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Breast cancers enduring treatment with chemotherapy may be enriched for cancer stem cells or tumor-initiating cells, which have an enhanced capacity for self-renewal, tumor initiation, and/or metastasis. Breast cancer cells that express the type I tyrosine kinaselike orphan receptor ROR1 also may have such features. Here we find that the expression of ROR1 increased in breast cancer cells following treatment with chemotherapy, which also enhanced expression of genes induced by the activation of Rho-GTPases, Hippo-YAP/TAZ, or B lymphoma Mo-MLV insertion region 1 homolog (BMI1). Expression of ROR1 also enhanced the capacity of breast cancer cells to invade Matrigel, form spheroids, engraft in Rag2 − / − γ c − / − mice, or survive treatment with paclitaxel. Treatment of mice bearing breast cancer patient-derived xenografts (PDXs) with the humanized anti-ROR1 monoclonal antibody cirmtuzumab repressed expression of genes associated with breast cancer stemness, reduced activation of Rho-GTPases, Hippo-YAP/TAZ, or BMI1, and impaired the capacity of breast cancer PDXs to metastasize or reengraft Rag2 − / − γ c − / − mice. Finally, treatment of PDX-bearing mice with cirmtuzumab and paclitaxel was more effective than treatment with either alone in eradicating breast cancer PDXs. These results indicate that targeting ROR1 may improve the response to chemotherapy of patients with breast cancer.

Salinomycin, an antibiotic potassium ionophore, has been reported recently to act as a selective breast cancer stem cell inhibitor, but the biochemical basis for its anticancer effects is not clear. The Wnt/β-catenin signal transduction pathway plays a central role in stem cell development, and its aberrant activation can cause cancer. In this study, we identified salinomycin as a potent inhibitor of the Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomycin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Nigericin, another potassium ionophore with activity against cancer stem cells, exerted similar effects. In otherwise unmanipulated chronic lymphocytic leukemia cells with constitutive Wnt activation nanomolar concentrations of salinomycin down-regulated the expression of Wnt target genes such as LEF1, cyclin D1, and fibronectin, depressed LRP6 levels, and limited cell survival. Normal human peripheral blood lymphocytes resisted salinomycin toxicity. These results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal Wnt signaling by interfering with LPR6 phosphorylation, and thus impair the survival of cells that depend on Wnt signaling at the plasma membrane.

Although initially responsive to chemotherapy, many patients with ovarian cancer subsequently develop relapsed and potentially fatal metastatic disease, which is thought to develop from cancer stem cells (CSCs) that are relatively resistant to conventional therapy. Here, we show that CSCs express a type I receptor tyrosine kinase-like orphan receptor (ROR1), which is expressed during embryogenesis and by many different cancers, but not normal postpartum tissues. Ovarian cancers with high levels of ROR1 had stem cell-like gene-expression signatures. Furthermore, patients with ovarian cancers with high levels of ROR1 had higher rates of relapse and a shorter median survival than patients with ovarian cancers that expressed low-to-negligible amounts of ROR1 . We found that ROR1-positive (ROR1 ⁺) cells isolated from primary tumor-derived xenografts (PDXs) also expressed aldehyde dehydrogenase 1 (ALDH1) and had a greater capacity to form spheroids and to engraft immune-deficient mice than did ROR1-negative (ROR1 ᴺᵉᵍ) ovarian cancer cells isolated from the same tumor population. Treatment with UC-961, an anti-ROR1 mAb, or shRNA silencing of ROR1 inhibited expression of the polycomb ring-finger oncogene, Bmi-1, and other genes associated with the epithelial–mesenchymal transition. Moreover, shRNA silencing of ROR1, depletion of ROR1 ⁺ cells, or treatment with UC-961 impaired the capacity of ovarian cancer cells to form spheroids or tumor xenografts. More importantly, treatment with anti-ROR1 affected the capacity of the xenograft to reseed a virgin mouse, indicating that targeting ROR1 may affect CSC self-renewal. Collectively, these studies indicate that ovarian CSCs express ROR1, which contributes to their capacity to form tumors, making ROR1 a potential target for the therapy of patients with ovarian cancer. Significance This study demonstrates that the oncoembryonic surface antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR1), is expressed on human ovarian cancer stem cells (CSCs), on which it seems to play a functional role in promoting migration/invasion or spheroid formation in vitro and tumor engraftment in immune-deficient mice. Treatment with a humanized mAb specific for ROR1 (UC-961) could inhibit the capacity of ovarian cancer cells to migrate, form spheroids, or engraft immune-deficient mice. Moreover, such treatment inhibited the growth of tumor xenografts, which in turn had a reduced capacity to engraft immune-deficient mice and were relatively depleted of cells with features of CSC, suggesting that treatment with UC-961 could impair CSC renewal. Collectively, these studies indicate that ovarian CSCs express ROR1, which may be targeted for anti-CSC therapy.

WNT signaling is involved in maintaining stem cells in an undifferentiated state; however, it is often unclear which WNTs and WNT receptors are mediating these activities. Here we examined the role of the WNT receptor FZD7 in maintaining human embryonic stem cells (hESCs) in an undifferentiated and pluripotent state. FZD7 expression is significantly elevated in undifferentiated cells relative to differentiated cell populations, and interfering with its expression or function, either by short hairpin RNA-mediated knockdown or with a fragment antigen binding (Fab) molecule directed against FZD7, disrupts the pluripotent state of hESCs. The FZD7-specific Fab blocks signaling by Wnt3a protein by down-regulating FZD7 protein levels, suggesting that FZD7 transduces Wnt signals to activate Wnt/β-catenin signaling. These results demonstrate that FZD7 encodes a regulator of the pluripotent state and that hESCs require endogenous WNT/β-catenin signaling through FZD7 to maintain an undifferentiated phenotype.

Significance Identifying molecules that are specific to tumors for use in early detection, diagnosis, prognosis, and therapy is both a primary goal and a key discovery challenge across diverse areas of oncology. To discover ovarian tumor-specific molecules, we developed custom bioinformatics algorithms to analyze transcriptome sequence data of 296 ovarian cancer and 1,839 normal tissues and validated putative tumor-specific mRNA isoforms by RT–quantitative PCR. The results revealed multiple candidate diagnostic and therapeutic targets with unique sequences that were expressed in most of the cancers examined but not in normal tissues. The process we developed can be readily applied to identify diagnostic and therapeutic targets for any of the 30 or more tumor types for which large amounts of transcriptome data now exist. Tumor-specific molecules are needed across diverse areas of oncology for use in early detection, diagnosis, prognosis and therapy. Large and growing public databases of transcriptome sequencing data (RNA-seq) derived from tumors and normal tissues hold the potential of yielding tumor-specific molecules, but because the data are new they have not been fully explored for this purpose. We have developed custom bioinformatic algorithms and used them with 296 high-grade serous ovarian (HGS-OvCa) tumor and 1,839 normal RNA-seq datasets to identify mRNA isoforms with tumor-specific expression. We rank prioritized isoforms by likelihood of being expressed in HGS-OvCa tumors and not in normal tissues and analyzed 671 top-ranked isoforms by high-throughput RT-qPCR. Six of these isoforms were expressed in a majority of the 12 tumors examined but not in 18 normal tissues. An additional 11 were expressed in most tumors and only one normal tissue, which in most cases was fallopian or colon. Of the 671 isoforms, the topmost 5% ( n = 33) ranked based on having tumor-specific or highly restricted normal tissue expression by RT-qPCR analysis are enriched for oncogenic, stem cell/cancer stem cell, and early development loci—including ETV4, FOXM1, LSR, CD9, RAB11FIP4, and FGFRL1. Many of the 33 isoforms are predicted to encode proteins with unique amino acid sequences, which would allow them to be specifically targeted for one or more therapeutic strategies—including monoclonal antibodies and T-cell–based vaccines. The systematic process described herein is readily and rapidly applicable to the more than 30 additional tumor types for which sufficient amounts of RNA-seq already exist.

Vaccines are highly effective in preventing the spread of communicable diseases and are critical to overall public health. As immune stimulants vaccine adjuvants augment the level and longevity of these protective responses. Seeking novel adjuvants using parallel high throughput screens and subsequent systematic structure-activity relationship studies we identified an analogue of a hit compound, , that in screening assays retained induction of calcium (Ca ) influx, tetraspanin CD63 EV reporter activity and cell viability. Here, we further our analyses of the biological activity of related its potential use as a vaccine adjuvant. was tested for activation of murine bone marrow-derived dendritic cells (mBMDC) by flow cytometry for Ca entry, levels of CD80 and CD86 expression, and stimulation of antigen-specific T cell proliferation. Cytokines and IgG responses from BALB/c mice injected with as a single agent or as an adjuvant with ovalbumen were measured by ELISA. triggered store-operated Ca entry in mBMDC as well as increases in CD80 and CD86 surface expression. In co-culture experiments, this compound amplified the stimulation of cognate T cell proliferation. Intramuscular injection of elicited minimal systemic cytokine and chemokine release. When used as an adjuvant with ovalbumen, generated a significant antigen-specific IgG1 response with a higher splenocyte T helper 2 (Th2) cytokine response. activated mBMDCs associated with EV release and a store-operated calcium entry response. Enhanced cognate T cell proliferation was mediated either through direct engagement with compound-stimulated mBMDCs or indirectly via immunostimulatory extracellular vesicles released by -activated mBMDCs. elicited minimal systemic cytokine and chemokine release, demonstrating a promising safety profile. When used as an adjuvant in a murine vaccination model, enhanced the IgG1 response with an associated T helper 2 cytokine profile. Hence this compound shows promise as an adjuvant if a Th2 response is beneficial or in combination with other agents to provide a balanced immune response in vaccines.

We examined the sera of six patients before and after i.v. infusions of autologous chronic lymphocytic leukemia (CLL) cells transduced ex vivo with an adenovirus encoding CD154 (Ad-CD154). Five patients made high-titer antibodies against adenovirus and three made IgG reactive with a leukemia-associated surface antigen, which we identified as ROR1. Anti-ROR1 antibodies were not detected in the sera of untreated patients. We generated anti-ROR1 mAbs and found they reacted specifically with the CLL cells of all patients, but not with nonleukemic leukocytes, a wide variety of normal adult tissues, or blood mononuclear cells, including CD5⁺ B cells of healthy adults. ROR1 could bind Wnt5a, which induced activation of NF-κB when coexpressed with ROR1 in HEK293 cells and enhanced the survival of CLL cells in vitro, an effect that could be neutralized by posttreatment anti-ROR1 antisera. We conclude that patients with CLL can break immune tolerance to ROR1, which is an oncofetal surface antigen and survival-signaling receptor in this neoplastic disease.

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z′ factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.

Osteoarthritis (OA) is a leading cause of disability in Western society with multiple risk factors, including a complex genetic pattern. Identifying loci involved in the heredity of OA might lead to insights into the molecular pathogenesis of this common disorder. A previous genome scan mapped a primary hip OA susceptibility locus to chromosome 2q with a maximum multipoint logarithm of odds score of 1.6 in 378 affected sibling pair families. Here, microsatellite targeting of eight candidate genes in this region from 2q23-2q32 demonstrated significant associations with the tumor necrosis factor α-induced protein 6 gene in all probands and the integrin α6 and frizzled motif associated with bone development (FRZB) genes in female probands. However, genotyping showed lack of association for a nonsynonymous single-nucleotide polymorphism in tumor necrosis factor α-induced protein 6, whereas a single-nucleotide polymorphism in FRZB resulting in an Arg324Gly substitution at the carboxyl terminus was associated with hip OA in the female probands (P = 0.04). This association was confirmed in an independent cohort of female hip cases (n = 338; P = 0.04). In addition, a haplotype coding for substitutions of two highly conserved arginine residues (Arg200Trp and Arg324Gly) in FRZB was a strong risk factor for primary hip OA, with an odds ratio of 4.1 (P = 0.004). FRZB encodes secreted frizzled-related protein 3, which is a soluble antagonist of wingless (wnt) signaling. Variant secreted frizzled-related protein 3 with the Arg324Gly substitution had diminished ability to antagonize wnt signaling in vitro. Hence, functional polymorphisms within FRZB confer susceptibility for hip OA in females and implicate the wnt signaling pathway in the pathogenesis of this disease.

Recent studies show that redox-active small molecules are selectively cytotoxic to chronic lymphocytic leukemia (CLL). Although elevated levels of reactive oxygen species in CLL cells have been implicated, the molecular mechanism underlying this selectivity is unclear. In other cell types, the nuclear factor erythroid 2—related factor 2 (Nrf2) signaling pathway regulates the oxidative stress response. We found elevated Nrf2 signaling in untreated CLL cells compared with normal lymphocytes. Therefore, we tested 27 known electrophilic and antioxidant compounds with drug-like properties and determined their CLL-selective cytotoxicity and effect on Nrf2 signaling. The selected compounds were from five distinct structural classes; α-β unsaturated carbonyls, isothiocyanates, sulfhydryl reactive metals, flavones, and polyphenols. Our results show that compounds containing α-β unsaturated carbonyls, sulfhydryl reactive metals, and isothiocyanates are strong activators of Nrf2 in a reporter assay system and in primary human CLL based on increased expression of the Nrf2 target heme oxygenase—1. α-β Unsaturated carbonyl-containing compounds were selectively cytotoxic to CLL, and loss of the α-β unsaturation abrogated Nrf2 activity and CLL toxicity. The α-β unsaturated carbonyl containing compounds ethacrynic acid and parthenolide activated Nrf2 in normal peripheral blood mono-nuclear cells, but had a less potent effect in CLL cells. Furthermore, ethacrynic acid bound directly to the Nrf2-negative regulator Kelch-like ECH-associated protein 1 (Keap1) in CLL cells. These experiments document the presence of Nrf2 signaling in human CLL and suggest that altered Nrf2 responses may contribute to the observed selective cytotoxicity of electrophilic compounds in this disease.