Title: HAX-1 overexpression, splicing and cellular localization in tumors.

Transcription

1 Author's response to reviews Authors: Alicja Trebinska Alina Rembiszewska Karolina Ciosek Konrad Ptaszynski Sebastian Rowinski Jolanta Kupryjanczyk Janusz A Siedlecki (jas@coi.waw.pl) Ewa A Grzybowska (ewag@coi.waw.pl) Version: 3 Date: 14 December 2009 Author's response to reviews: see over

2 MS: Alicja Trebinska, Alina Rembiszewska, Karolina Ciosek, Konrad Ptaszynski, Sebastian Rowinski, Jolanta Kupryjanczyk, Janusz A Siedlecki and Ewa A Grzybowska Dear Dr Alam, I am submitting a revised manuscript in the hope that comments and changes are satisfying and address all the demands of the referees. Here I present a list of explanations and changes: Reviewer's report Version: 1 Date: 8 October 2009 Reviewer: Delphine Lees Reviewer's report: In the manuscript entitled HAX-1 overexpression, splicing and cellular localization in tumors Trebinska et al. are attempting to assess the mrna levels of five HAX1 variants in normal and tumour samples, and HAX1 protein expression in sections by immunohistochemistry. This is a very interesting question, as it is becoming more and more likely that different HAX1 variants perform different functions in cells. Unfortunately, the technical conditions used for semi-quantitative and quantitative PCR are not specific for all the variants and in particular for the variant I, which considerably limits the interest of the study. Two observations are interesting however and would deserve further investigations. Unlike most results published to date which show widespread expression, the variant III mrna is predominantly detected in tumour samples. It would be very interesting to confirm this result at the protein level. The HAX1 nuclear localisation observed in tissue sections is intriguing, but is in contradiction with previous reports in normal or cancer tissues using the same technique. It would be interesting to confirm this result using alternative antibodies, and to increase the number of samples included in the study and extend it to other cancer types. Major compulsory revisions The authors have used the same primers pairs to determine HAX1 variant I mrna levels by RT-PCR and by qpcr in Figures 1, 2, 4, 5 and 6. This primers pair is not specific for variant I. It is predicted to generate an identical 126bp product from variants I, V and 006 (Lees et al. J Mol Biol : ). The melting curve analysis described in the quantitative PCR methods section is not appropriate as the different amplicons, being identical, will have the same melting characteristics. In this study, this is the only means the authors have used to monitor variant I levels. Therefore, the interpretation of results presented Figures 1, 2, 4, 5 and 6 relating to variant I levels may not actually represent changes in variant I levels, but changes in steady-state levels of the three variants. Unfortunately, for the same reason, all the statements and the conclusions in the manuscript regarding HAX1 variant I levels may not actually relate to variant I. Unless the authors can discriminate between these three different variants the study is of limited interest as the levels of the major HAX1 variant are not reliably

3 quantified. Figure 7 and Table 4. The authors show high HAX1 mrna levels by RT-PCR in Fig 4 in all normal tissues studied, yet no HAX1 protein is detected in 10 out of 15 normal samples by immunohistochemistry This should be addressed. In 13 out of 15 tumour samples HAX1 is detected by IHC, and in four of these it is also detected in the nucleus. This may an interesting observation but to avoid the possibility of it being artefactual it should be confirmed using one of the other commercially available HAX1 antibodies. Minor essential revisions Page 6 line 7. In ref [14 ] HAX1 overexpression was shown in oral squamous cell carcinoma samples by immunohistochemistry, and not in cell lines as stated twice line 7. Page 6 line 12. in ref 14, HAX1 has been shown to be expressed at higher levels in samples of oral squamous cell carcinoma by immunohistochemistry not only in a few cancer cells lines, please modify the text accordingly. Page 6 line 20. The major problem with SAGE and microarray analysis data is not their lack of reliability. The HAX1 gene codes for several variants, and none of this is clearly taken into account in these analyses Page 13 line 8. Expression of splice variant I. The whole point of the study is to determine expression levels of the different HAX1 isoforms, so why state that HAX1 variant I levels is representative for overall HAX1 expression? Page 16 line and are summarized in Table 2. Change for Table 3. Level of interest: An article of limited interest Quality of written English: Acceptable Statistical review: No, the manuscript does not need to be seen by a statistician. Declaration of competing interests: I declare that I have no competing interests Reviewer 1: 1. Primers design to amplify variant I are not specific The data obtained in this study, but also in the previous publication addressing the expression level of the variants I and II (Carlsson et al., 2008) indicate that variant I expression is about 20 times higher than variant II in most of the analyzed tissues. Our extended analysis has shown that expression levels of the other variants are also considerably lower than for variant I, producing ambiguous and unreliable results in qpcr. Since these differences were not sufficiently demonstrated in our manuscript, we have added the results of RT-PCR performed on the same material (normal human breast tissue cdna) for a different number of cycles, showing that the expression of variant II, but also III, IV and V is considerably lower than the expression of variant I (Fig 4A in the revised manuscript). This is in agreement with the observation made by our group while performing RACE analysis (Carlsson et al., 2008), which indicated that variants I and II can be cloned relatively easily, but III and IV are quite difficult to clone from normal human tissues. Variants V and 006 were not detected in RACE. These results suggest indirectly that the expression levels of the variants differ considerably, with the prevalence of variants I and II.

4 Variant 006 was not included in the analysis since in this case alternative splicing generates frameshift, hence the putative protein is different in sequence and potential functionality (Lees et al., 2008). Nevertheless, we present here the result of RT-PCR, showing that 006 expression is also low, comparing to variant I (Figure 1). Summarizing, variants I and II seem to represent the main players in HAX-1 functional significance in the cell and since we took an effort to discriminate between these two variants, the overlapping expression of the other variants should be minimal (which does not exclude the possibility that some of the variants are over-expressed in some diseases, like variant III in case of breast cancer, although on a small scale). Another difficulty with satisfying this demand arises from the fact, that there are no features characteristic to variant I, which are not present in other variants, so designing strictly variant I specific primers is practically impossible. However, in the light of the above arguments, assessing the expression of the splice variant I with the primers used, seems to represent a good approximation of its overall expression. 30 cycles 40 cycles I II III IV/V 006 I II III IV/V 006 I Figure 1. Expression of the six splice variants assessed by RT-PCR with 30 and 40 cycles, from the same template: normal breast tissue cdna. Expression of the splice variant I is visibly predominant, splice variants V and 006 are expressed at a very low level. 2. Figure 7 and Table 4. The authors show high HAX1 mrna levels by RT-PCR in Fig 4 in all normal tissues studied, yet no HAX1 protein is detected in 10 out of 15 normal samples by immunohistochemistry The main purpose of this work is to demonstrate the difference in HAX1 expression levels between normal and cancer tissues. Accordingly, immunohistochemical reaction conditions were optimized to obtain clear detection of HAX1 in cancer tissues with the minimal reaction in normal tissues, in order to bring out the difference in the expression levels. With the procedure optimized to obtain clear HAX1 detection in normal tissues, the difference would be diminished.

5 Also, patients differ in HAX1 expression in normal tissues, so optimizing the staining in an arbitrary way is quite hard, and should probably be established as an outcome of some collective effort in the future. For the purposes of our manuscript we have decided that the chosen conditions ensure the best representation of our data. 3. In 13 out of 15 tumour samples HAX1 is detected by IHC, and in four of these it is also detected in the nucleus. This may an interesting observation but to avoid the possibility of it being artefactual it should be confirmed using one of the other commercially available HAX1 antibodies. Hitherto, HAX1 nuclear localization was reported twice, by Kawaguchi et al., 2006 (in systemic sclerosis fibroblasts, immunofluorescence) and by our group (Sarnowska et al., 2007, cellular and nuclear fractionation). HAX1 nuclear localization in some specific breast cancer cells are not exactly contradictory to the previous reports, since, to date, HAX1 localization in tumor tissue was reported only by Ramsay et al., 2007 in oral SCC. Tissue- and disease-specificity (e.g. ER-positive or negative) might represent critical factors, since in the current manuscript we indicate HAX1 differential expression in several types of cancer. Even in our breast cancer samples nuclear localization is present only in about 27% of cases (coinciding with high ER status), which points to the existence of some, so far unidentified stimulus/stimuli, influencing HAX1 cellular localization. Moreover, we are convinced that nuclear localization is not artefactual, because of the results obtained in our immunofluorescence analysis performed in MCF-7 breast cancer cell line. We ve observed nuclear localization of GFP-fused HAX1 in up to 20% of the nuclei. It seems possible, that some specific signal may enhance HAX1 nuclear presence, and right now we are trying to identify this signal, but the mechanism seems to be complex and beyond the scope of the current manuscript. Estradiol treatment of the cells do enhance HAX1 nuclear presence, but only in about 5%, so it s not enough to cause the observed effects. Nevertheless, nuclear presence is confirmed in the breast cancer cells by the independent method. The results obtained by IF analysis were added to the current manuscript as the Additional files 1 and Page 6 line 7. In ref [14] HAX1 overexpression was shown in oral squamous cell carcinoma samples by immunohistochemistry, and not in cell lines The text was modified. 5. Page 6 line 12. in ref 14, HAX1 has been shown to be expressed at higher levels in samples of oral squamous cell carcinoma by immunohistochemistry not only in a few cancer cell lines The text was modified. 6. Page 6 line 20. The major problem with SAGE and microarray analysis data is not their lack of reliability. The HAX1 gene codes for several variants, and none of this is clearly taken into account in these analyses

6 As stated previously, different expression levels for HAX1 splice variants, with a prevalence of splice variant I, causes that this objection is not really of major importance. However, the text was modified to conform to this comment. 7. Page 13 line 8. Expression of splice variant I The whole point of this study is to determine expression levels of the different HAX1 isoforms, so why state that HAX1 variant I levels is representative for overall HAX1 expression? As stated in the manuscript, and several times in this text, expression of splice variant I was considered as representative on the basis of its prevalence in respect to other variants. 8. Page 16 line and are summarized in Table 2. Change for Table 3. The text was changed.

7 Reviewer's report Version: 1 Date: 4 November 2009 Reviewer: Karl Welte Reviewer's report: This is an interesting manuscript on HAX-1 overexpression, splicing and cellular localization in tumors. Major critique: This is a more descriptive study. It would be important to demonstrate the functional relevance of overexpression of the HAX-1 protein, e.g. apoptosis of tumor cells depending on HAX-1 splice variants or expression levels, mitochondrial membrane potential or cytochrome-c release, F-actin polymerization, etc. Level of interest: An article of importance in its field Quality of written English: Acceptable Statistical review: No, the manuscript does not need to be seen by a statistician. Declaration of competing interests: I declare that I have no competing interests Reviewer 2: The main purpose of this study is to determine HAX1 over-expression in cancer versus normal cells derived from patients. Hitherto, only scanty reports indicating such over-expression were available. In this study we have identified three solid tumors showing significant HAX1 over-expression and characterized differential expression of the five HAX1 splice variants in the breast cancer samples. Additionally, we ve analyzed the connections between HAX1 expression and estrogen presence (on the mrna and protein levels), obtaining some novel data pointing to different HAX1 localization in the cell. These results might be descriptive to some extent, but also point to the role of HAX1 in carcinogenesis, indicate specific contribution of some splice variants in the process and suggest that cellular localization might be a factor. This may lead to some clinical applications or at least represents a good starting point. HAX1 is described as involved in regulation of apoptosis and cell migration, but the mechanisms are still unknown and the proposed models are disputable. The proposed studies (measurements of the levels of apoptosis and/or migration in tumor cells depending on HAX-1 splice variants expression levels) are currently under investigation in our lab, but they represent different kinds of analysis and cannot be performed on the frozen/paraffin embedded tissue samples from patients. They also represent quite a different scope and try to answer different questions than studies presented in the current manuscript. I totally agree that defining mechanisms behind HAX-1 role in apoptosis and cell migration represents a fascinating task, but, taking into account the complexity of these processes and the number of the involved factors, that s probably a material for several publications.