Downregulation of Histone H3.3 Induces p53-Dependent Cellular Senescence in Human Diploid Fibroblasts.

Cellular senescence is an irreversible growth arrest that acts as a barrier to cancer initiation and progression. Histone alteration is one of the major events during replicative senescence. However, little is known about the function of H3.3 in cellular senescence. Here we found that the downregulation of H3.3 induced growth suppression with senescence-like phenotypes such as senescence-associated heterochromatin foci (SAHF) and ß-galactosidase (SA-ß-gal) activity. Furthermore, H3.3 depletion induced senescence-like phenotypes with the p53/p21-depedent pathway. In addition, we identified miR-22-3p, tumor suppressive miRNA, as an upstream regulator of the H3F3B (H3 histone, family 3B) gene which is the histone variant H3.3 and replaces conventional H3 in active genes. Therefore, our results reveal for the first time the molecular mechanisms for cellular senescence which are regulated by H3.3 abundance. Taken together, our studies suggest that H3.3 exerts functional roles in regulating cellular senescence and is a promising target for cancer therapy.

Association Between Telomere G-Tail Length and Coronary Artery Disease or Statin Treatment in Patients With Cardiovascular Risks　- A Cross-Sectional Study.

Background: The utility of telomere G-tail length to predict coronary artery disease (CAD) remains controversial. CAD results from coronary artery narrowing due to cholesterol and lipid accumulation, augmented by inflammatory cells and other factors. This study explored the significance of telomere G-tail length in suspected CAD patients. Methods and Results: In all, 95 patients with suspected CAD or ≥1 cardiac risk factor underwent coronary computed tomography angiography (CCTA). We measured leukocyte telomere length and G-tail length using a hybrid protection method, and diagnosed the presence of CAD using CCTA. Associations between G-tail length and the presence of CAD, the number of stenosed coronary arteries, and brachial-ankle pulse wave velocity (baPWV) were analyzed. No significant difference was observed in G-tail length when comparing groups with or without CAD or statin treatment. However, in the non-statin group, G-tail length was significantly shorter in patients with 3-vessel disease compared with 1-vessel disease. Dividing the group using a baPWV of 1,300 cm/s, telomere G-tail length was significantly shorter in the high-risk (baPWV ≥1,300 cm/s) group. Conclusions: The clinical utility of telomere G-tail length as a CAD risk indicator seems limited. There was a trend for longer telomere G-tail length in the statin-treated group. Moreover, telomere G-tail length was reduced in patients at high-risk of cardiovascular events, aligning with the trend of a shortening in telomere G-tail length with CAD severity.

Immunocyte Derived Exosomes: Insight into the Potential Chemo-immunotherapeutic Nanocarrier Targeting the Tumor Microenvironment.

"Cancer" is a dreadful immune-pathological condition that is characterized by anti-inflammatory and tumorigenic responses, elicited by the infiltrating immune cells in the vicinity of an uncontrollably proliferative tumor in the tumor microenvironment (TME). The TME offers a conducive microenvironment that supports cancer cell survival by modulating the host immune defense. Recent advancement in exosomal research has shown exosomes, originating from immune cells as well as the cancer cells, have immense potential for suppressing cancer progression and survival in the TME. Additionally, exosomes, irrespective of their diverse sources, have been reported to be efficient nanocarriers for cancer therapeutics with the ability for targeted delivery due to their biogenic nature, ease of cellular uptake, and scope for functionalization with biomolecules like peptides, aptamers, targeting ligands, etc. Immune cell-derived exosomes per se have been found efficacious against cancer owing to their immune-stimulant properties (in either naive or antigen primed form) even without loading any of cancer therapeutics or targeting ligand conjugation. Nevertheless, exosomes are being primarily explored as nanovesicular carriers for therapeutic molecules with different loading and targeting strategies, and the synergism between immunotherapeutic behavior of exosomes and the anticancer effect of the therapeutic molecules is yet to be explored. Hence, this review focuses specifically on the possible strategies to modulate the immunological nature of the source immune cells to obtain immune stimulant exosomes and bring these into the spotlight as chemo-immunotherapeutic nanovesicles, that can easily target and modulate the TME.

Leukocyte Telomeric G-Tail Length Shortening Is Associated with Esophageal Cancer Recurrence.

Despite significant advances in therapeutics for esophageal cancer (ESC) in the past decade, it remains the sixth most fatal malignancy, with a poor 5-year survival rate (approximately 10%). There is an urgent need to improve the timely diagnosis to aid the prediction of the therapeutic response and prognosis of patients with ESC. The telomeric G-tail plays an important role in the chromosome protection. However, aging and age-related diseases lead to its shortening. Therefore, the G-tail length has been proposed as a novel potential biomarker. In the present study, to examine the possibility of G-tail shortening in patients with ESC, we measured the leukocyte telomere length (LTL) and the G-tail length using a hybridization protection assay in 147 patients with ESC and 170 age-matched healthy controls. We found that the G-tail length in patients with ESC was shorter than that in the healthy controls (p = 0.02), while the LTL shortening was not correlated with the ESC incidence and recurrence. Our results suggest that the G-tail length reflects the physiological status of patients with ESC and is a promising biomarker for the diagnosis and prognosis of ESC.

Comprehensive serum and tissue microRNA profiling in dedifferentiated liposarcoma.

Sarcoma is a rare cancer with several subtypes; therefore, our understanding of the pathogenesis of sarcoma is limited, and designing effective treatments is difficult. Circulating microRNAs (miRNAs), including exosomal miRNAs, have attracted attention as biomarkers in cancer. However, the roles of miRNAs and exosomes in sarcoma remain unclear. The present analysis of tissue and serum miRNA expression in osteosarcoma, Ewing's sarcoma and dedifferentiated liposarcoma (DDLPS) identified miR-1246, -4532, -4454, -619-5p and -6126 as biomarkers for DDLPS. These miRNAs were highly expressed in human DDLPS cell lines and exosomes, suggesting that they are secreted from DDLPS tissues. The present results suggested that specific miRNAs may be used as biomarkers for early diagnosis or treatment targets in DDLPS.

PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA.

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53-p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.

Identification of a Selective RelA Inhibitor Based on DSE-FRET Screening Methods.

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA-DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.

Quercetin Inhibits Lef1 and Resensitizes Docetaxel-Resistant Breast Cancer Cells.

Drug resistance is a major problem for breast cancer patients. Docetaxel is an anti-mitotic agent that serves as first line of treatment in metastatic breast cancer, however it is susceptible to cellular drug resistance. Drug-resistant cells are able to spread during treatment, leading to treatment failure and eventually metastasis, which remains the main cause for cancer-associated death. In previous studies, we used single-cell technologies and identified a set of genes that exhibit increased expression in drug-resistant cells, and they are mainly regulated by Lef1. Furthermore, upregulating Lef1 in parental cells caused them to become drug resistant. Therefore, we hypothesized that inhibiting Lef1 could resensitize cells to docetaxel. Here, we confirmed that Lef1 inhibition, especially on treatment with the small molecule quercetin, decreased the expression of Lef1 and resensitized cells to docetaxel. Our results demonstrate that Lef1 inhibition also downregulated ABCG2, Vim, and Cav1 expression and equally decreased Smad-dependent TGF-ß signaling pathway activation. Likewise, these two molecules worked in a synergetic manner, greatly reducing the viability of drug-resistant cells. Prior studies in phase I clinical trials have already shown that quercetin can be safely administered to patients. Therefore, the use of quercetin as an adjuvant treatment in addition to docetaxel for the treatment of breast cancer may be a promising therapeutic approach.

Bacterial SOS Genes mucAB/umuDC Promote Mouse Tumors by Activating Oncogenes Nedd9/Aurkb via a miR-145 Sponge.

The mechanism of cancer induction involves an aberrant expression of oncogenes whose functions can be controlled by RNAi with miRNA. Even foreign bacterial RNA may interfere with the expression of oncogenes. Here we show that bacterial plasmid mucAB and its Escherichia coli genomic homolog umuDC, carrying homologies that match the mouse anti-miR-145, sequestered the miR-145 function in mouse BALB 3T3 cells in a tetracycline (Tet)-inducible manner, activated oncogene Nedd9 and its downstream Aurkb, and further enhanced microcolony formation and cellular transformation as well as the short fragments of the bacterial gene containing the anti-miR-145 sequence. Furthermore, mucAB transgenic mice showed a 1.7-fold elevated tumor incidence compared with wild-type mice after treatments with 3-methylcolanthrene. However, the mutation frequency in intestinal stem cells of the mucAB transgenic mice was unchanged after treatment with X-rays or ethyl-nitrosourea, indicating that the target of mucAB/umuDC is the promotion stage in carcinogenesis. IMPLICATIONS: Foreign bacterial genes can exert oncogenic activity via RNAi, if endogenously expressed. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/9/1271/F1.large.jpg.

CD44s Induces miR-629-3p Expression in Association with Cisplatin Resistance in Head and Neck Cancer Cells.

Cisplatin (cis-diamminedichloroplatinum II [CDDP] ) is a well-known chemotherapeutic drug that has been used for the treatment of various types of human cancers, including head and neck cancer. Cisplatin exerts anticancer effects by causing DNA damage, replication defects, transcriptional inhibition, cell cycle arrest, and the induction of apoptosis. However, drug resistance is one of the most serious problems with cancer chemotherapy, and it causes expected therapeutic effects to not always be achieved. Here, we analyzed global microRNA (miRNA) expression in CD44 standard form (CD44s)-expressing SAS cells, and we identified miR-629-3p as being responsible for acquiring anticancer drug resistance in head and neck cancer. The introduction of miR-629-3p expression inhibited apoptotic cell death under cisplatin treatment conditions, and it promoted cell migration. Among the computationally predicted target genes of miR-629-3p, we found that a number of gene expressions were suppressed by the transfection with miR-629-3p. Using a xenografting model, we showed that miR-629-3p conferred cisplatin resistance to SAS cells. Clinically, increased miR-629-3p expression tended to be associated with decreased survival in head and neck cancer patients. In conclusion, our data suggest that the increased expression of miR-629-3p provides a mechanism of cisplatin resistance in head and neck cancer and may serve as a therapeutic target to reverse chemotherapy resistance.

Diagnostic performance of peripheral leukocyte telomere G-tail length for detecting breast cancer.

The telomere G-tail (G-tail) plays an essential role in maintaining chromosome stability. In this study, we assessed the leukocyte G-tail length of breast cancer (BC) patients and cancer-free individuals and evaluated the association between the G-tail length and the presence of BC. A significant shortening of the median G-tail length was observed in BC patients compared with cancer-free individuals and was found in the early phase of BC. Our study indicated that the leukocyte G-tail length might be a potential biomarker for BC detection.

Predicting the presence of breast cancer using circulating small RNAs, including those in the extracellular vesicles.

Emerging evidence indicates that small RNAs, including microRNAs (miRNAs) and their isoforms (isomiRs), and transfer RNA fragments (tRFs), are differently expressed in breast cancer (BC) and can be detected in blood circulation. Circulating small RNAs and small RNAs in extracellular vesicles (EVs) have emerged as ideal markers in small RNA-based applications for cancer detection. In this study, we first undertook small RNA sequencing to assess the expression of circulating small RNAs in the serum of BC patients and cancer-free individuals (controls). Expression of 3 small RNAs, namely isomiR of miR-21-5p (3' addition C), miR-23a-3p and tRF-Lys (TTT), was significantly higher in BC samples and was validated by small RNA sequencing in an independent cohort. Our constructed model using 3 small RNAs showed high diagnostic accuracy with an area under the receiver operating characteristic curve of 0.92 and discriminated early-stage BCs at stage 0 from control. To test the possibility that these small RNAs are released from cancer cells, we next examined EVs from the serum of BC patients and controls. Two of the 3 candidate small RNAs were identified, and shown to be abundant in EVs of BC patients. Interestingly, these 2 small RNAs are also more abundantly detected in culture media of breast cancer cell lines (MCF-7 and MDA-MB-231). The same tendency in selective elevation seen in total serum, serum EV, and EV derived from cell culture media could indicate the efficiency of this model using total serum of patients. These findings indicate that small RNAs serve as significant biomarkers for BC detection.

Targeting DNA binding proteins for cancer therapy.

Dysregulation or mutation of DNA binding proteins such as transcription factors (TFs) is associated with the onset and progression of various types of disease, including cancer. Alteration of TF activity occurs in numerous cancer tissues due to gene amplification, deletion, and point mutations, and epigenetic modification. Although cancer-associated TFs are promising targets for cancer therapy, development of drugs targeting these TFs has historically been difficult due to the lack of high-throughput screening methods. Recent advances in technology for identification and selective inhibition of DNA binding proteins enable cancer researchers to develop novel therapeutics targeting cancer-associated TFs. In the present review, we summarize known cancer-associated TFs according to cancer type and introduce recently developed high-throughput approaches to identify selective inhibitors of cancer-associated TFs.

Recent Advances in Liquid Biopsy Based on Circulating Tumor DNA.

Many types of cells secrete DNA, RNA, and proteins through microvesicles, such as exosomes and apoptotic bodies, for the purpose of extracellular communication [...].

Single-Cell Analysis Reveals a Preexisting Drug-Resistant Subpopulation in the Luminal Breast Cancer Subtype.

Drug resistance is a major obstacle in the treatment of breast cancer. Surviving cells lead to tumor recurrence and metastasis, which remains the main cause of cancer-related mortality. Breast cancer is also highly heterogeneous, which hinders the identification of individual cells with the capacity to survive anticancer treatment. To address this, we performed extensive single-cell gene-expression profiling of the luminal-type breast cancer cell line MCF7 and its derivatives, including docetaxel-resistant cells. Upregulation of epithelial-to-mesenchymal transition and stemness-related genes and downregulation of cell-cycle-related genes, which were mainly regulated by LEF1, were observed in the drug-resistant cells. Interestingly, a small number of cells in the parental population exhibited a gene-expression profile similar to that of the drug-resistant cells, indicating that the untreated parental cells already contained a rare subpopulation of stem-like cells with an inherent predisposition toward docetaxel resistance. Our data suggest that during chemotherapy, this population may be positively selected, leading to treatment failure. SIGNIFICANCE: This study highlights the role of breast cancer intratumor heterogeneity in drug resistance at a single-cell level.

Cancer extracellular vesicles contribute to stromal heterogeneity by inducing chemokines in cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs), one of the major components of a tumour microenvironment, comprise heterogeneous populations involved in tumour progression. However, it remains obscure how CAF heterogeneity is governed by cancer cells. Here, we show that cancer extracellular vesicles (EVs) induce a series of chemokines in activated fibroblasts and contribute to the formation of the heterogeneity. In a xenograft model of diffuse-type gastric cancer, we showed two distinct fibroblast subpopulations with alpha-smooth muscle actin (α-SMA) expression or chemokine expression. MicroRNAs (miRNAs) profiling of the EVs and the transfection experiment suggested that several miRNAs played a role in the induction of chemokines such as CXCL1 and CXCL8 in fibroblasts, but not for the myofibroblastic differentiation. Clinically, aberrant activation of CXCL1 and CXCL8 in CAFs correlated with poorer survival in gastric cancer patients. Thus, this link between chemokine expression in CAFs and tumour progression may provide novel targets for anticancer therapy.

Development of miRNA-based therapeutic approaches for cancer patients.

Over the past few decades, siRNA and miRNA have attracted a great deal of attention from researchers and clinicians. These molecules have been extensively studied from the standpoint of developing biopharmaceuticals against various diseases, including heart disease, diabetes and cancers. siRNA suppresses only a single target, whereas each miRNA regulates the expression of multiple target genes. More importantly, because miRNA are also secreted from cancer cells, and their aberrant expression is associated with tumor development and progression, they represent not only therapeutic targets but also promising biomarkers for diagnosis and prognosis. Therefore, miRNA may be more effective tools against cancers, in which multiple signal pathways are dysregulated. In this review, we summarize recent progress in the development of miRNA therapeutics for the treatment of cancer patients, and describe delivery systems for oligonucleotide therapeutics.

[MicroRNA in Body Fluids - Development of the Novel Plat Form for Cancer Therapeutics and Diagnosis].

MicroRNAs(miRNAs)are small non-coding RNAs that function in diverse biological processes and are approximately 20-22 nucleotide RNAs that regulate the expression of target genes, mainly at the post-transcriptional level. A number of studies report that miRNAs are involved in homeostatic maintenance such as cell cycle regulation, cell division and apoptosis, and that aberrant expression of miRNAs is often detected in various types of diseases, including cancer. In cancer biology, miRNAs play functional roles in tumor seeding, drug sensitivity, and metastasis. MiRNAs are also secreted through the small vesicles called exosomes, which are endosome-derived vesicles from various cell types including immune and tumor cells. In addition to cellular miRNAs, secreted miRNAs also play important roles in cancer development and metastasis. Therefore, secreted miRNAs in body fluids have been investigated as a promising biomarkers and therapeutic targets for the treatment of cancer patients. In this review, we introduce the current knowledge of miRNA functions in cancer development and discuss the clinical applications of se-miRNAs, eg, as diagnostic markers and therapeutic targets.

CD44 exerts a functional role during EMT induction in cisplatin-resistant head and neck cancer cells.

A number of studies report that epithelial to mesenchymal transition (EMT) supports the generation and maintenance of cancer stem cells (CSCs), which show tumor seeding ability and drug resistance; however, the molecular mechanisms underlying induction of EMT-associated tumor malignancy remain unclear. The present study reports that oral cancer cells switch from expressing the CD44 variant form (CD44v) to expressing the standard form (CD44s) during acquisition of cisplatin-resistance, which resulted in EMT induction. CD44s induced an EMT phenotype in cisplatin resistant cells by up-regulating ZEB1, a transcriptional repressor of E-cadherin. More importantly, CD44s up-regulated ZEB1 by suppressing microRNA-200c, which is a non-coding RNA that directly represses the ZEB1 gene. These results demonstrate the importance of the association between platinum resistance and CD44s during EMT induction in oral cancer cells.

Drug Resistance Driven by Cancer Stem Cells and Their Niche.

Drug resistance represents one of the greatest challenges in cancer treatment. Cancer stem cells (CSCs), a subset of cells within the tumor with the potential for self-renewal, differentiation and tumorigenicity, are thought to be the major cause of cancer therapy failure due to their considerable chemo- and radioresistance, resulting in tumor recurrence and eventually metastasis. CSCs are situated in a specialized microenvironment termed the niche, mainly composed of fibroblasts and endothelial, mesenchymal and immune cells, which also play pivotal roles in drug resistance. These neighboring cells promote the molecular signaling pathways required for CSC maintenance and survival and also trigger endogenous drug resistance in CSCs. In addition, tumor niche components such as the extracellular matrix also physically shelter CSCs from therapeutic agents. Interestingly, CSCs contribute directly to the niche in a bilateral feedback loop manner. Here, we review the recent advances in the study of CSCs, the niche and especially their collective contribution to resistance, since increasingly studies suggest that this interaction should be considered as a target for therapeutic strategies.