The crossroads of breast cancer progression: insights into the modulation of major signaling pathways.

Cancer is the disease with highest public health impact in developed countries. Particularly, breast cancer has the highest incidence in women worldwide and the fifth highest mortality in the globe, imposing a significant social and economic burden to society. The disease has a complex heterogeneous etiology, being associated with several risk factors that range from lifestyle to age and family history. Breast cancer is usually classified according to the site of tumor occurrence and gene expression profiling. Although mutations in a few key genes, such as BRCA1 and BRCA2, are associated with high breast cancer risk, the large majority of breast cancer cases are related to mutated genes of low penetrance, which are frequently altered in the whole population. Therefore, understanding the molecular basis of breast cancer, including the several deregulated genes and related pathways linked to this pathology, is essential to ensure advances in early tumor detection and prevention. In this review, we outline key cellular pathways whose deregulation has been associated with breast cancer, leading to alterations in cell proliferation, apoptosis, and the delicate hormonal balance of breast tissue cells. Therefore, here we describe some potential breast cancer-related nodes and signaling concepts linked to the disease, which can be positively translated into novel therapeutic approaches and predictive biomarkers.

A p57 conditional mutant allele that allows tracking of p57-expressing cells.

p57Kip2 (p57) is a maternally expressed imprinted gene regulating growth arrest which belongs to the CIP/KIP family of cyclin-dependent kinase inhibitors. While initially identified as a cell cycle arrest protein through inhibition of cyclin and cyclin-dependent kinase complexes, p57 activity has also been linked to differentiation, apoptosis, and senescence. In addition, p57 has recently been shown to be involved in tumorigenesis and cell fate decisions in stem cells. Yet, p57 function in adult tissues remains poorly characterized due to the perinatal lethality of p57 knock-out mice. To analyze p57 tissue-specific activity, we generated a conditional mouse line (p57FL-ILZ/+ ) by flanking the coding exons 2-3 by LoxP sites. To track p57-expressing or mutant cells, the p57FL-ILZ allele also contains an IRES-linked ß-galactosidase reporter inserted in the 3' UTR of the gene. Here, we show that the ß-galactosidase reporter expression pattern recapitulates p57 tissue specificity during development and in postnatal mice. Furthermore, we crossed the p57FL-ILZ/+ mice with PGK-Cre mice to generate p57cKO-ILZ/+ animals with ubiquitous loss of p57. p57cKO-ILZ/+ mice display developmental phenotypes analogous to previously described p57 knock-outs. Thus, p57FL-ILZ/+ is a new genetic tool allowing expression and functional conditional analyses of p57.

Gene Expression Profiling of Muscle Stem Cells Identifies Novel Regulators of Postnatal Myogenesis.

Skeletal muscle growth and regeneration require a population of muscle stem cells, the satellite cells, located in close contact to the myofiber. These cells are specified during fetal and early postnatal development in mice from a Pax3/7 population of embryonic progenitor cells. As little is known about the genetic control of their formation and maintenance, we performed a genome-wide chronological expression profile identifying the dynamic transcriptomic changes involved in establishment of muscle stem cells through life, and acquisition of muscle stem cell properties. We have identified multiple genes and pathways associated with satellite cell formation, including set of genes specifically induced (EphA1, EphA2, EfnA1, EphB1, Zbtb4, Zbtb20) or inhibited (EphA3, EphA4, EphA7, EfnA2, EfnA3, EfnA4, EfnA5, EphB2, EphB3, EphB4, EfnBs, Zfp354c, Zcchc5, Hmga2) in adult stem cells. Ephrin receptors and ephrins ligands have been implicated in cell migration and guidance in many tissues including skeletal muscle. Here we show that Ephrin receptors and ephrins ligands are also involved in regulating the adult myogenic program. Strikingly, impairment of EPHB1 function in satellite cells leads to increased differentiation at the expense of self-renewal in isolated myofiber cultures. In addition, we identified new transcription factors, including several zinc finger proteins. ZFP354C and ZCCHC5 decreased self-renewal capacity when overexpressed, whereas ZBTB4 increased it, and ZBTB20 induced myogenic progression. The architectural and transcriptional regulator HMGA2 was involved in satellite cell activation. Together, our study shows that transcriptome profiling coupled with myofiber culture analysis, provides an efficient system to identify and validate candidate genes implicated in establishment/maintenance of muscle stem cells. Furthermore, tour de force transcriptomic profiling provides a wealth of data to inform for future stem cell-based muscle therapies.

The A17 enhancer directs expression of Myf5 to muscle satellite cells but Mrf4 to myonuclei.

The myogenic regulatory factors, Myf5 and Mrf4, play key roles in the specification and differentiation of skeletal muscle, respectively. Many cis-acting regulatory elements at the Mrf4/Myf5 locus have been identified, including the A17 enhancer. During development, A17 in conjunction with the Mrf4 or Myf5 promoter, directs transgene expression either to embryonic or fetal muscles. We now show that this enhancer also regulates Mrf4/Myf5 transcription in the adult. A17 linked to the Myf5 promoter drives expression in muscle satellite cells, whereas with the Mrf4 promoter, A17 directs transgene expression to myonuclei. Interestingly, expression of A17-Mrf4-nlacZ transgenes in myonuclei varies between muscles, revealing muscle autonomous transcriptional regulation. During muscle repair, satellite cells are induced to proliferate and differentiate to provide new myonuclei. A17 directs Myf5 expression in satellite cell progeny while it only drives the Mrf4 promoter after differentiation. Importantly, therefore, this promoter-specific activity directed by A17 reflects aspects of the expression profiles of the endogenous Myf5 and Mrf4 genes.

An enhancer directs differential expression of the linked Mrf4 and Myf5 myogenic regulatory genes in the mouse.

The myogenic regulatory factors, Mrf4 and Myf5, play a key role in skeletal muscle formation. An enhancer trap approach, devised to isolate positive-acting elements from a 200-kb YAC covering the mouse Mrf4-Myf5 locus in a C2 myoblast assay, yielded an enhancer, A17, which mapped at -8 kb 5' of Mrf4 and -17 kb 5' of Myf5. An E-box bound by complexes containing the USF transcription factor is critical for enhancer activity. In transgenic mice, A17 gave two distinct and mutually exclusive expression profiles before birth, which correspond to two phases of Mrf4 transcription. Linked to the Tk or Mrf4 minimal promoters, the nlacZ reporter was expressed either in embryonic myotomes, or later in fetal muscle, with the majority of Mrf4 lines showing embryonic expression. When linked to the Myf5 minimal promoter, only fetal muscle expression was detected. These observations identify A17 as a sequence that targets sites of myogenesis in vivo and raise questions about the mutually exclusive modes of expression and possible promoter/enhancer interactions at the Mrf4-Myf5 locus.