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SMAD4 loss limits the vulnerability of pancreatic cancer cells to complex I inhibition via promotion of mitophagy

Abstract

Pancreatic cancer is one of the deadliest forms of cancer, which is attributed to lack of effective treatment options and drug resistance. Mitochondrial inhibitors have emerged as a promising class of anticancer drugs, and several inhibitors of the electron transport chain (ETC) are being clinically evaluated. We hypothesized that resistance to ETC inhibitors from the biguanide class could be induced by inactivation of SMAD4, an important tumor suppressor involved in transforming growth factor β (TGFβ) signaling, and associated with altered mitochondrial activity. Here we show that, paradoxically, both TGFβ-treatment and the loss of SMAD4, a downstream member of TGFβ signaling cascade, induce resistance to biguanides, decrease mitochondrial respiration, and fragment the mitochondrial network. Mechanistically, the resistance of SMAD4-deficient cells is mediated by increased mitophagic flux driven by MAPK/ERK signaling, whereas TGFβ-induced resistance is autophagy-independent and linked to epithelial-to-mesenchymal transition (EMT). Interestingly, mitochondria-targeted tamoxifen, a complex I inhibitor under clinical trial, overcomes resistance mediated by SMAD4-deficiency or TGFβ signaling. Our data point to differential mechanisms underlying the resistance to treatment in PDAC arising from TGFβ signaling and SMAD4 loss, respectively. The findings will help the development of mitochondria-targeted therapy for pancreatic cancer patients with SMAD4 as a plausible predictive marker.

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Fig. 1: PDAC cells without SMAD4 have lower level of mitochondrial respiration and are resistant to biguanides.
Fig. 2: SMAD4 deletion results in changes in mitochondrial morphology.
Fig. 3: SMAD4 deletion leads to fragmentation of mitochondria and changed cristae morphology.
Fig. 4: Mitophagy is increased by SMAD4 deletion.
Fig. 5: Induction of mitophagy in SMAD4-deficient cells is associated with increased phosphorylation of MAPK/ERK kinase.
Fig. 6: Increased mitophagy promotes resistance to MitoMet in PDAC cells.
Fig. 7: MitoTam overcomes the TGFβ- and SMAD4-mediated resistance in PDAC cells.
Fig. 8: Schematic model of signaling and phenotypic changes resulting from SMAD4 loss in PDAC cells.

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Acknowledgements

We thank the Laboratory of Electron Microscopy, Faculty of Science, Charles University, for the possibility to use the transmission electron microscope JEOL JEM-1011 with Veleta CCD camera and Dr. Petr Jezek for providing us with the Mito-Keima vector.

Funding

This work was supported by Czech Health Research Council grants (NV16-31604A) to JN, Czech Science Foundation grants (19-20553S, 20-18513S, 20-11724Y and 18–02550S), and Grant Agency of Charles University (GAUK1100217) to ZE. SB was supported by a project International mobility grant of researchers of the Institute of Biotechnology CAS, v. v. i. (CZ.02.2.69/0.0/0.0/16_027/0008353) from the ESF. Further support was provided by BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF, RVO: 86652036 and the Ministry of Education, Youth and Sports of the Czech Republic (LO1220) at the CZ-OPENSCREEN: National infrastructure for chemical biology. We acknowledge the Imaging Methods Core Facility at BIOCEV and Microscopy Centre - Electron Microscopy Core Facility, IMG AS CR, institutions supported by the Czech-BioImaging large RI projects (LM2015062 and CZ.02.1.01/0.0/0.0/16_013/0001775, funded by MEYS CR) for their support with obtaining scientific data presented in this paper. Access to computing and storage facilities owned by parties and projects contributing to the National Grid Infrastructure MetaCentrum provided under the program “Projects of Large Research, Development, and Innovations Infrastructures” (CESNET LM2015042), is greatly appreciated. Research data were collected in cooperation with the employees of the Czech Centre for Phenogenomics supported by the Czech Academy of Sciences RVO 68378050 and by the project of support program for large infrastructures for research, experimental development and innovation LM2018126 Czech Centre for Phenogenomics provided by MEYS CR. Further support was provided by MEYS and ESIF (CZ.02.1.01/0.0/0.0/16_013/0001789, CZ.02.1.01/0.0/0.0/18_046/0015861, and CZ.1.05/2.1.00/19.0395).

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Ezrova, Z., Nahacka, Z., Stursa, J. et al. SMAD4 loss limits the vulnerability of pancreatic cancer cells to complex I inhibition via promotion of mitophagy. Oncogene 40, 2539–2552 (2021). https://doi.org/10.1038/s41388-021-01726-4

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