuscript. Those highlighted in yellow relates to some reviewers’ specific requests, while
those highlighted in magenta results from new added references or changes on reference
numbering, according to reviewers’ suggestion. The only request that we did not accede to
relates to performing additional experiments in other androgen-responsive cell lines, and
the reasons for this were extensively and appropriately explained in the previous point-bypoint letter. We invite the editor to request an additional final revision, especially
considering that one of the reviewers accepted our first revision and considered that our
responses to the requests were satisfactory. As explained in the attached cover letter,
performing these additional experiments would further delay the publication of these data
for at least five more months. We also judge, as explained in the previous point-by-point
letter, that these additional data will not necessarily provide enough evidence to further
validate our results, which demonstrate the involvement of PCA3 in prostate-cancer cell
survival and androgen-receptor signaling. The reasons why we believe this were presented
previously. In our opinion, the manuscript has been greatly improved by these additional
revisions, and we hope that you will now find it suitable for publication in the journal
2
“BMC Cancer”. Please find our point-by-point responses to comments on the following
pages.
Queries/critiques are numbered and in blue Arial font. Responses follow in black
Times Roman font. Revised prose from within the manuscript is in red Times Roman font.
Major Compulsory Revisions
Point 1:
1) “In the attempt to improve the language, in the first paragraph of Results the
authors changed the sequence in which the experiments are presented.
Specifically, in the present re-submission of the paper the authors claim that they
first performed in vitro PCA3 knockdown assays in prostate and non prostate cell
lines and only then they evaluated PCA3 expression levels in such cell lines, thus
making the report lacking of logical connections. Thus, the authors are invited to
correct such an incongruity.
Furthermore, the manuscript needs a deeper language revision. For example,
the sentence "Our data indicated the LNCaP the most suitable cell model to
investigate the PCA3 function in PCa cell biology by using small interfering RNA
to silence this ncRNA, which is upregulated in these cells, in comparison to the
remaining prostate cells evaluated" sounds bad to me; "...cell transfection of with
siPCA3..." (page 15-line 7) is incorrect...
We sincerely thank the reviewers for these comments. In this second revised version
we made an additional language revision, which is highlighted throughout the manuscript.
The reviewer is absolutely right when mentioning the error regarding first mentioning the
knockdown experiments and then the expression analysis assays. After English corrections
were made, we wrongly changed the previously submitted sentence. Now the correct
3
information has been included, as shown here and also included in the second revised
manuscript:
“ …we first analyzed the PCA3 transcript expression in different prostate (LNCaP, PrEC,
RWPE-1, DU145 and PC3) and non-prostate (NIH3T3 and HeLa) cell lines. We analyzed
the PCA3 transcript levels in order to determine which was the most appropriate model for
further analysis.”
In relation to the second sentence, we now corrected it and this second revised
version now includes the following phrase:
“Our data have indicated LNCaP cells as the most suitable model to investigate the PCA3
function in PCa biology by using small interfering RNA to silence this ncRNA, which was
upregulated in these cells when compared to other tested cells lines.”
On page 17, we also made the recommended correction, which is now:
“Our first approach to investigate the putative roles of PCA3 in PCa biology was to
determine LNCaP cell growth rates by using crystal-violet assays over a 100-h time course
after LNCaP cell transfection with siPCA3 (Figure 2A).”
Point 2:
2) The authors did not perform the main experiments in another
androgen-dependent cell line as suggested to validate the functional role of
PCA3 in prostate cancer biology. They commented that different PCa cell lines
and sublines might display different behaviour after exposure to the same
treatments and that is true in general (not only for PCa cell lines) but that is not a
good reason not to try. Even if the role of PCA3 would result different for any
other androgen-responsive cell line the authors could reason, comment and
justify such eventual results. Since, they also observed that the experiments
required need intense efforts and probably long time to be performed, I suggest
to give them more time to satisfy the request.
4
Again we thank the reviewer for these comments. However, in our opinion, we have
already extensively and appropriately explained why we did not perform these experiments,
and why new data would not affect our interpretation regarding the role of PCA3 in
modulating AR signaling. In addition, as explained before, performing these assays would
enormously delay the publication of these unprecedented results regarding the
characterization of the role of this important prostate-specific ncRNA. Further emphasizing
the need to promptly publish these results, we inform the editors of BMC Cancer that these
data were recently presented orally at international scientific meetings that focused
especially on ncRNAs and their role in cancer biology. In these events, other groups that
are also attempting to describe the role of PCA3 in prostate-cancer cells had access to our
developed strategy and data. Considering the opposite decision made from the two
reviewers, we invite the editors to request an urgent third opinion and a reconsideration of
the reasons for not performing these assays as a prerequisite to publish this paper.
Point 3
3) Furthermore, I suggest to reserve a separate paragraph (for example at the
end of Results) to describe all the experiments carried out on PC-3 cell line by
explaining (as introduction to the paragraph) the rationale for the use of PC-3
cells. In the same context, they are invited to extensively discuss about the
expression, the function and the regulation of Androgen Receptor in PC-3 cells.
Thanks to the reviewer for this additional comment. Reserving a separate section for
presenting PC3 experiments would necessitate an extensive change in the structure of the
paper. We prefer to maintain the present organization, using specific sections, in
association with the LNCaP experiments. We first describe the analysis of PCA3
expression in PC3 cells, on page 13. Then, we present the effects of PCA3 knockdown in
PC3 cells, demonstrating downregulation of PCA3 after using the siPCA3 molecule (page
14). Finally, we present the effects of PCA3 knockdown on PC3 viability, on page 16.
5
Although we already provided explanations for our use of PC3 cell assays to further
determine the role of PCA3 in PCa cell survival, on pages 14 and 16 of the previously
submitted manuscript, we expanded and detailed the rationale for performing these assays
in PC3 cells in the Discussion section (on pages 23 and 24 of this newly revised version),
as requested by the reviewer. In our opinion, this is the best manuscript section to reinforce
this information. Text including this new information is copied herein, in addition to data
regarding AR expression, function and role in PC3 cells, according to the reviewer’s
recommendation.
“In order to further emphasize the role of PCA3 in modulating PCa cell survival, we
also investigated the effect of PCA3 knockdown in an androgen-independent PCa cell line,
which simulates a more aggressive PCa disease. However, conflicting results have been
reported regarding the androgen independence of these cells andAR protein expression in
the PC3 and DU145 cell lines, as well in castration-resistant prostate cancer tumors [36,37].
Although the majority of human prostate-cancer cell lines are reported to be AR-negative
[38,39], several studies have indicated that the DU-145 and PC-3 prostate-cancer cell lines
express detectable levels of the AR mRNA [40-44]. For this reason, the exact role of AR in
PC3 cells is still controversial [36]. Although PC3 cells showed lower PCA3 expression
than did the LNCaP cells, we asked whether PCA3 also regulates cell survival in these
cells. Considering the heterogeneity of PCa tumors regarding gene expression profiling and
tumor progression behavior, we then wanted to evaluate survival features after PCA3
knockdown even in cell lines that express low PCA3 levels, and that in principle are
androgen-independent. Since PC3 is a well-established cell-line model representing a more
aggressive stage of prostate cancer tumors, our data provide additional evidence for a role
of PCA3 in modulating PCa cell survival. We also wanted to investigate how these
6
androgen-independent cells would respond to PCA3 knockdown. Our results demonstrated
that PCA3 knockdown, as demonstrated by the significant decrease in PCA3 levels after
transfecting PC3 cells with the siPCA3 molecule, increased the proportion of cells with
pyknotic nuclei (compared to PC3 transfected with siScr control), which was indicative of a
larger number of cells undergoing apoptosis, further evidencing the role of PCA3 in
modulating PCa cell survival, even in an androgen-independent cell-line model that
expresses lower PCA3 levels
compared to LNCaP cells. However, how PCA3 can
modulate PCa cell survival in these cells and whether it is mediated by different
mechanisms rom those observed in siPCA3-LNCaP transfected cells, should be further
investigated.”
In accordance with these changes in the Discussion section and due to the inclusion
of additional references, the reference numbering has been changed in this second revised
manuscript. Changes are highlighted in magenta. Added references are highlighted in red
letters and after these included references numbering has been appropriately changes in the
reference list and at the second revised manuscript section.