Response to reviewer comments:
We would like to thank the reviewer for providing most helpful comments on our manuscript. Please
find below an answer and clarification to each of the points raised.
Reviewer #1:
Comment 1: The article by Banerjee et al. focused on the expression and OATPs, STS and 17betahydroxysteroid dehydrogenase type-1 in hormone-dependent and -independent breast cancer. The
results are of some interest, although there are a lack of functional studies that would have provided
much needed insight into E1S transport and metabolism.
Response: We appreciate the reviewer finding the study interesting. To the best of our knowledge the
comparative clinical expression of the OATPs among hormone-dependent and -independent breast
cancer tissues has not been previously reported. These data provide further understanding of the role of
the transporters (i.e. OATPs, BCRP, MRP-1) and enzymes (STS, 17β-HSD1) in E3S uptake and metabolism,
respectively, in hormone dependent breast cancers. The expression of the OATPs, STS and 17β-HSD-1
were determined in sections obtained from 40 tumour blocks. We agree with the reviewer that
additional functional data in the clinical sample would be valuable. However, we would like to
emphasize that it was extremely challenging to obtain these human breast cancer tissues. The Research
Ethics Board only approved the use of 8-9 (3-5µm thick) sections from each block. Hence although
initially planned, we were not able to conduct functional transport/enzyme assays using the clinical
samples. In order to address this limitation, we conducted functional transport experiments in hormone
dependent (MCF-7, T47D, ZR-75) and independent (MDA-MB-231) breast cancer cells in the presence or
absence of inhibitors of OATP [(100µM Bromosulphophthalein (BSP)] and STS (20 μM STX64) activity.
Figure 8, in the manuscript (shown below) demonstrates the specificity of OATP mediated E3S uptake
and STS mediated metabolism in hormone dependent breast cancer cells as observed by the significant
reduction in E3S cellular uptake, and, increased cellular accumulation, of E3S in presence of BSP and
STX64, respectively. As there was no significant difference observed in 17β-HSD-1 expression between
the hormone dependent and independent clinical breast cancer samples, E3S cellular accumulation was
not investigated in the presence of a 17β-HSD-1 inhibitor.
To clarify this point we added the following statement to the manuscript:
Section: Introduction; Page: 4 paragraph 2
“It would be valuable to assess functional data in the clinical sample to compare the function of these
transporters and enzymes in HR+ and HR- tumour tissues. However, it was extremely challenging to
obtain these human breast cancer tissues. Hence, to better understand the observed differences in
expression of these transporters and enzymes in the HR+ and HR- clinical tissues, the functional roles of
OATP (in E3S uptake) and STS (in E3S metabolism) were examined in HR+ (MCF-7, T47D, ZR-75) and HR(MDA-MB-231) breast cancer cell lines.”
Figure 8: Time course of [3H] E3S uptake by HR+/- breast cancer cells. Total uptake (closed circles) of E3S
by the cells was evaluated over 30 min at pH 7.4 and 37˚C. The non- specific uptake (closed squares) and
the non- specific metabolism (closed diamonds) were calculated by determining uptake in the presence
of an excess concentration of transport inhibitor (BSP 100µM) and enzyme inhibitor (STX64 20mM) as
described in the Materials and Methods section. A. MCF7, B. T47D, C. ZR75 and D. MDA-MB-231 cells.
*p<0.05 is considered to be statistically significant.
Comment 2: The reviewer questions the novelty of some of this work, especially with regards to the
STS/17b-HSD-1 work, and the published work from our group on OATP expression in MCF-7 and MDAMB-231.
Response: We agree with the reviewer that clinical expression of STS [1,2] and 17β-HSD-1 [3] has been
previously reported. The novelty of this study was to compare the expression of the OATPs among the
early and advanced stages (determined by lymph node status) of hormone dependent and independent
breast cancer tissues. As STS and 17β-HSD-1 have already been identified as a therapeutic target for
hormone dependent breast cancers, we wanted to include them in our study to compare the expression
patterns of STS to that of the OATPs. As the expression patterns (i.e. significantly higher expression in
hormone dependent breast cancers) are similar between STS and OATPs, this helped establish that
similar to STS, OATPs are a potential novel target for hormone dependent breast cancer patients. We
have addressed this in the manuscript in the “Introduction” section on page 4: paragraph 2
“While STS and 17β-HSD-1 are currently considered therapeutic targets for ER+ breast cancers, this
study helps to elucidate the potential of OATPs as novel molecular targets for breast cancer and
provides a better understanding of the intra-tumoral fate of E3S.”
OATP expression has been previously reported by our group in hormone dependent (MCF-7) and
hormone independent (MDA-MB-231, MDA-MB-468 and MDA/LLC-435) breast cancer cells. However,
expression of OATPs had not been previously studied in clinical breast tumour tissues. Additionally, this
study includes expression of OATPs in T47D and ZR75, two hormone dependent breast cancer cells that
had not been previously investigated. This further confirms that expression of OATPs (i.e. OATP1A2,
OATP2B1, OATP3A1 and OATP4A1) is indeed significantly higher in the hormone dependent breast
cancer cells as compared to the hormone independent cells. Figure 7 of the manuscript (as shown
below) demonstrates the significantly higher expression of the OATPs and STS in the hormone
dependent cells, thereby supporting the use of these cells as a surrogate for conducting functional
studies.
Figure 7: Immunoblot and densitometric analysis of OATP transporters and STS enzyme in HR+ and HRbreast cancer cells. Protein expression of OATP1A2 (A), OATP3A1 (B), OATP4A1 (C) and STS (D) was
determined in HR+ (i.e. MCF-7, T47D, ZR-75) and HR- (i.e.MD-MB-231) breast cancer cells applying
standard western blot analysis as described in the Material and Methods section Results of the
densitometric analysis are expressed as mean ± SD of three separate experiments (1: positive control; 2:
MDA-MB-231; 3: MCF-7; 4: ZR-75; 5: T47-D). *** p<0.001, ** p<0.01 and *p<0.05 are considered to be
statistically significant.
Comment 3: Overall, the article results are very descriptive and the relative function of the OATP in
hormone-dependent and - independent is not examined. It would have been very interesting to have
done functional uptake studies in the various cell lines whilst transiently/stably knocking down
specific OATPs to examine the functional relevance of the various OATP/BCRP/MRP1 in E3S
influx/efflux in breast cancer cell lines.
Response: The function of the OATPs were compared among the hormone dependent (MCF-7, T47D
and ZR-75) and independent (MDA-MB-231) breast cancer cells (as shown in Figure 8) and specificity of
OATP mediated E3S uptake was observed in the hormone dependent breast cancer cells. We agree with
the reviewer that it would be of additional value to understand E3S transport kinetics for each specific
OATP isoform that were investigated. However, the scope of this study was primarily to determine the
differences in clinical expression of OATPs in tissues obtained from hormone dependent and
independent breast cancer patients, and determine the level of OATP expression specifically in the
tumor cells. If availability of additional human breast cancer tissues is possible, in the future, we could
conduct knock down experiments for each of the OATPs in the breast cancer cells and tissues.
Comment 4: Furthermore, with regards to the STS data, it is widely known that protein levels of STS
do not necessarily correlate to activity, and therefore STS activity studies (on both tissue sections and
cell lines) would have been more informative. As little is known about what regulates STS and 17bHSD-1 activity, with some thoughts suggesting perhaps post-translational modifications might be
involved, the immunohistochemical/WB, although of some interest, should be backed up with
enzyme activity assays.
Response: STS inhibitor, STX64 was used to compare E3S accumulation in the breast cancer cells in the
presence or absence of STS activity. Increased cellular accumulation of E3S in the presence of STX64
suggests the role of STS in intra-cellular metabolism of E3S (as shown in Figure 8). As E3S is lipophobic,
unlike estrone or estradiol, it is unable to diffuse out of the cell membrane. We agree with the reviewers
that protein expression is not always reflective of function. Hence, we conducted cellular assays in
presence of STS inhibitor.
While it will be interesting to further understand the regulation of STS and 17β-HSD-1, we believe that
this aspect will require extensive additional experimental work and is beyond the scope of this study.
Comment 5: The authors do not fully explain why addition of STX64 (Irusostat) results in an increase in
E1S-uptake. From my understanding, inhibition of STS will block E3S de-sulfation, leading to a greater
accumulation of intracellular E1S, as this is lipophobic. Do these results therefore imply that
BCRP/MRP1 are not effluxing E1S? Again, some functional studies, perhaps by knocking-down
BCRP/MRP1, would have been useful.
Response: We thank the reviewer for this comment. He/she appreciates the rationale for using STX64.
As it is an inhibitor for STS activity, increased cellular accumulation of E3S is observed in presence of
STX64.
We planned on using transport inhibitors for BCRP/MRP-1 in presence of STX64 to delineate the role of
the efflux transporters, unfortunately the challenge is that the transport inhibitors used for inhibiting
BCRP and MRP transport [i.e. (3S,6S,12aS)-1,2,3,4,6,7,12,12a-Octahydro-9-methoxy-6-(2-methylpropyl)1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), N-(4[2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4acridine carboxamide (GF120918, elacridar) or 3-[[[3-[(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-
(dimethylamino)-3-oxopropyl]thio]methyl]thio]propanoic acid (MK571)], are also inhibitors for OATP
transport. Hence in the presence of BCRP/MRP inhibitors, cellular uptake of E3S was low and nonspecific and the data was inconclusive.
This has been addressed in the manuscript in the “Discussion” section on page 11 paragraph 1
“We acknowledge that the tumoral fate of E3S could be better understood if the functional role of the
efflux transporters could also be established. However, cross reactivity between the inhibitors for BCRP,
MRP-1 and OATPs make it technically challenging to ensure OATP mediated cellular uptake in the
presence of BCRP and MRP-1 transport inhibitors.”
Comment 6: Finally, there are various typos within the document, and a lack of consistency regarding
English/American language (estrone and sulphate).
Response: We thank the reviewer for pointing this out. We have now conductrd a very thorough review
of the spelling of the text and throughout the document we have now maintained consistency in using
“estrone” and “sulphate/sulphatase”.
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