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Soft-tissue sarcomas (STS) are a rare and heterogeneous group of tumors that arise from connective tissue and can occur anywhere in the body. Among the plethora of over 50 different STS types, liposarcoma (LPS) is one of the most common. The subtypes of STS are characterized by distinct differences in tumor biology that drive responses to pharmacologic therapy and disparate oncologic outcomes. Small non-coding RNAs (sncRNA) are a heterogeneous class of regulatory RNAs involved in the regulation of gene expression by targeting mRNAs. Among the several types of sncRNAs, miRNAs and tRNA-derived ncRNAs are the most studied in the context of tumor biology, and we are learning more about the role of these molecules as important regulators of STS tumorigenesis and differentiation. However, challenges remain in translating these findings and no biomarkers or therapeutic approaches targeting sncRNAs have been developed for clinical use. In this review, we summarize the current landscape of sncRNAs in the context of STS with an emphasis on LPS, including the role of sncRNAs in the tumorigenesis and differentiation of these rare malignancies and their potential as novel biomarkers and therapeutic targets. Finally, we provide an appraisal of published studies and outline future directions to study sncRNAs in STS, including tRNA-derived ncRNAs.

Esophageal squamous cell carcinoma (ESCC) is a deadly disease with few prevention or treatment options. ESCC development in humans and rodents is associated with Zn deficiency (ZD), inflammation, and overexpression of oncogenic microRNAs: miR-31 and miR-21. In a ZD-promoted ESCC rat model with upregulation of these miRs, systemic antimiR-31 suppresses the miR-31-EGLN3/STK40-NF-κB–controlled inflammatory pathway and ESCC. In this model, systemic delivery of Zn-regulated antimiR-31, followed by antimiR-21, restored expression of tumor-suppressor proteins targeted by these specific miRs: STK40/EGLN3 (miR-31), PDCD4 (miR-21), suppressing inflammation, promoting apoptosis, and inhibiting ESCC development. Moreover, ESCC-bearing Zn-deficient (ZD) rats receiving Zn medication showed a 47% decrease in ESCC incidence vs. Zn-untreated controls. Zn treatment eliminated ESCCs by affecting a spectrum of biological processes that included downregulation of expression of the two miRs and miR-31-controlled inflammatory pathway, stimulation of miR-21-PDCD4 axis apoptosis, and reversal of the ESCC metabolome: with decrease in putrescine, increase in glucose, accompanied by downregulation of metabolite enzymes ODC and HK2. Thus, Zn treatment or miR-31/21 silencing are effective therapeutic strategies for ESCC in this rodent model and should be examined in the human counterpart exhibiting the same biological processes.

Although disruption of DNA repair capacity is unquestionably associated with cancer susceptibility in humans and model organisms, it remains unclear if the inherent tumor phenotypes of DNA repair deficiency syndromes can be regulated by manipulating DNA repair pathways. Loss-of-function mutations in BLM, a member of the RecQ helicase family, cause Bloom's syndrome (BS), a rare, recessive genetic disorder that predisposes to many types of cancer. BLM functions in many aspects of DNA homeostasis, including the suppression of homologous recombination (HR) in somatic cells. We investigated whether BLM overexpression, in contrast with loss-of-function mutations, attenuated the intestinal tumor phenotypes of ApcMin/+ and ApcMin/+;Msh2−/− mice, animal models of familial adenomatous polyposis coli (FAP). We constructed a transgenic mouse line expressing human BLM (BLM-Tg) and crossed it onto both backgrounds. BLM-Tg decreased adenoma incidence in a dose-dependent manner in our ApcMin/+ model of FAP, although levels of GIN were unaffected and concomitantly increased animal survival over 50%. It did not reduce intestinal tumorigenesis in ApcMin/+;Msh2−/− mice. We used the pink-eyed unstable (pun) mouse model to demonstrate that increasing BLM dosage in vivo lowered endogenous levels of HR by 2-fold. Our data suggest that attenuation of the Min phenotype is achieved through a direct effect of BLM-Tg on the HR repair pathway. These findings demonstrate that HR can be manipulated in vivo to modulate tumor formation at the organismal level. Our data suggest that lowering HR frequencies may have positive therapeutic outcomes in the context of specific hereditary cancer predisposition syndromes, exemplified by FAP. Cancer Prev Res; 8(7); 650–6. ©2015 AACR.

Supplementary Figure S1. qPCR discriminates between mice hemizygous and homozygous for the BLM-Tg.

Osteosarcoma is a highly metastatic form of bone cancer in adolescents and young adults that is resistant to existing treatments. Development of an effective therapy has been hindered by very limited understanding of the mechanisms of osteosarcomagenesis. Here, we used genetically engineered mice to investigate the effects of deleting the tumor suppressor Wwox selectively in either osteoblast progenitors or mature osteoblasts. Mice with conditional deletion of Wwox in preosteoblasts (WwoxΔosx1) displayed a severe inhibition of osteogenesis accompanied by p53 upregulation, effects that were not observed in mice lacking Wwox in mature osteoblasts. Deletion of p53 in WwoxΔosx1 mice rescued the osteogenic defect. In addition, the Wwox;p53Δosx1 double knockout mice developed poorly differentiated osteosarcomas that resemble human osteosarcoma in histology, location, metastatic behavior, and gene expression. Strikingly, the development of osteosarcomas in these mice was greatly accelerated compared with mice lacking p53 only. In contrast, combined WWOX and p53 inactivation in mature osteoblasts did not accelerate osteosarcomagenesis compared with p53 inactivation alone. These findings provide evidence that a WWOX–p53 network regulates normal bone formation and that disruption of this network in osteoprogenitors results in accelerated osteosarcoma. The Wwox;p53Δosx1 double knockout establishes a new osteosarcoma model with significant advancement over existing models. Cancer Res; 76(20); 6107–17. ©2016 AACR.

Supplemental Tables are: ST1: Detailed information about DDLPS patients; ST2: Detailed information about healthy control; ST3: MDM2 primer sequences; ST4: DDLPS patients information; ST5: Healthy control clinical information; ST6: RT-PCR quality control on isolated EVs; ST7: Cell cycle distribution analysis regarding P-a incubated with Lipo246-EVs; ST8: Additional DDLPS patients information; ST9: Additional healthy control clinical information.

MicroRNAs are strongly implicated in such processes as development, carcinogenesis, cell survival, and apoptosis. It is likely, therefore, that they can also modulate sensitivity and resistance to anticancer drugs in substantial ways. To test this hypothesis, we studied the pharmacologic roles of three microRNAs previously implicated in cancer biology (let-7i, mir-16, and mir-21) and also used in silico methods to test pharmacologic microRNA effects more broadly. In the experimental system, we increased the expression of individual microRNAs by transfecting their precursors (which are active) or suppressed the expression by transfection of antisense oligomers. In three NCI-60 human cancer cell lines, a panel of 60 lines used for anticancer drug discovery, we assessed the growth-inhibitory potencies of 14 structurally diverse compounds with known anticancer activities. Changing the cellular levels of let-7i, mir-16, and mir-21 affected the potencies of a number of the anticancer agents by up to 4-fold. The effect was most prominent with mir-21, with 10 of 28 cell-compound pairs showing significant shifts in growth-inhibitory activity. Varying mir-21 levels changed potencies in opposite directions depending on compound class; indicating that different mechanisms determine toxic and protective effects. In silico comparison of drug potencies with microRNA expression profiles across the entire NCI-60 panel revealed that ∼30 microRNAs, including mir-21, show highly significant correlations with numerous anticancer agents. Ten of those microRNAs have already been implicated in cancer biology. Our results support a substantial role for microRNAs in anticancer drug response, suggesting novel potential approaches to the improvement of chemotherapy. [Mol Cancer Ther 2008;7(1):1–9]

TCGA TNBC samples

Supplementary Figure S1. Effects of combination treatment of both bortezomib and oHSV on cancer cell killing. Supplementary Figure S2. Necrostatin-1 (Nec-1) treatment and RIPK1 knock down did not reduce the bortezomib-induced increase in virus replication. Supplementary Figure S3. Single treatment with bortezomib or oHSV enhances NK cell mediated glioma killing. Supplementary Figure S4. Combination treatment with bortezomib and oHSV enhance mice survival in vivo.

Supplementary Methods, Figures and Tables

Supplemental Figures are: SF1: Determination of number of molecules of MDM2 in the serum-EVs using Standard Curve Methodology. SF2: Quality control of cell line-isolated vesicles, performed through nanosight and western blot analyses. SF3: Nanosight analysis performed on serum-derived EVs. SF4: Original film corresponding to Figure 3. SF5: WB showing a compensatory increase in MDM2 expression depending on SAR405838 treatment. SF6: WB showing MDM2 protein levels in Lipo863 and Lipo863 OE. SF7: Number of MDM2 molecules assessed in healthy serum EVs versus DDLPS serum EVs.

Purpose: We conducted genome-wide miRNA-sequencing (miRNA-seq) in primary cancer tissue from patients of lung adenocarcinoma to identify markers for the presence of lymph node metastasis.Experimental Design: Markers for lymph node metastasis identified by sequencing were validated in a separate cohort using quantitative PCR. After additional validation in the The Cancer Genome Atlas (TCGA) dataset, functional characterization studies were conducted in vitro.Results: MiR-31 was upregulated in lung adenocarcinoma tissues from patients with lymph node metastases compared with those without lymph node metastases. We confirmed miR-31 to be upregulated in lymph node-positive patients in a separate patient cohort (P = 0.009, t test), and to be expressed at higher levels in adenocarcinoma tissue than in matched normal adjacent lung tissues (P < 0.0001, paired t test). MiR-31 was then validated as a marker for lymph node metastasis in an external validation cohort of 233 lung adenocarcinoma cases of the TCGA (P = 0.031, t test). In vitro functional assays showed that miR-31 increases cell migration, invasion, and proliferation in an ERK1/2 signaling-dependent manner. Notably, miR-31 was a significant predictor of survival in a multivariate cox regression model even when controlling for cancer staging. Exploratory in silico analysis showed that low expression of miR-31 is associated with excellent survival for T2N0 patients.Conclusions: We applied miRNA-seq to study microRNomes in lung adenocarcinoma tissue samples for the first time and potentially identified a miRNA predicting the presence of lymph node metastasis and survival outcomes in patients of lung adenocarcinoma. Clin Cancer Res; 19(19); 5423–33. ©2013 AACR.

Supplementary Figure Legend

PDF file, 34K, Supplementary Table 1. Clinical demographic information of 43 lung ADC cases. NAT is normal adjacent tissue. Supplementary Table 2. Clinical demographic information of lung adenocarcinoma TCGA patients. Supplementary Table 3. IPA analysis for the downstream targets for miR-31. Supplementary Table 4. Genetic alteration overview of the cell lines used in this study. Supplementary Table 5. Correlation coefficient between EMT-related genes and miR-31 expression.

Nanostring microRNAs data

Dedifferentiated liposarcoma (DDLPS) is frequently diagnosed late, and patients typically respond poorly to treatments. DDLPS is molecularly characterized by wild-type p53 and amplification of the MDM2 gene, which results in overexpression of MDM2 protein, a key oncogenic process in DDLPS. In this study, we demonstrate that extracellular vesicles derived from patients with DDLPS or from DDLPS cell lines are carriers of MDM2 DNA that can be transferred to preadipocytes, a major and ubiquitous cellular component of the DDLPS tumor microenvironment, leading to impaired p53 activity in preadipocytes and increased proliferation, migration, and production of matrix metalloproteinase 2; treatment with MDM2 inhibitors repressed these effects. Overall, these findings indicate that MDM2 plays a crucial role in DDLPS by enabling cross-talk between tumor cells and the surrounding microenvironment and that targeting vesicular MDM2 could represent a therapeutic option for treating DDLPS.Significance:Extracellular vesicles derived from dedifferentiated liposarcoma cells induce oncogenic properties in preadipocytes.

Supplementary Table S1: Molecular and Pathological variables of patients included in the discovery cohort.

Supplementary Data from Pan-Cancer Analysis of Canonical and Modified miRNAs Enhances the Resolution of the Functional miRNAome in Cancer

Purpose: The dismal outcome of esophageal cancer patients highlights the need for novel prognostic biomarkers, such as microRNAs (miRNA). Although recent studies have established the role of miRNAs in esophageal carcinoma, a comprehensive multicenter study investigating different histologic types, including squamous cell carcinoma (SCC) and adenocarcinoma with or without Barrett's, is still lacking.Experimental Design: miRNA expression was measured in cancerous and adjacent noncancerous tissue pairs collected from 100 adenocarcinoma and 70 SCC patients enrolled at four clinical centers from the United States, Canada, and Japan. Microarray-based expression was measured in a subset of samples in two cohorts and was validated in all available samples.Results: In adenocarcinoma patients, miR-21, miR-223, miR-192, and miR-194 expression was elevated, whereas miR-203 expression was reduced in cancerous compared with noncancerous tissue. In SCC patients, we found elevated miR-21 and reduced miR-375 expression levels in cancerous compared with noncancerous tissue. When comparing cancerous tissue expression between adenocarcinoma and SCC patients, miR-194 and miR-375 were elevated in adenocarcinoma patients. Significantly, elevated miR-21 expression in noncancerous tissue of SCC patients and reduced levels of miR-375 in cancerous tissue of adenocarcinoma patients with Barrett's were strongly associated with worse prognosis. Associations with prognosis were independent of tumor stage or nodal status, cohort type, and chemoradiation therapy.Conclusions: Our multicenter-based results highlight miRNAs involved in major histologic types of esophageal carcinoma and uncover significant associations with prognosis. Elucidating miRNAs relevant to esophageal carcinogenesis is potentially clinically useful for developing prognostic biomarkers and identifying novel drug targets and therapies. (Clin Cancer Res 2009;15(19):6192–200)

Purpose: Inflammatory genes and microRNAs have roles in colon carcinogenesis; therefore, they may provide useful biomarkers for colon cancer. This study examines the potential clinical utility of an inflammatory gene expression signature as a prognostic biomarker for colon cancer in addition to previously examined miR-21 expression.Experimental Design: Quantitative reverse transcriptase-PCR. was used to measure the expression of 23 inflammatory genes in colon adenocarcinomas and adjacent noncancerous tissues from 196 patients. These data were used to develop models for cancer-specific mortality on a training cohort (n = 57), and this model was tested in both a test (n = 56) and a validation (n = 83) cohort. Expression data for miR-21 were available for these patients and were compared and combined with inflammatory gene expression.Results: PRG1, IL-10, CD68, IL-23a, and IL-12a expression in noncancerous tissue, and PRG1, ANXA1, IL-23a, IL-17a, FOXP3, and HLA-DRA expression in tumor tissues were associated with poor prognosis based on Cox regression (|Z-score| >1.5) and were used to generate the inflammatory risk score (IRS). IRS was associated with cancer-specific mortality in the training, test (P = 0.01), and validation (P = 0.02) cohorts. This association was strong for stage II cases (P = 0.002). Expression of miR-21 was associated with IL-6, IL-8, IL-10, IL-12a, and NOS2a, providing evidence that the function of this microRNA and these inflammatory genes are linked. Both IRS and miR-21 expression were independently associated with cancer-specific mortality, including stage II patients alone.Conclusion: IRS and miR-21 expression are independent predictors of colon cancer prognosis and may provide a clinically useful tool to identify high-risk patients. (Clin Cancer Res 2009;15(18):5878–87)

Supplementary Data from Association of Inflammation-Related and microRNA Gene Expression with Cancer-Specific Mortality of Colon Adenocarcinoma

Supplementary Figure from Pan-Cancer Analysis of Canonical and Modified miRNAs Enhances the Resolution of the Functional miRNAome in Cancer

Figure S1. GFP-LC3 autophagosome puncta in MEF-iRas cells. Figure S2. X-gal staining for examining cell senescence. Figure S3. Comparing MEF-iRas, HOSE-iRas and MCF7-iRas cells for autophagic cell death. Figure S4. Cell proliferation and apoptosis in transformed MEFs with/without PTEN knockdown. Figure S5. The positions of PTEN sgRNAs in PTEN cDNA. Figure S6. Global somatic alterations of miR-130a/miR-130b. Figure S7. Correlation analysis between miR-130a and PTEN in breast cancer subtypes. Figure S8. The expression of miR-130a/miR-130b and PTEN in PR-positive and -negative breast cancer.

Supplementary Data from MicroRNA Expression in Squamous Cell Carcinoma and Adenocarcinoma of the Esophagus: Associations with Survival

Supplementary Figure 1: Relapse-free survival (RFS) analysis for WWOX in KM plotter. Supplementary Figure 2: Relapse-free survival (RFS) analysis for the WWOX gene in TCGA dataset. Supplementary Figure 3: Boxplots for each comparison of the expression of the specific gene between WWOX+ (>75th percentile) and WWOX- (

Epitranscriptomic studies of miRNAs have added a new layer of complexity to the cancer field. Although there is fast-growing interest in adenosine-to-inosine (A-to-I) miRNA editing and alternative cleavage that shifts miRNA isoforms, simultaneous evaluation of both modifications in cancer is still missing. Here, we concurrently profiled multiple miRNA modification types, including A-to-I miRNA editing and shifted miRNA isoforms, in >13,000 adult and pediatric tumor samples across 38 distinct cancer cohorts from The Cancer Genome Atlas and The Therapeutically Applicable Research to Generate Effective Treatments data sets. The differences between canonical miRNAs and the wider miRNAome in terms of expression, clustering, dysregulation, and prognostic standpoint were investigated. The combination of canonical miRNAs and modified miRNAs boosted the quality of clustering results, outlining unique clinicopathologic features among cohorts. Certain modified miRNAs showed opposite expression from their canonical counterparts in cancer, potentially impacting their targets and function. Finally, a shifted and edited miRNA isoform was experimentally validated to directly bind and suppress a unique target. These findings outline the importance of going beyond the well-established paradigm of one mature miRNA per miRNA arm to elucidate novel mechanisms related to cancer progression.Significance:Modified miRNAs may act as cancer biomarkers and function as allies or antagonists of their canonical counterparts in gene regulation, suggesting the concurrent consideration of canonical and modified miRNAs can boost patient stratification.

PDF file, 450K, Supplementary Figure 1. The length distribution of reads for ten miR-seq samples Supplementary Figure 2. Flow diagram illustrating microRNA quantification techniques used and sample sizes of each step of the analysis. NAT: normal adjacent tissue. Supplemental Figure 3. Identification of differentially expressed miRNAs between N0 and N1+ groups of patients. Supplementary Figure 4. The receiver operator characteristic (ROC) curve as well as area under curve (AUC) was determined using CART. The results show that AUC is 0.79 in 43 lung ADC samples (cohort 1 +2). Supplementary Figure 5. Higher miR-31 expression is associated with adverse outcome in T2N0 patients (n=75, log-rank p=0.0194) Supplementary Figure 6. IPA showing the top five canonical pathways for miR-31 target genes is the CDK5 (A), PTEN (B), p70S6K (C) , ERK/MAPK (D) and PI3K/AKT (E)signaling. Supplementary Figure 7. Ectopic expression of miR-31 increases migration/invasion capabilities of H2228 cells. (A) Expression of miR-31 following infection with Lenti-miR vector containing miR-31 precursor was confirmed by TaqMan real-time PCR. (B) Cell invasion assay for miR-31 overexpressing H2228 cells. (C) Cell migration assay for miR-31 overexpressing H2228 cells using transwell membranes (upper panel, representative pictures of migration chambers; bottom panel, average counts from 10 random microscopic fields). The average counts were derived from ten random microscopic fields. (D) Cell proliferation assay for miR-31 overexpressing H2228 cells. Data are presented as mean plus-minus S.D. *P

Supplementary Figure S1. Validation of differentially expressed miRs in the discovery dataset. Supplementary Figure S2. Design of a recurrence prediction model in HNSCC based on miRs expression using a bioinformatic approach. Supplementary Figure S3. In vitro validation of miR-9 putative targets Supplementary Figure S4. Validation of putative miRs seed sites in the 3'-UTR of SASH1 and SP1 genes Supplementary Figure S5. The combined expression of miRs-1, 133a and -150 is necessary to downregulate SP1 mRNA and protein levels. Supplementary Figure S6. Correlation analyses of miRs putative targets in primary HNSCC collected in our Institute and described in Supplementary table S2 as validation set. Supplementary Figure S7. Prognostic significance of 4 miRs-targets in the TCGA HNSCC dataset. Supplementary Table S3. Median miRs' expression (qRT-PCR) according to selected characteristics Supplementary Experimental Procedures

Supplementary Table S1. Detailed information about patients (A) and control group (B)used in the discovery set; Supplementary Table S2. Detailed information about patients (A) and control group (B) used in the validation set; Supplementary Table S3. Detailed information about cancer tissue (A) and normal adjacent tissue (B) analyzed; Supplementary Table S4. Detailed information about additional patients whose tissues have been analyzed by immunohistochemistry (not previously reported); Supplementary Figure S1. Peripheral blood-derived EVs characterization (Nanosight); Supplementary Figure S2. LPS- secreted EVs characterization (Nanosight); Supplementary Figure S3. Expression levels of miR-25-3p and miR-92a-3p in EVs and LPS tissues; Supplementary Figure S4. Western blot analysis for phospho-p65; Supplementary Figure S5. miR-25-3p and miR-92a-3p stimulate the secretion of TNFα from macrophages

Advances in the understanding of cancer cell biology and response to drug treatment have benefited from new molecular technologies and methods for integrating information from multiple sources. The NCI-60, a panel of 60 diverse human cancer cell lines, has been used by the National Cancer Institute to screen >100,000 chemical compounds and natural product extracts for anticancer activity. The NCI-60 has also been profiled for mRNA and protein expression, mutational status, chromosomal aberrations, and DNA copy number, generating an unparalleled public resource for integrated chemogenomic studies. Recently, microRNAs have been shown to target particular sets of mRNAs, thereby preventing translation or accelerating mRNA turnover. To complement the existing NCI-60 data sets, we have measured expression levels of microRNAs in the NCI-60 and incorporated the resulting data into the CellMiner program package for integrative analysis. Cell line groupings based on microRNA expression were generally consistent with tissue type and with cell line clustering based on mRNA expression. However, mRNA expression seemed to be somewhat more informative for discriminating among tissue types than was microRNA expression. In addition, we found that there does not seem to be a significant correlation between microRNA expression patterns and those of known target transcripts. Comparison of microRNA expression patterns and compound potency patterns showed significant correlations, suggesting that microRNAs may play a role in chemoresistance. Combined with gene expression and other biological data using multivariate analysis, microRNA expression profiles may provide a critical link for understanding mechanisms involved in chemosensitivity and chemoresistance. [Mol Cancer Ther 2007;6(5):1483–91]