Axl contributes to efficient migration and invasion of melanoma cells

Axl, a member of the TAM receptor family has been broadly suggested to play a key role in tumor metastasis. However, the function of Axl in the invasion and metastasis of melanoma, the most lethal skin cancer, remains largely unknown. In the present study, we found that melanoma cell lines present variable protein levels of Axl and Tyro3; interestingly, MerTK is not noted at detectable levels in any of tested MGP (metastatic growth phase) cell lines. Treatment with recombinant human Gas6 significantly activates Akt in the Axl-expressing WM852 and IgR3 lines but just slightly in WM1158. IgR3, WM852 and WM1158 demonstrate different autocrine signaling. Knockdown of Axl by siRNA or the treatment with Axl-specific inhibitor R428 dramatically inhibits the migration and invasion of both IgR3 and WM852 in vitro. These findings suggest that Axl enhances the invasion of melanoma cells.

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Response: We now provide these data in the revised manuscript to show that the invasion of IgR3 in 1% FBS medium is significantly higher than that in 0.1% FBS medium.
8. Please ensure that you refer to Figure 5 in your text as, if accepted, production will need this reference to link the reader to the figure.
Response: We now address the data in Figure 5 in the revised text. 9. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.
Response: We have updated the references as suggested by the reviewers.

Review Comments to the Author
Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript by Shao and colleagues describes investigation into the effect of AXL stimulation and level on the migration and invasion of melanoma cells. The authors demonstrate clearly that stimulation of Axl signalling with Gas6 increases melanoma migration and invasion in lines with Axl expression. Further, knockdown of Axl expression either using transient or stable means reduces melanoma migration and invasion, as does treatment with a specific inhibitor. While the data arising from the stable knockdown is novel, much of the other data presented in the manuscript is confirmatory. Use of shAXL to reduce invasion in matrigel/boyden chamber Response: We apologize for these oversights. We now added these data and references with statement in the discussion.
Comment: In summary, the data presented in Figure 2A and B, Figure 3A-D and Figure 4A-D has been previously published by others.
Response: We include these data as part of the complete story as these are new data sets but refer to earlier works in the text. We used Gas6 to check if the interaction between Axl and EGFR is involved in the activation of Gas6-depependent of Axl in available melanoma cell lines. These data also confirmed that Gas6 functions in Axl positive melanoma cells.
Comment: Figure 2A is currently very confusing. It would be best to group the western blot images by cell line rather than antibody. Further, the figure legend states 10 nM EGF was used whereas the results text states 2 nM. Which is correct?
Response: We now reorganized Fig. 2A by cell line according to reviewer's suggestion. For short stimulation of cells with EGF, we usually use a concentration of 10 nM. We mistyped the concentration of EGF in text but that is now corrected.
Comment: There is currently no text in the results section that addresses the data in Figure 5.
Response: We apologize for this oversight. We now address the data in Figure 5.
Comment: The reference to figures throughout the manuscript needs revision. Further, the labelling of Supplementary Figure 2 A, B -A, B, C and D is confusing and should be revised.
Response: Figure S2 now is reorganized, relabeled and renumbered as Figure S3 in the revised manuscript.
Reviewer #2: Eight melanoma lines were evaluated for expression of EGFR and AXL family members. IgR3 and WM852 were then stimulated with Gas6 and EGF and evaluated for pAKT, pAXL, p38, pEGFR, and pERK. Knockdown of AXL blocked Gas6 induced pAKT in both lines.as well as a line lacking AXL (WM983b) although TYRO3 was present, indicating that AXL is a mediating of Gas6 induced Akt phosphorylation. WM852 showed stronger relative migration speeds induced by Gas6 or EGF compared to IgR3. With respect to invasion, IgR3 showed higher basal migration than WM852 but there was a similar absolute increase induced by Gas6. Basal migration and invasion of the two lines was reduced either by an AXL inhibitor or siRNA. The main claims of the paper are that AXL can mediate motility and invasion of melanoma cell lines. This has not been demonstrated previously for melanoma and thus adds to the body of knowledge for this disease.
Comment: The authors should discuss the current knowledge regarding AXL mediating Gas6 induced migration/invasion in other tumor types. There are some concerns regarding selected claims noted below. Detailed protocols are not required but more details regarding specific reagents and the initial width in the scratch assay should be indicated. The manuscript is written clearly and well organized with no dual use concerns.
Response: We thank the reviewer for the suggestion. We now discussed this in the part of discussion of our revised manuscript. The width of the wound scratch area is about 3 mm. It is now stated in the revised manuscript.
Other comments: Comment: On page 5, there is reference to "all 4 MGP" and "all 4 VGP". Figure 1 however does not clearly indicate 4 VGP lines -please correct the inconsistency. Is WM983B a VGP, MGP or neither? In referring to Figure 2 on page 5, it is stated" Surprisingly, we did not observe an increase in pERK in either IgR3 or WM852." However, the blot seems to clearly show increased pERK for IgR3 in response to EGF and possibly also for GM852. Similarly, though weaker, there is EGFR phosphorylation in response to EGF although it is claimed that there is not.
Response: We apologize for our imprecise statements. As the labeling in Figure 1, there are three VGP cell lines and five MGP cell lines; we now corrected this in the revised manuscript. For the reference to Figure 2 on page 5, we stated that "Surprisingly, we did not observe an increase in pERK in either IgR3 or WM852" referred to no significant pERK increase when cells are treated only by Gas6. The reviewer is correct in that EGF treatment significantly increased the pERK which has been mentioned on page 6. We also stated that "the EGFR was functioning as shown by phosphorylation when exposed to 10 nM EGF for 10 min" on page 6. This meant that the EGF treatment triggers the phosphorylation of EGFR in both IgR3 and WM852 cells. It is stronger in WM852 and weaker in IgR3. These statements are now clarified.
Comment: The statement on page 6 in the middle: "These findings indicate that Axl signaling is cell-line specific" is confusing -one possibly interpretation is there are lines that express axl but do not respond to Gas6. Please clarify or remove.
Response: We thank the review for pointing out this issue. We now deleted the statement in the revised manuscript.
Comment: Catalog numbers for siRNAs, antibodies, transwells, growth media, growth factors (Gas6, EGF) and all other biological reagents should be provided.
Response: We now provide catalog numbers for all reagents.
Comment: For Figure 3, please indicate the absolute basal migration speeds for all the lines. Does the smaller relative increase for IgR3 simply reflect a higher basal migration speed?
Response: The reviewer is correct in that IgR3 presents much high basal migration. We now updated Figure 3C with relative migratory units instead of normalized data to more clearly show this.
Comment: The statement at the top of page 7 "…suggest that pAkt plays important role in Gas6-mediated enhanced migration of IgR3 and WM852" does not seem justified since no inhibitor of Akt phosphorylation was tested.
Response: We thank the reviewer for pointing out this misstatement. We now corrected it to "suggest that Axl plays an important role in Gas6-mediated enhanced migration of IgR3 and WM852.
Comment: Figure S2 legend refers only to panels A and B while the figure is labeled A-D.
Response: We thank the reviewer for noting this omission. We now reorganized this figure and corrected the figure legend as shown in figure S3 in the revised manuscript.
Comment: In the discussion it is stated that ASL siRNA eliminated Gas6 induced p38 phosphorylation -that does not seem supported by Figure 2 since there is little gas6 induced p38 phosphorylation, especially in IgR3. Quantitative analysis and statistics should be used to show this point.
Response: We thank the reviewer for noting the lack of significance of this and have corrected this in the revised manuscript.