Axl mediates tumor invasion and chemosensitivity through PI3K/Akt signaling pathway and is transcriptionally regulated by slug in breast carcinoma
Authors: Chen Liu
Dongliang Ren
Yanyan Li
Yongfu Zhao
Ning Wang
Li Jia
Yanxin Gong
Xu Zhang
Journal: IUBMB life (2014)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Axl; GSK3β; PI3K/Akt; chemosensitivity; invasion
Affiliations: Department of General Surgery, Dalian Medical University, Dalian, Liaoning Province, China.

Abstract:  The invasion and chemoresistance are crucial causes of morbidity and relapse for cancer patients. Axl is implicated in the modulation of cell invasion, cancer metastasis, and chemosensitivity in human breast carcinoma cell lines. Both breast cancer cell lines and tissues displayed increased expression of Axl, and it over expressed in highly metastatic breast cancer. The altered expression level of Axl was corresponding to the changed invasive phenotype and chemosensitivity of MDA-MB-231 cells both in vitro and in vivo. Further data indicated that experimental inhibition of Axl by RNAi assay inhibited phosphatidylinositol 3-kinase (PI3K)/Akt/GSK3β signaling pathway, resulted in the decrease of Slug expression, and further suppressed cell invasion properties and chemosensitivity. What is more, after the detection and statistics in human breast cancer specimens, we found the Axl expression was closely correlated with histological grade, lymph node metastasis, and clinical stage (P &lt; 0.01). Taken together, these findings indicate that Axl exerts the role of tumor metastasis and chemosensitivity through activation of the PI3K/Akt/GSK3β signaling pathway, which is transcriptionally regulated by Slug. Our findings support the possibility that Axl is a novel regulator. It means by targeting Axl or its related signaling pathways, we can reduce the invasion and chemosensitivity of breast tumor. 

Retracted: No

PubPeer comments:
 Concern about Figure 5G. Two panels (representing different cells and treatments) appear to look identical. Shown with blue boxes. ![file] , 
 Fig 3(B).  Reduced regulation of **Axl** was also shown by IHC staining in xenograft tumors derived from Axl shRNA cells (x400). Below: Fig 3(B)  B: Reduced regulation of **ST6GalNAcII** was also shown by IHC staining in xenograft tumors derived from MDA-MB-231-ST6GalNAcII shRNA1 cells (400x) , from  Axl as a downstream effector of TGF-u03b21 via PI3K Akt-PAK1 signaling pathway promotes tumor invasion and chemoresistance in breast carcinoma [(Li et al 2014)] . Different shRNA targets seem to produce xenograft tumors differing only by a 180-deg rotation. ![file] , 
 Figure 5(C):  After MDA-MB-231 cells been treated with 20 mM Akt shRNA (T308A S473A) for 48 H, we found that the protein levels of p-Akt, GSK3b, and Slug have a tendency to descend. Surprisingly little difference between Slug and P-Akt S473 panels. ![file] Fig 5(A):  Cells were treated with 20 mM LY294002 and further subjected to Akt, GSK3b, and Slug analysis, respectively, in MDA-MB-231 cells and MDA-MB-231 Axl shRNA cells. The phosphorylation levels of Akt and GSK3b, Slug were found to be down-regulated in cells treated with LY294002. This image seems to duplicate Supplemental Figure 1a from [Li et al (2014)] , except that the P-Gsk3-beta panels at 46 kDa become p-PAK1 at 72 kDa, or vice versa. ![] , 
 Fig 6 (B).  MDA-MB-231-control shRNA and MDA-MB-231-Slug shRNA cells were treated with or without Gas6, respectively. The invasion property was appraised by counting the average number of cells that invaded through the filter described as ECMatrix gel analysis. Comparing with Figures 2(c) and (d) from [Li et al (2014)] :  **c** In vitro ECMatrix gel analysis was performed. The average number of cells that invaded through the filter was counted. MDA-MB-231-TGF-u03b21 shRNA cells were significantly less invasive than the MDA-MB231 and MDA-MB-231-control shRNA cells. **d** ... MDA-MB-231- Axl shRNA cells were significantly less invasive than the untreated and control cells. ![file] Concerns arise as to the correct identification of the images. , 
 #2 Whoops, comparing with the wrong paper.  Below: Fig 3(B)  B: Reduced regulation of ST6GalNAcII was also shown by IHC staining in xenograft tumors derived from MDA-MB-231-ST6GalNAcII shRNA1 cells (400x) , from  Axl as a downstream effector of TGF-u03b21 via PI3K Akt-PAK1 signaling pathway promotes tumor invasion and chemoresistance in breast carcinoma (Li et al 2014). should read Below: Fig 3(B)  B: Reduced regulation of ST6GalNAcII was also shown by IHC staining in xenograft tumors derived from MDA-MB-231-ST6GalNAcII shRNA1 cells (400x) , from ** ST6GalNAcII Mediates the Invasive Properties of Breast Carcinoma Through PI3K Akt NF-jB Signaling Pathway ** [(Ren et al 2014)] . , 
 [Right]: Fig 4 B  With western blot analysis, the main signal molecules of PI3K Akt GSK3b signal pathway were found to be down-regulated at protein phosphorylation level (p-Akt, p-GSK3b) in MDA-MB-231 cells with Axl shRNA, as compared with those in control shRNA cells . [Left] Fig 5 (b):  With Western blot analysis, the protein phosphorylation levels of PI3K Akt-PAK1 signaling molecules were found to be down-regulated in MDA-MB231 cells with Axl shRNA, as compared with those in control shRNA cells . From  Axl as a downstream effector of TGF-u03b21 via PI3K Akt-PAK1 signaling pathway promotes tumor invasion and chemoresistance in breast carcinoma [(Li et al 2015)] . ![file] Three lanes of PI3K match, implying that the two figures came from the same blot, and that the shRNA variant of this paper is the shRNA-1 variant from Li et al (2015). Yet the Akt and GAPDH panels do not match. It is a mystery. , 
 Fig 2 (B):  After shRNA transfection, distinct reduction of Axl was observed at protein levels by western blot analysis. GAPDH was also examined and served as controls for sample loading . Here at right. The protein measurements also appear in Fig 2(b) of [Li et al (2015)] . :  The mRNA and protein levels of Axl gene remarkably declined in MDAMB-231 cells with Axl shRNA comparing to untreated and control cells. Here at left. Which is fair enough, for the conditions are the same. Odd, though, that the GAPDH panels for normalising are **not** the same in the two studies. ![file] ,