Induction of ZEB Proteins by Inactivation of RB Protein Is Key Determinant of Mesenchymal Phenotype of Breast Cancer*

Background: Inactivation of RB is a key event for induction of EMT in cancers. Results: ZEB proteins are markedly up-regulated through the reduction of miR-200 family of microRNAs in RB-inactive cancer cells. Conclusion: RB/ZEB pathway plays a pivotal role in mesenchymal and aggressive phenotype in breast cancers. Significance: Suppressing ZEB1 by cyclin-dependent kinase inhibitors provides a novel therapeutic strategy for RB-inactive breast cancers. We previously showed that depletion of the retinoblastoma protein (RB) induces down-regulation of the adhesion molecule E-cadherin and thereby triggers the epithelial-mesenchymal transition. To further characterize the effect of RB inactivation on the phenotype of cancer cells, we have now examined RB expression in human breast cancer cell lines and clinical specimens. We found that RB-inactive cells exhibit a mesenchymal-like morphology and are highly invasive. We also found that ZEB proteins, transcriptional repressors of the E-cadherin gene, are markedly up-regulated in these cells in a manner sensitive to the miR-200 family of microRNAs. Moreover, depletion of ZEB in RB-inactive cells suppressed cell invasiveness and proliferation and induced epithelial marker expression. These results implicate ZEB in induction of the epithelial-mesenchymal transition, as well as in maintenance of the mesenchymal phenotype in RB-inactive cells. We also developed a screening program for inhibitors of ZEB1 expression and thereby identified several cyclin-dependent kinase inhibitors that blocked both ZEB1 expression and RB phosphorylation. Together, our findings suggest that RB inactivation contributes to tumor progression not only through loss of cell cycle control but also through up-regulation of ZEB expression and induction of an invasive phenotype.

[1]  K. Wilner,et al.  Phase I study of PD 0332991, a cyclin-dependent kinase inhibitor, administered in 3-week cycles (Schedule 2/1) , 2011, British Journal of Cancer.

[2]  B. Pockaj,et al.  Triple-Negative Breast Cancers: Unique Clinical Presentations and Outcomes , 2010, Annals of Surgical Oncology.

[3]  Simone Brabletz,et al.  The ZEB/miR‐200 feedback loop—a motor of cellular plasticity in development and cancer? , 2010, EMBO reports.

[4]  Dexin Kong,et al.  Discovery of phosphatidylinositol 3-kinase inhibitory compounds from the Screening Committee of Anticancer Drugs (SCADS) library. , 2010, Biological & pharmaceutical bulletin.

[5]  J. Zhang,et al.  miR-200bc/429 cluster targets PLCγ1 and differentially regulates proliferation and EGF-driven invasion than miR-200a/141 in breast cancer , 2010, Oncogene.

[6]  C. Caldas,et al.  Triple negative breast cancers: clinical and prognostic implications. , 2009, European journal of cancer.

[7]  F. Portillo,et al.  Transcriptional regulation of cell polarity in EMT and cancer , 2008, Oncogene.

[8]  M. F. Shannon,et al.  A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. , 2008, Cancer research.

[9]  M. Korpal,et al.  The emerging role of miR-200 family of MicroRNAs in epithelial-mesenchymal transition and cancer metastasis , 2008, RNA biology.

[10]  T. Shibata,et al.  Rb depletion results in deregulation of E-cadherin and induction of cellular phenotypic changes that are characteristic of the epithelial-to-mesenchymal transition. , 2008, Cancer research.

[11]  T. Brabletz,et al.  A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells , 2008, EMBO reports.

[12]  G. Goodall,et al.  The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1 , 2008, Nature Cell Biology.

[13]  Sun-Mi Park,et al.  The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. , 2008, Genes & development.

[14]  A. Osunkoya,et al.  Insulin-like growth factor-I-dependent up-regulation of ZEB1 drives epithelial-to-mesenchymal transition in human prostate cancer cells. , 2008, Cancer research.

[15]  G. Moreno-Bueno,et al.  Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. , 2008, Cancer research.

[16]  Karla Kerlikowske,et al.  Abrogated response to cellular stress identifies DCIS associated with subsequent tumor events and defines basal-like breast tumors. , 2007, Cancer cell.

[17]  J. Padmanabhan,et al.  Rb function in the apoptosis and senescence of non-neuronal and neuronal cells: role in oncogenesis. , 2006, Current molecular medicine.

[18]  Pl Chen,et al.  RB, the conductor that orchestrates life, death and differentiation , 2006, Oncogene.

[19]  C. Simone,et al.  pRb: master of differentiation. Coupling irreversible cell cycle withdrawal with induction of muscle-specific transcription , 2006, Oncogene.

[20]  M. Nakao,et al.  Transcriptional Blockade Induces p53-dependent Apoptosis Associated with Translocation of p53 to Mitochondria* , 2005, Journal of Biological Chemistry.

[21]  G. Berx,et al.  DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells , 2005, Oncogene.

[22]  S. Ramaswamy,et al.  Twist, a Master Regulator of Morphogenesis, Plays an Essential Role in Tumor Metastasis , 2004, Cell.

[23]  J. Thiery Epithelial–mesenchymal transitions in tumour progression , 2002, Nature Reviews Cancer.

[24]  E. Fearon,et al.  The SLUG zinc-finger protein represses E-cadherin in breast cancer. , 2002, Cancer research.

[25]  G. Berx,et al.  The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. , 2001, Molecular cell.

[26]  A. G. Herreros,et al.  The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells , 2000, Nature Cell Biology.

[27]  Francisco Portillo,et al.  The transcription factor Snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression , 2000, Nature Cell Biology.