Publications by authors named "Stephen J Kron"

99 Publications

Lipid-derived electrophiles mediate the effects of chemotherapeutic topoisomerase I poisons.

Cell Chem Biol 2020 Dec 16. Epub 2020 Dec 16.

University of Chicago, Department of Molecular Genetics and Cell Biology, 929 E. 57th Street W522A, Chicago, IL 60637, USA. Electronic address:

Topoisomerase 1 (Top1) reversibly nicks chromosomal DNA to relax strain accumulated during transcription, replication, chromatin assembly, and chromosome condensation. The Top1 poison camptothecin targets cancer cells by trapping the enzyme in the covalent complex Top1, tethered to cleaved DNA by a tyrosine-3'-phosphate bond. In vitro mechanistic studies point to interfacial inhibition, where camptothecin binding to the Top1-DNA interface stabilizes Top1. Here we present a complementary covalent mechanism that is critical in vivo. We observed that camptothecins induce oxidative stress, leading to lipid peroxidation, lipid-derived electrophile accumulation, and Top1 poisoning via covalent modification. The electrophile 4-hydroxy-2-nonenal can induce Top1 on its own and forms a Michael adduct to a cysteine thiol in the Top1 active site, potentially blocking tyrosine dephosphorylation and 3' DNA phosphate release. Thereby, camptothecins may leverage a physiological cysteine-based redox switch in Top1 to mediate their selective toxicity to rapidly proliferating cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2020.11.011DOI Listing
December 2020

Subcellular localization of the J-protein Sis1 regulates the heat shock response.

J Cell Biol 2021 Jan;220(1)

Whitehead Institute for Biomedical Research, Cambridge, MA.

Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.202005165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748816PMC
January 2021

Small-molecule drug repurposing to target DNA damage repair and response pathways.

Semin Cancer Biol 2021 Jan 27;68:230-241. Epub 2020 Feb 27.

Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL, United States; Ludwig Center for Metastasis Research, University of Chicago, Chicago, IL, United States. Electronic address:

For decades genotoxic therapy has been a mainstay in the treatment of cancer, based on the understanding that the deregulated growth and genomic instability that drive malignancy also confer a shared vulnerability. Although chemotherapy and radiation can be curative, only a fraction of patients benefit, while nearly all are subjected to the harmful side-effects. Drug repurposing, defined here as retooling existing drugs and compounds as chemo or radiosensitizers, offers an attractive route to identifying otherwise non-toxic agents that can potentiate the benefits of genotoxic cancer therapy to enhance the therapeutic ratio. This review seeks to highlight recent progress in defining cellular mechanisms of the DNA damage response including damage sensing, chromatin modification, DNA repair, checkpoint signaling, and downstream survival and death pathways, as a framework to determine which drugs and natural products may offer the most potential for repurposing as chemo- and/or radiosensitizers. We point to classical examples and recent progress that have identified drugs that disrupt cellular responses to DNA damage and may offer the greatest clinical potential. The most important next steps may be to initiate prospective clinical trials toward translating these laboratory discoveries to benefit patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.semcancer.2020.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483256PMC
January 2021

Targeted Covalent Inhibition of Telomerase.

ACS Chem Biol 2020 03 24;15(3):706-717. Epub 2020 Feb 24.

Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States.

Telomerase is a ribonuceloprotein complex responsible for maintaining telomeres and protecting chromosomal integrity. The human telomerase reverse transcriptase (hTERT) is expressed in ∼90% of cancer cells where it confers the capacity for limitless proliferation. Along with its established role in telomere lengthening, telomerase also serves noncanonical extra-telomeric roles in oncogenic signaling, resistance to apoptosis, and enhanced DNA damage response. We report a new class of natural-product-inspired covalent inhibitors of telomerase that target the catalytic active site.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.9b00945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7233141PMC
March 2020

Genetic analysis of Hsp70 phosphorylation sites reveals a role in Candida albicans cell and colony morphogenesis.

Biochim Biophys Acta Proteins Proteom 2020 03 10;1868(3):140135. Epub 2018 Sep 10.

Department of Molecular Microbiology, B. Rappaport Faculty of Medicine, Technion - I.I.T. and the Rappaport Institute for Research in the Medical Sciences, Haifa 31096, Israel. Electronic address:

Heat shock proteins are best known for their role as chaperonins involved in general proteostasis, but they can also participate in specific cellular regulatory pathways, e.g. via their post-translational modification. Hsp70/Ssa1 is a central cytoplasmic chaperonin in eukaryotes, which also participates in cell cycle regulation via its phosphorylation at a specific residue. Here we analyze the role of Ssa1 phosphorylation in the morphogenesis of the fungus Candida albicans, a common human opportunistic pathogen. C. albicans can assume alternative yeast and hyphal (mold) morphologies, an ability that contributes to its virulence. We identified 11 phosphorylation sites on C. albicans Ssa1, of which 8 were only detected in the hyphal cells. Genetic analysis of these sites revealed allele-specific effects on growth or hyphae formation at 42 °C. Colony morphology, which is normally wrinkled or crenellated at 37 °C, reverted to smooth in several mutants, but this colony morphology phenotype was unrelated to cellular morphology. Two mutants exhibited a mild increase in sensitivity to the cell wall-active compounds caspofungin and calcofluor white. We suggest that this analysis could help direct screens for Ssa1-specific drugs to combat C. albicans virulence. The pleiotropic effects of many Ssa1 mutations are consistent with the large number of Ssa1 client proteins, whereas the lack of concordance between the phenotypes of the different alleles suggests that different sites on Ssa1 can affect interaction with specific classes of client proteins, and that modification of these sites can play cellular regulatory roles, consistent with the "chaperone code" hypothesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbapap.2018.09.001DOI Listing
March 2020

Mevalonate pathway activity as a determinant of radiation sensitivity in head and neck cancer.

Mol Oncol 2019 09 26;13(9):1927-1943. Epub 2019 Jul 26.

Department of Molecular Genetics and Cell Biology, The University of Chicago, IL, USA.

Radioresistance is a major hurdle in the treatment of head and neck squamous cell carcinoma (HNSCC). Here, we report that concomitant treatment of HNSCCs with radiotherapy and mevalonate pathway inhibitors (statins) may overcome resistance. Proteomic profiling and comparison of radioresistant to radiosensitive HNSCCs revealed differential regulation of the mevalonate biosynthetic pathway. Consistent with this finding, inhibition of the mevalonate pathway by pitavastatin sensitized radioresistant SQ20B cells to ionizing radiation and reduced their clonogenic potential. Overall, this study reinforces the view that the mevalonate pathway is a promising therapeutic target in radioresistant HNSCCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1878-0261.12535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717759PMC
September 2019

Repair-independent functions of DNA-PKcs protect irradiated cells from mitotic slippage and accelerated senescence.

J Cell Sci 2019 07 1;132(13). Epub 2019 Jul 1.

Department of Molecular Genetics and Cell Biology and Ludwig Center for Metastasis Research, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA

The binding of DNA-dependent protein kinase catalytic subunit (DNA-PKcs, also known as PRKDC) to Ku proteins at DNA double-strand breaks (DSBs) has long been considered essential for non-homologous end joining (NHEJ) repair, providing a rationale for use of DNA-PKcs inhibitors as cancer therapeutics. Given lagging clinical translation, we reexamined mechanisms and observed instead that DSB repair can proceed independently of DNA-PKcs. While repair of radiation-induced DSBs was blocked in cells expressing shRNAs targeting Ku proteins or other NHEJ core factors, DSBs were repaired on schedule despite targeting DNA-PKcs. Although we failed to observe a DSB repair defect, the γH2AX foci that formed at sites of DNA damage persisted indefinitely after irradiation, leading to cytokinesis failure and accumulation of binucleated cells. Following this mitotic slippage, cells with decreased DNA-PKcs underwent accelerated cellular senescence. We identified downregulation of ataxia-telangiectasia mutated kinase (ATM) as the critical role of DNA-PKcs in recovery from DNA damage, insofar as targeting ATM restored γH2AX foci resolution and cytokinesis. Considering the lack of direct impact on DSB repair and emerging links between senescence and resistance to cancer therapy, these results suggest reassessing DNA-PKcs as a target for cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.229385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633392PMC
July 2019

Phosphoregulation of the oncogenic protein regulator of cytokinesis 1 (PRC1) by the atypical CDK16/CCNY complex.

Exp Mol Med 2019 04 16;51(4):1-17. Epub 2019 Apr 16.

Faculty of Medicine and Health Sciences, International University of Catalonia, 08195, Sant Cugat del Vallès, Barcelona, Spain.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s12276-019-0242-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467995PMC
April 2019

A cmap-enabled gene expression signature-matching approach identifies small-molecule inducers of accelerated cell senescence.

BMC Genomics 2019 Apr 15;20(1):290. Epub 2019 Apr 15.

Department of Molecular Genetics and Cell Biology and Ludwig Center for Metastasis Research, The University of Chicago, 929 East 57th Street, GCIS W522A, Chicago, IL, 60637, USA.

Background: Diverse stresses including genotoxic therapy can induce proliferating cancer cells to undergo cellular senescence and take on the characteristic phenotypes of replicative cellular aging. This accelerated or therapy-induced senescence has been alternatively proposed to contribute to therapeutic efficacy or resistance. Toward better understanding this cell state, we sought to define the core transcriptome of accelerated senescence in cancer cells.

Results: We examined senescence induced by ionizing irradiation or ectopic overexpression of the stoichiometric cyclin-dependent kinase (CDK) inhibitor p21 in the human breast cancer cell line MCF7. While radiation produces a strong DNA damage response, ectopic expression of p21 arrests cell cycle progression independently of DNA damage. Both conditions promoted senescence within 5 days. Microarray analysis revealed 378 up- and 391 down-regulated genes that were shared between the two conditions, representing a candidate signature. Systems analysis of the shared differentially expressed genes (DEGs) revealed strong signals for cell cycle control and DNA damage response pathways and predicted multiple upstream regulators previously linked to senescence. Querying the shared DEGs against the Connectivity Map (cmap) database of transcriptional responses to small molecules yielded 20 compounds that induce a similar gene expression pattern in MCF7 cells. Of 16 agents evaluated, six induced senescence on their own. Of these, the selective estrogen receptor degrader fulvestrant and the histone acetyltransferase inhibitor vorinostat did so without causing chromosomal damage.

Conclusions: Using a systems biology approach with experimental validation, we have defined a core gene expression signature for therapy-induced senescence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-019-5653-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466706PMC
April 2019

Targeted antibody and cytokine cancer immunotherapies through collagen affinity.

Sci Transl Med 2019 04;11(487)

Institute for Molecular Engineering, University of Chicago, Chicago, IL 60637, USA.

Cancer immunotherapy with immune checkpoint inhibitors (CPIs) and interleukin-2 (IL-2) has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T lymphocyte antigen 4 antibody (αCTLA4) + anti-programmed death ligand 1 antibody (αPD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain, harnessing the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously administered CBD protein accumulated mainly in tumors. CBD conjugation or fusion decreases the systemic toxicity of both αCTLA4 + αPD-L1 combination therapy and IL-2, for example, eliminating hepatotoxicity with the CPI molecules and ameliorating pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8 T cells. In an orthotopic breast cancer model, combination treatment with CPI and IL-2 eradicated tumors in 9 of 13 animals with the CBD-modified drugs, whereas it did so in only 1 of 13 animals with the unmodified drugs. Thus, the A3 domain of VWF can be used to improve safety and efficacy of systemically administered tumor drugs with high translational promise.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aau3259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541444PMC
April 2019

Repurposing Drugs for Cancer Radiotherapy: Early Successes and Emerging Opportunities.

Cancer J 2019 Mar/Apr;25(2):106-115

From the Winship Cancer Institute, Emory University, Atlanta, GA; and Ludwig Center for Metastasis Research, The University of Chicago, Chicago, IL.

It has long been recognized that combining radiotherapy with cytotoxic drugs such as cisplatin can improve efficacy. However, while concurrent chemoradiotherapy improves patient outcomes, it comes at costs of increased toxicity. A tremendous opportunity remains to investigate drug combinations in the clinical setting that might increase the benefits of radiation without additional toxicity. This chapter highlights opportunities to apply repurposing of drugs along with a mechanistic understanding of radiation effects on cancer and normal tissue to discover new therapy-modifying drugs and help rapidly translate them to the clinic. We survey candidate radiosensitizers that alter DNA repair, decrease hypoxia, block tumor survival signaling, modify tumor metabolism, block growth factor signaling, slow tumor invasiveness, impair angiogenesis, or stimulate antitumor immunity. Promising agents include widely used drugs such as aspirin, metformin, and statins, offering the potential to improve outcomes, decrease radiation doses, and lower costs. Many other candidate drugs are also discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/PPO.0000000000000369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518392PMC
July 2020

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors.

Mol Cancer Res 2019 06 18;17(6):1338-1350. Epub 2019 Mar 18.

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL.

The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence . Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. IMPLICATIONS: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1541-7786.MCR-18-1025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548675PMC
June 2019

The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair.

Nucleic Acids Res 2019 04;47(6):2703-2715

Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA.

P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451129PMC
April 2019

STING Promotes Homeostasis via Regulation of Cell Proliferation and Chromosomal Stability.

Cancer Res 2019 04 27;79(7):1465-1479. Epub 2018 Nov 27.

Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, Illinois.

Given the integral role of stimulator of interferon genes (STING, ) in the innate immune response, its loss or impairment in cancer is thought to primarily affect antitumor immunity. Here we demonstrate a role for STING in the maintenance of cellular homeostasis through regulation of the cell cycle. Depletion of STING in human and murine cancer cells and tumors resulted in increased proliferation compared with wild-type controls. Microarray analysis revealed genes involved in cell-cycle regulation are differentially expressed in STINGko compared with WT MEFs. STING-mediated regulation of the cell cycle converged on NFκB- and p53-driven activation of p21. The absence of STING led to premature activation of cyclin-dependent kinase 1 (CDK1), early onset to S-phase and mitosis, and increased chromosome instability, which was enhanced by ionizing radiation. These results suggest a pivotal role for STING in maintaining cellular homeostasis and response to genotoxic stress. SIGNIFICANCE: These findings provide clear mechanistic understanding of the role of STING in cell-cycle regulation, which may be exploited in cancer therapy because most normal cells express STING, while many tumor cells do not..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-18-1972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445702PMC
April 2019

Quinic Acid-Conjugated Nanoparticles Enhance Drug Delivery to Solid Tumors via Interactions with Endothelial Selectins.

Small 2018 12 9;14(50):e1803601. Epub 2018 Nov 9.

Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN, 47907, USA.

Current nanoparticle (NP) drug carriers mostly depend on the enhanced permeability and retention (EPR) effect for selective drug delivery to solid tumors. However, in the absence of a persistent EPR effect, the peritumoral endothelium can function as an access barrier to tumors and negatively affect the effectiveness of NPs. In recognition of the peritumoral endothelium as a potential barrier in drug delivery to tumors, poly(lactic-co-glycolic acid) (PLGA) NPs are modified with a quinic acid (QA) derivative, synthetic mimic of selectin ligands. QA-decorated NPs (QA-NP) interact with human umbilical vein endothelial cells expressing E-/P-selectins and induce transient increase in endothelial permeability to translocate across the layer. QA-NP reach selectin-upregulated tumors, achieving greater tumor accumulation and paclitaxel (PTX) delivery than polyethylene glycol-decorated NPs (PEG-NP). PTX-loaded QA-NP show greater anticancer efficacy than Taxol or PTX-loaded PEG-NP at the equivalent PTX dose in different animal models and dosing regimens. Repeated dosing of PTX-loaded QA-NP for two weeks results in complete tumor remission in 40-60% of MDA-MB-231 tumor-bearing mice, while those receiving control treatments succumb to death. QA-NP can exploit the interaction with selectin-expressing peritumoral endothelium and deliver anticancer drugs to tumors to a greater extent than the level currently possible with the EPR effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/smll.201803601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361670PMC
December 2018

Nondestructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy.

Lab Invest 2019 09 6;99(9):1400-1413. Epub 2018 Nov 6.

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL, USA.

Enumeration of tumor-infiltrating lymphocytes (TILs) in H&E stained tissue sections has demonstrated limited value in predicting immune responses to cancer immunotherapy, likely reflecting the diversity of cell types and immune activation states among tumor infiltrates. Multiparametric flow cytometry enables robust phenotypic and functional analysis to distinguish suppression from activation, but tissue dissociation eliminates spatial context. Multiplex methods for immunohistochemistry (IHC) are emerging, but these interrogate only a single tissue section at a time. Here, we report transparent tissue tomography (T3) as a tool for three-dimensional (3D) imaging cytometry in the complex architecture of the tumor microenvironment, demonstrating multiplexed immunofluorescent analysis in core needle biopsies. Using T3 imaging, image processing and machine learning to map CD3CD8 cytotoxic T cells (CTLs) in whole core needle biopsies from Her2 murine mammary tumors and human head and neck surgical specimens revealed marked inhomogeneity within single needle cores, confirmed by serial section IHC. Applying T3 imaging cytometry, we discovered a strong spatial correlation between CD3CD8 CTLs and microvasculature in the EGFR parenchyma, revealing significant differences among head and neck cancer patients. These results show that T3 offers simple and rapid access to three-dimensional and quantitative maps of the tumor microenvironment and immune infiltrate, offering a new diagnostic tool for personalized cancer immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41374-018-0156-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6502706PMC
September 2019

Multiplex Three-Dimensional Mapping of Macromolecular Drug Distribution in the Tumor Microenvironment.

Mol Cancer Ther 2019 01 15;18(1):213-226. Epub 2018 Oct 15.

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois.

Macromolecular cancer drugs such as therapeutic antibodies and nanoparticles are well known to display slow extravasation and incomplete penetration into tumors, potentially protecting cancer cells from therapeutic effects. Conventional assays to track macromolecular drug delivery are poorly matched to the heterogeneous tumor microenvironment, but recent progress on optical tissue clearing and three-dimensional (3D) tumor imaging offers a path to quantitative assays with cellular resolution. Here, we apply transparent tissue tomography (T3) as a tool to track perfusion and delivery in the tumor and to evaluate target binding and vascular permeability. Using T3, we mapped anti-programmed cell death protein-ligand 1 (PD-L1) antibody distribution in whole mouse tumors. By measuring 3D penetration distances of the antibody drug out from the blood vessel boundaries into the tumor parenchyma, we determined spatial pharmacokinetics of anti-PD-L1 antibody drugs in mouse tumors. With multiplex imaging of tumor components, we determined the distinct distribution of anti-PD-L1 antibody drug in the tumor microenvironment with different PD-L1 expression patterns. T3 imaging revealed CD31 capillaries are more permeable to anti-PD-L1 antibody transport compared with the blood vessels composed of endothelium supported by vascular fibroblasts and smooth muscle cells. T3 analysis also confirmed that isotype IgG antibody penetrates more deeply into tumor parenchyma than anti-Her2 or anti-EGFR antibody, which were restrained by binding to their respective antigens on tumor cells. Thus, T3 offers simple and rapid access to 3D, quantitative maps of macromolecular drug distribution in the tumor microenvironment, offering a new tool for development of macromolecular cancer therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-18-0554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318001PMC
January 2019

Phospho-dependent recruitment of the yeast NuA4 acetyltransferase complex by MRX at DNA breaks regulates RPA dynamics during resection.

Proc Natl Acad Sci U S A 2018 10 17;115(40):10028-10033. Epub 2018 Sep 17.

St. Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Centre de Recherche du Centre Hospitalier Universitaire de Québec-Axe Oncologie, Québec City, QC G1R 3S3, Canada;

The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1806513115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176631PMC
October 2018

The atypical cyclin CNTD2 promotes colon cancer cell proliferation and migration.

Sci Rep 2018 08 7;8(1):11797. Epub 2018 Aug 7.

Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Barcelona, Spain.

Colorectal cancer (CRC) is one of the most common cancers worldwide, with 8-10% of these tumours presenting a BRAF (V600E) mutation. Cyclins are known oncogenes deregulated in many cancers, but the role of the new subfamily of atypical cyclins remains elusive. Here we have performed a systematic analysis of the protein expression levels of eight atypical cyclins in human CRC tumours and several cell lines, and found that CNTD2 is significantly upregulated in CRC tissue compared to the adjacent normal one. CNTD2 overexpression in CRC cell lines increases their proliferation capacity and migration, as well as spheroid formation capacity and anchorage-independent growth. Moreover, CNTD2 increases tumour growth in vivo on xenograft models of CRC with wild-type BRAF. Accordingly, CNTD2 downregulation significantly diminished the proliferation of wild-type BRAF CRC cells, suggesting that CNTD2 may represent a new prognostic factor and a promising drug target in the management of CRC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-30307-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6081371PMC
August 2018

Three-Dimensional Analysis of the Human Pancreas.

Endocrinology 2018 03;159(3):1393-1400

Department of Medicine, University of Chicago, Chicago, Illinois.

Pancreatic islets are endocrine micro-organs scattered throughout the exocrine pancreas. Islets are surrounded by a network of vasculature, ducts, neurons, and extracellular matrix. Three-dimensional imaging is critical for such structural analyses. We have adapted transparent tissue tomography to develop a method to image thick pancreatic tissue slices (1 mm) with multifluorescent channels. This method takes only 2 to 3 days from specimen preparation and immunohistochemical staining to clearing tissues and imaging. Reconstruction of the intact pancreas visualizes islets with β, α, and δ cells together with their surrounding networks. Capturing several hundred islets at once ensures sufficient power for statistical analyses. Further surface rendering provides clear views of the anatomical relationship between islets and their microenvironment as well as the basis for volumetric quantification. As a proof-of-principle demonstration, we show an islet size-dependent increase of intraislet capillary density and an inverse decrease in sphericity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2017-03076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839749PMC
March 2018

Radiation-enhanced delivery of plasmid DNA to tumors utilizing a novel PEI polyplex.

Cancer Gene Ther 2018 08 19;25(7-8):196-206. Epub 2017 Dec 19.

Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Dr., West Lafayette, IN, 47907, USA.

The excitement surrounding the potential of gene therapy has been tempered due to the challenges that have thus far limited its successful implementation in the clinic such as issues regarding stability, transfection efficiency, and toxicity. In this study, low molecular weight linear polyethyleneimine (2.5 kDa) was modified by conjugation to a lipid, lithocholic acid, and complexed with a natural polysaccharide, dermatan sulfate (DS), to mask extra cationic charges of the modified polymer. In vitro examination revealed that these modifications improved complex stability with plasmid DNA (pDNA) and transfection efficiency. This novel ternary polyplex (pDNA/3E/DS) was used to investigate if tumor-targeted radiotherapy led to enhanced accumulation and retention of gene therapy vectors in vivo in tumor-bearing mice. Imaging of biodistribution revealed that tumor irradiation led to increased accumulation and retention as well as decreased off-target tissue buildup of pDNA in not only pDNA/3E/DS, but also in associated PEI-based polyplexes and commercial DNA delivery vehicles. The DS-containing complexes developed in this study displayed the greatest increase in tumor-specific pDNA delivery. These findings demonstrate a step forward in nucleic acid vehicle design as well as a promising approach to overall cancer gene therapy through utilization of radiotherapy as a tool for enhanced delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41417-017-0004-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008165PMC
August 2018

Multiplex three-dimensional optical mapping of tumor immune microenvironment.

Sci Rep 2017 12 5;7(1):17031. Epub 2017 Dec 5.

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL, USA.

Recent developments in optical tissue clearing and microscopic imaging have advanced three-dimensional (3D) visualization of intact tissues and organs at high resolution. However, to expand applications to oncology, critical limitations of current methods must be addressed. Here we describe transparent tissue tomography (T3) as a tool for rapid, three-dimensional, multiplexed immunofluorescent tumor imaging. Cutting tumors into sub-millimeter macrosections enables simple and rapid immunofluorescence staining, optical clearing, and confocal microscope imaging. Registering and fusing macrosection images yields high resolution 3D maps of multiple tumor microenvironment components and biomarkers throughout a tumor. The 3D maps can be quantitatively evaluated by automated image analysis. As an application of T3, 3D mapping and analysis revealed a heterogeneous distribution of programmed death-ligand 1 (PD-L1) in Her2 transgenic mouse mammary tumors, with high expression limited to tumor cells at the periphery and to CD31 vascular endothelium in the core. Also, strong spatial correlation between CD45 immune cell distribution and PD-L1 expression was revealed by T3 analysis of the whole tumors. Our results demonstrate that a tomographic approach offers simple and rapid access to high-resolution three-dimensional maps of the tumor immune microenvironment, offering a new tool to examine tumor heterogeneity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-16987-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717053PMC
December 2017

A signature of enhanced lipid metabolism, lipid peroxidation and aldehyde stress in therapy-induced senescence.

Cell Death Discov 2017 30;3:17075. Epub 2017 Oct 30.

Department of Molecular Genetics and Cell Biology and Ludwig Center for Metastasis Research, The University of Chicago, Chicago, IL, USA.

At their proliferative limit, normal cells arrest and undergo replicative senescence, displaying large cell size, flat morphology, and senescence-associated beta-galactosidase (SA--Gal) activity. Normal or tumor cells exposed to genotoxic stress undergo therapy-induced senescence (TIS), displaying a similar phenotype. Senescence is considered a DNA damage response, but cellular heterogeneity has frustrated identification of senescence-specific markers and targets. To explore the senescent cell proteome, we treated tumor cells with etoposide and enriched SA--Gal cells by fluorescence-activated cell sorting (FACS). The enriched TIS cells were compared to proliferating or quiescent cells by label-free quantitative LC-MS/MS proteomics and systems analysis, revealing activation of multiple lipid metabolism pathways. Senescent cells accumulated lipid droplets and imported lipid tracers, while treating proliferating cells with specific lipids induced senescence. Senescent cells also displayed increased lipid aldehydes and upregulation of aldehyde detoxifying enzymes. These results place deregulation of lipid metabolism alongside genotoxic stress as factors regulating cellular senescence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/cddiscovery.2017.75DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661608PMC
October 2017

Simple strategies to enhance discovery of acetylation post-translational modifications by quadrupole-orbitrap LC-MS/MS.

Biochim Biophys Acta Proteins Proteom 2018 Feb 16;1866(2):224-229. Epub 2017 Oct 16.

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

Enzyme-dependent post-translational modifications (PTMs) mediate the cellular regulation of proteins and can be discovered using proteomics. However, even where the peptides of interest can be enriched for analysis with state-of-the-art LC-MS/MS tools and informatics, only a fraction of peptide ions can be identified confidently. Thus, many PTM sites remain undiscovered and unconfirmed. In this minireview, we use a case study to discuss how the use of inclusion lists, turning off isotopic exclusion, and manual validation significantly increased depth of coverage, facilitating discovery of acetylation sites in targets of an acetyltransferase virulence factor. These underutilized strategies have the potential to help answer many mechanistic biological questions that large-scale proteomic studies cannot.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbapap.2017.10.006DOI Listing
February 2018

HMG-CoA Reductase Inhibition Delays DNA Repair and Promotes Senescence After Tumor Irradiation.

Mol Cancer Ther 2018 02 13;17(2):407-418. Epub 2017 Oct 13.

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois.

Despite significant advances in combinations of radiotherapy and chemotherapy, altered fractionation schedules and image-guided radiotherapy, many cancer patients fail to benefit from radiation. A prevailing hypothesis is that targeting repair of DNA double strand breaks (DSB) can enhance radiation effects in the tumor and overcome therapeutic resistance without incurring off-target toxicities. Unrepaired DSBs can block cancer cell proliferation, promote cancer cell death, and induce cellular senescence. Given the slow progress to date translating novel DSB repair inhibitors as radiosensitizers, we have explored drug repurposing, a proven route to improving speed, costs, and success rates of drug development. In a prior screen where we tracked resolution of ionizing radiation-induced foci (IRIF) as a proxy for DSB repair, we had identified pitavastatin (Livalo), an HMG-CoA reductase inhibitor commonly used for lipid lowering, as a candidate radiosensitizer. Here, we report that pitavastatin and other lipophilic statins are potent inhibitors of DSB repair in breast and melanoma models both and When combined with ionizing radiation, pitavastatin increased persistent DSBs, induced senescence, and enhanced acute effects of radiation on radioresistant melanoma tumors. shRNA knockdown implicated HMG-CoA reductase, farnesyl diphosphate synthase, and protein farnesyl transferase in IRIF resolution, DSB repair, and senescence. These data confirm on-target activity of statins, although via inhibition of protein prenylation rather than cholesterol biosynthesis. In light of prior studies demonstrating enhanced efficacy of radiotherapy in patients taking statins, this work argues for clinical evaluation of lipophilic statins as nontoxic radiosensitizers to enhance the benefits of image-guided radiotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-17-0288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805623PMC
February 2018

Radiation-enhanced delivery of systemically administered amphiphilic-CpG oligodeoxynucleotide.

J Control Release 2017 Nov 5;266:248-255. Epub 2017 Oct 5.

Ludwig Center for Metastasis Research, The University of Chicago, 5758 South Maryland Avenue, MC 9006, Chicago, IL 60637, United States; Department of Molecular Genetics and Cellular Biology, The University of Chicago, 929 East 57th Street, GCIS W519, Chicago, IL 60637, United States. Electronic address:

Along with vaccines and checkpoint blockade, immune adjuvants may have an important role in tumor immunotherapy. Oligodeoxynucleotides containing unmethylated cytidyl guanosyl dinucleotide motifs (CpG ODN) are TLR9 ligands with attractive immunostimulatory properties, but intratumoral administration has been required to induce an effective anti-tumor immune response. Following on recent studies with radiation-targeted delivery of nanoparticles, we examined enhanced tumor-specific delivery of amphiphile-CpG, an albumin-binding analog of CpG ODN, following systemic administration 3days after tumor irradiation. The combination of radiation and CpG displayed superior tumor control over either treatment alone. Intravital imaging of fluorescently labeled amphiphilic-CpG revealed increased accumulation in irradiated tumors along with decreased off-target accumulation in visceral organs. Within 48h after amphiphile-CpG administration, immune activation could be detected by increased Granzyme B and Interferon gamma activity in the tumor as well as in circulating monocytes and activated CD8 T cells. Using radiotherapy to enhance the targeting of CpG to tumors may help advance this once promising therapy to clinical relevance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2017.09.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723529PMC
November 2017

Chemical inhibitors of Candida albicans hyphal morphogenesis target endocytosis.

Sci Rep 2017 07 18;7(1):5692. Epub 2017 Jul 18.

Department of Molecular Microbiology, B. Rappaport Faculty of Medicine, Technion - I.I.T. and the Rappaport Institute for Research in the Medical Sciences, Haifa, 31096, Israel.

Candida albicans is an opportunistic pathogen, typically found as a benign commensal yeast living on skin and mucosa, but poised to invade injured tissue to cause local infections. In debilitated and immunocompromised individuals, C. albicans may spread to cause life-threatening systemic infections. Upon contact with serum and at body temperature, C. albicans performs a regulated switch to filamentous morphology, characterized by emergence of a germ tube from the yeast cell followed by mold-like growth of branching hyphae. The ability to switch between growth morphologies is an important virulence factor of C. albicans. To identify compounds able to inhibit hyphal morphogenesis, we screened libraries of existing drugs for inhibition of the hyphal switch under stringent conditions. Several compounds that specifically inhibited hyphal morphogenesis were identified. Chemogenomic analysis suggested an interaction with the endocytic pathway, which was confirmed by direct measurement of fluid-phase endocytosis in the presence of these compounds. These results suggest that the activity of the endocytic pathway, which is known to be particularly important for hyphal growth, represents an effective target for hyphae-inhibiting drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-05741-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515890PMC
July 2017

Differential Growth of , Which Alters Expression of Virulence Factors, Dominant Antigens, and Surface-Carbohydrate Synthases, Governs the Apparent Virulence of SchuS4 to Immunized Animals.

Front Microbiol 2017 22;8:1158. Epub 2017 Jun 22.

Department of Immunology and Microbial Disease, Albany Medical CollegeAlbany, NY, United States.

The gram-negative bacterium () is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select virulence factors by an unknown mechanism. Here, we tested the hypotheses that grown in BHI (BHI-) accurately displays a full protein composition more similar to that reported for infection-derived and that this similarity would make BHI- more susceptible to pre-existing, vaccine-induced immunity than MHB-. We performed comprehensive proteomic analysis of grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published "omics" data derived from grown . Based on the abundance of ~1,000 proteins, the fingerprint of BHI- is one of nutrient-deprived bacteria that-through induction of a stringent-starvation-like response-have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI- would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2017.01158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5479911PMC
June 2017

Lipid-derived reactive aldehydes link oxidative stress to cell senescence.

Cell Death Dis 2016 09 8;7(9):e2366. Epub 2016 Sep 8.

Ludwig Center for Metastasis Research, Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/cddis.2016.275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059874PMC
September 2016