Functional Nucleic Acid-Based Immunomodulation for T Cell-Mediated Cancer Therapy.

T cell-mediated immunity plays a pivotal role in cancer immunotherapy. The anticancer actions of T cells are coordinated by a sequence of biological processes, including the capture and presentation of antigens by antigen-presenting cells (APCs), the activation of T cells by APCs, and the subsequent killing of cancer cells by activated T cells. However, cancer cells have various means to evade immune responses. Meanwhile, these vulnerabilities provide potential targets for cancer treatments. Functional nucleic acids (FNAs) make up a class of synthetic nucleic acids with specific biological functions. With their diverse functionality, good biocompatibility, and high programmability, FNAs have attracted widespread interest in cancer immunotherapy. This Review focuses on recent research progress in employing FNAs as molecular tools for T cell-mediated cancer immunotherapy, including corresponding challenges and prospects.

Generation of DNA Aptamers with Functional Activity in Mammalian Cells by Mimicking Retroviruses.

DNA aptamers are single-stranded DNA oligonucleotide sequences that bind to specific targets with high affinity. Currently, DNA aptamers can be produced only by in vitro synthesis. It is difficult for DNA aptamers to have a sustained impact on intracellular protein activity, which limits their clinical application. In this study, we developed a DNA aptamer expression system to generate DNA aptamers with functional activity in mammalian cells by mimicking retroviruses. Using this system, DNA aptamers targeting intracellular Ras (Ra1) and membrane-bound CD71 (XQ2) were successfully generated in cells. In particular, the expressed Ra1 not only specifically bound to the intracellular Ras protein but also inhibited the phosphorylation of downstream ERK1/2 and AKT. Furthermore, by inserting the DNA aptamer expression system for Ra1 into a lentivirus vector, the system can be delivered into cells and stably produce Ra1 over time, resulting in the inhibition of lung cancer cell proliferation. Therefore, our study provides a novel strategy for the intracellular generation of DNA aptamers with functional activity and opens a new avenue for the clinical application of intracellular DNA aptamers in disease treatment.

Aptamer-Mediated Enrichment of Rare Circulating Fetal Nucleated Red Blood Cells for Noninvasive Prenatal Diagnosis.

Isolation of circulating fetal nucleated red blood cells (cfNRBCs) from maternal peripheral blood provides a superior strategy for noninvasive prenatal genetic diagnosis. Recent technical advances in single-cell isolation and genetic analyses have promoted the clinical application of circulating fetal cell-based noninvasive prenatal diagnosis. However, the lack of highly specific ligands for rare circulating fetal cell enrichment from massive maternal cells significantly impedes the clinical transformation progress. In this work, aptamers specific to NRBCs were developed through clinical sample-based cell-SELEX. Herein, the complex clinical system provides natural selection stringency through binding competition between target and background cells, and it empowers aptamers with high specificity. An aptamer-based strategy was also established to isolate cfNRBCs from maternal peripheral blood. Results show the remarkable selectivity and affinity of developed aptamers, enabling efficient enrichment of cfNRBCs from abundant maternal cells. Moreover, screening for fetal sex and trisomy syndrome achieved high accuracy through chromosome analysis of enriched cfNRBCs. To the best of our knowledge, this is the first report to develop aptamer ligands for cfNRBC enrichment, providing an efficient strategy to screen cfNRBC-specific ligands and demonstrating broad application potential for cfNRBC-based noninvasive prenatal diagnosis.

Chemically Synthetic Membrane Receptors Establish Cells with Artificial Sense-and-Respond Signaling Pathways.

Chemically synthetic receptors that establish cells a new sense-and-respond capability to interact with outer worlds are highly desired, but rarely reported. In this work, we develop a membrane-anchored synthetic receptor (Ts-pHLIP-Pr) using DNA and peptide as the building block to equip cells with artificial signaling pathways. Upon sensing external pH stimuli, the Pr module can be translocated across the cell membrane via the conformation switch of pHLIP, enabling membrane-proximal recruitment of specific proteins to trigger downstream signaling cascades. Our experimental results demonstrate the capability of Ts-pHLIP-Pr for regulating PKCε-related signaling events upon responding to external pH reduction. With a modular feature, this receptor can be extended to elicit T cell activation through low-pH environment-induced directional movement of cytoplasmic ZAP70. Our work is expected to offer a new paradigm for intelligent synthetic biology and customized cell engineering.

ERK-mediated Cytoplasmic Retention of USP11 Contributes to Breast Cancer Cell Proliferation by Stabilizing Cytoplasmic p21.

Breast cancer ranks as the most frequently diagnosed cancer among women worldwide. Elevated cytoplasmic p21 levels are often found in breast cancer tissues and related to a poor prognosis. However, the underlying mechanisms that lead to the stabilization of cytoplasmic p21 protein, which normally has a very short half-life, remain obscure. In this study, we found that there was a strong correlation between p21 and USP11 in the cytoplasm of breast cancer tissues and cells. Furthermore, we revealed that ERK1/2 phosphorylated USP11 at the Ser905 site, which promoted the cytoplasmic localization of USP11. In the cytoplasm, USP11 colocalized and interacted with p21. As a result, USP11 catalyzed the removal of polyubiquitin chains bound to cytoplasmic p21 and resulted in its stabilization. Functionally, USP11-mediated stabilization of cytoplasmic p21 induced breast cancer cell proliferation in vitro and in vivo. Our findings provide the first evidence that ubiquitinated p21 in the cytoplasm can be recycled through USP11-mediated deubiquitination, and we identified the USP11-p21 axis in the cytoplasm as a potential therapeutic target for breast cancer control.

Nucleic acid aptamer controls mycoplasma infection for inhibiting the malignancy of esophageal squamous cell carcinoma.

Esophageal cancer is one of the most frequent malignant tumors of the digestive tract, among which esophageal squamous cell carcinoma (ESCC) is the main pathological type worldwide. Previous studies have shown microbial infections in the upper digestive tract to be a potential risk factor in ESCC etiology. In this study, we identified that Mycoplasma hyorhinis infection promoted the malignancy of ESCC. In response, we generated a single-stranded DNA aptamer, ZY3A, against M. hyorhinis using the cell-SELEX strategy. The underlying recognition mechanism of ZY3A on M. hyorhinis involves its binding to M. hyorhinis-specific p37 protein. This tool allowed us to provide the first proof-of-concept evidence using a nucleic acid aptamer to control mycoplasma infection. More specifically, we found that ZY3A could neutralize M. hyorhinis infection on ESCC cells by blocking the interaction between p37 protein and its receptor TLR4 on the ESCC cell membrane. As a result, ZY3A inhibited the migration and invasion of M. hyorhinis-infected ESCC cells in vitro and metastasis in vivo. Taken together, these findings indicate that aptamer ZY3A is a potential candidate for development into a novel molecular tool for treatment of M. hyorhinis infection and a safe first-in-class M. hyorhinis-targeting antitumor agent.

Development of a DNA Aptamer against Multidrug-Resistant Hepatocellular Carcinoma for In Vivo Imaging.

Hepatocellular carcinoma (HCC) is a type of cancer that has high rates of recurrence and mortality. One of the most important factors that lead to treatment failure of HCC is the acquisition of multidrug resistance (MDR). Development of specific ligands for multidrug-resistant HCC will provide useful molecular tools for precise diagnosis and targeted theranostics. Herein, a multidrug-resistant HCC cell (HepG2/MDR)-specific aptamer was developed through Cell-SELEX (systematic evolution of ligands by exponential enrichment) technology. With dissociation constants lying in the nanomolar range, the molecularly designed PS-ZL-7c aptamer showed great selectivity to drug-resistant cancer cells. The in vivo imaging results illustrated that the PS-ZL-7c specifically accumulated in the drug-resistant tumors but not in drug-sensitive tumors and normal tissues, indicating that the PS-ZL-7c aptamer possessed excellent potential as a targeting ligand for precise diagnosis and target theranostics of multidrug-resistant HCC.

The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells.

Ubiquitin-specific protease 11 (USP11) has been implicated in the regulation of DNA repair, apoptosis, signal transduction and cell cycle. It belongs to a USP subfamily of deubiquitinases. Although previous research has shown that USP11 overexpression is frequently found in melanoma and is correlated with a poor prognosis, the potential molecular mechanism of USP11 in melanoma remains indefinitive. Here, we report that USP11 and NONO colocalize and interact with each other in the nucleus of melanoma cells. As a result, the knockdown of USP11 decreases NONO levels. Whereas, overexpression of USP11 increases NONO levels in a dose-dependent manner. Furthermore, we reveal that USP11 protects NONO protein from proteasome-mediated degradation by removing poly-ubiquitin chains conjugated onto NONO. Functionally, USP11 mediated melanoma cell proliferation via the regulation of NONO levels because ablation of USP11 inhibits the proliferation which could be rescued by ectopic expression of NONO protein. Moreover, a significant positive correlation between USP11 and NONO concentrations was found in clinical melanoma samples. Collectively, these results demonstrate that USP11 is a new deubiquitinase of NONO and that the signalling axis of USP11-NONO is significantly involved in melanoma proliferation.

NONO and tumorigenesis: More than splicing.

The non-POU domain-containing octamer-binding protein NONO/p54nrb , which belongs to the Drosophila behaviour/human splicing (DBHS) family, is a multifunctional nuclear protein rarely functioning alone. Emerging solid evidences showed that NONO engages in almost every step of gene regulation, including but not limited to mRNA splicing, DNA unwinding, transcriptional regulation, nuclear retention of defective RNA and DNA repair. NONO is involved in many biological processes including cell proliferation, apoptosis, migration and DNA damage repair. Dysregulation of NONO has been found in many types of cancer. In this review, we summarize the current and fast-growing knowledge about the regulation of NONO, its biological function and implications in tumorigenesis and cancer progression. Overall, significant findings about the roles of NONO have been made, which might make NONO to be a new biomarker or/and a possible therapeutic target for cancers.

Deubiquitinase DUB3 Regulates Cell Cycle Progression via Stabilizing Cyclin A for Proliferation of Non-Small Cell Lung Cancer Cells.

The deubiquitinase DUB3 is frequently overexpressed in non-small cell lung cancer (NSCLC) and contributes to its malignant phenotype. However, the underlying molecular mechanism of DUB3 in NSCLC is largely unknown. In this study, we report that DUB3 regulates cell cycle progression by deubiquitinating cyclin A that links to proliferation of NSCLC cells. We found that knockdown of DUB3 decreases cyclin A levels, whereas overexpression of DUB3 strongly increases cyclin A levels. Mechanistically, DUB3 interacts with cyclin A, which removes the polyubiquitin chains conjugated onto cyclin A and stabilizes the cyclin A protein. Furthermore, we demonstrate that DUB3 regulates cell cycle progression by stabilizing cyclin A, because ablation of DUB3 arrests cell cycle from G0/G1 to S phase and the resulting effect can be rescued by introducing cyclin A into NSCLC cells. Functionally, we found that the effect of DUB3 on cyclin A mediates proliferation of NSCLC cells. Moreover, a significant correlation between DUB3 abundance and cyclin A expression levels were also found in NSCLC samples. Taken together, these results reveal that DUB3 functions as a novel cyclin A regulator through maintaining cyclin A stability, and that the DUB3-cyclin A signaling axis plays a critical role in cell cycle progression for proliferation of NSCLC.