Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Translational Therapeutics

PFKFB3 works on the FAK-STAT3-SOX2 axis to regulate the stemness in MPM

Abstract

Background

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm and often acquires chemoresistance by increasing stemness in tumour tissue, thereby generating cancer stem cells (CSCs). CSCs escape treatment by deploying metabolic pathways to trigger dormancy or proliferation, also gaining the ability to exit and re-enter the cell cycle to hide their cellular identity.

Methods

We employed various cellular and biochemical assays to identify the role of the glycolytic enzyme PFKFB3, by knocking it down and pharmacologically inhibiting it with PFK158, to determine its anticancer effects in vitro and in vivo by targeting the CSC population in MPM.

Results

Here, we have identified PFKFB3 as a strategic player to target the CSC population in MPM and demonstrated that both pharmacologic (PFK158) and genetic inhibition of PFKFB3 destroy the FAK-Stat3-SOX2 nexus resulting in a decline in conspicuous stem cell markers viz. ALDH, CD133, CD44, SOX2. Inhibition of PFKFB3 accumulates p21 and p27 in the nucleus by decreasing SKP2. Lastly, PFK158 diminishes tumour-initiating cells (TICs) mediated MPM xenograft in vivo.

Conclusions

This study confers a comprehensive and mechanistic function of PFKFB3 in CSC maintenance that may foster exceptional opportunities for targeted small molecule blockade of the TICs in MPM.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: PFKFB3 inhibition downregulates stem cell markers in MPM.
Fig. 2: PFKFB3 inhibition diminishes the ALDH+ CD133+ cell population.
Fig. 3: PFK158 works on the FAK-Stat3-SOX2 axis.
Fig. 4: MPM cells show the nuclear localisation and function of PFKFB3.
Fig. 5: Enrichment and characterisation of MPM-CSC population.
Fig. 6: Repression of p21/p27 and overexpression of PFKFB3 in spheroid MPM cells regulate the stemness.
Fig. 7: Tumour-initiating stem-like cells (TIC) in human pleural mesothelioma exhibit upregulation of PFKFB3.
Fig. 8: A summarised illustration (BioRender, https://biorender.com) showing the contribution of PFKFB3 in the enrichment of MPM stemness and cell cycle regulation.

Data availability

The data that support the findings of this study are available from the corresponding author on a reasonable request.

References

  1. De Francesco EM, Sotgia F, Lisanti MP. Cancer stem cells (CSCs): metabolic strategies for their identification and eradication. Biochem J. 2018;475:1611–34.

    PubMed  Article  Google Scholar 

  2. Colombo SL, Palacios-Callender M, Frakich N, Carcamo S, Kovacs I, Tudzarova S, et al. Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells. Proc Natl Acad Sci USA. 2011;108:21069–74.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. Clem BF, O’Neal J, Tapolsky G, Clem AL, Imbert-Fernandez Y, Kerr DA 2nd, et al. Targeting 6-phosphofructo-2-kinase (PFKFB3) as a therapeutic strategy against cancer. Mol Cancer Ther. 2013;12:1461–70.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Shi L, Pan H, Liu Z, Xie J, Han W. Roles of PFKFB3 in cancer. Signal Transduct Target Ther. 2017;2:17044.

    PubMed  PubMed Central  Article  Google Scholar 

  5. Kessler R, Bleichert F, Warnke J-P, Eschrich K. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is up-regulated in high-grade astrocytomas. J Neuro-Oncol. 2008;86:257–64.

    CAS  Article  Google Scholar 

  6. Li H-M, Yang J-G, Liu Z-J, Wang W-M, Yu Z-L, Ren J-G, et al. Blockage of glycolysis by targeting PFKFB3 suppresses tumor growth and metastasis in head and neck squamous cell carcinoma. J Exp Clin Cancer Res. 2017;36:7.

    PubMed  PubMed Central  Article  Google Scholar 

  7. Bobarykina AY, Minchenko DO, Opentanova IL, Moenner M, Caro J, Esumi H, et al. Hypoxic regulation of PFKFB-3 and PFKFB-4 gene expression in gastric and pancreatic cancer cell lines and expression of PFKFB genes in gastric cancers. Acta Biochim Pol. 2006;53:789–99.

    CAS  PubMed  Article  Google Scholar 

  8. Atsumi T, Chesney J, Metz C, Leng L, Donnelly S, Makita Z, et al. High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3) in human cancers. Cancer Res. 2002;62:5881–7.

    CAS  PubMed  Google Scholar 

  9. Mondal S, Roy D, Sarkar Bhattacharya S, Jin L, Jung D, Zhang S, et al. Therapeutic targeting of PFKFB3 with a novel glycolytic inhibitor PFK158 promotes lipophagy and chemosensitivity in gynecologic cancers. Int J Cancer. 2019;144:178–89.

    CAS  PubMed  Article  Google Scholar 

  10. Sarkar Bhattacharya S, Thirusangu P, Jin L, Roy D, Jung D, Xiao Y, et al. PFKFB3 inhibition reprograms malignant pleural mesothelioma to nutrient stress-induced macropinocytosis and ER stress as independent binary adaptive responses. Cell Death Dis. 2019;10:725.

    PubMed  PubMed Central  Article  Google Scholar 

  11. Yalcin A, Clem BF, Simmons A, Lane A, Nelson K, Clem AL, et al. Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases. J Biol Chem. 2009;284:24223–32.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Gustafsson NMS, Färnegårdh K, Bonagas N, Ninou AH, Groth P, Wiita E, et al. Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination. Nat Commun. 2018;9:3872.

    PubMed  PubMed Central  Article  Google Scholar 

  13. Li F-L, Liu J-P, Bao R-X, Yan G, Feng X, Xu Y-P, et al. Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis. Nat Commun. 2018;9:508.

    PubMed  PubMed Central  Article  Google Scholar 

  14. Folmes CD, Dzeja PP, Nelson TJ, Terzic A. Metabolic plasticity in stem cell homeostasis and differentiation. Cell Stem Cell. 2012;11:596–606.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Jang H, Yang J, Lee E, Cheong JH. Metabolism in embryonic and cancer stemness. Arch Pharm Res. 2015;38:381–8.

    CAS  PubMed  Article  Google Scholar 

  16. Snyder V, Reed-Newman TC, Arnold L, Thomas SM, Anant S. Cancer stem cell metabolism and potential therapeutic targets. Front Oncol. 2018;8:203.

    PubMed  PubMed Central  Article  Google Scholar 

  17. Cancer.Net. Mesothelioma: Statistics, 2019; https://www.cancer.net/cancer-types/mesothelioma/statistics.

  18. Villanova F, Procopio A, Rippo MR. Malignant mesothelioma resistance to apoptosis: recent discoveries and their implication for effective therapeutic strategies. Curr Med Chem. 2008;15:631–41.

    CAS  PubMed  Article  Google Scholar 

  19. Cortes-Dericks L, Carboni GL, Schmid RA, Karoubi G. Putative cancer stem cells in malignant pleural mesothelioma show resistance to cisplatin and pemetrexed. Int J Oncol. 2010;37:437–44.

    CAS  PubMed  Google Scholar 

  20. Song K, Kwon H, Han C, Zhang J, Dash S, Lim K, et al. Active glycolytic metabolism in CD133(+) hepatocellular cancer stem cells: regulation by MIR-122. Oncotarget. 2015;6:40822–35.

    PubMed  PubMed Central  Article  Google Scholar 

  21. Okamoto H, Fujishima F, Nakamura Y, Zuguchi M, Ozawa Y, Takahashi Y, et al. Significance of CD133 expression in esophageal squamous cell carcinoma. World J Surg Oncol. 2013;11:51.

    PubMed  PubMed Central  Article  Google Scholar 

  22. Chu IM, Hengst L, Slingerland JM. The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer. 2008;8:253–67.

    CAS  PubMed  Article  Google Scholar 

  23. Li H, Collado M, Villasante A, Matheu A, Lynch CJ, Cañamero M, et al. p27Kip1 directly represses SOX2 during embryonic stem cell differentiation. Cell Stem Cell. 2012;11:845–52.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Jung D, Khurana A, Roy D, Kalogera E, Bakkum-Gamez J, Chien J, et al. Quinacrine upregulates p21/p27 independent of p53 through autophagy-mediated downregulation of p62-Skp2 axis in ovarian cancer. Sci Rep. 2018;8:2487.

    PubMed  PubMed Central  Article  Google Scholar 

  25. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell. 2007;1:555–67.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Sarkar S, Dutta D, Samanta SK, Bhattacharya K, Pal BC, Li J, et al. Oxidative inhibition of Hsp90 disrupts the super‐chaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo. Int J cancer. 2013;132:695–706.

    CAS  PubMed  Article  Google Scholar 

  27. Mandal C, Sarkar S, Chatterjee U, Schwartz-Albiez R, Mandal C. Disialoganglioside GD3-synthase over expression inhibits survival and angiogenesis of pancreatic cancer cells through cell cycle arrest at S-phase and disruption of integrin-beta1-mediated anchorage. Int J Biochem Cell Biol. 2014;53:162–73.

    CAS  PubMed  Article  Google Scholar 

  28. Sarkar S, Mandal C, Sangwan R, Mandal C. Coupling G2/M arrest to the Wnt/β-catenin pathway restrains pancreatic adenocarcinoma. Endocr Relat Cancer. 2014;21:113–25.

    CAS  PubMed  Article  Google Scholar 

  29. Thakur B, Ray P. Cisplatin triggers cancer stem cell enrichment in platinum-resistant cells through NF-kappaB-TNFalpha-PIK3CA loop. J Exp Clin Cancer Res. 2017;36:164.

    PubMed  PubMed Central  Article  Google Scholar 

  30. Phi LTH, Sari IN, Yang YG, Lee SH, Jun N, Kim KS, et al. Cancer stem cells (CSCs) in drug resistance and their therapeutic implications in cancer treatment. Stem Cells Int. 2018;2018:5416923.

    PubMed  PubMed Central  Article  Google Scholar 

  31. Toledo-Guzman ME, Hernandez MI, Gomez-Gallegos AA, Ortiz-Sanchez E. ALDH as a stem cell marker in solid tumors. Curr Stem Cell Res Ther. 2019;14:375–88.

    PubMed  Article  Google Scholar 

  32. Clark DW, Palle K. Aldehyde dehydrogenases in cancer stem cells: potential as therapeutic targets. Ann Transl Med. 2016;4:518.

    PubMed  PubMed Central  Article  Google Scholar 

  33. Mori Y, Yamawaki K, Ishiguro T, Yoshihara K, Ueda H, Sato A, et al. ALDH-dependent glycolytic activation mediates stemness and paclitaxel resistance in patient-derived spheroid models of uterine endometrial cancer. Stem Cell Rep. 2019;13:730–46.

    CAS  Article  Google Scholar 

  34. Li Z. CD133: a stem cell biomarker and beyond. Exp Hematol Oncol. 2013;2:17.

    PubMed  PubMed Central  Article  Google Scholar 

  35. Zhang J, Gao Q, Zhou Y, Dier U, Hempel N, Hochwald SN. Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis. Oncogene. 2016;35:1926–42.

    CAS  PubMed  Article  Google Scholar 

  36. News in brief. FAK inhibitor kills mesothelioma cells. Cancer Discov. 2014;4:OF3. https://doi.org/10.1158/2159-8290.CD-NB2014-093.

  37. Wang L, Liu X, Ren Y, Zhang J, Chen J, Zhou W, et al. Cisplatin-enriching cancer stem cells confer multidrug resistance in non-small cell lung cancer via enhancing TRIB1/HDAC activity. Cell Death Dis. 2017;8:e2746–e2746.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Visvader JE, Lindeman GJ. Cancer stem cells: current status and evolving complexities. Cell Stem Cell. 2012;10:717–28.

    CAS  PubMed  Article  Google Scholar 

  39. Ciavardelli D, Rossi C, Barcaroli D, Volpe S, Consalvo A, Zucchelli M, et al. Breast cancer stem cells rely on fermentative glycolysis and are sensitive to 2-deoxyglucose treatment. Cell Death Dis. 2014;5:e1336–e1336.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Liao J, Qian F, Tchabo N, Mhawech-Fauceglia P, Beck A, Qian Z, et al. Ovarian cancer spheroid cells with stem cell-like properties contribute to tumor generation, metastasis and chemotherapy resistance through hypoxia-resistant metabolism. PLoS ONE. 2014;9:e84941.

    PubMed  PubMed Central  Article  Google Scholar 

  41. Zhou Y, Zhou Y, Shingu T, Feng L, Chen Z, Ogasawara M, et al. Metabolic alterations in highly tumorigenic glioblastoma cells: preference for hypoxia and high dependency on glycolysis. J Biol Chem. 2011;286:32843–53.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Shen Y-A, Wang C-Y, Hsieh Y-T, Chen Y-J, Wei Y-H. Metabolic reprogramming orchestrates cancer stem cell properties in nasopharyngeal carcinoma. Cell Cycle. 2015;14:86–98.

    PubMed  Article  Google Scholar 

  43. Chen CL, Uthaya Kumar DB, Punj V, Xu J, Sher L, Tahara SM, et al. NANOG metabolically reprograms tumor-initiating stem-like cells through tumorigenic changes in oxidative phosphorylation and fatty acid metabolism. Cell Metab. 2016;23:206–19.

    CAS  PubMed  Article  Google Scholar 

  44. Kondoh H, Lleonart ME, Gil J, Wang J, Degan P, Peters G, et al. Glycolytic enzymes can modulate cellular life span. Cancer Res. 2005;65:177–85.

    CAS  PubMed  Article  Google Scholar 

  45. Nam K, Oh S, Shin I. Ablation of CD44 induces glycolysis-to-oxidative phosphorylation transition via modulation of the c-Src-Akt-LKB1-AMPKalpha pathway. Biochem J. 2016;473:3013–30.

    CAS  PubMed  Article  Google Scholar 

  46. Tamada M, Nagano O, Tateyama S, Ohmura M, Yae T, Ishimoto T, et al. Modulation of glucose metabolism by CD44 contributes to antioxidant status and drug resistance in cancer cells. Cancer Res. 2012;72:1438–48.

    CAS  PubMed  Article  Google Scholar 

  47. Schober M, Fuchs E. Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-beta and integrin/focal adhesion kinase (FAK) signaling. Proc Natl Acad Sci USA. 2011;108:10544–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Laszlo V, Valko Z, Ozsvar J, Kovacs I, Garay T, Hoda MA, et al. The FAK inhibitor BI 853520 inhibits spheroid formation and orthotopic tumor growth in malignant pleural mesothelioma. J Mol Med. 2019;97:231–42.

    CAS  PubMed  Article  Google Scholar 

  49. Pachter JA, Kolev VN, Schunselaar L, Shapiro IM, Bueno R, Baas P, et al. Abstract 4236: FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells, which are enriched by standard of care chemotherapy. Cancer Res. 2015;75:4236–4236.

    Article  Google Scholar 

  50. Kolev VN, Tam WF, Wright QG, McDermott SP, Vidal CM, Shapiro IM, et al. Inhibition of FAK kinase activity preferentially targets cancer stem cells. Oncotarget. 2017;8:51733–47.

    PubMed  PubMed Central  Article  Google Scholar 

  51. Zhang J, Gao Q, Zhou Y, Dier U, Hempel N, Hochwald SN. Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis. Oncogene. 2016;35:1926–42.

    CAS  PubMed  Article  Google Scholar 

  52. Zhu W, Ye L, Zhang J, Yu P, Wang H, Ye Z, et al. PFK15, a small molecule inhibitor of PFKFB3, induces cell cycle arrest, apoptosis and inhibits invasion in gastric cancer. PLoS ONE. 2016;11:e0163768.

    PubMed  PubMed Central  Article  Google Scholar 

  53. Banerjee K, Keasey MP, Razskazovskiy V, Visavadiya NP, Jia C, Hagg T. Reduced FAK-Stat3 signaling contributes to ER stress-induced mitochondrial dysfunction and death in endothelial cells. Cell Signal. 2017;36:154–62.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Visavadiya NP, Keasey MP, Razskazovskiy V, Banerjee K, Jia C, Lovins C, et al. Integrin-FAK signaling rapidly and potently promotes mitochondrial function through Stat3. Cell Commun Signal. 2016;14:32.

    PubMed  PubMed Central  Article  Google Scholar 

  55. Xiao F, Connolly DC. Abstract 2095: FAK mediates Stat3 activation, migration and invasion in ovarian carcinoma cells. Cancer Res. 2014;74:2095–2095.

    Article  Google Scholar 

  56. Thakur R, Trivedi R, Rastogi N, Singh M, Mishra DP. Inhibition of Stat3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer. Sci Rep. 2015;5:10194.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. Lin L, Fuchs J, Li C, Olson V, Bekaii-Saab T, Lin J. Stat3 signaling pathway is necessary for cell survival and tumorsphere forming capacity in ALDH+/CD133+ stem cell-like human colon cancer cells. Biochem Biophys Res Commun. 2011;416:246–51.

    CAS  PubMed  Article  Google Scholar 

  58. Won C, Kim BH, Yi EH, Choi KJ, Kim EK, Jeong JM, et al. Signal transducer and activator of transcription 3-mediated CD133 up-regulation contributes to promotion of hepatocellular carcinoma. Hepatology. 2015;62:1160–73.

    CAS  PubMed  Article  Google Scholar 

  59. Garg N, Bakhshinyan D, Venugopal C, Mahendram S, Rosa DA, Vijayakumar T, et al. CD133+ brain tumor-initiating cells are dependent on Stat3 signaling to drive medulloblastoma recurrence. Oncogene. 2017;36:606–17.

    CAS  PubMed  Article  Google Scholar 

  60. Wei Z, Jiang X, Qiao H, Zhai B, Zhang L, Zhang Q, et al. Stat3 interacts with Skp2/p27/p21 pathway to regulate the motility and invasion of gastric cancer cells. Cell Signal. 2013;25:931–8.

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

We sincerely acknowledge Dr. Tobias Peikert, Mayo Clinic, Rochester, MN for providing the EMMeso cell line. Our acknowledgement towards the personnel of Microscopy and Cell Analysis Core and Pathology Research Cores Mayo Clinic, Rochester, MN.

Funding

This work is supported (in part) by the Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN (JM and VS) and a generous gift from Samuel and Ilda Conde to JM—Mayo Clinic, Rochester.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualisation: SSB and VS; formal analysis: SSB; funding acquisition: VS and JRM; investigation: SSB and VS; methodology: SSB and PT; project administration: VS; supervision: JRM and VS; writing—original draft: SSB; writing—review and editing: SSB, PT, JS and VS.

Corresponding authors

Correspondence to Viji Shridhar or Julian R. Molina.

Ethics declarations

Ethics approval and consent to participate

This work was approved by the Mayo Clinic Institutional Review Board (IRB).

Consent to publish

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sarkar Bhattacharya, S., Thirusangu, P., Jin, L. et al. PFKFB3 works on the FAK-STAT3-SOX2 axis to regulate the stemness in MPM. Br J Cancer 127, 1352–1364 (2022). https://doi.org/10.1038/s41416-022-01867-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41416-022-01867-7

Search

Quick links