SUMMARY ID genes are required for breast cancer colonization of the lungs, but the mechanism remains poorly understood. Here, we show that Id1 expression induces a stem-like phenotype in breast cancer cells, while retaining epithelial properties, contrary to the notion that cancer stem-like properties are inextricably linked to the mesenchymal state. During metastatic colonization, Id1 induces a mesenchymal-to-epithelial transition (MET), specifically in cells whose mesenchymal state is dependent on the Id1 target protein Twist1 but not at the primary site, where this state is controlled by the zinc-finger protein Snail1. Knockdown of Id expression in metastasizing cells prevents MET and dramatically reduces lung colonization. Furthermore, Id1 is induced by TGFβ only in cells that have first undergone EMT, demonstrating that EMT is a pre-requisite for subsequent Id1-induced MET during lung colonization. Collectively, these studies underscore the importance of Id-mediated phenotypic switching during distinct stages of breast cancer metastasis.
The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1؉1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1؉1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element.Adeno-associated virus type 2 (AAV2) is a nonpathogenic single-stranded DNA parvovirus that requires coinfection with a helper virus (adenovirus [Ad] or herpes virus) to stimulate progression through the viral replicative cycle. The Ad helper virus expresses the immediate early E1A gene products which have been shown to transactivate the AAV2 p5 promoter (1, 2), resulting in expression of two alternatively spliced p5 transcripts that yield the p68 and p78 (p68/78) Rep polypeptides (8,33). The large Rep polypeptides possess endonuclease, helicase, and ATPase enzymatic activities (reviewed in reference 21). In addition to enzymatic properties, the N-terminal domain of Rep p68/78 has sequence-specific DNA binding capabilities that recognize a GAGC sequence motif (3,18,24,28). This motif is imperfectly repeated four times in the AAV2 origin element located in the hairpin inverted terminal repeat (ITR) present at both ends of the viral genome (18,24). Both the enzymatic and DNA binding functions of p68/78 are essential for full-length re...
Endocrine resistance is a major clinical issue. AP-1 is a transcription factor downstream of different growth factor receptors (GFR) and stress-related signaling cascades implicated in endocrine resistance. We have previously shown that acquired endocrine resistance is associated with increased AP-1 activity. Moreover, AP-1 modulates the estrogen receptor (ER) transcriptional program, especially upon high GFR signaling. We therefore hypothesized that interfering with AP-1 could circumvent endocrine resistance. Methods and results: AP-1 was genetically inhibited by siRNA or by stable expression of an inducible dominant-negative (DN) c-Jun in MCF7 cells. In vitro, siRNA c-Jun significantly inhibited the growth of acquired tamoxifen resistant (TamR) MCF7 derivatives (>95% inhibition, p = .001) but not of parental cells ( p = .06). Xenografts of two inducible DN c-Jun clones were established in nude mice. Mice were randomized to continued estrogen (E2) supplementation or to either estrogen deprivation (ED) or Tam, all in the presence or absence of DN c-Jun. AP-1 blockade significantly reduced time to tumor response ( p = .014 and p = .006 for the two clones) and time to tumor disappearance ( p = .001 and p = .0034) in the Tam group, with similar results in the ED group. In addition, AP-1 blockade significantly delayed TamR by increasing time to tumor doubling ( p = .002). Furthermore, induction of DN c-Jun resulted in dramatic tumor shrinkage after long-term Tam treatment, suggesting reversal of endocrine resistance with AP-1 blockade. Interestingly, no significant effect was observed on E2-stimulated tumor growth. Immunohystochemistry showed that AP-1 blockade reduced proliferation and induced apoptosis. A gene signature of our TamR MCF7 xenografts significantly overlapped ( p < 2E-16) with a putative gene list associated with EGF-induced ER-DNA binding sites that mostly contain the AP-1 motif. Pathway analysis of these genes identified the AP-1 member c-Fos as the most represented transcription factor. Conclusions: We show that AP-1 blockade increases tumor sensitivity and circumvents resistance to endocrine therapy. We suggest that AP-1 is critical in a switch in the ER transcriptional program and may be a new hallmark of endocrine resistance. Disclosure: All authors have declared no conflicts of interest. Background: Triple negative breast cancer (TNBC), defined by estrogen, progesterone and HER2 negativity, is a heterogeneous disease with limited targeted therapy. Molecular and immunohistochemical stratification have already identified several TNBC subgroups, characterised by different biological processes with possible implication for therapy. A clear picture of the various TNBC entities and their relationship(s), however, is still missing. Aim: To shed light on this problem we have analysed a collection of 111 needle-macrodissected, clinically-annotated TNBCs using an approach based on the integration of DNA copy-number aberrations, transcriptional data and publically available gene signatures. Methods: Allel...
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