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Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control

Nature volume 430, pages 797–802 (2004)Cite this article

Abstract

Advanced human cancers are invariably aneuploid, in that they harbour cells with abnormal chromosome numbers1,2. However, the molecular defects underlying this trait, and whether they are a cause or a consequence of the malignant phenotype, are not clear. Mutations that disable the retinoblastoma (Rb) pathway are also common in human cancers1. These mutations promote tumour development by deregulating the E2F family of transcription factors leading to uncontrolled cell cycle progression3. We show that the mitotic checkpoint protein Mad2 is a direct E2F target and, as a consequence, is aberrantly expressed in cells with Rb pathway defects. Concordantly, Mad2 is overexpressed in several tumour types, where it correlates with high E2F activity and poor patient prognosis. Generation of Rb pathway lesions in normal and transformed cells produces aberrant Mad2 expression and mitotic defects leading to aneuploidy, such that elevated Mad2 contributes directly to these defects. These results demonstrate how chromosome instability can arise as a by-product of defects in cell cycle control that compromise the accuracy of mitosis, and suggest a new model to explain the frequent appearance of aneuploidy in human cancer.

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Figure 1: Mad2 is an E2F target.
Figure 2: Deregulating the Rb/E2F pathway produces aberrant Mad2 expression.
Figure 3: Abnormal Mad2 expression in human tumours.
Figure 4: Cells with defective Rb or elevated Mad2 display chromosomal instability.
Figure 5: Aberrant Mad2 expression contributes to the mitotic defects associated with Rb loss.

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Acknowledgements

We thank M. Narita for providing the shRb construct, M. Lu for the bladder tissue microarray, S. Menendez for technical assistance and A. Kel for the use of the SiteScan program. We thank the Molecular Cytology and the Flow Cytometry Core Laboratories at Memorial Sloan-Kettering and P. McCloskey from CSHL flow cytometry facility for technical assistance. Also, we thank S. Gangadharan, R. Dickins, S. Gonzalez, N. Abumrad, B. Stillman and P. P. Pandolfi for reading the manuscript. We also thank all the members of the Lowe and Cordon-Cardo laboratories for discussions. This work was supported by program project grants from the NCI (S.W.L. and C.C.C.), Clinical Investigator Research Development Awards (L.M. and R.B.), DOD-Breast Cancer Research Program fellowship award (Z.N.), a Fundacion Caja Madrid-CNIO-MSKCC fellowship (E.H. and M.A.), a Fellowship from the Spanish Ministry of Education, Culture and Sports (E.D.R.), a US NCI postdoctoral training grant (M.T.H.) and gifts from the Laurie Strauss Leukemia Foundation and the Herbert J. Siegel philanthropic fund.

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Author notes

  1. Zaher Nahlé

    Present address: Department of Internal Medicine, Washington University in St. Louis, Saint Louis, Missouri, Missouri, 63110, USA

  2. Miguel Alaminos

    Present address: Department of Histology, School of Medicine, University of Granada, Avenida de Madrid, 11, E-18012, Granada, Spain

  3. Eva Hernando and Zaher Nahlé: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York, 10021, USA

    Eva Hernando, Gloria Juan, Miguel Alaminos, William Gerald & Carlos Cordon-Cardo

  2. Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York, 10021, USA

    Elena Diaz-Rodriguez, Loren Michel & Robert Benezra

  3. Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA

    Zaher Nahlé, Michael Hemann, Vivek Mittal & Scott W. Lowe

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  1. Eva Hernando
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Correspondence to Scott W. Lowe.

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Supplementary information

Supplementary Figure S1

a, Schematic diagrams of the MAD2L1 and Cyclin A (CCNA) promoter regions, indicating the theoretical E2F binding sites (black boxes). b, Table containing the nucleotide sequence and location of the predicted E2F binding sites in the Mad2 promoter (PDF 48 kb)

Supplementary Figure S2

Immunohistochemistry of Mad2 and Ki-67 in highly proliferating organs (a), and in some transitional cell carcinomas of the bladder (b), illustrating that Mad2 expression is low or negative in some proliferative tissues - it is not simply a proliferation marker (PDF 1618 kb)

Supplementary Figure S3

a, Mad2 immunofluorescence staining (FITC) of Mad2-transduced IMR90 fibroblasts, showing a highly positive multinucleated cell. b, g-tubulin staining (FITC) in Mad2- and vector-infected IMR90 cells, demonstrating that Mad2-transduced cells don’t display major centrosomal amplification (PDF 1588 kb)

Supplementary Figure S4

a, Table containing the average time required to complete two defined mitotic stages in vector, shRB, E1A, and Mad2 transduced cells. Western Blot analyses of cyclin B1 (cycB) (b) and securin/Pds1 (c) in HCT116 and IMR90, released after a double thymidine blockade, showing that cyclin B and securin degradation are delayed in shRb, E1A or Mad2 transduced cells. d, Western blot for E2F and E1A in HCT116 cells ectopically expressing E2F, E1A or a vector control together with either a Mad2 hairpin (sh-Mad2) or a hairpin control (sh-con). E1A or E2F levels are not altered in the presence of shMad2 (PDF 156 kb)

Supplementary Video A

Normal cell division in vector-transduced NIH-3T3 cells by phase-contrast. (MOV 3133 kb)

Supplementary Video B

Normal chromosomal segregation in vector-transduced NIH-3T3 cells by green fluorescence. (MOV 272 kb)

Supplementary Video C

Prolonged or failed cytokinesis in 3T3 cells transduced with shRb. (MP4 231 kb)

Supplementary Video D

Prolonged chromosome segregation due to a cytokinesis problem in 3T3 cells transduced with shRb. (MP4 103 kb)

Supplementary Video E

Cytokinesis failure in Mad2-transduced 3T3 cells. (MP4 605 kb)

Supplementary Video F

Cytokinesis failure followed by reattachment and multinucleation in a Mad2-transduced 3T3 (MP4 309 kb)

Supplementary Video G

Small fragment of DNA material lost during chromosome segregation in an E1A-expressing NIH-3T3 cell . (MOV 381 kb)

Supplementary Video H

Asymmetrical chromosome segregation in Mad2-transduced 3T3 cells. (MOV 808 kb)

Supplementary Video I

Problems in chromosome alignment followed by abnormal segregation in shRb-expressing HCT116 cells. (MOV 631 kb)

Supplementary Video J

Chromosomes pulling towards the poles of the spindle for a prolonged period of time, ending in a completely aberrant segregation. Mad2-transduced HCT116 cells. (MOV 972 kb)

Supplementary Figure and Video Legends (DOC 23 kb)

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Hernando, E., Nahlé, Z., Juan, G. et al. Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature 430, 797–802 (2004). https://doi.org/10.1038/nature02820

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