TGLI1 transcription factor mediates breast cancer brain metastasis via activating metastasis-initiating cancer stem cells and astrocytes in the tumor microenvironment
Authors: Fei Xing
Austin Arrigo
Jimmy Ruiz
Richard L Carpenter
Kounosuke Watabe
Waldemar Debinski
Linda J Metheny-Barlow
Daniel Doheny
Roy Strowd
Marlyn Anguelov
Boris Pasche
Angelina T Regua
Hui-Wen Lo
Michael Chan
Sherona R Sirkisoon
Jiayuh Lin
Dongqin Zhu
Tadas Rimkus
Noah R Aguayo
Journal: Oncogene (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Breast cancer metastasis; TGLI1; GLI1; Brain metastasis; Metastasis-initiating cells
Affiliations: Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA., Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Bloomington, IN, USA., Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, NC, USA., Department of Radiation Oncology, Wake Forest University School of Medicine, Winston-Salem, NC, USA., Department of Hematology and Oncology, Wake Forest University School of Medicine, Winston-Salem, NC, USA., Department of Neurology, Wake Forest University School of Medicine, Winston-Salem, NC, USA., Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA., Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA. ., Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, NC, USA. .

Abstract:  Mechanisms for breast cancer metastasis remain unclear. Whether truncated glioma-associated oncogene homolog 1 (TGLI1), a transcription factor known to promote angiogenesis, migration and invasion, plays any role in metastasis of any tumor type has never been investigated. In this study, results of two mouse models of breast cancer metastasis showed that ectopic expression of TGLI1, but not GLI1, promoted preferential metastasis to the brain. Conversely, selective TGLI1 knockdown using antisense oligonucleotides led to decreased breast cancer brain metastasis (BCBM) in vivo. Immunohistochemical staining showed that TGLI1, but not GLI1, was increased in lymph node metastases compared to matched primary tumors, and that TGLI1 was expressed at higher levels in BCBM specimens compared to primary tumors. TGLI1 activation is associated with a shortened time to develop BCBM and enriched in HER2-enriched and triple-negative breast cancers. Radioresistant BCBM cell lines and specimens expressed higher levels of TGLI1, but not GLI1, than radiosensitive counterparts. Since cancer stem cells (CSCs) are radioresistant and metastasis-initiating cells, we examined TGLI1 for its involvement in breast CSCs and found TGLI1 to transcriptionally activate stemness genes CD44, Nanog, Sox2, and OCT4 leading to CSC renewal, and TGLI1 outcompetes with GLI1 for binding to target promoters. We next examined whether astrocyte-priming underlies TGLI1-mediated brain tropism and found that TGLI1-positive CSCs strongly activated and interacted with astrocytes in vitro and in vivo. These findings demonstrate, for the first time, that TGLI1 mediates breast cancer metastasis to the brain, in part, through promoting metastasis-initiating CSCs and activating astrocytes in BCBM microenvironment. 

Retracted: No

PubPeer comments:
 Figure 1C appears to show a sharp transition. Could the authors post the original scan? Below: contrast enhanced image *** ![file] , 
 I agree with the concerned raised here. In addition, in Figure 1I, there are discontinuities in the presented IVIS images. It is important than the samples are imaged with the identical setup in order to compare. Discontinuities indicates that samples are taken from different experiments conditions. ![file] , 
 #1 Thank you for the inquiry. I apologize for my delayed response as I just became aware of this inquiry today (9 4 20) through your email. Here is my response below. About Fig. 1A, the original images are shown in attached file. About Fig. 1C, the original images are shown in attached file. We removed BCM-3887 because we wanted to show 2 TNBC and 2 HER2+ PDXs instead of 3 TNBC and 2 HER2+. All four lines shown in the manuscript were on the same original blots. Thank you for bring this manner into my attention and I appreciate you for reading our paper. ![file] , 
 #2 Thank you for your inquiry and I apologize for my delayed response. I only became aware of the inquiry today. Here is my response. About the western blots in Fig. 1C, the original images are shown in attached file. As you can see from the original images, all four lines shown in the manuscript were on the same original blots. We removed BCM-3887 (in the second lane) because we wanted to show 2 TNBC and 2 HER2+ PDXs instead of 3 TNBC and 2 HER2+. About Fig. 1I, we showed only representative organ images (3 images out of 10-12) as stated in the manuscript. When we imaged these organs harvested from mice, we had to image them in batches because the imager has a limited capacity. In some cases, the 3 representative images were not taken together in the imager. For these reasons, the organ images in the publication had to be assembled. But we ensured the images show in the figure were under the same bioluminescent scale for fair comparisons (scale bar included). Thank you for brining this manner to my attention. Please feel free to reach out to me for further clarifications. ![file] , 
 #3 Thank you for posting these images. I would suggest that to address the concerns about undisclosed splices in 1C that you may wish to provide uncropped original scans rather than a previous version of the figure. Additionally, the author may wish to consult the journal about their preferred method of indicating splices to readers and peer reviewers. However, before doing so, I would suggest that the senior author more closely examine the uncropped blot as it appears the u201coriginalu201d provided to me via email may also include an undisclosed splice. *** Closeup ![file] , 
 #5 Thank you. We found the original scans of western blots (see below). For TGLI1, lane 2 (an empty lance) and lane 3 (BCM-3887) were removed. For tubulin, lane 2 was removed. Two different gels membranes were used in this western blots because these two proteins have very different molecular weight (145 kD versus 50 kD). But same cell lysates were run in both gels. In the future, we will be sure we avoid splicing and if it is necessary, we will indicate that in the figure. Thank you again for your comments! ![file] , 
 #6 I agree that splicing should be disclosed. The journal [editorial policies] make this point clearly. I would go so far to suggest that a correction should be published to disclose to readers that the original figure did not adhere to these policies, namely:  Quantitative comparisons between samples on different gels blots are discouraged; if this is unavoidable, the figure legend must state that the samples derive from the same experiment and that gels blots were processed in parallel. Vertically sliced gels that juxtapose lanes that were not contiguous in the experiment must have a clear separation or a black line delineating the boundary between the gels. Loading controls (e.g. GAPDH, actin) must be run on the same blot. Sample processing controls run on different gels must be identified as such, and distinctly from loading controls. , 
 I would also like to note that it appears that the image dimensions and contrast were altered from the original. So much so that I had difficulty matching the  original scans to the published figure. Immediately below, the band outlined in magenta appears to have a different shape, and the purple ones appear somewhat different as well. ![file] However, when I attempted to make the images appear the same, I found that by stretching horizontally and vertically (independently) and adjusting the contrast, I was able to approach the appearance in the published figure. At first glance the SKBR3 bands do not match, but after reviewing this for a while, I think they likely match. See below. ![file] I illustrated this to express concern that the original image dimensions and contrast may have be altered, which, if true, is unlikely to be considered a best practice. , 
 #7 Thanks for the suggestion. We will contact the journal for a correction request in which we will provide the original scans and also provide a new figure panel with the original dimension, as well as, a dividing line to indicate the splice. In the future, we will certainly carefully check journal guidelines and adhere to the guidelines. Thank you again for the comments, which are helpful when we prepare new manuscripts in the future. , 
 When comparing the provided raw data and Figure 1C, it is clear that the two blots were run on different gels (different number of wells), which makes it impossible to compare the two blots. Furthermore, the blots in Figure 1C show different exposure and are more stretched horizontally, compared to the provided raw blots. ![file] , 
 March 2, 2021 Correction to: Oncogene 39, 64u201378(2019) https: doi.org 10.1038 s41388-019-0959-3 Article published online 28 August 2019 Following the publication of this article, it was brought to the attention of the authors that dividing lines between lanes 1 and 2 in Fig. 1c were inadvertently omitted. Dividing lines are necessary to indicate that two irrelevant lanes were removed. The addition of dividing lines in the corrected figure does not change the conclusion but provides clarity. The corrected Fig. 1c (left) and the original images (right) are shown below. ![file] It was also noted that the source of a cell line was incorrectly stated (page 77; Materials and Methods; first paragraph; 2nd sentence). This has been corrected to read u201cThe brain metastatic cell line (SKBRM) and radio-resistant cell lines (SKBRM-RR and 231BRM-RR) were kind gifts from Drs. Fei Xing and Kounosuke Watabe; E6 E7 hTERT immortalized human astrocytes were kind gifts from Dr. Russell Pieper (University of California-San Francisco).u201d This has been corrected in the HTML and PDF versions of the article. ,