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There is increasing evidence that breast tumors are organized in a hierarchy, with a subpopulation of tumorigenic cancer cells, the cancer stem cells (CSCs), which sustain tumor growth. The characterization of protein networks that govern CSC behavior is paramount to design new therapeutic strategies targeting this subpopulation of cells. We have sought to identify specific molecular pathways of CSCs isolated from 13 different breast cancer cell lines of luminal or basal/mesenchymal subtypes. We compared the gene expression profiling of cancer cells grown in adherent conditions to those of matched tumorsphere cultures. No specific pathway was identified to be commonly regulated in luminal tumorspheres, resulting from a minor CSC enrichment in tumorsphere passages from luminal cell lines. However, in basal/mesenchymal tumorspheres, the enzymes of the mevalonate metabolic pathway were overexpressed compared to those in cognate adherent cells. Inhibition of this pathway with hydroxy-3-methylglutaryl CoA reductase blockers resulted in a reduction of breast CSC independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Further modulation of this metabolic pathway demonstrated that protein geranylgeranylation (GG) is critical to breast CSC maintenance. A small molecule inhibitor of the geranylgeranyl transferase I (GGTI) enzyme reduced the breast CSC subpopulation both in vitro and in primary breast cancer xenografts. We found that the GGTI effect on the CSC subpopulation is mediated by inactivation of Ras homolog family member A (RHOA) and increased accumulation of P27(kip1) in the nucleus. The identification of protein GG as a major contributor to CSC maintenance opens promising perspectives for CSC targeted therapy in basal breast cancer., (Copyright © 2012 AlphaMed Press.)

The aim of the present study was to characterize human side population (SP) epidermal keratinocytes isolated from primary cell cultures. For that purpose, keratinocytes were isolated from normal adult breast skin samples and the Hoechst 33342 exclusion assay described for hematopoietic cells was adapted to keratinocytes. Three types of keratinocytes were studied: the SP, the main population (MP), and the unsorted initial population. SP keratinocytes represented 0.16% of the total population. In short-term cultures, they exhibited an increased colony-forming efficiency and produced more actively growing colonies than did unsorted and MP keratinocytes. In long-term cultures, SP cells exhibited an extensive expansion potential, performing a mean of 44 population doublings for up to 12 successive passages after cell sorting. Moreover, even in long-term cultures, SP keratinocytes were able to form a pluristratified epidermis when seeded on a dermal substrate. Unsorted and MP keratinocytes promoted a reduced expansion: mean values of 14 population doublings for five passages and 12 population doublings for four successive passages, respectively. To further characterize SP cells, cDNA microarrays were used to identify their molecular signature. Transcriptome profiling showed that 41 genes were differentially expressed in SP (vs. MP) cells, with 37 upregulated genes and only four downregulated genes in SP cells. The majority of these genes were functionally related to the regulation of transcription and cell signaling. In conclusion, SP human keratinocytes isolated from primary cultures exhibited both short- and long-term high proliferative potential, formed a pluristratified epidermis, and were characterized by a specific gene expression profile.

Connexins (Cx) are the protein subunits of gap junctions, which play an important role in cell-to-cell communication. We characterized the genomic structure of the human GJB6 gene, encoding connexin 30 (C x 30), and showed that it differs from most connexin-encoding genes. GJB6 presents six different exons, some of which can be alternatively spliced. We also mapped a basal promoter sequence active in a human keratinocyte cell line which responds to the activation of the EGF receptor. One of the non-encoding exons of GJB6, which has been described in brain C x 30 cDNA, was not found in cDNA obtained from human keratinocytes, suggesting tissue-specific splicing.

Arrhythmogenic right ventricular dysplasia type 2 (ARVD2, OMIM 600996) is an autosomal dominant cardiomyopathy, characterized by partial degeneration of the myocardium of the right ventricle, electrical instability and sudden death. The disease locus was mapped to chromosome 1q42--q43. We report here on the physical mapping of the critical ARVD2 region, exclusion of two candidate genes (actinin 2 and nidogen), elucidation of the genomic structure of the cardiac ryanodine receptor gene (RYR2) and identification of RYR2 mutations in four independent families. In myocardial cells, the RyR2 protein, activated by Ca(2+), induces the release of calcium from the sarcoplasmic reticulum into the cytosol. RyR2 is the cardiac counterpart of RyR1, the skeletal muscle ryanodine receptor, involved in malignant hyperthermia (MH) susceptibility and in central core disease (CCD). The RyR2 mutations detected in the present study occurred in two highly conserved regions, strictly corresponding to those where mutations causing MH or CCD are clustered in the RYR1 gene. The detection of RyR2 mutations causing ARVD2, reported in this paper, opens the way to pre-symptomatic detection of carriers of the disease in childhood, thus enabling early monitoring and treatment.