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F-box and leucine-rich repeat protein 16 controls tamoxifen sensitivity via regulation of mitochondrial respiration in estrogen receptor-positive breast cancer cells

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Abstract

Tamoxifen is one of the most effective therapeutic tools for estrogen receptor-positive (ER +) breast cancer. However, the intrinsic insensitivity and resistance to tamoxifen remains a significant hurdle for achieving optimal responses and curative therapy. In this study, we report that F-box and leucine-rich repeat protein 16 (FBXL16) is located in the mitochondria of ER + breast cancer cells. The mitochondrial FBXL16 plays an essential role in sustaining mitochondrial respiration and thereby regulates the sensitivity of ER + breast cancer cells to tamoxifen treatment. Importantly, high FBXL16 expression is significantly correlated with poor overall survival of ER + breast cancer patients. Moreover, mitochondrial inhibition phenocopies FBXL16 depletion in terms of sensitizing the ER + breast cancer cells to tamoxifen treatment. Together, our study demonstrates that FBXL16 acts as a novel regulator of tamoxifen sensitivity. Thus, targeting FBXL16 may serve as a promising approach for improving the therapeutic efficacy of tamoxifen in ER + breast cancer cells.

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Data availability

Publicly available dataset analyzed in this study can be found here: TCGA (https://portal.gdc.cancer.gov/).

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Funding

This present study was funded by a grant from the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 82203425), and a grand from National Natural Science Foundation of China (Grant No. 82002490).

Author information

Author notes

  1. Feng Chen and Lu Wang have contributed equally to this work and share first authorship.

Authors and Affiliations

  1. Department of Medical Oncology, Ordos Central Hospital, Ordos, China

    Feng Chen

  2. Department of Radiation Oncology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China

    Lu Wang & Yao Song

  3. Ordos School of Clinical Medicine, Inner Mongolia Medical University, Hohhot, China

    Yanrong Feng, Wenxin Ma, Junqin Liu & Qianyao Bi

  4. Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, China

    Rui Gao

  5. Sichuan Cancer Hospital and Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China

    Yanhan Jia

  6. Radiation Oncology Key Laboratory of Sichuan Province, Chengdu, China

    Yanhan Jia

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  1. Feng Chen
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Contributions

Conceptualization: FC, LW, RG, and YJ; Resources: FC, LW, RG, and YJ; Data curation: RG, and YJ; Formal analysis: FC, LW, RG, and YJ; Supervision: RG, and YJ; Validation: FC, LW, YF, WM, JL, QB, and YS; Investigation: FC, LW, YF, WM, JL, QB, YS, RG, and YJ; Visualization: FC, LW, RG, and YJ; Methodology: FC, LW, YF, WM, JL, QB, YS, RG, and YJ; Project administration: RG, and YJ; Writing—original draft: FC and LW; Writing—review and editing: FC, LW, RG, and YJ.

Corresponding authors

Correspondence to Rui Gao or Yanhan Jia.

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Conflict of interest

The authors declare no conflict of interest.

Ethical approval

All the patients’ clinical data used in this study were obtained from The Cancer Genome Atlas (TCGA) database. Since the TCGA is an open public database, additional approval by an ethics committee was not required.

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Supplementary Information

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13577_2023_961_MOESM1_ESM.jpg

Supplementary file1 (JPG 8992 KB) Immunofluorescence analysis of ER-α in various breast cancer cell lines. Representative immunofluorescence images for ER-α in the indicated breast cancer cell lines confirming that MDA-MB-231, SKBR3, MDA-MB-453, and MDA-MB-468 cells are ER negative, while BT474, T-47D, MCF7, and MDA-MB-415 cells are ER positive. Scale bar, 20 µm

13577_2023_961_MOESM2_ESM.jpg

Supplementary file2 (JPG 3027 KB) FBXL16 depletion does not affect the cell viabilities or colony formation capacities in most of the ER+ breast cancer cells. (A) Western blot analysis of FBXL16 expression in indicated breast cancer cells transduced with shCtrl or two different shRNAs targeting FBXL16. (B) Cell viabilities were determined using SRB assay in a variety of ER+ breast cancer cells transduced with shCtrl or two different FBXL16-targeting shRNAs. (C) Colony formation capacities of shCtrl or shFBXL16-transduced breast cancer cells were assessed using colony formation assay. The number of colonies was normalized to the mean number of colonies of shCtrl transduced cells. (D) Immunoblotting analysis confirmed the knockout (KO) of FBXL16 in indicated breast cancer cells. (E and F) Cell viabilities (E) and colony formation capacities (F) were examined in WT and FBXL16 KO breast cancer cells. (B, C, E, F) **, p< 0.01; ***, p < 0.001; ns, not significant versus shCtrl by two-tailed Student’s t test. Data are presented as mean + SD. n = 3

13577_2023_961_MOESM3_ESM.jpg

Supplementary file3 (JPG 10511 KB) Immunofluorescence analysis of FBXL16 in various breast cancer cell lines. Representative immunofluorescence images for FBXL16 in the indicated breast cancer cell lines. MitoTracker and DAPI were used to stain mitochondria and nuclei, respectively. Arrows depict the overlap of FBXL16 immunoreactivity with mitochondria area indicating the localization of FBXL16 in the mitochondria. Scale bar, 10 µm

13577_2023_961_MOESM4_ESM.jpg

Supplementary file4 (JPG 2964 KB) FBXL16 knockdown does not influence the mitochondrial respiration of normal breast epithelial cells or ER- breast cancer cells. (A-H) Mitochondrial respiration profiles of indicated normal breast epithelial cells and ER- breast cancer cells transduced with shCtrl or two different FBXL16-targeting shRNAs. O, oligomycin; F, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP); R+A, rotenone and antimycin A. (B, D, F, and H) Basal, ATP-linked, and maximal respiration were calculated based on the respiration profiles. ns, not significant versus shCtrl by two-tailed Student’s t test. (A-H) Data are presented as mean + SD. n = 6

13577_2023_961_MOESM5_ESM.jpg

Supplementary file5 (JPG 9322 KB) Loss of FBXL16 does not affect mitochondrial morphology or dynamics. (A) Representative images of mitochondria labeled by MitoTracker in the indicated cells. Scale bar = 10 μm. (B and C) The mitochondrial mean fluorescence intensity of MitoTracker fluorescence was quantified using Fiji-ImageJ software in each cell and then normalized to the mean fluorescence intensity of shCtrl transduced cells (D and E) The average length of mitochondria in each cell was measured using Fiji-ImageJ software to determine the average size of mitochondria in the indicated cells. (F and G) The number of mitochondria in each cell was counted using Fiji-ImageJ software. The number of mitochondria of each cell was normalized to the average number of mitochondria in shCtrl transduced cells (B-G) Cells from each group were randomly selected and quantified (n = 20 per group). ns, not significant by two-tailed Student’s t test

13577_2023_961_MOESM6_ESM.jpg

Supplementary file6 (JPG 3514 KB) Mitochondrial subpopulation of FBXL16 is responsible for the regulation of tamoxifen sensitivity in ER+ breast cancer cells via modulating mitochondrial respiration and ATP production. (A-D) FBXL16 knockout (KO) MDA-MB-415 (A and B) and BT474 (C and D) cells were transduced with MTS-FBXL16 lentiviral vector to generate MTS-FBXL16-expressing MDA-MB-415 (A and B) and BT474 (C and D) cells. WT, FBXL16 KO, and MTS-FBXL16-expressing cells were subjected to determination of mitochondrial respiration profiles. O, oligomycin; F, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP); R+A, rotenone and antimycin A. (B and D) Basal, ATP-linked, and maximal respiration were calculated based on the respiration profiles. (E) Cellular ATP level was determined in WT, FBXL16 KO, and MTS-FBXL16-expressing MDA-MB-415 and BT474 cells. (F and G) Representative dose–response curves of tamoxifen treatment in WT, FBXL16 KO, and MTS-FBXL16-expressing MDA-MB-415 (F) and BT474 cells (G). (B, D, and E) **, p< 0.01; ***, p < 0.001 by two-tailed Student’s t test. Data are presented as mean + SD. (A-D) n = 6. (E-G) n=3

13577_2023_961_MOESM7_ESM.jpg

Supplementary file7 (JPG 1958 KB) Loss of FBXL16 does not affect the level of cellular or mitochondrial ROS production. (A and B) shCtrl or shFBXL16-transduced (A), as well as WT and FBXL16 knockout (B) BT474 and MDA-MB-415 cells were subjected to DCFDA assay for measuring the cellular ROS production using a commercial kit (Abcam). (C and D) Mitochondrial ROS production of indicated cells was examined by MitoROS 580 assay using a commercial kit (Abcam). (A-D) ns, not significant versus shCtrl (A and C) or WT (B and D) by two-tailed Student’s t test. Data are presented as mean + SD. n = 3

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Chen, F., Wang, L., Feng, Y. et al. F-box and leucine-rich repeat protein 16 controls tamoxifen sensitivity via regulation of mitochondrial respiration in estrogen receptor-positive breast cancer cells. Human Cell 36, 2087–2098 (2023). https://doi.org/10.1007/s13577-023-00961-z

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