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Too Much Cleavage of Cyclin E Promotes Breast Tumorigenesis

  • Keith R. Loeb ,

    kloeb@fhcrc.org

    Affiliations Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, United States of America, Department of Pathology, University of Washington School of Medicine, Seattle, Washington, United States of America, Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America

  • Xueyan Chen

    Affiliation Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington, United States of America

Too Much Cleavage of Cyclin E Promotes Breast Tumorigenesis

  • Keith R. Loeb, 
  • Xueyan Chen
PLOS
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Cyclin E, together with cyclin-dependent kinase 2 (CDK2), functions as a gatekeeper to promote G1/S transitions and the initiation of DNA replication. In normal cells, cyclin E–associated kinase activity is exquisitely regulated, with activity being limited to a brief time interval between late G1 and early S phase. Human cancers frequently exhibit deregulated cyclin E–associated kinase activity resulting from overexpression of cyclin E and loss of cyclin-dependent kinase inhibition (via p53 mutations) promoting genetic instability and cell proliferation [1]. Increased levels of cyclin E correlate with tumorigenesis and are a poor prognostic indicator independent of proliferation rate, suggesting that cyclin E's role in tumorigenesis is not limited to promoting increased cell proliferation [2], [3]. By eliminating regulatory constraints using p53 null cells, we and others have shown that overexpression or endogenous expression of stabilizing mutant forms of cyclin E can lead to hyperproliferation, genetic instability, and malignancy in cell culture and murine models [4], [5]. Normal cells suppress the effects of excess/stabilized cyclin E via the G1/S checkpoint involving the p53/p21 pathway.

Five isoforms of cyclin E, ranging in size from 33 to 44 kDa, have been identified in tumors over-expressing cyclin E. These low molecular weight forms of cyclin E (LMW-E) are generated through post-translational cleavage of full-length cyclin E by the elastase family of serine proteases in tumor cells [6], [7]. In comparison to full-length cyclin E (50 kDa), LMW-E forms are uniquely expressed in tumor cells, exhibit enhanced CDK2-associated kinase activity, have increased affinity for CDK2 [7][9], and exhibit decreased inhibition by CDK2 inhibitors, p21 and p27 (Figure 1) [10], [11]. Ectopic expression of LMW-E isoforms promotes cell proliferation, genetic instability, centrosome amplification, and malignancy [12], [13]. In addition, clinical studies have shown that high LMW-E is strongly associated with poor survival in breast cancer [2], colorectal cancer [14], [15], ovarian cancer [16], and melanomas [17]. Given its unique properties and distinct function in human cancers, targeting LMW-E could have important therapeutic implications.

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Figure 1. Low molecular weight cyclin E promotes tumorigenesis.

In normal cells, cyclin E/CDK2 is tightly regulated and triggers the onset of S phase. In tumors, cyclin E undergoes proteolytic processing generating low molecular weight species that exhibit increased kinase activity and resistance to inhibition by cyclin kinase inhibitors p21/p27. The expression of LMW-E promotes aberrant acinar morphogenesis, centrosome amplification, and tumors associated with activation of the bRaf/ERK/mTOR pathway. LMW-E, low molecular weight cyclin E.

https://doi.org/10.1371/journal.pgen.1002623.g001

The study by Duong et al. in this issue of PLoS Genetics [18] convincingly uncovers the tumorigenic potential of LMW-E. The authors used three different model systems—3D acinar cultures, xenograft transplantation, and transgenic mice—to show that overexpression of LMW-E is sufficient to induce aberrant acinar morphogenesis in culture and mammary tumors in mice (Figure 1). When grown on Matrigel, immortalized human mammary epithelial cells (hMECs) expressing LMW-E exhibit large misshapen multiacinar structures resulting from defective growth arrest and apoptosis that mimic morphologic features of breast carcinomas. Further, ectopic expression of LMW-E in immortalized hMECs promotes tumorigenesis in xenografts and transgenic mice to a much greater extent than full-length cyclin E. Consistent with the reports by Akli et al. and Nanos-Webb et al., tumorigenesis associated with LMW-E is dependent on CDK2 [19], [20]. Furthermore, in vivo passaging of tumor cells increases the expression of LMW-E, suggesting that LMW-E provides a selective growth advantage to the tumor. Duong et al. also took advantage of a proteomic analysis termed reverse-phase protein array assay (RPPA) to examine protein expression patterns in cultured tumor cells and in breast tumors expressing high LMW-E levels. Their analyses revealed that multiple components of the b-RAF-ERK1/2-mTOR pathway are elevated in these cells. Activation of the b-RAF-ERK1/2-mTOR pathway normally promotes cell division and cell survival. Consistent with this, the authors observed that endogenous cyclin E levels are also increased in cells expressing high LMW-E, indicative of the existence of a positive feedback loop that promotes cell proliferation. Both high LMW-E levels and up-regulation of the b-RAF-ERK1/2-mTOR signaling pathway are associated with poor survival, suggesting functional correlation of these events in aggressive tumors. Importantly, the authors demonstrated that combination therapy targeting LMW-E/CDK2 and the b-RAF-ERK1/2-mTOR pathway has a synergistic effect in abrogating the tumorigenic effect of LMW-E. Thus, the identification of these downstream regulators may provide novel biomarkers and/or potential therapeutic targets for LMW-E–expressing tumors.

The report that LMW-E potentiates tumorigenesis in three independent model systems associated with activation of the b-RAF-ERK1/2-mTOR pathway is intriguing. However, there are many important questions about the role of LMW-E in tumorigenesis that need to be addressed. 1) What is the functional relationship between LMW-E and full-length cyclin E? In each tumor model reported by Duong et al., the effect was examined by over-expressing LMW-E in a background of endogenous full-length cyclin E. Further, the authors show that ectopic expression of LMW-E in transplanted xenografts triggers tumor evolution and results in increased levels of endogenous cyclin E. Thus, the contribution of endogenous full-length cyclin E in tumorigenesis cannot be excluded. In addition, Spruck et al. reported that the level of LMW-E correlates with full-length cyclin E, suggesting that LMW-E reflects the total cyclin E protein in primary breast tumors, cell lines, and even normal breast tissue [21]. To examine the effect of LMW-E in the absence of over-expression, and in the absence of full-length cyclin E, it will be important to use a knock-in model in which expression of LMW-E is driven from the endogenous cyclin E promoter. 2) What is the relationship between LMW-E and the b-Raf-ERK1/2-mTOR signaling pathway? The authors demonstrated that the b-Raf-ERK1/2-mTOR signaling pathway is activated in tumors expressing high levels of LMW-E. The b-Raf-ERK1/2-mTOR pathway may be a downstream signaling pathway deregulated by LMW-E, or it could be a parallel survival pathway selected in LMW-E–expressing tumors. In particular, the fact that only combinational therapy targeting both cyclin E–associated kinase activity and the b-Raf-ERK1/2-mTOR pathway generates an anti-tumor effect argues against a direct cause–effect relationship and is suggestive of a parallel pathway. 3) Is LMW-E expression required for tumor growth and does down-regulation of LMW-E alter tumor growth, invasion, or metastasis? The authors have generated an inducible model that should facilitate these studies. 4) Is the tumor-promoting activity of LMW-E due to enhanced deregulated kinase activity or to alternative target specificity? The LMW-E construct used in these studies has an N-terminal deletion (40 amino acids) that eliminates the proposed nuclear localization signal (NLS) and potentially affects the intracellular localization and substrate specificity [22], [23]. 5) How is LMW-E generated in tumors, and is it tumor-type specific? It has been proposed and demonstrated by Caruso et al. that many tumors have elevated protease activity and decreased levels of protease inhibitors such as elafin [24] that may contribute to the generation of LMW-E. Further characterization of the proteolytic pathways that target cyclin E in tumors may provide alternative therapeutic targets.

References

  1. 1. Hwang HC, Clurman BE (2005) Cyclin E in normal and neoplastic cell cycles. Oncogene 24: 2776–2786.HC HwangBE Clurman2005Cyclin E in normal and neoplastic cell cycles.Oncogene2427762786
  2. 2. Keyomarsi K, Tucker SL, Buchholz TA, Callister M, Ding Y, et al. (2002) Cyclin E and survival in patients with breast cancer. N Engl J Med 347: 1566–1575.K. KeyomarsiSL TuckerTA BuchholzM. CallisterY. Ding2002Cyclin E and survival in patients with breast cancer.N Engl J Med34715661575
  3. 3. Porter PL, Malone KE, Heagerty PJ, Alexander GM, Gatti LA, et al. (1997) Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nat Med 3: 222–225.PL PorterKE MalonePJ HeagertyGM AlexanderLA Gatti1997Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients.Nat Med3222225
  4. 4. Loeb KR, Kostner H, Firpo E, Norwood T, Tsuchiya KD, et al. (2005) A mouse model for cyclin E-dependent genetic instability and tumorigenesis. Cancer Cell 8: 35–47.KR LoebH. KostnerE. FirpoT. NorwoodKD Tsuchiya2005A mouse model for cyclin E-dependent genetic instability and tumorigenesis.Cancer Cell83547
  5. 5. Minella AC, Swanger J, Bryant E, Welcker M, Hwang H, et al. (2002) p53 and p21 form an inducible barrier that protects cells against cyclin E-cdk2 deregulation. Curr Biol 12: 1817–1827.AC MinellaJ. SwangerE. BryantM. WelckerH. Hwang2002p53 and p21 form an inducible barrier that protects cells against cyclin E-cdk2 deregulation.Curr Biol1218171827
  6. 6. Harwell RM, Porter DC, Danes C, Keyomarsi K (2000) Processing of cyclin E differs between normal and tumor breast cells. Cancer Res 60: 481–489.RM HarwellDC PorterC. DanesK. Keyomarsi2000Processing of cyclin E differs between normal and tumor breast cells.Cancer Res60481489
  7. 7. Porter DC, Zhang N, Danes C, McGahren MJ, Harwell RM, et al. (2001) Tumor-specific proteolytic processing of cyclin E generates hyperactive lower-molecular-weight forms. Mol Cell Biol 21: 6254–6269.DC PorterN. ZhangC. DanesMJ McGahrenRM Harwell2001Tumor-specific proteolytic processing of cyclin E generates hyperactive lower-molecular-weight forms.Mol Cell Biol2162546269
  8. 8. Harwell RM, Mull BB, Porter DC, Keyomarsi K (2004) Activation of cyclin-dependent kinase 2 by full length and low molecular weight forms of cyclin E in breast cancer cells. J Biol Chem 279: 12695–12705.RM HarwellBB MullDC PorterK. Keyomarsi2004Activation of cyclin-dependent kinase 2 by full length and low molecular weight forms of cyclin E in breast cancer cells.J Biol Chem2791269512705
  9. 9. Wingate H, Puskas A, Duong M, Bui T, Richardson D, et al. (2009) Low molecular weight cyclin E is specific in breast cancer and is associated with mechanisms of tumor progression. Cell Cycle 8: 1062–1068.H. WingateA. PuskasM. DuongT. BuiD. Richardson2009Low molecular weight cyclin E is specific in breast cancer and is associated with mechanisms of tumor progression.Cell Cycle810621068
  10. 10. Akli S, Zheng PJ, Multani AS, Wingate HF, Pathak S, et al. (2004) Tumor-specific low molecular weight forms of cyclin E induce genomic instability and resistance to p21, p27, and antiestrogens in breast cancer. Cancer Res 64: 3198–3208.S. AkliPJ ZhengAS MultaniHF WingateS. Pathak2004Tumor-specific low molecular weight forms of cyclin E induce genomic instability and resistance to p21, p27, and antiestrogens in breast cancer.Cancer Res6431983208
  11. 11. Wingate H, Zhang N, McGarhen MJ, Bedrosian I, Harper JW, et al. (2005) The tumor-specific hyperactive forms of cyclin E are resistant to inhibition by p21 and p27. J Biol Chem 280: 15148–15157.H. WingateN. ZhangMJ McGarhenI. BedrosianJW Harper2005The tumor-specific hyperactive forms of cyclin E are resistant to inhibition by p21 and p27.J Biol Chem2801514815157
  12. 12. Akli S, Van Pelt CS, Bui T, Multani AS, Chang S, et al. (2007) Overexpression of the low molecular weight cyclin E in transgenic mice induces metastatic mammary carcinomas through the disruption of the ARF-p53 pathway. Cancer Res 67: 7212–7222.S. AkliCS Van PeltT. BuiAS MultaniS. Chang2007Overexpression of the low molecular weight cyclin E in transgenic mice induces metastatic mammary carcinomas through the disruption of the ARF-p53 pathway.Cancer Res6772127222
  13. 13. Bagheri-Yarmand R, Biernacka A, Hunt KK, Keyomarsi K (2010) Low molecular weight cyclin E overexpression shortens mitosis, leading to chromosome missegregation and centrosome amplification. Cancer Res 70: 5074–5084.R. Bagheri-YarmandA. BiernackaKK HuntK. Keyomarsi2010Low molecular weight cyclin E overexpression shortens mitosis, leading to chromosome missegregation and centrosome amplification.Cancer Res7050745084
  14. 14. Zhou YJ, Xie YT, Gu J, Yan L, Guan GX, et al. (2011) Overexpression of cyclin E isoforms correlates with poor prognosis in rectal cancer. Eur J Surg Oncol 37: 1078–1084.YJ ZhouYT XieJ. GuL. YanGX Guan2011Overexpression of cyclin E isoforms correlates with poor prognosis in rectal cancer.Eur J Surg Oncol3710781084
  15. 15. Corin I, Di Giacomo MC, Lastella P, Bagnulo R, Guanti G, et al. (2006) Tumor-specific hyperactive low-molecular-weight cyclin E isoforms detection and characterization in non-metastatic colorectal tumors. Cancer Biol Ther 5: 198–203.I. CorinMC Di GiacomoP. LastellaR. BagnuloG. Guanti2006Tumor-specific hyperactive low-molecular-weight cyclin E isoforms detection and characterization in non-metastatic colorectal tumors.Cancer Biol Ther5198203
  16. 16. Davidson B, Skrede M, Silins I, Shih Ie M, Trope CG, et al. (2007) Low-molecular weight forms of cyclin E differentiate ovarian carcinoma from cells of mesothelial origin and are associated with poor survival in ovarian carcinoma. Cancer 110: 1264–1271.B. DavidsonM. SkredeI. SilinsM. Shih IeCG Trope2007Low-molecular weight forms of cyclin E differentiate ovarian carcinoma from cells of mesothelial origin and are associated with poor survival in ovarian carcinoma.Cancer11012641271
  17. 17. Bales E, Mills L, Milam N, McGahren-Murray M, Bandyopadhyay D, et al. (2005) The low molecular weight cyclin E isoforms augment angiogenesis and metastasis of human melanoma cells in vivo. Cancer Res 65: 692–697.E. BalesL. MillsN. MilamM. McGahren-MurrayD. Bandyopadhyay2005The low molecular weight cyclin E isoforms augment angiogenesis and metastasis of human melanoma cells in vivo.Cancer Res65692697
  18. 18. Duong MT, Akli S, Wei C, Wingate HF, Liu W, et al. (2012) LMW-E/CDK2 deregulates acinar morphogenesis, induces tumorigenesis and associates with the activated b-Raf-ERK1/2-mTOR pathway in breast cancer patients. PLoS Genet 8: e1002538.MT DuongS. AkliC. WeiHF WingateW. Liu2012LMW-E/CDK2 deregulates acinar morphogenesis, induces tumorigenesis and associates with the activated b-Raf-ERK1/2-mTOR pathway in breast cancer patients.PLoS Genet8e1002538
  19. 19. Akli S, Van Pelt CS, Bui T, Meijer L, Keyomarsi K (2011) Cdk2 is required for breast cancer mediated by the low-molecular-weight isoform of cyclin E. Cancer Res 71: 3377–3386.S. AkliCS Van PeltT. BuiL. MeijerK. Keyomarsi2011Cdk2 is required for breast cancer mediated by the low-molecular-weight isoform of cyclin E.Cancer Res7133773386
  20. 20. Nanos-Webb A, Jabbour NA, Multani AS, Wingate H, Oumata N, et al. (2011) Targeting low molecular weight cyclin E (LMW-E) in breast cancer. Breast Cancer Res Treat. A. Nanos-WebbNA JabbourAS MultaniH. WingateN. Oumata2011Targeting low molecular weight cyclin E (LMW-E) in breast cancer.Breast Cancer Res TreatE-pub ahead of print 22 June 2011. E-pub ahead of print 22 June 2011.
  21. 21. Spruck C, Sun D, Fiegl H, Marth C, Mueller-Holzner E, et al. (2006) Detection of low molecular weight derivatives of cyclin E1 is a function of cyclin E1 protein levels in breast cancer. Cancer Res 66: 7355–7360.C. SpruckD. SunH. FieglC. MarthE. Mueller-Holzner2006Detection of low molecular weight derivatives of cyclin E1 is a function of cyclin E1 protein levels in breast cancer.Cancer Res6673557360
  22. 22. Delk NA, Hunt KK, Keyomarsi K (2009) Altered subcellular localization of tumor-specific cyclin E isoforms affects cyclin-dependent kinase 2 complex formation and proteasomal regulation. Cancer Res 69: 2817–2825.NA DelkKK HuntK. Keyomarsi2009Altered subcellular localization of tumor-specific cyclin E isoforms affects cyclin-dependent kinase 2 complex formation and proteasomal regulation.Cancer Res6928172825
  23. 23. Jackman M, Kubota Y, den Elzen N, Hagting A, Pines J (2002) Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm. Mol Biol Cell 13: 1030–1045.M. JackmanY. KubotaN. den ElzenA. HagtingJ. Pines2002Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm.Mol Biol Cell1310301045
  24. 24. Caruso JA, Hunt KK, Keyomarsi K (2010) The neutrophil elastase inhibitor elafin triggers rb-mediated growth arrest and caspase-dependent apoptosis in breast cancer. Cancer Res 70: 7125–7136.JA CarusoKK HuntK. Keyomarsi2010The neutrophil elastase inhibitor elafin triggers rb-mediated growth arrest and caspase-dependent apoptosis in breast cancer.Cancer Res7071257136