Oncogenic fibulin-5 promotes nasopharyngeal carcinoma cell metastasis through the FLJ10540/AKT pathway and correlates with poor prognosis
Authors: Li-Jen Su
Yen-Yi Zhen
Shun-Chen Huang
Tai-Jan Chiu
Tai-Lin Huang
Li-Yen Shiu
Chao-Cheng Huang
Hsin-Ting Tsai
Chung-Feng Hwang
Chang-Han Chen
Fu-Min Fang
Yu-Fang Yin
Wei-Sheng Wang
Journal: PloS one (2013)
Discipline: Multidisciplinary
Keywords: Nasopharyngeal carcinoma; fibulin-5; metastasis; survival; clinicopathological variables
Affiliations: Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan ; Kaohsiung Chang Gung Head and Neck Oncology Group, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan., Department of Medical Research and Development, Show Chwan Memorial Hospital and Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan., Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli, Taiwan., Department of Pathology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan., Department of Hematology-Oncology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan., Kaohsiung Chang Gung Head and Neck Oncology Group, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan ; Department of Pathology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan., Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan., Kaohsiung Chang Gung Head and Neck Oncology Group, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan ; Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan., Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan ; Kaohsiung Chang Gung Head and Neck Oncology Group, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan ; Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.

Abstract:  Background: Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential and locoregional recurrence, although the molecular alterations that are driving NPC metastasis remain unclear at this time. This study aimed to examine the expression of fibulin-5 in NPC, correlate the results with clinicopathological variables and survival, and to investigate the role of fibulin-5 in human NPC cell lines. Material and methods: Standard semi-quantitative-RT-PCR, quantitative-RT-PCR, immunoblotting, and immunohistochemistry were used to investigate the mRNA and protein expression profiles of fibulin-5 in normal and NPC tissues. Immunohistochemistry of fibulin-5 was correlated with clinicopathological characteristics by univariate analyses. NPC cells overexpressing fibulin-5 or fibulin-5-siRNA cells were generated by stable transfection to characterize the molecular mechanisms of fibulin-5-elicited cell growth and metastasis. Results: Our results demonstrated that fibulin-5 overexpression in NPC specimens and significantly correlated with advanced tumor metastasis indicating a poor 5-year overall survival. Fibulin-5 was mainly expressed in the nucleus in human NPC specimens and cell lines. Functionally, fibulin-5 overexpression yielded fast growth in NPC cells. In addition, fibulin-5 promotes cell metastasis in NPC cells through increased FLJ10540 and phosphor-AKT activity. In contrast, siRNA depletion of fibulin-5 suppressed FLJ10540 expression and phosphor-AKT activity. Suppression of either fibulin-5 or FLJ10540 can cause significant inhibition with regards to cell motility in NPC cells. Finally, immunohistochemical analysis of human aggressive NPC specimens showed a significant and positive correlation between fibulin-5 and FLJ10540 expression. Conclusion: Higher fibulin-5 expression is not only an important indicator of poor survival, but also contributes to the development of new therapeutic strategies in the FLJ10540/AKT pathway for NPC treatment. 

Retracted: Yes

PubPeer comments:
 2020 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0227931 After publication of this article [1], it was reported that the Hone1 cell line, used in the experiments shown in Figs 3u20139, is contaminated with HeLa cells [2]. In light of this issue the results reported for this cell line may not be representative of nasopharyngeal carcinoma cell biology. In the PLOS ONE article, the authors provided results from experiments on TW01 cells in Supporting Information files S1-S3 to support the Hone1 results shown in Figs 4, 6, and 8A, respectively [1]. TW01 data for other experiments reported in the article are available from the authors. The authors noted that in preparing the figures for this publication [1], the wrong images were presented for the U2-OS cell panel of Fig 3B and shFibulin-5-Hone1 invasion panel of Fig 6D. In Fig 3B, the images shown as representing fibulin-5 expression in U2-OS cells were instead obtained when examining fibulin-5 expression in a different head and neck cancer cell line. In Fig 6D, the image shown as representing invasion results for shFibulin-5-Hone1 cells reports data from a different experiment (invasion in siRap-1A-FaDu cells) reported in [3], published 2013 [copyright American Society for Investigative Pathology; published by Elsevier Inc.], and which are not offered under a CC-BY license. This image is therefore excluded from this article s [1] license. At the time of retraction, the article [1] was republished to note this exclusion in the Fig 6 legend and the articleu2019s copyright statement. Replication data for this experiment and the raw data underlying graphs in Fig 6D are available from the authors; the authors noted that quantification data shown in the original version of Fig 6D were obtained using the correct data. There are several concerns about the western blot figures reported in [1]: In some cases (e.g. in Fig 9), it is difficult to evaluate the data due to the poor image quality. For all western blot panels in the article, the background is unexpectedly uniform. In Fig 1C, when adjusted for brightness and contrast, there appear to be vertical discontinuities between lane 3 and 4 of the Fibulin-5 blot, and there appear to be horizontal discontinuities above and below the bands in all lanes of the Fibulin-5 blot, suggesting that a blot image fragment was superimposed on a gray background image. In Fig 3C, there are visible splice lines around the bands for the Fibulin-5 Cytoplasm panel. In Fig 4A, when levels are adjusted there appears to be a vertical discontinuity before lane 2 and we cannot verify whether image data is present in lane 1 (Vehicle control). In Fig 6A, when adjusted for brightness and contrast, there appear to be vertical and horizontal discontinuities around the band in lane 1 of the Fibulin-5 blot, and we cannot verify whether image data is present in lanes 2, 3. There also appear to be horizontal discontinuities above and below the u03b2-actin bands. In Fig 7B, when adjusted for brightness and contrast there appear to be vertical discontinuities in the image between lanes 3 and 4 in all three Hone1 blots, and there also appear to be horizontal discontinuities above and below the bands at different positions in lanes 1u20133 versus lane 4 for each panel. In Fig 8B, when levels are adjusted, there are discontinuities in background above and below the bands, and the background for lane 1 appears different than the background for lanes 2, 3. There also appear to be discontinuities above and below the u03b2-actin bands. The authors noted that the original image data to support the western blot results are no longer available, and that the original data were cropped and or rearranged, and were superimposed on a gray background image for the purpose of presentation. In light of the data unavailability, we are unable to resolve the above concerns and as such, the reliability and validity of the results are in question. The authors provided data for a replication of the Fig 3C experiment, but the data provided for this experiment did not appear to be complete uncropped and unadjusted blot images, did not include a supernatant fraction, and the results of nuclear vs. cytoplasm blots did not demonstrate an enrichment in the nucleus as was reported in the Results of [1]. In light of the unresolved concerns discussed above, the PLOS ONE Editors retract this article. CC and FF agreed with the retraction. LYS, LJS, TC, YY, WW, SY, CCH, CFH, YZ, HT, TH did not reply or could not be reached. 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