PERSPECTIVE PERSPECTIVE Cell Cycle 10:10, 1-8; May 15, 2011; © 2011 Landes Bioscience RB1 and p53 at the crossroad of EMT and triple negative breast cancer Zhe Jiang,1 Robert Jones,1 Jeff C. Liu,1 Tao Deng,1 Tyler Robinson,1 Philip E.D. Chung,1 Sharon Wang,1 Jason I. Herschkowitz,2 Sean E. Egan,3 Charles M. Perou4 and Eldad Zacksenhaus1,* 1Division of Cell and Molecular Biology; Toronto General Research Institute; University Health Network; Toronto, Ontario, Canada; 2Department of Molecular and Cellular Biology; Baylor College of Medicine; Houston, TX USA; 3Program in Developmental and Stem Cell Biology; The Hospital for Sick Children; Department of Molecular Genetics; University of Toronto; Toronto, Ontario, Canada; 4Lineberger Comprehensive Cancer Center; Department of Genetics and Pathology; University of North Carolina at Chapel Hill; Chapel Hill, NC USA riple negative breast cancer (TNBC) NEU-positive and Triple Negative (TN) Tis a heterogeneous disease that tumors, the latter of which do not express includes Basal-like and Claudin-low hormone receptors or HER2.1-7 TNBC tumors. The Claudin-low tumors are affects 15–30% of patients. By IHC it can enriched for features associated with be further divided into Basal-like breast epithelial-to-mesenchymal transition cancer and non-basal tumors, some of (EMT) and possibly for tumor initiating which exhibit features of EMT.8 Basal-like cells. Primary TNBCs respond relatively BCs express the basal cytokeratins (CK) well to conventional chemotherapy; CK5/6, CK14, CK17, and/or epidermal © 2011 Landes Bioscience. Landes ©2011 however, metastatic disease is virtually growth factor receptor (EGFR), whereas incurable. Thus, there is a great inter- non-basal TNBCs do not express these Do not distribute. not Do est in identifying specific therapeutic markers (reviewed in ref. 8–10). targets for TNBC. The tumor suppres- Transcriptional profiling identified five sor RB1 is frequently lost in Basal-like BC subtypes that overlap but are not nec- breast cancer. To test for a causative role essarily identical to the IHC-based clas- of RB1 gene loss in BC and for its effect sification. This includes ERα+ luminal A on specific subtypes, we deleted mouse and luminal B, HER2+, Basal-like and Rb in mammary stem/bipotent progeni- Claudin-low BC.7,9,11-16 The latter exhibits tor cells. This led to diverse mammary low expression of epithelial junction pro- tumors including TNBC, with a subset teins such as Claudin 3, 4 and 7, as well as of the latter containing p53 mutations low levels of E-Cadherin and high levels and exhibiting features of Basal-like BC of EMT markers, including Zeb1, Twist or EMT. Combined mutation of Rb and and Snail. Basal-like tumors and cell lines p53 in mammary stem/bipotent progeni- (referred to as Basal-A) express low levels tors induced EMT type tumors. Here, we of the tumor suppressor pRb and high review our findings and those of others, levels of the tumor suppressor p53, indica- which connect Rb and p53 to EMT in tive of stabilizing mutations.17,18 The status TNBC. Furthermore, we discuss how by of RB1 in Claudin-low BC and cell lines This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly. the issue is complete and page numbers have Once to printing. has been published online, prior This manuscript understanding this circuit and its vulner- (Basal-B) is yet to be established. abilities, we may identify novel therapy Breast tumors that form in Brca1 car- Key words: retinoblastoma, p53, AP-2, for TNBC. riers constitute a third group of TNBC, basal-like breast cancer, claudin low, which, like basal-like BC express high EMT Triple Negative Breast Cancer level of basal-cytokeratins and EGFR and Submitted: 04/01/11 (TNBC) display high incidence of p53 mutation. Brca1 tumors, however, do not typically Accepted: 04/04/11 Breast cancer (BC) is a heterogeneous dis- show loss of pRb.8 Thus, inactivation of DOI: ease that can be classified by immunohis- BRCA1 or RB1 may divide TNBC into *Correspondence to: Eldad Zacksenhaus; tochemistry (IHC) into Estrogen Receptor two major non-overlapping subclasses, Email: [email protected] alpha (ERα)-positive, HER2/ERBB2/ both with p53 mutations. Notably, pRb www.landesbioscience.com Cell Cycle 1 and BRCA1 interact with each other,19 as Snail1/2(Slug), Zeb1/2, Twist1/2 and as Basal-like/Basal-A, whereas other and this interaction has been recently Ladybird homeobox 1 (LBX1).32,36-38 (BT549, MDA-MB436) as mesenchy- confirmed in a non-biased genome-wide Activation of the EMT program enables mal/Claudin-low/Basal-B.16,18 Low RB1 mammalian protein-protein interaction transformed epithelial cells to invade gene expression and loss of heterozygos- analysis.20 BRCA1-induced growth arrest locally, to disseminate via blood or lym- ity (LOH) at the RB1 locus were subse- is pRb-dependent.21 It is therefore possible phatic vessels and to establish microme- quently identified in a high percentage of that these tumor suppressors act on a com- tastasis.34,35 Thus, EMT may account for luminal-B (61.5%) and Basal-like (72%) mon genetic pathway linking genomic major steps in the metastatic cascade, with subtypes.49,50 LOH does not always cor- stability with cell cycle progression; inacti- the exception of growth at the secondary relate with loss of immuno-reactivity. vation of either gene may suffice to disrupt site. The basis for this is not known, and this checkpoint and induce tumorigenic- Although most BCs do not appear mes- direct sequencing of RB1 in these tumors ity. Although Brca1 is not mutated in enchymal, some not only express EMT is needed to clarify the issue. In a more sporadic Basal-like BC, the gene is often inducers but also depend on their expres- recent study, loss of pRb protein expres- downregulated due to LOH or promoter sion for continuous growth. For example, sion coupled with high expression of p53, methylation, or the protein is inactivated MMTV-neu mammary tumor cells, which indicative of a stabilized mutant form, by post-translation modifications.8,9,22 are not mesenchymal, express Twist, and were demonstrated in most basal-like Thus, in carriers of germ-line mutations, knockdown of this transcription factor TNBCs.17 RB1 LOH, with or without loss Brca1 is completely inactivated and this causes cell senescence.39,40 Expression of of protein expression, was typically asso- may be sufficient to drive cancer indepen- EMT-inducers in these tumors may prime ciated with increased p16ink4a expression. dent of RB1, whereas in sporadic Basal- them for EMT in response to stromal or This CDK4/6 inhibitor is a direct tran- like BC, both RB1 and Brca1 may be other, yet to be defined, signals at tumor scriptional target of pRb-E2F, which sup- partially inactivated, each contributing to margins. Such tumors may not undergo presses its expression by inducing histone the high proliferation rate of these tumors. full EMT but rather a limited conversion H3K27-methylation and thereby recruit- However, the frequency in which RB1 and that allows for detachment of cells from ment of BMI1 repression complexes to the BRCA1 are co-inactivated, albeit partially, the tumor mass, increasing their motility p16ink4a promoter.51 Thus, p16ink4a expres- in basal-like BC is yet to be determined. and invasion. Interestingly, in pancreatic sion serves as a surrogate for pRb loss in © 2011 Landes Bioscience. Landes ©2011 cancer, metastatic initiating cells (MICs) TNBC. Furthermore, in pre-neoplastic The CSC-EMT-Claudin-low Link represent a subset of TICs.41 Thus, the lesions (DCIS), high p16ink4a expression Do not distribute. not Do relationship between EMT, TICs and combined with high Ki67 index is a pre- Many cancers are organized in a hierarchy MICs in BC needs to be further explored. dictor for tumor progression.49 In contrast whereby relatively rare Tumor Initiating In 2008, Mani et al. showed that to basal-like BC, less is known about the Cells (TICs; also referred to as Cancer breast TICs exhibit spindle morphology status of RB1 (protein, mRNA, mutation Stem Cells, CSC) can initiate and main- and express EMT markers when cultured and LOH) in Claudin-low BC. This gap tain primary tumor growth. These cells, in vitro.36 Moreover, expression of EMT is likely to be filled soon. like normal stem cells, can self-renew and inducers increased the breast TIC fraction Interestingly, a recent survey of RB1 differentiate to generate cells that have lost in a heterogeneous tumor cell popula- status in metastatic breast cancer revealed their tumorigenic potential.23-29 A breast tion, rendering them highly tumorigenic two cases with duplication of the entire cancer tumorigenicity signature based on and resistant to conventional chemother- gene.52 This may be related to a phenomena differential gene expression in TIC versus apy.32,36 Indeed, following therapy, resid- observed in colorectal carcinoma where non-TIC fractions has been developed.30-32 ual BC cells display mesenchymal and high expression of pRb was shown, para- Claudin-low BCs exhibit high similarity TIC features.33 An EMT signature has doxically, to protect from E2F-induced to the TIC signature—whereas Basal-like, recently been generated; when compared apoptosis.53,54 In line with this, expression ERα+ and HER2+ tumors share only mar- to BC subtypes, the signature most closely of constitutively active phospho-mutant ginal overlap.12,31,33 resembled Claudin-low BC.37 Thus, Rb transgenes in mouse mammary epi- EMT is an embryonic program by Claudin-low breast tumors highly express thelium induces adenocarcinoma.55 Thus, which epithelial cells lose cell-cell con- genes associated with the breast cancer like overexpression and loss-of-function tact and polarity.32,34,35 EMT also involves tumorigenicity and EMT signatures.