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Supplementary Figures Legends. Supplementary Figures S1-S3 Legends

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Supplementary Data includes Supplementary Figures 1-6 and Supplementary Tables 1-3. Supplementary Figure 1 shows microscopic images for the characterization of exosomes. Supplementary Figure 2 shows the qPCR results of miR-6126 overexpression in miR-6126 mimic treated ovarian cancer cells (HeyA8, HeyA8-MDR, Skkov3-ip1, SKOV3-TR) Supplementary Figure 3 shows western blotting and qPCR results that shows the ingibition of Integrin beta-1 expression at protein and mRNA level upon miR-6126 mimic treatment. Supplementary Figure 4 shows the size distribution graphs of exosomes isolated from CTL mimic, miR-6126 mimic , CTL inhibitor, miR-6126 inhibitor treated SKOV3-IP1 cells. Supplementary Figure 5 shows ITGB1 mRNA levels in different ovarian cancer exosomes. Supplementary Figure 6 shows in vivo down regulation of ITGB1. Supplementary Table 1 shows demographic information of ovarian cancer patients whose tissue samples used in quantification of miR-6126 expression. Supplementary Table 2 shows raw microarray data for miR-6126 Supplementary Table 3 shows z-score values for miR-6126 for each exosome and cell sample.

In vivo nanoliposomal miR-34a treatment is safe and effectively suppresses eEF2K and FOXM1 protein expression in TNBC tumors.

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Supplementary Figure S2. Hierarchical clustering of microRNA expression

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Purpose: Recent studies indicated that dysregulation of noncoding RNAs (ncRNA) such as miRNAs is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC).Experimental Design: We performed in vitro functional assays on TNBC cell lines to investigate the role of miR-34a in FOXM1/eEF2K signaling axis. TNBC tumor xenograft models were used for in vivo therapeutic delivery of miR-34a.Results: In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival. We showed that miR-34a directly binds to the 3′-untranslated region of eEF2K and FOXM1 mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of eEF2K and FOXM1, the transcription factor for eEF2K and the direct target of p53, in TNBC cell lines, whereas overexpression of eEF2K and FOXM1 rescued the effects and signaling pathways mediated by miR-34a. Moreover, in vivo therapeutic delivery of miR-34a nanoparticles by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis.Conclusions: Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a–based gene therapy may be a potential therapeutic strategy in TNBC. Clin Cancer Res; 24(17); 4225–41. ©2018 AACR.

Related Article from microRNA-10b: A New Marker or the Marker of Pancreatic Ductal Adenocarcinoma?

Dose-response curves for MEC1 and MEC2 cells treated with fludarabine.

Figure Legends for Supplementary Figures

Purpose: Triple-negative breast cancers (TNBC) are associated with high risk of early tumor recurrence and poor outcome. Common prognostic biomarkers give very restricted predictive information of tumor recurrences in TNBC. Human serum contains stably expressed microRNAs (miRNAs), which have been discovered to predict prognosis in patients with cancer. The purpose of this study was to identify circulating biomarkers able to predict clinical outcome in TNBC.Experimental Design: We performed genome-wide serum miRNA expression and real-time PCR analyses to investigate the ability of miRNAs in predicting tumor relapse in serum samples from 60 primary TNBC. Patients were divided into training and testing cohorts.Results: By Cox regression analysis, we identified a four-miRNA signature (miR-18b, miR-103, miR-107, and miR-652) that predicted tumor relapse and overall survival. This miRNA signature was further validated in an independent cohort of 70 TNBC. A high-risk signature score was developed and significantly associated with tumor recurrence and reduced survival. Multivariate Cox regression models indicated that the risk score based on the four-miRNA signature was an independent prognostic classifier of patients with TNBC.Conclusions: This signature may serve as a minimally invasive predictor of tumor relapse and overall survival for patients with TNBC. This prediction model may ultimately lead to better treatment options for patients with TNBC. Clin Cancer Res; 21(5); 1207–14. ©2014 AACR.

Supplementary Table S1 Clinical characteristics of two chronic lymphocytic leukemia patient datasets; Supplementary Table S2 Clinical characteristics of two lung cancer patient datasets; Supplementary Table S3 Clinical characteristics of the ALL dataset; Supplementary Table S4 Integrated function and pathway analysis on 248 experimentally validated targets of miR-155; Supplementary Table S5 Univariate and multivariate analyses of survival with patient characteristics and miR-155 and TP53 expression as categorical and continuous variables in different patient cohorts; Supplementary Table S6 Estimate of Cox model and multivariate Cox model, as well as the HR estimated based on the model for miR-155 high and TP53 low vs. miR-155 low and TP53 high.

Supplementary Data from MicroRNA-221 Targets Bmf in Hepatocellular Carcinoma and Correlates with Tumor Multifocality

Purpose: The oncogenic miR-155 is upregulated in many human cancers, and its expression is increased in more aggressive and therapy-resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors.Experimental Design: We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.Results: We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism and that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer.Conclusions: Our findings support the existence of an miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death. Clin Cancer Res; 23(11); 2891–904. ©2016 AACR.

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Purpose: Characterization of colorectal cancer transcriptome by high-throughput techniques has enabled the discovery of several differentially expressed genes involving previously unreported miRNA abnormalities. Here, we followed a systematic approach on a global scale to identify miRNAs as clinical outcome predictors and further validated them in the clinical and experimental setting.Experimental Design: Genome-wide miRNA sequencing data of 228 colorectal cancer patients from The Cancer Genome Atlas dataset were analyzed as a screening cohort to identify miRNAs significantly associated with survival according to stringent prespecified criteria. A panel of six miRNAs was further validated for their prognostic utility in a large independent validation cohort (n = 332). In situ hybridization and functional experiments in a panel of colorectal cancer cell lines and xenografts further clarified the role of clinical relevant miRNAs.Results: Six miRNAs (miR-92b-3p, miR-188-3p, miR-221-5p, miR-331-3p, miR-425-3p, and miR-497-5p) were identified as strong predictors of survival in the screening cohort. High miR-188-3p expression proves to be an independent prognostic factor [screening cohort: HR = 4.137; 95% confidence interval (CI), 1.568–10.917; P = 0.004; validation cohort: HR = 1.538; 95% CI, 1.107–2.137; P = 0.010, respectively]. Forced miR-188-3p expression increased migratory behavior of colorectal cancer cells in vitro and metastases formation in vivo (P < 0.05). The promigratory role of miR-188-3p is mediated by direct interaction with MLLT4, a novel identified player involved in colorectal cancer cell migration.Conclusions: miR-188-3p is a novel independent prognostic factor in colorectal cancer patients, which can be partly explained by its effect on MLLT4 expression and migration of cancer cells. Clin Cancer Res; 23(5); 1323–33. ©2016 AACR.

Supplementary Figure Legend

Supplementary Table S2. The 71 microRNAs expressed in at least 75% of the samples in the training cohort

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Purpose: MicroRNAs are short noncoding RNAs that regulate gene expression and are over- or underexpressed in most tumors, including colorectal adenocarcinoma. MicroRNAs are potential biomarkers and therapeutic targets and agents, but limited information on microRNAome alterations during progression in the well-known adenoma-adenocarcinoma sequence is available to guide their usage.Experimental Design: We profiled 866 human microRNAs by microarray analysis in 69 matched specimens of microsatellite-stable adenocarcinomas, adjoining precursor adenomas including areas of high- and low-grade dysplasia, and nonneoplastic mucosa.Results: We found 230 microRNAs that were significantly differentially expressed during progression, including 19 not reported previously. Altered microRNAs clustered into two major patterns of early (type I) and late (type II) differential expression. The largest number (n = 108) was altered at the earliest step from mucosa to low-grade dysplasia (subtype IA) prior to major nuclear localization of β-catenin, including 36 microRNAs that had persistent differential expression throughout the entire sequence to adenocarcinoma. Twenty microRNAs were intermittently altered (subtype IB), and six were transiently altered (subtype IC). In contrast, 33 microRNAs were altered late in high-grade dysplasia and adenocarcinoma (subtype IIA), and 63 in adenocarcinoma only (subtype IIB). Predicted targets in 12 molecular pathways were identified for highly altered microRNAs, including the Wnt signaling pathway leading to low-grade dysplasia. β-catenin expression correlated with downregulated microRNAs.Conclusions: Our findings suggest that numerous microRNAs play roles in the sequence of molecular events, especially early events, resulting in colorectal adenocarcinoma. The temporal patterns and complexity of microRNAome alterations during progression will influence the efficacy of microRNAs for clinical purposes. Clin Cancer Res; 17(23); 7283–93. ©2011 AACR.

Supplemental Figure 1. Cell surface expression of IL-11R in AML patient derived samples and hematopoietic cells from healthy bone marrow as analyzed by flow cytometry.

Supplemental Table 1. Features of synthesized analogs of BMTP-11 for drug-lead optimization.

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Deregulated cell proliferation and apoptosis play a major role in hepatocellular carcinoma (HCC). MicroRNAs participate in the modulation of key molecules linked to hepatocarcinogenesis.Purpose: This study aims to investigate the role of miR-221 in the modulation of Bmf, a proapoptotic BH3-only protein, and to characterize miR-221 contribution to hepatocarcinogenesis through modulation of apoptosis.Experimental Design: Transfection of miR-221 and anti-miR-221 in HCC-derived cell lines and luciferase reporter assay were used to assess Bmf as a target of miR-221. Modulation of miR-221 and Bmf expression contributed to characterize their role in anoikis. Primary HCC tissues were analyzed to assess the clinical relevance of in vitro findings.Results: Enforced miR-221 expression caused Bmf down-regulation, whereas anti-miR-221 induced its up-regulation. A luciferase reporter assay confirmed Bmf as a target of miR-221. Following matrix detachment, miR-221 silencing led to increased apoptotic cell death. The analysis of HCC tissues revealed an inverse correlation between miR-221 and Bmf expression and a direct correlation between Bmf and activated caspase-3, as a marker of apoptosis. High miR-221 levels were associated with tumor multifocality and reduced time to recurrence after surgery.Conclusions: Our results indicate that miR-221, by targeting Bmf, inhibits apoptosis. Moreover, in HCC, miR-221 overexpression is associated with a more aggressive phenotype. These findings, together with the previously reported modulation of CDKN1B/p27 and CDKN1C/p57, show that miR-221 simultaneously affects multiple pro-oncogenic pathways and suggest miR-221 as a potential target for nonconventional treatment against HCC. (Clin Cancer Res 2009;15(16):5073–81)

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XLSX file - 33K, Features of CLL samples.

Clinical correlation of miR-155 expression with survival in leukemia. Kaplan-Meier survival analysis for patients expressing high levels of miR-155 vs. low levels of miR-155 in two CLL cohorts, CLL - NEJM (A) and CLL - Italy (B), and in one ALL cohort, ALL - MDACC (C).

Purpose: The IL11 receptor (IL11R) is an established molecular target in primary tumors of bone, such as osteosarcoma, and in secondary bone metastases from solid tumors, such as prostate cancer. However, its potential role in management of hematopoietic malignancies has not yet been determined. Here, we evaluated the IL11R as a candidate therapeutic target in human leukemia and lymphoma.Experimental Design and Results: First, we show that the IL11R protein is expressed in a variety of human leukemia– and lymphoma–derived cell lines and in a large panel of bone marrow samples from leukemia and lymphoma patients, whereas expression is absent from nonmalignant control bone marrow. Moreover, a targeted peptidomimetic prototype (termed BMTP-11), specifically bound to leukemia and lymphoma cell membranes, induced ligand–receptor internalization mediated by the IL11R, and resulted in a specific dose-dependent cell death induction in these cells. Finally, a pilot drug lead-optimization program yielded a new myristoylated BMTP-11 analogue with an apparent improved antileukemia cell profile.Conclusions: These results indicate (i) that the IL11R is a suitable cell surface target for ligand-directed applications in human leukemia and lymphoma and (ii) that BMTP-11 and its derivatives have translational potential against this group of malignant diseases. Clin Cancer Res; 21(13); 3041–51. ©2015 AACR.

Supplementary Material

Purpose: Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial.Experimental Design: We analyzed the expression of 723 mature miRNAs in 217 early-stage CLL cases and in various different normal B-cell subpopulations from tonsils and peripheral blood.Results: Our analyses indicated that CLL cells exhibited a miRNA expression pattern that was most similar to the subsets of antigen-experienced and marginal zone–like B cells. These normal subpopulations were used as reference to identify differentially expressed miRNAs in comparison with CLL. Differences related to the expression of 25 miRNAs were found to be independent from IGHV mutation status or cytogenetic aberrations. These differences, confirmed in an independent validation set, led to a novel comprehensive description of miRNAs potentially involved in CLL. We also identified miRNAs whose expression was distinctive of cases with mutated versus unmutated IGHV genes or cases with 13q, 11q, and 17p deletions and trisomy 12. Finally, analysis of clinical data in relation to miRNA expression revealed that miR26a, miR532-3p, miR146-5p, and miR29c* were strongly associated with progression-free survival.Conclusion: This study provides novel information on miRNAs expressed by CLL and normal B-cell subtypes, with implication on the cell of origin of CLL. In addition, our findings indicate a number of deregulated miRNAs in CLL, which may play a pathogenic role and promote disease progression. Collectively, this information can be used for developing miRNA-based therapeutic strategies in CLL. Clin Cancer Res; 20(15); 4141–53. ©2014 AACR.

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Supplementary Methods includes experimental details on western blotting, invasion, wound healing, tube formation, EdU cell proliferation assays, quantitativce RT-PCR, luciferase reporter assay, gene expression analysis and Ingenuity Pathway Analysis.

Contains the Methods and Material in all Details

In vivo orthotopic lung cancer model for the role of miR-155 in chemoresistance. (A) Injection and treatment schedule for CDDP (green arrows) and anti-miR negative control (NC) or anti-miR-155 liposomal nanoparticles (red stars) for four different treatment groups: mice that were injected with A549-LVEV cells and untreated (group 1), injected with A549-LVEV cells and treated with anti-miR-NC and CDDP (group 2), injected with A549-155LV cells and treated with anti-miR-NC and CDDP (group 3), and injected with A549-155LV cells and treated with anti-miR-155 and CDDP (group 4). (B) Representative pictures of dissected mice belonging to each of the treatment groups described in panel A of this Figure. Tumor nodules are marked by dotted white circles. (C-D) Graphs of the primary tumor size (C) and aggregate mass of nodules in mediastinum (D) of the four treatment groups mentioned above. (E) In situ hybridization for miR-155 for each of the four treatment groups mentioned above. (F) Immunohistochemical analysis for Ki-67 (proliferation) and CD31 (angiogenesis), as well as the TUNEL assay (apoptosis) and TP53 immunostaining for each of the four treatment groups mentioned above.

Supplemental Figure 3. Drug activity of BMTP-11 and BMTP#4 on a panel of established leukemia and lymphoma cell lines.

Clinical correlation of miR-155 and TP53 expression with survival in the lung adenocarcinoma - TCGA dataset when distinguishing between TP53 wild-type and TP53 mutated samples. (A-B) Kaplan-Meier survival analysis for patients expressing high levels of miR-155 vs. low levels of miR-155 in samples that express wild-typeTP53 or harbor TP53 mutations that do not affect TP53 function (A), and in samples expressing mutated TP53 that affects TP53 function (B). (C-D) Kaplan-Meier survival analysis for patients expressing high levels of miR-155 and low levels of TP53 vs. low levels of miR-155 and high levels of TP53 in samples that express wild-type TP53 or harbor TP53 mutations that do not affect TP53 function (C), and in samples expressing mutated TP53 that affects TP53 function (D).

Purpose: To evaluate the effects of single-nucleotide polymorphisms (SNP) in microRNA-related genes on clinical outcomes in patients with colorectal cancer (CRC) receiving first-line fluoropyrimidine-based chemotherapy.Experimental Design: Forty-one SNPs in 26 microRNA-related genes were genotyped in 1,097 patients with CRC recruited at the University of Texas MD Anderson Cancer Center (Houston, TX). Patients were enrolled between 1990 and 2008 and last follow-up was in 2010. The associations between genotypes and recurrence-free survival (RFS), progression-free survival (PFS), and overall survival (OS) stratified by clinical stage were analyzed in 741 newly diagnosed patients (diagnosed within 1 year) and replicated the findings in an additional 356 patients.Results: In patients with stage III disease, mir608: rs4919510 was associated with increased risk for both recurrence [HR, 2.72; 95% confidence interval (CI), 1.38–5.33] and death (HR, 3.53; 95%CI, 1.42–8.73). The associations were confirmed in the replication set, and the combined HRs for training and replication sets were 1.65 (95% CI, 1.13–2.41) for recurrence and 1.96 (95% CI, 1.19–3.21) for death, respectively. The mir219-1:rs213210 showed consistent association with death in the training set (HR, 3.86; 95% CI, 1.33–11.22), the replication set (HR, 3.33; 95% CI, 1.39–7.98), and combined data set (HR, 3.22; 95% CI, 1.70–6.10). In combined analysis of these two SNPs, patients carrying the variant genotypes at both sites exhibited a 5.6-fold increased risk of death.Conclusion: Genetic polymorphisms in the microRNA pathway may predict prognosis in patients with stage III CRC treated with fluoropyrimidine-based chemotherapy. Clin Cancer Res; 18(14); 3982–91. ©2012 AACR.

PDF file - 1024K, Supplementary Figure 1. Purification of normal human B cell subset populations. Supplementary Figure 2. CLL miRNA expression profile is homogeneous and distinct from multiple myeloma and Sezary syndrome. Supplementary Figure 3. Quantitative PCR validation of miRNAs differentially expressed between CLL and normal memory B cells. Supplementary Figure 4. Expression of miR-29c* in M-CLL and UM-CLL. Supplementary Figure 5. Validation of some miRNAs differentially expressed in IGHV or FISH classes. Supplementary Figure 6. Cluster analysis of averaged miRNA expression in the different FISH subgroups of CLL.

Supplemental Figure Legends

Supplementary Figure S1 In situ hybridization of U6 in normal mucosae (n = 3, upper panel) and corresponding tumor tissue (n = 3, lower panel). Supplementary Figure S2 (A) MiR-188-3p is significantly up-regulated in matched cancer tissue compared to the adjacent non-cancerous mucosa (p

CCR Translation for This Article from Complex Patterns of Altered MicroRNA Expression during the Adenoma-Adenocarcinoma Sequence for Microsatellite-Stable Colorectal Cancer

The ataxia-telangiectasia mutated (ATM) protein kinase is widely known for its function as a chief mobilizer of the DNA damage response (DDR) upon DNA double-strand breaks. ATM orchestrates the DDR by modulating the expression of various miRNAs through several mechanisms. On the other hand, a set of miRNAs contribute to tight regulation of ATM by directly targeting the 3′-untranslated region of ATM mRNA. This review addresses the therapeutic application and molecular mechanisms that underlie the intricate interactions between miRNAs and ATM. It also describes therapeutic delivery of miRNAs in different environments such as hypoxic tumor microenvironments.

Legends for the supplementary Figures

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XLSX file - 248K, microRNAs exhibiting differential expression between CLL and normal antigen-experienced B cells.

Supplemental Figure 2. Cell membrane and cytoplasmic expression of IL-11R.

Supplementary Figure 3 from Alterations of the Tumor Suppressor Gene ARLTS1 in Ovarian Cancer

Supplementary Figure Legends - PDF file 70K, Supplementary Figure Legends