Author s response to reviews Title: Expression of AE1/p16 promoted degradation of AE2 in gastric cancer cells Authors: Guo-Hui Fu (fuguhu@263.net) Ting Wang (wangting_tina@126.com) Hong-Jun Fei (feihongjun1@126.com) Ye Yang (yangye0611@126.com) Min Yan (yanmin0502@126.com) Jun Wu (jun.wu@shsmu.edu.cn) Lin-Jun Song (songlingjun1230@yahoo.cn) Hua Tian (huatian722@yahoo.com.cn) Zhi Zeng (6965410@qq.com) Xiao-Shu Jiang (jiangxiaoshu12345@163.com) Version: 1 Date: 13 Feb 2016 Author s response to reviews: Professor Dafne Solera Executive Editor BMC Cancer Dear Prof. Solera: I wish to express my sincere thanks to you and your colleagues for your critical comments on our paper entitled Gastrin blocks the AE1/p16-promoted degradation of AE2 in gastric cancer cells (BCAN-D-15-00035). My colleagues and I appreciate the opportunity to address the reviewers comments and submit the revised manuscript to BMC Cancer. The manuscript attached to this letter is the revised version. The revisions were highlighted in red in the new manuscript. We
dont indicate tiny changes. The point-by-point responses to the reviewers comments are provided below (with the reviewers comments). Point-by-point response For comments from reviewer #1 We are grateful to the reviewers comments. This manuscript attempts to understand the mechanism of the interplay between human anion exchanger 1 and 2 (AE1 and AE2) and tumor suppressor p16. Previously, authors have shown that expression of AE1 sequestrates p16 in the cytoplasm and interaction of AE1 and p16 play pivotal role in the progression of gastric cancer (GC). Herein authors propose that the formation of AE1/AE2/p16 complex results in the enhanced degradation of AE2 in poorly-differentiated GC cells. In addition, authors try to show that gastrin can inhibit the GC growth through blocking the AE1/p16-promoted degradation of AE2. Regrettably, throughout the study conclusions are overstated, inconsistent and/or unsupported. Many additional experiments would be required. The main issue of manuscript is that most of the conclusions are made by the overexpressed proteins, suggesting that studying the real "endogenous" relationship between AE1, p16 and AE2 is either technically impossible or may not be physiologically relevant. Thus, I am afraid here the "forest may have been missed for the trees". Consequently, the manuscript unfortunately falls short of the present stringent standards for publication in BMC Cancer. Thank you very much for your professional review on our manuscript. SGC7901 cells without any transfections were applied to the IP experiments to study the real "endogenous" relationship among AE1, p16 and AE2. Previous anti-p16 antibody (from Santa Cruz) was replaced by a new one (from Abcam). The results showed that anti-p16 antibody simultaneously interact with both endogenous AE1 and AE2 (Fig.1a).
1. AE1 and AE2 are over 80% identical in C-terminus. Antibodies raised against the recombinant protein (as is the case with the Sigma antibodies used in the study) of one paralog are likely to cross-react with the other paralog. To support their W-B and IF data, the authors need to provide evidence that these antibodies are indeed paralog-specific. The anti-ae2 antibody used in Western Blot was kindly provided by Dr. Alper, not from the Sigma (as shown in Acknowledgement). We agree with the reviewer that the AE2 antibody can cross react with the corresponding AE1 C-terminus (Panos Papageorgiou et al in Am J Physiol Heart Circ Physiol 2001). However, the molecular weight of AE2 (~160 kda) is much higher than that of AE1 (~90-100 kda). Therefore, the immunoreactive signal of AE2 (~160 kda) does not correspond to AE1. Besides, the anti-ae2 antibody used in inmunoflourences was purchased from Sigma-Aldrich. The immunogen for anti-ae2 antibody is synthetic peptide directed towards the human AE2 N- terminus. 2. a)figure 1a - The level of AE2 brought down by p16 Ab is very low and does not seem specific, as the probe with p16 Ab revealed the same low level of p16, suggesting that either this p16 Ab is not perfect for IP purpose or the interaction of endogenous AE2 and p16 is weak. On top of that, the statement "the results confirmed that the anti-p16 antibody interact with endogenous AE2" is inaccurate. Rather, anti-p16 Ab pulled-down p16, which hypothetically may form complex with AE2. Thanks again for the comments. We repeated the IP experiment by using new anti-p16 antibody (Abcam). The results showed that the anti-p16 antibody simultaneously interact with both endogenous AE1 and AE2 (Fig.1a). We added statements in the text (page 12, first paragraph of Results).
b)figure 1b - Similarly, even after overexpression of AE1, AE2 and p16 the complex formed between p16 and AE2 is not prominent, contrary to a predictable solid complex of AE1 and p16. We have obtained a solid evidence of the three proteins interaction in SGC7901 cells. To avoid confusion, the data from overexpression experiment (Fig.1b) was deleted (replaced by the endogenous IP). c)figure 1c - Consequently, the model of interaction cannot be interpreted based on only unconvincing IP results. To prove physical interaction between AE2 and p16, additional approaches would be useful: yeast two-hybrid system, pull-down by recombinant C-terminus constructs of AE2, PLA (Proximity ligation assay) etc. Also, it is not clear from the abstract and statements throughout the manuscript, whether authors think that interaction of p16 with AE2 and AE1 is sequential or simultaneous. Thanks for the reviewers suggestion. Interactions of AE1 and p16 have been identified by using yeast two-hybrid system, mammalian two-hybrid system, pull-down and IP experiment (Fu GH et al., 2005; Shen WW et al.,2007). In the present paper, we confirmed that the p16 interacts with AE1 and further performed the IP and IF to prove the interaction between AE2 and p16. We believe that the data were enough to confirm our conclusion. We believe that interaction of p16 with AE1 and AE2 is sequential or simultaneous based on that the AE1 and AE2 are over 80% identical in C-terminus.
d)figure 1d - There is the misinterpretation of p16 staining. The p16 specific puncta in the second row (mostly cytoplasmic) is inconsistent with third row (mostly intranuclear), thus giving wrong impression that under normal condition AE2 is not colocalized with p16. In addition, neither colocalization analysis non intracellular distribution has been statistically supported. In Fig.1d (Fig.1b in the revised version), the second row showed that the AE1 co-localized with p16 in HEK293T cell. In the third row, AE2 was expressed on the plasma membrane and the p16 was expressed on both membrane and nucleus (mainly in nucleus). In the last row, AE2 colocalized with p16 when the cells were transfected with AE1 constructs. These data suggested that the co-localization of AE2 and p16 was weak which was promoted by AE1. We and Professor proved that the AE1 and AE2 could not interact with each other. We added statement on page 13, first paragraph. 3. a) Figure 2 a and b: authors claim that knockdown of AE1 or p16 enhances redistribution of AE2 to the plasma membrane. The signal of AE2 in control cells looks very weak, however, based on Figure 2c, WT SGC7901 cells successfully express the reasonable amount of AE2. Therefore, it is hard to screen the shift of AE2-specific IF signal in both AE1-siRNA and p16- shrna treated cells. Again, where are the data for isolated plasma membrane fraction or flow cytometric analysis? Moreover, it looks that AE2 may still function in cells lacking AE1 and p16, which does not harmonize with the authors' previous statement that "AE1, AE2 and p16 might exist in the formation of complex".
We repeated the experiment, detected the signal by the confocal laser scanning microscopy and changed the data in Fig.2, which were well consistent with our statements. We agree with that AE2 may still function in cells lacking AE1 and p16, because the knockdown of AE1 and p16 enhanced AE2 trafficking to the plasma membrane. Moreover, under physiologic condition, the AE2 was abundantly expressed in parietal cells which lacking AE1 and expressing very low level of p16 (even no express). We changed the statement in the revised manuscript. b) beta-actin signal is underexposed on Figure 2c and e, and in Figure 2e the signal of AE1 in overexpressed samples is not sharp. We repeated the experiments and replaced the data in Fig. 2. 4. a) Figure 3a - it is unclear, why treatment of MKN28 cells with cyclohexamide enhances the expression of AE2, should be quite bit the opposite. We repeated this experiment with the CHX concentration of 50 g/ml and obtained perfect results. The Fig. 3a (lower panel) was replaced in the revised version.
b) Figure 3c - Authors suggest that p16 is necessary for stability of AE2 and overexpressed p16 may enhance proteasomal degradation of AE2. First, there is no essential control (GFP- AE2+HA-Ub+MG-132 vs that presented in the fourth line GFP-AE2+HA-Ub+p16+MG-132) which would allow us to conclude that indeed p16 facilitate ubiquitination of AE2. Second, typically, polyubiqitination appears either as the smearing or multiple (separated by 8kDa) bands. The signal presented in the fourth line does not seem the case. Third, in order to compare ubiquitination of AE2, the input for all samples need to be normalized by AE2, but not betaactin. Thanks again for the reviewers suggestion. The ubiquitination assay in this text was carried out essentially as described in the reports by Shuo Wei et al in PLoS One 2012 and Xinsong Xu et al in J Biol Chem 2012. In these two papers, the input for all samples was normalized by HSP90 or beta-actin. 5. a) Figure 4a-c: authors assume that the phenomenon of enhanced expression of AE2 under knockdown of AE1 or p16 can explain the reducing of phi, however, they completely ignore the contribution of other proteins which may be affected after silencing of AE1 and p16, and which contribution in maintaining of phi may be also significant. Indeed, the overexpression of AE2 reduces the phi, but these two events do not look sequential. Thus, the casual relationship is not well-established. The critical experiment to perform is the screening of phi under the coknockdown of p16, AE1 vs. combined knockdown of p16, AE1 followed by additional knockdown of AE2. If AE2 is the one and single protein regulating phi in these cells, then authors should not detect any decrease of phi in cells lacking all these three proteins.
AE2 protein is important for the regulation of phi. We agree with the reviewer that other proteins may affect the phi after silencing of AE1 and p16. We would like to study the relative mechanism in the further research work. As AE2 is a housekeeping protein, over- knockdown of AE2 protein could lead to the cell death, making it difficult to observe the changes in phi. b)figure 4d-e: it is not clear from the Figure legend and Methods section how authors measured survival ratio of cells. While downregulation of cyclin D1 upon overexpressed AE2 is the interesting data, I am afraid is not enough to conclude that AE2 suppressed GC growth. At least the invasion and migration activity of cells need to be performed to make such strong conclusion. We added statement in the manuscript on page 9 and highlighted in red color. In addition, the migration activity of cells was performed and the results were integrated to the revised version (Fig.4f). 6. The authors declare that low level of AE2 is correlated with poor differentiation and poor prognosis, however, there is no data of expression or immunohistochemistry analysis of AE2 in normal gastric tissue. Simple comparison of cancer area with para-cancer is not sufficient. Also, it seems authors did have a chance to monitor 5-year survival in patients with negative AE2, however, did not present any data of expression of AE1 and p16 in these patients. Thus, in spite of very big selection and nice opportunity, the authors did not provide any significant correlation between AE1 and p16 expression on the one hand and downregulation of AE2 on the other in GC patients. Indeed, many cytoplasmic and plasma membrane proteins are downregulate in cancer cells, but it does not mean that deficiency of all of them play a grand role in the survival of cancer cells.
We added the data of AE2 expression in normal gastric tissues (Fig.5a). In addition, expression of AE1 and AE2, and AE1 and p16 was identified by IHC in serial sections (Fig.5c and d). Correlations of AE1 and p16 with poor survival were statistically analysized. We added the data as Fig.5e. Again, the data are inconsistent. According to the Figure 6c, the level of AE2 in the WT cells is reasonable, which suggest that poorly differentiated SGC7901 still express AE2. If this is true, then what is the significance of AE2 for progression of GC and its prognosis? Finally, in Figure 6e, authors suggest that gastrin blocks (authors use the term "destroy") the complex between AE1 and p16, however did not provide any evidence supporting this speculation. Given that manuscript is entitled "Gastrin blocks the AE1/p16-promoted degradation of AE2 in gastric cancer cells", it looks unfair, why the contribution of gastrin was not in the main focus of the story. Data presented in this manuscript shown that the AE2 expression is decreased in poorly differentiated SGC7901 and correlated with poor differentiation and prognosis. Title of this manuscript has been changed. For comments from reviewer #2 The manuscript advances the molecular understanding of AE1 and p16 as oncogenes in gastric cancer proliferation. The biochemical interaction patient and animal in vivo data are convincing. However, the authors still need to come up with a more refined model on how p16/e1 affects the stability and localization of AE2.
Thank you very much for your positive comments on our manuscript. Fig. 1c: AE1 and AE2 are trans-membrane proteins, which do not exists in the cytosol as drawn but must be associated with membranous organelles in the cytoplasm if not at the cell surface. Also, you mention in the text the proteins possess one transmembrane domain but drew multispanning membrane proteins here. To avoid the confusion, we deleted the data in Fig.1. The IF in 1d does not allow any conclusion other than that all three proteins reside in the cytoplasm and that only p16 is present in the nucleus. The resolution and saturation of the micrographs does neither support any co-localization evaluation nor does it permit the assessment of how much of AE1 and AE2 are actually at the cell surface versus in the cytoplasm. We added more detailed statement in Results section to explain the interaction of the three proteins (page 13, first paragraph). In addition, the IP experiments were repeated and the results clearly showed that the p16 binds to both AE1 (strong) and AE2 (week) in Fig 1a. Fig. 2a: How does AE2 get its nuclear localization here? It is not detected (and expected) in the nucleus in any of the other figures. Figs. 2a and b do not allow the distinction between cytoplasmic and plasma membrane staining.
We repeated the experiment, detected the signal by the confocal laser scanning microscopy and changed the data in Fig. 2a and b, which were well consistent with our statements. Indeed, we are also puzzled by the results that part AE2 localized in nuclear in Fig 2.We would like to study the reason in our further work. Fig.2e: The AE2 panel here should be improved. The change is hard to appreciate in this particular blot (it is far better in a similar panel, corresponding to p16 O/E, in 2f). As suggested, we repeated the experiments, and changed the data in Fig.2. The authors should discuss their surprising finding that AE2 is degraded by the proteasome. Membrane proteins are usually degraded in the lysosome. Is there evidence for retrotranslocation out of the ER (an instance where proteasomal degradation of unfolded membrane proteins occurs)? Otherwise, some proteolytic cleave of the fully folded AE2 is expected for that to work. Thanks for the suggestion. In this manuscript, we focused on the interaction of AE2 and p16 in gastric cancer cells. The mechanism of AE2 degradation by the proteasome should be further studied by our colleagues.
For comments from reviewer #3 This paper examines Human anion exchanger 1 and 2, their interaction with p16, their ability to regulate intracellular ph, and their regulation by gastrin. The data presented in the manuscript extend previously knowledge of the AE1-p16 interaction to AE2-p16 interactions. Overall the conclusions are supported by the data presented, and the work is novel and relevant to gastric cancer. Thank you very much for your positive review on our manuscript. The manuscript does not state how many times each experiment was performed when a figure is shown, and the legends to not state the statistical tests used to determine significance. In this manuscript, each experiment was performed at least three times when a figure in shown. As suggested, we added the statistical tests used to determine significance in Figure legends. There are a few minor issues that should be addressed, including: Grammatical errors in the paper that should be corrected. We corrected the grammatical errors in the paper as much as possible.
In the introduction, it is stated that gastric cancer is the leading cause of cancer-related deaths in the world, this should be corrected, most references now put it as the 3rd leading cause of cancer-related deaths. I'm not sure if the 5-year survival rate that is listed (10-15%) is correct, the references is from 2005. As the suggestion, we corrected the introduction (page 5) and updated the references. Sincerely Yours Guo-Hui Fu Chief & Professor Pathology Center, Shanghai First Peoples Hospital / Faculty of Basic Medicine, Shanghai Jiao Tong University School of Medicine Number 280, South Chong-Qing Road,Shanghai, 200025, P.R. China E-mail: guohuifu@shsmu.edu.cn;fuguhu@263.net Tel: 86-21-63846590 Ex. 776601