Title: MYBBP1A suppresses breast cancer tumorigenesis by enhancing the p53 dependent anoikis

Transcription

1 Author's response to reviews Title: MYBBP1A suppresses breast cancer tumorigenesis by enhancing the p53 dependent anoikis Authors: Kensuke Akaogi Wakana Ono Hiroyuki Kishimoto Junn Yanagisawa Yuki Hayashi Version: 2 Date: 21 December 2012 Author's response to reviews: see over

2 December 21, 2012 The Editor of BMC Cancer Dear Editor, RE: MS: "MYBBP1A suppresses breast cancer tumorigenesis by enhancing the p53 dependent anoikis" Thank you very much for your generally positive letter dated November 22 in We have finished additional experiments to address the concerns of Referees 1 and 2. We would like to update you about our progress and provide point-by-point responses to the Referee s comments. We believe that the changes made in response to the comments from the Referees have resulted in a stronger paper. We hope that the revised manuscript is acceptable for publication in BMC Cancer. Yours sincerely, Junn Yanagisawa Ph.D. junny@agbi.tsukuba.ac.jp University of Tsukuba Graduate school of Life and Environmental Sciences Tennodai Tsukuba Science City, Ibaraki-ken JAPAN

3 A detailed list of the revisions that were suggested by the Referee 1 Referee 1 (Comments to the Author): In this manuscript, Akaogi et al. describes tumor suppressor role of MYBBP1A protein via increase in p53-dependent anoikis in MCF7 cells. Authors performed in-silico analysis of breast cancer patients microarray databases, and carried out various in vitro transformation assays to address the potential tumor suppressor role of MYBBP1A. This is a relatively straight-forward study with few concerns as noted below- 1) Authors analyzed Oncomine database to infer the tumor suppressor role of MYBBP1A. Specifically authors use data from three studies. Authors should provide some details of breast carcinoma samples used in these studies. Are they all invasive or from different stages of breast cancer progression. If possible authors should also confirm results from Oncomine database using commercially available TMAs and IHC studies for p53 and MYBBP1A. To address the Referee s comments, we have added the following descriptions in the Methods section. Data sets obtained from TCGA Breast, Finak Breast, and Richardson Breast 2 included various stages, and all cancer samples were invasive. In addition, we performed IHC examinations using tissue microarrays and assessed MYBP1A protein levels in normal and breast tumor tissues. The description of the tissue microarrays is given in the Methods section. Decreased immunoreactivity was observed in breast tumor tissues compared with normal tissues (Figure 1B). These results supported the findings in the Oncomine database. 2) Fig. 2: Authors use a single MYBBP1A shrna, which appears to be very effective. However, there are no data presented to show the specificity of the shrna to rule out its off-target effects. To demonstrate the specificity of the shrna used and to rule out its off-target effects, we have shown the data obtained with two shrnas for MYBBP1A in Figure 2. Both shrnas for MYBBP1A effectively reduced the expression of MYBBP1A and increased the number of colonies. 3) Fig 3 needs a western blot analysis of p53 in indicated set of cells. As shown Figures 3 and 4, we showed the western blot results for p53 in the indicated set of cells.

4 4) Authors should also examine cell death markers to better define the role of MYBB1A in anoikis. To address this, we performed Annexin/PI staining and FACS analysis using cells cultured under detached conditions. p53-dependent cell death was regulated by MYBBP1A under detached conditions (Figure 5). 5) Data obtained with MCF7 cells should be confirmed using another p53 positive or a p53 reconstituted breast cancer cell line. To address this, we performed anoikis assay and real time-pcr using ZR-75-1 cells (p53-positive breast cancer cell line). The results obtained were same as those with MCF-7 cells (Figures 3C, 4B, and 6B). 6) In general, authors do not provide enough experimental details. In response to the Referee s comment, we have rewritten part of the Figure legends and the Methods section. 7) Authors need to follow guidelines to prepare manuscript properly, and have it carefully edited for typos and grammatical mistakes. We have carefully re-checked the instructions for authors and have corrected our manuscript. We have also had a scientific writer check our manuscript.

5 A detailed list of the revisions that were suggested by the Referee 2 Referee 2 (Comments to the Author): This manuscript by Akaogi et al., investigated the role and mechanism of MYBBP1A in breast cancer oncogenesis. The authors report that MYBBP1A through p53 induces anoikis in a breast cancer cell line. Although these are interesting observations, the authors need to address following concerns. Major points 1) The evidence presented for decrease in MYBBP1A expression with breast cancer progression is based on the Oncomine database analysis. The authors need to assess levels of MYBBP1A protein in different breast cancer cell lines and in tumor tissue arrays to validate the finding. In response to the Referee s comments, we compared the MYBBP1A protein levels in MCF-10A, MCF-7, and ZR-75-1 cells. As shown in Figure 1C, immunoblotting results revealed that the MYBBP1A protein levels in MCF-10A cells (mammary epithelial cells) were higher than those in the MCF-7 and ZR-75-1 cells (p53-positive breast cancer cells). In addition, we performed IHC studies using tissue microarrays and assess the levels of MYBP1A protein in normal breast tissues or breast cancer tissues. The decreased immunoreactivity was observed in breast cancer tissues comparing to normal tissues (Figure 1B). These data supports the findings in Oncomine database. 2) Authors have presented the data in one cell line MCF7. The authors should use at least one more cancer cell line and normal cell line MCF10A (as control) for their analysis to address cancer specificity concern. To address this comment, we performed the anoikis assay and Real time-pcr using ZR-75-1 cells and same results as the MCF-7 were obtained (Figures 3C, 4B and 6B). In addition, the anoikis assay using MCF-10A cells showed that p53 and MYBBP1A knockdown increased the number of viable cells under detached conditions (Figure 3A). These results indicate that MYBBP1A regulates anoikis in both normal and cancer cells. 3) The qrt-pcr primers mentioned for GAPDH are not GAPDH but are beta-actin primers-authors please correct. We apologize for this overlook. We have corrected the sequence in the Methods section.

6 4) Is MYBBP1A only induces p53-dependent anoikis? Did the authors assess for apoptosis? We previously showed that MYBBP1A was involved in apoptosis after DNA damage or glucose starvation (Kuroda et al., 2011 and Kumazawa et al., 2011). In this study, we focused on the role of MYBBP1A in p53-dependent anoikis (apoptosis under detached conditions). In response to the Referee s comment, we assessed cellular apoptosis by FACS analysis. This demonstrated that MYBBP1A induced p53-dependent apoptosis under detached conditions (Figure 5). 5) Authors state that detached condition through MYBBP1A accumulates and acetylates p53. There is no experimental proof provided for this statement. As shown in Figure 7A, detached conditions induced the accumulation and acetylation of p53 (lane 3). The accumulation and acetylation of p53 were reduced by MYBBP1A knockdown (lane 4). These results provide evidence for MYBBP1A regulation of p53 accumulation and acetylation. Minor points 6) The figure legends as well as methodology are written relatively brief and need more explanation for conditions of Anoikis assay. The authors should either describe the method in detail or reference an article if it is already published. In response to the Referee s comments, we have rewritten part of the Methods section. 7) Overall the paper is not written well and has several typographic errors and needs to be written in a more scientific language. Authors, please have a scientific writer correct the manuscript. As suggested by the referee, we have had a scientific writer check our manuscript. 8) In the background paragraph five, lane no 8, the authors have mentioned role of MYBBP1A in cancer prevention. Is there any study in which MYBBP1A was studied in relation to cancer prevention? We have rewritten this part of the background paragraph and have cited a previous study. We have added the following sentences. In addition, Sanhueza et al. recently reported that MYBBP1A regulated the proliferation and migration of head and neck squamous cell carcinoma cells [17].

7 However, the role of MYBBP1A in breast cancer prevention and detailed mechanisms underlying these activities has not been determined. 9) The authors have mentioned anoikis assay just by counting cells. Use of more quantitative method, such as MTT assay will be better. To address this, we have placed the quantitative results obtained by MTT assay next to the cell counting results.