Cellular and Molecular Biology

Ascites-derived ALDH+CD44+ tumour cell subsets endow stemness, metastasis and metabolic switch via PDK4-mediated STAT3/AKT/NF-κB/IL-8 signalling in ovarian cancer

Abstract

Background

Ovarian cancer is characterised by frequent recurrence due to persistent presence of residual cancer stem cells (CSCs). Here, we identify and characterise tumour subsets from ascites-derived tumour cells with stemness, metastasis and metabolic switch properties and to delineate the involvement of pyruvate dehydrogenase kinase 4 (PDK4) in such process.

Methods

Ovarian cancer cells/cell lines derived from ascites were used for tumourspheres/ALDH+CD44+ subset isolation. The functional roles and downstream signalling of PDK4 were explored. Its association with clinical outcome of ovarian cancer was analysed.

Results

We demonstrated enhanced CSC characteristics of tumour cells derived from ovarian cancer ascites, concomitant with ALDH and CD44 subset enrichment and high PDK4 expression, compared to primary tumours. We further showed tumourspheres/ALDH+CD44+ subsets from ascites-derived tumour cells/cell lines with CSC properties and enhanced glycolysis. Clinically, PDK4 expression was correlated with aggressive features. Notably, blockade of PDK4 in tumourspheres/ALDH+CD44+ subsets led to inhibition of CSC characteristics, glycolysis and activation of STAT3/AKT/NF-κB/IL-8 (signal transducer and activator of transcription 3/protein kinases B/nuclear factor-κB/interleukin-8) signalling. Conversely, overexpression of PDK4 in ALDH−CD44– subsets exerted the opposite effects.

Conclusion

Ascites-derived ALDH+CD44+ tumour cell subsets endow stemness, metastatic and metabolic switch properties via PDK4-mediated STAT3/AKT/NF-κB/IL-8 signalling, suggesting PDK4 as a viable therapeutic molecular target for ovarian cancer management.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Ascites-derived tumour cells and tumourspheres show CSC properties and express higher levels of PDK4.
Fig. 2: ALDH+CD44+ cells derived from ovarian cancer cells shows enhanced CSC properties and PDK4 expression.
Fig. 3: PDK4 is overexpressed in ovarian cancer and correlates with metastasis and poor prognosis.
Fig. 4: PDK4 shifts the mode of energy metabolism and is crucial for ovarian CSC maintenance.
Fig. 5: DCA hinders ovarian CSC properties in vitro and in vivo.
Fig. 6: PDK4 mediates ovarian CSC characteristics through STAT3/AKT/NF-κB/IL-8 signalling.

References

  1. 1.

    Pattabiraman, D. R. & Weinberg, R. A. Tackling the cancer stem cells—what challenges do they pose? Nat. Rev. Drug Discov. 13, 497–512 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Kwon, M. J. & Shin, Y. K. Regulation of ovarian cancer stem cells or tumor-initiating cells. Int. J. Mol. Sci. 14, 6624–6648 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Lengyel, E. Ovarian cancer development and metastasis. Am. J. Pathol. 177, 1053–1064 (2010).

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Condello, S., Morgan, C. A., Nagdas, S., Cao, L., Turek, J., Hurley, T. D. et al. Beta-catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids. Oncogene 34, 2297–2308 (2015).

    CAS  PubMed  Google Scholar 

  5. 5.

    Alvero, A. B., Chen, R., Fu, H. H., Montagna, M., Schwartz, P. E., Rutherford, T. et al. Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance. Cell Cycle 8, 158–166 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Koppenol, W. H., Bounds, P. L. & Dang, C. V. Otto Warburg’s contributions to current concepts of cancer metabolism. Nat. Rev. Cancer 11, 325–337 (2011).

    CAS  PubMed  Google Scholar 

  7. 7.

    Chen, C. T., Hsu, S. H. & Wei, Y. H. Upregulation of mitochondrial function and antioxidant defense in the differentiation of stem cells. Biochim. Biophys. Acta 1800, 257–263 (2010).

    CAS  PubMed  Google Scholar 

  8. 8.

    Wong, T. L., Che, N. & Ma, S. Reprogramming of central carbon metabolism in cancer stem cells. Biochim. Biophys. Acta 1863, 1728–1738 (2017).

    CAS  Google Scholar 

  9. 9.

    Sutendra, G. & Michelakis, E. D. Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology. Front. Oncol. 3, 38 (2013).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Bonnet, S., Archer, S. L., Allalunis-Turner, J., Haromy, A., Beaulieu, C., Thompson, R. et al. A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell 11, 37–51 (2007).

    CAS  PubMed  Google Scholar 

  11. 11.

    Walter, W., Thomalla, J., Bruhn, J., Fagan, D. H., Zehowski, C., Yee, D. et al. Altered regulation of PDK4 expression promotes antiestrogen resistance in human breast cancer cells. Springerplus 4, 689 (2015).

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Leclerc, D., Pham, D. N., Levesque, N., Truongcao, M., Foulkes, W. D., Sapienza, C. et al. Oncogenic role of PDK4 in human colon cancer cells. Br. J. Cancer 116, 930–936 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Trinidad, A. G., Whalley, N., Rowlinson, R., Delpuech, O., Dudley P., Rooney C. et al. Pyruvate dehydrogenase kinase 4 exhibits a novel role in the activation of mutant KRAS, regulating cell growth in lung and colorectal tumour cells. Oncogene 36, 6164–6176 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Zhang, S., Balch, C., Chan, M. W., Lai, H. C., Matei, D., Schilder, J. M. et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res. 68, 4311–4320 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Rajendran, V. & Jain, M. V. In vitro tumorigenic assay: colony forming assay for cancer stem cells. Methods Mol. Biol. 1692, 89–95 (2018).

    CAS  PubMed  Google Scholar 

  16. 16.

    Shaw, F. L., Harrison, H., Spence, K., Ablett, M. P., Simoes, B. M., Farnie, G. et al. A detailed mammosphere assay protocol for the quantification of breast stem cell activity. J. Mammary Gland Biol. Neoplasia 17, 111–117 (2012).

    PubMed  Google Scholar 

  17. 17.

    Michelakis, E. D., Webster, L. & Mackey, J. R. Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer. Br. J. Cancer 99, 989–994 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Saed, G. M., Fletcher, N. M., Jiang, Z. L., Abu-Soud, H. M. & Diamond, M. P. Dichloroacetate induces apoptosis of epithelial ovarian cancer cells through a mechanism involving modulation of oxidative stress. Reprod. Sci. 18, 1253–1261 (2011).

    CAS  PubMed  Google Scholar 

  19. 19.

    Qin, H. R., Kim, H. J., Kim, J. Y., Hurt, E. M., Klarmann, G. J., Kawasaki, B. T. et al. Activation of signal transducer and activator of transcription 3 through a phosphomimetic serine 727 promotes prostate tumorigenesis independent of tyrosine 705 phosphorylation. Cancer Res. 68, 7736–7741 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Hazan-Halevy, I., Harris, D., Liu, Z., Liu, J., Li, P., Chen, X. et al. STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells. Blood 115, 2852–2863 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Korkaya, H., Liu, S. & Wicha, M. S. Regulation of cancer stem cells by cytokine networks: attacking cancer’s inflammatory roots. Clin. Cancer Res. 17, 6125–6129 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Bradford, J. W. & Baldwin, A. S. IKK/nuclear factor-kappaB and oncogenesis: roles in tumor-initiating cells and in the tumor microenvironment. Adv. Cancer Res. 121, 125–145 (2014).

    CAS  PubMed  Google Scholar 

  23. 23.

    Wang, S., Liu, Z., Wang, L. & Zhang, X. NF-kappaB signaling pathway, inflammation and colorectal cancer. Cell. Mol. Immunol. 6, 327–334 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Merritt, W. M., Lin, Y. G., Spannuth, W. A., Fletcher, M. S., Kamat, A. A., Han, L. Y. et al. Effect of interleukin-8 gene silencing with liposome-encapsulated small interfering RNA on ovarian cancer cell growth. J. Natl Cancer Inst. 100, 359–372 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Ginestier, C., Liu, S., Diebel, M. E., Korkaya, H., Luo, M., Brown, M. et al. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. J. Clin. Invest. 120, 485–497 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    He, M., Wang, D., Zou, D., Wang, C., Lopes-Bastos, B., Jiang, W. G. et al. Re-purposing of curcumin as an anti-metastatic agent for the treatment of epithelial ovarian cancer: in vitro model using cancer stem cell enriched ovarian cancer spheroids. Oncotarget 7, 86374–86387 (2016).

    PubMed  PubMed Central  Google Scholar 

  27. 27.

    Weiswald, L. B., Bellet, D. & Dangles-Marie, V. Spherical cancer models in tumor biology. Neoplasia 17, 1–15 (2015).

    PubMed  PubMed Central  Google Scholar 

  28. 28.

    Yilmazer, A. Evaluation of cancer stemness in breast cancer and glioblastoma spheroids in vitro. 3 Biotech 8, 390 (2018).

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Leung, H. W., Ko, C. H., Yue, G. G., Herr, I. & Lau, C. B. The natural agent 4-vinylphenol targets metastasis and stemness features in breast cancer stem-like cells. Cancer Chemother. Pharm. 82, 185–197 (2018).

    CAS  Google Scholar 

  30. 30.

    Young, M. J., Wu, Y. H., Chiu, W. T., Weng, T. Y., Huang, Y. F. & Chou, C. Y. All-trans retinoic acid downregulates ALDH1-mediated stemness and inhibits tumour formation in ovarian cancer cells. Carcinogenesis 36, 498–507 (2015).

    CAS  PubMed  Google Scholar 

  31. 31.

    Lin, J. & Ding, D. The prognostic role of the cancer stem cell marker CD44 in ovarian cancer: a meta-analysis. Cancer Cell Int. 17, 8 (2017).

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    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 9, e84941 (2014).

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Kamarajugadda, S., Stemboroski, L., Cai, Q., Simpson, N. E., Nayak, S., Tan, M. et al. Glucose oxidation modulates anoikis and tumor metastasis. Mol. Cell. Biol. 32, 1893–1907 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Liu, Z., Chen, X., Wang, Y., Peng, H., Wang, Y., Jing, Y. et al. PDK4 protein promotes tumorigenesis through activation of cAMP-response element-binding protein (CREB)-Ras homolog enriched in brain (RHEB)-mTORC1 signaling cascade. J. Biol. Chem. 289, 29739–29749 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Zhang, Y., Zhang, Y., Geng, L., Yi, H., Huo, W., Talmon, G. et al. Transforming growth factor beta mediates drug resistance by regulating the expression of pyruvate dehydrogenase kinase 4 in colorectal cancer. J. Biol. Chem. 291, 17405–17416 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Rellinger, E. J., Romain, C., Choi, S., Qiao, J. & Chung, D. H. Silencing gastrin-releasing peptide receptor suppresses key regulators of aerobic glycolysis in neuroblastoma cells. Pediatr. Blood Cancer 62, 581–586 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Lee, S. J., Jeong, J. Y., Oh, C. J., Park, S., Kim, J. Y., Kim, H. J. et al. Pyruvate dehydrogenase kinase 4 promotes vascular calcification via SMAD1/5/8 phosphorylation. Sci. Rep. 5, 16577 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    House, C. D., Jordan, E., Hernandez, L., Ozaki, M., James, J. M., Kim, M. et al. NFkappaB promotes ovarian tumorigenesis via classical pathways that support proliferative cancer cells and alternative pathways that support ALDH(+) cancer stem-like cells. Cancer Res. 77, 6927–6940 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Burgos-Ojeda, D., Wu, R., McLean, K., Chen, Y. C., Talpaz, M., Yoon, E. et al. CD24+ ovarian cancer cells are enriched for cancer-initiating cells and dependent on JAK2 signaling for growth and metastasis. Mol. Cancer Ther. 14, 1717–1727 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Egusquiaguirre, S. P., Yeh, J. E., Walker, S. R., Liu, S. & Frank, D. A. The STAT3 target gene TNFRSF1A modulates the NF-kappaB pathway in breast cancer cells. Neoplasia 20, 489–498 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    McFarland, B. C., Hong, S. W., Rajbhandari, R., Twitty, G. B. Jr., Gray, G. K., Yu, H. et al. NF-kappaB-induced IL-6 ensures STAT3 activation and tumor aggressiveness in glioblastoma. PLoS ONE 8, e78728 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Singh, J. K., Simoes, B. M., Howell, S. J., Farnie, G. & Clarke, R. B. Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells. Breast Cancer Res. 15, 210 (2013).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Velpula, K. K., Bhasin, A., Asuthkar, S. & Tsung, A. J. Combined targeting of PDK1 and EGFR triggers regression of glioblastoma by reversing the Warburg effect. Cancer Res. 73, 7277–7289 (2013).

    CAS  PubMed  Google Scholar 

  44. 44.

    Lin, G., Hill, D. K., Andrejeva, G., Boult, J. K., Troy, H., Fong, A. C. et al. Dichloroacetate induces autophagy in colorectal cancer cells and tumours. Br. J. Cancer 111, 375–385 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Shen, Y. C., Ou, D. L., Hsu, C., Lin, K. L., Chang, C. Y., Lin, C. Y. et al. Activating oxidative phosphorylation by a pyruvate dehydrogenase kinase inhibitor overcomes sorafenib resistance of hepatocellular carcinoma. Br. J. Cancer 108, 72–81 (2013).

    CAS  PubMed  Google Scholar 

  46. 46.

    Morfouace, M., Lalier, L., Bahut, M., Bonnamain, V., Naveilhan, P., Guette, C. et al. Comparison of spheroids formed by rat glioma stem cells and neural stem cells reveals differences in glucose metabolism and promising therapeutic applications. J. Biol. Chem. 287, 33664–33674 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Michelakis, E. D. S. G., Dromparis, P., Webster, L., Haromy, A. & Niven, E. Metabolic modulation of glioblastoma with dichloroacetate. Sci. Transl. Med. 2, 31ra34 (2010).

    CAS  PubMed  Google Scholar 

  48. 48.

    Sun, L., Moritake, T., Ito, K., Matsumoto, Y., Yasui, H., Nakagawa, H. et al. Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets. PLoS ONE 12, e0176162 (2017).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    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 6, 40822–40835 (2015).

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Zhang, Y., Guo, G., Ma, B., Du, R., Xiao, H., Yang, X. et al. A hybrid platinum drug dichloroacetate-platinum(II) overcomes cisplatin drug resistance through dual organelle targeting. Anticancer Drugs 26, 698–705 (2015).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Ms. Erica Yau and Dr. Emily Pang from Core Facility, for providing and maintaining the equipment needed for flow cytometry.

Author information

Affiliations

Authors

Contributions

Y.-X.J., M.K.-Y.S. and K.K.-L.C. conceived the project. Y.-X.J., M.K.-Y.S. and J.-J.W. performed the experiments. X.-T.M., T.H.-Y.L., D.W.C. and A.N.-Y.C. contributed new reagents/analytic tools; Y.-X.J., M.K.-Y.S., H.Y.-S.N. and K.K.-L.C. analysed the data. Y.-X.J. and M.K.-Y.S. wrote the manuscript. All authors provided critical revision and approved the final manuscript.

Corresponding author

Correspondence to Karen Kar-Loen Chan.

Ethics declarations

Ethics approval and consent to participate

Sample collection was under the approval of Human Research Ethics Committee of the University of Hong Kong. Consented patients were informed of the use of their clinical information before sample collection. The authors confirm that they have obtained written consent from each patient to publish the manuscript. The study was performed in accordance with the Declaration of Helsinki. Animal experiments were conducted following protocols approved by the Committee of the Use of Live Animals in Teaching and Research (CULATR) and were carried out under an approved CULATR licence (No. 4598-18).

Consent to publish

This manuscript does not contain any individual person’s data.

Data availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Funding information

The work was jointly funded by the University of Hong Kong (201711159248) and by the Hong Kong Research Grants Council General Research Fund (HKU 17101414), and the Research Fund from the Department of Obstetrics and Gynaecology.

Additional information

Note This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0).

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

Jiang, Y., Siu, M.K., Wang, J. et al. Ascites-derived ALDH+CD44+ tumour cell subsets endow stemness, metastasis and metabolic switch via PDK4-mediated STAT3/AKT/NF-κB/IL-8 signalling in ovarian cancer. Br J Cancer 123, 275–287 (2020). https://doi.org/10.1038/s41416-020-0865-z

Download citation