Tumor Biology

, Volume 37, Issue 7, pp 9731–9738 | Cite as

ING3 is associated with increased cell invasion and lethal outcome in ERG-negative prostate cancer patients

  • Amal Almami
  • Samar A. Hegazy
  • Arash Nabbi
  • Mohammed Alshalalfa
  • Asma Salman
  • Hatem Abou-Ouf
  • Karl Riabowol
  • Tarek A. Bismar
Original Article

Abstract

The inhibitor of growth family member 3 (ING3) is a member of the ING tumor suppressor family. Although its expression has been reported in various types of cancers, the role of ING3 and its prognostic value in prostate cancer (PCa) has not been investigated. ING3 expression and prognostic value was assessed in a cohort of PCa patients (n = 312) treated with transurethral resection of prostate using immumoflourescent automated quantitative analysis (AQUA) system. In vitro studies were carried out in conjunction to investigate its expression in various PCa cell lines. ING3 knockdown was also carried out in DU145 cell lines to assess for any changes in invasion and migration. ING3 expression was highest in benign prostate tissues (mean 3.2 ± 0.54) compared to PCa (mean 2.5 ± 0.26) (p = 0.437), advanced prostate cancer (AdvPCa) (mean 1.5 ± 0.32) (p = 0.004), and castration-resistant prostate cancer (CRPC) (mean 2.28 ± 0.32) (p = 0.285). ING3 expression was inversely correlated to Gleason score (p = 0.039) and ETS-related gene (ERG) expression (p = 0.019). Higher ING3 expression was marginally associated with lethal disease (p = 0.052), and this was more pronounced in patients with ERG-negative status (p = 0.018). Inhibition of ING3 in DU145 PCa cells using small interfering RNA (siRNA) was associated with decreased cell invasion (p = 0.0016) and cell migration compared to control cells. ING3 is significantly associated with PCa disease progression and cancer-specific mortality. To our knowledge, this is the first report suggesting an oncogenic function of ING3, previously well known as a tumor suppressor protein. Further studies should investigate potential-related pathways in association to ING3.

Keywords

Prostate cancer Tumor suppressor Oncogene Progression ING3 Invasion Metastasis 

Abbreviations

ING3

Inhibitor of growth member 3

CRPC

Castration-resistant prostate cancer

GS

Gleason score

PCa

Prostate cancer

ERG

ETS-related gene

PCK

Cytokeratin

DAPI

4′,6-Diamidino-2-phenylindole dihydrochloride

Notes

Acknowledgments

The authors acknowledge the Antibody Facility at University of Calgary especially Ms. Donna Boland for ING3 antibody and Mr. Brant Pohorelic for AQUA images. Amal Almami is a graduate student sponsored by King Abdullah Scholarship, Saudi Arabia, and supported by Saudi Cultural Bureau in Canada. This work was supported in part by the Prostate Cancer Foundation Young Investigator Award (T.A.B). This work was also supported by Prostate Cancer Canada and is proudly funded by the Movember Foundation-Grant # B2013-01.

Compliance with ethical standards

Conflicts of interest

None

Supplementary material

13277_2016_4802_Fig6_ESM.gif (84 kb)
Supplementary Fig. 1

Overexpression of ERG in prostate cell lines. ERG was cloned from VCaP cell lines and then stably transfected in RWPE-1, LNCaP and PC3 cell lines. Transfection was confirmed with qRT-PCR and Western blot, by comparing the transfected cells to its control. A) Relative expression of ERG mRNA levels in ERG overexpressed RWPE1-ERG (P = 0.013), LNCaP-ERG (P = 0.0005), and PC-ERG (P = 0.001) cell lines to it controls (RWPE1-GFP, LNCaP-GFP and PC3-Luc). GUSB was used as internal control. The ERG and GUSB primers that were used are shown in in Supplementary Table 1. The figure represents experiments run in triplicates (n = 3). (B) represents protein levels of ERG in RWPE-1, LNCaP and PC3, respectively, depicting overexpression of ERG compared to their controls. GAPDH were used as loading controls for Western blot. Bars indicate quantification of three separate Western blots experiments. P values < 0.05 were considered significant. (GIF 83 kb)

13277_2016_4802_MOESM1_ESM.tif (117 kb)
High resolution image (TIF 117 kb)
13277_2016_4802_Fig7_ESM.gif (48 kb)
Supplementary Fig. 2

Correlation between ING3 percentage level and Gleason score. The percentage of ING3 positive nuclear cells based on AQUA analysis in relation to Gleason score. Representative percentage of cells with ING3 expression in tissue samples with more specific Gleason Scores. GS <7 gray, GS7 (3 + 4) narrow strikes and GS7 (4 + 3) broad dark strikes. Chi-square statistical test using SPSS software and the value of P <0.05 was considered statistically significant. (GIF 47 kb)

13277_2016_4802_MOESM2_ESM.tif (147 kb)
High resolution image (TIF 146 kb)
13277_2016_4802_Fig8_ESM.gif (40 kb)
Supplementary Fig. 3

ING3 mRNA expression in prostate cell lines. Quantitative Real time PCR experiments of ING3 mRNA expression in four prostate cancer cell lines LNCaP (P = 0.00017), VCaP (P = 0.008), PC3 (P = 0.0007) and DU145 (P = 0.0003) relative to the expression of ING3 in RWPE-1 cells. GUSB was used as an internal control. The ING3 and GUSB primers that were used are shown in Supplementary Table 1. The figure represents experiments repeated three times (n = 3). (GIF 40 kb)

13277_2016_4802_MOESM3_ESM.tif (54 kb)
High resolution image (TIF 54 kb)
13277_2016_4802_Fig9_ESM.gif (15 kb)
Supplementary Fig. 4

Successful knockdown of ING3 in DU-145 cells. ING3 was knocked down in DU145 cell lines compared to DU145 cells transfected with scrambled siRNA. The mRNA levels of ING3 in DU145 cells transfected with siRNA ING3 down-regulated relative to cells transfected with control siRNA quantified by qRT-PCR. GUSB was used as an internal control. Student t-test used to calculate the P value where ** p < 0.01 was considered significant. The figure represents three times independent experiments (n = 3). ING3 and GUSB used primers are described in Supplementary Table 1. (GIF 14 kb)

13277_2016_4802_MOESM4_ESM.tif (20 kb)
High resolution image (TIF 19 kb)
13277_2016_4802_MOESM5_ESM.docx (18 kb)
Supplementary Table 1 (DOCX 18 kb)

References

  1. 1.
    Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63(1):11–30. doi: 10.3322/caac.21166.CrossRefPubMedGoogle Scholar
  2. 2.
    Society AC (2013) Prostate cancer overview American Cancer Society, Inc. http://www.cancer.org/cancer/prostatecancer/overviewguide/prostate-cancer-overview-key-statisticsGoogle Scholar
  3. 3.
    Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64(1):9–29. doi: 10.3322/caac.21208.CrossRefPubMedGoogle Scholar
  4. 4.
    He GH, Helbing CC, Wagner MJ, Sensen CW, Riabowol K. Phylogenetic analysis of the ING family of PHD finger proteins. Mol Biol Evol. 2005;22(1):104–16. doi: 10.1093/molbev/msh256.CrossRefPubMedGoogle Scholar
  5. 5.
    Gunduz M, Beder LB, Gunduz E, Nagatsuka H, Fukushima K, Pehlivan D, et al. Downregulation of ING3 mRNA expression predicts poor prognosis in head and neck cancer. Cancer Sci. 2008;99(3):531–8. doi: 10.1111/j.1349-7006.2007.00708.x.CrossRefPubMedGoogle Scholar
  6. 6.
    Wang Y, Dai DL, Martinka M, Li G. Prognostic significance of nuclear ING3 expression in human cutaneous melanoma. Clin Cancer Res. 2007;13(14):4111–6. doi: 10.1158/1078-0432.CCR-07-0408.CrossRefPubMedGoogle Scholar
  7. 7.
    Yang HY, Liu HL, Tian LT, Song RP, Song X, Yin DL, et al. Expression and prognostic value of ING3 in human primary hepatocellular carcinoma. Exper Biol Med. 2012;237(4):352–61. doi: 10.1258/ebm.2011.011346.CrossRefGoogle Scholar
  8. 8.
    Nagashima M, Shiseki M, Pedeux RM, Okamura S, Kitahama-Shiseki M, Miura K, et al. A novel PHD-finger motif protein, p47ING3, modulates p53-mediated transcription, cell cycle control, and apoptosis. Oncogene. 2003;22(3):343–50. doi: 10.1038/sj.onc.1206115.CrossRefPubMedGoogle Scholar
  9. 9.
    Gunduz M, Ouchida M, Fukushima K, Ito S, Jitsumori Y, Nakashima T, et al. Allelic loss and reduced expression of the ING3, a candidate tumor suppressor gene at 7q31, in human head and neck cancers. Oncogene. 2002;21(28):4462–70. doi: 10.1038/sj.onc.1205540.CrossRefPubMedGoogle Scholar
  10. 10.
    Lu M, Chen F, Wang Q, Wang K, Pan Q, Zhang X. Downregulation of inhibitor of growth 3 is correlated with tumorigenesis and progression of hepatocellular carcinoma. Oncol lett. 2012;4(1):47–52. doi: 10.3892/ol.2012.685.PubMedPubMedCentralGoogle Scholar
  11. 11.
    Laudet V, Hanni C, Stehelin D, Duterque-Coquillaud M. Molecular phylogeny of the ETS gene family. Oncogene. 1999;18(6):1351–9. doi: 10.1038/sj.onc.1202444.CrossRefPubMedGoogle Scholar
  12. 12.
    Ichikawa H, Shimizu K, Hayashi Y, Ohki M. An RNA-binding protein gene, TLS/FUS, is fused to ERG in human myeloid leukemia with t(16;21) chromosomal translocation. Cancer Res. 1994;54(11):2865–8.PubMedGoogle Scholar
  13. 13.
    Birdsey GM, Dryden NH, Amsellem V, Gebhardt F, Sahnan K, Haskard DO, et al. Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin. Blood. 2008;111(7):3498–506. doi: 10.1182/blood-2007-08-105346.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Demichelis F, Fall K, Perner S, Andren O, Schmidt F, Setlur SR, et al. TMPRSS2:ERG gene fusion associated with lethal prostate cancer in a watchful waiting cohort. Oncogene. 2007;26(31):4596–9. doi: 10.1038/sj.onc.1210237.CrossRefPubMedGoogle Scholar
  15. 15.
    Teng LH, Wang C, Dolph M, Donnelly B, Bismar TA. ERG protein expression is of limited prognostic value in men with localized prostate cancer. ISRN Urology. 2013;2013:786545. doi: 10.1155/2013/786545.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Nagle RB, Algotar AM, Cortez CC, Smith K, Jones C, Sathyanarayana UG, et al. ERG overexpression and PTEN status predict capsular penetration in prostate carcinoma. Prostate. 2013;73(11):1233–40. doi: 10.1002/pros.22675.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Hoogland AM, Jenster G, van Weerden WM, Trapman J, van der Kwast T, Roobol MJ, et al. ERG immunohistochemistry is not predictive for PSA recurrence, local recurrence or overall survival after radical prostatectomy for prostate cancer. Mod Pathol. 2012;25(3):471–9. doi: 10.1038/modpathol.2011.176.CrossRefPubMedGoogle Scholar
  18. 18.
    Bismar TA, Dolph M, Teng LH, Liu S, Donnelly B. ERG protein expression reflects hormonal treatment response and is associated with Gleason score and prostate cancer specific mortality. Eur J Cancer. 2012;48(4):538–46. doi: 10.1016/j.ejca.2012.01.001.CrossRefPubMedGoogle Scholar
  19. 19.
    Minner S, Enodien M, Sirma H, Luebke AM, Krohn A, Mayer PS, et al. ERG status is unrelated to PSA recurrence in radically operated prostate cancer in the absence of antihormonal therapy. Clin Cancer Res. 2011;17(18):5878–88. 1078–0432.CCR-11-1251.CrossRefPubMedGoogle Scholar
  20. 20.
    Korenchuk S, Lehr JE LMC, Lee YG, Whitney S, Vessella R, Lin DL, et al. VCaP, a cell-based model system of human prostate cancer. In vivo. 2001;15(2):163–8.PubMedGoogle Scholar
  21. 21.
    Nupponen NN, Hyytinen ER, Kallioniemi AH, Visakorpi T. Genetic alterations in prostate cancer cell lines detected by comparative genomic hybridization. Cancer Genet Cytogenet. 1998;101(1):53–7.CrossRefPubMedGoogle Scholar
  22. 22.
    Chen TR. Chromosome identity of human prostate cancer cell lines, PC-3 and PPC-1. Cytogenet Cell Genet. 1993;62(2–3):183–4.CrossRefPubMedGoogle Scholar
  23. 23.
    Zhu X, Kumar R, Mandal M, Sharma N, Sharma HW, Dhingra U, et al. Cell cycle-dependent modulation of telomerase activity in tumor cells. Proc Natl Acad Sci U S A. 1996;93(12):6091–5.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Mahmood T, Yang PC. Western blot: technique, theory, and trouble shooting. N Am J Med Sci. 2012;4(9):429–34. doi: 10.4103/1947-2714.100998.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Epstein JI, Allsbrook Jr WC, Amin MB, Egevad LL. The 2005 International Society of Urological Pathology (ISUP) Consensus Conference on Gleason Grading of Prostatic Carcinoma. Am J Surg Pathol. 2005;29(9):1228–42.CrossRefPubMedGoogle Scholar
  26. 26.
    Budczies J, Klauschen F, Sinn BV, Gyorffy B, Schmitt WD, Darb-Esfahani S, et al. Cutoff Finder: a comprehensive and straightforward web application enabling rapid biomarker cutoff optimization. PLoS One. 2012;7(12):e51862. doi: 10.1371/journal.pone.0051862.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Shah S, Smith H, Feng X, Rancourt DE, Riabowol K. ING function in apoptosis in diverse model systems. Biochem Cell Biol. 2009;87(1):117–25. doi: 10.1139/O08-107.CrossRefPubMedGoogle Scholar
  28. 28.
    Kumamoto K, Fujita K, Kurotani R, Saito M, Unoki M, Hagiwara N, et al. ING2 is upregulated in colon cancer and increases invasion by enhanced MMP13 expression. Int J Cancer. 2009;125(6):1306–15. doi: 10.1002/ijc.24437.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Zhang HK, Pan K, Wang H, Weng DS, Song HF, Zhou J, et al. Decreased expression of ING2 gene and its clinicopathological significance in hepatocellular carcinoma. Cancer Lett. 2008;261(2):183–92. doi: 10.1016/j.canlet.2007.11.019.CrossRefPubMedGoogle Scholar
  30. 30.
    Li X, Kikuchi K, Takano Y. ING genes work as tumor suppressor genes in the carcinogenesis of head and neck squamous cell carcinoma. J Oncol. 2011;2011:963614. doi: 10.1155/2011/963614.CrossRefPubMedGoogle Scholar
  31. 31.
    Lu F, Dai DL, Martinka M, Ho V, Li G. Nuclear ING2 expression is reduced in human cutaneous melanomas. Br J Cancer. 2006;95(1):80–6. doi: 10.1038/sj.bjc.6603205.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Yu J, Yu J, Mani RS, Cao Q, Brenner CJ, Cao X, et al. An integrated network of androgen receptor, polycomb, and TMPRSS2-ERG gene fusions in prostate cancer progression. Cancer Cell. 2010;17(5):443–54. doi: 10.1016/j.ccr.2010.03.018.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Amal Almami
    • 1
    • 4
  • Samar A. Hegazy
    • 2
  • Arash Nabbi
    • 1
    • 4
  • Mohammed Alshalalfa
    • 5
  • Asma Salman
    • 2
  • Hatem Abou-Ouf
    • 2
  • Karl Riabowol
    • 2
    • 3
    • 4
  • Tarek A. Bismar
    • 2
    • 3
    • 4
    • 5
    • 6
  1. 1.Medical Science Department Faculty of MedicineUniversity of CalgaryCalgaryCanada
  2. 2.Department of Pathology and Laboratory MedicineUniversity of Calgary and Calgary Laboratory ServicesCalgaryCanada
  3. 3.Departments of Oncology, Biochemistry and Molecular BiologyCalgaryCanada
  4. 4.Arnie Charbonneau Cancer Institute and Tom Baker Cancer CenterCalgaryCanada
  5. 5.The Prostate CentreCalgaryCanada
  6. 6.Departments of Pathology & Laboratory Medicine and OncologyUniversity of Calgary-Cumming School of MedicineCalgaryCanada

Personalised recommendations