Enhanced plant-derived vesicles for nucleotide delivery for cancer therapy.

Small RNAs (microRNAs [miRNAs] or small interfering RNAs [siRNAs]) are effective tools for cancer therapy, but many of the existing carriers for their delivery are limited by low bioavailability, insufficient loading, impaired transport across biological barriers, and low delivery into the tumor microenvironment. Extracellular vesicle (EV)-based communication in mammalian and plant systems is important for many physiological and pathological processes, and EVs show promise as carriers for RNA interference molecules. However, some fundamental issues limit their use, such as insufficient cargo loading and low potential for scaling production. Plant-derived vesicles (PDVs) are membrane-coated vesicles released in the apoplastic fluid of plants that contain biomolecules that play a role in several biological mechanisms. Here, we developed an alternative approach to deliver miRNA for cancer therapy using PDVs. We isolated vesicles from watermelon and formulated a hybrid, exosomal, polymeric system in which PDVs were combined with a dendrimer bound to miRNA146 mimic. Third generation PAMAM was chosen due to its high branching structure and versatility for loading molecules of interest. We performed several in vivo experiments to demonstrate the therapeutic efficacy of our compound and explored in vitro biological mechanisms underlying the anti-tumor effects of miRNA146, which are mostly related to its anti-angiogenic activity.

EphA2- and HDAC-Targeted Combination Therapy in Endometrial Cancer.

Endometrial cancer is the most frequent malignant tumor of the female reproductive tract but lacks effective therapy. EphA2, a receptor tyrosine kinase, is overexpressed by various cancers including endometrial cancer and is associated with poor clinical outcomes. In preclinical models, EphA2-targeted drugs had modest efficacy. To discover potential synergistic partners for EphA2-targeted drugs, we performed a high-throughput drug screen and identified panobinostat, a histone deacetylase inhibitor, as a candidate. We hypothesized that combination therapy with an EphA2 inhibitor and panobinostat leads to synergistic cell death. Indeed, we found that the combination enhanced DNA damage, increased apoptosis, and decreased clonogenic survival in Ishikawa and Hec1A endometrial cancer cells and significantly reduced tumor burden in mouse models of endometrial carcinoma. Upon RNA sequencing, the combination was associated with downregulation of cell survival pathways, including senescence, cyclins, and cell cycle regulators. The Axl-PI3K-Akt-mTOR pathway was also decreased by combination therapy. Together, our results highlight EphA2 and histone deacetylase as promising therapeutic targets for endometrial cancer.

In situ profiling reveals metabolic alterations in the tumor microenvironment of ovarian cancer after chemotherapy.

In this study, we investigated the metabolic alterations associated with clinical response to chemotherapy in patients with ovarian cancer. Pre- and post-neoadjuvant chemotherapy (NACT) tissues from patients with high-grade serous ovarian cancer (HGSC) who had poor response (PR) or excellent response (ER) to NACT were examined. Desorption electrospray ionization mass spectrometry (DESI-MS) was performed on sections of HGSC tissues collected according to a rigorous laparoscopic triage algorithm. Quantitative MS-based proteomics and phosphoproteomics were performed on a subgroup of pre-NACT samples. Highly abundant metabolites in the pre-NACT PR tumors were related to pyrimidine metabolism in the epithelial regions and oxygen-dependent proline hydroxylation of hypoxia-inducible factor alpha in the stromal regions. Metabolites more abundant in the epithelial regions of post-NACT PR tumors were involved in the metabolism of nucleotides, and metabolites more abundant in the stromal regions of post-NACT PR tumors were related to aspartate and asparagine metabolism, phenylalanine and tyrosine metabolism, nucleotide biosynthesis, and the urea cycle. A predictive model built on ions with differential abundances allowed the classification of patients' tumor responses as ER or PR with 75% accuracy (10-fold cross-validation ridge regression model). These findings offer new insights related to differential responses to chemotherapy and could lead to novel actionable targets.

Monitoring the spatial distribution pattern according to urban land use and health risk assessment on potential toxic metal contamination via street dust in Ankara, Türkiye.

The urbanization processes with growing vehicle numbers cause heavy metal pollution in street dust, and high populations in metropolitan cities are exposed to pollutants. This paper aims to monitor the spatial distribution of heavy metals and evaluate the concentrations via health risk assessment of HMs (Cu, Ni, Cd, Co, Pb, and Zn) that expose the inhabitants to health hazards. According to the results of the current study, sixty street dust samples were applied to the acid digestion technique and quantification by inductively coupled plasma-mass spectrometry (ICP-MS). The spatial distribution of the selected heavy metals in the street dust was investigated using the spatial analysis tool in ArcGIS 10.0 according to population density and land use. In the present study, we used hazard index and cancer risk methods to estimate the public health risk of the pollutants exposed to street dust in Ankara. The concentrations range of the elements in street dust over the study area ranged from 3.34-4.50, 31.69-42.87, 16.09-21.54, 42.85-57.55, 0.00-3.51, and 23.03-30.79, respectively. The overall decreasing order of mean concentration of metals was observed as follows: Pb > Cu > Ni > Co > Cd > Zn. Vehicle traffic and industrial activities seem to be the most critical anthropogenic sources responsible for dust pollution in the study area. The risk assessment of Pb and Ni exposure was the highest, and the hazard index values were 2.42E + 00 and 2.28E + 00 mg/kg/day for children. However, the effect on adults was 2.62E-01 and 2.37E-02 mg/kg/day, followed by inhalation and dermal contact with street dust was almost negligible. The decreasing concentration is modeled spatially along the western development corridor of the city. The risk to public health is high in areas with high densities close to the city center and the main artery.

Enhancing oral delivery of plant-derived vesicles for colitis.

Plant-derived vesicles (PDVs) are attractive for therapeutic applications, including as potential nanocarriers. However, a concern with oral delivery of PDVs is whether they would remain intact in the gastrointestinal tract. We found that 82% of cabbage PDVs were destroyed under conditions mimicking the upper digestive tract. To overcome this limitation, we developed a delivery method whereby lyophilized Eudragit S100-coated cabbage PDVs were packaged into a capsule (Cap-cPDVs). Lyophilization and suspension of PDVs did not have an appreciable impact on PDV structure, number, or therapeutic effect. Additionally, packaging the lyophilized Eudragit S100-coated PDVs into capsules allowed them to pass through the upper gastrointestinal tract for delivery into the colon better than did suspension of PDVs in phosphate-buffered saline. Cap-cPDVs showed robust therapeutic effect in a dextran sulfate sodium-induced colitis mouse model. These findings could have broad implications for the use of PDVs as orally delivered nanocarriers of natural therapeutic plant compounds or other therapeutics.

Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility.

Targeting miRNAs and Other Non-Coding RNAs as a Therapeutic Approach: An Update.

Since the discovery of the first microRNAs (miRNAs, miRs), the understanding of miRNA biology has expanded substantially. miRNAs are involved and described as master regulators of the major hallmarks of cancer, including cell differentiation, proliferation, survival, the cell cycle, invasion, and metastasis. Experimental data indicate that cancer phenotypes can be modified by targeting miRNA expression, and because miRNAs act as tumor suppressors or oncogenes (oncomiRs), they have emerged as attractive tools and, more importantly, as a new class of targets for drug development in cancer therapeutics. With the use of miRNA mimics or molecules targeting miRNAs (i.e., small-molecule inhibitors such as anti-miRS), these therapeutics have shown promise in preclinical settings. Some miRNA-targeted therapeutics have been extended to clinical development, such as the mimic of miRNA-34 for treating cancer. Here, we discuss insights into the role of miRNAs and other non-coding RNAs in tumorigenesis and resistance and summarize some recent successful systemic delivery approaches and recent developments in miRNAs as targets for anticancer drug development. Furthermore, we provide a comprehensive overview of mimics and inhibitors that are in clinical trials and finally a list of clinical trials based on miRNAs.

Exploiting metabolic vulnerabilities after anti-VEGF antibody therapy in ovarian cancer.

Despite modest clinical improvement with anti-vascular endothelial growth factor antibody (AVA) therapy in ovarian cancer, adaptive resistance is ubiquitous and additional options are limited. A dependence on glutamine metabolism, via the enzyme glutaminase (GLS), is a known mechanism of adaptive resistance and we aimed to investigate the utility of a GLS inhibitor (GLSi). Our in vitro findings demonstrated increased glutamine abundance and a significant cytotoxic effect in AVA-resistant tumors when GLSi was administered in combination with bevacizumab. In vivo, GLSi led to a reduction in tumor growth as monotherapy and when combined with AVA. Furthermore, GLSi initiated after the emergence of resistance to AVA therapy resulted in a decreased metabolic conversion of pyruvate to lactate as assessed by hyperpolarized magnetic resonance spectroscopy and demonstrated robust antitumor effects with a survival advantage. Given the increasing population of patients receiving AVA therapy, these findings justify further development of GLSi in AVA resistance.

Combination of EphA2- and Wee1-Targeted Therapies in Endometrial Cancer.

EphA2 tyrosine kinase is upregulated in many cancers and correlated with poor survival of patients, including those with endometrial cancer. EphA2-targeted drugs have shown modest clinical benefit. To improve the therapeutic response to such drugs, we performed a high-throughput chemical screen to discover novel synergistic partners for EphA2-targeted therapeutics. Our screen identified the Wee1 kinase inhibitor, MK1775, as a synergistic partner to EphA2, and this finding was confirmed using both in vitro and in vivo experiments. We hypothesized that Wee1 inhibition would sensitize cells to EphA2-targeted therapy. Combination treatment decreased cell viability, induced apoptosis, and reduced clonogenic potential in endometrial cancer cell lines. In vivo Hec1A and Ishikawa-Luc orthotopic mouse models of endometrial cancer showed greater anti-tumor responses to combination treatment than to either monotherapy. RNASeq analysis highlighted reduced cell proliferation and defective DNA damage response pathways as potential mediators of the combination's effects. In conclusion, our preclinical findings indicate that Wee1 inhibition can enhance the response to EphA2-targeted therapeutics in endometrial cancer; this strategy thus warrants further development.

The effect of laughter yoga on loneliness, psychological resilience, and quality of life in older adults: A pilot randomized controlled trial.

This study aims to examine how laughter yoga affects the loneliness, psychological resilience, and quality of life of older adults living in a nursing home. The sample of this intervention study, made using a control group with a pretest/posttest design, consists of 65 older adults living in Turkey. The data were collected in September 2022 using the Personal Information Form, the Loneliness Scale for the Elderly, the Brief Psychological Resilience Scale, and the Quality of Life Scale for the Elderly. The intervention group (n=32) took part in laughter yoga twice a week for four weeks. No intervention was made with the control group (n=33). A statistically significant difference was found between the groups' mean post-test scores for loneliness, psychological resilience, and quality of life (p < 0.05) after the laughter yoga sessions. The eight-session laughter yoga program was found to reduce loneliness and increase resilience and quality of life in older adults.

Molecular Analysis of Short- versus Long-Term Survivors of High-Grade Serous Ovarian Carcinoma.

Despite having similar histologic features, patients with high-grade serous ovarian carcinoma (HGSC) often experience highly variable outcomes. The underlying determinants for long-term survival (LTS, ≥10 years) versus short-term survival (STS, <3 years) are largely unknown. The present study sought to identify molecular predictors of LTS for women with HGSC. A cohort of 24 frozen HGSC samples was collected (12 LTS and 12 STS) and analyzed at DNA, RNA, and protein levels. OVCAR5 and OVCAR8 cell lines were used for in vitro validation studies. For in vivo studies, we injected OVCAR8 cells into the peritoneal cavity of female athymic nude mice. From RNAseq analysis, 11 genes were found to be differentially expressed between the STS and LTS groups (fold change > 2; false discovery rate < 0.01). In the subsequent validation cohort, transmembrane protein 62 (TMEM62) was found to be related to LTS. CIBERSORT analysis showed that T cells (follicular helper) were found at higher levels in tumors from LTS than STS groups. In vitro data using OVCAR5 and OVCAR8 cells showed decreased proliferation with TMEM62 overexpression and positive correlation with a longevity-regulating pathway (KEGG HSA04213) at the RNA level. In vivo analysis using the OVCAR8-TMEM62-TetON model showed decreased tumor burden in mice with high- vs. low-expressing TMEM62 tumors. Our results demonstrate that restoring TMEM62 may be a novel approach for treatment of HGSC. These findings may have implications for biomarker and intervention strategies to help improve patient outcomes

Targeting CCR2+ macrophages with BET inhibitor overcomes adaptive resistance to anti-VEGF therapy in ovarian cancer.

PURPOSE: Tumor-associated macrophages (TAMs) are known to contribute to adaptive resistance to anti-vascular endothelial growth factor (VEGF) antibody (AVA) therapy in ovarian cancer. BET (bromodomain and extra-terminal domain) inhibitors (BETi) may have unique roles in targeting TAMs. Our objective was to examine the effects of BETi on TAMs, especially in the context of enhancing the efficacy of AVA therapy. METHODS: We conducted a series of in vitro (MTT assay, apoptosis, flow cytometry, and RNA sequencing) and in vivo (xenograft ovarian cancer model) experiments to determine the biological effects of BETi combined with AVA in ovarian cancer. For statistical analysis, a two-tailed Student's t test (equal variance) or ANOVA was used for multiple groups' comparison, and p < 0.05 was considered significant. RESULTS: BETi resulted in a dose-dependent decrease in cell viability and induced apoptosis (p < 0.01) in ovarian cancer cells (SKOV3ip1, OVCAR5, and OVCAR8). Treatment with BETi significantly increased apoptosis in THP-1 monocytes and macrophages (PMA-differentiated THP-1; p < 0.01). Furthermore, BETi selectively induced greater apoptosis in M2-like macrophages (PMA and IL-4, IL-13-differentiated THP-1) (31.3%-36.1%) than in M1-like macrophages (PMA and LPS-differentiated THP-1) (12.4%-18.5%) (p < 0.01). Flow cytometry revealed that the percentage of M1-like macrophages (CD68+/CD80+) was significantly increased after treatment with low-dose BETi (ABBV-075 0.1 µM; p < 0.05), whereas the percentage of CD68+/CCR2+ macrophages was significantly decreased (p < 0.001); these findings suggest that BETi may selectively inhibit the survival of CCR2+ macrophages and re-polarize the macrophages into an M1-like phenotype. RNA-seq analysis revealed that BETi selectively targeted macrophage infiltration-related cytokines/chemokines in ovarian cancer (adjusted p < 0.05 and Log2 fold change ≥ 1.5). Finally, using in vivo ovarian cancer models, compared with control or monotherapy, the combination of BETi (ABBV-075) and bevacizumab resulted in greater inhibition of tumor growth and macrophage infiltration (p < 0.05) and longer survival of tumor-bearing mice (p < 0.001). CONCLUSIONS: Our findings indicate a previously unrecognized role for BETi in selectively targeting CCR2+ TAMs and enhancing the efficacy of AVA therapy in ovarian cancer.

Rational Combination of CRM1 Inhibitor Selinexor and Olaparib Shows Synergy in Ovarian Cancer Cell Lines and Mouse Models.

CRM1 inhibitors have demonstrated antitumor effects in ovarian and other cancers; however, rational combinations are largely unexplored. We performed a high-throughput drug library screen to identify drugs that might combine well with selinexor in ovarian cancer. Next, we tested the combination of selinexor with the top hit from the drug screen in vitro and in vivo Finally, we assessed for mechanisms underlying the identified synergy using reverse phase protein arrays (RPPA). The drug library screen assessing 688 drugs identified olaparib (a PARP inhibitor) as the most synergistic combination with selinexor. Synergy was further demonstrated by MTT assays. In the A2780luc ip1 mouse model, the combination of selinexor and olaparib yielded significantly lower tumor weight and fewer tumor nodules compared with the control group (P < 0.04 and P < 0.03). In the OVCAR5 mouse model, the combination yielded significantly fewer nodules (P = 0.006) and markedly lower tumor weight compared with the control group (P = 0.059). RPPA analysis indicated decreased expression of DNA damage repair proteins and increased expression of tumor suppressor proteins in the combination treatment group. Collectively, our preclinical findings indicate that combination with selinexor to expand the utility and efficacy of PARP inhibitors in ovarian cancer warrants further exploration.

Gene Body Methylation of the Lymphocyte-Specific Gene CARD11 Results in Its Overexpression and Regulates Cancer mTOR Signaling.

Investigations into the function of nonpromoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated nonpromoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 260 genes exhibiting a correlation of > 0.5 between gene body methylation and gene expression in at least one tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in kidney renal cell carcinoma (KIRC) and lung adenocarcinoma (LUAD) tumor tissues in comparison with respective control samples; and is significantly associated with lower overall survival in KIRC. Contrary to its canonical function in lymphocyte NFκB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy. Furthermore, demethylation of a CpG island within the gene body of CARD11 decreases gene expression. Collectively, our study highlights how DNA methylation outside the promoter region can impact tumor progression. IMPLICATIONS: Our study describes a novel regulatory role of gene body DNA methylation-dependent CARD11 expression on mTOR signaling and its impact on tumor progression.

Assessment of In Vivo siRNA Delivery in Cancer Mouse Models.

RNA interference (RNAi) has rapidly become a powerful tool for target discovery and therapeutics. Small interfering RNAs (siRNAs) are highly effective in mediating sequence-specific gene silencing. However, the major obstacle for using siRNAs for cancer therapeutics is their systemic delivery from the administration site to target cells in vivo. This chapter describes approaches to deliver siRNA effectively for cancer treatment and discusses in detail the current methods to assess pharmacokinetics and biodistribution of siRNAs in vivo.

Inactivating Mutations of the IK Gene Weaken Ku80/Ku70-Mediated DNA Repair and Sensitize Endometrial Cancer to Chemotherapy.

IK is a mitotic factor that promotes cell cycle progression. Our previous investigation of 271 endometrial cancer (EC) samples from the Cancer Genome Atlas (TCGA) dataset showed IK somatic mutations were enriched in a cluster of patients with high-grade and high-stage cancers, and this group had longer survival. This study provides insight into how IK somatic mutations contribute to EC pathophysiology. We analyzed the somatic mutational landscape of IK gene in 547 EC patients using expanded TCGA dataset. Co-immunoprecipitation and mass spectrometry were used to identify protein interactions. In vitro and in vivo experiments were used to evaluate IK's role in EC. The patients with IK-inactivating mutations had longer survival during 10-year follow-up. Frameshift and stop-gain were common mutations and were associated with decreased IK expression. IK knockdown led to enrichment of G2/M phase cells, inactivation of DNA repair signaling mediated by heterodimerization of Ku80 and Ku70, and sensitization of EC cells to cisplatin treatment. IK/Ku80 mutations were accompanied by higher mutation rates and associated with significantly better overall survival. Inactivating mutations of IK gene and loss of IK protein expression were associated with weakened Ku80/Ku70-mediated DNA repair, increased mutation burden, and better response to chemotherapy in patients with EC.