VEGFA activates an epigenetic pathway upregulating ovarian cancer‐initiating cells

Abstract The angiogenic factor, VEGFA, is a therapeutic target in ovarian cancer (OVCA). VEGFA can also stimulate stem‐like cells in certain cancers, but mechanisms thereof are poorly understood. Here, we show that VEGFA mediates stem cell actions in primary human OVCA culture and OVCA lines via VEGFR2‐dependent Src activation to upregulate Bmi1, tumor spheres, and ALDH1 activity. The VEGFA‐mediated increase in spheres was abrogated by Src inhibition or SRC knockdown. VEGFA stimulated sphere formation only in the ALDH1+ subpopulation and increased OVCA‐initiating cells and tumor formation in vivo through Bmi1. In contrast to its action in hemopoietic malignancies, DNA methyl transferase 3A (DNMT3A) appears to play a pro‐oncogenic role in ovarian cancer. VEGFA‐driven Src increased DNMT3A leading to miR‐128‐2 methylation and upregulation of Bmi1 to increase stem‐like cells. SRC knockdown was rescued by antagomir to miR‐128. DNMT3A knockdown prevented VEGFA‐driven miR‐128‐2 loss, and the increase in Bmi1 and tumor spheres. Analysis of over 1,300 primary human OVCAs revealed an aggressive subset in which high VEGFA is associated with miR‐128‐2 loss. Thus, VEGFA stimulates OVCA stem‐like cells through Src‐DNMT3A‐driven miR‐128‐2 methylation and Bmi1 upregulation.

Thank you for the submission of your manuscript to EMBO Molecular Medicine. We are very sorry that it has taken longer than usual to get back to you on your manuscript.
As I had anticipated, due to the holiday season we experienced unusual difficulties in securing three willing and appropriate reviewers. As a further delay cannot be justified I have decided to proceed based on the two available evaluations.
Although #1 is more reserved, both reviewers are largely positive but raise a few of issues that require your action. I will not go into detail, as their comments are quite clear.
Reviewer 1 feels that a mechanistic analysis upgrade is required to required to strengthen the findings and increase their impact. We agree, especially considered the novelty issue mentioned by reviewer 2. I should also mention that, notwithstanding the overall interest of your findings, we shared the same concern while initially deciding on your manuscript. Reviewer 2 also raises a very interesting general point concerning the cellular origin of ovarian cancer(s) and suggests that your manuscript be reworded to reflect that it is mostly focused on the HGSC type.
In conclusion, while publication of the paper cannot be considered at this stage, we would be pleased to consider a revised submission, with the understanding that the Reviewers' concerns must be addressed including with additional experimental data where appropriate and that acceptance of the manuscript will entail a second round of review. responses included in the next, final version of the manuscript.
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I look forward to seeing a revised form of your manuscript as soon as possible.
***** Reviewer's comments ***** Referee #1 (Remarks): In this manuscript the authors show that VEGF favours the expansion of aldehyde dehydrogenase positive ovarian cancer stem cells. The authors define a mechanisms connecting VEGFR2 with src, which in turn positively regulates the expression of Bmi-1. The effect of VEGF on Bmi1 expression is post-transcriptional. Actually VEGF promotes the methylation of miR-128 resulting in its repression. miR-128 is known to negatively regulate Bmi1 expression. Interestingly, high levels of VEGF with loss of miR128 select a subpopulation of ovarian cancer with a poor prognosis. The paper contains relevant information but the present version requires new experiments to explain better the mechanisms involved and some controls.

CRITICISMS
The effect of VEGF on the expansion of CSF is detectable after 7 days of treatment and the authors show that the expression of phosphorylated src and Bmi1 started after 6 hours and persisted up to day 7. This prolonged stimulation of src requires a deeper analysis than that proposed by the present version of the manuscript. Do the cells undergo a VEGF autocrine pathway? Does this putative mechanism contribute in the prolonged src activation? Which is the minimal time required to VEGF to trigger src activation, increased expression of Bmi1 and DNMT3A. Actually the authors stimulate spheres with VEGFA every days. Which is the time course of src activation, VEGFR2 phosphorylation, Bmi1 and DNMT3A expression in the 2 days frame? In the experimental conditions described by the authors, which are the VEGFR2 phosphorylation sites crucial for the described activities?
The authors show that VEGFA does not modify the cell cycle in the sphere? Does it has an antiapoptotic effect? Does the lack of effect on cell cycle occur in 2D culture conditions?
The role of src in the VEGF-mediated expansion of CSF is based on the use of a specific inhibitor (Fig 2). In other experiments the ( Fig 6) the authors use a specific siRNA. I suggest planning an experiment on sphere formation by showing the effect of src silencing.
The authors demonstrate the pivotal role of miR128. Does antagomir128 mimic the effect of VEGFA?
Referee #2 (Comments on Novelty/Model System): The paper uses accepted methodology for studying cancer stem cells, CSCs therefore I conclude that the model systems are adequate and the technical quality high. I would rate the novelty as medium as previous papers have shown that VEGF stimulates CSCs. The medical impact is probably high as there are supporting data from large numbers of patient samples and an effect on CSC expansion may in part explain the brevity of responses to anti-VEGF therapies.

Referee #2 (Remarks):
This paper investigates the molecular mechanisms by which VEGF stimulates expansion of cancer stem cells, CSCs in ovarian cancer cell lines and relates the data to ovarian cancer patient data. In general the work is carefully performed but the manuscript requires revision to reflect our current knowledge of the human cancers collectively termed 'ovarian'. We now know that invasive 'ovarian' cancers are actually four different cancers, none of which are thought to arise in the ovary, high grade serous, HGSC, (fallopian tube origin) endometriod and clear cell (arise in the endometrium) mucinous (probably a gut metastasis). In this paper PEO1 cells are HGSC I believe as are the OC1-C5X cells. Domcke et al in a Nature Communications paper in 2013 also described OVCAR8 as 'possibly HGSC' and they do have a TP53 mutation which is characteristic of HGSC. Therefore I suggest that to reflect our current knowledge OVCA should be replaced with HGSC and all patient data used should be from HGSC patients. The other 'ovarian' cancers have different genetic drivers and it is possible that their CSCs may have different markers and different responses to growth factors.

The effect of VEGF on the expansion of CSF is detectable after 7 days of treatment and the authors show that the expression of phosphorylated Src and Bmi1 started after 6 hours and persisted up to day 7. This prolonged stimulation of Src requires a deeper analysis than that
proposed by the present version of the manuscript. Do the cells undergo a VEGF autocrine pathway? Does this putative mechanism contribute in the prolonged Src activation? Which is the minimal time required to VEGF to trigger Src activation, increased expression of Bmi1 and DNMT3A. Actually the authors stimulate spheres with VEGFA every days. Which is the time course of Src activation, VEGFR2 phosphorylation, Bmi1 and DNMT3A expression in the 2 days frame?
First, we would like to correct the referee's statement that "the authors stimulate spheres with VEGFA every days." We treated cells in 2D culture with VEGFA for a 7 day period with 10ng/ml VEGFA, which was replenished every 2 days prior to plating cells for sphere formation assay. Cells received no further VEGFA during the 2 week interval of sphere formation after seeding onto ultralow adhesion plates.
As requested by Referee 1, we present a more detailed kinetic time course in new data in Fig 6 showing that short term VEGFA exposure rapidly activates VEGFR2pY1175 leading to increased SrcpY416. Both DNMT3 and BMI1 increased gradually within the first 6 hrs after VEGFA addition. Notably, miR128 levels fell significantly within the first 6-12 hrs, and the decline was sustained and progressive over the next 7 days. Thus, while VEGFR2 and Src signaling is triggered within minutes, the signaling increases progressively and the epigenetic consequences of Srcdependent DNMT3A upregulation, and miR128 loss were felt progressively over the successive 3-4 DNA replication cycles over 7 days. In keeping with an epigenetic effect and not merely a signaling dependent mechanism, the increase in sphere forming ability was not fully felt until after several cell divisions: VEGFA exposure for 48 hrs was not sufficient to increase spheres and a 7 day exposure was required. These data support that long term VEGFA exposure is required increase the abundance of CSC. VEGFA treatment does indeed upregulate VEGFA gene expression, which would leading to a VEGFA-driven feed-forward mechanism to sustain and amplify Src activation with prolonged exposure.
2. In the experimental conditions described by the authors, which are the VEGFR2 phosphorylation sites crucial for the described activities? We assayed VEGFR2 activity using an antibody reactive to VEGFR2pTyr1175, a major phosphorylation site required for receptor activation (See Fig 6A).
The authors show that VEGFA does not modify the cell cycle in the sphere? Does it have an antiapoptotic effect? Does the lack of effect on cell cycle occur in 2D culture conditions? In ExtraView Fig 1A we showed VEGFA exposure did not change the cell cycle in 2D culture conditions, nor did it affect cell cycle distribution when cells were grown as spheres for 48 or 7 days (48 hr exposure cell cycle effects, Extra View Fig 1A). The short term increase in cell number over 7 days was not affected by cell death (ExtraView Fig 1C). We have added new date to show that Annexin V staining for apoptotic cells was unchanged by VEGFA exposure (see ExtraView Fig  1B). Notably VEGFA also did not change cell viability after 7 days (ExtraView Fig 3B).

The role of Src in the VEGF-mediated expansion of CSF is based on the use of a specific
inhibitor (Fig 2). In other experiments the (Fig 6) the authors use a specific siRNA. I suggest planning an experiment on sphere formation by showing the effect of src silencing. The referee correctly points out that AZD0530 is a pan-Src family kinase inhibitor. As requested by Referee 1, we present new data in the revised Figure 4E showing that siRNA of SRC prevented VEGFA mediated increase in sphere formation.
4. The authors demonstrate the pivotal role of miR128. Does antagomir128 mimic the effect of VEGFA? As requested by Referee 1, we present new data in the revised Figure 4E showing that antagomiR-128 decreased miRNA128 levels by QPRC and also stimulated sphere formation, even without added VEGFA. In addition, While SiRNA Src prevented the upregulation of spheres by VEGFA, siRNA Src could be rescued by miR-128 antagomir, confirming that miR-128 is regulated downstream of Src.

REFEREE 2.
Minor point While PEO1R and OVCAR8 are of high grade serous ovarian cancer (HGSC) origin, the primary ovarian cancer culture, OCI-C5X, was derived from a high grade Clear Cell cancer. All of the mechanistic data in the paper were carried out with not lonely the HGSC cell lines but confirmed in primary Clear cell cOVCA-derived OCI-C5X. Thus we respectfully submit that our findings of a VEGFA-dependent stem cell expansion pathway induced may be more broadly applicable, and not just apply to HGSC.

Page 20 Methods -is 'mammospheres' the correct term
Because the critiques were straightforward and the new data address their concerns simply, we ask that you consider making an editorial decision in favor of accepting the manuscript. We thank you for considering this work and look forward to hearing from you. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) Reviewer 2 notes that the data in the paper suggest that VEGF can have effects on cancer initiating cells from multiple cancer types. S/he therefore would like you to mention this in the discussion and to better explain the different origins of the cell lines in the results section.

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***** Reviewer's comments ***** Referee #1 (Remarks): The authors did a whole revision Referee #2 (Comments on Novelty/Model System): The authors have used two cell lines of high grade serous ovarian cancer and one cell line of clear cell cancer origin. All information we have now shows that these are completely different cancers with different sites of origin and different genetic drivers. The data in the paper therefore suggest that VEGF can have effects on cancer initiating cells from at least two different cancer types. I think this should be mentioned in the discussion and the different origins of the cell lines made more clear in the results section.
2nd Revision -authors' response 16 December 2016 We submit now a version with the minor revisions requested by Reviewer 2.
1. As requested, the different origins of ovarian cancer lines used in the paper are made clearer in the revised results section. We also make the comment in the last paragraph of the discussion that since ovarian cancers of clear cell and high grade serous origin are very different, our data may have application to other epithelial cancers of different tissue origin also. The revisions are tracked in the word version uploaded. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
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