piggyBac-mediated genomic integration of linear dsDNA-based library for deep mutational scanning in mammalian cells.

Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here, we developed two novel strategies for DMS library construction in mammalian cells, namely 'piggyBac-in vitro ligation' and 'piggyBac-in vitro ligation-PCR'. For the first strategy, we took the 'in vitro ligation' approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore-randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino-acid sequences. And we further identified a novel transcriptional activator peptide with the 'piggyBac-in vitro ligation-PCR' strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future.

Construction and Applications of Mammalian Cell-Based DNA-Encoded Peptide/Protein Libraries.

DNA-encoded peptide/protein libraries are the starting point for protein evolutionary modification and functional peptide/antibody selection. Different display technologies, protein directed evolution, and deep mutational scanning (DMS) experiments employ DNA-encoded libraries to provide sequence variations for downstream affinity- or function-based selections. Mammalian cells promise the inherent post-translational modification and near-to-natural conformation of exogenously expressed mammalian proteins and thus are the best platform for studying transmembrane proteins or human disease-related proteins. However, due to the current technical bottlenecks of constructing mammalian cell-based large size DNA-encoded libraries, the advantages of mammalian cells as screening platforms have not been fully exploited. In this review, we summarize the current efforts in constructing DNA-encoded libraries in mammalian cells and the existing applications of these libraries in different fields.

Construction of Peptide Library in Mammalian Cells by dsDNA-Based Strategy.

While different display technologies, represented by phage display, have been widely used in drug discovery, they still can hardly achieve function-based peptide screening, which in most cases is performed in mammalian cells. And most attempts to screen functional peptides with mammalian platforms utilized plasmids to store coding information. Our previous work established double-stranded DNAs (dsDNAs) as innovative biological parts to implement AND-gate genetic circuits in mammalian cells. In the current study, we employ dsDNAs with terminal NNK degenerate codons to implement AND-gate genetic circuits and generate peptide libraries in mammalian cells. This dsDNA-based AND-gate (DBAG) peptide library construction strategy is easy to perform, requiring only PCR reaction and cell transfection. High-throughput sequencing (HTS) and single-cell sequencing results revealed both peptide length and amino acid sequence diversity of DBAG peptide libraries. Moreover, as a feasibility test of this strategy, we identified an MDM2-interacting peptide by applying the DBAG peptide library to a mammalian cell-based two-hybrid system. Our work establishes dsDNAs with terminal degenerate codons as biological parts to build peptide libraries in mammalian cells, which may have great application potential in the future.

MZF1 mediates oncogene-induced senescence by promoting the transcription of p16INK4A.

Oncogene induced senescence is a tumor suppressing defense mechanism, in which the cell cycle-dependent protein kinase (CDK) inhibitor p16INK4A (encoded by the CDKN2A gene) plays a key role. We previously reported that a transcriptional co-activator chromodomain helicase DNA binding protein 7 (CHD7) mediates oncogenic ras-induced senescence by inducing transcription of the p16INK4A gene. In the current study, we identified myeloid zinc finger 1 (MZF1) as the transcriptional factor that recruits CHD7 to the p16INK4A promoter, where it mediates oncogenic ras-induced p16INK4A transcription and senescence through CHD7, in primary human cells from multiple origins. Moreover, the expression of MZF1 is induced by oncogenic ras in senescent cells through the c-Jun and Ets1 transcriptional factors upon their activation by the Ras-Raf-1-MEK-ERK signaling pathway. In non-small cell lung cancer (NSCLC) and pancreatic adenocarcinoma (PAAD) where activating ras mutations occur frequently, reduced MZF1 expression is observed in tumors, as compared to corresponding normal tissues, and correlates with poor patient survival. Analysis of single cell RNA-sequencing data from PAAD patients revealed that among the tumor cells with normal RB expression levels, those with reduced levels of MZF1 are more likely to express lower p16INK4A levels. These findings have identified novel signaling components in the pathway that mediates induction of the p16INK4A tumor suppressor and the senescence response, and suggested that MZF1 is a potential tumor suppressor in at least some cancer types, the loss of which contributes to the inactivation of the p16INK4A/RB pathway and disruption of senescence in tumor cells with intact RB.

Construction of Synthetic Nanobody Library in Mammalian Cells by dsDNA-Based Strategies*.

A nanobody is an antibody fragment consisting of a single monomeric variable antigen-binding domain. Mammalian cells are ideal platforms for identifying nanobodies targeting hard-to-display transmembrane proteins and nanobodies that function as modulators of cellular phenotypes. However, the introduction of a high-diversity nanobody library into mammalian cells is challenging. We have developed two novel methods for constructing a nanobody library in mammalian cells. Complementarity-determining region (CDR) random sequences were first incorporated into upstream and downstream dsDNAs by PCR. In the first method, named dsDNA-HR, upstream and downstream dsDNAs containing an identical overlapping sequence were co-transfected into cultured mammalian cells for intracellular homologous recombination that resulted in the formation of an intact nanobody library expression cassette. In the second method, named in vitro ligation, we generated full-length nanobody expression dsDNAs via ligation of restriction digested upstream and downstream dsDNAs. The obtained full-length dsDNAs were transfected into mammalian cells for nanobody library expression. Using both methods, we generated over a million unique nanobody sequences, as revealed by high-throughput sequencing. Single-cell sequencing was employed to resolve the diversity of the dsDNA-HR nanobody library. We also identified a small molecule, Nocodazole, which could enhance the efficacy of dsDNA-HR.

Development of a CRISPR-Cas9 Based Luciferase Turn-On System as Nonhomologous End Joining Pathway Reporter.

There is a need of a non-homologous end joining (NHEJ) pathway reporter system that facilitates screening and discovery of NHEJ chemical inhibitors. In this study, we developed a CRISPR-Cas9 based luciferase turn-on system as a NHEJ pathway reporter. By substituting nucleotide 205C with ATC, we introduced a reading-frame shift and a pre-stop codon into the luciferase coding region and thereby generated a bioluminescent signal mute HEK293T reporter cell line. Then, a CRISPR-Cas9 plasmid expressing a guide RNA targeting luciferase coding region was introduced into the reporter cell line to generate NHEJ-associated indel to restore the reading frame and subsequently turn on the bioluminescent signal. We observed over three-thousand fold increase in signal after CRISPR-Cas9 vector transfection. Different known chemical inhibitors of the NHEJ pathway, such as NU7441, KU0060648, and KU55933, could significantly inhibit the bioluminescent signal generated by CRISPR-Cas9 targeting. In addition, we validated our system by high throughput sequencing.

Ferroptosis and Its Potential Role in Human Diseases.

Ferroptosis is a novel regulated cell death pattern discovered when studying the mechanism of erastin-killing RAS mutant tumor cells in 2012. It is an iron-dependent programmed cell death pathway mainly caused by an increased redox imbalance but with distinct biological and morphology characteristics when compared to other known cell death patterns. Ferroptosis is associated with various diseases including acute kidney injury, cancer, and cardiovascular, neurodegenerative, and hepatic diseases. Moreover, activation or inhibition of ferroptosis using a variety of ferroptosis initiators and inhibitors can modulate disease progression in animal models. In this review, we provide a comprehensive analysis of the characteristics of ferroptosis, its initiators and inhibitors, and the potential role of its main metabolic pathways in the treatment and prevention of various diseased states. We end the review with the current knowledge gaps in this area to provide direction for future research on ferroptosis.

CD137 promotes bone metastasis of breast cancer by enhancing the migration and osteoclast differentiation of monocytes/macrophages.

Rationale: Bone is one of the most common metastatic sites of breast cancer. CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor superfamily, is mainly expressed in activated leukocytes. Previous study demonstrates the effect of CD137-CD137L bidirectional signaling pathway on RANKL-mediated osteoclastogenesis. However, the role of CD137 in bone metastasis of breast cancer needs further study. Methods: Stable monocyte/macrophage cell lines with Cd137 overexpression and silencing were established. Western blot, real-time PCR, transwell and tartrate-resistant acid phosphatase staining were used to detect the regulatory effect of CD137 on migration and osteoclastogenesis of monocytes/macrophages in vitro. Spontaneous bone metastasis mouse model was established, bioluminescent images, immunohistochemistry and histology assay were performed to detect the function of CD137 in bone metastasis in vivo. Results: We found that CD137 promotes the migration of monocytes/macrophages to tumor microenvironment by upregulating the expression of Fra1. It also promoted the differentiation of monocytes/macrophages into osteoclasts at the same time, thus providing a favorable microenvironment for the colonization and growth of breast cancer cells in bone. Based on these findings, a novel F4/80-targeted liposomal nanoparticle encapsulating the anti-CD137 blocking antibody (NP-αCD137 Ab-F4/80) was synthesized. This nanoparticle could inhibit both bone and lung metastases of 4T1 breast cancer cells with high efficacy in vivo. In addition, it increased the therapeutic efficacy of Fra1 inhibitor on tumor metastasis. Conclusions: Taken together, these findings reveal the promotion effect of macrophage/monocyte CD137 on bone metastases and provide a promising therapeutic strategy for metastasis of breast cancer.

Linear double-stranded DNAs as innovative biological parts to implement genetic circuits in mammalian cells.

Synthetic biology employs engineering principles to redesign biological systems for biomedical or industrial purposes. Innovation and application of original biological parts for genetic circuit construction will significantly facilitate and expedite the development of synthetic biology. Here, we built two- or three-input linear double-stranded DNA (ldsDNA)-based Boolean AND gate genetic circuits in mammalian cells. Bioluminescence imaging revealed the feasibility of ldsDNA-based Boolean AND gate circuits in vivo. Inhibition of DNA-PKcs, a pivotal enzyme in nonhomologous end joining, significantly attenuated the output signals from ldsDNA-based Boolean AND gate circuits. We further showed that ldsDNA with additional terminal random nucleotide(s) could undergo end nucleotide deletion and generate in-frame proteins via the Boolean AND gate response. Additionally, ldsDNAs or plasmids with identical overlapping sequences could also serve as input signals for Boolean AND gate genetic circuits. Our work establishes ldsDNAs as innovative biological parts for building low noise-signal ratio Boolean AND gate circuits with application potential in biomedical engineering fields.

PFKFB4 Promotes Breast Cancer Metastasis via Induction of Hyaluronan Production in a p38-Dependent Manner.

BACKGROUND/AIMS: The bi-functional enzyme 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase-4 (PFKFB4) is highly expressed in many types of cancer and its requirement for tumor survival has been demonstrated in glioma, lung, and prostate cancers. However, whether PFKFB4 plays a role in the tumor metastasis remains uncertain. This study explores the role of PFKFB4 in tumor metastasis and its underlying mechanisms in breast cancer cells. METHODS: The expression of PFKFB4 was first analyzed using the Cancer Genome Atlas (TCGA) dataset, and confirmed by immunohistochemical staining of tissue microarray and breast cancer tissues from patient samples. Gain- and loss-of- function approaches were used to investigate the effects of PFKFB4 on breast cancer cell migration in vitro. Orthotopic xenograft model and experimental metastasis model were used to assess the effects of PFKFB4 on breast cancer cell metastasis in vivo. ELISA and immunofluorescence staining were used to examine HA production. Quantitative RT-PCR and western blotting were used to explore the mRNA and protein levels of HAS2, respectively. RESULTS: We found that PFKFB4 enhances the migration/invasiveness of breast cancer cells in vitro as well as in vivo. Notably, the effects of PFKFB4 on migration are mediated by induction of HAS2 expression and HA production. Moreover, PFKFB4-induced HAS2 up-regulation depends upon the activation of p38 signaling. CONCLUSION: PFKFB4 promotes the metastasis of breast cancer cells via induction of HAS2 expression and HA production in a p38-dependent manner. Therefore, the PFKFB4/p38/HAS2 signaling pathway may serve as a potential therapeutic target for metastatic breast cancer.

miR-30 disrupts senescence and promotes cancer by targeting both p16INK4A and DNA damage pathways.

miR-30 is a microRNA frequently overexpressed in human cancers. However, the biological consequence of miR-30 overexpression in cancer has been unclear. In a genetic screen, miR-30 was found to abrogate oncogenic-induced senescence, a key tumor-suppressing mechanism that involves DNA damage responses, activation of p53 and induction of p16INK4A. In cells and mouse models, miR-30 disrupts senescence and promotes cancer by suppressing 2 targets, CHD7 and TNRC6A. We show that while CHD7 is a transcriptional coactivator essential for induction of p16INK4A in senescent cells, TNRC6A, a miRNA machinery component, is required for expression and functionality of DNA damage response RNAs (DDRNAs) that mediate DNA damage responses and p53 activation by orchestrating histone modifications, chromatin remodeling and recruitment of DNA damage factors at damaged sites. Thus, miR-30 inhibits both p16INK4A and p53, 2 key senescence effectors, leading to efficient senescence disruption. These findings have identified novel signaling pathways mediating oncogene-induced senescence and tumor-suppression, and revealed the molecular and cellular mechanisms underlying the oncogenic activity of miR-30. Thus, the miR-30/CHD7/TNRC6A pathway is potentially a novel diagnostic biomarker and therapeutic target for cancer.

Inflammatory Human Umbilical Cord-Derived Mesenchymal Stem Cells Promote Stem Cell-Like Characteristics of Cancer Cells in an IL-1ß-Dependent Manner.

To ensure the safety of clinical applications of MSCs, thorough understanding of their impacts on tumor initiation and progression is essential. Here, to further explore the complex dialog between MSCs and tumor cells, umbilical cord-derived mesenchymal stem cells (UC-MSCs) were employed to be cocultured with either breast or ovarian cancer cells. Though having no obvious influence on proliferation or apoptosis, UC-MSCs exerted intense stem cell-like properties promoting effects on both cancer models. Cocultured cancer cells showed enriched side population, enhanced sphere formation ability, and upregulated pluripotency-associated stem cell markers. Human cytokine array and real-time PCR revealed a panel of MSC-derived prostemness cytokines CCL2, CXCL1, IL-8, and IL-6 which were induced upon coculturing. We further revealed IL-1ß, a well-characterized proinflammatory cytokine, to be the inducer of these prostemness cytokines, which was generated from inflammatory UC-MSCs in an autocrine manner. Additionally, with introduction of IL-1RA (an IL-1 receptor antagonist) into the coculturing system, the stem cell-like characteristics promoting effects of inflammatory UC-MSCs were partially blocked. Taken together, these findings suggest that transduced inflammatory MSCs work as a major source of IL-1ß in tumor microenvironment and initiate the formation of prostemness niche via regulating their secretome in an IL-1ß-dependent manner.

Inactivation of p38 MAPK contributes to stem cell-like properties of non-small cell lung cancer.

Cancer stem cells (CSCs) are recognized as the major source for cancer initiation and recurrence. Yet, the mechanism by which the cancer stem cell properties are acquired and maintained in a cancer cell population is not well understood. In the current study, we observed that the level of active p38 MAPK is downregulated, while the level of the stemness marker SOX2 is upregulated in lung cancer tissues as compared to normal tissues. We further demonstrated that inactivation of p38 is a potential mechanism contributing to acquisition and maintenance of cancer stem cell properties in non-small cell lung cancer (NSCLC) cells. p38, in particular the p38γ and p38δ isoforms, suppresses the cancer stem cell properties and tumor initiating ability of NSCLC cells by promoting the ubiquitylation and degradation of stemness proteins such as SOX2, Oct4, Nanog, Klf4 and c-Myc, through MK2-mediated phosphorylation of Hsp27 that is an essential component of the proteasomal degradation machinery. In contrast, inactivation of p38 in lung cancer cells leads to upregulation of the stemness proteins, thus promoting the cancer stem cell properties of these cells. These findings have demonstrated a novel mechanism by which cancer stem cell properties are acquired and maintained in a cancer cell population, and have revealed a new function of the p38 pathway in suppressing cancer development. These studies have also identified a new pathway that can potentially serve as a target for cancer therapies aimed at eliminating CSCs.

Artificial Virus Delivers CRISPR-Cas9 System for Genome Editing of Cells in Mice.

CRISPR-Cas9 has emerged as a versatile genome-editing platform. However, due to the large size of the commonly used CRISPR-Cas9 system, its effective delivery has been a challenge and limits its utility for basic research and therapeutic applications. Herein, a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) was constructed for the delivery of CRISPR-Cas9 system. The artificial virus could efficiently load with the CRISPR-Cas9 system, accelerate the endosomal escape, and promote the penetration into the nucleus without additional nuclear-localization signal, thus enabling targeted gene disruption. Notably, the artificial virus is more efficient than SuperFect, Lipofectamine 2000, and Lipofectamine 3000. When loaded with a CRISPR-Cas9 plasmid, it induced higher targeted gene disruption efficacy than that of Lipofectamine 3000. Furthermore, the artificial virus effectively targets the ovarian cancer via dual-receptor-mediated endocytosis and had minimum side effects. When loaded with the Cas9-hMTH1 system targeting MTH1 gene, RRPHC showed effective disruption of MTH1 in vivo. This strategy could be adapted for delivering CRISPR-Cas9 plasmid or other functional nucleic acids in vivo.

Paclitaxel and Tacrolimus Coencapsulated Polymeric Micelles That Enhance the Therapeutic Effect of Drug-Resistant Ovarian Cancer.

The combination of chemotherapy drugs and multidrug-resistant reversing agents for treating multidrug resistance in tumors has attracted increasing attention. However, the poor water solubility of some anticancer drugs restricted their clinical application. In this work, we prepared poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles as a codelivery system to load the chemotherapy drug paclitaxel (PTX) and the multidrug-resistant reversing agent tacrolimus (FK506). The PTX- and FK506-coloaded MPEG-PCL micelles (P-F/M) were prepared by a one-step solid dispersion method without any surfactants, toxic organic solvent, or severe experimental conditions. P-F/M had small particle size (28.7 ± 3.2 nm) and high encapsulation efficiency (99.3 ± 0.5%). Compared with A2780s cells (PTX-sensitive human ovarian cancer cells), P-F/M showed a stronger cytotoxicity and an improving intracellular drug concentration of PTX than PTX-loaded micelles (PTX/M) in A2780/T cells (PTX-resistant human ovarian cancer cells). Furthermore, a P-F/M codelivery system showed a more significant G2/M arrest and apoptosis induction effects, as well as activating apoptosis protein signaling pathway, in A2780/T cells than in A2780s cells. In summary, the results suggested that the codelivery micelles of PTX and FK506 may serve as a potential candidates against MDR human ovarian cancer.

Activating transcription factor 4 promotes angiogenesis of breast cancer through enhanced macrophage recruitment.

Angiogenesis plays an important role in the progression of tumor. Besides being regulated by tumor cells per se, tumor angiogenesis is also influenced by stromal cells in tumor microenvironment (TME), for example, tumor associated macrophages (TAMs). Activating transcription factor 4 (ATF4), a member of the ATF/CREB family, has been reported to be related to tumor angiogenesis. In this study, we found that exogenous overexpression of ATF4 in mouse breast cancer cells promotes tumor growth via increasing tumor microvascular density. However, ATF4 overexpression failed to increase the expression level of a series of proangiogenic factors including vascular endothelial growth factor A (VEGFA) in tumor cells in this model. Thus, we further investigated the infiltration of proangiogenic macrophages in tumor tissues and found that ATF4-overexpressing tumors could recruit more macrophages via secretion of macrophage colony stimulating factor (M-CSF). Overall, we concluded that exogenous overexpression of ATF4 in breast cancer cells may facilitate the recruitment of macrophages into tumor tissues and promote tumor angiogenesis and tumor growth indirectly.

Biodegradable polymeric micelle-encapsulated doxorubicin suppresses tumor metastasis by killing circulating tumor cells.

Circulating tumor cells (CTCs) play a crucial role in tumor metastasis, but it is rare for any chemotherapy regimen to focus on killing CTCs. Herein, we describe doxorubicin (Dox) micelles that showed anti-metastatic activity by killing CTCs. Dox micelles with a small particle size and high encapsulation efficiency were obtained using a pH-induced self-assembly method. Compared with free Dox, Dox micelles exhibited improved cytotoxicity, apoptosis induction, and cellular uptake. In addition, Dox micelles showed a sustained release behavior in vitro, and in a transgenic zebrafish model, Dox micelles exhibited a longer circulation time and lower extravasation from blood vessels into surrounding tissues. Anti-tumor and anti-metastatic activities of Dox micelles were investigated in transgenic zebrafish and mouse models. In transgenic zebrafish, Dox micelles inhibited tumor growth and prolonged the survival of tumor-bearing zebrafish. Furthermore, Dox micelles suppressed tumor metastasis by killing CTCs. In addition, improved anti-tumor and anti-metastatic activities were also confirmed in mouse tumor models, where immunofluorescent staining of tumors indicated that Dox micelles induced more apoptosis and showed fewer proliferation-positive cells. There were decreased side effects in transgenic zebrafish and mice after administration of Dox micelles. In conclusion, Dox micelles showed stronger anti-tumor and anti-metastatic activities and decreased side effects both in vitro and in vivo, which may have potential applications in cancer therapy.