Skip to main content

Isorhapontigenin (ISO) inhibits stem cell-like properties and invasion of bladder cancer cell by attenuating CD44 expression

Cellular and Molecular Life Sciences volume 77, pages 351–363 (2020)Cite this article

Abstract

Cancer stem cells (CSC) are highly associated with poor prognosis in cancer patients. Our previous studies report that isorhapontigenin (ISO) down-regulates SOX2-mediated cyclin D1 induction and stem-like cell properties in glioma stem-like cells. The present study revealed that ISO could inhibit stem cell-like phenotypes and invasivity of human bladder cancer (BC) by specific attenuation of expression of CD44 but not SOX-2, at both the protein transcription and degradation levels. On one hand, ISO inhibited cd44 mRNA expression through decreases in Sp1 direct binding to its promoter region-binding site, resulting in attenuation of its transcription. On the other hand, ISO also down-regulated USP28 expression, which in turn reduced CD44 protein stability. Further studies showed that ISO treatment induced miR-4295, which specific bound to 3′-UTR activity of usp28 mRNA and inhibited its translation and expression, while miR-4295 induction was mediated by increased Dicer protein to enhance miR-4295 maturation upon ISO treatment. Our results provide the first evidence that ISO has a profound inhibitory effect on human BC stem cell-like phenotypes and invasivity through the mechanisms distinct from those previously noted in glioma stem-like cells.

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

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

ActD:

Actinomycin D

AP:

Alkaline phosphatase

BAF:

Bafilomycin A1

BC:

Bladder cancer

ChIP:

Chromatin immunoprecipitation

CHX:

Cycloheximide

CSC:

Cancer stem cells

DMSO:

Dimethyl sulfoxide

DUB:

Deubiquitinating enzymes

FBS:

Fetal bovine serum

IgG:

Immunoglobulin G

ISO:

Isorhapontigenin

MIBC:

Muscle-invasive bladder cancer

miR-4295:

Hsa-microRNA-4295

ncRNA:

Non-coding RNA

NMIBC:

Non-muscle-invasive bladder cancer

RT-PCR:

Reverse transcription-polymerase chain reaction

WT:

Wide type

3′UTR:

3′-Untranslated regions

References

  1. Choi W, Czerniak B, Ochoa A, Su X, Siefker-Radtke A, Dinney C et al (2014) Intrinsic basal and luminal subtypes of muscle-invasive bladder cancer (Review). Nat Rev Urol 11(7):400–410. https://doi.org/10.1038/nrurol.2014.129

    Article  CAS  PubMed  Google Scholar 

  2. American Cancer Society (2019) Cancer facts & figures 2019. American Cancer Society, Atlanta. https://doi.org/10.3322/caac.21551

    Book  Google Scholar 

  3. Magee JA, Piskounova E, Morrison SJ (2012) Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 21(3):283–296

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Khandelwal P, Abraham SN, Apodaca G (2009) Cell biology and physiology of the uroepithelium. Am J Physiol Renal Physiol 297(6):F1477–F1501

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Chan KS, Espinosa I, Chao M, Wong D, Ailles L, Diehn M et al (2009) Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells. Proc Natl Acad Sci 106(33):14016–14021

    CAS  PubMed  Google Scholar 

  6. Britton KM, Kirby JA, Lennard TW, Meeson AP (2011) Cancer stem cells and side population cells in breast cancer and metastasis. Cancers 3(2):2106–2130

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Dou J, Gu N (2010) Emerging strategies for the identification and targeting of cancer stem cells. Tumor Biol 31(4):243–253

    Google Scholar 

  8. Deng S, Wong CKC, Lai H-C, Wong AST (2017) Ginsenoside-Rb1 targets chemotherapy-resistant ovarian cancer stem cells via simultaneous inhibition of Wnt/β-catenin signaling and epithelial-to-mesenchymal transition. Oncotarget 8(16):25897

    PubMed  Google Scholar 

  9. Huang K-S, Wang Y-H, Li R-L, Lin M (2000) Stilbene dimers from the lianas of Gnetum hainanense. Phytochemistry 54(8):875–881

    CAS  PubMed  Google Scholar 

  10. Fang Y, Yu Y, Hou Q, Zheng X, Zhang M, Zhang D et al (2012) The Chinese herb isolate isorhapontigenin induces apoptosis in human cancer cells by down-regulating overexpression of antiapoptotic protein XIAP. J Biol Chem 287(42):35234–35243. https://doi.org/10.1074/jbc.M112.389494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jiang G, Wu AD, Huang C, Gu J, Zhang L, Huang H et al (2016) Isorhapontigenin (ISO) inhibits invasive bladder cancer (BC) formation in vivo and human BC invasion in vitro by targeting STAT1/FOXO1 Axis. Cancer Prevent Res. https://doi.org/10.1158/1940-6207.capr-15-0338

    Article  Google Scholar 

  12. Zeng X, Xu Z, Gu J, Huang H, Gao G, Zhang X et al (2016) Induction of miR-137 by isorhapontigenin (ISO) directly targets Sp1 protein translation and mediates its anticancer activity both in vitro and in vivo. Mol Cancer Therapeut 15:512–522

    CAS  Google Scholar 

  13. Kimura Y, Goi T, Nakazawa T, Hirono Y, Katayama K, Urano T et al (2013) CD44variant exon 9 plays an important role in colon cancer initiating cells. Oncotarget 4(5):785

    PubMed  PubMed Central  Google Scholar 

  14. Culty M, Nguyen HA, Underhill CB (1992) The hyaluronan receptor (CD44) participates in the uptake and degradation of hyaluronan. J Cell Biol 116(4):1055–1062

    CAS  PubMed  Google Scholar 

  15. Iczkowski KA (2010) Cell adhesion molecule CD44: its functional roles in prostate cancer. Am J Transl Res 3(1):1–7

    PubMed  PubMed Central  Google Scholar 

  16. Lokeshwar B, Lokeshwar V, Block N (1995) Expression of CD44 in prostate cancer cells: association with cell proliferation and invasive potential. Anticancer Res 15(4):1191–1198

    CAS  PubMed  Google Scholar 

  17. Savani RC, Cao G, Pooler PM, Zaman A, Zhou Z, DeLisser HM (2001) Differential involvement of the hyaluronan (HA) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis. J Biol Chem 276(39):36770–36778

    CAS  PubMed  Google Scholar 

  18. McFarlane S, Coulter JA, Tibbits P, O’Grady A, McFarlane C, Montgomery N et al (2015) CD44 increases the efficiency of distant metastasis of breast cancer. Oncotarget 6(13):11465

    PubMed  PubMed Central  Google Scholar 

  19. Okamoto I, Kawano Y, Murakami D, Sasayama T, Araki N, Miki T et al (2001) Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway. J Cell Biol 155(5):755–762

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Thorne RF, Legg JW, Isacke CM (2004) The role of the CD44 transmembrane and cytoplasmic domains in co-ordinating adhesive and signalling events. J Cell Sci 117(3):373–380

    CAS  PubMed  Google Scholar 

  21. Zhu J, Huang G, Hua X, Li Y, Yan H, Che X et al (2019) CD44s is a crucial ATG7 downstream regulator for stem-like property, invasion, and lung metastasis of human bladder cancer (BC) cells. Oncogene. https://doi.org/10.1038/s41388-018-0664-7

    Article  PubMed  PubMed Central  Google Scholar 

  22. Ding J, Li J, Xue C, Wu K, Ouyang W, Zhang D et al (2006) Cyclooxygenase-2 induction by arsenite is through a nuclear factor of activated T-cell-dependent pathway and plays an antiapoptotic role in Beas-2B cells. J Biol Chem 281(34):24405–24413

    CAS  PubMed  Google Scholar 

  23. Zhang D, Li J, Zhang M, Gao G, Zuo Z, Yu Y et al (2012) The requirement of c-Jun N-terminal kinase2 in regulation of hypoxia inducing factor-1α mRNA stability. J Biol Chem JBC M112:365882

    Google Scholar 

  24. Jin H, Yu Y, Hu Y, Lu C, Li J, Gu J et al (2015) Divergent behaviors and underlying mechanisms of cell migration and invasion in non-metastatic T24 and its metastatic derivative T24T bladder cancer cell lines. Oncotarget 6(1):522

    PubMed  Google Scholar 

  25. Gildea JJ, Golden WL, Harding MA, Theodorescu D (2000) Genetic and phenotypic changes associated with the acquisition of tumorigenicity in human bladder cancer. Genes Chromosom Cancer 27(3):252–263

    CAS  PubMed  Google Scholar 

  26. Song L, Li J, Zhang D, Liu Z-G, Ye J, Zhan Q et al (2006) IKKβ programs to turn on the GADD45α–MKK4–JNK apoptotic cascade specifically via p50 NF-κB in arsenite response. J Cell Biol 175(4):607–617. https://doi.org/10.1083/jcb.200602149

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Song L, Li J, Ye J, Yu G, Ding J, Zhang D et al (2007) p85α acts as a novel signal transducer for mediation of cellular apoptotic response to UV radiation. Mol Cell Biol 27(7):2713–2731

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Huang C, Zeng X, Jiang G, Liao X, Liu C, Li J et al (2017) XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell. J Hematol Oncol 10(1):6

    PubMed  PubMed Central  Google Scholar 

  29. Song L, Gao M, Dong W, Hu M, Li J, Shi X et al (2011) p85α mediates p53 K370 acetylation by p300 and regulates its promoter-specific transactivity in the cellular UVB response. Oncogene 30(11):1360

    CAS  PubMed  Google Scholar 

  30. Jiang G, Wu AD, Huang C, Gu J, Zhang L, Huang H et al (2016) Isorhapontigenin (ISO) inhibits invasive bladder cancer (BC) formation in vivo and human BC invasion in vitro by targeting STAT1/FOXO1 Axis. Cancer Prevent Res Canprevres 0338:2015

    Google Scholar 

  31. Wang X, Robbins J (2014) Proteasomal and lysosomal protein degradation and heart disease. J Mol Cell Cardiol 71:16–24

    CAS  PubMed  Google Scholar 

  32. Kim DH, Sætrom P, Snøve O, Rossi JJ (2008) MicroRNA-directed transcriptional gene silencing in mammalian cells. Proc Natl Acad Sci 105:16230–16235

    CAS  PubMed  Google Scholar 

  33. Winter J, Jung S, Keller S, Gregory RI, Diederichs S (2009) Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol 11(3):228

    CAS  PubMed  Google Scholar 

  34. Ha M, Kim VN (2014) Regulation of microRNA biogenesis (Review Article). Nat Rev Mol Cell Biol 15:509. https://doi.org/10.1038/nrm3838

    Article  CAS  Google Scholar 

  35. Enderling H, Hlatky L, Hahnfeldt P (2013) Cancer stem cells: a minor cancer subpopulation that redefines global cancer features. Front Oncol 3:76. https://doi.org/10.3389/fonc.2013.00076

    Article  PubMed  PubMed Central  Google Scholar 

  36. Kondo T (2007) Stem cell-like cancer cells in cancer cell lines. Cancer Biomark 3(4–5):245–250

    CAS  PubMed  Google Scholar 

  37. Ohishi T, Koga F, Migita T (2015) Bladder cancer stem-like cells: their origin and therapeutic perspectives. Int J Mol Sci 17(1):43

    PubMed Central  Google Scholar 

  38. Wang P, Gao Q, Suo Z, Munthe E, Solberg S, Ma L et al (2013) Identification and characterization of cells with cancer stem cell properties in human primary lung cancer cell lines. PLoS One 8(3):e57020–e57020. https://doi.org/10.1371/journal.pone.0057020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100(7):3983–3988. https://doi.org/10.1073/pnas.0530291100

    Article  CAS  PubMed  Google Scholar 

  40. Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P et al (2008) Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 13(2):153–166

    CAS  PubMed  Google Scholar 

  41. Dalerba P, Dylla SJ, Park I-K, Liu R, Wang X, Cho RW et al (2007) Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci 104(24):10158–10163

    CAS  PubMed  Google Scholar 

  42. Wu K, Ning Z, Zeng J, Fan J, Zhou J, Zhang T et al (2013) Silibinin inhibits β-catenin/ZEB1 signaling and suppresses bladder cancer metastasis via dual-blocking epithelial–mesenchymal transition and stemness. Cell Signal 25(12):2625–2633

    CAS  PubMed  Google Scholar 

  43. Xu Z, Zeng X, Xu J, Xu D, Li J, Jin H et al (2015) Isorhapontigenin suppresses growth of patient-derived glioblastoma spheres through regulating miR-145/SOX2/cyclin D1 axis. Neuro-oncology 18(6):830–839

    PubMed  PubMed Central  Google Scholar 

  44. Hannon GJ (2002) RNA interference. Nature 418(6894):244

    CAS  PubMed  Google Scholar 

  45. Jafarnejad SM, Ardekani GS, Ghaffari M, Martinka M, Li G (2013) Sox4-mediated Dicer expression is critical for suppression of melanoma cell invasion. Oncogene 32(17):2131

    CAS  PubMed  Google Scholar 

  46. Tokumaru S, Suzuki M, Yamada H, Nagino M, Takahashi T (2008) let-7 regulates Dicer expression and constitutes a negative feedback loop. Carcinogenesis 29(11):2073–2077

    CAS  PubMed  Google Scholar 

  47. Rigbolt KT, Prokhorova TA, Akimov V, Henningsen J, Johansen PT, Kratchmarova I et al (2011) System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation. Sci Signal 4(164):rs3

    PubMed  Google Scholar 

  48. Gross TJ, Powers LS, Boudreau RL, Brink B, Reisetter A, Goel K et al (2014) A microRNA processing defect in smokers’ macrophages is linked to SUMOylation of the endonuclease DICER. J Biol Chem 289(18):12823–12834. https://doi.org/10.1074/jbc.M114.565473

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Nan Y-H, Wang J, Wang Y, Sun P-H, Han Y-P, Fan L et al (2016) MiR-4295 promotes cell growth in bladder cancer by targeting BTG1. Am J Transl Res 8(11):4892

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Jin H, Xu J, Guo X, Huang H, Li J, Peng M et al (2016) XIAP RING domain mediates miR-4295 expression and subsequently inhibiting p63α protein translation and promoting transformation of bladder epithelial cells. Oncotarget 7(35):56540

    PubMed  PubMed Central  Google Scholar 

  51. Zhang L, Xu B, Qiang Y, Huang H, Wang C, Li D et al (2015) Overexpression of deubiquitinating enzyme USP 28 promoted non-small cell lung cancer growth. J Cell Mol Med 19(4):799–805

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhen Y, Knobel PA, Stracker TH, Reverter D (2014) Regulation of USP28 de-ubiquitinating activity by SUMO conjugation. J Biol Chem JBC M114:601849

    Google Scholar 

  53. Valero R, Bayés M, Francisca Sánchez-Font M, González-Angulo O, Gonzàlez-Duarte R, Marfany G (2001) Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25. Genome Biol 2(10):research0043.1. https://doi.org/10.1186/gb-2001-2-10-research0043

    Article  Google Scholar 

  54. Lim K-H, Baek K-H (2013) Deubiquitinating enzymes as therapeutic targets in cancer. Curr Pharm Des 19(22):4039–4052

    CAS  PubMed  Google Scholar 

  55. Nicholson B, Marblestone JG, Butt TR, Mattern MR (2007) Deubiquitinating enzymes as novel anticancer targets. Fut Oncol (Lond Engl) 3(2):191–199. https://doi.org/10.2217/14796694.3.2.191

    Article  CAS  Google Scholar 

  56. Wang Z, Song Q, Xue J, Zhao Y, Qin S (2016) Ubiquitin-specific protease 28 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity by regulating MYC expression. Exper Biol Med 241(3):255–264

    CAS  Google Scholar 

  57. Guo G, Xu Y, Gong M, Cao Y, An R (2014) USP28 is a potential prognostic marker for bladder cancer. Tumor Biol 35(5):4017–4022

    CAS  Google Scholar 

Download references

Funding

This work was partially supported by Grants from NIH/NCI CA177665, CA217923, CA229234, CA165980, and NIH/NIEHS ES000260.

Author information

Author notes

  1. Yisi Luo and Zhongxian Tian contributed equally to this work.

Authors and Affiliations

  1. Nelson Institute and Department of Environmental Medicine, New York University School of Medicine, 341 East 25th Street, New York, NY, 10100, USA

    Yisi Luo, Zhongxian Tian, Xiaohui Hua, Maowen Huang, Jiheng Xu, Jingxia Li, Haishan Huang, Mitchell Cohen & Chuanshu Huang

Authors
  1. Yisi Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhongxian Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaohui Hua
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maowen Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiheng Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jingxia Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Haishan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mitchell Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chuanshu Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chuanshu Huang.

Additional information

Publisher's Note

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

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig S1.

T24T(vector) and T24T(CD44) cells were treated with ISO or DMSO at indicated concentrations for 7 days to determine impact on sphere formation ability (JPEG 650 kb)

Fig S2.

(A) T24T cells were treated with Bafomycin A1 (5 nM) or ISO (20 μM) alone or Bafomycin A1+ ISO, which tested whether CD44 protein accumulated by Bafomycin A1. (B) Cell sphere formation by cells transfected with Flag-USP28 or vector. Transfectants were exposed to 20 μM ISO for 7 days before analyses using microscopy (JPEG 778 kb)

Fig S3.

(A) Schematic representation of transcription factor binding sites in human CD44 promoter region from -1133 to -1. (B & C) FOXO1 or c-MYC did not affect CD44 protein expression. (D) T24T(vector) and T24T(GFP-Sp1) cells were treated with/without ISO (20 μM) to determine sphere formation ability. (E) T24T(nonsense) and T24T(shSp1) cells were used in sphere formation assay as described in Methods (JPEG 900 kb)

Fig S4.

(A) usp28 mRNA expression levels in T24T cells were determined by RT-PCR after cells were treated with 20 μM ISO for indicated time periods. (B) T24T cells were treated with/without 20 μM ISO in the presence of CHX for indicated time periods, then underwent extraction; extracts were then analyzed by Western blot to assess USP28 protein expression. (C) Schematic representation of potential miRNA-binding sites in USP28 3’UTR were analyzed with TargetScan software. (D) Schematic sequence of intact miR-4295-binding site in wide-type USP28 3’UTR and its mutation of USP28 3’UTR luciferase reporter (JPEG 912 kb)

Fig S5.

Invasivity of T24T(vector) and T24T(miR-4295 inhibitor) cells treated with or without 20 µM ISO for 24 hours was evaluated using a BD BioCoat TM Matrigel TM Invasion Chamber. After incubation for 24 hours, cells were fixed and stained and took pictures as described in Methods (JPEG 838 kb)

Fig S6.

(A) T24T(miR-4295 promoter) stable transfectants were treated with 20 μM ISO for indicated time periods; thereafter, miR-4295 promoter luciferase activity was measured (via Dual-Luciferase Reporter Assay System). (B) T24T cells treated with 20 μM ISO or vehicle control - both along with ActD - for the time periods indicated. Total RNA was isolated and subjected to qRT-PCR evaluation of miR-4295 expression levels. (C) Cell sphere formation abilities of T24T(vector) and T24T(shDicer) cells treated with or without 20 µM ISO were determined as described in Methods (JPEG 811 kb)

Fig S7.

(A) Kaplan-Meier estimation about has-mir-4295 of overall survival (OS) in bladder cancer (BC) patients from the kmplot.com database. Overall survival (OS) curves showing that patients with high miR-4295 expression (n=564) was related with better OS, compared with lower expression of miR-4295 (n=186). (B) Kaplan-Meier estimation about USP28 of overall survival (OS) in bladder cancer (BC) patients from the kmplot.com database. Overall survival (OS) curves showing that patients with high USP28 expression (n=294) were positively associated with decreased survival of BC patients, lower expression of miR-4295 (n=191) was associated with longer survival (JPEG 833 kb)

Supplementary material 8 (JPEG 712 kb)

Supplementary material 9 (JPEG 917 kb)

Supplementary material 10 (JPEG 864 kb)

Supplementary material 11 (JPEG 931 kb)

Supplementary material 12 (JPEG 847 kb)

Supplementary material 13 (JPEG 986 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Luo, Y., Tian, Z., Hua, X. et al. Isorhapontigenin (ISO) inhibits stem cell-like properties and invasion of bladder cancer cell by attenuating CD44 expression. Cell. Mol. Life Sci. 77, 351–363 (2020). https://doi.org/10.1007/s00018-019-03185-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-019-03185-3

Keywords

  • Isorhapontigenin
  • Bladder cancer
  • Stem cell-like properties
  • CD44
  • USP28