CCL16 maintains stem cell-like properties in breast cancer by activating CCR2/GSK3Œ≤/Œ≤-catenin/OCT4 axis
Authors: Wenzhi Shen
Yanping Li
Rong Xiang
Shanshan Wang
Jianjun Li
Xiaoran Liu
Na Luo
Renle Du
Jiaping Tang
Xiaoyuan Zhang
Wancai Yang
Yunping Luo
Zhixin Zhang
Dehong Luo
Yong Xia
Siyuan Yan
Journal: Theranostics (2021)
Discipline: CLINICAL MEDICINE
Keywords: CCL16; CCR2; CSCs; OCT4; breast cancer
Affiliations: Dept. of Pathology and Institute of Precision Medicine, Jining Medical University, Jining 272067, China., Institute of Breast Research, Jining Medical University, Jining 272067, China., Dept. of Anatomy and Histology, School of Medicine, Nankai University, Tianjin 300071, China., Dept. of Gastrointestinal Surgery, Affiliated Hospital of Jining Medical University, Jining 272029, China., Dept. of Immunology, School of Medicine, Nankai University, Tianjin 300071, China., The First People's Hospital of Zunyi, Zunyi, 563002, China., Dept. of Immunology, Institute of Basic Medical Science, Chinese Academy of Medical Science, School of Basic Medicine Peking Union Medical College, Beijing, 100005, China.

Abstract:  Rationale: Considerable evidence suggests that breast cancer metastasis and recurrence occur due to emergence of cancer stem cells (CSCs). In our previous study, we designed a high-throughput siRNA screening platform that identifies inflammation genes involved in the regulation of cancer cell stemness. We reported that CCL16 protein decreases OCT4 expression and reduces the ALDH+ subpopulation. However, the mechanism by which CCL16 maintains stem cell-like properties remains unclear. Methods: Tissue microarrays were used to evaluate CCL16 expression. Cancer stemness assays were performed in CCL16 knockdown and overexpressing cells in vitro and in a xenograft model in vivo. Human phosphokinase array, immunofluorescence and chromatin immunoprecipitation assays were performed to explore the underlying mechanism. Results: We report that CCL16 was overexpressed in breast tumors and significantly correlated with clinical progression. We found that silencing CCL16 in MDA-MB-231 and BT549 cells diminished CSC properties including ALDH+ subpopulation, side population, chemo-resistance, and sphere formation. Furthermore, mice bearing CCL16-silenced MDA-MB-231 xenografts had lower tumorigenic frequency and developed smaller tumors. Exploration of the underlying mechanism found that CCL16 selects CCR2 to activate p-AKT/GSK3Œ≤ signaling and facilitate Œ≤-catenin nuclear translocation. Further, CCL16 binds to the OCT4 promoter and promotes OCT4 expression. In addition, shRNAs targeting CCR2 and XAV939 targeting Œ≤-catenin abolished CCL16-mediated cancer stemness. Upstream, IL10 mediates STAT3 activation, which binds to the CCL16 promoter and enhances its expression. The STAT3-targeted inhibitor Stattic suppressed CCL16 expression in vitro and restrained tumor progression in vivo. Conclusions: We identified a potential CSC regulator and suggest a novel mechanism for how CCL16 governs cancer cell stemness. We propose that CCL16 could be an effective target for breast cancer therapy. 

Retracted: No

PubPeer comments:
 Figure 7E appears to have 2 images which overlap. ***  , 
 At least two images in Figure 8D share some similar internal features, but the images do not appear to be identical. Could the authors comment? ***  , 
 #2 Thanks for your concern. We checked the analysis record as soon as possible, we found that the two images were actually from different samples as below, to compare the tumor volume difference between each group, we regulated the color bar scale to the same range.  #1 Thank you for your pointing this out. We checked the original data as soon as possible, as every slides we captured several images for analysis, we are very sorry that we used the wrong images (from one group) unintentionally, we have contacted the journal and applied for correction. , 
 #3 Thank you for moving to correct Figure 7E. Regarding the two images in #2, thank you for providing higher resolution photos. I believe these images are of the same animal. You will see below, especially on the left side, the two images unexpectedly share many similarities. *** False color  *** Inverse  *** However, the number of other images in 8D which seem to appear multiple times is concerning. ***  , 
 Thank you for your pointing this out. We are very sorry for these mistakes. We checked the analysis record carefully and finally found some reason caused the images seem to appear multiple times as below: Our first grouped data was arranged in the PPT (see image 1), but when we copied the data and paste them in Illustrator CS6 (see image 2), some of them were changed and became the same, we have marked them in different color, we performed this process several times and got the same results. We really donu2019t know why. To record this, we made a video by others but failed to upload. Anyway, Thanks again for pointing these mistakes out, we have applied for correction.   , 
 May 15, 2021 In our article [1], there were misplaced images in Figure 7E and Figure 8D, respectively. The corrected version is provided here: ***  ***  The correction made in this erratum does not affect the original conclusions. The authors apologize for any inconvenience or misunderstanding that this error may have caused. , 
 At least one image of an animal in Corrected Figure 8D seems to have been used to represent different conditions in a previously published paper with at least one shared author (WS). Corrected Figure 8D - excerpt, Theranostics (2021), doi: 10.7150 thno.51000, Received: July 23, 2020, https: pubpeer.com publications 8B69E99936A19CA66690AE906036EC Figure 2E, Stem Cell Research Therapy (2020), doi: 10.1186 s13287-020-01884-4, Received: December 7, 2019, https: pubpeer.com publications 80FE8CA2ADFDCFE7BDB78213647819 The unusual black boxes in Figure 2E seem to be present in all versions of the paper I could find online. ***  ,