Membrane Bound Peroxiredoxin-1 Serves as a Biomarker for In Vivo Detection of Sessile Serrated Adenomas.

Aim: Sessile serrated adenomas (SSAs) are premalignant lesions driven by the BRAFV600E mutation to give rise to colorectal cancers (CRCs). They are often missed during white light colonoscopy because of their subtle appearance. Previously, a fluorescently labeled 7mer peptide KCCFPAQ was shown to detect SSAs in vivo. We aim to identify the target of this peptide. Results: Peroxiredoxin-1 (Prdx1) was identified as the binding partner of the peptide ligand. In vitro binding assays and immunofluorescence staining of human colon specimens ex vivo supported this result. Prdx1 was overexpressed on the membrane of cells with the BRAFV600E mutation, and this effect was dependent on oxidative stress. RKO cells harboring the BRAFV600E mutation and human SSA specimens showed higher oxidative stress as well as elevated levels of Prdx1 on the cell membrane. Innovation and Conclusion: These results suggest that Prdx1 is overexpressed on the cell surface in the presence of oxidative stress and can serve as an imaging biomarker for in vivo detection of SSAs. Antioxid. Redox Signal. 36, 39-56.

Combinatorial ETS1-dependent control of oncogenic NOTCH1 enhancers in T-cell leukemia.

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to co-bind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis, but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and points to Ets1-mediated enucleation of Notch-Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition.

Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents.

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1WTversus Kir2.1Δ314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.

Integrated Imaging Methodology Detects Claudin-1 Expression in Premalignant Nonpolypoid and Polypoid Colonic Epithelium in Mice.

OBJECTIVES: Conventional colonoscopy with white light illumination detects colonic adenomas based on structural changes alone and is limited by a high miss rate. We aim to demonstrate an integrated imaging strategy that combines wide-field endoscopy and confocal endomicroscopy in real time to visualize molecular expression patterns in vivo to detect premalignant colonic mucosa. METHODS: A peptide specific for claudin-1 is labeled with Cy5.5 and administrated intravenously in genetically engineered mice that develop adenomas spontaneously in the distal colon. Wide-field endoscopy is used to identify the presence of nonpolypoid and polypoid adenomas. Anatomic landmarks are used to guide placement of a confocal endomicroscope with side-view optics to visualize claudin-1 expression patterns with subcellular resolution. RESULTS: Wide-field fluorescence images show peak uptake in colon adenoma at ∼1 hour after systemic peptide administration, and lesion margins are clearly defined. Further examination of the lesion using a confocal endomicroscope shows dysplastic crypts with large size, elongated shape, distorted architecture, and variable dimension compared with normal. The mean fluorescence intensity is significantly higher for dysplasia than normal. Increased claudin-1 expression in dysplasia vs normal is confirmed ex vivo, and the binding pattern is consistent with the in vivo imaging results. DISCUSSION: Wide-field endoscopy can visualize molecular expression of claudin-1 in vivo to localize premalignant colonic mucosa, and confocal endomicroscopy can identify subcellular feature to distinguish dysplasia from normal.

Murine Oviductal High-Grade Serous Carcinomas Mirror the Genomic Alterations, Gene Expression Profiles, and Immune Microenvironment of Their Human Counterparts.

Robust preclinical models of ovarian high-grade serous carcinoma (HGSC) are needed to advance our understanding of HGSC pathogenesis and to test novel strategies aimed at improving clinical outcomes for women with the disease. Genetically engineered mouse models of HGSC recapitulating the likely cell of origin (fallopian tube), underlying genetic defects, histology, and biologic behavior of human HGSCs have been developed. However, the degree to which the mouse tumors acquire the somatic genomic changes, gene expression profiles, and immune microenvironment that characterize human HGSCs remains unclear. We used integrated molecular characterization of oviductal HGSCs arising in the context of Brca1, Trp53, Rb1, and Nf1 (BPRN) inactivation to determine whether the mouse tumors recapitulate human HGSCs across multiple domains of molecular features. Targeted DNA sequencing showed the mouse BPRN tumors, but not endometrioid carcinoma-like tumors based on different genetic defects (e.g., Apc and Pten), acquire somatic mutations and widespread copy number alterations similar to those observed in human HGSCs. RNA sequencing showed the mouse HGSCs most closely resemble the so-called immunoreactive and mesenchymal subsets of human HGSCs. A combined immuno-genomic analysis demonstrated the immune microenvironment of BPRN tumors models key aspects of tumor-immune dynamics in the immunoreactive and mesenchymal subtypes of human HGSC, with enrichment of immunosuppressive cell subsets such as myeloid-derived suppressor cells and regulatory T cells. The findings further validate the BPRN model as a robust preclinical experimental platform to address current barriers to improved prevention, diagnosis, and treatment of this often lethal cancer. SIGNIFICANCE: The acquired gene mutations, broad genomic alterations, and gene expression and immune cell-tumor axis changes in a mouse model of oviductal serous carcinoma closely mirror those of human tubo-ovarian high-grade serous carcinoma.

Detection of colonic neoplasia in vivo using near-infrared-labeled peptide targeting cMet.

White light colonoscopy is widely used to detect colorectal polyps, but flat and depressed lesions are often missed. Here, we report a molecular imaging strategy to potentially improve diagnostic performance by developing a fluorescently-labeled peptide specific for cMet. This 7mer is conjugated to Cy5.5, a near-infrared (NIR) cyanine dye. Specific binding to cMet was confirmed by cell staining, knockdown, and competition assays. The probe showed high binding affinity (kd = 57 nM) and fast onset (k = 1.6 min) to support topical administration in vivo. A mouse model (CPC;Apc) that develops spontaneous adenomas that overexpress cMet was used to demonstrate feasibility for real time in vivo imaging. This targeting ligand showed significantly higher target-to-background (T/B) ratio for polypoid and non-polypoid lesions by comparison with a scrambled control peptide. Immunofluorescence staining on human colon specimens show significantly greater binding to tubular and sessile serrated adenomas versus hyperplastic polyps and normal mucosa. These results demonstrate a peptide specific for cMet that is promising for endoscopic detection of pre-malignant lesions and guiding of tissue biopsy.

Trp53 null and R270H mutant alleles have comparable effects in regulating invasion, metastasis, and gene expression in mouse colon tumorigenesis.

Somatic APC (adenomatous polyposis coli), TP53, KRAS mutations are present in roughly 80%, 60%, and 40%, respectively, of human colorectal cancers (CRCs). Most TP53 mutant alleles in CRCs encode missense mutant proteins with loss-of-function (LOF) of p53's transcriptional activity and dominant negative (DN) effects on wild-type p53 function. Missense mutant p53 proteins have been reported to exert gain-of-function (GOF) effects in cancer. We compared the phenotypic effects of the common human cancer-associated TP53 R273H missense mutation to p53 null status in a genetically engineered mouse CRC model. Inactivation of one allele of Apc together with activation of a Kras mutant allele in mouse colon epithelium instigated development of serrated and hyperplastic epithelium and adenomas (AK mice). Addition of a Trp53R270H or Trp53null mutant allele to the model (AKP mice) led to markedly shortened survival and increased tumor burden relative to that of AK mice, including adenocarcinomas in AKP mice. Comparable life span and tumor burden were seen in AKP mice carrying Trp53R270H or Trp53null alleles, along with similar frequencies of spontaneous metastasis to lymph nodes, lung, and liver. The fraction of adenocarcinomas with submucosa or deeper invasion was higher in AKP270/fl mice than in AKPfl/fl mice, but the incidence of adenocarcinomas per mouse did not differ significantly between AKPfl/fl and AKP270/fl mice. In line with their comparable biological behaviors, mouse primary tumors and tumor-derived organoids with the Trp53R270H or Trp53null alleles had highly similar gene expression profiles. Human CRCs with TP53 R273 missense mutant or null alleles also had essentially homogeneous gene expression patterns. Our findings indicate the R270H/R273H p53 mutant protein does not manifest definite GOF biological effects in mouse and human CRCs, suggesting possible GOF effects of mutant p53 in cancer phenotypes are likely allele-specific and/or context-dependent.

Lineage tracing suggests that ovarian endosalpingiosis does not result from escape of oviductal epithelium.

Most high-grade serous carcinomas are thought to arise from Fallopian tube epithelium (FTE), but some likely arise outside of the tube, perhaps from ectopic tubal-type epithelium known as endosalpingiosis. Importantly, the origin of endosalpingiosis is poorly understood. The proximity of the tubal fimbriae to the ovaries has led to the proposal that disruptions in the ovarian surface that occur during ovulation may allow detached FTE to implant in the ovary and form tubal-type glands and cysts. An alternative model suggests that cells present in ectopic locations outside the Müllerian tract retain the capacity for multi-lineage differentiation and can form glands with tubal-type epithelium. We used double transgenic Ovgp1-iCreERT2 ;R26RLSL-eYFP mice, which express an eYFP reporter protein in OVGP1-positive tissues following transient tamoxifen (TAM) treatment, to track the fate of oviductal epithelial cells. Cohorts of adult mice were given TAM to activate eYFP expression in oviductal epithelium, and ovaries were examined at time points ranging from 2 days to 12 months post-TAM. To test whether superovulation might increase acquisition of endosalpingiosis, additional cohorts of TAM-treated mice underwent up to five cycles of superovulation and ovaries were examined at 1, 6, and 12 months post-TAM. Ovaries were sectioned in their entirety to identify endosalpingiosis. Immunohistochemical staining for PAX8, tubulin, OVGP1, and eYFP was employed to study endosalpingiosis lesions. Ovarian endosalpingiosis was identified in 14.2% of TAM-treated adult mice. The endosalpingiotic inclusion glands and cysts were lined by secretory and ciliated cells and expressed PAX8, tubulin, OVGP1, and eYFP. Neither age nor superovulation was associated with a significant increase in endosalpingiosis. Endosalpingiosis was also occasionally present in the ovaries of pre-pubertal mice. The findings imply that ovarian endosalpingiosis in the mouse does not likely arise as a consequence of detachment and implantation of tubal epithelium and other mechanisms may be relevant. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identification of Tumor Specific Peptide as EpCAM Ligand and Its Potential Diagnostic and Therapeutic Clinical Application.

Tumor targeting agents are being developed for early tumor detection and therapeutics. We previously identified the peptide SNFYMPL (SNF*) and demonstrated its specific binding to human esophageal specimens of high-grade dysplasia (HGD) and adenocarcinoma with imaging ex vivo. Here, we aim to identify the target for this peptide and investigate its potential applications in imaging and drug delivery. With SNF* conjugated affinity chromatography, mass spectrum, Western blot, enzyme-linked immunosorbent assay (ELISA), and molecular docking, we found that the epithelial cell adhesion molecule (EpCAM) was the potential target of SNF*. Next, we showed that FITC-labeled SNF* (SNF*-FITC) colocalized with EpCAM antibody on the surface of esophageal adenocarcinoma cells OE33, and SNF*-FITC binding patterns significantly changed after EpCAM knockdown or exogenous EpCAM transfection. With the data from TCGA, we demonstrated that EpCAM was overexpressed in 17 types of cancers. Using colon and gastric adenocarcinoma cells and tissues as examples, we found that SNF*-FITC bound in a pattern was colocalized with EpCAM antibody, and the SNF* binding did not upregulate the EpCAM downstream Wnt signals. Subsequently, we conjugated SNF* with our previously constructed poly(histidine)-PEG/DSPE copolymer micelles. SNF* labeling significantly improved the micelle binding with colon and gastric adenocarcinoma cells in vitro, and enhanced the antitumor effects and decreased the toxicities of the micelles in vivo. In conclusion, we identified and validated SNF* as a specific peptide for EpCAM. The future potential use of SNF* peptide in multiple tumor surveillance and tumor-targeted therapeutics was demonstrated.

Constitutively Higher Level of GSTT2 in Esophageal Tissues From African Americans Protects Cells Against DNA Damage.

BACKGROUND & AIMS: African American and European American individuals have a similar prevalence of gastroesophageal reflux disease (GERD), yet esophageal adenocarcinoma (EAC) disproportionately affects European American individuals. We investigated whether the esophageal squamous mucosa of African American individuals has features that protect against GERD-induced damage, compared with European American individuals. METHODS: We performed transcriptional profile analysis of esophageal squamous mucosa tissues from 20 African American and 20 European American individuals (24 with no disease and 16 with Barrett's esophagus and/or EAC). We confirmed our findings in a cohort of 56 patients and analyzed DNA samples from patients to identify associated variants. Observations were validated using matched genomic sequence and expression data from lymphoblasts from the 1000 Genomes Project. A panel of esophageal samples from African American and European American subjects was used to confirm allele-related differences in protein levels. The esophageal squamous-derived cell line Het-1A and a rat esophagogastroduodenal anastomosis model for reflux-generated esophageal damage were used to investigate the effects of the DNA-damaging agent cumene-hydroperoxide (cum-OOH) and a chemopreventive cranberry proanthocyanidin (C-PAC) extract, respectively, on levels of protein and messenger RNA (mRNA). RESULTS: We found significantly higher levels of glutathione S-transferase theta 2 (GSTT2) mRNA in squamous mucosa from African American compared with European American individuals and associated these with variants within the GSTT2 locus in African American individuals. We confirmed that 2 previously identified genomic variants at the GSTT2 locus, a 37-kb deletion and a 17-bp promoter duplication, reduce expression of GSTT2 in tissues from European American individuals. The nonduplicated 17-bp promoter was more common in tissue samples from populations of African descendant. GSTT2 protected Het-1A esophageal squamous cells from cum-OOH-induced DNA damage. Addition of C-PAC increased GSTT2 expression in Het-1A cells incubated with cum-OOH and in rats with reflux-induced esophageal damage. C-PAC also reduced levels of DNA damage in reflux-exposed rat esophagi, as observed by reduced levels of phospho-H2A histone family member X. CONCLUSIONS: We found GSTT2 to protect esophageal squamous cells against DNA damage from genotoxic stress and that GSTT2 expression can be induced by C-PAC. Increased levels of GSTT2 in esophageal tissues of African American individuals might protect them from GERD-induced damage and contribute to the low incidence of EAC in this population.

Loss of AMPKα2 Impairs Hedgehog-Driven Medulloblastoma Tumorigenesis.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that has a dual role in cancer, i.e., pro- or anti-tumorigenic, depending on the context. In medulloblastoma, the most frequent malignant pediatric brain tumor, several in vitro studies previously showed that AMPK suppresses tumor cell growth. The role of AMPK in this disease context remains to be tested in vivo. Here, we investigate loss of AMPKα2 in a genetically engineered mouse model of sonic hedgehog (SHH)-medulloblastoma. In contrast to previous reports, our study reveals that AMPKα2 KO impairs SHH medulloblastoma tumorigenesis. Moreover, we performed complementary molecular and genomic analyses that support the hypothesis of a pro-tumorigenic SHH/AMPK/CNBP axis in medulloblastoma. In conclusion, our observations further underline the context-dependent role of AMPK in cancer, and caution is warranted for the previously proposed hypothesis that AMPK agonists may have therapeutic benefits in medulloblastoma patients. Note: an abstract describing the project was previously submitted to the American Society for Investigative Pathology PISA 2018 conference and appears in The American Journal of Pathology (Volume 188, Issue 10, October 2018, Page 2433).

Stage-specific roles for Zmiz1 in Notch-dependent steps of early T-cell development.

Notch1 signaling must elevate to high levels in order to drive the proliferation of CD4-CD8- double-negative (DN) thymocytes and progression to the CD4+CD8+ double-positive (DP) stage through ß-selection. During this critical phase of pre-T-cell development, which is also known as the DN-DP transition, it is unclear whether the Notch1 transcriptional complex strengthens its signal output as a discrete unit or through cofactors. We previously showed that the protein inhibitor of activated STAT-like coactivator Zmiz1 is a context-dependent cofactor of Notch1 in T-cell leukemia. We also showed that withdrawal of Zmiz1 generated an early T-lineage progenitor (ETP) defect. Here, we show that this early defect seems inconsistent with loss-of-Notch1 function. In contrast, at the later pre-T-cell stage, withdrawal of Zmiz1 impaired the DN-DP transition by inhibiting proliferation, like withdrawal of Notch. In pre-T cells, but not ETPs, Zmiz1 cooperatively regulated Notch1 target genes Hes1, Lef1, and Myc. Enforced expression of either activated Notch1 or Myc partially rescued the Zmiz1-deficient DN-DP defect. We identified residues in the tetratricopeptide repeat (TPR) domain of Zmiz1 that bind Notch1. Mutating only a single residue impaired the Zmiz1-Notch1 interaction, Myc induction, the DN-DP transition, and leukemic proliferation. Similar effects were seen using a dominant-negative TPR protein. Our studies identify stage-specific roles of Zmiz1. Zmiz1 is a context-specific cofactor for Notch1 during Notch/Myc-dependent thymocyte proliferation, whether normal or malignant. Finally, we highlight a vulnerability in leukemic cells that originated from a developmentally important Zmiz1-Notch1 interaction that is hijacked during transformation from normal pre-T cells.

Multiplexed Targeting of Barrett's Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft in Vivo and Human Specimens ex Vivo.

Esophageal adenocarcinoma (EAC) is a molecularly heterogeneous disease that is rising rapidly in incidence and has poor prognosis. We developed a heterobivalent peptide to target detection of early Barrett's neoplasia by combining monomer heptapeptides specific for either EGFR or ErbB2 in a heterodimer configuration. The structure of a triethylene glycol linker was optimized to maximize binding interactions to the surface receptors on cells. The Cy5.5-labeled heterodimer QRH*-KSP*-E3-Cy5.5 demonstrated specific binding to each target and showed 3-fold greater fluorescence intensity and 2-fold higher affinity compared with those of either monomer alone. Peak uptake in xenograft tumors was observed at 2 h postinjection with systemic clearance by ∼24 h in vivo. Furthermore, ligand binding was evaluated on human esophageal specimens ex vivo, and 88% sensitivity and 87% specificity were found for the detection of either high-grade dysplasia (HGD) or EAC. This peptide heterodimer shows promise for targeted detection of early Barrett's neoplasia in clinical study.

Granulocyte-derived TNFα promotes vascular and hematopoietic regeneration in the bone marrow.

Endothelial cells are a critical component of the bone marrow (BM) stromal network, which maintains and regulates hematopoietic cells. Vascular regeneration precedes, and is necessary for, successful hematopoietic stem cell (HSC) transplantation, the only cure for most hematopoietic diseases. Recent data suggest that mature hematopoietic cells regulate BM stromal-cell function. Whether a similar cross-talk regulates the BM vasculature is not known. Here we found that donor hematopoietic cells act on sinusoidal endothelial cells and induce host blood vessel and hematopoietic regeneration after BM transplantation in mice. Adoptive transfer of BM, but not peripheral, granulocytes prevented the death of mice transplanted with limited numbers of HSCs and accelerated recovery of host vessels and hematopoietic cells. Moreover, selective granulocyte ablation in vivo impaired vascular and hematopoietic regeneration after BM transplantation. Gene expression analyses indicated that granulocytes are the main source of the cytokine TNFα, whereas its receptor TNFR1 is selectively upregulated in regenerating blood vessels. In adoptive transfer experiments, wild type, but not Tnfa-/-, granulocytes induced vascular recovery, and wild-type granulocyte transfer did not prevent death or promote vascular regeneration in Tnfr1-/-; Tnfr2-/- mice. Thus, by delivering TNFα to endothelial cells, granulocytes promote blood vessel growth and hematopoietic regeneration. Manipulation of the cross-talk between granulocytes and endothelial cells may lead to new therapeutic approaches to improve blood vessel regeneration and increase survival and hematopoietic recovery after HSC transplantation.

Identification and validation of FGFR2 peptide for detection of early Barrett's neoplasia.

The incidence of esophageal adenocarcinoma (EAC) is rising rapidly, and early detection within the precursor state of Barrett's esophagus (BE) is challenged by flat premalignant lesions that are difficult detect with conventional endoscopic surveillance. Overexpression of cell surface fibroblast growth factor receptor 2 (FGFR2) is an early event in progression of BE to EAC, and is a promising imaging target. We used phage display to identify the peptide SRRPASFRTARE that binds specifically to the extracellular domain of FGFR2. We labeled this peptide with a near-infrared fluorophore Cy5.5, and validated the specific binding to FGFR2 overexpressed in cells in vitro. We found high affinity kd = 68 nM and rapid binding k = 0.16 min-1 (6.2 min). In human esophageal specimens, we found significantly greater peptide binding to high-grade dysplasia (HGD) versus either BE or normal squamous epithelium, and good correlation with anti-FGFR2 antibody. We also observed significantly greater peptide binding to excised specimens of esophageal squamous cell carcinoma and gastric cancer compared to normal mucosa. These results demonstrate potential for this FGFR2 peptide to be used as a clinical imaging agent to guide tissue biopsy and improve methods for early detection of EAC and potentially other epithelial-derived cancers.

In vivo near-infrared imaging of ErbB2 expressing breast tumors with dual-axes confocal endomicroscopy using a targeted peptide.

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in large patient populations. Accurate assessment with physical biopsy and conventional pathology can be limited by tumor heterogeneity. We aim to demonstrate real-time optical sectioning using a near-infrared labeled ErbB2 peptide that generates tumor-specific contrast in human xenograft breast tumors in vivo. We used IRDye800CW as the fluorophore, validated performance characteristics for specific peptide binding to cells in vitro, and investigated peak peptide uptake in tumors using photoacoustic tomography. We performed real-time optical imaging using a handheld dual-axes confocal fluorescence endomicroscope that collects light off-axis to reduce tissue scattering for greater imaging depths. Optical sections in either the vertical or horizontal plane were collected with sub-cellular resolution. Also, we found significantly greater peptide binding to pre-clinical xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. We used a scrambled peptide for control. Peptide biodistribution showed high tumor uptake by comparison with other organs to support safety. This novel integrated imaging strategy is promising for visualizing ErbB2 expression in breast tumors and serve as an adjunct during surgery to improve diagnostic accuracy, identify tumor margins, and stage early cancers.

RBPJ/CBF1 interacts with L3MBTL3/MBT1 to promote repression of Notch signaling via histone demethylase KDM1A/LSD1.

Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.

Ultrasmall Paramagnetic Iron Oxide Nanoprobe Targeting Epidermal Growth Factor Receptor for In Vivo Magnetic Resonance Imaging of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common worldwide cancer that is rising rapidly in incidence. MRI is a powerful noninvasive imaging modality for HCC detection, but lack of specific contrast agents limits visualization of small tumors. EGFR is frequently overexpressed in HCC and is a promising target. Peptides have fast binding kinetics, short circulatory half-life, low imaging background, high vascular permeability, and enhanced tissue diffusion for deep tumor penetration. We demonstrate a peptide specific for EGFR labeled with an ultrasmall paramagnetic iron oxide (UPIO) nanoparticle with 3.5 nm dimensions to target HCC using T1-weighted MRI. We modified the hydrophobic core with oleic acid and capped with PEGylated phospholipids DSPE-PEG and DSPE-PEG-Mal. The EGFR peptide is attached via thioether-mediated conjugation of a GGGSC linker to the maleimide-terminated phospholipids. On in vivo MR images of HCC xenograft tumors, we observed peak nanoprobe uptake at 2 h post-injection followed by a rapid return to baseline by ∼24 h. We measured significantly greater MR signal in tumor with the targeted nanoprobe versus scrambled peptide, blocked peptide, and Gadoteridol. Segmented regions on MR images support rapid renal clearance. No significant difference in animal weight, necropsy, hematology, and chemistry was found between treatment and control groups at one month post-injection. Our nanoprobe based on an EGFR specific peptide labeled with UPIO designed for high stability and biocompatibility showed rapid tumor uptake and systemic clearance to demonstrate safety and promise for clinical translation to detect early HCC.

High-grade serous carcinomas arise in the mouse oviduct via defects linked to the human disease.

Recent studies have suggested that the most common and lethal type of 'ovarian' cancer, i.e. high-grade serous carcinoma (HGSC), usually arises from epithelium on the fallopian tube fimbriae, and not from the ovarian surface epithelium. We have developed Ovgp1-iCreERT2 mice in which the Ovgp1 promoter controls expression of tamoxifen-regulated Cre recombinase in oviductal epithelium - the murine equivalent of human fallopian tube epithelium (FTE). We employed Ovgp1-iCreERT2 mice to show that FTE-specific inactivation of several different combinations of tumour suppressor genes that are recurrently mutated in human HGSCs - namely Brca1, Trp53, Rb1, and Nf1 - results in serous tubal intraepithelial carcinomas (STICs) that progress to HGSC or carcinosarcoma, and to widespread metastatic disease in a subset of mice. The cancer phenotype is highly penetrant and more rapid in mice carrying engineered alleles of all four tumour suppressor genes. Brca1, Trp53 and Pten inactivation in the oviduct also results in STICs and HGSCs, and is associated with diffuse epithelial hyperplasia and mucinous metaplasia, which are not observed in mice with intact Pten. Oviductal tumours arise earlier in these mice than in those with Brca1, Trp53, Rb1 and Nf1 inactivation. Tumour initiation and/or progression in mice lacking conditional Pten alleles probably require the acquisition of additional defects, a notion supported by our identification of loss of the wild-type Rb1 allele in the tumours of mice carrying only one floxed Rb1 allele. Collectively, the models closely recapitulate the heterogeneity and histological, genetic and biological features of human HGSC. These models should prove useful for studying the pathobiology and genetics of HGSC in vivo, and for testing new approaches for prevention, early detection, and treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.