Compensatory Increase of Transglutaminase 2 Is Responsible for Resistance to mTOR Inhibitor Treatment
Authors: Jingwen Cao
Wenlong Huang
Journal: PLoS ONE (2016)
Discipline: Multidisciplinary
Keywords: mTORC1; rapamycin; cancer therapy; transglutaminase 2; inhibitor resistance
Affiliations: China Pharmaceutical University, Nanjing, Jiangsu Province, People's Republic of China., Chinese Academy of Medical Sciences, Beijing, People's Republic of China.

Abstract:  The mechanistic target of rapamycin complex 1 (mTORC1) plays a crucial role in controlling cell growth and homeostasis. Deregulation of mTOR signaling is frequently observed in some cancers, making it an attractive drug target for cancer therapy. Although mTORC1 inhibitor rapalog-based therapy has shown positive results in various pre-clinical animal cancer studies, tumors rebound upon treatment discontinuation. Moreover, several recent clinical trials showed that the mTORC1 inhibitors rapamycin and rapalog only reduce the capacity for cell proliferation without promoting cell death, consistent with the concept that rapamycin is cytostatic and reduces disease progression but is not cytotoxic. It is imperative that rapamycin-regulated events and additional targets for more effective drug combinations be identified. Here, we report that rapamycin treatment promotes a compensatory increase in transglutaminase 2 (TGM2) levels in mTORC1-driven tumors. TGM2 inhibition potently sensitizes mTORC1-hyperactive cancer cells to rapamycin treatment, and a rapamycin-induced autophagy blockade inhibits the compensatory TGM2 upregulation. More importantly, tumor regression was observed in MCF-7-xenograft tumor-bearing mice treated with both mTORC1 and TGM2 inhibitors compared with those treated with either a single inhibitor or the vehicle control. These results demonstrate a critical role for the compensatory increase in transglutaminase 2 levels in promoting mTORC1 inhibitor resistance and suggest that rational combination therapy may potentially suppress cancer therapy resistance. 

Retracted: No

PubPeer comments:
 2020 expression of concern. https: journals.plos.org plosone article?id=10.1371 journal.pone.0227851 After publication of this article [1], the authors notified the journal office of concerns about the reliability of results presented in Fig 6. They had attempted to replicate the in vivo tumor results, and results of these post-publication experiments did not support the original findings that combinatorial treatment with Rapamycin and KCC009 had a greater effect than Rapamycin alone (Supporting Information S1 and S2 Files). The authors subsequently noted that they had found evidence of mycoplasma contamination in the MCF-7-Luciferase cell line used in the initial post-publication replication experiments. They repeated the experiments using a fresh vial of MCF-7 cells, although different methods were used (e.g. chemical versus shRNA inhibition) and outcome data were collected over a shorter timecourse (4 weeks in the replication experiments versus 6 or 9 weeks in the original experiments). The results of these experiments are reported here in an updated version of Fig 6. The Methods used for the replication experiments are included below. The authors summarized the results of the Fig 6 replication experiments as follows: To reconcile the efficacy of combining mTORC1 and TGM2 inhibition in tumor cells with mTORC1-hyperactive cells in vivo, an MCF-7 xenograft tumor model was used. Mice bearing MCF-7 xenograft tumors were treated with rapamycin and KCC009 either singly or in combination, and tumor growth was monitored at various time points during treatment by caliper measurement. Compared with the vehicle control, rapamycin reduced tumor growth capacity while KCC009 did not affect tumor growth. In contrast, treatment with both rapamycin and KCC009 fully suppressed xenograft tumor progression (new Fig 6A). This benefit was also evidenced by statistical analysis of tumor volume and weight in response to combination treatment (new Fig 6Bu20136C). The combinational treatment was shown not to have toxic effect according to body weight monitoring (new Fig 6D). Immunohistochemical staining revealed that the combined rapamycin and KCC009 treatment reduced the cell-proliferation marker proliferating cell nuclear antigen (PCNA), suggesting reduced tumor compared with the single treatments (new Fig 6E). To further validate that blocking TGM2 could benefit mTORC1 inhibitors treatment, we performed xenograft model using TGM2 knockdown MCF-7 cell line instead of KCC009 treatment. It showed that dual targeting TGM2 and mTORC1 for four weeks post-inoculation caused tumor arrest by analysis of tumor volume and weight (new Fig 6F and 6G). We also performed TGM2 protein immunohistochemical staining in xenograft tumors. Rapamycin promotes expression level of TGM2 in the tumors of mice inoculated with the control shRNA MCF-7 cell line. However, TGM2 shRNA could fully suppress the expression level of TGM2 in tumors treated with rapamycin (new Fig 6H). A member of PLOS ONEu2019s Editorial Board advised that the results of these experiments support the conclusions reported in [1]. In addition, a reader raised concerns that the lower panels of Fig 3B (TGM2 shRNA, Control and Rapamycin panels) in [1] appear to duplicate the left panels of Fig 1F (Control, Vehicle and H2O2 panels, respectively) in [2]. The authors noted that this was due to an error in preparing the PLOS ONE figure, and provide here an updated version of Fig 3 in which all four panels have been replaced with data from a replication experiment. The raw image data from the original experiment and the replication experiment are in S4 File. As the duplicated panels are not licensed for distribution under the terms of the Creative Commons Attribution License, this content (original Fig 3B TGM2 shRNA, Control and Rapamycin panels in [1]) has been removed from the original version of the PLOS ONE article. The primary data underlying results in this article were not included with the published article although the Data Availability Statement for this article stated, u201cAll relevant data are within the paper and its Supporting Information files.u201d The authors provide in S1u2013S8 Files the underlying data supporting results reported in [1] and in this notice. In addition, a reader questioned the origin of results reported in the original published versions of Figs 1B and 6A. We attempted to contact the corresponding authoru2019s institution about this issue but to date this remains unresolved. The PLOS ONE Editors issue this Expression of Concern to notify readers of the above concerns and relay the supporting data provided by authors post-publication. Methods for replication studies 1 x 106 MCF-7 cells with a luciferase tag were inoculated in both flanks of mice. Four weeks post inoculation, mice bearing subcutaneous tumors were randomized into four groups: vehicle control (n = 5; 10% DMSO i.p.), rapamycin (n = 5; 1 mg kg day i.p.), KCC009 (n = 5; 50 mg kg day in 10% DMSO i.p.) and rapamycin plus KCC009 (n = 5; 1 mg kg day and 50 mg kg day, respectively, i.p.). Drug treatments were initiated four weeks post-inoculation, and tumor growth was monitored by tumor diameters (L and W) using caliper for six weeks. Tumor volume was calculated using the formula V = (W2 u00d7 L) 2. MCF7 cells with a luciferase tag were tested for mycoplasma using PCR. In brief, 100 u03bcl of cell culture supernatant from a dense culture (80u2013100% confluent) were taken into a 1.5ml tube. Heat the sample for 5 min at 95u00b0C and spin it for 2 min in a bench centrifuge at maximum speed. Tag polymerase was used to amplify the mycoplasma DNA. Primers were shown below: Forward 5u2019CGCCTGAGTAGTACGTTCGC3u2019, Reverse 5u2019GCGGTGTGTACAAGACCCGA3u2019. Further test for mycoplasma was done using LONZA MycoAlertTM Mycoplasma Detection Kit followed by manufactureu2019s instruction. A fresh vial of mycoplasma free MCF7 cells (1 x 106) were inoculated into both flanks of mice. Two weeks post inoculation, mice bearing subcutaneous tumors were randomized into four groups: vehicle control (n = 5; 10% DMSO i.p.), rapamycin (n = 5; 1 mg kg day i.p.), KCC009 (n = 5; 50 mg kg day in 10% DMSO i.p.) and rapamycin plus KCC009 (n = 5; 1 mg kg day and 50 mg kg day, respectively, i.p.). Drug treatments were initiated two weeks post-inoculation, and tumor growth was monitored by tumor diameters (L and W) using caliper for four weeks. Tumor volume was calculated using the formula V = (W2 u00d7 L) 2. References 1.Cao J, Huang W (2016) Compensatory Increase of Transglutaminase 2 Is Responsible for Resistance to mTOR Inhibitor Treatment. PLoS ONE 11(2): e0149388. pmid:26872016 r 2.Sun Y, Gu X, Zhang E, Park M-A, Pereira AM, Wang S, et al. (2014) Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells. Cell Death Disease ,