Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):E1301-10. doi: 10.1073/pnas.1210353110.

Met synergizes with p53 loss to induce mammary tumors that possess features of claudin-low breast cancer.

Jennifer F. Knight1*, Robert Lesurf1,2*, Dushanthi Pinnaduwage3, Ryan Davis4, Sadiq M. Saleh1,2, Dongmei Zuo1, Hong Zhao1, Monica Naujokas1, Naila Chughtai1, Jason I. Herschkowitz5, Aleix Prat6,7, Anna Marie Mulligan8,9,William J. Muller1,2, Robert D. Cardiff4, Jeffrey Gregg4, Irene L. Andrulis3,8,9,  Michael Hallett1 and Morag Park1,2,11

1Goodman Cancer Research Centre, McGill University, Montreal, QC, H3A 1A3; 2Department of Biochemistry, McGill University, H2W 1S6; 3Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; 4Centre for Comparative Medicine, University of California, Davis, CA 95616; 5Baylor College of Medicine, Houston, TX; 6Lineberger Comprehensive Cancer Center University of North Carolina, Chapel Hill, NC; 7Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain, Department of Molecular and Cellular Biology; 8Department of Laboratory Medicine, St. Michael’s Hospital, Toronto, Ontario M5B 1W8; 9Department of Laboratory Medicine and Pathobiology, University of Toronto; 10Department of Molecular Genetics, University of Toronto; 11Department of Oncology, McGill University, Montreal, QC, Canada H2W 1S6. *These authors contributed equally to this work


Abstract

Triple negative breast cancer (TNBC) accounts for ~20% of cases, and contributes to basal and claudin-low molecular subclasses. TNBCs have poor prognosis, display frequent mutations in tumor suppressor gene p53 (TP53) and lack targeted therapies. The MET receptor tyrosine kinase is elevated in TNBC, and transgenic Met models (Metmt) develop ‘basal-like’ tumors. To investigate collaborating events in the genesis of TNBC, we generated Metmt mice with conditional loss of murine p53 (Trp53) in mammary epithelia. Somatic Trp53 loss, in combination with Metmt, significantly increased tumor penetrance over Metmt or Trp53 loss alone. Unlike Metmt tumors, which are histologically diverse and enriched in a basal-like molecular signature, the majority of Metmt tumors with Trp53 loss displayed a spindloid pathology with a distinct molecular signature that resembles the human claudin-low subtype of TNBC, including diminished claudins,  an epithelial-to-mesenchymal transition signature and decreased expression of the microRNA-200 family. Moreover, although mammary specific loss of Trp53 promotes tumors with diverse pathologies, those with spindloid pathology and claudin-low signature display genomic Met amplification. In both models, MET activity is required for maintenance of the claudin-low morphological phenotype, where MET inhibitors restore cell-cell junctions, rescue Claudin-1 expression, and abrogate growth and dissemination of cells in vivo. Among human breast cancers, elevated levels of MET and stabilised TP53, indicative of mutation, correlate with highly proliferative TNBCs of poor outcome. This work shows synergy between MET and TP53 loss for claudin-low breast cancer, identifies a restricted claudin-low gene signature and provides a rationale for anti-MET therapies in TNBC.

Keywords: Met RTK, EMT, mouse model, breast cancer

PMID: 23509284

 

Supplement

Breast cancer is a heterogeneous disease, defined by biomarker positivity into three broad subclasses: 1) Estrogen receptor (ER), or 2) Her2-receptor positive, which determine targeted treatment strategies, and 3) triple negative (ER/HER2 and progesterone receptor (PR) negative), associated with poor outcome and for which no targeted therapies are available. As determined through gene expression profiling, the triple negative (TN) subclass itself is divided into multiple molecular subtypes of disease (1), the two most studied of which are known as ‘basal-like’ and ‘claudin-low’. (2, 3). The molecular mechanisms that give rise to the subtypes of TN breast cancer are poorly understood.

Elevated expression of the receptor tyrosine kinase MET, has been associated with poor outcome and TN breast cancers by several groups including ours (4), suggesting that MET could be a candidate therapeutic target. We have previously shown that expression of MET in the mouse mammary gland (MMTV-Metmt) stimulates the formation of mammary tumors that model human basal-like tumors (5).  In our current study, we have used our mouse model to study additional events that may collaborate with Met in the development of TNBC and have identified that combining Met expression with mammary gland specific loss of p53 (MMTV-Metmt;Trp53fl/+;Cre), leads to a dramatic increase in tumor incidence and the development of tumors which model the claudin-low phenotype.

Claudin-low breast cancers are characterised by a process referred to as epithelial-to-mesenchymal transition (EMT), in which differentiated epithelial cells become less polarised and adopt a mesenchymal, migratory phenotype (3).  Importantly, we show that the claudin-low phenotype in our mouse model is dependent upon Met protein activity, as the use of small molecule inhibitors of MET leads to reduction in tumor cell survival and proliferation, in addition to the  re-organisation of cell-cell junctions.

Analysis of a tissue microarray comprised of 618 node-negative breast cancer patients identified a significant correlation between elevated expression of MET and TP53 proteins, the latter of which being indicative of loss of TP53 function. Moreover, breast tumors that showed co-positivity for both MET and TP53 were more frequently hormone receptor negative than breast tumors that were positive for only one or the other. MET/TP53 co-positive tumors were associated with significantly worse patient outcome and MET/TP53 co-positivity could predict patient outcome independently of other clinical parameters, providing further support for MET as a clinically relevant target, particularly in a background with loss of function of TP53.

Finally, we use a bioinformatics approach to generate a 36-gene core signature, capable of identifying claudin-low tumors from those of other subtypes, with a degree of accuracy equivalent to that of a published predictor comprising over 700 genes (3).

The transgenic mouse model of human claudin-low breast cancer described here provides the first evidence for cooperation between MET and loss of function of a common tumor suppressor gene, p53. It will be indispensable in aiding our understanding of the molecular mechanisms causing claudin-low breast cancer and has the potential to be used in pre-clinical screening of new therapeutics.

Jennifer Knight-fig1

Acknowledgment

This research was supported with funds from a Terry Fox New Frontier grant (MP) in addition to the Canadian Institutes for Health Research (ILA, MP). JFK is funded by a Susan G. Komen for the Cure post-doctoral fellowship, SMS by a McGill Faculty of Medicine Studentship and RL by a CIHR studentship.

 

Reference:

1.         Lehmann BD, et al. (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 121(7):2750-2767.

2.         Herschkowitz JI, et al. (2007) Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol 8(5):R76.

3.         Prat A, et al. (2010) Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 12(5):R68.

4.         Ponzo MG & Park M (2010) The met receptor tyrosine kinase and basal breast cancer. Cell Cycle 9(6).

5.         Ponzo MG, et al. (2009) Met induces mammary tumors with diverse histologies and is associated with poor outcome and human basal breast cancer. Proc Natl Acad Sci U S A 106(31):12903-12908.

 

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