Cell injury releases nucleic acids supporting inflammation and stem cell activation. Here, the impact of extracellular ribonucleic acid, especially transfer RNA (ex-tRNA), on vasculogenesis and leukopoiesis of mouse embryonic stem (ES) cells was investigated.

Angiogenic switch is a hallmark feature of transition from low-grade to high-grade cervical intraepithelial neoplasia (CIN) in cervical cancer progression. Therefore, early events leading to locally-advanced cervical metastatic lesions demand a greater understanding of the underlying mechanisms. Recent leads indicate the role of tumor-derived exosomes in altering the functions of endothelial cells in cervical cancer, which needs further investigation.

Extracellular vesicles (EV) within the cellular secretome are emerging as modulators of pathological processes involved in tumor growth through their ability to transfer donor-derived RNA into recipient cells. While the effects of tumor and stromal cell EVs within the tumor microenvironment have been studied, less is known about the contributions of normal, nontransformed cells. We examined the impact of EVs within the cellular secretome from nonmalignant cells on transformed cell growth and behavior in cholangiocarcinoma cells. These effects were enhanced in the presence of the pro-fibrogenic mediator TGF-β. We identified miR-195 as a TGF-β responsive miRNA in normal cells that can be transferred via EV to tumor cells and regulate cell growth, invasion, and migration. The effects of miR-195 involve modulation of the epithelial-mesenchymal transition through direct effects on the transcription factor Snail. These studies provide in vitro and in vivo evidence for the impact of normal cellular secretome on transformed cell growth, show the importance of EV RNA transfer, and identify mechanisms of EV-mediated transfer of miRNA as a contributor to tumor development, which may provide new therapeutic opportunities for targeting human cholangiocarcinoma.

Hypopharyngeal squamous cell carcinoma (HPSCC) has the worst prognosis among all head-and-neck cancers, and treatment options are limited. Tumor microenvironment (TME) analysis can help identify new therapeutic targets and combined treatment strategies.

Accelerated cell apoptosis with dysregulated long noncoding RNAs is the crucial pathogenesis in lupus nephritis (LN). Pro-apoptotic lincRNA-p21 was studied in LN patients, cell lines with lentivirus-mediated overexpression and CRISPR interference (CRISPRi)-conducted repression, and a mouse model. Clinical samples were from patients and age/sex-matched controls. Expression of lincRNA-p21 and endogenous RNA target miR-181a, were examined in mononuclear and urine cells. Guide RNA sequences targeting lincRNA-p21 were cloned into CRISPRi with dCas9/ Krüppel-associated box (KRAB) domain. LincRNA-p21-silened transfectants were investigated for apoptosis and miR-181a expression. LincRNA-p21-overexpressed cells were evaluated for apoptosis and p53-related down-stream molecules. Balb/C mice were injected with pristane to induce LN and examined for apoptosis and lincRNA-p21. Higher lincRNA-p21 levels were found in LN mononuclear and urine cells, positively correlated with activity. There were lower miR-181a levels in LN mononuclear cells, negatively correlated with activity. Doxorubicin-induced apoptotic cells had up-regulated lincRNA-p21 levels. CRISPRi with dCas9/KARA domain showed efficient repression ability on transcription initiation/elongation. CRISPRi-conducted lincRNA-p21-silenced transfectants displayed reduced apoptosis with up-regulated miR-181a levels, whereas lentivirus-mediated lincRNA-p21-overexpressed cells revealed enhanced apoptosis with up-regulated downstream PUMA/Bax expression. LN mice had glomerular apoptosis with progressive increased lincRNA-p21 levels. Our results demonstrate up-regulated lincRNA-p21 expression in LN, implicating a potential diagnostic marker and therapeutic target.

Intraluminal vesicles (ILVs) are released into the extracellular space as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane. miRNAs are delivered to the raft-like region of MVB by RNA-binding proteins (RBPs). RNA loading into exosomes can be either through direct interaction between RNA and the raft-like region of the MVB membrane, or through interaction between an RBP-RNA complex with this raft-like region. Selection of RNA aptamers that bind to lipid raft region of liposomal membranes was performed using the selection-amplification (SELEX) method. The pool of RNA aptamers was isolated, and the binding of this pool to lipid-raft regions was demonstrated. Sequencing of clones from rafted liposome-eluted RNAs showed sequences apparently of independent origin. Bioinformatics analysis revealed the most frequent raft-motifs present within these sequences. Four raft RNA motifs, one of them an EXO motif, have been identified. These motifs appear to be most frequent both in the case of raft RNA aptamers and in the case of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells, thus suggesting that the selection for incorporation of these miRNAs into ILVs is based on their affinity to the raft-like region of the MVB membrane.

Previous studies have analyzed transfer of RNA-encoded tumor-associated antigens (TAAs) into immature dendritic cells (DCs) because of their exceptional ability to internalize antigens. Concerns have been raised regarding the use of immature DCs in clinical studies because of their capacity to tolerize T cells. Therefore, we focused on optimizing RNA transfer into mature DCs using the method of electroporation and obtained high protein expression in 90% of mature DCs. Particular emphasis was placed on quantifying RNA transfer. Reconstitution of peptide-MHC (pMHC) ligands on RNA-pulsed DCs was measured with the help of effector-memory cytotoxic T lymphocytes (CTLs) specific for the melanoma-associated antigens tyrosinase and mutated cyclin-dependent kinase 4. In contrast to single-species RNA, transfer of native tumor-derived RNA or amplified tumor RNA into DCs resulted in very low or no capacity to reactivate these CTLs. A correlation was found between TAA message levels in tumor-derived RNA and pMHC ligand reconstitution on DCs. These results demonstrate that even TAAs with highly immunogenic mutated epitopes are not necessarily transferred when tumor-derived RNA is transfected into the DCs, thereby resulting in a lack of DC capacity to reactivate some preexisting effector-memory CTLs.

Currently, resistance to tyrosine kinase inhibitors, such as gefitinib, has become a major obstacle in improving the clinical outcome of patients with metastatic and advanced-stage esophageal squamous cell carcinoma (ESCC). While cell behavior can be modulated by long non-coding RNAs (lncRNAs), the roles of lncRNAs within extracellular vesicles (exosomes) are largely unknown. Therefore, we investigated the involvement and regulatory functions of potential lncRNAs enclosed in exosomes during formation of chemoresistance in human ESCC.

Clear cell renal cell carcinoma (ccRCC) represents the most common type of renal cell carcinoma (RCC) in adults, in addition to the worst prognosis among the common epithelial kidney tumors. Inflammation and angiogenesis seem to potentiate tumor growth and metastasis of the malignancy. The current study explored the contributions of the lncRNA MCM3AP-AS1 in tumor-associated inflammation and angiogenesis in ccRCC with a specific focus on its transcriptional regulation and its interactions with transcription factor E2F1 and DPP4. Tumor tissues and matched adjacent non-tumor tissues were collected from 78 ccRCC patients. Methylation-specific PCR and ChIP assays were applied to detect the methylation at the promoter region of MCM3AP-AS1. Dual-luciferase reporter assay, RIP, RNA pull-down, and ChIP assays were employed to confirm the interactions between MCM3AP-AS1, E2F1, and DPP4. Nude mice were subcutaneously xenografted with human ccRCC cells. Cell proliferation was evaluated by CCK-8 assays and EDU staining in ccRCC cells in vitro and by immunohistochemical staining of Ki67 in vivo. Inflammation was examined by detecting the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). Pro-angiogenic ability of ccRCC cells was assessed by the co-culture with human umbilical vein endothelial cells (HUVEC) in vitro and by microvessel density (MVD) measurements and angiogenesis in the chicken chorioallantoic membrane. MCM3AP-AS1 was highly-expressed in ccRCC and associated with poor patient survival. Demethylation of MCM3AP-AS1 was noted in ccRCC tissues and cells. Over-expression of MCM3AP-AS1 enhanced cell proliferation, the release of pro-inflammatory cytokines, and the tube formation of HUVECs in cultured human Caki-1 and 786-O cells. MCM3AP-AS1 was shown to enhance the E2F1 enrichment at the DPP4 promoter, to further increase the expression of DPP4. Knockdown of DPP4 could abate pro-angiogenic and pro-inflammatory abilities of MCM3AP-AS1 in ccRCC cells. Pro-angiogenic and pro-inflammatory abilities of MCM3AP-AS1 in vivo were confirmed in mice subcutaneously xenografted with human ccRCC cells. Our findings demonstrate a novel mechanism by which lncRNA MCM3AP-AS1 exerts pro-angiogenic and pro-inflammatory effects, highlighting the potential of MCM3AP-AS1 as a promising target for treating ccRCC.

Candida albicans is the major fungal cause of healthcare-associated bloodstream infections worldwide with a 40% mortality rate. The scarcity of antifungal treatments due to the eukaryotic origin of fungal cells has challenged the development of selectively antifungal drugs. In an attempt to identify novel antifungal agents, aurones SH1009 and SH9051, as synthetically bioactive compounds, have been recently documented as anti-Candida agents. Since the molecular mechanisms behind the inhibitory activities of these aurones in C. albicans are unclear, this study aimed to determine the comprehensive cellular processes affected by these aurones and their molecular targets. Genome-wide transcriptional analysis of SH1009- and SH9051-treated C. albicans revealed uniquely repressed expression in different metabolic pathways, particularly trehalose and sulfur amino acid metabolic processes for SH1009 and SH9051, respectively. In contrast, the most commonly enriched process for both aurones was the up-regulation of RNA processing and ribosomal cleavages as an indicator of high oxidative stress, suggesting that a common aspect in the chemical structure of both aurones led to pro-oxidative properties. Additionally, uniquely induced responses (iron ion homeostasis for SH1009 and arginine biosynthesis for SH9051) garnered attention on key roles for the aurone functional groups. Deletion of the transcription factor for the trehalose biosynthesis pathway, Tye7p, resulted in an SH1009-resistant mutant, which also exhibited low trehalose content, validating the primary molecular target of SH1009. Aurone SH9051 uniquely simulated an exogenous supply of methionine or cysteine, leading to sulfur amino acid catabolism as evidenced by quantifying an overproduction of sulfite. Phenyl aurone, the common structure of aurones, contributed proportionally in the pro-oxidative activity through ferric ion reduction effects leading to high ROS levels. Our results determined selective and novel molecular mechanisms for aurone SH1009 and also elucidated the diverse cellular effects of different aurones based on functional groups.

The tumor suppressor gene TP53 is the most frequently mutated gene in numerous cancer types, including prostate cancer (PCa). Specifically, missense mutations in TP53 are selectively enriched in PCa, and cluster to particular "hot spots" in the p53 DNA binding domain with mutation at the R273 residue occurring most frequently. While this residue is similarly mutated to R273C-p53 or R273H-p53 in all cancer types examined, in PCa selective enrichment of R273C-p53 is observed. Importantly, examination of clinical datasets indicated that TP53 heterozygosity can either be maintained or loss of heterozygosity (LOH) occurs. Thus, to mimic tumor-associated mutant p53, R273C-p53 and R273H-p53 isogenic PCa models were developed in the presence or absence of wild-type p53. In the absence of wild-type p53, both R273C-p53 and R273H-p53 exhibited similar loss of DNA binding, transcriptional profiles, and loss of canonical tumor suppressor functions associated with wild-type p53. In the presence of wild-type p53 expression, both R273C-p53 and R273H-p53 supported canonical p53 target gene expression yet elicited distinct cistromic and transcriptional profiles when compared to each other. Moreover, heterozygous modeling of R273C-p53 or R273H-p53 expression resulted in distinct phenotypic outcomes in vitro and in vivo. Thus, mutant p53 acts in a context-dependent manner to elicit pro-tumorigenic transcriptional profiles, providing critical insight into mutant p53-mediated prostate cancer progression.

Elucidating the transitional stages that define the pathway stem cells progress through during differentiation advances our understanding of biology and fosters the identification of therapeutic opportunities. However, distinguishing progenitor cells from other cell types and placing them in an epistatic pathway is challenging. This is exemplified in the adipocyte lineage, where the stromal vascular fraction (SVF) from adipose tissue is enriched for progenitor cells but also contains heterogeneous populations of cells. Single-cell RNA sequencing (scRNA-seq) has begun to facilitate the deconvolution of cell types in the SVF, and a hierarchical structure is emerging. Here, we use scRNA-seq to discover a population of CD31- CD45- cells in the SVF that are distinguished by a specific expression profile. Further, we place this population on an epistatic pathway upstream of the previously defined preadipocyte population. Finally, we discover functional properties of this population with broad implications, including revealing physiological mechanisms that regulate adipogenesis.

Despite the ongoing progress in diagnosis and treatments, cancer remains a threat to more than one-third of the human population. The emerging data indicate that many Krüppel-associated box zinc finger proteins (KRAB-ZNF) belonging to a large gene family may be involved in carcinogenesis. Our previous study identified Zinc Finger Protein 714 (ZNF714), a KRAB-ZNF gene of unknown function, as being commonly overexpressed in many tumors, pointing to its hypothetical oncogenic role. Here, we harnessed The Cancer Genome Atlas (TCGA)-centered databases and performed functional studies with transcriptomic and methylomic profiling to explore ZNF714 function in cancer. Our pan-cancer analyses confirmed frequent ZNF714 overexpression in multiple tumors, possibly due to regional amplification, promoter hypomethylation, and Nuclear Transcription Factor Y Subunit Beta (NFYB) signaling. We also showed that ZNF714 expression correlates with tumor immunosuppressive features. The in vitro studies indicated that ZNF714 expression positively associates with proliferation, migration, and invasion. The transcriptomic analysis of ZNF714 knocked-down cells demonstrated deregulation of cell adhesion, migration, proliferation, apoptosis, and differentiation. Importantly, we provided evidence that ZNF714 negatively regulates the expression of several known TSGs indirectly via promoter methylation. However, as ZNF714 did not show nuclear localization in our research model, the regulatory mechanisms exerted by ZNF714 require further investigation. In conclusion, our results reveal, for the first time, that ZNF714 may support pro-oncogenic features in lung cancer cells.

Scant understanding of the glioblastoma microenvironment and molecular bases hampers development of efficient treatment strategies. Analyses of gene signatures of human gliomas demonstrate that the SETD2 mutation is correlated with poor prognosis of IDH1/2 wild-type (IDH-WT) adult glioblastoma patients. To better understand the crosstalk between SETD2 mutant (SETD2-mut) glioblastoma cells and the tumor microenvironment, we leverage single-cell transcriptomics to comprehensively map cellular populations in glioblastoma. In this study, we identify a specific subtype of high-grade glioma-associated microglia (HGG-AM). Further analysis shows that transforming growth factor (TGF)-β1 derived from SETD2-mut/IDH-WT tumor cells activates HGG-AM, exhibiting pro-inflammation and proliferation signatures. Particularly, HGG-AM secretes interleukin (IL)-1β via the apolipoprotein E (ApoE)-mediated NLRP1 inflammasome, thereby promoting tumor progression. HGG-AM present extensive proliferation and infiltration to supplement the activated microglia pool. Notably, TGF-β1/TβRI depletion dramatically reduces HGG-AM density and suppresses tumor growth. Altogether, our studies identify a specific microglia subpopulation and establish the cellular basis of interactions between HGG-AM and glioblastoma cells.

Glioblastomas are hierarchically organised tumours driven by glioma stem cells that retain partial differentiation potential. Glioma stem cells are maintained in specialised microenvironments, but whether, or how, they undergo lineage progression outside of these niches remains unclear. Here we identify the white matter as a differentiative niche for glioblastomas with oligodendrocyte lineage competency. Tumour cells in contact with white matter acquire pre-oligodendrocyte fate, resulting in decreased proliferation and invasion. Differentiation is a response to white matter injury, which is caused by tumour infiltration itself in a tumoursuppressive feedback loop. Mechanistically, tumour cell differentiation is driven by selective white matter upregulation of SOX10, a master regulator of normal oligodendrogenesis. SOX10 overexpression or treatment with myelination-promoting agents that upregulate endogenous SOX10, mimic this response, leading to niche-independent pre-oligodendrocyte differentiation and tumour suppression in vivo. Thus, glioblastoma recapitulates an injury response and exploiting this latent programme may offer treatment opportunities for a subset of patients.

BH3-mimetic drugs are an anti-cancer therapy that can induce apoptosis in malignant cells by directly binding and inhibiting pro-survival proteins of the BCL-2 family. The BH3-mimetic drug venetoclax, which targets BCL-2, has been approved for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia by regulatory authorities worldwide. However, while most patients initially respond well, resistance and relapse while on this drug is an emerging and critical issue in the clinic. Though some studies have begun uncovering the factors involved in resistance to BCL-2-targeting BH3-mimetic drugs, little focus has been applied to pre-emptively tackle resistance for the next generation of BH3-mimetic drugs targeting MCL-1, which are now in clinical trials for diverse blood cancers. Therefore, using pre-clinical mouse and human models of aggressive lymphoma, we sought to predict factors likely to contribute to the development of resistance in patients receiving MCL-1-targeting BH3-mimetic drugs. First, we performed multiple whole genome CRISPR/Cas9 KO screens and identified that loss of the pro-apoptotic effector protein BAX, but not its close relative BAK, could confer resistance to MCL-1-targeting BH3-mimetic drugs in both short-term and long-term treatment regimens, even in lymphoma cells lacking the tumour suppressor TRP53. Furthermore, we found that mouse Eµ-Myc lymphoma cells selected for loss of BAX, as well as upregulation of the untargeted pro-survival BCL-2 family proteins BCL-XL and A1, when made naturally resistant to MCL-1 inhibitors by culturing them in increasing doses of drug over time, a situation mimicking the clinical application of these drugs. Finally, we identified therapeutic approaches which could overcome these two methods of resistance: the use of chemotherapeutic drugs or combined BH3-mimetic treatment, respectively. Collectively, these results uncover some key factors likely to cause resistance to MCL-1 inhibition in the clinic and suggest rational therapeutic strategies to overcome resistance that should be investigated further.

The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development.

Argininosuccinate lyase (ASL) belongs to the hepatic urea cycle detoxifying ammonia, and the citrulline-nitric oxide (NO) cycle producing NO. ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia, multiorgan disease and neurocognitive impairment despite treatment aiming to normalise ammonaemia without considering NO imbalance. Here we show that cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress independent of hyperammonaemia. Intravenous injection of AAV8 vector into adult or neonatal ASL-deficient mice demonstrates long-term correction of the hepatic urea cycle and the cerebral citrulline-NO cycle, respectively. Cerebral disease persists if ammonaemia only is normalised but is dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This correlates with behavioural improvement and reduced cortical cell death. Thus, neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Disease amelioration by simultaneous brain and liver gene transfer with one vector, to treat both metabolic pathways, provides new hope for hepatocerebral metabolic diseases.

Mutant p53s (mutp53) increase cancer invasiveness by upregulating Rab-coupling protein (RCP) and diacylglycerol kinase-α (DGKα)-dependent endosomal recycling. Here we report that mutp53-expressing tumour cells produce exosomes that mediate intercellular transfer of mutp53's invasive/migratory gain-of-function by increasing RCP-dependent integrin recycling in other tumour cells. This process depends on mutp53's ability to control production of the sialomucin, podocalyxin, and activity of the Rab35 GTPase which interacts with podocalyxin to influence its sorting to exosomes. Exosomes from mutp53-expressing tumour cells also influence integrin trafficking in normal fibroblasts to promote deposition of a highly pro-invasive extracellular matrix (ECM), and quantitative second harmonic generation microscopy indicates that this ECM displays a characteristic orthogonal morphology. The lung ECM of mice possessing mutp53-driven pancreatic adenocarcinomas also displays increased orthogonal characteristics which precedes metastasis, indicating that mutp53 can influence the microenvironment in distant organs in a way that can support invasive growth.

Radiation-induced fibrosis is a common side effect of radiotherapy, which is the most common treatment for cancer. However, radiation also causes p53-mediated cell cycle arrest, prolonged expression of p21, and the development of senescence in normal cells that reside in irradiated tissues. Bone marrow-derived mesenchymal stem cells (MSCs) accumulate in primary tumor sites because of their natural tropism for inflammatory and fibrotic tissues. MSCs are extremely sensitive to low doses of ionizing radiation and acquire senescence as a result of bystander radiation effects. Senescent cells remain metabolically active but develop a potent senescence-associated secretory phenotype (SASP) that correlates to hyperactive secretion of cytokines, pro-fibrotic growth factors, and exosomes (EXOs). Integrative pathway analysis highlighted that radiation-induced senescence significantly enriched cell-cycle, extracellular matrix, transforming growth factor-β (TGF-β) signaling, and vesicle-mediated transport genes in MSCs. EXOs are cell-secreted nanovesicles (a subclass of small extracellular vesicles) that contain biomaterials-proteins, RNAs, microRNAs (miRNAs)-that are critical in cell-cell communication. miRNA content analysis of secreted EXOs further revealed that radiation-induced senescence uniquely altered miRNA profiles. In fact, several of the standout miRNAs directly targeted TGF-β or downstream genes. To examine bystander effects of radiation-induced senescence, we further treated normal MSCs with senescence-associated EXOs (SA-EXOs). These modulated genes related to TGF-β pathway and elevated both alpha smooth muscle actin (protein increased in senescent, activated cells) and Ki-67 (proliferative marker) expression in SA-EXO treated MSCs compared to untreated MSCs. We revealed SA-EXOs possess unique miRNA content that influence myofibroblast phenotypes via TGF-β pathway activation. This highlights that SA-EXOs are potent SASP factors that play a large role in cancer-related fibrosis.