Interleukin-4 enhances prostate-specific antigen expression by activation of the androgen receptor and Akt pathway
Authors: Min Hou
Wei Lou
Sergio A Onate
Allen C Gao
Soo Ok Lee
Journal: Oncogene (2003)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Androgen receptor; Prostate cancer; IL-4; PSA expression; Akt pathway
Affiliations: Department of Medicine and Pharmacology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.

Abstract:  Androgen receptor (AR) plays an important role in the development and progression of prostate cancer upon the action of androgen through the binding of the androgen-responsive elements (AREs) on the target genes. Abnormal activation of the AR by nonandrogen has been implicated in the progression of androgen-independent prostate cancer. The levels of interleukin-4 (IL-4) are significantly elevated in sera of patients with hormone refractory prostate cancer. The potential role of IL-4 on the activation of AR was investigated in prostate cancer cells. IL-4 enhances AR-mediated prostate-specific antigen (PSA) expression and ARE-containing gene activity through activation of the AR in the androgen ablation condition in human prostate cancer cells. The AR can also be sensitized by IL-4 and activated by significantly lower levels of androgen (10 pM of R1881) in prostate cancer cells. IL-4 enhances nuclear translocation of AR and increases binding of the AR to the ARE in LNCaP prostate cancer cells. Blocking of the Akt pathway by an Akt-specific inhibitor LY294002 abrogates IL-4-induced PSA expression and AR signaling. These results demonstrate that IL-4 enhances PSA expression through activation of the AR and Akt signaling pathways in LNCaP prostate cancer cells. Understanding IL-4-induced signaling leading to abnormal activation of AR will provide insights into the molecular mechanisms of androgen-independent progression of prostate cancer cells. 

Retracted: No

PubPeer comments:
 Figure 6C. * Red arrows: The far-right lane in the  Nuclear AR panel looks remarkably similar to the left-most lane in the  Total AR panel. Note the position and shape of a dot above the top band, and the spacing between the upper and lower bands. Would the authors still be able to pull out the original blots to check, please? ![file] , 
 Figure 1B. Some lanes look similar to lanes in Figure 4A of an older paper by the same research group. Could the authors please check? * Blue boxes: Lanes 2-5 in the R1881 PSA panel of Figure 1B look similar to lanes 1-4 in the PSA panel of Figure 4A of Fernando DeMiguel et al., The Prostate 52:123-129 (2002), DOI 10.1002 pros.10110. * Green boxes: Lanes 1-4 in GAPDH panel of Figure 1B look similar to lanes 1-4 in the GAPDH panel of Figure 4A of Fernando DeMiguel et al., The Prostate 52:123-129 (2002), DOI 10.1002 pros.10110. Note that in this paper, the lanes represent a time series within LNCaP cells, while in the older paper these lanes represent different cell lines. ![file] , 
 Figure 6B. * Pink boxes: Figure 6B (supershift assay of LNCaP cells after R1881 treatment) looks similar to Figure 3B from Soo Ok Lee et al., Clin Cancer Res 2003, PMID: 12538490 (supershift assay of LN-17 cells). Could the authors please clarify if these are the same experiments? ![file] , 
 GAPDH panel in another paper also looks more similar than you would expect (vertical re-sizing). https: imgur.com YC4CRt2 , 
 Dear Lucinda. With regards to your concerns with image issues in the 2003 publications, I agree only in part with the findings. The following should be considered before publishing the editorial comments: 1) In Figure 6C, upon enlarged inspection, the shape of the dot is distinct, and distance between the dot and the immune detected nuclear AR bands is greater than the one in Total AR. It is important to mention that the immune detected AR bands are always quite similar between experiments as Western blots are very well standardized. An experiment done one week will appear similar to the next. 2) In Figure 1B and 4A, the GAPDH in green is also different. The immune detection is similar but different. I, however, do agree that the PSA expression (in blue) Looks very similar, and will concur with the editorial comment postulated; 3) The gel-shift assays shown in Figures 3B and 6B are not the same experiment. The pattern of the super-shifted bands in the developed film are clearly different. The standardized technique for gel-shift assays uses similar extracts and the same antibody, which may give the appearance of similarity at first glance.. However, closer inspection will show that the aforementioned figures are not exact duplicates. It is a similar experiment conducted under different conditions. When enlarged and placed side by side, the figures show that the AR complex with the ARE is completely different, due to the different running conditions of the electrophoreses of the gel. In addition, the super-shifted AR complex with added AR antibody is distinct. Again, reading and counting silver grains in the developed bands demonstrates a difference. As one of the developers of the gel-shift assay for nuclear receptor in the early 1990s, I have years of experience to detect the similarities and differences in the nuclear receptor and DNA interaction; and 4) I agree that the images for PSA expression ( in blue) are similar, with different exposure time. The same cell extracts can be used for different experiments. Western blotted membranes are used several times with different antibodies. Therefore, Western blot can provide very similar images. This specific observation from the editorial board is well considered. However, the GAPDH expression images between 1B (with four overlapping bands due to degradation of the mRNA encoding GAPDH) is distinct from Figure 4A, which appears with 3 developed overlapped bands, as shown in green. Specifically, when enlarging to the maximum possible, it appears that the silver grains and number of bands resulting from the developed film are greater in 1B, compared to 4A. I do, however, find that Figure 2A in the clinical cancer research from 2003 (in light green) seems similar. However, to some extent different from the other two described. Again, the number of bands is greater in Figure 2A, which show 6 overlapping bands when developed. The  smile resulting from running the gel at high voltage is always very similar, if not the same, because the loading buffer is high in ions (cations) and salts. I am aware that it is highly improbable, almost 20 years later, that the films used in these publications are still in storage. It is important to mention that the findings presented in these publications have been substantiated using massive DNA sequencing expression systems in several other labs. Therefore, at this point, there is little to be gained by required the repeat of experiments used in the publications. Regards, Sergio -- Sergio A. Onate, PhD Molecular Endocrinology and Oncology Lab. Pine Cone Hill, La Paz, Florida Bio Bio, Chile Zip Code 4170000 Phone 56-9-79582490 , 
 Dear Dr. Onate, Thank you for your reply here. It is always greatly appreciated if an author responds to concerns raised about their articles. You write that the bands dots in Figure 6C, marked in red, are different, but I actually think they are remarkably similar, allowing for some pixel-level difference because of compression issues. You also write that the GAPDH blots, marked in green, are different. Again, these blots still look similar to me. You then write that the gel-shift assays in Figures 3B and 6B (marked by me in pink) are  clearly different , and that you have years of experience to detect similarities and differences in these blots. But I have years of experience in comparing gel and blot images, and I do not agree with you that these blots are clearly different. In fact, I will stand by my initial concern that these remarkably similar. The argument that other people have reproduced your findings is not very relevant when it comes to potential image reuse. A sports analogy would be that an athlete cannot walk free from a positive doping test if someone else completed the race in the same time. I d love to see the original blots - if you could find them - and be proven wrong. , 
 Expression of Concern, [1 October 2021] . -------------------------- The Editors-in-Chief are issuing an Expression of Concern for this article [1] due to concerns about the images. The specific concerns are as follows: - For Fig. 6C, the far-right lane in the u201cNuclear ARu201d panel is remarkably similar to the left-most lane in the u201cTotal ARu201d panel. - Despite representing different cell lines, lanes 2u20135 in the R1881 PSA panel of Fig. 1B look similar to lanes 1u20134 in the PSA panel of Fig. 4A of DeMiguel et al. [2]. - Additionally, lanes 1u20134 in GAPDH panel of Fig. 1B appear to overlap with lanes 1u20134 in the GAPDH panel of Fig. 4A of DeMiguel et al. [2] and lanes 1u20134 in the GAPDH panel of Fig. 2A of Lee et al. [3]. - Figure 6B, which is supposed to show the results of the supershift assay of LNCaP cells after R1881 treatment, exhibits remarkable similarities with Fig. 3B from Lee et al. [3], which is supposed to show the results of the supershift assay of LN-17 cells using anti-AR antibody. The original images are no longer available, so readers are advised to interpret the findings related to these figures with caution. Authors Allen C. Gao and Sergio A. Onate do not agree to this Editorial Expression of Concern. Soo Ok Lee, Wei Lou, and Min Hou have not responded to any correspondence from the Publisher about this Editorial Expression of Concern. ,