Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC 50 in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.

1Cx36 is the predominant connexin isoform expressed by pancreatic ␤-cells. However, little is known about the role of this protein in the functioning of insulin-secreting cells. To address this question, we searched for a cell line expressing Cx36 and having glucose-induced insulin secretion comparable to that of primary ␤-cells. T he well coordinated functioning of cells within tissues is ensured by multiple mechanisms, including the direct exchange of ions, second messengers, and other metabolites through connexin channels, clustered at gap junctions (1,2). These channels connect almost all types of vertebrate cells, including those of pancreatic islets (3). Previous studies suggest that connexin-mediated communication between ␤-cells is required for the control of insulin secretion. Thus, glucose stimulation of single ␤-cells is reduced compared with that of cell clusters (4), pharmacological blockade of gap junction channels markedly decreases insulin release (5), and transgenic mice whose ␤-cells express Cx32 show altered insulin secretion in response to glucose (6). While these findings provide evidence that connexin-dependent communication contributes to the control of insulin release, they do not specifically address the function of native connexins expressed by pancreatic islet cells.We recently found that Cx36 is the predominant isoform expressed by ␤-cells (7). As yet, however, the specific role of this somewhat unusual connexin isoform (8,9) and, in particular, its possible contribution to insulin secretion have not been investigated. Moreover, no data are available to show how connexin-mediated signaling may influence ␤-cell function even though gap junctions have been implicated in the synchronization of [Ca 2ϩ ] i oscillations in ␤-cells (10 -13).To address these questions, we screened MIN6 cells, which, unlike some other cell lines, retain glucose-induced insulin secretion and express Cx36 like primary ␤-cells (14 -19). We decreased Cx36 expression via the stable transfection of an antisense construct. Here, we show that the loss of Cx36 resulted in impaired electrical coupling, desynchronization of [Ca 2ϩ ] i oscillations, and altered insulin release of MIN6 cells. These results support the hypothesis that Cx36 plays a critical role in glucoseinduced insulin secretion. RESEARCH DESIGN AND METHODSCell lines and tissues. RIN2A (14), INS1 (15), and HIT cells (16) were cultured in RPMI-1640 medium, whereas ␤TC3 (17) and MIN6 cells (18) were cultured in Dulbecco's modified Eagle's medium as indicated in the original reports. Control tissues were obtained from OFA rats (olfactory bulb) or C57BL/6 mice (liver and heart), which were frozen in liquid nitrogen and stored at Ϫ80°C until use. RNA analysis. Samples of total RNA were extracted, reverse-transcribed, and amplified as previously described (7). To detect native Cx36, we amplified either a 980-bp or a 559-bp fragment, using the oligonucleotide pairs 5Ј-CACAGCGATGGGGGAATGGA-3Ј/5Ј-TGCCCTTTCACACATAGGCA-3Ј and 5Ј-GAGCCCAGGCCAAGAGGAAGTC-3Ј/5Ј-GG...

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Connexin channels clustered at gap junctions are obligatory attributes of all macroscopic endocrine and exocrine glands investigated so far and also connect most types of cells which produce secretory products in other tissues. Increasing evidence indicates that connexins, and the cell-to-cell communications that these proteins permit, contribute to control the growth of secretory cells, their expression of specific genes and their differentiated function, including their characteristic ability to biosynthetize and release secretory products in a regulated manner. Since the previous reviews which have been published on this topic, several lines of evidence have been added in support of multiple regulatory roles of gland connexins. Here, we review this novel evidence, point to the many questions which are still open and discuss some interesting perspectives of the field.

Transforming growth factor ␤ (TGF-␤) and platelet-derived growth factor A (PDGF〈) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-␤ signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C ؊/؊ and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-␤1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C ؊/؊ mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C ؊/؊ mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-␤ in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Triple-negative breast cancer (TNBC) is a heterogeneous group of tumors characterized by aggressive behavior, high risk of distant recurrence, and poor survival. Chemotherapy is still the main therapeutic approach for this subgroup of patients, therefore, progress in the treatment of TNBC remains an important challenge. Data derived from molecular technologies have identified TNBCs with different gene expression and mutation profiles that may help developing targeted therapies. So far, however, only a few of these have shown to improve the prognosis and outcomes of TNBC patients. Robust predictive biomarkers to accelerate clinical progress are needed. Herein, we review prognostic and predictive biomarkers in TNBC, discuss the current evidence supporting their use, and look at the future of this research field.

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