High Levels Of miR-21 Research Articles

FoxO1-negative cells are cancer stem-like cells in pancreatic ductal adenocarcinoma.

Flow cytometry assays using aldehyde dehydrogenase (ALDH) activity or CD133 positivity to isolate cancer stem cells (CSCs) are widely applied but have limitations. Thus, characterization of CSC makers for a specific cancer is potentially important. We have previously shown that miR-21 regulates cancer cell growth via FoxO1 in pancreatic ductal adenocarcinoma (PDAC). Here, we areported evidence of FoxO1-negative PDAC cells as CSCs in PDAC. Both ALDH-high and CD133-high cell fractions isolated from PDAC of the patients expressed high levels of miR-21 and null FoxO1. Cultured PDAC cells were virally transduced with GFP under FoxO1 promoter. GFP (FoxO1)-null PDAC cells expressed high levels of miR-21, and grew more quickly than FoxO1-positive PDAC cells. Moreover, the fold increases in growth of FoxO1-negative vs FoxO1-positive cells were greater than CD133-high vs CD133-low cells, or ALDH-high vs ALDH-low cells. Further, FoxO1-negative cells formed tumor spheres in culture and developed tumors after serial adoptive transplantation into NOD/SCID mice, while the FoxO1-positive cells did not. Finally, selective elimination of FoxO1-negative cells completely inhibited the growth of PDAC cells. Together, these data suggest that FoxO1-negative cells as CSCs in PDAC, and targeting FoxO1-negative cells in PDAC may provide better therapeutic outcome.

Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924 (Pevonedistat) in FLT3-ITD acute myeloid leukemia

High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-κB, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluated in clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients.

Arg⁹⁷² insulin receptor substrate-1 inhibits endothelial nitric oxide synthase expression in human endothelial cells by upregulating microRNA-155.

The dysregulation of nitric oxide (NO) synthesis attributable to the abnormal expression/activity of endothelial NO synthase (eNOS) is considered to be a major characteristic of insulin-resistant states, as well as an essential contributor to the pathogenesis of cardiovascular diseases. The Arg972 insulin receptor substrate-1 (IRS-1) is associated with insulin resistance. In the present study, we investigated the association between Arg972 IRS-1 and eNOS expression/activity in human subjects and in primary cultures of human endothelial cells. Data from 832 human subjects revealed that heterozygous and homozygous Arg972 IRS-1 carriers had significantly lower levels of plasma eNOS and nitrite/nitrate than the homozygous wild-type (WT) IRS-1 carriers. Human umbilical vein endothelial cells (HUVECs) established from delivering mothers expressing heterozygous Arg972 IRS-1 had significantly lower eNOS expression/activity and higher miR-155 levels than those expressing WT homozygous IRS-1. The overexpression of IRS-1 and Arg972 IRS-1 in the HUVECs, respectively, decreased and increased the miR-155 expression level. In addition, the overexpression of IRS-1 in the HUVECs significantly increased eNOS expression; this effect was reversed by transfection with mature miR-155 mimic or treatment with the selective phosphatidylinositol-3 kinase (PI3K) inhibitor, BKM120. On the other hand, the overexpression of Arg972 IRS-1 markedly decreased eNOS expression and this effect was reversed by transfection with antagomir-155. On the whole, our in vivo data demonstrate that Arg972 IRS-1 is associated with decreased plasma eNOS and nitrite/nitrate levels in human subjects. Our in vitro data demonstrate that Arg972 IRS-1 inhibits eNOS expression in human endothelial cells by upregulating miR-155 expression through the impairment of PI3K signaling. The present study provides new insight into the pathophysiological role of Arg972 IRS-1 in cardiovascular diseases.

The Oncogenic MicroRNA-21 Inhibits the Tumor Suppressive Activity of FBXO11 to Promote Tumorigenesis

The microRNA miR-21 is overexpressed in most human cancers and accumulating evidence indicates that it functions as an oncogene. Since miRNAs suppress the expression of their target genes, we hypothesized that some miR-21 targets may act as tumor suppressors, and thus their expression would be anticipated to be reduced by the high miR-21 levels observed in various human cancers. By microarray analysis and quantitative PCR we identified and validated FBXO11 (a member of the F-box subfamily lacking a distinct unifying domain) as a miR-21 target gene. FBXO11 is a component of the SKP1-CUL1-F-box ubiquitin ligase complex that targets proteins for ubiquitination and proteosomal degradation. By loss of function and gain of function studies, we show that FBXO11 acts as a tumor suppressor, promotes apoptosis and mediates the degradation of the oncogenic protein BCL6. The critical role that FBXO11 plays in miR-21-mediated tumorigenesis was demonstrated by a rescue experiment, in which silencing FBXO11 in miR-21KD cancer cells restored their high tumorigenicity. Expression of miR-21 and FBXO11 are inversely correlated in tumor tissue, and their expression correlates with patient survival and tumor grade. High FBXO11 expression correlates with better patient survival and lower tumor grade consistent with its tumor suppressor activity. In contrast high miR-21 expression, which correlates with poor patient survival and higher tumor grade, is consistent with its oncogenic activity. Our results identify FBXO11 as a novel miR-21 target gene, and demonstrate that the oncogenic miRNA miR-21 decreases the expression of FBXO11, which normally acts as a tumor suppressor, and thereby promotes tumorigenesis.

MiR-21 promotes intrahepatic cholangiocarcinoma proliferation and growth in vitro and in vivo by targeting PTPN14 and PTEN.

Intrahepatic cholangiocarcinoma (ICC) constitutes the second-most common primary hepatic malignancy. MicroRNAs (miRNAs) play important roles in the pathogenesis of ICC. However, the clinical significance of miR-21 levels in ICC remains unclear. Here, we investigated the role of miR-21 in ICC and found that its expression was significantly upregulated in serum of ICC patients. Serum miR-21 levels robustly distinguished ICC patients from control subjects. Further experiments showed that inhibition of miR-21 suppressed ICC cell proliferation in vitro and tumor growth in vivo. Specifically, inhibition of miR-21 induced cell cycle arrest and apoptosis. Moreover, PTPN14 and PTEN were identified as direct and functional targets of miR-21. Finally, we showed high expression levels of miR-21 were closely related to adverse clinical features, diminished survival, and poor prognosis in ICC patients. This study revealed functional and mechanistic links between miR-21 and tumor suppressor genes, PTPN14 and PTEN, in the pathogenesis of ICC. MiR-21 not only plays important roles in the regulation of cell proliferation and tumor growth in ICC, but is also a diagnostic and prognostic marker, and a potential therapeutic target for ICC.

Sulindac has strong antifibrotic effects by suppressing STAT3-related miR-21.

Pulmonary fibrosis (PF) is a disease with an unknown cause and a poor prognosis. In this study, we aimed to explore the pathogenesis of PF and the mechanism of sulindac in attenuating bleomycin (BLM)-induced PF. The rat PF model was induced by BLM and verified through histological studies and hydroxyproline assay. The severity of BLM-induced PF in rats and other effects, such as the extent of the wet lung to bw ratios, thickening of alveolar interval or collagen deposition, was obviously ameliorated in sulindac-treated rat lungs compared with BLM-induced lungs. Sulindac also reversed the epithelial mesenchymal transition (EMT) and inhibited the PF process by restoring the levels of E-cadherin and α-smooth muscle actin (SMA) in A549 cells. Our results further demonstrated that the above effects of sulindac might be related to regulating of interferon gamma (IFN-γ) expression, which further affects signal transducers and activators of transcription 3 (STAT3) and phosphorylated STAT3 (p-STAT3) levels. Moreover, higher miR-21 levels with the decreased E-cadherin and increased α-SMA expressions were found in transforming growth factor-β1-treated A549 cells, which can be reversed by sulindac. Collectively, our results demonstrate that by decreasing IFN-γ-induced STAT3/p-STAT3 expression to down-regulate miR-21, sulindac could significantly reverse EMT in A549 cells and prevent BLM-induced PF.

A role for microRNA-155 expression in microenvironment associated to HPV-induced carcinogenesis in K14-HPV16 transgenic mice.

Human Papillomavirus cause a number of diseases most notably cervical cancer. K14-HPV16 transgenic mice expressing the HPV16 early genes in squamous epithelial cells provide a suitable experimental model for studying these diseases. MicroRNAs are small non-coding RNAs that play an important role in regulating gene expression and have been suggested to play an important role in cancer development. The role of miR-155 in cancer remains controversial and there is limited evidence linking this miRNA to HPV- associated diseases. We hypothesized that miR-155 expression modulates each tissue’s susceptibility to develop HPV-associated carcinogenesis. In this study, we analyzed miR-155 expression in ear and chest skin samples from 22-26 weeks old, female K14-HPV16 transgenic (HPV16+/-) and wild-type (HPV-/-) mice. Among wild-type mice the expression of miR-155 was lower in ear skin compared with chest skin (p = 0.028). In transgenic animals, in situ carcinoma was present in all ear samples whereas chest tissues only showed epidermal hyperplasia. Furthermore, in hyperplastic chest skin samples, miR-155 expression was lower than in normal chest skin (p = 0,026). These results suggest that miR-155 expression may modulate the microenvironmental susceptibility to cancer development and that high miR155 levels may be protective against the carcinogenesis induced by HPV16.

Stromal expression of miR-21 in T3-4a colorectal cancer is an independent predictor of early tumor relapse.

BackgroundMicroRNA-21 (miR-21) is an oncogenic microRNA that regulates the expression of multiple cancer-related target genes. miR-21 has been associated with progression of some types of cancer. Metastasis-associated protein1 expression and loss of E-cadherin expression are correlated with cancer progression and metastasis in many cancer types. In advanced colorectal cancer, the clinical significance of miR-21 expression remains unclear. We aimed to investigate the impact of miR-21 expression in advanced colorectal cancer and its correlation with target proteins associated with colorectal cancer progression.MethodsFrom 2004 to 2007, 277 consecutive patients with T3-4a colorectal cancer treated with R0 surgical resection were included. Patients with neoadjuvant therapy and distant metastasis at presentation were excluded. The expression of miR-21 was investigated by in situ hybridization. Immunohistochemistry was used to detect E-cadherin and metastasis-associated protein1 expression.ResultsHigh stromal expression of miR-21 was found in 76 of 277 (27.4%) colorectal cancer samples and was correlated with low E-cadherin expression (P = 0.019) and high metastasis-associated protein1 expression (P = 0.004). T3-4a colorectal cancer patients with high miR-21 expression had significantly shorter recurrence-free survival than those with low miR-21 expression. When analyzing colon and rectal cancer separately, high expression of miR-21 was an independent prognostic factor of unfavorable recurrence-free survival in T3-4a colon cancer patients (P = 0.038, HR = 2.45; 95% CI = 1.05-5.72) but not in T3-4a rectal cancer patients. In a sub-classification analysis, high miR-21 expression was associated with shorter recurrence-free survival in the stage II cancer (P = 0.001) but not in the stage III subgroup (P = 0.267).ConclusionsStromal miR-21 expression is related to the expression of E-cadherin and metastasis-associated protein1 in colorectal cancer. Stage II colorectal cancer patients with high levels of miR-21 are at higher risk for tumor recurrence and should be considered for more intensive treatment.

Suppression of transforming growth factor β receptor 2 and Smad5 is associated with high levels of microRNA miR-155 in the oral mucosa during chronic simian immunodeficiency virus infection.

Chronic human immunodeficiency virus and simian immunodeficiency virus (HIV and SIV) infections are characterized by mucosal inflammation in the presence of anti-inflammatory cytokines such as transforming growth factor β (TGFβ). The mechanisms for refractiveness to TGFβ are not clear. Here we show that the expression of microRNA miR-155 was significantly upregulated in the oropharyngeal mucosa during chronic SIV infection and was coincident with downregulation of TGFβ receptor 2 (TGFβ-R2) and SMAD5, key TGFβ signaling genes that harbor putative target sites for miR-155. Ectopic expression of miR-155 in vitro was found to significantly downregulate TGFβ-R2 and Smad5 expression, suggesting a role for miR-155 in the suppression of TGFβ-R2 and SMAD5 genes in vivo. The downregulation of TGFβ signaling genes by miR-155 likely contributes to the nonresponsiveness to TGFβ during SIV infection and may inadvertently aid in increased immune activation during HIV and SIV infections.

Altered levels of miR-21, miR-125b-2*, miR-138, miR-155, miR-184, and miR-205 in oral squamous cell carcinoma and association with clinicopathological characteristics.

Previous studies have described the aberrantly expressed microRNAs (miRNAs) in oral squamous cell carcinoma (OSCC), and we reasoned that studying frequently deregulated candidate miRNAs in OSCC of Indian ethnicity could aid in better understanding of the genetic/environmental impact on the expression statuses of these miRNAs. Therefore, we evaluated the differential expression of six selected miRNAs namely hsa-miR-21, hsa-miR-125b2*, hsa-miR-138, hsa-miR-155, hsa-miR-184, and hsa-miR-205 in OSCC specimens of Indian ethnicity. Two-step Reverse transcriptase quantitative PCR using inventoried TaqMan single miRNA assays was employed to study the expression of the selected miRNAs in 42 OSCC tumors and eight adjacent normal specimens. The expression levels of the miRNAs were tested for any association with clinicopathological parameters. miR-21 was significantly elevated while miR-125b-2* was significantly downregulated in tumors compared to controls (P < 0.01 and P < 0.05 respectively). miR-138 and miR-184 were observed to be predominantly downregulated in the tumor samples. High levels of miR-155 were associated with the habit of chewing tobacco/betel quid. Our results corroborate the previous findings on the overexpression of mir-21 and downregulation of miR-138 in OSCC. As the expression of miR-184 is controversial in tongue/oral cancer, the downregulation may be specific to tumor anatomical localization. On the other hand, to the best of our knowledge, this is the first report to show the association of miR-155 with tobacco chewing and the downregulation of miR-125b-2* in OSCC. Computational predictions suggest that miR-125b-2* may have a role in alternative splicing.

Aberrant Overexpression and Regulatory Mechanism of Mir-21 in Diffuse Large B Cell Lymphoma

MicroRNAs (miRNAs) are critically responsible for lymphomagenesis. We found that miR-21 expression was significantly higher in diffuse large B cell lymphoma (DLBCL) line and paraffin-embedded tissue of primary lymphoma (n = 26) than that in normal lymph nodes (n = 10). To determine the functions of miR-21 in lymphomagenesis, we examined phosphatase and tensin homolog (PTEN) expression using immunohistochemical SP method in specimens of primary DLBCL. MiR-21 expression is negatively correlated with PTEN expression, but positively correlated with LDH. MiR-21 expression was significantly higher in stage III / IV patients than in stage I/II patients. Moreover, we examined the effects of antisense oligonucleotide (ASO) targeting miR-21 (ASO-21) in the DLCBL line overexpressing this miRNA. Electroporation was used to transfect ASO-21 into the cells, which showed slight endogenous miR-21 expression. Downregulating miR-21 expression resulted in increased incidence of apoptosis and PTEN upregulation. These results provide relevant new insight into the pathogenesis of DLBCL and suggest that targeting miR-21 may represent a useful approach for DLBCL treatment, and that high levels of miR-21 may be associated with increased poor prognosis for DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

Correlation of Mir-155 Expression with Clinical Outcome in Childhood AML: A Report from Children’s Oncology Group

Acute myeloid leukemia (AML) is characterized by genomic abnormalities that impair differentiation and promote proliferation. Karyotypic alterations and somatic mutations are associated with relapse risk but are rarely predictive of response to induction chemotherapy. Aberrant miRNA expression has been implicated in AML pathogenesis. We present association of altered miR-155 expression with specific molecular characteristics and show its association with high risk of induction failure and worse clinical outcome in children with normal karyotype (NK) AML treated on COG AML trial, AAML0531.Of 1022 patients enrolled, 233 were NK. 198 diagnostic specimens were available for testing. miR-155 expression was evaluated by quantitative TaqMan miRNA assays normalized against normal marrow. Expression varied by 4 log fold. Patients were divided into 4 quartiles (Q1-Q4) based on expression levels. Q1 had patients with the lowest and Q4, with the highest expression. We correlated disease characteristics and clinical outcome across quartiles. All comparisons and data presented is between high (Q4) vs. low (Q1-Q3) expressors.Males (66% vs. 48%, p=0.027), Asians (15% vs. 5%, p=0.048) and high median diagnostic WBC (59.4 vs. 22.6, p=0.002) were over-represented in high expressors.Molecular abnormalities were used to stratify risk. Low-risk patients were those with mutations in CEBPA/NPM and high-risk, in FLT3-ITD. Others were designated as standard-risk. High miR-155 expression was associated with a high prevalence of FLT3-ITD mutations (69% vs. 31%, p < 0.001) and high-risk disease (52% vs. 15%, p<0.001), and had an inverse association with low-risk (22% vs. 38%, p=0.041) and standard-risk disease (26% vs. 47%, p=0.008). There was no association with mutations in CEBPA (p=0.710) or NPM (p=0.826).We evaluated expression levels and response to induction chemotherapy, morphologic complete remission (CR). CR rates for high vs. low expressors was 46% vs. 83% (p<0.001). As high expressors were more likely FLT3-ITD+, we computed CR for those without FLT3-ITD mutations. High vs. low expressors who were FLT3-ITD-, had CR rates of 47% vs. 86% (p=0.002), suggesting that miR-155 expression may independently provide data on response to chemotherapyWe evaluated overall survival (OS) and event-free survival (EFS) at 3 years. OS for high vs. low expressors was 51% vs. 75% (p=0.002) and EFS was 32% vs. 59% (p<0.001). Association of high miR-155 and worse survival was also verified against RNASeq data, in an independent cohort of children with NK-AML (p=0.005). We performed Cox regression analyses to evaluate the impact of miR-155 expression level as a predictor of clinical outcome in the context of prognostic factors (FLT3-ITD, NPM, CEBPA mutations, treatment arm, risk group, age, WBC) and molecular risk groups using them as covariates in both univariate and multivariate models. In the univariate model, high expression was a prognostic factor for inferior OS (HR of 2.19, p= 0.002) and EFS (HR 2.24, p<0.001). Patients with low-risk molecular features also had improved OS (HR 0.29, p<0.001) and EFS (HR 0.22, p<0.001). In a multivariate model that included the aforementioned prognostic features, high miR-155 expression retained prognostic significance for OS (HR 1.92, p=0.022) and EFS (HR 1.75, p= 0.019). High risk molecular features were no longer an independent predictor of outcome. Low-risk molecular features remained an independent prognostic factor for improved OS (HR 0.34, p=0.005) and EFS (HR 0.22, p < 0.0001).We extended our study to identify target genes whose expression was impacted or regulated by miR-155 expression. Comparison of the mRNA expression profile for the 23 highest vs. 23 lowest expressors identified anti-correlation of miR-155 and 15 target genes. High miR-155 levels were associated with down-regulation of 9 genes, including E2F2 and ETS1. Low miR-155 levels were associated with up-regulation of 6 genes, including DET1, GATM and SMAD1.We demonstrate association of miR-155 with specific disease characteristics and clinically significant mutations in pediatric AML. We also show that high expression is highly predictive of induction failure and outcome providing potential biomarkers for identifying patients at high risk of poor response prior to therapy. miRNA expression could provide clinically important information for use in therapeutic allocation. DisclosuresNo relevant conflicts of interest to declare.

Serum inflammatory miRNAs predict radiation esophagitis in patients receiving definitive radiochemotherapy for non-small cell lung cancer

Background and purposeMicroRNAs (miRNAs) are small, highly conserved non-coding RNAs that regulate many biological processes. We sought to investigate whether three serum miRNAs related to immunity or inflammation were associated with esophagitis induced by chemoradiation therapy (CRT) for non-small cell lung cancer (NSCLC). Material and methodsWe measured serum miR-155, miR-221 and miR-21, before and during week 1–2 of CRT for 101 NSCLC patients by real-time PCR. Associations between miRNA and severe radiation-induced esophageal toxicity (RIET) were analyzed by logistic regression. ResultsWe found that patients with stage IIIB–IV disease, higher mean esophagus dose or esophageal V50 had higher rates of severe RIET. Furthermore, high levels of miR-155 and miR-221 at week 1–2 of CRT were also risk factors for severe RIET (miR-155: OR=1.53, 95% CI: 1.04–2.25, P=0.03; miR-221: OR=2.07, 95% CI: 1.17–3.64, P=0.012). In addition, the fold change of miR-221 was also predictive of severe RIET (OR=1.18, 95% CI: 1.02–1.37, P=0.026). However, pretreatment miRNAs was not predictive of severe RIET. ConclusionsHigh serum miR-155 and miR-221 during the first 2weeks of CRT were associated with the development of severe RIET, suggesting that these miRNAs may be useful as an early surrogate for this form of toxicity.

SF Treg cells transcribing high levels of Bcl-2 and microRNA-21 demonstrate limited apoptosis in RA.

The aim of this study was to investigate the turnover of Treg cells in the SF of RA patients. Treg cells were enumerated in peripheral blood and SF of RA patients and analysed by flow cytometry for expression of the proliferation marker Ki-67 and binding of the apoptosis marker annexin V. Sorted Treg cells of RA patients were analysed for expression of anti-apoptotic regulators Bcl-2 and microRNA-21 (miR-21) by RT-PCR. Treg cells displaying a memory phenotype were abundant in the SF of RA patients. SF Treg cells more frequently expressed the proliferation marker Ki-67 than conventional T cells. Only few SF Treg cells were apoptotic, as indicated by limited annexin V staining of these cells. SF Treg cells displayed high transcription levels of Bcl-2 and miR-21 in comparison with SF conventional T cells and peripheral blood Treg cells. Treg cells with a memory phenotype accumulate in the SF of RA patients. These Treg cells have a high proliferative activity and demonstrate little apoptosis. The latter finding could be explained by high transcription of Bcl-2 and miR-21 in SF Treg cells.

The PDCD4/miR-21 pathway in medullary thyroid carcinoma.

Programmed cell death 4 (PDCD4) is a tumor suppressor gene involved in tumorogenesis. MicroRNA-21 (miR-21) specifically targets PDCD4, and recent studies suggest that PDCD4 is also regulated by Akt (antiapoptotic regulator within phosphatidylinositol 3-kinase). Medullary thyroid carcinoma (MTC) is a rare neuroendocrine cancer, and disease stage at diagnosis represents the main prognostic indicator. A consecutive series of 64 MTCs was considered. REarranged during Transfection (RET) and rat sarcoma (RAS) mutation status was assessed by direct sequencing. Quantitative real-time polymerase chain reaction was used to quantify mature hsa-miR-21. PDCD4 and Ki-67 immunostaining was performed with an automated platform. Immunoblot analysis of PI3K/Akt pathway was done on thyroid tissues. MTCs were consistently associated with miR-21 up-regulation (P < .0016) and featured significant PDCD4 nuclear down-regulation. An inverse correlation emerged between miR-21 overexpression and PDCD4 down-regulation (P = .0013). At enrollment, high miR-21 levels were associated with high calcitonin levels (P = .0003), lymph node metastases (P = .001), and advanced stages (P = .0003). At the end of follow-up, high miR-21 levels were associated with biochemically persistent disease (P = .0076). At enrollment, instead, PDCD4 nuclear down-regulation was associated with high calcitonin levels (P = .04), more advanced stages of disease (P < .01), and persistent disease after the follow-up (P = .02). p-Akt was more expressed in RAS-mutated MTC than in nonmutated cancers and normal tissue. This study showed, in MTCs, that miR-21 regulates PDCD4 expression and also that the miR-21/PDCD4 pathway correlates with clinicopathological variables and prognosis. Further studies should investigate the role of miR-21 as a prognostic biomarker and the feasibility of using PDCD4-restoring strategies as a therapeutic approach to MTC.

Abstract 535: Expression of miR-21 in cancer associated fibroblasts in lung adenocarcinoma

Abstract Lung cancer is the leading cause of cancer mortality worldwide, and novel therapeutic targets and molecular diagnostic markers are highly warranted. Micro RNAs (miRNAs) are a class of small non-coding RNAs associated with a variety of cancer. miRNA-21 is an “oncomiR” and upregulated in many cancers, including lung cancer, and high miR-21 levels have been associated with poor prognosis. To determine the localization of miR-21 and to compare its expression levels, we performed in situ hybridization of miR-21 on 89 mixed subtypes of lung adenocarcinoma. miR-21 was predominantly expressed in cancer associated fibroblasts (CAF)s expressing α-SMA, with no difference in expression between non-invasive and invasive component. In addition, stromal miR-21 expression was significantly associated with decreased overall survival (p=0.0021). In functional analyses of miR-21 in vitro, miR-21 was overexpressed in lung fibroblasts to assess migration and invasion. miR-21 expression in lung fibroblasts promoted migration and invasion. Moreover, the proliferation of lung cancer cells was accelerated by conditioned medium of miR-21-expressing lung fibroblasts. These results suggest that miR-21 overexpression in CAFs of lung adenocarcinoma might serve as a prognostic biomarker and be involved in cancer cell proliferation through interactions with cancer cells. Citation Format: Akiko Kunita, Shigeki Morita, Masashi Fukayama. Expression of miR-21 in cancer associated fibroblasts in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 535. doi:10.1158/1538-7445.AM2014-535

Abstract 975: MicroRNA-155 In chronic lymphocytic leukemia influences B-cell receptor signaling

Abstract MicroRNAs (miRNAs) can influence the expression levels of genes that can affect the clinical outcome of patients with chronic lymphocytic leukemia (CLL). In a well-defined cohort of 86 patients, we observed heterogeneity in the relative leukemia-cell expression of miR-155. Cases that expressed high-levels of miR-155 more frequently expressed zeta-associated protein of 70 kD (ZAP-70), used unmutated Ig heavy-chain-variable-region genes (IGHV), and had shorter treatment-free survival (TFS) and overall survival (OS) than cases with low-level expression of miR-155. Recursive partitioning allowed us to define a threshold for “high-level” expression of miR-155 (in copy numbers per CLL cell) that best associates with adverse clinical outcome. We examined the potential basis for this association. One of the genes targeted by miR-155 encodes the phosphatidylinositol polyphosphate 5-phosphatase SHIP-1, which can dephosphorylate proteins that had been activated in response to B-cell receptor (BCR) ligation. We found that CLL cells with “high-level” miR-155 expressed significantly lower levels of SHIP-1 protein and were more sensitive to ligation of surface-µ than CLL cells with low-levels of miR-155. Transfecting CLL cells with miR-155 reduced their expression of SHIP-1 and enhanced the stimulatory-response to surface-µ ligation. Conversely, transfection of CLL cells with a miR-155 inhibitor had the opposite effects. The enhanced sensitivity to BCR-ligation of cells expressing “high-level” miR-155 may account for its association with adverse clinical outcome in patients with CLL. However, within any one patient we also find heterogeneity in the expression levels of miR-155. By GEO data analysis (GSE21029) we found that CLL cells in blood expressed lower levels of miR-155 than did CLL cells in lymphoid tissues, where they interact with supportive cells, such as Nurselike cells or activated T cells, which can provide survival- or growth-promoting signals via expression of BAFF/APRIL or CD40L, respectively. We found that stimulation of CLL cells in vitro via CD40 ligation or exogenous BAFF could induce higher expression of miR-155, leading to reduced leukemia-cell expression of SHIP-1 and enhanced sensitivity to surface-µ ligation. Moreover, we found that the “proliferative” subgroup of blood CD5brightCXCR4dim CLL cells, which represent CLL cells newly released from the tissue microenvironment, expressed higher level of miR-155 and lower levels of SHIP-1 protein, and were more sensitive to surface-µ ligation than the “resting” subgroup of blood CD5dimCXCR4bright CLL cells, which represent cells that may be due to enter the tissue microenvironment. As such, this study also demonstrates that miR-155 could be upregulated by signals in the CLL microenvironment, leading to lower-level expression of SHIP-1, and enhanced stimulation in response to BCR ligation. Citation Format: Liguang Chen, Bing Cui, Suping Zhang, Marek Mraz, Jessie-F. Fecteau, Jian Yu, Ling Zhang, Lei Bao, Laura Rassenti, Karen Messer, Carlo Croce, Thomas Kipps. MicroRNA-155 In chronic lymphocytic leukemia influences B-cell receptor signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 975. doi:10.1158/1538-7445.AM2014-975

MicroRNA-21-induced dissociation of PDCD4 from rictor contributes to Akt-IKKβ-mTORC1 axis to regulate renal cancer cell invasion

Renal cancer metastasis may result from oncogenic forces that contribute to the primary tumor. We have recently identified microRNA-21 as an oncogenic driver of renal cancer cells. The mechanism by which miR-21 controls renal cancer cell invasion is poorly understood. We show that miR-21 directly downregulates the proapoptotic protein PDCD4 to increase migration and invasion of ACHN and 786-O renal cancer cells as a result of phosphorylation/activation of Akt and IKKβ, which activate NFκB-dependent transcription. Constitutively active (CA) Akt or CA IKKβ blocks PDCD4-mediated inhibition and restores renal cancer cell migration and invasion. PDCD4 inhibits mTORC1 activity, which was reversed by CA IKKβ. Moreover, CA mTORC1 restores cell migration and invasion inhibited by PDCD4 and dominant negative IKKβ. Moreover, PDCD4 negatively regulates mTORC2-dependent Akt phosphorylation upstream of this cascade. We show that PDCD4 forms a complex with rictor, an exclusive component of mTORC2, and that this complex formation is reduced in renal cancer cells due to increased miR-21 expression resulting in enhanced phosphorylation of Akt. Thus our results identify a previously unrecognized signaling node where high miR-21 levels reduce rictor–PDCD4 interaction to increase phosphorylation of Akt and contribute to metastatic fitness of renal cancer cells.

MicroRNA-21 promotes tumour malignancy via increased nuclear translocation of β-catenin and predicts poor outcome in APC-mutated but not in APC-wild-type colorectal cancer.

MiR-21 has been associated with poor prognosis in colon adenocarcinomas. However, in our preliminary data, the prognostic value of miR-21 levels was observed only in adenomatous polyposis coli (APC)-mutated tumours, not in APC-wild-type tumours. We explored whether β-catenin nuclear translocation was synergistically promoted by miR-21 in APC-mutated cells but not in APC-wild-type cells. We enrolled 165 colorectal tumour to determine APC mutation, miR-21 levels and nuclear β-catenin expression by direct sequencing, real-time PCR and immunohistochemistry. Overall survival and relapse-free survival were analysed by Kaplan-Meier and Cox regression models. The mechanistic action of β-catenin nuclear translocation modulated by miR-21 and its effect on cell invasion were evaluated in a cell model. Positive nuclear β-catenin expression was more commonly occurred in APC-mutated tumours than in APC-wild-type tumours. High miR-21 levels were relatively more common in tumours with positive nuclear β-catenin expression than in those with negative nuclear β-catenin expression. APC-mutated tumours with high miR-21 levels had shorter overall survival and relapse-free survival periods compared with others. However, the prognostic value of miR-21 levels was not observed in APC-wild-type tumours. Phosphorylation of β-catenin at Ser552 via the miR-21-mediated PTEN/AKT axis plays a critical role in β-catenin nuclear translocation in APC-mutated cells but not in APC-wild-type cells. Moreover, nuclear β-catenin expression increased by miR-21 is responsible for the capability of invasiveness. In summary, nuclear translocation of β-catenin increased by miR-21 promotes tumour malignancy and a poor outcome in APC-mutated patients but not in APC-wild-type colorectal cancer.

MicroRNA-155 and MicroRNA-21 promote the expansion of functional myeloid-derived suppressor cells (IRC5P.473)

Abstract Myeloid-derived suppressor cells (MDSC) are one of the main cell populations that negatively regulate immune responses. However, the mechanism underlying the expansion of MDSC remains unclear. Using miRNA microarray and TaqMan probe-based quantitative RT-PCR assay, we identified microRNA (miR)-155 and miR-21 as the two most upregulated miRNAs during the induction of MDSC from the bone marrow cells by GM-CSF and IL-6. High levels of miR-155 and miR-21 also were detected in bone marrow and spleen MDSC isolated from tumor-bearing mice. Our results also showed that TGF-b promoted the induction of MDSC through upregulating miR-155 and miR-21 expression. Overexpression of miR-155 and miR-21 enhanced whereas depletion of miR-155 and miR-21 reduced the frequencies of cytokine-induced MDSC. Subpopulation analysis indicated that miR-21 and miR-155 induced the expansion of both monocytic and granulocytic MDSC. Furthermore, miR-155 and miR-21 showed a synergistic effect on MDSC induction via targeting SHIP-1 and phosphatase and tensin homolog, respectively, leading to STAT3 activation. Finally, dexamethasone treatment strongly enhanced MDSC expansion through upregulating miR-155 and miR-21 expression, and the effect of dexamethasone on MDSC induction was abolished by depleting cellular miR-155 and miR-21. These results demonstrate a novel miR-155/miR-21-based regulatory mechanism that modulates functional MDSC induction.