Marine diterpenoid targets STING palmitoylation in mammalian cells.

Natural products are important sources of therapeutic agents and useful drug discovery tools. The fused macrocycles and multiple stereocenters of briarane-type diterpenoids pose a major challenge to total synthesis and efforts to characterize their biological activities. Harnessing a scalable source of excavatolide B (excB) from cultured soft coral Briareum stechei, we generated analogs by late-stage diversification and performed structure-activity analysis, which was critical for the development of functional excB probes. We further used these probes in a chemoproteomic strategy to identify Stimulator of Interferon Genes (STING) as a direct target of excB in mammalian cells. We showed that the epoxylactone warhead of excB is required to covalently engage STING at its membrane-proximal Cys91, inhibiting STING palmitoylation and signaling. This study reveals a possible mechanism-of-action of excB, and expands the repertoire of covalent STING inhibitors.

CRISPR/Cas-Mediated Genome Editing of Streptomyces.

Streptomyces are an important source and reservoir of natural products with diverse applications in medicine, agriculture, and food. Engineered Streptomyces strains have also proven to be functional chassis for the discovery and production of bioactive compounds and enzymes. However, genetic engineering of Streptomyces is often laborious and time-consuming. Here we describe protocols for CRISPR/Cas-mediated genome editing of Streptomyces. Starting from the design and assembly of all-in-one CRISPR/Cas constructs for efficient double-strand break-mediated genome editing, we also present protocols for intergeneric conjugation, CRISPR/Cas plasmid curing, and validation of edited strains.

Chemoproteomic profiling reveals cellular targets of nitro-fatty acids.

Nitro-fatty acids are a class of endogenous electrophilic lipid mediators with anti-inflammatory and cytoprotective effects in a wide range of inflammatory and fibrotic disease models. While these beneficial biological effects of nitro-fatty acids are mainly attributed to their ability to form covalent adducts with proteins, only a small number of proteins are known to be nitro-alkylated and the scope of protein nitro-alkylation remains undetermined. Here we describe the synthesis and application of a clickable nitro-fatty acid probe for the detection and first global identification of mammalian proteins that are susceptible to nitro-alkylation. 184 high confidence nitro-alkylated proteins were identified in THP1 macrophages, majority of which are novel targets of nitro-fatty acids, including extended synaptotagmin 2 (ESYT2), signal transducer and activator of transcription 3 (STAT3), toll-like receptor 2 (TLR2), retinoid X receptor alpha (RXRα) and glucocorticoid receptor (NR3C1). In particular, we showed that 9-nitro-oleate covalently modified and inhibited dexamethasone binding to NR3C1. Bioinformatic analyses revealed that nitro-alkylated proteins are highly enriched in endoplasmic reticulum and transmembrane proteins, and are overrepresented in lipid metabolism and transport pathways. This study significantly expands the scope of protein substrates targeted by nitro-fatty acids in living cells and provides a useful resource towards understanding the pleiotropic biological roles of nitro-fatty acids as signaling molecules or as multi-target therapeutic agents.

Osteonectin Promoter-Mediated Suicide Gene Therapy of Prostate Cancer.

Suicide gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene, combined with the prodrug ganciclovir (GCV) medication, is a promising approach for the treatment of malignant tumors, including prostate cancer. The success of this therapeutic strategy requires tissue- or tumor-specific gene expression and efficient gene delivery. In this chapter, we describe the experimental protocols of key methodologies, including promoter construction, reporter assay, adenoviral vector construction and preparation, HSV-tk enzymatic assay and cytotoxicity assay to evaluate the specificity and efficacy of osteonectin promoter-mediated HSV-tk/GCV suicide gene therapy of prostate cancer.

Loss of let-7 microRNA upregulates IL-6 in bone marrow-derived mesenchymal stem cells triggering a reactive stromal response to prostate cancer.

Bone marrow-derived mesenchymal stem cells (MSCs) are able to migrate to tumors, where they promote tumorigenesis and cancer metastasis. However, the molecular phenotype of the recruited MSCs at the tumor microenvironment and the genetic programs underlying their role in cancer progression remains largely unknown. By using a three-dimensional rotary wall vessel coculture system in which human MSCs were grown alone or in close contact with LNCaP, C4-2 or PC3 prostate cancer cell lines, we established in vitro matched pairs of normal and cancer-associated MSC derivatives to study the stromal response of MSCs to prostate cancer. We observed that prostate cancer-associated MSCs acquired a higher potential for adipogenic differentiation and exhibited a stronger ability to promote prostate cancer cell migration and invasion compared with normal MSCs both in vitro and in experimental animal models. The enhanced adipogenesis and the pro-metastatic properties were conferred by the high levels of IL-6 secretion by cancer-associated MSCs and were reversible by functionally inhibiting of IL-6. We also found that IL-6 is a direct target gene for the let-7 microRNA, which was downregulated in cancer-associated MSCs. The overexpression of let-7 via the transfection of let-7 precursors decreased IL-6 expression and repressed the adipogenic potential and metastasis-promoting activity of cancer-associated MSCs, which was consistent with the inhibition of IL-6 3'UTR luciferase activity. Conversely, the treatment of normal MSCs with let-7 inhibitors resulted in effects similar to those seen with IL-6. Taken together, our data demonstrated that MSCs co-evolve with prostate cancer cells in the tumor microenvironment, and the downregulation of let-7 by cancer-associated MSCs upregulates IL-6 expression. This upregulation triggers adipogenesis and facilitates prostate cancer progression. These findings not only provide key insights into the molecular basis of tumor-stroma interactions but also pave the way for new treatments for metastatic prostate cancer.

Vitamin D3-inducible mesenchymal stem cell-based delivery of conditionally replicating adenoviruses effectively targets renal cell carcinoma and inhibits tumor growth.

Cell-based carriers were recently exploited as a tumor-targeting tool to improve systemic delivery of oncolytic viruses for cancer therapy. However, the slow clearance of carrier cells from normal organs indicates the need for a controllable system which allows viral delivery only when the carrier cells reach the tumor site. In this study, we sought to develop a pharmaceutically inducible cell-based oncolytic adenovirus delivery strategy for effective targeting and treatment of renal cell carcinoma (RCC), which is one of the most malignant tumor types with an unfavorable prognosis. Herein, we demonstrated the intrinsic tumor homing property of human bone marrow-derived mesenchymal stem cells (hMSCs) to specifically localize primary and metastatic RCC tumors after systemic administration in a clinically relevant orthotopic animal model. The platelet derived growth factor AA (PDGF-AA) secreted from RCC was identified as a chemoattractant responsible for the recruitment of hMSCs. Like endogenous osteocalcin whose barely detectable level of expression was dramatically induced by vitamin D(3), the silenced replication of human osteocalcin promoter-directed Ad-hOC-E1 oncolytic adenoviruses loaded in hMSCs was rapidly activated, and the released oncolytic adenoviruses sequentially killed cocultured RCC cells upon vitamin D(3) exposure. Moreover, the systemic treatment of RCC tumor-bearing mice with hMSC cell carriers loaded with Ad-hOC-E1 had very limited effects on tumor growth, but the loaded hMSCs combined with vitamin D(3) treatment induced effective viral delivery to RCC tumors and significant tumor regression. Therapeutic effects of hMSC-based Ad-hOC-E1 delivery were confirmed to be significantly greater than those of injection of carrier-free Ad-hOC-E1. Our results presented the first preclinical demonstration of a novel controllable cell-based gene delivery strategy that combines the advantages of tumor tropism and vitamin D(3)-regulatable human osteocalcin promoter-directed gene expression of hMSCs to improve oncolytic virotherapy for advanced RCC.

A novel targeting modality for renal cell carcinoma: human osteocalcin promoter-mediated gene therapy synergistically induced by vitamin C and vitamin D3.

BACKGROUND: Advanced renal cell carcinoma (RCC) frequently develops skeletal metastasis and is highly resistant to conventional therapies. We hypothesized that the osteocalcin (OC) promoter may be a promising gene delivery system for RCC targeted gene therapy because osteotropic tumors gain osteomimetic properties and thrive in the new environment by exhibiting a bone-like gene expression profile. Human OC (hOC) expression is highly regulated by vitamins and hormone. In the present study, we tested the feasibility of vitamin-regulatable hOC promoter for RCC-specific transcriptional targeting, and examined the anti-tumor effect of vitamins C and D3 with hOC-based adenoviral vectors towards RCC. METHODS: Real-time reverse transcriptase-polymerase chain reaction measured OC expression induced by vitamins C and D3, either alone or in combination, in RCC and normal human renal epithelial cells (HRE). The RCC-cytotoxic effects of concomitant vitamins and hOC promoter-based adenoviral vectors, Ad-hOC-TK and Ad-hOC-E1, were evaluated in both cell culture and a xenograft murine model. RESULTS: We found that high doses of vitamin C induced H2O2-dependent apoptosis in RCC but not HRE. Treatment of RCC cells with combined vitamins C and D3 treatment significantly increased OC promoter activity compared to single reagent treatment. Combined vitamin therapy reduced tumor size (85%) and complete tumor regression occurred in 38% of mice co-administrated Ad-hOC-E1. CONCLUSIONS: The results obtained in the present study demonstrate that vitamins C and D3 synergized with the anti-tumor effects of therapeutic genes driven by hOC promoter through direct cytotoxicity as well as transcriptional targeting. This combined gene therapy provides a promising modality for advanced RCC targeted therapy.