Breast Cancer Research and Treatment

, Volume 132, Issue 2, pp 575–588 | Cite as

Targeting low molecular weight cyclin E (LMW-E) in breast cancer

  • Angela Nanos-Webb
  • Natalie A. Jabbour
  • Asha S. Multani
  • Hannah Wingate
  • Nassima Oumata
  • Hervé Galons
  • Benoît Joseph
  • Laurent Meijer
  • Kelly K. Hunt
  • Khandan Keyomarsi
Preclinical Study

Abstract

Low molecular weight cyclin E (LMW-E) plays an important oncogenic role in breast cancer. LMW-E, which is not found in normal tissue, can promote the formation of aggressive tumors and can lead to increased genomic instability and tumorigenesis. Additionally, breast cancer patients whose tumors express LMW-E have a very poor prognosis. Therefore, we investigated LMW-E as a potential specific target for treatment either alone or in combination therapy. We hypothesized that because LMW-E binds to CDK2 more efficiently than full length cyclin E, resulting in increased activity, CDK inhibitors could be used to target tumors with LMW-E bound to CDK2. To test the hypothesis, an inducible full length and LMW-E MCF7-Tet-On system was established. Cyclin E (full length (EL) or LMW-E) is only expressed upon induction of the transgene. The doubling times of cells were unchanged when the transgenes were induced. However, upon induction, the kinase activity associated with LMW-E was much higher than that in the EL induced cells or any of the uninduced cells. Additionally only the LMW-E induced cells underwent chromosome aberrations and increased polyploidy. By examining changes in proliferation and survival in cells with induced full length and LMW-E, CDK inhibitors alone were determined to be insufficient to specifically inhibit LMW-E expressing cells. However, in combination with Doxorubicin, the CDK inhibitor, Roscovitine (Seliciclib, CYC202), synergistically led to increased cell death in LMW-E expressing cells. Clinically, the combination of CDK inhibitors and chemotherapy such as Doxorubicin provides a viable personalized treatment strategy for those breast cancer patients whose tumors express the LMW-E.

Keywords

Roscovitine Seliciclib CYC202 Doxorubicin LMW cyclin E CDK inhibitors 

Notes

Acknowledgments

We would like to thank Tuyen Bui for his assistance on the generation of the MCF7-Tet-On T2 cell line. Also, we would like to thank Yanna Liu for the immunofluorescence of the MCF7-Tet-On cells. We would also like to thank Jin Ma for the time spent preparing the MCF7-Tet-On samples for metaphase spread analysis. This research was supported by NIH grants CA87458, P50CA116199, Susan G. Komen grants KG100521 and KG100876 to Khandan Keyomarsi and Kelly K. Hunt, by the CCTS T32 grant to Natalie Jabbour through the NIH Clinical and Translational Award TL1 RR024147, by NCI CCSG grant CA16672 to M.D. Anderson Cancer Center, “Cancéropole Grand-Ouest”, the “Association pour la Recherche sur le Cancer” (ARC-1092), the “Ligue Nationale contre le Cancer (Comité Grand-Ouest)” to Laurent Meijer.

Supplementary material

10549_2011_1638_MOESM1_ESM.ppt (324 kb)
Supplemental Table 1. IC50 values according to the MTT Proliferation Assay. IC50 values were determined using the MTT proliferation assay in the presence or absence of cyclin EL, T1, and T2 induction. CDK2 inhibitors were categorized as HD (Hymenialdisine), Perharidines, Indirubins or purines. nd = not determined. No significant difference was seen between the induced and non-induced cyclin E samples. (PPT 319 kb)
10549_2011_1638_MOESM2_ESM.ppt (181 kb)
Supplemental Table 2. IC50 values according to HTCA Survival Assay. IC50 values were determined using the HTCA clonogenic assay in the presence or absence of cyclin EL, T1, and T2 induction. CDK2 inhibitors were categorized as Meriolins or purines. nd = not determined. Again, no significant difference was seen between the induced and non-induced cyclin E samples. (PPT 178 kb)
10549_2011_1638_MOESM3_ESM.ppt (560 kb)
Supplemental Figure 1. Cyclin E overexpression leads to an increase in elastase protein levels. MCF7-Tet-On EL, T1, and T2 cells were induced with doxycycline for expression of the cyclin E transgene (“ON”) compared to the non-induced controls (“OFF”). Cells were incubated with antibodies against cyclin E and elastase and examined using confocal microscopy. DAPI was used to stain the nucleus. Overexpression of cyclin E, both full length (EL) and LMW-E (T1 and T2) resulted in an increase in the protein levels of elastase. (PPT 555 kb)
10549_2011_1638_MOESM4_ESM.ppt (126 kb)
Supplemental Figure 2. Cyclin E LMW/CDK2 complexes are more sensitive to Roscovitine that the cyclin E full length/CDK2 complex. Sf9 insect cells were co-infected with baculovirus to cyclin E full length (EL) or the LMW forms (T1 or T2) with CDK2. Sixty hours post-infection, Histone H1 kinase analysis were done where 100 µg of each protein extract were immunoprecipitated with polyclonal antibody to CDK2-coupled protein A in the presence of increasing concentrations of Roscovitine for 30 min at 37°C. The samples were then subjected to SDS-PAGE, autoradiography and phosphor-image analysis. The histone H1 raw counts were normalized to the untreated controls and presented at% CDK2 activity. Black line indicates EL/CDK2, while the green and red lines refer to T1/CDK2 andT2/CDK2 complexes respectively.(PPT 122 kb)

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Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Angela Nanos-Webb
    • 1
  • Natalie A. Jabbour
    • 1
  • Asha S. Multani
    • 2
  • Hannah Wingate
    • 3
  • Nassima Oumata
    • 4
  • Hervé Galons
    • 5
  • Benoît Joseph
    • 6
  • Laurent Meijer
    • 7
  • Kelly K. Hunt
    • 3
  • Khandan Keyomarsi
    • 1
  1. 1.Department of Experimental Radiation OncologyM. D. Anderson Cancer CenterHoustonUSA
  2. 2.Department of GeneticsM. D. Anderson Cancer CenterHoustonUSA
  3. 3.Department of Surgical OncologyM. D. Anderson Cancer CenterHoustonUSA
  4. 4.ManRos TherapeuticsCentre de PerharidyRoscoffFrance
  5. 5.Laboratoire de Chimie Organique 2, CNRS UMR8601, INSERM U 648Université Paris-DescartesParis cedex 06France
  6. 6.Institut de Chimie et Biochimie Moléculaires et SupramoléculairesUniversité Claude Bernard-Lyon 1VilleurbanneFrance
  7. 7.CNRS, ‘Protein Phosphorylation & Human Disease’ GroupRoscoffFrance

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