Targeting regulator of G protein signaling 1 in tumor-specific T cells enhances their trafficking to breast cancer.

Reduced infiltration of anti-tumor lymphocytes remains a major cause of tumor immune evasion and is correlated with poor cancer survival. Here, we found that upregulation of regulator of G protein signaling (RGS)1 in helper TH1 cells and cytotoxic T lymphocytes (CTLs) reduced their trafficking to and survival in tumors and was associated with shorter survival of patients with breast and lung cancer. RGS1 was upregulated by type II interferon (IFN)-signal transducer and activator of transcription (STAT)1 signaling and impaired trafficking of circulating T cells to tumors by inhibiting calcium influx and suppressing activation of the kinases ERK and AKT. RGS1 knockdown in adoptively transferred tumor-specific CTLs significantly increased their infiltration and survival in breast and lung tumor grafts and effectively inhibited tumor growth in vivo, which was further improved when combined with programmed death ligand (PD-L)1 checkpoint inhibition. Our findings reveal RGS1 is important for tumor immune evasion and suggest that targeting RGS1 may provide a new strategy for tumor immunotherapy.

DNA of neutrophil extracellular traps promotes cancer metastasis via CCDC25.

Neutrophil extracellular traps (NETs), which consist of chromatin DNA filaments coated with granule proteins, are released by neutrophils to trap microorganisms1-3. Recent studies have suggested that the DNA component of NETs (NET-DNA) is associated with cancer metastasis in mouse models4-6. However, the functional role and clinical importance of NET-DNA in metastasis in patients with cancer remain unclear. Here we show that NETs are abundant in the liver metastases of patients with breast and colon cancers, and that serum NETs can predict the occurrence of liver metastases in patients with early-stage breast cancer. NET-DNA acts as a chemotactic factor to attract cancer cells, rather than merely acting as a 'trap' for them; in several mouse models, NETs in the liver or lungs were found to attract cancer cells to form distant metastases. We identify the transmembrane protein CCDC25 as a NET-DNA receptor on cancer cells that senses extracellular DNA and subsequently activates the ILK-ß-parvin pathway to enhance cell motility. NET-mediated metastasis is abrogated in CCDC25-knockout cells. Clinically, we show that the expression of CCDC25 on primary cancer cells is closely associated with a poor prognosis for patients. Overall, we describe a transmembrane DNA receptor that mediates NET-dependent metastasis, and suggest that targeting CCDC25 could be an appealing therapeutic strategy for the prevention of cancer metastasis.

Construction of cell factory capable of efficiently converting L-tryptophan into 5-hydroxytryptamine.

BACKGROUND: L-Tryptophan (L-Trp) derivatives such as 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT), N-Acetyl-5-hydroxytryptamine and melatonin are important molecules with pharmaceutical interest. Among, 5-HT is an inhibitory neurotransmitter with proven benefits for treating the symptoms of depression. At present, 5-HT depends on plant extraction and chemical synthesis, which limits its mass production and causes environmental problems. Therefore, it is necessary to develop an efficient, green and sustainable biosynthesis method to produce 5-HT. RESULTS: Here we propose a one-pot production of 5-HT from L-Trp via two enzyme cascades for the first time. First, a chassis cell that can convert L-Trp into 5-HTP was constructed by heterologous expression of tryptophan hydroxylase from Schistosoma mansoni (SmTPH) and an artificial endogenous tetrahydrobiopterin (BH4) module. Then, dopa decarboxylase from Harminia axyridis (HaDDC), which can specifically catalyse 5-HTP to 5-HT, was used for 5-HT production. The cell factory, E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH, which contains SmTPH and HaDDC, was constructed for 5-HT synthesis. The highest concentration of 5-HT reached 414.5 ± 1.6 mg/L (with conversion rate of 25.9 mol%) at the optimal conditions (substrate concentration,2 g/L; induced temperature, 25℃; IPTG concentration, 0.5 mM; catalysis temperature, 30℃; catalysis time, 72 h). CONCLUSIONS: This protocol provided an efficient one-pot method for converting. L-Trp into 5-HT production, which opens up possibilities for the practical biosynthesis of natural 5-HT at an industrial scale.

The Dawning of Translational Breast Cancer: From Bench to Bedside.

Breast cancer is one of the world's leading causes of death in women. Although tumor initiation and progression are predominantly driven by somatic or acquired (epi) genetic alterations that govern signaling abnormalities, growing evidence suggests that the inflammatory microenvironments of cancer also play a role. Molecular characterization of breast cancer biology is essential for high-efficient management of this disease in clinical practice. Translating basic research into clinically valuable biomarkers for diagnosis, prognosis, and prediction of response to treatment and into precisely targeted therapies is crucial for the development of precision medicine in breast cancer. Such a process is known as "from bench to bedside." In this chapter, we will present an overview of breast cancer pathogenesis and selected translational advances in multistage clinical settings and aim to illustrate the dawning of precision medicine implementation in managing human breast malignancies.

Noncoding RNAs: New Players in Cancers.

The world of noncoding RNAs (ncRNAs) has gained widespread attention in recent years due to their novel and crucial potency of biological regulation. Noncoding RNAs play essential regulatory roles in a broad range of developmental processes and diseases, notably human cancers. Regulatory ncRNAs represent multiple levels of structurally and functionally distinct RNAs, including the best-known microRNAs (miRNAs), the complicated long ncRNAs (lncRNAs), and the newly identified circular RNAs (circRNAs). However, the mechanisms by which they act remain elusive. In this chapter, we will review the current knowledge of the ncRNA field, discussing the genomic context, biological functions, and mechanisms of action of miRNAs, lncRNAs, and circRNAs. We also highlight the implications of the biogenesis and gene expression dysregulation of different ncRNA subtypes in the initiation and development of human malignancies.

ATR/Chk1 interacting lncRNA modulates DNA damage response to induce breast cancer chemoresistance.

The ATR-Chk1 pathway is essential in cellular responses to DNA damage and replication stress, whereas the role of long noncoding RNAs (lncRNAs) in regulating this pathway remains largely unknown. In this study, we identify an ATR and Chk1 interacting lncRNA (ACIL, also known as LRRC75A-AS1 or SNHG29), which promotes the phosphorylation of Chk1 by ATR upon DNA damages. High ACIL levels are associated with chemoresistance to DNA damaging agents and poor outcome of breast cancer patients. ACIL knockdown sensitizes breast cancer cells to DNA damaging drugs in vitro and in vivo. ACIL protects cancer cells against DNA damages by inducing cell cycle arrest, stabilizing replication forks and inhibiting unscheduled origin firing, thereby guarding against replication catastrophe and contributing to DNA damage repair. These findings demonstrate a lncRNA-dependent mechanism of activating the ATR-Chk1 pathway and highlight the potential of utilizing ACIL as a predictive biomarker for chemotherapy sensitivity, as well as targeting ACIL to reverse chemoresistance in breast cancer.

Activation of Bivalent Gene POU4F1 Promotes and Maintains Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) is the most aggressive molecular subtype of breast cancer with worse prognosis and fewer treatment options. The underlying mechanisms upon BLBC transcriptional dysregulation and its upstream transcription factors (TFs) remain unclear. Here, among the hyperactive candidate TFs of BLBC identified by bioinformatic analysis, POU4F1 is uniquely upregulated in BLBC and is associated with poor prognosis. POU4F1 is necessary for the tumor growth and malignant phenotypes of BLBC through regulating G1/S transition by direct binding at the promoter of CDK2 and CCND1. More importantly, POU4F1 maintains BLBC identity by repressing ERα expression through CDK2-mediated EZH2 phosphorylation and subsequent H3K27me3 modification in ESR1 promoter. Knocking out POU4F1 in BLBC cells reactivates functional ERα expression, rendering BLBC sensitive to tamoxifen treatment. In-depth epigenetic analysis reveals that the subtype-specific re-configuration and activation of the bivalent chromatin in the POU4F1 promoter contributes to its unique expression in BLBC, which is maintained by DNA demethylase TET1. Together, these results reveal a subtype-specific epigenetically activated TF with critical role in promoting and maintaining BLBC, suggesting that POU4F1 is a potential therapeutic target for BLBC.

LncRNA ZFPM2-AS1 promotes phyllodes tumor progression by binding to CDC42 and inhibiting STAT1 activation.

Breast phyllodes tumor (PT) is a rare fibroepithelial neoplasm with potential malignant behavior. Long non-coding RNAs (lncRNAs) play multifaceted roles in various cancers, but their involvement in breast PT remains largely unexplored. In this study, microarray was leveraged for the first time to investigate the role of lncRNA in PT. We identified lncRNA ZFPM2-AS1 was significantly upregulated in malignant PT, and its overexpression endowed PT with high tumor grade and adverse prognosis. Furthermore, we elucidated that ZFPM2-AS1 promotes the proliferation, migration, and invasion of malignant PT in vitro. Targeting ZFPM2-AS1 through nanomaterial-mediated siRNA delivery in patient-derived xenograft (PDX) model could effectively inhibit tumor progression in vivo. Mechanistically, our findings showed that ZFPM2-AS1 is competitively bound to CDC42, inhibiting ACK1 and STAT1 activation, thereby launching the transcription of TNFRSF19. In conclusion, our study provides evidence that ZFPM2-AS1 plays a pivotal role in the pathogenesis of breast PT, and suggests that ZFPM2-AS1 could serve as a prognostic indicator for patients with PT as well as a promising novel therapeutic target.

The theory of tumor ecosystem.

Cancer cells can be conceived as "living organisms" interacting with cellular or non-cellular components in the host internal environment, not only the local tumor microenvironment but also the distant organ niches, as well as the immune, nervous and endocrine systems, to construct a self-sustainable tumor ecosystem. With increasing evidence for the systemic tumor-host interplay, we predict that a new era of cancer therapy targeting the ecosystemic vulnerability of human malignancies has come. Revolving around the tumor ecosystem scoped as different hierarchies of primary, regional, distal and systemic onco-spheres, we comprehensively review the tumor-host interaction among cancer cells and their local microenvironment, distant organ niches, immune, nervous and endocrine systems, highlighting material and energy flow with tumor ecological homeostasis as an internal driving force. We also substantiate the knowledge of visualizing, modelling and subtyping this dynamically intertwined network with recent technological advances, and discuss ecologically rational strategies for more effective cancer therapies.

Characterization of a New Multifunctional GH20 ß-N-Acetylglucosaminidase From Chitinibacter sp. GC72 and Its Application in Converting Chitin Into N-Acetyl Glucosamine.

In this study, a gene encoding ß-N-acetylglucosaminidase, designated NAGaseA, was cloned from Chitinibacter sp. GC72 and subsequently functional expressed in Escherichia coli BL21 (DE3). NAGaseA contains a glycoside hydrolase family 20 catalytic domain that shows low identity with the corresponding domain of the well-characterized NAGases. The recombinant NAGaseA had a molecular mass of 92 kDa. Biochemical characterization of the purified NAGaseA revealed that the optimal reaction condition was at 40°C and pH 6.5, and exhibited great pH stability in the range of pH 6.5-9.5. The V ma x , K m, k cat, and k cat /K m of NAGaseA toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) were 3333.33 µmol min-1 l-1, 39.99 µmol l-1, 4667.07 s-1, and 116.71 ml µmol-1 s-1, respectively. Analysis of the hydrolysis products of N-acetyl chitin oligosaccharides (N-Acetyl COSs) indicated that NAGaseA was capable of converting N-acetyl COSs ((GlcNAc)2-(GlcNAc)6) into GlcNAc with hydrolysis ability order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6. Moreover, NAGaseA could generate (GlcNAc)3-(GlcNAc)6 from (GlcNAc)2-(GlcNAc)5, respectively. These results showed that NAGaseA is a multifunctional NAGase with transglycosylation activity. In addition, significantly synergistic action was observed between NAGaseA and other sources of chitinases during hydrolysis of colloid chitin. Finally, 0.759, 0.481, and 0.986 g/l of GlcNAc with a purity of 96% were obtained using three different chitinase combinations, which were 1.61-, 2.36-, and 2.69-fold that of the GlcNAc production using the single chitinase. This observation indicated that NAGaseA could be a potential candidate enzyme in commercial GlcNAc production.

Property and Function of a Novel Chitinase Containing Dual Catalytic Domains Capable of Converting Chitin Into N-Acetyl-D-Glucosamine.

A novel multifunctional chitinase (CmChi3)-encoding gene was cloned from Chitinolyticbacter meiyuanensis and actively expressed in Escherichia coli. Sequence analysis showed that CmChi3 contains two glycoside hydrolase family 18 (GH18) catalytic domains and exhibited low identity with well-characterized chitinases. The optimum pH and temperature of purified recombinant CmChi3 were 6.0 and 50°C, respectively. CmChi3 exhibited strict substrate specificity of 4.1 U/mg toward colloidal chitin (CC) and hydrolyzed it to yield N-acetyl-D-glucosamine (GlcNAc) as the sole end product. An analysis of the hydrolysis products toward N-acetyl chitooligosaccharides (N-acetyl COSs) and CC substrates revealed that CmChi3 exhibits endochitinase, N-acetyl-ß-d-glucosaminidase (NAGase), and transglycosylase (TGase) activities. Further studies revealed that the N-terminal catalytic domain of CmChi3 exhibited endo-acting and NAGase activities, while the C-terminal catalytic domain showed exo-acting and TGase activities. The hydrolytic properties and favorable environmental adaptations indicate that CmChi3 holds potential for commercial GlcNAc production from chitin.

Long noncoding RNA DIO3OS induces glycolytic-dominant metabolic reprogramming to promote aromatase inhibitor resistance in breast cancer.

Aromatase inhibition is an efficient endocrine therapy to block ectopic estrogen production for postmenopausal estrogen receptor (ER)-positive breast cancer patients, but many develop resistance. Here, we show that aromatase inhibitor (AI)-resistant breast tumors display features of enhanced aerobic glycolysis with upregulation of long noncoding RNA (lncRNA) DIO3OS, which correlates with poor prognosis of breast cancer patients on AI therapies. Long-term estrogen deprivation induces DIO3OS expression in ER-positive breast tumor cells, which further enhances aerobic glycolysis and promotes estrogen-independent cell proliferation in vitro and in vivo. Mechanistically, DIO3OS interacts with polypyrimidine tract binding protein 1 (PTBP1) and stabilizes the mRNA of lactate dehydrogenase A (LDHA) by protecting the integrity of its 3'UTR, and subsequently upregulates LDHA expression and activates glycolytic metabolism in AI-resistant breast cancer cells. Our findings highlight the role of lncRNA in regulating the key enzyme of glycolytic metabolism in response to endocrine therapies and the potential of targeting DIO3OS to reverse AI resistance in ER-positive breast cancer.

The IRENA lncRNA converts chemotherapy-polarized tumor-suppressing macrophages to tumor-promoting phenotypes in breast cancer.

Although chemotherapy can stimulate antitumor immunity by inducing interferon (IFN) response, the functional role of tumor-associated macrophages in this scenario remains unclear. Here, we found that IFN-activated proinflammatory macrophages after neoadjuvant chemotherapy enhanced antitumor immunity but promoted cancer chemoresistance. Mechanistically, IFN induced expression of cytoplasmic long noncoding RNA IFN-responsive nuclear factor-κB activator (IRENA) in macrophages, which triggered nuclear factor-κB signaling via dimerizing protein kinase R and subsequently increased production of protumor inflammatory cytokines. By constructing macrophage-conditional IRENA-knockout mice, we found that targeting IRENA in IFN-activated macrophages abrogated their protumor effects, while retaining their capacity to enhance antitumor immunity. Clinically, IRENA expression in post-chemotherapy macrophages was associated with poor patient survival. These findings indicate that lncRNA can determine the dichotomy of inflammatory cells on cancer progression and antitumor immunity and suggest that targeting IRENA is an effective therapeutic strategy to reversing tumor-promoting inflammation.

Turning foes to friends: targeting cancer-associated fibroblasts.

Current paradigms of cancer-centric therapeutics are usually not sufficient to eradicate the malignancy, as the cancer stroma may prompt tumour relapse and therapeutic resistance. Among all the stromal cells that populate the tumour microenvironment, cancer-associated fibroblasts (CAFs) are the most abundant and are critically involved in cancer progression. CAFs regulate the biology of tumour cells and other stromal cells via cell-cell contact, releasing numerous regulatory factors and synthesizing and remodelling the extracellular matrix, and thus these cells affect cancer initiation and development. The recent characterization of CAFs based on specific cell surface markers not only deepens our insight into their phenotypic heterogeneity and functional diversity but also brings CAF-targeting therapies for cancer treatment onto the agenda. In this Review, we discuss the current knowledge of biological hallmarks, cellular origins, phenotypical plasticity and functional heterogeneity of CAFs and underscore their contribution to cancer progression. Moreover, we highlight relevant translational advances and potential therapeutic strategies that target CAFs for cancer treatment.

Mammary stem cells: angels or demons in mammary gland?

A highly dynamic development process exits within the epithelia of mammary gland, featuring morphogenetic variation during puberty, pregnancy, lactation, and regression. The identification of mammary stem cells (MaSCs) via lineage-tracing studies has substantiated a hierarchical organization of the mammary epithelia. A single MaSC is capable of reconstituting the entirely functional mammary gland upon orthotopic transplantation. Although different mammary cell subpopulations can be candidate cells-of-origin for distinct breast tumor subtypes, it still lacks experimental proofs whether MaSCs, the most primitive cells, are the 'seeds' of malignant transformation during most, if not all, tumorigenesis in the breast. Here, we review current knowledge of mammary epithelial hierarchy, highlighting the roles of mammary stem/progenitor cells and breast cancer stem cells (BCSCs) along with their key molecular regulators in organ development and cancer evolution. Clarifying these issues will pave the way for developing novel interventions toward stem/progenitor cells in either prevention or treatment of breast cancer (BrCa).

Silibinin inhibits acetylcholinesterase activity and amyloid ß peptide aggregation: a dual-target drug for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by amyloid ß (Aß) peptide aggregation and cholinergic neurodegeneration. Therefore, in this paper, we examined silibinin, a flavonoid extracted from Silybum marianum, to determine its potential as a dual inhibitor of acetylcholinesterase (AChE) and Aß peptide aggregation for AD treatment. To achieve this, we used molecular docking and molecular dynamics simulations to examine the affinity of silibinin with Aß and AChE in silico. Next, we used circular dichroism and transmission electron microscopy to study the anti-Aß aggregation capability of silibinin in vitro. Moreover, a Morris Water Maze test, enzyme-linked immunosorbent assay, immunohistochemistry, 5-bromo-2-deoxyuridine double labeling, and a gene gun experiment were performed on silibinin-treated APP/PS1 transgenic mice. In molecular dynamics simulations, silibinin interacted with Aß and AChE to form different stable complexes. After the administration of silibinin, AChE activity and Aß aggregations were down-regulated, and the quantity of AChE also decreased. In addition, silibinin-treated APP/PS1 transgenic mice had greater scores in the Morris Water Maze. Moreover, silibinin could increase the number of newly generated microglia, astrocytes, neurons, and neuronal precursor cells. Taken together, these data suggest that silibinin could act as a dual inhibitor of AChE and Aß peptide aggregation, therefore suggesting a therapeutic strategy for AD treatment.