RETRACTED: Hypoxia induced E-cadherin involving regulators of Hippo pathway due to HIF-1Œ± stabilization/nuclear translocation in bone metastasis from breast carcinoma
Authors: Giuseppe Banfi
Paola Bendinelli
Paola Maroni
Maria Alfonsina Desiderio
Emanuela Matteucci
Lorenzo Drago
Journal: Experimental Cell Research (2015)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Bone metastasis; E-cadherin; HIF-1; Hypoxia; Wwox.
Affiliations: Istituto Ortopedico Galeazzi, IRCCS, Milano, Italy., Dipartiimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Universit√† degli Studi di Milano, Milano, Italy., Istituto Ortopedico Galeazzi, IRCCS, Milano, Italy; Dipartiimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Universit√† degli Studi di Milano, Milano, Italy., Dipartiimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Universit√† degli Studi di Milano, Milano, Italy. Electronic address: .

Abstract:  The present study deals with the molecular mechanisms involved in the regulation of E-cadherin expression under hypoxia, because the adjustment of the amount of E-cadherin due to physical stimuli of the microenvironment might influence the colonization of metastasis to skeleton. We analyzed the effect of 1% oxygen tension, that is similar to that encountered in the bone marrow by metastatic cells spreading from breast carcinoma. The purpose was to evaluate the hypoxia-orchestrated control of E-cadherin transactivation via hypoxia inducible factor-1 (HIF-1) and peroxisome proliferator activated receptor-Œ≥ (PPARŒ≥), and the involvement of Hippo pathway members, as regulators of transcription factors. To give a translational significance to the study, we took into consideration human pair-matched ductal breast carcinoma and bone metastasis: E-cadherin and Wwox were expressed in bone metastasis but not in breast carcinoma, while HIF-1Œ± and TAZ seemed localized principally in nuclei of metastasis and were found in all cell compartments of breast carcinoma. A close examination of the regulatory mechanisms underlying E-cadherin expression in bone metastasis was done in 1833 clone derived from MDA-MB231 cells. Hypoxia induced E-cadherin only in 1833 clone, but not in parental cells, through HIF-1 and PPARŒ≥ activities, while Wwox decreased. Since Wwox was highly expressed in bone metastasis, the effect of ectopic Wwox was evaluated, and we showed E-cadherin transactivation and enhanced invasiveness in WWOX transfected 1833 cells. Also, hypoxia was additive with ectopic Wwox remarkably enhancing HIF-1Œ± nuclear shuttle and accumulation due to the lengthening of the half-life of HIF-1Œ± protein; under this experimental condition HIF-1Œ± appeared as a slower migrated band compared with control, in agreement with the phosphorylation state. The in vitro data strongly supported the almost exclusive presence of HIF-1Œ± in nuclei of human-bone metastasis. Thus, we identified Wwox as a novel molecule in the HIF-1Œ±-HDM2 regulatory loop, necessary for the dynamic regulation of the HIF-1Œ± amount, and we suggested that the reduction of endogenous Wwox free pool under hypoxia might also be due to the interaction with HDM2, sequestering the E3 ubiquitin ligase. We highlighted the importance of nuclear HIF-1Œ± in the biology of metastasis for the mesenchymal-epithelial transition: this phenotype was regulated by Wwox plus hypoxia through E-cadherin target gene, playing a pivotal role in bone metastasis colonization. 

Retracted: Yes

PubPeer comments:
 - Figure 4C BBA 1813 and Figure 5 Mol Cancer Res 7 For HDAC1 we reported our results in the two papers (Mol Cancer Res 7 and BBA 1813). So, also the vinculins are repeated - For the other vinculins in the red and yellow squares, it looks like an inattention in the graphical assembling of the acquisitions, since the aspect of the vinculins is very similar. The vinculins used in the Figures originate from our experiments. In any case the data are calculated on a triplicate. , 
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 Data in this paper, Experimental Cell Research Volume 330, Issue 2, 15 January 2015, Pages 287-299, looks very similar the data which appeared in Mol Cancer Res. 2009 Aug;7(8):1328-41. See North and North East corner. https: imgur.com Pj2PE5f , 
 Earlier provenance data figure 2B this paper, Exp Cell Res. 2015 Jan 15;330(2):287-99, from figure 1E Mol Cancer Res. 2007 Aug;5(8):833-45.  , 
 Could the authors clarify whether the 4th lane in Fig 1D *[right]* is the result of  16 hours Hypoxia , or of *[at left]*  3 hours Hypoxia + NS398 ? (Fig 3B of [Bendinelli et al 2013)]  , 
 At top left here are are Figs 4C and (part of) 1D. At top right: (part of) Figure 2B from [Bendinelli et al (2013)] . The two B23 loading controls are unexpectedly similar. There are also surprising connections between two lanes in the HDM2 band *[total extract]* and lanes from  TAZ Nuclear extract band, after a 180-deg rotation in one case and a vertical flip in the other.  , 
 Another mystery for readers to ponder. Here comparing Fig 6B *[at left]* with *[right]* 2D from Bendinelli et al (2013).  Lanes 1 and 3 match between the two TAZ bands, and between the two HIF-1alpha bands. Yet the 2nd lanes do not match, nor do the 4th lanes. , 
 The marked slice of primary breast carcinoma is either *[below]* a negative, unstained control, at 20x magnification (part of Fig 5C); or *[above]* stained with E-cadherin antibody, to demonstrate the non-expression of E-cadherin, at 60x magnification (Fig 7 , [Bendinelli et al, 2013] )  , 
 Comparing *[right]* cytosolic extracts panel of Fig 4A with *[left]* Fig 5E, [Bendinelli et al (2014)] .  Apparent overlap between the vinculin loading controls (after a horizontal flip). , 
 More from the Department of Unexpected Similarities. At left: Fig 1B. At right: (part of) Fig 5A from [Matteucci, Bendinelli Desiderio (2009)] .  Note that Fig 1B had previously appeared as Fig 2E of [Matteucci et al (2013)] .  , 
 #11 Fig 1C *[at right]* had previously appeared as Fig 2D *[at left]* of Matteucci et al (2013).  , 
 #10 Further concerns with Fig 4A. Specifically, about the similarity of lanes 4, 5 and 6.  , 
 #6  Could the authors clarify whether the 4th lane in Fig 1D [right] is the result of  16 hours Hypoxia , or of [at left]  3 hours Hypoxia + NS398 ? (Fig 3B of Bendinelli et al 2013)  Also of interest: Fig 1(b) from [Maroni et al (2011)] .  , 
 #13 Darkening the C lane and flipping it horizontally (thanks to E. Bik for the suggestion):  , 
 *[Right]*, part of Fig 1D, for MDA-MB231 cells. Please compare with *[at left]* Figure 4B from [Maroni et al (2006)] -  Western blot analysis of total and nuclear proteins prepared from HGF- or hypoxia-treated MCF-7 cells (detail at lower right) .  The lanes should not be similar, yet they are. , 
 Fig 4C.  Control or WWOX transfected cells were used for Matrigel invasion assay. Representative images are shown, and the numbers at the bottom are the means7S.E. of the counts of ten selected fields for three independent experiments The resolution leaves much to be desired, but it is still possible to see a surprising number of identical cells.  , 
 2020 retraction. https: www.sciencedirect.com science article pii S0014482720304158?via%3Dihub This article has been retracted at the request of the Editor-in-Chief and the authors because of the similarity between various figures of this article and previous articles published by the authors et al. Lanes of Figure 1B are similar with lanes of Figure 5A of the article that some of the authors have previously published in Carcinogenesis 30 (2009) 937u2013945 https: doi.org 10.1093 carcin bgp080. Figure 1B is similar with Figure 2E of the article that some of the authors et al. have previously published in the European Journal of Cancer 49 (2013) 231u2013244 https: doi.org 10.1016 j.ejca.2012.05.006. A large number of similar features have been noticed within the panels of Figure 1C. Moreover, Figure 1C is similar with panels of Figure 2D of the article that some of the authors et al. have previously published in the European Journal of Cancer 49 (2013) 231u2013244. Lanes of Figure 1D are similar with lanes of Figure 3B of the article that some of the authors et al. have previously published in the European Journal of Cancer 49 (2013) 2608u20132618 https: doi.org 10.1016 j.ejca.2013.03.002 and Figure 4B of the article that some of the authors have previously published in Carcinogenesis 28 (2007) 267u2013279 https: doi.org 10.1093 carcin bgl129. Lanes of Figure 2B are similar with lanes of Figure 1E of the article that some of the authors et al. have published in Molecular Cancer Research 5 (2007) 833u2013845 https: doi.org 10.1158 1541-7786.MCR-07-0054. Similarities between lanes of Figure 4A have been noticed. Also, other lanes of Figure 4A are similar with lanes from Figure 5E of the article that some of the authors et al. have previously published in Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1843 (2014) 815u2013826 https: doi.org 10.1016 j.bbamcr.2013.12.015. Lanes of Figure 4C and 6B are similar with lanes of Figure 2B and 2D of the article that some of the authors et al. have previously published in the European Journal of Cancer 49 (2013) 2608u20132618. ,