TGF-Beta suppresses VEGFA-mediated angiogenesis in colon cancer metastasis
Authors: Geoffrey Talmon
Anathbandhu Chaudhuri
Liying Geng
James L Wisecarver
Jing Wang
Journal: PloS one (2013)
Discipline: Multidisciplinary
Keywords: FET cell line; TGF-α; EGFR/ErbB signaling; xenograft tumors; TGF-beta
Affiliations: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

Abstract:  The FET cell line, derived from an early stage colon carcinoma, is non-tumorigenic in athymic nude mice. Engineered FET cells that express TGF-α (FETα) display constitutively active EGFR/ErbB signaling. These cells readily formed xenograft tumors in athymic nude mice. Importantly, FETα cells retained their response to TGF-beta-mediated growth inhibition, and, like the parental FET cells, expression of a dominant negative TGF-beta type II receptor (DNRII) in FETα cells (FETα/DNRII) abrogated responsiveness to TGF-beta-induced growth inhibition and apoptosis under stress conditions in vitro and increased metastatic potential in an orthotopic model in vivo, which indicates metastasis suppressor activity of TGF-beta signaling in this model. Cancer angiogenesis is widely regarded as a key attribute for tumor formation and progression. Here we show that TGF-beta signaling inhibits expression of vascular endothelial growth factor A (VEGFA) and that loss of autocrine TGF-beta in FETα/DNRII cells resulted in increased expression of VEGFA. Regulation of VEGFA expression by TGF-beta is not at the transcriptional level but at the post-transcriptional level. Our results indicate that TGF-beta decreases VEGFA protein stability through ubiquitination and degradation in a PKA- and Smad3-dependent and Smad2-independent pathway. Immunohistochemical (IHC) analyses of orthotopic tumors showed significantly reduced TGF-beta signaling, increased CD31 and VEGFA staining in tumors of FETα/DNRII cells as compared to those of vector control cells. These results indicate that inhibition of TGF-beta signaling increases VEGFA expression and angiogenesis, which could potentially contribute to enhanced metastasis of those cells in vivo. IHC studies performed on human colon adenocarcinoma specimens showed that TGF-beta signaling is inversely correlated with VEGFA expression, indicating that TGF-beta-mediated suppression of VEGFA expression exists in colon cancer patients. 

Retracted: No

PubPeer comments:
 Readers on social media have expressed concern about apparent splicing in VEGFA band but not in Actin band in Figure 1A. *** ![file] *** Full Figure and caption ![file] , 
 The splicing is of course a concern, but this figure has another problem. The actin control in panel A reprised its appearance one year later in Oncogene. Of course, they are different experiments. It seems to me the figures in this paper and the Oncogene paper need some serious scrutiny. Being an old geezer, I do not follow social media, so I do not know what the buzz is about. Can someone fill me in? Below: figures as they appeared in PLOS one and Oncogene. The actin panelss are underlined in red. ![file] Actin controls with contrast and brightness adjusted. The image in Oncogene is at lower resolution, but it still appears to be the same image as that in PLOS one. ![file] , 
 #2 Thank you for your additional work. My reference to social media is only intended to convey that I checked with some more knowledgeable readers about the image I posted before raising the concern here on PubPeer. The article below explains this more thoroughly u2014 and wonu2019t require you to use Twitter :-) My amateur contributions are not always welcomed so I try to be careful. https: forbetterscience.com 2019 07 30 help-with-another-not-on-pubpeer-yet amp , 
 The first commentator was more observant than I. I am not sure that I would have detected the splices, so good call. This gel is actually quite an assemblage of stuff. If one adjusts the contrast and brightness, it looks to me that all the kings horses and all the kings men may have partly reassembled Humpty Dumpty. Appears to me that five different chunks of gel were put together. So perhaps we could call it  Five Easy Pieces . Difficult to see how they go with the actin lanes. ![file] , 
 #1 Does it bother anyone that they show a cell with a nucleus full of VEGF, and that both cells in the figure are dead or dying? , 
 It apparently did not bother the authors or the reviewers, for starters. , 
 #2 Dear Drs. Biskrensis and Labialis, Thank you for your comments. You are correct regarding Fig. 1A. The VEGFA panel was assembled from the same western blot with the relevant samples (in squares) put together. Please see the original blot shown below. The actin blot was mistakenly used. The correct one is shown below. We apologize for the mistake. Thank you. Best, Jing Wang ![file] , 
 #7 Thank you for your reply. Can you post a copy of the replacement figure here once it has been prepared for the corrigendum? Also, do you have any comment to make regarding the apparent appearance of the original actin blot in the Oncogene paper as mentioned in #2? , 
 Dear Actinopolyspora, Sorry for the delayed response. Thank you for your comment and suggestion. Please see the uploaded Fig. 1A below with indication of splicing. Two projects were carried out at the same time. A mistake was made when preparing the figures. We apologize again for the mistake. Thank you. Best, Jing Wang ![file] , 
 Am I correct in concluding that you are saying that the wrong actin blot was used in the Oncogene paper? , 
 #10 And a related question for the authors: will you be making a correction to the Oncogene paper as well? , 
 I have the same question as Actinopolyspora Biskrensis. So amen to the last comment. , 
 Just to document the correction on this paper. ![file] , 
 #11 We will make a correction. Thank you. , 
 #10 Yes, you are correct. Thank you. ,