Point-by-Point Response to Reviewers’ Comments
We appreciate the reviewers’ comments on our work entitled “CITED2 functions as a
molecular switch of cytokine-induced proliferation and quiescence”. We have
followed the reviewers’ comments to make appropriate changes in the paper. A
point-by-point response to the reviewers’ comments is described as follows.
Reviewer #1 (Reviewer Comments to the Author):
This manuscript by Chou et al. describes a series of experiments characterizing the
action of CITED2, a MYC-binding protein, with respect both to TGF-alpha induced
growth stimulation and to TGF-beta induced growth suppression. The authors
demonstrate in an extensive series of experiments that with respect to TGF-alpha,
production of CITED2 is induced in a manner that requires the EGF receptor, MYC is
a required positive regulator of CITED2 levels, CITED2 and MYC bind to each other,
CITED2 is a positive growth regulator that mediates E2F levels, and CITED2
transactivates MYC by recruiting p300. The authors also show that with respect to
TGF-beta growth suppression, TGF-beta downregulates CITED2 and there is up
regulation of p21CIPI via the enhanced interaction of MYC and HDAC1. Finally the
authors present data indicating the role of CITED2 in the growth of tumors in vivo as
well as expression in a panel of human tumors.
Overall the results are presented clearly and the hypothesis is supported by the data.
There are a few points to which the authors should address themselves, but in sum this
work would seem to be of interest to the readership.
Minor points
1. Some of the figures that contain more than one panel in a figure 4b, which contains
an upper and lower panel, require more description in the text in order for the reader
to know to which part of the figure the authors are discussing.
Response:
The upper (Western blot) and lower (Q-PCR) panels in original Figure 4b have
been separated into two subfigures as new Figure 4b and Figure 4c, respectively.
We have added more description in the text to make them clear.
“Since CITED2-silencing induced cellular quiescence of these cells (Figure 2, a
and b), we also examined whether CITED2 expression is affected by TGF-
stimulation. Upon TGF- treatment, nuclear CITED2 was decreased in A549
cells (Figure 4b). Furthermore, TGF- downregulated both MYC and CITED2
but induced p21CIP1 in A549 cells in a time dependent manner (Figure 4b). The
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Q-PCR data supported that TGF- induced MYC downregulation in A549 cells;
however, TGF-–mediated downregulation of MYC was not observed in H1975
cells (Figure 4c).” (Please see Page 8, line 20～Page 9, line 3.)
2. Figure 6d needs additional description in the text or figure legend. The reader is not
given any indication of what has really changed in some of the photos other than there
appears to be more staining, or more nuclear staining, or nothing.
Response:
Additional description is made in the text as follows:
“We found that CITED2, MYC and E2F3 were highly expressed in neoplastic
cells of lung xenografts (Figure 6d). In contrast, normal alveolar ducts and
alveoli were observed in the CITED2 knockdown xenografts, and high p21CIP1
expression was detected in normal alveolar cells (Figure 6d).” (Please see Page 12,
line 8～11.)
3. P.10, line 18 "others report" should be "other reports."
Response:
We have made the correction as suggested. (Please see Page 10, line 21.)
Referee #2 (Remarks to the Author):
Chou et al describe that CITED2 is a protein that controls MYC function in gene
transcription. They put forward a model in which CITED2, by directly binding to
MYC, regulates its association with either transcriptional co-activators or
co-repressors. What the molecular basis for this differential effect is remains open. In
addition the authors demonstrate that CITED2 mRNA and protein expression is under
the control of TGF-alpha and TGF-beta while the former activates, the latter represses
CITED2 expression. Moreover MYC also has the capacity to stimulate the CITED2
promoter by directly binding to E box sequences upstream of the start of transcription.
The authors suggest that CITED2 is important to mediate proliferation of epithelial
cells. Knockdown of CITED2 promotes a G1-S phase arrest while overexpression
overcomes the growth inhibitory activity of TGF-beta. A key effector in the cells
studied is p21CIP1. Its expression is enhanced by knockdown of CITED2, which
promotes senescence. Finally A549 tumor cells grow better in animals with enhanced
CITED2 levels, but are impaired when CITED2 expression is inhibited. Patients with
NSCLC have a considerably lower survival rate when the tumor cells express
CITED2, MYC and E2F3, while the differences are less pronounced when patient
survival was analyzed stratified for single proteins.
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Overall this is a nice paper in which a number of interesting findings are reported that
shed new light on how TGFs control cell proliferation and senescence.
Specific comments:
1. It is not clear whether the micrographs in Fig. 1a are confocal or not. Does CITED2
indeed enter the nucleus upon stimulation? It would be good to know how CITED2
distributes between the two compartments. The knockdown experiments would
suggest that also under non-stimulated conditions some CITED2 is nuclear. Also it is
unclear how good the antibodies against CITED2 are. Some control Western blots of
endogenous CITED2 would be good to see so that the reader can evaluate the
specificity of the antibodies used.
Response:
The original micrographs in Fig. 1a are not confocal. We have retaken
micrographs by confocal imaging (Figure 1a). To better understand how
CITED2 distributes between nucleus and cytoplasm, we isolated nuclei and
cytoplasmic protein fractions before and after cytokine treatment followed by
Western blot to monitor CITED2 distribution in the two compartments. We
found that CITED2 was predominantly localized in the nucleus of A549 cells and
TGF-stimulation further enhanced CITED2 expression in the nucleus (Figure
1a). (Please see Page 4, line 16～20 and Page 24, line 3～6.)
To compare the distribution of CITED2 between normal and CITED2
knockdown condition, A549 cells were treated with or without TGF- followed
by nuclei/cytoplasmic protein fractionation for Western blot analysis (Figure 4b).
Immunoblotting showed that CITED2 was mostly expressed in the nucleus and
TGF- stimulation effectively downregulated CITED2 expression in the nucleus
(Figure 4b). (Please see Page 8, line 21～22 and Page 27, line 7～9.)
2. The MYC knockdown is impressive, in fact this is the best knockdown I have ever
seen. Please indicate the precise sequence of the shMYC construct so that this can be
used by others.
Response:
The information now has been provided in the Supplemental Table S2.
3. In Figure 3 MYC, p300 and CITED2 cooperate in stimulating the expression of a
reporter construct. Several other groups have shown that p300 is sufficient to enhance
MYC transactivation. It is not clear why the findings here are different to published
observations. This should be discussed.
Response:
Feola et al. reported that p300 interacts with MYC, and MYC–p300 complex
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activates HTERT promoter 8-fold 1. In fact, our findings are consistent with this
report in that p300 enhances MYC transactivation of target promoters and that
MYC-p300 complex activates E2F3 promoter 7～8-fold (Figure 3e). Moreover,
we found that CITED2 expression boosted MYC-p300 mediated transactivation
of E2F3 promoter reporter to 25-fold (Figure 3e). Detail discussion is described
as follows:
“It is reported that MYC interacts with p300, and MYC-p300 interaction
enhances MYC mediated transactivation of hTERT promoter 1. Consistent with
this report, we observed that p300 enhanced MYC mediated transactivation of
E2F3 promoter, and CITED2 expression boosted MYC-p300 mediated
transactivation of E2F3 promoter (Figure 3e). CITED2 interacts with p300 2, and
MYC binds p300 through its N-terminus 1. In this study, we found MYC bound
CITED2 through its C-terminus, and knocking down CITED2 attenuated
MYC-p300 interaction. These findings indicate that CITED2 promotes
MYC-p300 complex formation and enhnaces MYC-p300 mediated
transactivation of E2F3.” (Please see Page 13, line 19～Page 14, line 3.)
4. It is unclear how the mRNA expression levels in Fig. 4c compare. Obviously it is
difficult to compare mRNA levels between different cell types because control
mRNAs may also differ. Here it would be worthwhile to determine copy numbers.
Also protein expression by FACS could be analyzed to validate the statements made.
Moreover it is unclear how extensive the methylation of the TGFBRII promoter is.
Are these the relevant CpGs that give a good readout of the expression of the
promoter?
Response:
a. Previously, we have used relative quantitative real-time PCR (Q-PCR) to
compare the relative expression of TGFBRI and TGFBRII in A549, H1975
and CL1-0 three cell lines. Though a number of reports used relative Q-PCR
to calculate differential gene expression between different cells or tissues 3, 4,
we have followed the reviewer’s suggestion to quantitate gene expression by
calculating the copy numbers of TGFBRI and TGFBRII transcript in three
different cell lines based on the method described by Warner et al.5 Data
normalization is carried out against an endogenous unregulated reference
gene transcript 18S rRNA since the CT (threshold cycle) value of 18S rRNA
from equal amount of total cDNA of theses cell lines often reaches a very
close level (△CT＜0.5) and is not affected by TGF- stimulation. Our
absolute Q-PCR analysis showed deficient expression of TGFBRII in CL1-0
cells (Figure 4d). (Please see Page 9, line 7～8 and Page 27, line 13～14. )
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b. Instead of FACS, we have performed Western blot to characterize TGFBRII
protein expression in these cell lines (Figure 4c). Data from Western blot
analysis further support the Q-PCR data that TGFBRII is not expressed in
CL1-0. (Please see Page 9, line 7～8 and Page 27, line 14～15.)
c. The methylation status of TGFBRII promoter in A549, H1975 and CL1-0
cells has been characterized by bisulfate sequencing (Supplementary Figure
S3). We found that the TGFBRII promoter in CL1-0 cells, but not in A549
and H1975 cells, was highly methylated (Supplementary Figure S3).
Furthermore, pretreatment of CL1-0 cells with TSA and 5Aza, two epigenetic
modulating drugs, enhanced TGFBRII expression and decreased methylation
level of TGFBRII promoter, suggesting that the methylation status of these
CpGs gives a good readout of the expression of the TGFBRII promoter in
lung cancer cells. (Please see Page 9, line 8～10 and Supplementary Figure
S3.)
References
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Faiola F, Liu X, Lo S, Pan S, Zhang K, Lymar E et al. Dual regulation of
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10220-34.
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Bhattacharya S, Michels CL, Leung MK, Arany ZP, Kung AL, Livingston
DM. Functional role of p35srj, a novel p300/CBP binding protein, during
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Twist, a master regulator of morphogenesis, plays an essential role in
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Chou YT, Lin HH, Lien YC, Wang YH, Hong CF, Kao YR et al. EGFR
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Warner KA, Crawford EL, Zaher A, Coombs RJ, Elsamaloty H,
Roshong-Denk SL et al. The c-myc x E2F-1/p21 interactive gene
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fine-needle aspirate specimens. J Mol Diagn 2003; 5(3): 176-83.
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