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Metabolic reprogramming of normal oral fibroblasts correlated with increased glycolytic metabolism of oral squamous cell carcinoma and precedes their activation into carcinoma associated fibroblasts

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Abstract

Cancers show a metabolic shift towards aerobic glycolysis. By “corrupting” their microenvironment, carcinoma cells are able to obtain energy substrates to “fuel” their mitochondrial metabolism and cell growth in an autophagy-associated, paracrine manner. However, the metabolic changes and role of normal fibroblasts in this process remain unclear. We devised a novel, indirect co-culture system to elucidate the mechanisms of metabolic coupling between stromal cells and oral squamous cell carcinoma (OSCC) cells. Here, we showed that normal oral fibroblasts (NOFs) and OSCC become metabolically coupled through several processes before acquiring an activated phenotype and without inducing senescence. We observed, for the first time, that NOFs export mitochondria towards OSCCs through both direct contact and via indirect mechanisms. NOFs are activated and are able to acquire a cancer-associated fibroblasts metabolic phenotype when co-cultivation with OSSC cells, by undergoing aerobic glycolysis, secreting more reactive oxygen species (ROS), high l-lactate and overexpressing lactate exporter MCT-4, leading to mitochondrial permeability transition pore (mPTP) opening, hypoxia, and mitophagy. On the other hand, Cav-1-low NOFs generate l-lactate to “fuel” mitochondrial metabolism and anabolic growth of OSCC. Most interestingly, the decrease in AMPK activity and PGC-1α expression might involve in regulation of ROS that functions to maintain final energy and metabolic homeostasis. This indicated, for the first time, the existence of ATP and ROS homeostasis during carcinogenesis. Our study suggests that an efficient therapeutical approach has to target the multiple mechanisms used by them to corrupt the normal surrounding stroma and metabolic homeostasis.

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Availability of data and material

The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

OSCC:

Oral squamous cell carcinoma

NOFs:

Normal oral fibroblasts

CAFs:

Cancer-associated fibroblasts

ROS:

Reactive oxygen species

mPTP:

Mitochondrial permeability transition pore

NOKs:

Normal oral epithelial cells

FBS:

Fetal bovine serum

BPE:

Bovine pituitary extract

KSFM:

Serum-free keratinocyte specific medium

DOK:

Dysplastic oral keratinocyte

EGF:

Epidermal growth factor

MTG:

MitoTracker Green

TMRE:

Tetramethylrhodamine, ethyl ester

FCCP:

Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone

PBS:

Phosphate-buffered saline

MTDR:

MitoTracker Deep Red

ROS:

Reactive oxygen stress

DCFDA:

2′,7′-Dichlorofluorescin diacetate

ATP:

Adenosine triphosphate

ATPases:

ATP-degrading enzymes

IHC:

Immunohistochemistry staining

EMU:

Epithelium–stroma unit

TNTs:

Tunneling nanotubes

mPTP:

Mitochondrial permeability transition pore

VDAC:

Voltage-dependent anion channel

ECM:

Extracellular matrix

MCTs:

Monocarboxylate transporters

Alpha-SMA:

Alpha-smooth muscle actin

FAP:

Fibroblast activation protein

References

  1. Johnson NW, Jayasekara P, Amarasinghe AA (2011) Squamous cell carcinoma and precursor lesions of the oral cavity: epidemiology and aetiology. Periodontology 57:19–37

    Article  Google Scholar 

  2. Schafer M, Werner S (2008) Cancer as an overhealing wound: an old hypothesis revisited. Nat Rev Mol Cell Biol 9:628–638

    Article  CAS  Google Scholar 

  3. Pavlides S, Whitaker-Menezes D, Castello-Cros R, Flomenberg N, Witkiewicz AK, Frank PG, Casimiro MC, Wang C, Fortina P, Addya S, Pestell RG, Martinez-Outschoorn UE, Sotgia F, Lisanti MP (2009) The reverse Warburg effect: aerobic glycolysis in cancer associated fibroblasts and the tumor stroma. Cell Cycle 8:3984–4001

    Article  CAS  Google Scholar 

  4. Lewis MP, Lygoe KA, Nystrom ML, Anderson WP, Speight PM, Marshall JF, Thomas GJ (2004) Tumour-derived TGF-beta1 modulates myofibroblast differentiation and promotes HGF/SF-dependent invasion of squamous carcinoma cells. Br J Cancer 90:822–832

    Article  CAS  Google Scholar 

  5. Davalos AR, Coppe JP, Campisi J, Desprez PY (2010) Senescent cells as a source of inflammatory factors for tumor progression. Cancer Metastasis Rev 29:273–283

    Article  Google Scholar 

  6. Rosenthal E, McCrory A, Talbert M, Young G, Murphy-Ullrich J, Gladson C (2004) Elevated expression of TGF-beta1 in head and neck cancer-associated fibroblasts. Mol Carcinog 40:116–121

    Article  CAS  Google Scholar 

  7. Costea DE, Hills A, Osman AH, Thurlow J, Kalna G, Huang X, Pena Murillo C, Parajuli H, Suliman S, Kulasekara KK, Johannessen AC, Partridge M (2013) Identification of two distinct carcinoma-associated fibroblast subtypes with differential tumor-promoting abilities in oral squamous cell carcinoma. Cancer Res 73:3888–3901

    Article  CAS  Google Scholar 

  8. Jensen DH, Therkildsen MH, Dabelsteen E (2015) A reverse Warburg metabolism in oral squamous cell carcinoma is not dependent upon myofibroblasts. J Oral Pathol Med 44:714–721

    Article  CAS  Google Scholar 

  9. Shi Y, Tan SH, Ng S, Zhou J, Yang ND, Koo GB, McMahon KA, Parton RG, Hill MM, Del Pozo MA, Kim YS, Shen HM (2015) Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy. Autophagy. 11:769–784

    Article  CAS  Google Scholar 

  10. Vered M, Lehtonen M, Hotakainen L, Pirila E, Teppo S, Nyberg P, Sormunen R, Zlotogorski-Hurvitz A, Salo T, Dayan D (2015) Caveolin-1 accumulation in the tongue cancer tumor microenvironment is significantly associated with poor prognosis: an in vivo and in vitro study. BMC Cancer 15:25

    Article  Google Scholar 

  11. Whitaker-Menezes D, Martinez-Outschoorn UE, Lin Z, Ertel A, Flomenberg N, Witkiewicz AK, Birbe RC, Howell A, Pavlides S, Gandara R, Pestell RG, Sotgia F, Philp NJ, Lisanti MP (2011) Evidence for a stromal-epithelial “lactate shuttle” in human tumors: mCT4 is a marker of oxidative stress in cancer-associated fibroblasts. Cell Cycle 10:1772–1783

    Article  CAS  Google Scholar 

  12. Pasquier J, Guerrouahen BS, Al Thawadi H, Ghiabi P, Maleki M, Abu-Kaoud N, Jacob A, Mirshahi M, Galas L, Rafii S, Le Foll F, Rafii A (2013) Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance. J Transl Med 11:94

    Article  CAS  Google Scholar 

  13. Chatterjee M, Ben-Josef E, Thomas DG, Morgan MA, Zalupski MM, Khan G, Andrew Robinson C, Griffith KA, Chen CS, Ludwig T, Bekaii-Saab T, Chakravarti A, Williams TM (2015) Caveolin-1 is associated with tumor progression and confers a multi-modality resistance phenotype in pancreatic cancer. Sci Rep 5:10867

    Article  CAS  Google Scholar 

  14. Mercier I, Casimiro MC, Wang C, Rosenberg AL, Quong J, Minkeu A, Allen KG, Danilo C, Sotgia F, Bonuccelli G, Jasmin J-F, Xu H, Bosco E, Aronow B, Witkiewicz AK, Pestell RG, Knudsen ES, Lisanti MP (2014) Human breast cancer-associated fibroblasts (CAFs) show caveolin-1 down-regulation and RB tumor suppressor functional inactivation: implications for the response to hormonal therapy. Cancer Biol Ther 7:1212–1225

    Article  Google Scholar 

  15. Pavlides S, Vera I, Gandara R, Sneddon S, Pestell RG, Mercier I, Martinez-Outschoorn UE, Whitaker-Menezes D, Howell A, Sotgia F, Lisanti MP (2012) Warburg meets autophagy: cancer-associated fibroblasts accelerate tumor growth and metastasis via oxidative stress, mitophagy, and aerobic glycolysis. Antioxid Redox Signal 16:1264–1284

    Article  CAS  Google Scholar 

  16. Wilde L, Roche M, Domingo-Vidal M, Tanson K, Philp N, Curry J, Martinez-Outschoorn U (2017) Metabolic coupling and the Reverse Warburg Effect in cancer: implications for novel biomarker and anticancer agent development. Semin Oncol 44:198–203

    Article  CAS  Google Scholar 

  17. Zorov DB, Juhaszova M, Sollott SJ (2014) Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev 94:909–950

    Article  CAS  Google Scholar 

  18. Rabinovitch RC, Samborska B, Faubert B, Ma EH, Gravel SP, Andrzejewski S, Raissi TC, Pause A, St-Pierre J, Jones RG (2017) AMPK maintains cellular metabolic homeostasis through regulation of mitochondrial reactive oxygen species. Cell reports. 21:1–9

    Article  CAS  Google Scholar 

  19. St-Pierre J, Drori S, Uldry M, Silvaggi JM, Rhee J, Jager S, Handschin C, Zheng K, Lin J, Yang W, Simon DK, Bachoo R, Spiegelman BM (2006) Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell 127:397–408

    Article  CAS  Google Scholar 

  20. Rabinovitch RC et al (2017) AMPK maintains cellular metabolic homeostasis through regulation of mitochondrial reactive oxygen species. Cell Rep 21(1):1–9

    Article  CAS  Google Scholar 

  21. Yang G et al (2006) The chemokine growth-regulated oncogene 1 (Gro-1) links RAS signaling to the senescence of stromal fibroblasts and ovarian tumorigenesis. Proc Natl Acad Sci USA 103(44):16472–16477

    Article  CAS  Google Scholar 

  22. Capparelli C et al (2012) CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions, without an increase in neo-angiogenesis. Cell Cycle 11(19):3599–3610

    Article  CAS  Google Scholar 

  23. Quijano C et al (2012) Oncogene-induced senescence results in marked metabolic and bioenergetic alterations. Cell Cycle 11(7):1383–1392

    Article  CAS  Google Scholar 

  24. James EL et al (2015) Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease. J Proteome Res 14(4):1854–1871

    Article  CAS  Google Scholar 

  25. Audet-Walsh E, Papadopoli DJ, Gravel SP, Yee T, Bridon G, Caron M, Bourque G, Giguere V, St-Pierre J (2016) The PGC-1alpha/ERRalpha axis represses one-carbon metabolism and promotes sensitivity to anti-folate therapy in breast cancer. Cell Rep 14:920–931

    Article  CAS  Google Scholar 

  26. Krtolica A, Parrinello S, Lockett S, Desprez PY, Campisi J (2001) Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci USA 98:12072–12077

    Article  CAS  Google Scholar 

  27. Para R et al (2019) Metabolic reprogramming as a driver of fibroblast activation in pulmonary fibrosis. Am J Med Sci 357(5):394–398

    Article  Google Scholar 

  28. Hassona Y, Cirillo N, Lim KP, Herman A, Mellone M, Thomas GJ, Pitiyage GN, Parkinson EK, Prime SS (2013) Progression of genotype-specific oral cancer leads to senescence of cancer-associated fibroblasts and is mediated by oxidative stress and TGF-beta. Carcinogenesis 34:1286–1295

    Article  CAS  Google Scholar 

  29. Berridge MV, Crasso C, Neuzil J (2015) Mitochondrial genome transfer to tumor cells breaks the rules and establishes a new precedent in cancer biology. Mol Cell Oncol 5:e1023929

    Article  Google Scholar 

  30. Iglesias-Bartolome R, Patel V, Cotrim A, Leelahavanichkul K, Molinolo AA, Mitchell JB, Gutkind JS (2012) mTOR inhibition prevents epithelial stem cell senescence and protects from radiation-induced mucositis. Cell Stem Cell 11:401–414

    Article  CAS  Google Scholar 

  31. Dimmer KS, Friedrich B, Lang F, Deitmer JW, Broer S (2000) The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells. Biochem J 350(Pt 1):219–227

    Article  CAS  Google Scholar 

  32. Shan T, Lu H, Ji H, Li Y, Guo J, Chen X, Wu T (2014) Loss of stromal caveolin-1 expression: a novel tumor microenvironment biomarker that can predict poor clinical outcomes for pancreatic cancer. PLoS One 9:e97239

    Article  Google Scholar 

  33. Folgueira MA, Maistro S, Katayama ML, Roela RA, Mundim FG, Nanogaki S, de Bock GH, Brentani MM (2013) Markers of breast cancer stromal fibroblasts in the primary tumour site associated with lymph node metastasis: a systematic review including our case series. Biosci Rep 33:e00085

    Article  Google Scholar 

  34. Ha TK, Her NG, Lee MG, Ryu BK, Lee JH, Han J, Jeong SI, Kang MJ, Kim NH, Kim HJ, Chi SG (2012) Caveolin-1 increases aerobic glycolysis in colorectal cancers by stimulating HMGA1-mediated GLUT3 transcription. Cancer Res 72:4097–4109

    Article  CAS  Google Scholar 

  35. Zhao X, He Y, Gao J, Fan L, Li Z, Yang G, Chen H (2013) Caveolin-1 expression level in cancer associated fibroblasts predicts outcome in gastric cancer. PLoS One 8:e59102

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Hanne Linda Nakkestad for excellent technical assistance. We also thank Molecular Imaging Centre (MIC) for outstanding facilities.

Funding

This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme (DEC, project number 22325), the Western Health Authority (DEC grant nr. 911902/2013).

Author information

Author notes

  1. Zhuoyuan Zhang and Zhenjie Gao are the first co-authors and contributed equally.

  2. Daniela Elena Costea and Xiao Liang are the last corresponding authors and contributed equally.

Authors and Affiliations

  1. State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China

    Zhuoyuan Zhang, Zhenjie Gao & Longjiang Li

  2. Department of Head and Neck Cancer Surgery, West China School of Stomatology, Sichuan University, Chengdu, China

    Zhuoyuan Zhang & Longjiang Li

  3. Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

    Zhenjie Gao

  4. Gade Laboratory for Pathology, Department of Clinical Medicine, Faculty of Medicine and Dentistry, University of Bergen, Bergen, Norway

    Saroj Rajthala, Harsh Dongre, Himalaya Parajuli, Salwa Suliman, Ridhima Das & Daniela Elena Costea

  5. Centre for Cancer Biomarkers (CCBIO), Faculty of Medicine and Dentistry, University of Bergen, Bergen, Norway

    Saroj Rajthala, Harsh Dongre, Himalaya Parajuli, Ridhima Das & Daniela Elena Costea

  6. Department of Oral Biology, Faculty of Dentistry, University of Oslo, Postboks 1052, Blindern, 0316, Oslo, Norway

    Dipak Sapkota

  7. Department of Global Public Health and Primary Care, Centre for International Health, University of Bergen, Bergen, Norway

    Himalaya Parajuli

  8. Department of Clinical Dentistry, Center for Clinical Dental Research, University of Bergen, Bergen, Norway

    Salwa Suliman

  9. The Mitochondrial Medicine and Neurogenetics (MMN) Group, Department of Clinical Medicine, University of Bergen, PO Box 7804, 5020, Bergen, Norway

    Laurence A. Bindoff, Daniela Elena Costea & Xiao Liang

  10. Department of Neurology, Haukeland University Hospital, Bergen, Norway

    Zhuoyuan Zhang, Laurence A. Bindoff & Xiao Liang

  11. Department of Pathology, Haukeland University Hospital, Bergen, Norway

    Daniela Elena Costea

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  1. Zhuoyuan Zhang
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Contributions

XL, DEC, ZYZ, and JZG: conceptualization; XL, DEC, ZYZ, and JZG: methodology; DS, ZYZ, ZJG, LAB, RD, and SR: investigation; ZYZ and ZJG: writing—original draft; XL, DEC, ZYZ, LAB, and LJL: writing—review and editing; DEC and LAB: funding acquisition; HD, HP, SS, and LJL: resources; XL and DEC: supervision.

Corresponding authors

Correspondence to Daniela Elena Costea or Xiao Liang.

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No potential conflicts of interest were disclosed.

Ethics approval

The project was approved by the Committee for Ethics in Health Research of West Norway (REK nr. 2010/481); the study was performed in accordance with the Declaration of Helsinki. Tissues were acquired with written informed consent from all patients.

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Zhang, Z., Gao, Z., Rajthala, S. et al. Metabolic reprogramming of normal oral fibroblasts correlated with increased glycolytic metabolism of oral squamous cell carcinoma and precedes their activation into carcinoma associated fibroblasts. Cell. Mol. Life Sci. 77, 1115–1133 (2020). https://doi.org/10.1007/s00018-019-03209-y

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  • DOI: https://doi.org/10.1007/s00018-019-03209-y

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