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

Oncogene volume 40, pages 2539–2552 (2021)Cite this article

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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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References

  1. Rawla P, Sunkara T, Gaduputi V. Epidemiology of pancreatic cancer: global trends, etiology and risk factors. World J Oncol. 2019;10:10–27.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Colak S, Ten Dijke P. Targeting TGF-beta signaling in cancer. Trends Cancer. 2017;3:56–71.

    Article  CAS  PubMed  Google Scholar 

  3. Katsuno Y, Meyer DS, Zhang Z, Shokat KM, Akhurst RJ, Miyazono K, et al. Chronic TGF-beta exposure drives stabilized EMT, tumor stemness, and cancer drug resistance with vulnerability to bitopic mTOR inhibition. Sci Signal. 2019;12:eaau8544.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature. 2015;527:525–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Shibue T, Weinberg RA. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol. 2017;14:611–29.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Xia X, Wu W, Huang C, Cen G, Jiang T, Cao J, et al. SMAD4 and its role in pancreatic cancer. Tumour Biol. 2015;36:111–9.

    Article  CAS  PubMed  Google Scholar 

  7. David CJ, Huang YH, Chen M, Su J, Zou Y, Bardeesy N, et al. TGF-beta tumor suppression through a lethal EMT. Cell. 2016;164:1015–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Blackford A, Serrano OK, Wolfgang CL, Parmigiani G, Jones S, Zhang X, et al. SMAD4 gene mutations are associated with poor prognosis in pancreatic cancer. Clin Cancer Res. 2009;15:4674–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Papageorgis P, Cheng K, Ozturk S, Gong Y, Lambert AW, Abdolmaleky HM, et al. Smad4 inactivation promotes malignancy and drug resistance of colon cancer. Cancer Res. 2011;71:998–1008.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wasserman I, Lee LH, Ogino S, Marco MR, Wu C, Chen X, et al. SMAD4 loss in colorectal cancer patients correlates with recurrence, loss of immune infiltrate, and chemoresistance. Clin Cancer Res. 2019;25:1948–56.

    Article  CAS  PubMed  Google Scholar 

  11. Ozawa H, Ranaweera RS, Izumchenko E, Makarev E, Zhavoronkov A, Fertig EJ, et al. SMAD4 loss is associated with cetuximab resistance and induction of MAPK/JNK activation in head and neck cancer cells. Clin Cancer Res. 2017;23:5162–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sica V, Bravo-San Pedro JM, Stoll G, Kroemer G. Oxidative phosphorylation as a potential therapeutic target for cancer therapy. Int J Cancer. 2019;146:10–7.

    Article  PubMed  Google Scholar 

  13. Rohlenova K, Sachaphibulkij K, Stursa J, Bezawork-Geleta A, Blecha J, Endaya B, et al. Selective disruption of respiratory supercomplexes as a new strategy to suppress Her2(high) breast cancer. Antioxid Redox Signal. 2017;26:84–103.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Molina JR, Sun Y, Protopopova M, Gera S, Bandi M, Bristow C, et al. An inhibitor of oxidative phosphorylation exploits cancer vulnerability. Nat Med. 2018;24:1036–46.

    Article  CAS  PubMed  Google Scholar 

  15. Chae YK, Arya A, Malecek MK, Shin DS, Carneiro B, Chandra S, et al. Repurposing metformin for cancer treatment: current clinical studies. Oncotarget. 2016;7:40767–80.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Leone A, Di Gennaro E, Bruzzese F, Avallone A, Budillon A. New perspective for an old antidiabetic drug: metformin as anticancer agent. Cancer Treat Res. 2014;159:355–76.

    Article  CAS  PubMed  Google Scholar 

  17. Pecinova A, Brazdova A, Drahota Z, Houstek J, Mracek T. Mitochondrial targets of metformin-are they physiologically relevant? BioFactors. 2019;45:703–11.

    Article  CAS  PubMed  Google Scholar 

  18. Bridges HR, Sirvio VA, Agip AN, Hirst J. Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP-kinase. BMC Biol. 2016;14:65.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Boukalova S, Stursa J, Werner L, Ezrova Z, Cerny J, Bezawork-Geleta A, et al. Mitochondrial targeting of metformin enhances its activity against pancreatic cancer. Mol Cancer Ther. 2016;15:2875–86.

    Article  CAS  PubMed  Google Scholar 

  20. Cheng G, Zielonka J, Ouari O, Lopez M, McAllister D, Boyle K, et al. Mitochondria-targeted analogues of metformin exhibit enhanced antiproliferative and radiosensitizing effects in pancreatic cancer cells. Cancer Res. 2016;76:3904–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chen Y, Di C, Zhang X, Wang J, Wang F, Yan JF, et al. Transforming growth factor beta signaling pathway: a promising therapeutic target for cancer. J Cell Physiol. 2020;235:1903–14.

    Article  CAS  PubMed  Google Scholar 

  22. Rodriguez-Garcia A, Samso P, Fontova P, Simon-Molas H, Manzano A, Castano E, et al. TGF-beta1 targets Smad, p38 MAPK, and PI3K/Akt signaling pathways to induce PFKFB3 gene expression and glycolysis in glioblastoma cells. FEBS J. 2017;284:3437–54.

    Article  CAS  PubMed  Google Scholar 

  23. Zhang C, Zhang X, Xu R, Huang B, Chen AJ, Li C, et al. TGF-beta2 initiates autophagy via Smad and non-Smad pathway to promote glioma cells’ invasion. J Exp Clin Cancer Res. 2017;36:162.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Yalcin A, Solakoglu TH, Ozcan SC, Guzel S, Peker S, Celikler S, et al. 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 is required for transforming growth factor beta1-enhanced invasion of Panc1 cells in vitro. Biochem Biophys Res Commun. 2017;484:687–93.

    Article  CAS  PubMed  Google Scholar 

  25. Costanza B, Rademaker G, Tiamiou A, De Tullio P, Leenders J, Blomme A, et al. Transforming growth factor beta-induced, an extracellular matrix interacting protein, enhances glycolysis and promotes pancreatic cancer cell migration. Int J Cancer. 2019;145:1570–84.

    Article  CAS  PubMed  Google Scholar 

  26. Shi S, Ji S, Qin Y, Xu J, Zhang B, Xu W, et al. Metabolic tumor burden is associated with major oncogenomic alterations and serum tumor markers in patients with resected pancreatic cancer. Cancer Lett. 2015;360:227–33.

    Article  CAS  PubMed  Google Scholar 

  27. Basso D, Gnatta E, Padoan A, Fogar P, Furlanello S, Aita A, et al. PDAC-derived exosomes enrich the microenvironment in MDSCs in a SMAD4-dependent manner through a new calcium related axis. Oncotarget. 2017;8:84928–44.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Liang C, Shi S, Qin Y, Meng Q, Hua J, Hu Q, et al. Localisation of PGK1 determines metabolic phenotype to balance metastasis and proliferation in patients with SMAD4-negative pancreatic cancer. Gut. 2020;69:888–900.

    Article  CAS  PubMed  Google Scholar 

  29. Cottet-Rousselle C, Ronot X, Leverve X, Mayol JF. Cytometric assessment of mitochondria using fluorescent probes. Cytom A. 2011;79:405–25.

    Article  Google Scholar 

  30. Katayama H, Kogure T, Mizushima N, Yoshimori T, Miyawaki A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem Biol. 2011;18:1042–52.

    Article  CAS  PubMed  Google Scholar 

  31. Georgakopoulos ND, Wells G, Campanella M. The pharmacological regulation of cellular mitophagy. Nat Chem Biol. 2017;13:136–46.

    Article  CAS  PubMed  Google Scholar 

  32. Hernandez G, Thornton C, Stotland A, Lui D, Sin J, Ramil J, et al. MitoTimer: a novel tool for monitoring mitochondrial turnover. Autophagy. 2013;9:1852–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Liang C, Xu J, Meng Q, Zhang B, Liu J, Hua J, et al. TGFB1-induced autophagy affects the pattern of pancreatic cancer progression in distinct ways depending on SMAD4 status. Autophagy. 2019;16:1–15.

    Google Scholar 

  34. Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, et al. Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis. 2013;4:e838.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wang F, Xia X, Yang C, Shen J, Mai J, Kim HC, et al. SMAD4 gene mutation renders pancreatic cancer resistance to radiotherapy through promotion of autophagy. Clin Cancer Res. 2018;24:3176–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Springer W, Kahle PJ. Regulation of PINK1-Parkin-mediated mitophagy. Autophagy. 2011;7:266–78.

    Article  CAS  PubMed  Google Scholar 

  37. Urra FA, Munoz F, Lovy A, Cardenas C. The mitochondrial complex(I)ty of cancer. Front Oncol. 2017;7:118.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Chen YW, Hsiao PJ, Weng CC, Kuo KK, Kuo TL, Wu DC, et al. SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells. BMC Cancer. 2014;14:181.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Fullerton PT Jr., Creighton CJ, Matzuk MM. Insights into SMAD4 loss in pancreatic cancer from inducible restoration of TGF-beta signaling. Mol Endocrinol. 2015;29:1440–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Arumugam T, Ramachandran V, Fournier KF, Wang H, Marquis L, Abbruzzese JL, et al. Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer. Cancer Res. 2009;69:5820–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, et al. The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol. 2009;11:1487–95.

    Article  CAS  PubMed  Google Scholar 

  42. Wang Z, Li Y, Kong D, Banerjee S, Ahmad A, Azmi AS, et al. Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway. Cancer Res. 2009;69:2400–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Mao L, Li Y, Zhao J, Li Q, Yang B, Wang Y, et al. Transforming growth factor-beta1 contributes to oxaliplatin resistance in colorectal cancer via epithelial to mesenchymal transition. Oncol Lett. 2017;14:647–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Zhang YE. Non-Smad signaling pathways of the TGF-beta family. Cold Spring Harb Perspect Biol. 2017;9:128–39.

    Article  Google Scholar 

  45. Dagda RK, Zhu J, Kulich SM, Chu CT. Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress: implications for Parkinson’s disease. Autophagy. 2008;4:770–82.

    Article  CAS  PubMed  Google Scholar 

  46. Naguib A, Mathew G, Reczek CR, Watrud K, Ambrico A, Herzka T, et al. Mitochondrial complex I inhibitors expose a vulnerability for selective killing of Pten-null cells. Cell Rep. 2018;23:58–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hubackova S, Davidova E, Rohlenova K, Stursa J, Werner L, Andera L, et al. Selective elimination of senescent cells by mitochondrial targeting is regulated by ANT2. Cell Death Differ. 2019;26:276–90.

    Article  CAS  PubMed  Google Scholar 

  48. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343:84–87.

    Article  CAS  PubMed  Google Scholar 

  49. Bajzikova M, Kovarova J, Coelho AR, Boukalova S, Oh S, Rohlenova K, et al. Reactivation of dihydroorotate dehydrogenase-driven pyrimidine biosynthesis restores tumor growth of respiration-deficient cancer cells. Cell Metab. 2019;29:399–416.e310.

    Article  CAS  PubMed  Google Scholar 

  50. Valente AJ, Maddalena LA, Robb EL, Moradi F, Stuart JA. A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture. Acta Histochem. 2017;119:315–26.

    Article  CAS  PubMed  Google Scholar 

  51. Tan AS, Baty JW, Dong LF, Bezawork-Geleta A, Endaya B, Goodwin J, et al. Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA. Cell Metab. 2015;21:81–94.

    Article  CAS  PubMed  Google Scholar 

  52. Tauber J, Dlaskova A, Santorova J, Smolkova K, Alan L, Spacek T, et al. Distribution of mitochondrial nucleoids upon mitochondrial network fragmentation and network reintegration in HEPG2 cells. Int J Biochem Cell Biol. 2013;45:593–603.

    Article  CAS  PubMed  Google Scholar 

  53. Um JH, Kim YY, Finkel T, Yun J. Sensitive measurement of mitophagy by flow cytometry using the pH-dependent fluorescent reporter mt-Keima. J Vis Exp. 2018;138:58099.

    Google Scholar 

  54. Laker RC, Xu P, Ryall KA, Sujkowski A, Kenwood BM, Chain KH, et al. A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo. J Biol Chem. 2014;289:12005–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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Authors and Affiliations

  1. Institute of Biotechnology, Czech Academy of Sciences, Vestec, Prague-West, Czech Republic

    Zuzana Ezrova, Zuzana Nahacka, Jan Stursa, Lukas Werner, Renata Zobalova, Jakub Rohlena, Stepana Boukalova & Jiri Neuzil

  2. Faculty of Science, Charles University, Prague, Czech Republic

    Zuzana Ezrova

  3. Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic

    Erik Vlcak

  4. Czech Centre of Phenogenomics, Institute of Molecular Genetics, Czech Academy of Sciences, Vestec, Prague-West, Czech Republic

    Petra Kralova Viziova & Radislav Sedlacek

  5. Malaghan Institute of Medical Research, Wellington, New Zealand

    Michael V. Berridge

  6. School of Medical Science, Griffith University, Southport, QLD, Australia

    Jiri Neuzil

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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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