Oncogenic microRNA Research Articles

Hsa-miR-155-5p Up-Regulation in Breast Cancer and Its Relevance for Treatment With Poly[ADP-Ribose] Polymerase 1 (PARP-1) Inhibitors.

miR-155-5p is a well-known oncogenic microRNA, showing frequent overexpression in human malignancies, including breast cancer. Here, we show that high miR-155-5p levels are associated with unfavorable prognostic factors in two independent breast cancer cohorts (CSS cohort, n = 283; and TCGA-BRCA dataset, n = 1,095). Consistently, miR-155-5p results as differentially expressed in the breast cancer subgroups identified by the surrogate molecular classification in the CSS cohort and the PAM50 classifier in TCGA-BRCA dataset, with the TNBC and HER2-amplified tumors carrying the highest levels. Since the analysis of TCGA-BC dataset also demonstrated a significant association between miR-155-5p levels and the presence of mutations in homologous recombination (HR) genes, we hypothesized that miR-155-5p might affect cell response to the PARP-1 inhibitor Olaparib. As expected, miR-155-5p ectopic overexpression followed by Olaparib administration resulted in a greater reduction of cell viability as compared to Olaparib administration alone, suggesting that miR-155-5p might induce a synthetic lethal effect in cancer cells when coupled with PARP-1-inhibition. Overall, our data point to a role of miR-155-5p in homologous recombination deficiency and suggest miR-155-5p might be useful in predicting response to PARP1 inhibitors in the clinical setting.

MiR-26a-5p Serves as an Oncogenic MicroRNA in Non-Small Cell Lung Cancer by Targeting FAF1.

PurposeNon-small cell lung cancer (NSCLC) accounts for approximately 80–85% of all lung cancers, with the FAS-associated factor 1 (FAF1) acting as a tumor suppressor. MicroRNAs (miRNAs) can influence cancer progression by targeting oncogenes or anti-oncogenes. In this study, we aimed to reveal the influence of miR-26a-5p on the regulation of FAF1 expression and NSCLC progression, with the motivation of identifying a potential therapeutic target for NSCLC treatment.MethodsA dual-luciferase reporter assay was used to check for the direct targeting of FAF1 by miR-26a-3p. The miR-26a-5p inhibitor or FAF1 shRNA plasmid was transfected into A549 and H1299 cells to modulate FAF1 expression. Then, the effect of miR-26a-5p/FAF1 on cellular functions was investigated. MTT assay was used to evaluate cell viability. EdU proliferation assay and cell cycle assay were performed to analyze the effect of miR-26a-5p on cell replication and cell cycle. We used annexin V-FITC and PI to stain apoptotic cells, followed by flow cytometric analysis. Transwell and wound healing assays were performed to investigate metastasis. Moreover, the effect of miR-26a-5p/FAF1 on cancer progression was examined in vivo. Lastly, the underlying mechanism was uncovered using RT-qPCR, Western blotting, and TOP/FOP flash assay.ResultsmiR-26a-5p was found to directly target FAF1 and downregulate its expression. Blocking miR-26a-5p inhibited the cell growth, migration, and invasion, but promoted cell apoptosis. In addition, this inhibited the growth of tumor in mice. FAF1 knockdown reversed the functions of miR-26a-5p. Further, miR-26a-5p/FAF1 was observed to play an important role in the Wnt signaling pathway, regulating the expression of genes such as AXIN, c-Myc, and cyclin-D1.ConclusionTaken together, we show that miR-26a-5p functions as an oncogenic microRNA in NSCLC by targeting FAF1 and may serve as a potential target for NSCLC treatment.

Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer.

In aggressively growing tumors, hypoxia induces HIF-1α expression promoting angiogenesis. Previously, we have shown that overexpression of oncogenic microRNAs (miRNAs, miRs) miR526b/miR655 in poorly metastatic breast cancer cell lines promotes aggressive cancer phenotypes in vitro and in vivo. Additionally, miR526b/miR655 expression is significantly higher in human breast tumors, and high miR526b/miR655 expression is associated with poor prognosis. However, the roles of miR526b/miR655 in hypoxia are unknown. To test the relationship between miR526b/miR655 and hypoxia, we used various in vitro, in silico, and in situ assays. In normoxia, miRNA-high aggressive breast cancer cell lines show higher HIF-1α expression than miRNA-low poorly metastatic breast cancer cell lines. To test direct involvement of miR526b/miR655 in hypoxia, we analyzed miRNA-high cell lines (MCF7-miR526b, MCF7-miR655, MCF7-COX2, and SKBR3-miR526b) compared to controls (MCF7 and SKBR3). CoCl2-induced hypoxia in breast cancer further promotes HIF-1α mRNA and protein expression while reducing VHL expression (a negative HIF-1α regulator), especially in miRNA-high cell lines. Hypoxia enhances oxidative stress, epithelial to mesenchymal transition, cell migration, and vascular mimicry more prominently in MCF7-miR526b/MCF7-miR655 cell lines compared to MCF7 cells. Hypoxia promotes inflammatory and angiogenesis marker (COX-2, EP4, NFκB1, VEGFA) expression in all miRNA-high cells. Hypoxia upregulates miR526b/miR655 expression in MCF7 cells, thus observed enhancement of hypoxia-induced functions in MCF7 could be attributed to miR526b/miR655 upregulation. In silico bioinformatics analysis shows miR526b/miR655 regulate PTEN (a negative regulator of HIF-1α) and NFκB1 (positive regulator of COX-2 and EP4) expression by downregulation of transcription factors NR2C2, SALL4, and ZNF207. Hypoxia-enhanced functions in miRNA-high cells are inhibited by COX-2 inhibitor (Celecoxib), EP4 antagonist (ONO-AE3-208), and irreversible PI3K/Akt inhibitor (Wortmannin). This establishes that hypoxia enhances miRNA functions following the COX-2/EP4/PI3K/Akt pathways and this pathway can serve as a therapeutic target to abrogate hypoxia and miRNA induced functions in breast cancer. In situ, HIF-1α expression is significantly higher in human breast tumors (n = 96) compared to non-cancerous control tissues (n = 20) and is positively correlated with miR526b/miR655 expression. In stratified tumor samples, HIF-1α expression was significantly higher in ER-positive, PR-positive, and HER2-negative breast tumors. Data extracted from the TCGA database also show a strong correlation between HIF-1α and miRNA-cluster expression in breast tumors. This study, for the first time, establishes the dynamic roles of miR526b/miR655 in hypoxia.

Grape Powder Supplementation Attenuates Prostate Neoplasia Associated with Pten Haploinsufficiency in Mice Fed High-Fat Diet.

Previous studies have identified potent anticancer activities of polyphenols in preventing prostate cancer. The aim of the current study is to evaluate the chemopreventive potential of grape powder (GP) supplemented diets in genetically predisposed and obesity-provoked prostate cancer. Prostate-specific Pten heterozygous (Pten+/f ) transgenic mice are fed low- and high-fat diet (LFD and HFD, respectively) supplemented with 10% GP for 33 weeks, ad libitum. Prostate tissues are characterized using immunohistochemistry and western blots, and sera are analyzed by ELISA and qRT-PCR. Pten+/f mice fed LFD and HFD supplemented with 10% GP show favorable histopathology, significant reduction of the proliferative rate of prostate epithelial cells (Ki67), and rescue of PTEN expression. The most potent protective effect of GP supplementation is detected against HFD-induced increase in inflammation (IL-1β; TGF-β1), activation of cell survival pathways (Akt, AR), and angiogenesis (CD31) in Pten+/f mice. Moreover, GP supplementation reduces circulating levels of oncogenic microRNAs (miR-34a; miR-22) in Pten+/f mice. There are no significant changes in body weight and food intake in GP supplemented diet groups. GP diet supplementation can be a beneficial chemopreventive strategy for obesity-related inflammation and prostate cancer progression. Monitoring serum miRNAs can facilitate the non-invasive evaluation of chemoprevention efficacy.

NCL Inhibition Exerts Antineoplastic Effects against Prostate Cancer Cells by Modulating Oncogenic MicroRNAs.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men and second most common cause of cancer-related deaths in the United States. Androgen deprivation therapy (ADT) is only temporarily effective for advanced-stage PCa, as the disease inevitably progresses to castration-resistant prostate cancer (CRPC). The protein nucleolin (NCL) is overexpressed in several types of human tumors where it is also mislocalized to the cell surface. We previously reported the identification of a single-chain fragment variable (scFv) immuno-agent that is able to bind NCL on the surface of breast cancer cells and inhibit proliferation both in vitro and in vivo. In the present study, we evaluated whether NCL could be a valid therapeutic target for PCa, utilizing DU145, PC3 (CRPC), and LNCaP (androgen-sensitive) cell lines. First, we interrogated the publicly available databases and noted that higher NCL mRNA levels are associated with higher Gleason Scores as well as with recurrent and metastatic tumors. Then, using our anti-NCL scFv, we demonstrated that NCL is expressed on the surface of all three tested cell lines and that NCL inhibition results in reduced proliferation and migration. We also measured the inhibitory effect of NCL targeting on the biogenesis of oncogenic microRNAs such as miR-21, -221 and -222, which was cell context dependent. Taken together, our data provide evidence that NCL targeting inhibits the key hallmarks of malignancy in PCa cells and may provide a novel therapeutic option for patients with advanced-stage PCa.

Prenatal particulate air pollution exposure and expression of the miR-17/92 cluster in cord blood: Findings from the ENVIRONAGE birth cohort

BackgroundAir pollution exposure during pregnancy is an important environmental health issue. Epigenetics mediate the effects of prenatal exposure and could increase disease predisposition in later life. The oncogenic miR-17/92 cluster is involved in normal development and disease. ObjectivesHere, for the first time the potential prenatal effects of particulate matter with a diameter<2.5 μm (PM2.5) exposure on expression of the miR-17/92 cluster in cord blood are explored. MethodsIn 370 mother-newborn pairs from the ENVIRONAGE birth cohort, expression of three members of the miR-17/92 cluster was measured in cord blood by qRT-PCR. Expression of C-MYC and CDKN1A, a cluster activator and a target gene, respectively, was also analyzed. Multivariable linear regression models were used to associate the relative m(i)RNA expression with prenatal PM2.5 exposure. ResultsPM2.5 exposure averaged (10th-90th percentile) 11.7 (9.0–14.4) µg/m3 over the entire pregnancy. In cord blood, miR-17 and miR-20a showed a −45.0% (95%CI: −55.9 to −31.4, p < 0.0001) and a –33.7% (95%CI: −46.9 to −17.2, p = 0.0003), decrease in expression in association with first trimester PM2.5 exposure, and a –32.5% (95%CI: −45.6 to −16.3, p = 0.0004) and –23.3% (95%CI: −38.1 to −4.8, p = 0.02), respectively, decrease in expression in association with PM2.5 exposure during the entire pregnancy. In association with third trimester PM2.5 exposure, a reduction of −25.8% (95%CI: −40.2 to −8.0, p = 0.007) and −14.2% (95%CI: −27.7 to 1.9, p = 0.08), for miR-20a and miR-92a expression, respectively, was identified. Only miR-92a expression (-15.7%, 95%CI: −27.3 to −2.4, p = 0.02) was associated with PM2.5 exposure during the last month of pregnancy. C-MYC expression was downregulated in cord blood in association with prenatal PM2.5 exposure during the first trimester and the entire pregnancy, in the adjusted model. DiscussionLower expression levels of the miR-17/92 cluster in cord blood in association with increased prenatal PM2.5 exposure were observed. Whether this oncogenic microRNA cluster plays a role in trans-placental carcinogenesis remains to be elucidated.

Long-Term Detection of Oncogenic MicroRNA in Living Human Cancer Cells by Gold@ Polydopamine-Shell Nanoprobe.

Oncogenic microRNAs (miRNA), for example, miR-155, are key tumor biomarkers in cancer cells that drive tumorigenesis, and the miRNA profile signature can predict cancer development and aggressiveness. Hence, timely detection of oncogenic miRNA in living cells is highly attractive to the diagnosis of cancer at an early stage. Herein, we report a highly sequence-specific gold@polydopamine-based nanoprobe for long-term detection of miRNA in human cancer cell lines in vitro. A single administration of the nanoprobe enables continuous detection of the miR-155 expression level in living cancer cells for up to 5 days. We believe that our nanoprobe is highly promising for both oncology research and translational applications.

Bottom-Up Fabrication of Plasmonic Nanoantenna-Based High-throughput Multiplexing Biosensors for Ultrasensitive Detection of microRNAs Directly from Cancer Patients' Plasma.

There is an unmet need in clinical point-of-care (POC) cancer diagnostics for early state disease detection, which would greatly increase patient survival rates. Currently available analytical techniques for early stage cancer diagnosis do not meet the requirements for POC of a clinical setting. They are unable to provide the high demand of multiplexing, high-throughput, and ultrasensitive detection of biomarkers directly from low volume patient samples ("liquid biopsy"). To overcome these current technological bottle-necks, herein we present, for the first time, a bottom-up fabrication strategy to develop plasmonic nanoantenna-based sensors that utilize the unique localized surface plasmon resonance (LSPR) properties of chemically synthesized gold nanostructures, gold triangular nanoprisms (Au TNPs), gold nanorods (Au NRs), and gold spherical nanoparticles (Au SNPs). Our Au TNPs, NRs, and SNPs display refractive index unit (RIU) sensitivities of 318, 225, and 135 nm/RIU respectively. Based on the RIU results, we developed plasmonic nanoantenna-based multiplexing and high-throughput biosensors for the ultrasensitive assay of microRNAs. MicroRNAs are directly linked with cancer development, progression, and metastasis, thus they hold promise as next generation biomarkers for cancer diagnosis and prognosis. The developed biosensors are capable of assaying five different types of microRNAs at an attomolar detection limit. These sets of microRNAs include both oncogenic and tumor suppressor microRNAs. To demonstrate the efficiency as a POC cancer diagnostic tool, we analyzed the plasma of 20-bladder cancer patients without any sample processing steps. Importantly, our liquid biopsy-based biosensing approach is capable of differentiating healthy from early ("non-metastatic") and late ("metastatic") stage cancer with a p value <0.0001. Further, receiver operating characteristic analysis shows that our biosensing approach is highly specific, with an area under the curve of 1.0. Additionally, our plasmonic nanoantenna-based biosensors are regenerative, allowing multiple measurements using the same biosensors, which is essential in low- and middle-income countries. Taken together, our multiplexing and high-throughput biosensors have the unmatched potential to advance POC diagnostics and meet global needs for early stage detection of cancer and other diseases (e.g., infectious, autoimmune, and neurogenerative diseases).

Hypomethylation Causes MIR21 Overexpression in Tumors

miR-21 is an oncogenic microRNA (miRNA) that is upregulated in many solid tumors. However, the effect of MIR21 hypomethylation on miR-21 expression in tumors and the mechanism of miR-21 DNA demethylation remain unclear. In this study, we confirmed that the expression of miR-21 was significantly increased in multiple tumors. We analyzed eight types of cancer, including breast cancer (BRCA), lung adenocarcinoma (LUAD), renal and renal clear cell carcinoma (KIRC), bladder urothelial carcinoma (BLCA), hepatocellular carcinoma (LIHC), lung squamous cell cancer (LUSC), renal papillary cell carcinoma (KIRP), and pancreatic adenocarcinoma (PAAD). MIR21 DNA methylation levels were elevated in these cancers. CpG loci located approximately 200 bp upstream of the transcription initiation site strongly affect MIR21 expression. We also confirmed MIR21 hypomethylation by pyrosequencing of fresh clear cell renal cell carcinoma (ccRCC) samples. Demethylating agent was proved to increase hsa-miR-21-5p level in HEK293T cells, while knockdown of DNA demethylases TET3 and TDG decreased MIR21 expression. In addition, we showed that the cg02515217 CpG locus in MIR21 promoter was a conserved binding site of transcription factors CEBPB, MEIS3, and TEAD4, which were co-expressed with miR-21 in tumors. These observations identified that gene hypomethylation regulated the expression of MIR21 in tumors.

Exosomal circular RNAs derived from serum: Promising biomarkers for therapeutic targets and prognosis of triple-negative breast cancer (TNBC).

3528 Background: Exosomes are well known by the "exosomal shuttle" that delivers oncogenic microRNAs (miRNAs), mRNAs, circular RNAs (circRNAs) and proteins to the recipient cells and tumor microenvironment, and may be used as promising biomarkers for disease diagnosis. This study aims to provide a theoretical basis to use stable exosomal circRNAs as new biomarkers for predicting the development, metastasis and therapeutic targets of TNBC. Methods: A strategy combining RNA-sequencing technique, bioinformatic analysis and RT-qPCR was used to determine the level of differential expressed circRNAs in serum exosomes samples (n = 43) from TNBC patients compared with non-TNBC patients. The expression of circHSDL2 were also detected in tumor tissues (n = 20) from TNBC patients and breast cancer cell lines by qRT-PCR. Cell cycle analysis, the wound healing assays and transwell assays were used to investigate the function of circHSDL2 in proliferation, invasion and metastasis of TNBC cells. FISH, dual-luciferase reporter and functional assays were performed to confirm the interaction between circHSDL2 and let-7a-2-3p in TNBC cells. Results: We profiled the circRNAs in the serum exosomes samples from TNBC patients and non-TNBC patients by RNA sequencing and detected 803 significantly differentially-expressed circRNAs. After bioinformatic analysis, circHSDL2 was chose to further study. RT-qPCR results showed that higher expression of circHSDL2 in TNBC cell lines and tumor tissues from TNBC patients. Moreover, overexpression of circHSDL2 promoted TNBC cells proliferation and invasion, while knockdown of circHSDL2 inhibited TNBC cells proliferation and invasion. Mechanistically, circHSDL2 acted as a "miRNAs sponge" to absorb let-7a-2-3p; let-7a-2-3p inhibited TNBC cell invasion and metastasis. Kaplan-Meier plots showed lower expression of let-7a-2-3p was connected to poor prognosis in TNBC metastasis patients from TCGA database. Conclusions: The expression of circHSDL2 was found significantly upregulated in serum exosomes and tumor tissues from TNBC patients. Moreover, circHSDL2 could promote cell proliferation, invasion and metastasis in TNBC cells. CircHSDL2 might be function as competing endogenous RNAs (ceRNAs) by targeting let-7a-2-3p in the progression of TNBC. Therefore, this study provides a fresh perspective on novel therapeutic targets and potential biomarkers for TNBC from exosomal circRNAs.

Circ_0000526 Blocks the Progression of Breast Cancer by Sponging miR-492.

Aim: Breast cancer is one of the leading causes of cancer-associated death. This work aimed to explore the expression, function, and mechanism of circ_0000526 on the progression of breast cancer. Materials and Methods: The expressions of circ_0000526, miR-492, and suppressor of cytokine signaling 2 (SOCS2) in breast cancer samples were measured using quantitative real-time polymerase chain reaction. The correlation between pathological indexes of patients and the expression of circ_0000526 was also analyzed. Human breast cancer cell lines MCF-7 and MDA-MB-231 were applied to investigate the associated mechanism. Cell counting kit-8 assays were used to assess the effect of circ_0000526 on proliferation, and together with the wound healing assay, transwell assay was conducted to detect the effects of circ_0000526 on migration and invasion. Furthermore, luciferase reporter assay was used to verify the targeting relationship between miR-492 and circ_0000526. In addition, Western blot was performed to detect the regulatory effects of circ_0000526 on SOCS2. Results: Circ_0000526 expression in breast cancer samples was downregulated, which was correlated with unfavorable pathological indexes. It significantly inhibited the proliferation and metastasis of breast cancer cells, and facilitated apoptosis. The overexpression of circ_0000526 remarkably inhibited the expression of miR-492 by sponging it, and in turn promoted the expression of SOCS2. Conclusions: Circ_0000526 served as a tumor suppressor, which could function as the sponge of miR-492, an oncogenic microRNA, in breast cancer, and enhance the expression of tumor suppressor SOCS2 indirectly.

SAT-LB130 Tamoxifen Affects MiRNA Expression in Uterus and Breast Tamoxifen Affects MiRNA Expression in Uterus and Breast Tamoxifen Affects MiRNA Expression in Uterus and Breast

In addition to genetic factors, environmental factors and lifestyle can play a significant role in the development of hormone-dependent tumors, such as endometrial cancer (EC) and breast cancer (BC). The discovery of microRNAs (miRs) involved in the post-transcriptional regulation of many genes, including those of hormonal carcinogenesis, namely, steroid receptors and their target genes, strengthened the epigenetic direction in the study of carcinogenesis mechanisms. A critical event in the development of hormone-dependent human tumors is violation in the metabolism of steroid hormones, primarily estradiol. An interesting aspect of the problem of ERα inhibition is the use of tamoxifen (TAM) in clinical practice in the treatment of hormone-dependent BC. A well-known side effect of TAM is increased proliferation in the endometrium and an elevated risk of EC. One of the mechanisms explaining such differences in the effects of TAM is formation of DNA adducts in endometrial cells, but this mechanism has not yet been substantiated. Therefore, the problem of carcinogenesis of the uterus with this drug remains unresolved and requires further research. The aim of our study was to evaluate the expression of miRs and target genes for hormonal carcinogenesis in the uterus and mammary gland under the exposure with TAM. As an object of study, we used female rats, primary human cell cultures and tissues of TAM-induced human endometrial hyperplasia. The results showed that estradiol enhances the expression of oncogenic microRNAs miR-21-, 221, -222 by three-ten times, both in the rat mammary gland and endometrium, which confirms its oncogenic properties. In the rat endometrium, TAM, to a greater extent than estradiol, increased the expression of oncogenic miRs, especially miR-419, -23a, 24-2,- 27, and significantly reduced the expression of their target genes. In addition, TAM caused a multiple (8-fold) increase in the expression of cyclin D in uterus compared with mammary gland. In most cases, TAM reduced expression of oncogenic miR-21,-221,-222 by 50% in BC primary cell culture whereas in EC primary cell culture expression of oncogenic 190a was increased. We also investigated the activity of estrogen-metabolizing enzymes in tamoxifen-induced human endometrial hyperplasia. A significant difference was found in the expression of estrogen-metabolizing genes (CYP1A,1B, CYP19, SULT1A1, SULT1E1, GSTP1,2, COMT, STS) in TAM-induced endometrial hyperplasia, which may be due to the difference in miRNA expression. Thus, both for the animal model and human cell cultures, it was shown that TAM causes other changes in the expression of microRNAs in the endometrium compared with the breast. Further studies with the identification of target miRNA genes will help identify molecular targets of TAM-induced endometrial hyperplasia. This work was supported by Russian Science Foundation, grant # 19-15-00319.

MicroRNA-155-5p suppresses PD-L1 expression in lung adenocarcinoma.

MiR‐155‐5p is a key oncogenic microRNA that maintains immune homeostasis and mediates cross‐talk between inflammation and tumorigenesis. High expression of programmed death ligand‐1 (PD‐L1) also plays an important role in immune tolerance in tumors. The present study aimed to explore the relationship between miR‐155‐5p and PD‐L1 in lung adenocarcinoma (LUAD) cells A549 and H1650. The expression levels of miR‐155‐5p and PD‐L1 in LUAD patients were detected by a quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) and mimics of miR‐155‐5p were used to model increased expression in A549 or H1650 cells. After 24 h, we measured levels of PD‐L1 by qRT‐PCR, western blotting and flow cytometry. In addition, we identified two sites in the PD‐L1 3′‐UTR (5′‐AGCAUUA‐3′ and 5′‐GCAUUAA‐3′) that can be bound by miR‐155‐5p using TargetScan (http://www.targetscan.org). Compared to normal tissue, miR‐155‐5p was overexpressed in tumor tissue (P = 0.0456), whereas the expression of PD‐L1 was not significantly different (P = 0.1349). The expression levels of miR‐155‐5p and PD‐L1 were negatively correlated (r = −0.6409, P = 0.0459 and r = −0.7544, P = 0.0117). Exogenous overexpression of miR‐155‐5p decreased the mRNA, total protein and membrane protein expression levels of PD‐L1 both in A549 and H1650 cells (P < 0.05). Taken together, our data suggest that miR‐155‐5p may suppress the expression of PD‐L1 in LUAD.

Overexpression of miR-21-5p in colorectal cancer cells promotes self-assembly of E-cadherin-dependent multicellular tumor spheroids

Three-dimensional (3D) multicellular tumor spheroid (MCTS) cultures are increasingly popular as an in vitro tumor model for drug screening because they can mimic the complexity and heterogeneity of tumors compared to 2D monolayer cell cultures. The oncogenic microRNA, miR-21−5p (hereafter denoted as miR-21), is one of the most upregulated miRNAs in colorectal cancer (CRC). Herein, we established a stable miR-21-overexpressing clone in the DLD-1 human CRC cell line to investigate its impact on MCTS formation. We found that miR-21 overexpression enhanced cell-cell interactions/aggregations in both 2D monolayer and 3D suspension cultures. Cell aggregates in 3D suspension culture further formed MCTSs in miR-21-overexpressing cells. miR-21 overexpression was associated with the upregulation of proteins involved in E-cadherin-associated cell-cell adhesion. Furthermore, miR-21 induction of MCTSs could be reversed by the antibody-induced blockade of E-cadherin. Our results showed that miR-21 overexpression promoted MCTS formation through enhancing E-cadherin-dependent cell-cell interactions, which represents an advance in vitro model for investigating CRC biology.

MicroRNA-527 Induces Proliferation and Cell Cycle in Esophageal Squamous Cell Carcinoma Cells by Repressing PH Domain Leucine-Rich-Repeats Protein Phosphatase 2.

Increasing evidence indicated that microRNAs served dominant roles in carcinogenesis and cancer progression by targeting potential downstream genes. In our study, we found that miR-527 was an upregulated expression in human esophageal squamous cell carcinoma (ESCC) cells and tissues. Furthermore, overexpression of miR-527 promoted cell proliferation and colony formation, enhanced anchorage-independent growth ability, and contributed to cell cycle. In addition, protein phosphatase 2 (PHLPP2) was identified as the direct downstream target gene of miR-527 and was confirmed by luciferase gene reporter assay. In summary, we concluded that miR-527 acted as an oncogenic microRNA in ESCC development by directly targeting PHLPP2 might be a novel therapeutic target for the treatment of ESCC.

Targeted Degradation of the Oncogenic MicroRNA 17-92 Cluster by Structure-Targeting Ligands.

Many RNAs are processed into biologically active transcripts, the aberrant expression of which can contribute to disease phenotypes. For example, the primary microRNA-17-92 (pri-miR-17-92) cluster contains six microRNAs (miRNAs) that collectively act in several disease settings. Herein, we used sequence-based design of structure-specific ligands to target a common structure in the Dicer processing sites of three miRNAs in the cluster, miR-17, miR-18a, and miR-20a, thereby inhibiting their biogenesis. The compound was optimized to afford a dimeric molecule that binds the Dicer processing site and an adjacent bulge, affording a 100-fold increase in potency. The dimer's mode of action was then extended from simple binding to direct cleavage by conjugation to bleomycin A5 in a manner that imparts RNA-selective cleavage or to indirect cleavage by recruiting an endogenous nuclease, or a ribonuclease targeting chimera (RIBOTAC). Interestingly, the dimer-bleomycin conjugate cleaves the entire pri-miR-17-92 cluster and hence functionally inhibits all six miRNAs emanating from it. The compound selectively reduced levels of the cluster in three disease models: polycystic kidney disease, prostate cancer, and breast cancer, rescuing disease-associated phenotypes in the latter two. Further, the bleomycin conjugate exerted selective effects on the miRNome and proteome in prostate cancer cells. In contrast, the RIBOTAC only depleted levels of pre- and mature miR-17, -18a, and 20a, with no effect on the primary transcript, in accordance with the cocellular localization of RNase L, the pre-miRNA targets, and the compound. These studies demonstrate a strategy to tune RNA structure-targeting compounds to the cellular localization of the target.

MiR-1285-3p Controls Colorectal Cancer Proliferation and Escape from Apoptosis through DAPK2.

MicroRNAs are tiny but powerful regulators of gene expression at the post-transcriptional level. Aberrant expression of oncogenic and tumor-suppressor microRNAs has been recognized as a common feature of human cancers. Colorectal cancer represents a major clinical challenge in the developed world and the design of innovative therapeutic approaches relies on the identification of novel biological targets. Here, we perform a functional screening in colorectal cancer cells using a library of locked nucleic acid (LNA)-modified anti-miRs in order to unveil putative oncogenic microRNAs whose inhibition yields a cytotoxic effect. We identify miR-1285-3p and further explore the effect of its targeting in both commercial cell lines and primary colorectal cancer stem cells, finding induction of cell cycle arrest and apoptosis. We show that DAPK2, a known tumor-suppressor, is a novel miR-1285 target and mediates both the anti-proliferative and the pro-apoptotic effects of miR-1285 depletion. Altogether, our findings uncover a novel oncogenic microRNA in colorectal cancer and lay the foundation for further studies aiming at the development of possible therapeutic strategies based on miR-1285 targeting.

The “Big Five” Phytochemicals Targeting Cancer Stem Cells: Curcumin, EGCG, Sulforaphane, Resveratrol and Genistein

Cancer stem cells (CSCs) constitute a subpopulation of tumor cells that possess self-renewal and tumor initiation capacity, and the ability to give rise to the heterogeneous lineages of cancer cells that comprise the tumor. CSCs exhibit intrinsic mechanisms of resistance to virtually all conventional cancer therapeutics, allowing them to survive current cancer therapies and to initiate tumor recurrence and metastasis. Different pathways and mechanisms that confer resistance and survival of CSCs, including activation of the Wnt/β- catenin, Sonic Hedgehog, Notch, PI3K/Akt/mTOR and STAT3 signaling pathways, expression of aldehyde dehydrogenase 1 (ALDH1) and oncogenic microRNAs, and acquisition of epithelial-mesenchymal transition (EMT), have been identified recently. Certain phytochemicals, in particular curcumin, epigallocatechin-3-gallate (EGCG), sulforaphane, resveratrol and genistein have been shown to interfere with these intrinsic CSC pathways in vitro and in human xenograft mice, leading to elimination of CSCs. Moreover, recent clinical trials have demonstrated the therapeutic efficacy of five phytochemicals, alone or in combination with modern cancer therapeutics, and in various types of cancer. Since current cancer therapies fail to eradicate CSCs, leading to cancer recurrence and progression, targeting of CSCs with phytochemicals such as curcumin, EGCG, sulforaphane, resveratrol and genistein, combined with each other and/or in combination with conventional cytotoxic drugs and novel cancer therapeutics, may offer a novel therapeutic strategy against cancer.

Abstract P1-05-04: miR551b-3p promotes Oncostatin M signaling in breast cancer

Abstract An increased expression of oncogenic microRNA miR551b-3p (also known as miR551b) occurs in a subset of breast cancer due to genomic amplification of 3q26.2 locus. Using in silico and clinical sample analysis of breast cancer patient datasets, we identified that those breast cancer samples that encompass miR551b amplification express high levels of STAT3 mRNA and Oncostatin (OSM) family genes in triple-negative breast cancer (TNBC). Furthermore, high expression of Oncostatin family genes associates with poor outcome in estrogen receptor- negative breast cancer patients. In this study, we identified a previously unexplored mechanism that miR551b-3p promotes breast cancer progression and tumor growth by direct interaction of miR551b-3p with STAT3 promoter thereby activating STAT3 transcription. As a consequence, STAT3 upregulates a cytokine Oncostatin and its receptor Oncostatin M receptor (OSMR) and their interaction further induces STAT3 phosphorylation and an autocrine self-reinforcing loop in TNBC cells. In this study, we demonstrate that overexpression of miR551b-3p promoted proliferation, invasion and migration, epithelial to mesenchymal transition (EMT) and spheroid formation of breast cancer cells and its inhibition decreased the expression of miR551b-3p by inhibiting OSM, OSMR, IL6ST, IL31RA, phosphorylated and total STAT3 in breast cancer cells and reduced tumor cell growth. Overall, our results provide the first evidence that miR551b regulates OSM family gene expression by activating STAT3 and thereby promoting tumor cell growth and metastasis in breast cancer cells. Therefore, targeting miR-551b-3p-STAT3-OSMR axis and inhibiting their levels will provide an unprecedented therapeutic opportunity in breast cancer. Citation Format: Anjali Geethadevi, Deepak Parashar, Miriam Ragle Aure, Jasmine George, Changliang Chen, Jyotsna Mishra, Andliena Tahiri, Bindu Nair, Amadou Camara, Vessela N Kristensen, Sunila Pradeep, Pradeep Chaluvally Raghavan. miR551b-3p promotes Oncostatin M signaling in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-04.

MicroRNA-378a-5p iS a novel positive regulator of melanoma progression

Evaluating the expression levels of miR-378a-5p both in a large melanoma patient cohort from The Cancer Genome Atlas database and in melanoma patients from our Institute, we found that miR-378a-5p is upregulated in metastatic melanoma specimens. miR-378a-5p expression was also increased in melanoma cells resistant to target therapy, and decreased in response to drug treatment. We also demonstrated that overexpression of miR-378a-5p enhances in vitro cell invasion and migration, and facilitates the ability of melanoma cells to form de novo vasculogenic structures. While performing downstream targeting studies, we confirmed the ability of miR-378a-5p to modulate the expression of known target genes, such as SUFU, FUS-1, and KLF9. Luciferase-3′UTR experiments also identified STAMBP and HOXD10 as new miR-378a-5p target genes. MMP2 and uPAR, two HOXD10 target genes, were positively regulated by miR-378a-5p. Genetic and pharmacologic approaches inhibiting uPAR expression and activity evidenced that the in vitro tumor-promoting functions of miR-378a-5p, were in part mediated by uPAR. Of note miR-378a-5p was also able to increase VEGF, as well as in vitro and in vivo angiogenesis. Finally, genetic and pharmacologic modulation of Bcl-2 evidenced Bcl-2 ability to regulate miR-378a-5p expression. In conclusion, to the best of our knowledge, this is the first study demonstrating that miR-378a-5p acts as an oncogenic microRNA in melanoma.