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Molecular profiling of anastatic cancer cells: potential role of the nuclear export pathway

Cellular Oncology volume 42, pages 645–661 (2019)Cite this article

Abstract

Purpose

Anastasis is newly discovered process by which cells recover from late-stage apoptosis upon removal of a death stimulus. Recent reports suggest that cells may recover, even after the initiation of mitochondrial outer-membrane permeabilization (MOMP) and caspase activation. Here, we specifically studied the reversibility of late-stage apoptosis in cervical (HeLa) and breast (MDA-MB-231) cancer cells in relation to the extent of MOMP (limited or widespread). In addition, we explored the molecular factors involved in the anastatic process.

Methods

The extent of MOMP was assessed using time lapse confocal microscopic imaging, considering mitochondrial cytochrome c-GFP release as a marker for MOMP. Anastatic cells were generated by specifically recovering late-stage apoptotic (annexin V/PI positive) cervical and breast cancer cells. Molecular signaling events involved in death reversal were assessed using LC-MS/MS and qRT-PCR. Targeted chemical inhibition and shRNA-based gene silencing studies were employed to explore the role of the nuclear export pathway in anastasis and increased oncogenicity.

Results

Time-lapse imaging of drug-treated Cyt-c-GFP expressing cancer cells revealed cell recovery despite widespread MOMP. A few recovered anastatic cells were noted and these were found to proliferate through a selection-type of survival. They showed increased drug-resistance, migration and invasive potential compared to non-anastatic cancer cells. Network analysis using 49 proteins uniquely expressed in anastatic cells indicated upregulation of nuclear export/import, redox and Ras signaling pathways in both HeLa and MDA-MB-231 anastatic cells, indicating common molecular mechanisms in different cell types. Inhibition of XPO1 significantly reduced the recovery of apoptotic cells and abrogated acquired oncogenic transformation in the anastatic cancer cells.

Conclusions

Our study indicates that cancer cells can revert from apoptosis even after the induction of widespread MOMP. We noted a significant role of the nuclear-export pathway in the anastatic process of cancer cells. Inhibition of anastasis through the nuclear export pathway may be a potential therapeutic strategy for targeting drug-resistance, metastasis and recurrence problems during cancer treatment.

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Abbreviations

AKR1C1–3:

Aldo-Keto Reductase Family 1 Member C1–3

SH3BGRL3:

SH3 domain-Binding Glutamic acid-Rich-Like protein 3

TXNRD1:

Thioredoxin Reductase 1

ALDH1A3:

Aldehyde Dehydrogenase 1 Family Member A3

XPO1:

Exportin 1

NUTF2:

Nuclear Transport Factor 2

RANBP:

RAN Binding Protein 1

RAC2:

Ras-Related C3 Botulinum Toxin Substrate 2

RAC3:

Ras-Related C3 Botulinum Toxin Substrate 3

PIK3R1:

Phosphoinositide-3-Kinase Regulatory Subunit 1

PTX:

Paclitaxel

FACS:

Fluorescence Activated Cell Sorting

MOMP:

Mitochondrial Outer-Membrane Permeabilization

LC-MS:

Liquid Chromatography–Mass Spectrometry

EGFP:

Enhanced Green Fluorescent Protein

AIF:

Apoptosis inducing factor

ENDO G:

Endonuclease G

tBid:

Truncated BH3 domain-only death agonist protein

References

  1. A.R. Delbridge, L.J. Valente, A. Strasser, The role of the apoptotic machinery in tumor suppression. Cold Spring Harb. Perspect. Biol. 4, a008789 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. S. Baig, I. Seevasant, J. Mohamad, A. Mukheem, H.Z. Huri, T. Kamarul, Potential of apoptotic pathway-targeted cancer therapeutic research: Where do we stand? Cell Death Dis. 7, e2058 (2017)

    Article  Google Scholar 

  3. S. Gupta, G.E. Kass, E. Szegezdi, B. Joseph, The mitochondrial death pathway: A promising therapeutic target in diseases. J. Cell. Mol. Med. 13, 1004–1033 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. S. Elmore, Apoptosis: A review of programmed cell death. Toxicol. Pathol. 35, 495–516 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. R. Khosravi-Far, Death receptor signals to the mitochondria. Canc Biol Ther 3, 1051–1057 (2004)

    Article  CAS  Google Scholar 

  6. H.L. Tang, H.M. Tang, K.H. Mak, S. Hu, S.S. Wang, K. M. Wong, C. S. Wong, H.Y. Wu, H.T. Law, K. Liu, C.C. Talbo Jr, W.K. Lau, D. J. Montell, M.C. Fung, Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response. Mol. Biol. Cell 23, 2240–2252 (2012)

  7. X. Liu, Y. He, F. Li, Q. Huang, T.A. Kato, R.P. Hall, C.Y. Li, Caspase-3 promotes genetic instability and carcinogenesis. Mol. Cell 58, 284–296 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. A.X. Ding, G. Sun, Y.G. Argaw, J.O. Wong, S. Easwaran, D.J. Montell, CasExpress reveals widespread and diverse patterns of cell survival of caspase-3 activation during development in vivo. Elife 5, e10936 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. G. Ichim, J. Lopez, S.U. Ahmed, N. Muthalagu, E. Giampazolias, M.E. Delgado, M. Haller, J.S. Riley, S.M. Mason, D. Athineos, M.J. Parsons, B. van de Kooij, L. Bouchier-Hayes, A.J. Chalmers, R.W. Rooswinkel, A. Oberst, K. Blyth, M. Rehm, D.J. Murphy, S.W.G. Tait, Limited mitochondrial permeabilization causes DNA damage and genomic instability in the absence of cell death. Mol. Cell 57, 860–872 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. B.A. Weaver, How Taxol/paclitaxel kills cancer cells. Mol. Biol. Cell 25, 2677–2681 (2014)

    Article  PubMed  PubMed Central  Google Scholar 

  11. A. Montecucco, F. Zanetta, G. Biamonti, Molecular mechanisms of etoposide. EXCLI J. 14, 95–108 (2015)

    PubMed  PubMed Central  Google Scholar 

  12. I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutellingsperger, A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J. Immunol. Methods 184, 39–51 (1995)

    Article  CAS  PubMed  Google Scholar 

  13. A.L. Hong, Y.Y. Tseng, G.S. Cowley, O. Jonas, J.H. Cheah, B.D. Kynnap, M.B. Doshi, C. Oh, S.C. Meyer, A.J. Church, S. Gill, C.M. Bielski, P. Keskula, A. Imamovic, S. Howell, G.V. Kryukov, P.A. Clemons, A. Tsherniak, F. Vazquez, B.D. Crompton, A.F. Shamji, C. Rodriguez-Galindo, K.A. Janeway, C.W.M. Roberts, K. Stegmaier, P. van Hummelen, M.J. Cima, R.S. Langer, L.A. Garraway, S.L. Schreiber, D.E. Root, W.C. Hahn, J.S. Boehm, Integrated genetic and pharmacologic interrogation of rare cancers. Nat. Commun. 7, 11987 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. M.R. Dalman, A. Deeter, G. Nimishakavi, Z.H. Duan, Fold change and p-value cutoffs significantly alter microarray interpretations. BMC Bioinformatics 13, S11 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  15. K. Raza, A. Mishra, A novel anticlustering filtering algorithm for the prediction of genes as a drug target. Am J Biomed Eng 2, 206–211 (2012)

    Article  Google Scholar 

  16. P.D. Thomas, M.J. Campbell, A. Kejariwal, H. Mi, B. Karlak, R. Daverman, K. Diemer, A. Muruganujan, A. Narechania, PANTHER: A library of protein families and subfamilies indexed by function. Genome Res. 13, 2129–2141 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. D.W. Huang, B.T. Sherman, R.A. Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44 (2008)

    Article  CAS  Google Scholar 

  18. L.J. Jensen, M. Kuhn, M. Stark, S. Chaffron, C. Creevey, J. Muller, T. Doerks, P. Julien, A. Roth, M. Simonovic, P. Bork, STRING 8-a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res. 37(suppl_1), D412–D416 (2008)

    PubMed  PubMed Central  Google Scholar 

  19. M. Seervi, J. Joseph, P.K. Sobhan, B.C. Bhavya, T.R. Santhoshkumar, Essential requirement of cytochrome c release for caspase activation by procaspase-activating compound defined by cellular models. Cell Death Dis. 2, e207 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. J.C. Goldstein, C. Munoz-Pinedo, J.E. Ricci, S.R. Adams, A. Kelekar, M. Schuler, R.Y. Tsien, D.R. Green, Cytochrome c is released in a single step during apoptosis. Cell Death Differ. 12, 453–462 (2005)

    Article  CAS  PubMed  Google Scholar 

  21. N.V. Krakhmal, M.V. Zavyalova, E.V. Denisov, S.V. Vtorushin, V.M. Perelmuter, Cancer invasion: Patterns and mechanisms. Acta Nat. 7, 17–28 (2015)

    Article  CAS  Google Scholar 

  22. T.M. Penning, J.E. Drury, Human aldo-keto reductases: Function, gene regulation, and single nucleotide polymorphisms. Arch. Biochem. Biophys. 464, 241–250 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. C.Y. Chiang, C.C. Pan, H.Y. Chang, M.D. Lai, T.S. Tzai, Y.S. Tsai, P. Ling, H.S. Liu, B.F. Lee, H.L. Cheng, C.L. Ho, S.H. Chen, N.H. Chow, SH3BGRL3 protein as a potential prognostic biomarker for urothelial carcinoma: A novel binding partner of epidermal growth factor receptor. Clin. Cancer Res. 21, 5601–5611 (2015)

    Article  CAS  PubMed  Google Scholar 

  24. E.S. Arnér, A. Holmgren, Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267, 6102–6109 (2000)

    Article  PubMed  Google Scholar 

  25. J.J. Duan, J. Cai, Y.F. Guo, X.W. Bian, S.C. Yu, ALDH1A3, a metabolic target for cancer diagnosis and therapy. Int. J. Cancer 139, 965–975 (2016)

    Article  CAS  PubMed  Google Scholar 

  26. M. Fukuda, S. Asano, T. Nakamura, M. Adachi, M. Yoshida, M. Yanagida, E. Nishida, CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308–311 (1997)

    Article  CAS  PubMed  Google Scholar 

  27. K. Van Impe, T. Hubert, V. De Corte, B. Vanloo, C. Boucherie, J. Vandekerckhove, J. Gettemans, A new role for nuclear transport factor 2 and ran: Nuclear import of CapG. Traffic 9, 695–707 (2008)

    Article  CAS  PubMed  Google Scholar 

  28. C.C. Campa, E. Ciraolo, A. Ghigo, G. Germena, E. Hirsch, Crossroads of PI3K and Rac pathways. Small GTPases 6, 71–80 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. M. Garg, D. Kanojia, A. Mayakonda, T.S. Ganesan, B. Sadhanandhan, S. Suresh, R.P. Nagare, J.W. Said, N.B. Doan, L.W. Ding, E. Baloglu, S. Shacham, M. Kauffman, H.P. Koeffler, Selinexor (KPT-330) has antitumor activity against anaplastic thyroid carcinoma in vitro and in vivo and enhances sensitivity to doxorubicin. Sci. Rep. 7(9749), 9749 (2017)

    Article  PubMed  PubMed Central  Google Scholar 

  30. J. Yang, M.A. Bill, G.S. Young, K. La Perle, Y. Landesman, S. Shacham, M. Kauffman, W. Senapedis, T. Kashyap, J.R. Saint-Martin, K. Kendra, G.B. Lesinski, Novel small molecule XPO1/CRM1 inhibitors induce nuclear accumulation of TP53, phosphorylated MAPK and apoptosis in human melanoma cells. PLoS One 9, e102983 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. S.W. Tait, D.R. Green, Mitochondrial regulation of cell death. Cold Spring Harb. Perspect. Biol. 5, a008706 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. G. Sun, E. Guzman, V. Balasanyan, C.M. Conner, K. Wong, H.R. Zhou, K.S. Kosik, D.J. Montell, A molecular signature for anastasis, recovery from the brink of apoptotic cell death. J. Cell Biol. 216, 3355–3368 (2017)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. S. Asthana, S. Chauhan, S. Labani, Breast and cervical cancer risk in India: An update. Indian J. Public Health 58, 5 (2014)

    Article  PubMed  Google Scholar 

  34. X. Gào, B. Schöttker, Reduction–oxidation pathways involved in cancer development: A systematic review of literature reviews. Oncotarget 8(51888) (2017)

  35. A. Fernández-Medarde, E. Santos, Ras in cancer and developmental diseases. Genes & Cancer 2, 344–358 (2011)

    Article  CAS  Google Scholar 

  36. M. El-Tanani, E.H. Dakir, B. Raynor, R. Morgan, Mechanisms of nuclear export in cancer and resistance to chemotherapy. Cancers 8, E 35 (2016)

  37. G.L. Gravina, W. Senapedis, D. McCauley, E. Baloglu, S. Shacham, C. Festuccia, Nucleo-cytoplasmic transport as a therapeutic target of cancer. J. Hematol. Oncol. 7(85), 85 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Y.M. Chook, K.E. Süel, Nuclear import by karyopherin-βs: Recognition and inhibition. Biochim. Biophys. Acta 1813, 1593–1606 (2011)

    Article  CAS  PubMed  Google Scholar 

  39. S.C. Mutka, W.Q. Yang, S.D. Dong, S.L. Ward, D.A. Craig, P.B. Timmermans, S. Murli, Identification of nuclear export inhibitors with potent anticancer activity in vivo. Cancer Res. 69, 510–517 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Y. Chen, S.C. Camacho, T.R. Silvers, A.R. Razak, N.Y. Gabrail, J.F. Gerecitano, E. Kalir, E. Pereira, B.R. Evans, S.J. Ramus, F. Huang, N. Priedigkeit, E. Rordriguez, M. Donovan, F. Khan, T. Kalir, R. Sebra, A. Uzilov, R. Chen, R. Sinha, R. Hm, J.N. Alpert, S. Billaud, D. Shacham, Y. McCauley, T. Landesman, M. Rashal, M.R. Kauffman, M. mirza, P. Mau-Sørensen, J. Dottino, A. Martignetti, Inhibition of the nuclear export receptor XPO1 as a therapeutic target for platinum-resistant ovarian cancer. Clin. Cancer Res. 23, 1552–1563 (2017)

    Article  CAS  PubMed  Google Scholar 

  41. A.S. Azmi, Unveiling the role of nuclear transport in epithelial-to-mesenchymal transition. Curr. Cancer Drug Targets 13, 906–914 (2013)

    Article  CAS  PubMed  Google Scholar 

  42. Y. Xu, C. So, H.M. Lam, M.C. Fung, S.Y. Tsang, Apoptosis reversal promotes Cancer stem cell-like cell formation. Neoplasia 20, 295–303 (2018)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. C. Ginestier, M.H. Hur, E. Charafe-Jauffre, F. Monville, J. Dutcher, M. Brown, J. Jacquemier, P. Viens, C.G. Kleer, S. Liu, A. Schott, D. Hayes, D. Birnbaum, M.S. Wicha, G. Dontu, ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The corresponding author (Seervi M.) acknowledges financial support from a DST-SERB (YSS/2015/000755) Young Scientist Research Grant and the DBT-PU-IPLS program (BT/PR4577/INF/22/149/2012) by the Department of Science and Technology and the Department of Biotechnology, Government of India. The authors acknowledge Dr. Abdul Jaleel and Mr. Arun Surendran, Proteomic Facility Cell, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, for providing LC-MS/MS services. Dr. Santhosh Kumar T. R. acknowledges financial support from the Department of Science and Technology (NFDDDT VI D&P/535/2015-16/TDT).

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Authors and Affiliations

  1. DBT-PU-IPLS, Department of Botany/Biotechnology, Patna University, Patna, Bihar, India

    Mahendra Seervi & Abhay K. Sharma

  2. Department of Biological Sciences, P. D. Patel Institute of Applied Sciences, Charotar University of Science and Technology (CHARUSAT), Changa, Anand, Gujarat, India

    Mahendra Seervi

  3. Cardiovascular Diseases and Diabetes Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India

    S. Sumi

  4. Cancer Research Division 1, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India

    Aneesh Chandrasekharan & T. R. SanthoshKumar

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  1. Mahendra Seervi
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  2. S. Sumi
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Contributions

The experimental conception, design and manuscript preparation were performed by MS. Experiments and analyses were carried out by MS, AC, AKS, SS and TRSK. The proteomic data was analyzed by SS. AC and TRSK performed time-lapse confocal microscopic imaging. TRSK provided Cytochrome c-GFP expressing cells.

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Correspondence to Mahendra Seervi.

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Electronic supplementary material

Supplementary Video 1

Reversal of apoptosis after widespread MOMP (MP4 15,909 kb)

Supplementary Video 2

Time-lapse images demonstrating recovery of PI positive HeLa cells after removal of drug. Briefly, HeLa cells were treated with Etoposide (50 μM, for 48 h) and cells were stained with 1 mg/ml Propidium iodide (PI). After staining, medium was removed and replaced with fresh medium and imaging performed under fluorescent microscope. The cells shown with arrow marks are showing PI positive cells which are able to recover and proliferate (AVI 84824 kb)

Supplementary Video 3

Simultaneous tracking of the recovery of PI positive and Cyt-c-GFP expressing HeLa cells. HeLa Cyt-c-GFP cells treated with Etoposide were used for demonstrating anastatic process as explained in Materials and methods. PI positive cell with Cyt-c-GFP release regained its morphology and granular pattern of Cyt-c-GFP (mitochondrial localization) upon removal of apoptotic stimuli and replacement with fresh media (AVI 426183 kb)

Supplementary Table 1

List of primers used for quantitative real time PCR (PDF 61 kb)

Supplementary Table 2

Uniquely expressed proteins in anastatic (recovered Annexin V/PI positive) HeLa cells (PDF 100 kb)

Supplementary Table 3

Genes upregulated (excluding unique proteins) in anastatic HeLa cells compared to Control, apoptotic and sorted Annexin V/PI negative cells (PDF 8 kb)

Supplementary Table 4

Genes downregulated in anastatic HeLa cells compared to Control, apoptotic and sorted annexin V/PI negative cells (PDF 7 kb)

Supplementary Table 5

List of proteins detected from each cell fraction (Control, apoptotic, anastatic cells and sorted annexin V/PI negative cells) after screening for minimum 2 peptides (represented as gene symbols) (XLS 226 kb)

Supplementary Fig. 1

Tile view of confocal microscopic time-lapse live images of PTX (100 nM, 48 h) treated MDA-MB-231 Cyt-c-GFP cells after the removal of PTX to represent the recovery of cells with widespread MOMP (JPG 838 kb)

Supplementary Fig 2

Time-points images of tracking the recovery of PI positive and Cyt-c-GFP expressing HeLa cells at 0, 24, 56 and 66 h after the removal of Etoposide containing medium. The cell shown with arrow indicate PI positive HeLa Cyt-c-GFP cell which recovered from apoptosis (JPG 691 kb)

Supplementary Fig. 3a

Protein-protein interaction of upregulated (including unique) proteins in anastatic HeLa cells as predicted by STRING database. b String pathway analysis of downregulated proteins in anastatic cells. Only eight proteins were found to be significantly downregulated in anastatic HeLa cells. c Gene ontology assignment of anastatic cell specific proteins related to cellular components (JPG 661 kb)

Supplementary Fig. 4

Gene ontology assignment of anastatic cell specific proteins related to a biological processes, b molecular function, and c. protein class. Functional categories were obtained using GO annotations from PANTHER classification system (JPG 516 kb)

Supplementary Fig. 5

MTT viability assay for LMB optimum dose determination. a HeLa and anastatic HeLa cell viability is shown at different concentrations (0.25 to 16 nM) of LMB treatment for 24 h (n = 3). 1 nM LMB was selected as the non-lethal dose which does not affect viability and proliferation of control non-anastatic and anastatic cells in HeLa. b MDA-MB-231 and anastatic MDA-MB-231 cell viability is shown at different concentrations (1 to 30 nM) of LMB treatment for 24 h (n = 3). 5 nM LMB selected as the non-lethal dose which does not affect viability and proliferation of MDA-MB-231 control non-anastatic and anastatic cells (JPG 264 kb)

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Seervi, M., Sumi, S., Chandrasekharan, A. et al. Molecular profiling of anastatic cancer cells: potential role of the nuclear export pathway. Cell Oncol. 42, 645–661 (2019). https://doi.org/10.1007/s13402-019-00451-1

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Keywords

  • Anastasis
  • Apoptosis
  • MOMP
  • Proteomics
  • Nuclear export/import pathway
  • XPO1