The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tettransactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets.lentivirus | vector L oss-of-function studies represent a powerful means by which to gain insight into the molecular mechanisms behind complex biological processes. Until recently, these studies were practical for only a small set of genetically tractable model organisms (1, 2). The discovery of gene silencing through RNAi has made it possible to carry out loss-of-function studies in mammals. RNAi has transformed the way in which gene function can be investigated and has quickly become a versatile tool for a wide range of applications, including reverse genetics, highthroughput screens, and therapeutics (3). The application of RNAi technology both in vitro and in vivo has tremendous potential to further our knowledge of the molecular mechanisms that underpin both normal biology and human disease.To achieve efficient and long-term gene silencing, we have previously generated the retroviral-encoded microRNA-based shRNA PRIME (potent RNA interference using microRNA expression) vectors that are effective in suppressing expression of their intended gene targets from single proviral integrations (4). These vectors use the miR30 backbone and are expressed from RNA polymerase II promoters, which allows for exquisite regulation of shRNA expression. We have further generated genomewide shRNA libraries in these vectors that cover large numbers of mammalian transcripts for systematic study of gene function (5, 6). These shRNA libraries, as well as shRNA libraries generated by other laboratories (7,8), have been used to perform genetic screens in mammalian cell culture models for a variety of phenotypes, including cell transformation, synthetic lethal interactions, and resistance to chemotherapeutic treatments (9-15).Despite the success demonstrated by the above constitutive shRNA vectors, an inducible shRNA system would have obvious advantages in many experimental settings. First, the inducible shRNA system allows for the study of essential genes. Because constitutively expressed shRNAs targeting essential genes will be ...

Summary Molecular programs that mediate normal cell differentiation are required for oncogenesis and tumor cell survival in certain cancers. How cell lineage restricted genes specifically influence metastasis is poorly defined. In lung cancers, we uncovered a transcriptional program that is preferentially associated with distal airway epithelial differentiation and lung adenocarcinoma (ADC) progression. This program is regulated in part by the lineage transcription factors GATA6 and HOPX. These factors can cooperatively limit the metastatic competence of ADC cells, by modulating overlapping alveolar differentiation and invasogenic target genes. Thus, GATA6 and HOPX are critical nodes in a lineage-selective pathway that directly links effectors of airway epithelial specification to the inhibition of metastasis in the lung ADC subtype.

Interpenetrating polymer network (IPN), random copolymer, and homopolymer nanoparticles of acrylamide and acrylic acid were prepared using an inverse emulsion polymerization technique. Differential scanning calorimetry and Fourier-transform infrared spectroscopy were used to examine the molecular structure of the prepared polymeric nanoparticles. The spherical morphology and size (∼250 nm diameter) of the nanoparticles was confirmed using scanning electron microscopy. Dynamic light scattering was used to determine the monodispersity of the particle size distribution and examine the thermally responsive swelling properties of the polymeric nanoparticle structures. Of the particle systems studied, only the IPN nanoparticles exhibited a unique, rapid sigmoidal swelling transition with temperature. These systems also achieved a much larger relative swelling volume compared to random copolymer and homopolymer particles comprised of acrylamide and acrylic acid. Increased cross-linker density resulted in an overall decrease in the maximum relative swelling volume that was obtained.

Local symptoms, ECOG PS, cN stage and lactate dehydrogenase were independent predictors of lymph node metastasis in patients who underwent retroperitoneal lymph node dissection. Our predictive nomogram using these factors showed excellent discrimination and calibration.

SUMMARY Defining the molecular networks that drive breast cancer has led to therapeutic interventions and improved patient survival. However, the aggressive triple-negative breast cancer subtype (TNBC) remains recalcitrant to targeted therapies because its molecular etiology is poorly defined. In this study, we used a forward genetic screen to discover an oncogenic network driving human TNBC. SCYL1, TEX14, and PLK1 (“STP axis”) cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event in human TNBC. The STP axis induces REST degradation by phosphorylating a conserved REST phospho-degron and bridging REST interaction with the ubiquitin-ligase βTRCP. Inhibition of the STP axis leads to increased REST protein levels and impairs TNBC transformation, tumor progression, and metastasis. Expression of the STP axis correlates with low REST protein levels in human TNBCs and poor clinical outcome for TNBC patients. Our findings demonstrate that the STP-REST axis is a new molecular driver of human TNBC.