uPAR/cathepsin B overexpression reverse angiogenesis by rescuing FAK phosphorylation in uPAR/cathepsin B down regulated meningioma
Authors: Andrew J Tsung
Jasti S Rao
Sanjeeva Mohanam
Jeffrey D Klopfenstein
Venkateswara Rao Gogineni
Chandramu Chetty
Praveen Bhoopathi
Reshu Gupta
Arun Kumar Nalla
Journal: PLoS ONE (2011)
Discipline: Multidisciplinary
Keywords: Meningiomas; RNAi; uPA; uPAR; cathepsin B
Affiliations: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.

Abstract:  Background: Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression. Methodology/principal findings: In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model. Conclusion/significance: These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma. 

Retracted: Yes

PubPeer comments:
 Figure 2C. In the top row, the pUC and pU2 panels appear to be showing unexpected similar tissue sections, although they appear to show a different horizontal vertical ratio. Shown with green rectangles. ![file] , 
 Figure 2E. In the right VEGF panel, two lanes show a very similar looking band. Could the authors please confirm the authenticity of the bands by showing the uncropped blot? Highlighted here in blue. ![file] , 
 Figure 3A: * Shown in green: Two lanes in the top right panel look more similar than expected. The bands have a slightly different shape, but pay attention to the background patterns * Shown in red: two lanes in the left and right GAPDH panels look more similar than expected. Note the darker  trail on the left, and a sharp vertical white stripe in the right panel. ![file] , 
 The -IR Ang-2 panel in Figure 5A shows unexpected similarities with the +IR MEK1 panel in Figure 6A. As shown by purple rectangles, the blots appear to have similar background markings, in particular in the 1st and 3rd lanes. ![file] , 
 This brings us to Figure 3C. Very unexpectedly, the same cellular structures appear to be visible within or between different photos. These are highlighted here in yellow, pink, cyan and orange circles in the middle column, but they are also visible in the right column. ![file] , 
 #5 The locations of cell nuclei do not match between the DAPI and the Merged columns. ![file] , 
 Figure 1A and 1D and 4C appear to show unexpectedly similar-looking panels that represent different experiments. * In orange, the -IR pSV IOMM-Lee panel looks similar to the +IR pUC+FLpU C CH-157MN panel. * In tan, the 5gy CH-157MN panel looks similar to the +IR pUC+FLpU C CH-157MN panel. In addition, the bottom right tissue panel appears to be showing repeating elements, as circled with dark brown and blue ellipses. ![file] , 
 Here is a zoomed in version of the bottom right panel in Figure 4C, to appreciate the details. ![file] , 
 Supplemental Figure 1 also has some potential issues that I hope the authors could address. Apart from some potential splices, more attention should be given to the FAK panels, each of which appear to show the same band twice. Of course, let s hope that the authors can produce the uncropped blots to prove this hypothesis to be wrong. Shown here with a nice autumn-appropriate burgundy moss-green combination. ![file] , 
 Some lanes in Figure 5A and 6A of this paper also look unexpectedly similar to lanes in Figure 5C of another paper by the same author(s), i.e. Gupta R et al. International Journal of Oncology 38: 733-744, DOI: 10.3892 ijo.2010.883, https: pubpeer.com publications 292374A2B9A995F5D9725E1677D132 ![file] , 
 2020 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0228681 After this article [1] was published, concerns were raised about the following results: Fig 1A, CH-157MN, 5gy panel appears similar to Fig 4C, CH-157MN, +IR, pUC+FLpU C panel. Fig 1D, IOMM-Lee, -IR, pSV panel appears similar to Fig 4C, IOMM-Lee, -IR, pUC+FLpU C panel. Fig 2C, the -IR images for pUC and pU2 appear to report overlapping data. Fig 2D, Ang-1, +IR panel, lanes 2 and 3 (pUC, pU2) appear similar. Figs 2A, 2D, 2E, 3A, 4A, 5A, 6A, S1A: there are image splicing lines between the -IR and +IR data in each figure and it is unclear whether the plus and minus treatment results were obtained in the same experiments using the same blots and exposures. Fig 2E u25cb. Ang-1(60): There appears to be a vertical discontinuity after lane 4 (+IR, pSV). u25cb. VEGF(15): There appear to be similarities between data in lanes 5 and 6 (+IR data for pUC and pU2), although the main bands appear at different vertical positions. Fig 3A u25cb. IOMM-Lee, p-FAK (Y925)(125): There appear to be vertical discontinuities before the bands in lanes 3 and 6; lanes 5 and 6 appear similar; and the background appears to be of a different intensity in lane 5 versus lane 6. u25cb. IOMM-Lee, GAPDH(37): Lanes 1 and 4 appear similar. Fig 3C: There appear to be repeated image elements in the p-FAK(Y925) panels for pSV, pSV+IR, pU2, and pU2+IR, and the images in the DAPI, p-FAK(Y925), and Merged panels do not align in some cases. Fig 4C: There appear to be repeated image elements in the in-vivo, +IR, pUC+FLpU C panel. The in-vivo GAPDH panels in Figs 4A and 5A appear similar, although with different aspect ratios. Data shown in Fig 5A for in-vivo, -IR, Ang-1 (60), appear similar to data reported for a different experiment (DAOY, Bax lanes 1u20133) in Fig 5C of [2]. Also, there appears to be a vertical discontinuity after lane 1 in this panel of [1]. Fig 5A, IOMM-Lee, -IR, Ang-2(70) panel appears similar to the Fig 6A in-vivo, +IR, MEK1(45) panel. Fig 5A in-vivo, +IR, Ang-1(60) panel appears similar to the Fig 6A in-vivo, +IR, Y925(125) panel. Lane 1 of Fig 5A, IOMM-Lee, Ang-2(70) and lane 1 of Fig 6A, in-vivo, +IR, MEK1(45) appear similar to data reported for a different experiment (D283, Bcl2, lane 4) in Fig 5C of [2] Fig S1A, in vivo FAK panel, data appear similar in lanes 2 and 3 and in lanes 5 and 6, and there appears to be a vertical discontinuity before lane 6. Also, Fig S1A includes only a single GAPDH control panel although the figure reports three different experiments (IOMM-Lee, CH-157MN, in vivo). The above concerns call into question the integrity and reliability of the published results, and so the PLOS ONE Editors retract this article. We attempted to contact the articleu2019s authors to discuss these issues, request the underlying data, and notify them of the retraction decision, but the authors either did not reply or could not be reached. The IOMM-Lee, Ang-2(70) panel in Fig 5A, the in-vivo, -IR, Ang-1 panel in Fig 5A, and the in-vivo, +IR, MEK1 panel in Fig 6A [1] appear to report material from [2], published in 2011 [Spandidos Publications] which are not offered under a CC-BY license. These panels are therefore excluded from the PLOS ONE articleu2019s [1] license. At the time of retraction, the article [1] was republished to note this exclusion in the Figs 5 and 6 legends and the articleu2019s copyright statement. References 1.Gupta R, Nalla AK, Gogineni VR, Chetty C, Bhoopathi P, Klopfenstein JD, et al. (2011) uPAR Cathepsin B Overexpression Reverse Angiogenesis by Rescuing FAK Phosphorylation in uPAR Cathepsin B Down Regulated Meningioma. PLoS ONE 6(2): e17123. https: doi.org 10.1371 journal.pone.0017123 pmid:21347260 2.Gupta R, Chetty C, Bhoopathi P, Lakka S, Mohanam S, Rao JS, Dinh DE. (2011) Downregulation of uPA uPAR inhibits intermittent hypoxia-induced epithelial-mesenchymal transition (EMT) in DAOY and D283 medulloblastoma cells. Int J Oncol. 38(3):733u201344. pmid:21181094 ,