Flores-Pérez A et al Suppression of cell migration is promoted by mir-944 through targeting of SIAH1 and PTP4A1 in breast cancer cells

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1 Author s response to reviews Title: Suppression of cell migration is promoted by mir-944 through targeting of SIAH1 and PTP4A1 in breast cancer cells Authors: Cesar Lopez-Camarillo (genomicas@yahoo.com.mx) Ali Flores-Pérez (qfb_ali@hotmail.com) Laurence Marchat (lmarchat@gmail.com) Sergio Rodríguez-Cuevas (sergiorocue@gmail.com) Verónica Piña Bautista (verobau2002@yahoo.com.mx) Lizeth Fuentes-Mera (lizeth46@hotmail.com) Diana Romero-Zamora (haruko_21@hotmail.com) Anabel Maciel-Dominguez (anabel.macield@gmail.com) Olga Hernández de la Cruz (ediacara79@yahoo.com.mx) Miguel Fonseca-Sánchez (mfonseca_79@hotmail.com) Erika Ruiz-García (betzabe100@yahoo.com.mx) Horacio Astudillo-de la Vega (hastud2@aol.com) Version: 1 Date: 14 Mar 2016 Author s response to reviews: Reply to new reviewer BCAN-D , Review Flores-Pérez A et al Suppression of cell migration is promoted by mir-944 through targeting of SIAH1 and PTP4A1 in breast cancer cells

2 My first impression of this paper is the absence of coordination between the experimental design and the goal to be achieved, namely the role played by mir-944 in the invasion/metastasis phenotype. Let s start with the cell lines. In Fig 1C, we have breast cancer cell lines (MCF-7, T47-D, MDA-MB-453, ZR-75 and MDA-MB-231) and what the authors call Normal tissues. It is not appropriate to compare established breast cancer cell lines and normal tissues taken from tumors. Here there is need for a normal breast cell line like HMEC from ATCC. Reply: We acknowledge for the reviewer comments. As reviewer suggested we performed additional experiments to shown the expression of mir-944 in a normal breast cancer cell line. We performed qrt-pcr for mir-944 using total RNA from non-tumorigenic MCF-10A (ATTC CRL-10317). Results show that mir-944 expression was higher in MCF-10A cells in comparison to panel of breast cancer cell lines (MCF-7, T47-D, MDA-MB-453, ZR-75 and MDA-MB-231). In addition, mir-944 expression data obtained from normal breast tissues was removed in the new figure 1 as the reviewer suggested. In the Results section MiR-944 is suppressed in breast cancer cell lines and clinical tumors, the authors compared the expression of mir-944 in 40 patient tumors (the majority of them are triple negative) and breast cancer cell lines (MCF-7, T47-D, MDA-MB-453, ZR-75 and MDA-MB- 231). The breast cancer cell lines are a mixture of hormone dependent like MCF-7 and hormone independent like MDA-MB-231. The conclusion is the low expression of mir-944 by breast cancer cell lines and breast tumors regardless of their hormonal status. To strengthen their conclusion the authors validated their data by using 776 matched normal/tumor samples from The Cancer Genome Atlas (TCGA) (validation cohort). So we have results obtained from three types of material (breast cancer cell lines, clinical tumors and a TCGA validation cohort) to conclude that mir-944 is poorly expressed. Then the authors candidly stated We did not find significant differences in mir-944 expression when tumors were stratified according to the expression of estrogen, progesterone and HER2 receptors (data not shown). Why the authors didn t see the contradiction in their successive statements. According to the published literature breast cancer cell lines MDA-MB-231 and MDA-MB-453 are metastatic (Ref Cancer Res 1990, 50, ) and MCF-7 and T47-D are not metastatic (Ref Oncology Reports 2011, 25: ) and the reason is their hormonal status.

3 Cell Line Hormonal Status Metastatic Potential mir-944 Expression MDA-MB-231 independent Yes Very low MCF-7 dependent No Very low Reply: We apologize if the redaction of results caused confusion. Our data clearly indicate that mir-944 expression was low in a panel of breast cancer cell lines, independent of hormonal receptors status, thus we did not see the contradiction mentioned by the reviewer. We agree with reviewer that breast cell lines tested here for mir-944 expression have different hormonal receptors expression. However, as we found that mir-944 expression is suppressed in all cell lines regardless of their hormonal status, the contribution of hormonal receptors in gene expression of mir-944 was irrelevant in this case, and not discussed. It s important to note that our findings were validated by analyzing a large cohort of clinical breast tumors which also displayed different status of hormonal receptors. Again, the results obtained ware the same as observed in breast cell lines, i.e. the low expression of mir-944 was independent of hormonal receptors expression. Therefore, to avoid confusion in the interpretation of data, in this revised version of manuscript we tuned down our conclusions and only mentioned that mir-944 expression was low in all the samples analyzed (i.e., breast cancer cell lines vs normal cell line; clinical tumors vs normal tissues, and TGCA cohort vs normal tissues). In addition, to avoid confusion and misinterpretation we have remove the statement We did not find significant differences in mir-944 expression when tumors were stratified according to the expression of estrogen, progesterone and HER2 receptors (data not shown) in this revised version of manuscript. We agree with the reviewer comments about the low metastatic potential of MCF-7 cells and other cell lines. The original idea to include MCF-7 cells in our experiments was to compare the mir-944 effects in metastatic (MDA-MB-231) and poor-invasive (MCF-7) cell lines. As expected, we found a deeper effect of mir-944 in cell migration of MD-MB-231 cells in comparison to MCF-7. However, the role of hormonal receptors in migration inhibition by mir- 944 is out of the purposes of this study. In fact, the metastatic potential of cancer cells in vivo is not always influenced by hormonal receptors, but a complex genetic program which is activated and shaped by tumor environment.

4 This is not an exercise of philosophy but an accurate analysis of scientific results. The already available knowledge based on the hormonal status of breast cancer (such a status has implications in therapeutic decisions for patients) the metastatic potential depends on the hormonal status. The new knowledge: the mir-944 status of breast cancer cell lines does not depend on the hormonal status. The mir-944 sequence is located in the intron 4 of p63 gene (Ref Nucl. Acids Res. 2015, 43, ). The p63 inhibits metastasis according to this paper PNAS USA 2012, 109, Therefore the low expression of mir-944 may be due to the deletion of p63 gene or a lack of expression of p63 and perhaps mir-944 (epigenetic modification?). We have no idea about the expression mechanism of mir-944 and I didn t find a reference about the status of p63 in MDA-MB-231 and MCF-7. We don t know how the endocrine system in the body is coordinated with the micro RNA regulation. We are in a new area of knowledge, caution is the rule. Reply We thank the reviewer for his interest in our work. However, we do not understand what is the question?. The new knowledge indicates that epithelial-to-mesenchymal transition (EMT, a well-accepted marker of metastasis) is not always related to metastasis in vivo (Beerling et al. Plasticity between Epithelial and Mesenchymal States Unlinks EMT from Metastasis-Enhancing Stem Cell Capacity. Cell Rep Mar 2). Here we did not found alterations if EMT markers (data not shown) in mir-944 transfected cells suggesting that at least in our in vitro model, EMT is no related to mir-944 suppression of migration and additional mechanism may be responsible of effects on cell migration. As we known, in genetic studies where a mirna or gene is transfected, a fixed state is established and did not reveals all the mechanisms operating in vivo, thus we take caution of our observations and conclusions. We strongly disagree with reviewer comments about the dependence of metastatic potential with hormones receptor status. The mechanism responsible for metastatic potential of breast cancer cells are more complex and not always depends on the hormonal receptors status, but its depends on a transcriptional and epigenetic landscape that is shaped during the invasion progression, where the loss of hormonal receptors is frequent (Grewal et al. Isolated loss of hormonal receptors in leptomeningeal metastasis from estrogen receptor- and progesterone receptorpositive lobular breast cancer. J Clin Oncol. 2010, 1;28(13):e200-2).

5 In addition, change in expression of hormone receptors and HER-2 status and lack of concordance between primary tumour and corresponding local recurrence or distant metastasis has been well documented (Bogina et al. Comparison of hormonal receptor and HER-2 status between breast primary tumours and relapsing tumours: clinical implications of progesterone receptor loss. Virchows Arch Jul;459(1):1-10 and, Ibrahim et al. Hormonal receptor, HER2, and Ki67 discordance between primary breast cancer and paired metastases: clinical impact. Oncology. 2013;84(3):150-7). In particular, in this study we suggested that mir-944 low expression and effects on cell migration may be not influenced by hormonal receptors, as we did not find concordance between receptors status and mir-944 expression. We know that mir-944 gene is located into an intron of p63 gene. However, the role of p63 in cancer is more complex as it exhibits tumor suppressor and oncogene functions. Indeed it has been reported that p63 may have a dual function in metastasis: p63 enhances (PNAS. 2012;109(38): ) or inhibit (Cancer Lett. 2014, 10;353(1):124-32) metastasis of tumors. Whit this knowledge in mind, during the preparation of this study we explored if the p63 and mir-944 expression could be coregulated in breast tumors. However, our data indicate a lack of relationship between the expression of p63 and mir-944, at least in the tissue samples analyzed here (n=10 samples). This may be explained by the fact that mir-944 expression is controlled by an independent and own promoter (Kim et al. ΔNp63 intronic mir-944 is implicated in the ΔNp63-mediated induction of epidermal differentiation. Nucleic Acids Res. 2015;43(15): ), indicating that p63 and mir-944 expression is not always coupled. Therefore, at this point we cannot stablish a relation between the low expression of mir-944 and deletion/low expression of p63 gene. In my opinion this discrepancy between the metastatic potential of both cell lines MDA-MB-231 and MCF-7 in regard of their hormonal status and the mir-944 expression status is the main finding of this paper. Unfortunately it was missed by the authors because of rapid thinking and lack of knowledge in cancer research. As a consequence of this the next Results section MiR- 944 inhibits tumor cell migration and invasion was not done well. The mir-944 ectopic expression in both cell lines showed that in scratch/wound-healing assays cell monolayers restoration was delayed in both cell lines and that in transwell chamber assays the number of migratory cells was significantly (p<0.05) reduced in MDA-MB-231 (4-fold) and MCF-7 (8- fold) cells that ectopically express mir-944 (Fig. 2C and G). Moreover, mir-944 significantly (p<0.05) inhibited the ability of metastatic MDA-MB-231 cells to invade matrigel in vitro (Fig. 2D). The three tests should have been done for both cell lines. What is the consequence of mir- 944 ectopic expression on the metastatic potential of MCF-7? We never know.

6 Reply: We performed the scratch/wound-healing and transwell chambers assays using both MCF-7 and MDA-MB-231 cells, but invasion assays using matrigel in transwell chambers were done only for metastatic MDA-MB-23 cells. It is well known that MCF-7 cells do not traverse the matrigel in invasion assays using transwell chambers coated with extracellular matrix, thus MCF-7 cells were not included in these invasion assays. The effects of mir-944 restoration in cell migration were compared between MCF-7 and MDA-MB-231 cells; in contrast we cannot compare the effects in cell invasion between these cell lines as MCF-7 is poorly invasive and not suitable for invasion assays in matrigel systems. In addition a better test to investigate the effect of ectopic mir-944 expression on the metastatic phenotype should have been done in vivo. There are both MDA-MB-231 and MCF-7 cell lines transfected with luciferase and their growth and metastasis potential assessed by Bioluminescence Imagery (BLI) as it is explained in the this paper: J. Clin. Invest. 2005, 115: Furthermore the scratch/wound-healing assays even it is very widely used, is not very appropriate. Here is why for two reasons. First when you scratch a monolayer you take the cells but not the extracellular matrix which will enhance the growth of cells. Second the invasion in vivo is not done in empty space with only extracellular matrix, cancer cells invade tissue structures containing cells, extracellular matrix which nature depends on the tissue. The in vivo studies are more suited for this paper. Reply We agree with reviewer comments. However, in vivo studies are beyond the aim of this study as it s not possible to perform them in a short time period (editor gave us only 1 month to reply; by march 11). As we known, in vivo analysis is difficult to carried out and the logistic to make these experiments take at least 3 months, as the animal protocols need to be reviewed and approved by the Institutional Ethical committee. A complete in vivo analysis to extend our findings need more time to do, thus we consider that at this point in vivo assays are matter for a new manuscript. We acknowledge to reviewer for these suggestions, and we will take into account for a new upcoming project. In the Results section MiR-944 alters cytoskeleton organization both cell lines MDA-MB-231 and MCF-7 should be explored the same way.

7 Reply As reviewer suggested we performed additional experiments to shown the effect of mir-944 in the cytoskeleton of MCF-7 cells. As in MDA-MB-231 cells we examined the organization of cytoskeleton by analyzing the distribution of F-actin labeled with rhodamine-phalloidin using confocal microscopy and the subcellular distribution of a-actinin-1, an actin -crosslinking protein that reinforces focal adhesions. Our data showed that similar changes in actin cytoskeleton organization in MCF-7 cells after transfection of mir-944 precursor, although in less extent in comparison with MDA-MB-23 cells. MCF-7 cells also showed an axial F-actin cytoskeleton organization. In contrast, the ectopic expression of mir-944 produced a dramatic effect on overall cell morphology since spread area was increased. Moreover, F-actin was redistributed in a radial mode towards the periphery of the cell, as well as in the central zone; and the incipient membrane ruffles and filopodia structures were loss. In pre-mir-944 transfected cells, we observed a robust signal of α-actinin-1 and a slight increase in the number of contact points with F-actin in multiple points of cell body, indicative of the reinforcement of focal adhesions. All these data was added to the new version of figure 3. The authors didn t walk the extra mile and therefore they are missing interesting results. When we do research we should be prepared for everything, I mean what we expect to see and also what we do not expect to see. Two of the most important discoveries of all time are in that category: the discovery of Penicillin by Alexandre Fleming and the discovery of radioactivity by Henri Becquerel. I have two other minor remarks. First Ref 14 is PNAS USA 2012, 109, and not PNAS USA 2012, 18, Second: since its first description in 1983 by Tim Mossman (J. of lmmunol. Methods 1983, 65: 55-63) or in 2015 (J. of Cancer Ther. 2015, 6: ) the MTT should be dissolved in PBS at 5mg/ml and not 1mg/ml. The authors should say in what liquid they dissolved the MTT. Reply We apologize for the confusion. We prepare the MTT stock solution at 1mg/ml in DMEM culture media without serum fetal bovine complementation, but working solution was prepared to 0.5 ml/mg. In the above references, the same working solution (MTT 0.5 mg/ml) was used. MTT was dissolved in non-complete medium, as describe several reports ( ). In our hands we did not observed problems in solubility of MTT in culture media. Conclusion: this is an interesting work worthy of publication. If the authors take care of my recommendations the value of this paper will increase.

8 Reply We acknowledged to reviewer for these important observations which clarify specific points and conclusions. We take care of your recommendations. Abdelkrim Alileche MD, PhD. Boise State University University Drive. Biology Department. Boise, ID USA. Phone: