Browsing by Subject "Cell Line, Tumor"
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Item Open Access A CRISPRi/a screening platform to study cellular nutrient transport in diverse microenvironments.(Nature cell biology, 2024-05) Chidley, Christopher; Darnell, Alicia M; Gaudio, Benjamin L; Lien, Evan C; Barbeau, Anna M; Vander Heiden, Matthew G; Sorger, Peter KBlocking the import of nutrients essential for cancer cell proliferation represents a therapeutic opportunity, but it is unclear which transporters to target. Here we report a CRISPR interference/activation screening platform to systematically interrogate the contribution of nutrient transporters to support cancer cell proliferation in environments ranging from standard culture media to tumours. We applied this platform to identify the transporters of amino acids in leukaemia cells and found that amino acid transport involves high bidirectional flux dependent on the microenvironment composition. While investigating the role of transporters in cystine starved cells, we uncovered a role for serotonin uptake in preventing ferroptosis. Finally, we identified transporters essential for cell proliferation in subcutaneous tumours and found that levels of glucose and amino acids can restrain proliferation in that environment. This study establishes a framework for systematically identifying critical cellular nutrient transporters, characterizing their function and exploring how the tumour microenvironment impacts cancer metabolism.Item Open Access A curative combination cancer therapy achieves high fractional cell killing through low cross-resistance and drug additivity.(eLife, 2019-11) Palmer, Adam C; Chidley, Christopher; Sorger, Peter KCurative cancer therapies are uncommon and nearly always involve multi-drug combinations developed by experimentation in humans; unfortunately, the mechanistic basis for the success of such combinations has rarely been investigated in detail, obscuring lessons learned. Here, we use isobologram analysis to score pharmacological interaction, and clone tracing and CRISPR screening to measure cross-resistance among the five drugs comprising R-CHOP, a combination therapy that frequently cures Diffuse Large B-Cell Lymphomas. We find that drugs in R-CHOP exhibit very low cross-resistance but not synergistic interaction: together they achieve a greater fractional kill according to the null hypothesis for both the Loewe dose-additivity model and the Bliss effect-independence model. These data provide direct evidence for the 50 year old hypothesis that a curative cancer therapy can be constructed on the basis of independently effective drugs having non-overlapping mechanisms of resistance, without synergistic interaction, which has immediate significance for the design of new drug combinations.Item Open Access A genetic memory initiates the epigenetic loop necessary to preserve centromere position.(The EMBO journal, 2020-10) Hoffmann, Sebastian; Izquierdo, Helena M; Gamba, Riccardo; Chardon, Florian; Dumont, Marie; Keizer, Veer; Hervé, Solène; McNulty, Shannon M; Sullivan, Beth A; Manel, Nicolas; Fachinetti, DanieleCentromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP-A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP-A (CENP-AOFF/ON ). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP-B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP-A deposition. Indeed, lack of CENP-B favors neocentromere formation under selective pressure. Occasionally, CENP-B triggers centromere re-activation initiated by CENP-C, but not CENP-A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP-A-based epigenetic loop. Finally, we identify a population of CENP-A-negative, CENP-B/C-positive resting CD4+ T cells capable to re-express and reassembles CENP-A upon cell cycle entry, demonstrating the physiological importance of the genetic memory.Item Open Access A genetically engineered thermally responsive sustained release curcumin depot to treat neuroinflammation.(J Control Release, 2013-10-10) Sinclair, S Michael; Bhattacharyya, Jayanta; McDaniel, Jonathan R; Gooden, David M; Gopalaswamy, Ramesh; Chilkoti, Ashutosh; Setton, Lori ARadiculopathy, a painful neuroinflammation that can accompany intervertebral disc herniation, is associated with locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in the local delivery of anti-inflammatory drugs to treat this pathology as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNFα in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation. ELPs are biopolymers capable of thermally-triggered in situ depot formation that have been successfully employed as drug carriers and biomaterials in several applications. ELP-curcumin conjugates were shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNFα-induced cytotoxicity and monocyte activation with IC50 only two-fold higher than curcumin. When injected proximal to the sciatic nerve in mice via intramuscular (i.m.) injection, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4days post-injection and decreased plasma AUC 7-fold.Item Open Access A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.(Sci Signal, 2013-05-07) Kurokawa, Manabu; Kim, Jiyeon; Geradts, Joseph; Matsuura, Kenkyo; Liu, Liu; Ran, Xu; Xia, Wenle; Ribar, Thomas J; Henao, Ricardo; Dewhirst, Mark W; Kim, Wun-Jae; Lucas, Joseph E; Wang, Shaomeng; Spector, Neil L; Kornbluth, SallyIn the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.Item Open Access A novel human endogenous retroviral protein inhibits cell-cell fusion.(Scientific reports, 2013-01) Sugimoto, Jun; Sugimoto, Makiko; Bernstein, Helene; Jinno, Yoshihiro; Schust, DannyWhile common in viral infections and neoplasia, spontaneous cell-cell fusion, or syncytialization, is quite restricted in healthy tissues. Such fusion is essential to human placental development, where interactions between trophoblast-specific human endogenous retroviral (HERV) envelope proteins, called syncytins, and their widely-distributed cell surface receptors are centrally involved. We have identified the first host cell-encoded protein that inhibits cell fusion in mammals. Like the syncytins, this protein, called suppressyn, is HERV-derived, placenta-specific and well-conserved over simian evolution. In vitro, suppressyn binds to the syn1 receptor and inhibits syn1-, but not syn2-mediated trophoblast syncytialization. Suppressyn knock-down promotes cell-cell fusion in trophoblast cells and cell-associated and secreted suppressyn binds to the syn1 receptor, ASCT2. Identification of the first host cell-encoded inhibitor of mammalian cell fusion may encourage improved understanding of cell fusion mechanisms, of placental morphogenesis and of diseases resulting from abnormal cell fusion.Item Open Access A PK2/Bv8/PROK2 antagonist suppresses tumorigenic processes by inhibiting angiogenesis in glioma and blocking myeloid cell infiltration in pancreatic cancer.(2011) Curtis, Valerie ForbesIn many cancer types, infiltration of bone marrow-derived myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis. The polypeptide chemokine PK2 (Bv8) regulates myeloid cell mobilization from the bone marrow, leading to activation of angiogenesis as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. However, antibody-based therapies can be too large to treat certain diseases and too expensive to manufacture while small molecule therapeutics are not prohibitive in these ways. In this study, we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the contexts of glioblastoma and pancreatic cancer xenograft tumor models. In the highly vascularized glioblastoma, PKRA7 decreased blood vessel density while increasing necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 is mediated by the blockage of myeloid cell migration and infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of several pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both glioblastoma and pancreatic tumors. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy.Item Open Access ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via α-ketoglutarate.(PLoS Genet, 2015-10) Keenan, Melissa M; Liu, Beiyu; Tang, Xiaohu; Wu, Jianli; Cyr, Derek; Stevens, Robert D; Ilkayeva, Olga; Huang, Zhiqing; Tollini, Laura A; Murphy, Susan K; Lucas, Joseph; Muoio, Deborah M; Kim, So Young; Chi, Jen-TsanIn order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. To systematically identify genes that modulate cancer cell survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Additionally, the loss of ACLY or ACC1 reduced levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate is paradoxically increased under hypoxia when ACC1 or ACLY are depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulate the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.Item Open Access Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.(Oncotarget, 2015-02) Chao, Ting-Ting; Wang, Cheng-Yi; Chen, Yen-Lin; Lai, Chih-Cheng; Chang, Fang-Yu; Tsai, Yi-Ting; Chao, Chung-Hao H; Shiau, Chung-Wai; Huang, Yuh-Chin T; Yu, Chong-Jen; Chen, Kuen-FengAfatinib has anti-tumor effect in non-small cell lung carcinoma (NSCLC) with epidermal growth factor receptor (EGFR) mutation. We found afatinib can also induce apoptosis in NSCLC cells without EGFR mutation through CIP2A pathway. Four NSCLC cell lines (H358 H441 H460 and A549) were treated with afatinib to determine their sensitivity to afatinib-induced cell death and apoptosis. The effects of CIP2A on afatinib-induced apoptosis were confirmed by overexpression and knockdown of CIP2A expression in the sensitive and resistant cells, respectively. Reduction of Elk-1 binding to the CIP2A promoter and suppression of CIP2A transcription were analyzed. In vivo efficacy of afatinib against H358 and H460 xenografts tumors were also determined in nude mice. Afatinib induced significant cell death and apoptosis in H358 and H441 cells, but not in H460 or A549 cells. The apoptotic effect of afatinib in sensitive cells was associated with downregulation of CIP2A, promotion of PP2A activity and decrease in AKT phosphorylation. Afatinib suppressed CIP2A at the gene transcription level by reducing the promoter binding activity of Elk-1. Clinical samples showed that higher CIP2A expression predicted a poor prognosis and Elk-1 and CIP2A expressions were highly correlated. In conclusion, afatinib induces apoptosis in NSCLC without EGFR mutations through Elk-1/CIP2A/PP2A/AKT pathway.Item Open Access African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast.(PloS one, 2018-01) Sheridan, Megan A; Balaraman, Velmurugan; Schust, Danny J; Ezashi, Toshihiko; Roberts, R Michael; Franz, Alexander WEZika virus (ZIKV) drew worldwide attention when a recent epidemic was linked to fetal microcephaly. Here we used human embryonic stem cell derived trophoblasts as a model for primitive placental trophoblast to test the hypothesis that there are differences in how the two genetically distinct ZIKV lineages, African (AF) and Asian (AS), target the human placenta. Upon infection with three AF (ib-H30656, SEN/1984/41525-DAK, and MR-766) and three AS (FSS13025, MexI-44, and PANcdc259249) ZIKV strains, we observed that severe placental cell lysis was only induced after infection with AF strains, while viral replication rates remained similar between both lineages. Differences in cytopathic effects (CPE) were not observed in Vero cells, indicating that the AF strains were not inherently superior at cell lysis. Taken together, we propose that infection with AF strains of ZIKV early in pregnancy would likely result in pregnancy loss, rather than allow further fetal development with accompanying brain damage. Our results also suggest that the long term laboratory-adapted MR-766 strain does not behave aberrantly in cell culture relative to other AF lineage strains.Item Open Access An aberrant SREBP-dependent lipogenic program promotes metastatic prostate cancer.(Nature genetics, 2018-02) Chen, Ming; Zhang, Jiangwen; Sampieri, Katia; Clohessy, John G; Mendez, Lourdes; Gonzalez-Billalabeitia, Enrique; Liu, Xue-Song; Lee, Yu-Ru; Fung, Jacqueline; Katon, Jesse M; Menon, Archita Venugopal; Webster, Kaitlyn A; Ng, Christopher; Palumbieri, Maria Dilia; Diolombi, Moussa S; Breitkopf, Susanne B; Teruya-Feldstein, Julie; Signoretti, Sabina; Bronson, Roderick T; Asara, John M; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Pandolfi, Pier PaoloLipids, either endogenously synthesized or exogenous, have been linked to human cancer. Here we found that PML is frequently co-deleted with PTEN in metastatic human prostate cancer (CaP). We demonstrated that conditional inactivation of Pml in the mouse prostate morphs indolent Pten-null tumors into lethal metastatic disease. We identified MAPK reactivation, subsequent hyperactivation of an aberrant SREBP prometastatic lipogenic program, and a distinctive lipidomic profile as key characteristic features of metastatic Pml and Pten double-null CaP. Furthermore, targeting SREBP in vivo by fatostatin blocked both tumor growth and distant metastasis. Importantly, a high-fat diet (HFD) induced lipid accumulation in prostate tumors and was sufficient to drive metastasis in a nonmetastatic Pten-null mouse model of CaP, and an SREBP signature was highly enriched in metastatic human CaP. Thus, our findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis.Item Open Access An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.(J Exp Med, 2005-03-07) Ait-Azzouzene, Djemel; Verkoczy, Laurent; Peters, Jorieke; Gavin, Amanda; Skog, Patrick; Vela, José Luis; Nemazee, DavidUnderstanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.Item Metadata only An integrated approach to the prediction of chemotherapeutic response in patients with breast cancer.(PLoS One, 2008-04-02) Salter, Kelly H; Acharya, Chaitanya R; Walters, Kelli S; Redman, Richard; Anguiano, Ariel; Garman, Katherine S; Anders, Carey K; Mukherjee, Sayan; Dressman, Holly K; Barry, William T; Marcom, Kelly P; Olson, John; Nevins, Joseph R; Potti, AnilBACKGROUND: A major challenge in oncology is the selection of the most effective chemotherapeutic agents for individual patients, while the administration of ineffective chemotherapy increases mortality and decreases quality of life in cancer patients. This emphasizes the need to evaluate every patient's probability of responding to each chemotherapeutic agent and limiting the agents used to those most likely to be effective. METHODS AND RESULTS: Using gene expression data on the NCI-60 and corresponding drug sensitivity, mRNA and microRNA profiles were developed representing sensitivity to individual chemotherapeutic agents. The mRNA signatures were tested in an independent cohort of 133 breast cancer patients treated with the TFAC (paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide) chemotherapy regimen. To further dissect the biology of resistance, we applied signatures of oncogenic pathway activation and performed hierarchical clustering. We then used mRNA signatures of chemotherapy sensitivity to identify alternative therapeutics for patients resistant to TFAC. Profiles from mRNA and microRNA expression data represent distinct biologic mechanisms of resistance to common cytotoxic agents. The individual mRNA signatures were validated in an independent dataset of breast tumors (P = 0.002, NPV = 82%). When the accuracy of the signatures was analyzed based on molecular variables, the predictive ability was found to be greater in basal-like than non basal-like patients (P = 0.03 and P = 0.06). Samples from patients with co-activated Myc and E2F represented the cohort with the lowest percentage (8%) of responders. Using mRNA signatures of sensitivity to other cytotoxic agents, we predict that TFAC non-responders are more likely to be sensitive to docetaxel (P = 0.04), representing a viable alternative therapy. CONCLUSIONS: Our results suggest that the optimal strategy for chemotherapy sensitivity prediction integrates molecular variables such as ER and HER2 status with corresponding microRNA and mRNA expression profiles. Importantly, we also present evidence to support the concept that analysis of molecular variables can present a rational strategy to identifying alternative therapeutic opportunities.Item Open Access Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics.(Journal of proteome research, 2012-02) Yang, W; Thompson, JW; Wang, Z; Wang, L; Sheng, H; Foster, MW; Moseley, MA; Paschen, WTransient cerebral ischemia dramatically activates small ubiquitin-like modifier (SUMO2/3) conjugation. In cells exposed to 6 h of transient oxygen/glucose deprivation (OGD), a model of ischemia, SUMOylation increases profoundly between 0 and 30 min following re-oxygenation. To elucidate the effect of transient OGD on SUMO conjugation of target proteins, we exposed neuroblastoma B35 cells expressing HA-SUMO3 to transient OGD and used stable isotope labeling with amino acids in cell culture (SILAC) to quantify OGD-induced changes in levels of specific SUMOylated proteins. Lysates from control and OGD-treated cells were mixed equally, and HA-tagged proteins were immunoprecipitated and analyzed by 1D-SDS-PAGE-LC-MS/MS. We identified 188 putative SUMO3-conjugated proteins, including numerous transcription factors and coregulators, and PIAS2 and PIAS4 SUMO ligases, of which 22 were increased or decreased more than ±2-fold. In addition to SUMO3, the levels of protein-conjugated SUMO1 and SUMO2, as well as ubiquitin, were all increased. Importantly, protein ubiquitination induced by OGD was completely blocked by gene silencing of SUMO2/3. Collectively, these results suggest several mechanisms for OGD-modulated SUMOylation, point to a number of signaling pathways that may be targets of SUMO-based signaling and recovery from ischemic stress, and demonstrate a tightly controlled crosstalk between the SUMO and ubiquitin conjugation pathways.Item Open Access Associations of genotypes and haplotypes of IL-17 with risk of gastric cancer in an eastern Chinese population.(Oncotarget, 2016-12) Zhou, Fei; Qiu, Li-Xin; Cheng, Lei; Wang, Meng-Yun; Li, Jin; Sun, Meng-Hong; Yang, Ya-Jun; Wang, Jiu-Cun; Jin, Li; Wang, Ya-Nong; Wei, Qing-YiInterleukin-17 plays a crucial role in inflammation-related carcinogenesis. We hypothesize that genetic variants in IL-17 are associated with gastric cancer (GCa) risk, and we genotyped five potentially functional single nucleotide polymorphisms (SNPs) (rs1974226 G > A, rs2275913 A > G, rs3819024 A > G, rs4711998 A > G, and rs8193036 C > T) of IL-17 in 1121 GCa patients and 1216 cancer-free controls in an eastern Chinese population. Logistic regression analysis was used to calculate odds ratios (OR) and 95% confidence intervals (CI). Meta-analysis and genotype-mRNA expression correlation were performed to further validate positive associations. We found that an increased GCa risk was independently associated with rs1974226 (adjusted OR = 2.60, 95% CI = 1.27-5.32 for AA vs. GG + GA) and rs2275913 (adjusted OR = 1.33, 95% CI = 1.03-1.72 for GA + AA vs. GG), while a decreased GCa risk was independently associated with rs3819024 (adjusted OR = 0.72, 95% CI = 0.54-0.96 for GG vs. AA + AG). Additional meta-analyses confirmed the observed risk association with rs2275913. We also found that two IL-17 haplotypes (G-G-G-A-C) and (A-G-G-A-C) (in the order of rs1974226, rs2275913, rs3819024, rs4711998 and rs8193036) were associated with a reduced GCa risk (adjusted OR = 0.64, 95% CI = 0.46-0.89 and adjusted OR = 0.38, 95% CI = 0.17-0.81, respectively). However, the expression Quantitative Trait Locus (eQTL) analysis for the genotype-phenotype correlation did not find mRNA expression changes associated with either the genotypes. In conclusions, genetic variants of IL-17 are likely to be associated with risk of GCa, and additional larger studies with functional validation are needed to explore the molecular mechanisms underlying the observed associations.Item Open Access B7-H3-redirected chimeric antigen receptor T cells target glioblastoma and neurospheres.(EBioMedicine, 2019-09) Nehama, Dean; Di Ianni, Natalia; Musio, Silvia; Du, Hongwei; Patané, Monica; Pollo, Bianca; Finocchiaro, Gaetano; Park, James JH; Dunn, Denise E; Edwards, Drake S; Damrauer, Jeffrey S; Hudson, Hannah; Floyd, Scott R; Ferrone, Soldano; Savoldo, Barbara; Pellegatta, Serena; Dotti, GianpietroBackground
The dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM.Methods
We compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models.Findings
B7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines.Interpretation
We demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM. FUND: Alex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.Item Open Access BCL2 inhibits cell adhesion, spreading, and motility by enhancing actin polymerization.(Cell Res, 2010-04) Ke, Hengning; Parron, Vandy I; Reece, Jeff; Zhang, Jennifer Y; Akiyama, Steven K; French, John EBCL2 is best known as a multifunctional anti-apoptotic protein. However, little is known about its role in cell-adhesive and motility events. Here, we show that BCL2 may play a role in the regulation of cell adhesion, spreading, and motility. When BCL2 was overexpressed in cultured murine and human cell lines, cell spreading, adhesion, and motility were impaired. Consistent with these results, the loss of Bcl2 resulted in higher motility observed in Bcl2-null mouse embryonic fibroblast (MEF) cells compared to wild type. The mechanism of BCL2 regulation of cell adhesion and motility may involve formation of a complex containing BCL2, actin, and gelsolin, which appears to functionally decrease the severing activity of gelsolin. We have observed that the lysate from MCF-7 and NIH3T3 cells that overexpressed BCL2 enhanced actin polymerization in cell-free in vitro assays. Confocal immunofluorescent localization of BCL2 and F-actin during spreading consistently showed that increased expression of BCL2 resulted in increased F-actin polymerization. Thus, the formation of BCL2 and gelsolin complexes (which possibly contain other proteins) appears to play a critical role in the regulation of cell adhesion and migration. Given the established correlation of cell motility with cancer metastasis, this result may explain why the expression of BCL2 in some tumor cell types reduces the potential for metastasis and is associated with improved patient prognosis.Item Open Access Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation.(J Transl Med, 2010-01-27) Liu, Fang; Hu, Zhenlin; Qiu, Lei; Hui, Chun; Li, Chao; Zhong, Pei; Zhang, JunpingBACKGROUND: The conventional treatment protocol in high-intensity focused ultrasound (HIFU) therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs) inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation. METHODS: An experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-gamma-secreting cells in HIFU-treated mice. RESULTS: HIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an enhanced anti-tumor immune response than dense-scan HIFU, while their suppressive effects on the treated primary tumor were maintained at the same level. Flow cytometry analysis showed that sparse-scan HIFU was more effective than dense-scan HIFU in enhancing DC infiltration into tumor tissues and promoting their maturation in situ. CONCLUSION: Optimizing scan strategy is a feasible way to boost HIFU-induced anti-tumor immunity by more effectively promoting DC maturation.Item Open Access CCDC62/ERAP75 functions as a coactivator to enhance estrogen receptor beta-mediated transactivation and target gene expression in prostate cancer cells.(Carcinogenesis, 2009-05) Chen, Ming; Ni, Jing; Chang, Hong-Chiang; Lin, Chen-Yong; Muyan, Mesut; Yeh, ShuyuanHuman prostate cancer (PCa) and prostate epithelial cells predominantly express estrogen receptor (ER) beta, but not ERalpha. ERbeta might utilize various ER coregulators to mediate the E2-signaling pathway in PCa. Here, we identified coiled-coil domain containing 62 (CCDC62)/ERAP75 as a novel ER coactivator. CCDC62/ERAP75 is widely expressed in PCa cell lines and has low expression in MCF7 cells. Both in vitro and in vivo interaction assays using mammalian two-hybrid, glutathione S-transferase pull-down and coimmunoprecipitation methods proved that ERbeta can interact with the C-terminus of CCDC62/ERAP75 via the ligand-binding domain. The first LXXLL motif within CCDC62/ERAP75 is required for the interaction between ERbeta and CCDC62/ERAP75. Electrophoretic mobility shift assay showed that CCDC62/ERAP75 can be recruited by the estrogen response element-ER complex in the presence of ligand. Furthermore, a chromatin immunoprecipitation assay demonstrated the hormone-dependent recruitment of CCDC62/ERAP75 within the promoter of the estrogen-responsive gene cyclin D1. In addition, using silencing RNA (siRNA) against endogeneous CCDC62/ERAP75, we demonstrated that inhibition of endogenous CCDC62/ERAP75 results in the suppression of ERbeta-mediated transactivation as well as target gene expression in LNCaP cells. More importantly, using the tet-on overexpression system, we showed that induced expression of CCDC62/ERAP75 can enhance the E2-regulated cyclin D1 expression and cell growth in LNCaP cells. Together, our results revealed the role of CCDC62/ERAP75 as a novel coactivator in PCa cells that can modulate ERbeta transactivation and receptor function.Item Open Access Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells.(Bioorganic & medicinal chemistry letters, 2011-09) Hubbard, Sarah C; Boyce, Michael; McVaugh, Cheryl T; Peehl, Donna M; Bertozzi, Carolyn RMost clinically approved biomarkers of cancer are glycoproteins, and those residing on the cell surface are of particular interest in biotherapeutics. We report a method for selective labeling, affinity enrichment, and identification of cell-surface glycoproteins. PC-3 cells and primary human prostate cancer tissue were treated with peracetylated N-azidoacetylgalactosamine, resulting in metabolic labeling of cell surface glycans with the azidosugar. We used mass spectrometry to identify over 70 cell surface glycoproteins and biochemically validated CD146 and integrin beta-4, both of which are known to promote metastatic behavior. These results establish cell-surface glycoproteomics as an effective technique for discovery of cancer biomarkers.